The Myocardial Cell: Structure, Function, and Modification by Cardiac Drugs [Reprint 2016 ed.] 9781512814798

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The Myocardial Cell Structure, Function, and Modification by Cardiac Drugs

Heart Association of Southeastern Pennsylvania Third International Symposium

The Myocardial Cell Structure, Function, and Modification by Cardiac Drugs

EDITED B Y STANLEY A. BRILLER AND HADLEY L. CONN, JR.

Philadelphia University of Pennsylvania Press

© 1966 by the Trustees of the University of Pennsylvania

Published in Great Britain, India, and Pakistan by the Oxford University Press London, Bombay, and Karachi

Library of Congress Catalogue Card Number : 66 - 1 0 8 69

7516 Printed in the United States of America

Foreword FOR THE PAST S E V E R A L YEARS, T H E HEART ASSOCIATION OF SOUTH-

EASTERN Pennsylvania has sponsored symposia dealing with a variety of topics in the field of the circulation. T h e third international symposium was devoted to the structure and function of the myocardial cell and to the action of cardiac drugs at the cellular level. T h e lectures were enthusiastically received and discussed by a large audience, a considerable number of whom requested a printed record of the proceedings. This monograph is intended to meet those requests and to provide a means of disseminating the material to those who could not attend the meeting. The present editors, chairmen of that meeting, were fortunate to be able to secure definitive manuscripts of the topics presented from all but two of the original speakers. T h e authors have supplemented their original presentations and have cast them in a format more suitable for inclusion in a monograph. New material from additional authors has been included in this volume as a means of covering all the subjects originally explored at the meeting and in order to expand consideration of the cellular actions of the common cardiac drugs. Unquestionably the disciplines of Biophysics and Biochemistry have been responsible for considerable progress in accounting for the structure and function of, and pharmacologic effects on, the myocardium at a nearly molecular level. T h e prospect of future growth along these lines is exciting to many. Our hope is that this monograph, and the symposium from which it grew, will stimulate such growth. This volume contains a fund of current information that is now widely scattered in the literature but which, considered as an integrated whole, should provoke new ideas and experimental approaches to existing problems. The Heart Association of Southeastern Pennsylvania and the editors are deeply indebted to the contributors for the time and effort taken from the rigors of busy schedules for purposes of our enlightenment.

List of Contributors Robert M. Berne, M.D., Professor of Physiology, Western Reserve University, Cleveland, Ohio. H a d l e y L . Conn, Jr., M.D., Professor of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. Francis D. Carlson, Ph.D., Professor of Biophysics, Johns Hopkins University, Baltimore, Maryland. Robert E. Davies, Ph.D., Chairman, Department of Animal Biology, Professor of Biochemistry, Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Brian F. Hoffman, M.D., Professor and Chairman, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York. Richard D. Keynes, Ph.D., F.R.S., Deputy Director, Institute of Animal Physiology, Babraham, Cambridge, England. Ralph Lazzara, M.D., Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York.

Robert J. Luchi, M.D., Assistant Professor of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. Neil C. Moran, M.D., Professor and Chairman, Department of Pharmacology, Emory University, Atlanta, Georgia. Edmund H. Sonnenblick, M.D., Senior Investigator, Cardiology Branch, National Heart Institute, Bethesda, Maryland. Donald H. Singer, M.D., Assistant Professor of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York. David Spiro, M.D., Ph.D., Professor of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York. Daniel C. Tosteson, M.D., Professor and Chairman, Department of Physiology, Duke University School of Medicine, Durham, N. Carolina. Annemarie Weber, M.D., Associate Member, Institute for Muscle Disease Inc., New York, New York. W. Wilbrandt, M.D., Professor of Pharmacology. Head, Department of Pharmacology, University of Bern, Bern, Switzerland.

Contents Foreword

5

T h e F i n e S t r u c t u r e a n d C o n t r a c t i l e M e c h a n i s m of H e a r t Muscle, by David Spiro, M . D . , P h . D .

13

T h e F u n c t i o n of the Cell M e m b r a n e , by D r . R. D . K e y n e s

63

T r a n s m e m b r a n e Potentials of C a r d i a c Cells a n d T h e i r Ionic Basis, by D o n a l d H. Singer, R a l p h L a z z a r a , and Brian F. H o f f m a n

73

E n e r g y Sources in Ionic M o v e m e n t s , by Daniel C . Tosteson, M.D.

Ill

T h e Role of C a in the R e g u l a t i o n of M u s c l e Activity, by A n n e m a r i e W e b e r

131

T h e r m o c h e m i c a l A s p e c t s of Muscle C o n t r a c t i o n , by F r a n c e s D. C a r l s o n , P h . D .

145

T h e Role of A T P in C o n t r a c t i o n , by R. E . Davies

157

T h e M e c h a n i c s of M y o c a r d i a l C o n t r a c t i o n , by E d m u n d H. S o n n e n b l i c k , M . D .

173

T h e Possible M o d e of A c t i o n of A n t i a r r h y t h m i c A g e n t s , by Brian F. H o f f m a n , M . D .

251

Some C o n s i d e r a t i o n s of Q u i n i d i n e and P r o c a i n e A m i d e A c t i o n at the Cellular Level, by H a d l e y L. C o n n , Jr., M . D .

269

T h e M e c h a n i s m of A c t i o n of C a r d i a c Glycosides, by W . W i l b r a n d t

297

Some Biochemical R e a c t i o n s Influenced by the Digitalis Glycosides, by R o b e r t J. Luchi, M . D .

309

N e u r o h u m o r a l A g e n t s — A P h a r m a c o l o g i c a l Analysis of the Actions of C a t e c h o l a m i n e s u p o n the H e a r t , by Neil C. M o r a n , M . D .

331

A g e n t s M o d i f y i n g M y o c a r d i a l Blood F l o w , by R o b e r t M . Berne, M . D .

355

The Myocardial Cell Structure, Function, and Modification by Cardiac Drugs

The Fine Structure and Contractile Mechanism of Heart Muscle D A V I D SPIRO, M.D., Ph.D. Department of Pathology College of Physicians and Surgeons of Columbia New York, New York

University,

SEVERAL REVIEWS DEALING W I T H THE F I N E S T R U C T U R E O F HEART

muscle have recently appeared (1, 2, 3 ) . This paper will therefore emphasize some of the more recent advances correlating fine structure with function. Mammalian myocardium is largely a muscular tissue and is composed of elongated muscle cells arranged in columns. At the light microscope level these myocardial cells have centrally located nuclei and a cross striated appearance which is due to the organization of the contractile units or myofibrils (Fig. 1). The myofibrils are oriented parallel to the long axis of the cells and exhibit a repeating band pattern which accounts for the cross striations. The repeating unit of the myofibril is the sarcomere, its longitudinal limits being two adjacent Z lines. The sarcomere is subdivided into dark central A bands (which are strongly birefrigent Supported in part by the General Research Support Grant, Grant H - 5 9 0 6 of the United States Public Health Service and by the Health Research Council of the City of N e w York Grant U - 1 0 7 5 .

13

THE MYOCARDIAL

14

CELL

mm

uM>

Figure 1. Phase contrast photomicrograph. This figure reveals the characteristic cross-striated band pattern of heart muscle. The broad dark lines represent the A band: the light zones, the I bands; and the narrow dark lines transecting the I bands, the Z lines. A centrally located heart muscle nucleus (N) is present. Several intercalated discs (unlabelled arrows) are seen traversing the muscle fibers. The apparent branching of fibers is also noted. Mag. X 1000.

or anisotropic under polarized light) and lighter I bands (which are relatively non-birefrigent or isotropic under polarized light) located between either end of the A band and the Z lines (Figs, 1, 2,4-7,9-11). The elongated surfaces of the heart muscle cell are invested by the sarcolemma consisting of an external layer of basement

THE

MYOCARDIAL

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15

Figure 2. This figure and all subsequent micrographs are electron micrographs. The cell surfaces of two adjacent heart muscle cells are seen at the top of the figure. The cell surface membranes and the closely apposed external basement membranes (arrows) which together form the sarcolemma, are evident. Numerous vesicular structures, some continuous with the cell-lining membrane, are seen near the surface. Many mitochondria (M) are also noted adjacent to the longitudinally oriented myofibrils (myo.) Mag. X 49,000. Insert in lower left hand corner discloses a portion of cell membrane at high resolution, demonstrating the triple-layered or unit membrane structure consisting of an inner and outer dense line plus a less dense central zone. Mag. X 120,000. (Courtesy of Dr. Joseph Wiener)

16

THE MYOCARDIAL

CELL

membrane in addition to the cell surface membrane (Fig. 2 ) . T h e latter displays the typical tri-layered unit membrane structure which reflects the macromolecular organization of all cell membranes (4, 5, 6 ) (Fig. 2 inset and Fig. 3 ) . Numerous membrane limited vesicles which appear to be derived from the cell surface membrane are present in the peripherial portions of the cell (Fig. 2 ) . These vesicles are probably involved in the bulk transport of certain solutes into the cell, a process which is termed pinocytosis ( 7 ) . The cell membrane also participates in the active transport of various ions and substrates and in the electrical phenomena associated with myocardial excitation ( 8 , 9, 10, 1 1 ) . Various drugs may have the surface membrane as their sites of action ( 1 2 ) . Numerous capillaries, abundant collagen fibers and occasional unmyelinated nerve fibers are present between the columns of muscle cells. Heart muscle is not a syncytium from the structural point of view. T h e individual muscle cells measure approximately 10-20« in diameter and 50-100M in length. The so-called intercalated discs which are more or less perpendicular to the long axis of the muscle

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Figure 3. Schematic representation of the molecular organization of a cell membrane, or so-called "unit membrane." (After J. D. Robertson)

THE MYOCARDIAL

CELL

17

columns, delimit the longer dimensions of individual myocardial cells. These intercalated discs consist of the two cell surface membranes which invest the ends of two adjacent muscle cells within the same column, as well as an intercellular space ( 1 3 , 14, 15, 16, 1 7 ) (Figs. 1, 4 - 6 ) . These surface membranes are, of course, similar in structure to and continuous with the sarcolemmal cell membrane.

Figure 4. An intercalated disc is seen coursing in an irregular fashion from the lower right hand portion of the figure to the upper left. Two types of disc structure are present. The first consists of two adjacent cells separated by an intercellular space of variable width plus an associated electron-dense deposit (I). The second variant consists of rather dense, closely approximated cell membranes (II). Mag. X 49,000.

18

THE MYOCARDIAL

CELL

A l o n g the m a j o r portion of the discs the two apposing cell membranes are separated by an intercellular space which varies in width ( I in Figs. 4 - 6 ) . Dense cytoplasmic material adjacent to the cell m e m b r a n e s is present in these portions of the intercalated disc. A t other regions along the discs the extracellular space is very narrow and uniform in width ( I I in Figs. 4 and 6 ) . M o r e recently even more intimate contact between adjacent cells has been observed where there is actual fusion of the two cell membranes with obliteration of the intercellular space ( 1 8 ) . The basement membranes of the sarcolemma do not penetrate the intercellular space of the intercalated discs. T h e areas of close contact between the cell membrans of the intercalated disc probably account for the relatively low electrical impedance between adjacent muscle cells ( 9 ) . Paired

Figure 5. The first type of disc structure is shown (I). In addition, a desmosome (D), representing a third variant of disc structure is noted in the upper left. Mag. X 66,000.

THE MYOCARDIAL

CELL

19

dense bodies known as desmosomes which are believed to aid in maintaining cell to cell contacts are also f o u n d along the intercalated discs (Fig. 5 ) . It is of interest that even with marked degrees of muscle stretch, the integrity of the intercalated discs is maintained. N u m e r o u s mitochondria with a b u n d a n t cristae are present between and in close apposition to the myofibrills ( 1 9 ) (Figs. 2. 4 - 1 0 ) . These mitochondria are, of course, the m a j o r site of the energy yielding reactions of intermediary metabolism which result in the synthesis of adenosine triphosphate ( A T P ) , the ubiquitous energy source for various cell functions ( 2 0 , 2 1 ) . T h e large n u m b e r of highly structured mitochondria reflects the very active oxidative

Figure 6. The first two types of intercalated disc structure (I and II) are shown. Note that the second variant (II) is usually oriented parallel to the long axis of the myofibrils. Mag. X 38,000.

20

THE MYOCARDIAL

CELL

Figure 7. Numerous mitochondria (M) are seen in close approximation to the myofibrils (myo). Note the numerous internal mitochondrial membranes or cristae. In addition, intermediary vesicles (IV) and associated tubules (arrows) of the longitudinal component of the sarcoplasmic reticulum are present. A is A band and Z is Z line. Mag. X 56,000.

THE

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21

metabolism of myocardium ( 1 9 ) . Abundant glycogen granules and occasional fat droplets within the sarcoplasm represent substrate stores for the metabolic reactions. Heart muscle like skeletal muscle has an extensive intracytoplasmic system of membrane limited channels known as the sarcoplasmic reticulum (16, 17, 22, 23) (Figs. 7-10). The sarcoplasmic reticulum is a two component system. The first component known as the transverse or T system consists of deep invaginations of the sarcolemmal cell surface membrane (24, 25, 26, 2 7 ) . These invaginations are located at the level of the Z lines of the sarcomere and penetrate deeply into the sarcoplasm between the myofibrils. The second component of the sarcoplasmic reticulum, known as the longitudinal component, consists of a system of anastomosing channels intimately engirdling the myofibrils, and is limited to one sarcomere (Figs. 9 and 10). Thus there is no continuity between the longitudinal systems of adjacent sarcomeres across the Z lines. The tubules of the longitudinal system are in close apposition to those of the T system at the Z line level. Frequently an expanded T system tubule known as an intermediary vesicle is flanked by two longitudinal tubules, one from each sarcomere (Figs. 7 and 10). Such complexes are known as triads ( 2 2 ) . It should be emphasized that while the two systems are in close proximity they do not intercommunicate. Two very important functions have been demonstrated for the sarcoplasmic reticulum. The first of these is a function of the T system and concerns excitation—contraction coupling, providing for almost simultaneous activation of all the contractile elements in the cell ( 2 8 ) . The sarcoplasmic reticulum of heart muscle is less well developed than that of certain types of skeletal muscle (16, 17, 2 5 ) . This is probably related to the relatively small dimensions of heart muscle cells which do not therefore require as elaborate a system for internal activation. The second function which may be a property of the longitudinal component is relaxation of the contractile system (29, 30, 31, 32, 33, 3 4 ) . This relaxing function appears to result from the active accumulation and sequestration of calcium by the sarcoplasmic reticulum (35, 36, 37, 38, 39, 40, 4 1 ) . Recent evidence suggests that cardiac glycosides are also bound by the sarcoplasmic reticulum ( 4 2 ) .

22

THE MYOCARDIAL

CELL

Figure 8. Transverse section of heart muscle which shows the profiles of the myofibrils (myo) and mitochondria (M) in this plane. Note concentric arrangement of the cristae and the double peripheral membranes which delimit the mitochondria. Arrow points to an intermediary vesicle and associated tubule of the longitudinal sarcoplasmic reticulum (SR). Mag. X 32,000

Other structures which are found in heart muscle cells include secretion droplets of unknown nature and lipofuscin or lysosomal bodies ( 4 3 , 2 ) . As mentioned previously the myofibrils are the contractile elements and contain the contractile proteins. The band pattern of the myofibrils reflects the distribution of these contracile proteins.

THE MYOCARDIAL

CELL

23

Figure 9. Tubular components of the longitudinal sarcoplasmic reticulum are noted. These are best seen in areas where the plane of section has just grazed the myofibril (arrows). I is I band. Mag. X 39,000. T h e sarcomere, the repeating unit of the myofibril (delineated longitudinally by two adjacent Z lines) (Fig. 11) is the f u n d a m e n tal unit of contraction and its length is a f u n c t i o n of overall muscle length. In heart muscle fixed at diastolic length, sarcomeres measure 2.2u, the A b a n d 1.5«. and the I b a n d s 0.35M each ( 4 4 ) . T h e A b a n d is bisected by a central dark line, the M line on either side of which is a light line or L line ( 4 5 ) (Fig. 1 1 ) . T h e M line together with the two L lines comprises the M - L complex which measures a b o u t 0 . 2 u in length. T h e myofibrils are composed of subunits or myofilaments which lie parallel to the long axis of the myofibrils. T h e structural organization of the myofibril was definitively established for skeletal

24

THE MYOCARDIAL

CELL

Figure 10. The intímate relationship between the myofibrils and encircling tubules of the sarcoplasmic reticulum is seen (arrow). A small intermediary vesicle (IV) with adjacent tubular elements is also present. Mag. X 40,000. muscle by H. E. Huxley (31, 4 6 ) . Subsequently it was shown to be essentially similar for heart muscle by Stenger and Spiro (1, 17). The sarcomeres of both heart and skeletal muscle consist of two different sets of filaments which show varying degrees of overlap dependent on muscle and therefore on sarcomere length. Thick filaments measuring 100A in diameter are present within the confines of the A band (Figs. 12-14). The length of the thick filaments which defines the length of the A band is 1.5u. The second set of filaments are thinner measuring about 60 A in diameter. These

THE MYOCARDIAL

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25

Figure 11. The band patterns of several myofibrils are illustrated. The bands of the central sarcomere (Z to Z lines) are labeled. The M line shows lateral interconnections between filaments. The paired light lines adjacent to the M line are the L lines. M-L is the complex consisting of the central M and the paired L lines. The dark central A band measures 1.5 microns. Mag. X 33,000.

Figure 12. High resolution electron micrograph showing filamentous fine structure of several heart muscle sarcomeres. Only in the A band are both thick and thin filaments (arrows) evident. In most areas, pairs of thin filaments are present between the thick filaments. However, occasionally individual thin filaments are interposed between thick ones. The M-L complex contains only thick filaments. Mag. X 76,000.

Figure 13. As in Fig. 12, single or pairs of thin filaments are seen interdigitated between thick filaments in the A band. Mag. X 76,000.

28

THE MYOCARDIAL

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thin filaments which are 1M in length extend from the Z lines through the I bands and into the A bands where they are interposed between the thick A band filaments. The thin filaments in muscle at diastolic length do not penetrate the center of the A band but stop short of the edge of the M-L complex (Figs. 1 2 - 1 4 ) . Thus there is a partial overlapping of thin and thick filaments within the A band. The I band contains only thin filaments, the A band exclusive of its central M - L complex contains both thick and thin filaments and the M - L complex only thick filaments. The dark M line in the center of the sarcomere is the result of lateral interconnections or bridges between the thick filaments in this region ( 4 7 , 4 8 ) (Fig. 1 1 ) . Huxley has demonstrated another type of bridge connection between filaments in the A bands of skeletal muscle ( 3 1 , 4 6 ) . These latter bridges are of great importance and extend from the thick filaments to the neighboring thin ones. The A band bridges between thick and thin filaments are integral parts of the thick filaments and are not present in the M - L complex. The M line bridges form a different system of lateral inter-connections which do not appear to be derived from the thick filaments, inasmuch as isolated thick filaments have the A band bridges but not the central M line bridges ( 4 9 ) . The relative lightness of the L lines is due to the fact that they lack both the bridges and thin filaments of the adjacent A bands as well as the other bridge system of the M line. A band bridges have not as yet been satisfactorily demonstrated in heart muscle for either technical or other reasons that are not clear. The organization of the sarcomere filament lattice in three dimensions had been suggested by the earlier x-ray diffraction studies on skeletal muscle ( 5 0 ) and confirmed by more recent electron microscopic studies of transverse sections of skeletal ( 3 1 , 4 6 ) and heart muscle (1, 1 7 ) . In transverse sections the various sarcomere bands can be readily identified (Fig. 1 5 ) . In such sections through the level of the M line only thick filaments with their interconnecting bridges are seen ( 4 7 , 4 8 ) (Figs. 16 and 1 7 ) . The thick filaments form an hexagonal array i.e. each filament is surrounded by six close neighbors about 4 0 0 Angstrom units apart ( 5 0 ) . Transverse sections through the A band lateral to the M-L complex disclose the same hexagonal array of thick filaments as well as the

THE MYOCARDIAL

Z

CELL

I

29

A

M- L A

Figure 14. Similar to Figs. 12 and 13, thicker filaments (upper arrows) are confined to the A band, including the central M-L complex. Thinner filaments (lower arrows) are seen in the A band (but not in the M-L complex) between adjacent thick filaments. Mag. X 107,000. second set of interposed thin filaments (Figs. 18 and 1 9 ) . T h e thin filaments occupy the trigonal points of the primary hexagonal lattices i.e. a thin filament is equidistant f r o m each of 3 neighboring thick filaments. This results in each thick filament being surrounded by six thin filaments and in turn by six neighboring thick filaments within the A band which thus contains two thin filaments f o r each thick filament. The I b a n d contains profiles of only thin filaments

30

THE MYOCARDIAL

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•mm

Figure 1 5 . Slightly oblique transverse section of heart muscle showing appearances of the various bands in this plane of section. Thick filaments are noted in both the A and M - L region. The I bands and Z line contain only thin filaments. Thin filaments in addition to thick filaments may also be discerned in A band. Mag. X 3 7 , 0 0 0 .

THE

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31

(Fig. 15) which are r a n d o m l y a r r a y e d except at the Z line where they f o r m a square array ( 5 1 ) . T h e filament n u m b e r ratios ( 1 7 , 3 1 ) f o r the various b a n d s are as follows: ML 1 (Thick)

:

A 3 ( 1 thick) ( 2 thin)

:

I 2 (2

thin)

A schematic representation of the a r r a n g e m e n t of filaments in the myofibril is depicted in Figure 2 0 . It is k n o w n that m u s c u l a r c o n t r a c t i o n involves the reversible association of the two contractile proteins actin a n d myosin, a n d

Figure 16. Transverse section through the M-L complex which reveals the hexagonal packing of the primary array of thick filaments. The thin filaments are not noted at this level. Mag. X 94,000.

32

THE MYOCARDIAL

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Figure 17. Similar to Fig. 16. Note hexagonal array of thick filaments and the presence of bridges between them. Mag. X 178,000. the splitting of A T P in the presence of C a + + (52, 53, 5 4 ) . In activated muscle actin and myosin are combined and can thus interact to produce shortening or tension. In biochemical terms the thick filaments have been identified as being composed primarily of the protein myosin by a variety of techniques (55, 56, 57, 5 8 ) while the thin filaments are probably largely composed of actin ( 5 5 ) . Studies of actin derived from a number of different muscles indicate that the thin actin filaments consist of a double helix of 2 chains of actin monomers polymerized end to end (59, 6 0 ) . Myosin can be broken into two types of subunits termed light or L and heavy or H meromyosins (38, 6 1 ) . The H meromyosin is the myosin subunit that interacts with actin and also has ATP'ase activity.

THE

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33

Studies of thick filaments and purified myosin from skeletal muscle indicate that the thick filaments consist of aggregates of myosin molecules oriented so that the L meromyosin portions of the molecules form the back bone of the thick filament, while the H meromyosin portions project from this backbone and constitute the A band cross bridges (49, 6 2 ) . Huxley has demonstrated that both the thick and thin filaments (due to the arrangement of their constituent myosin and actin molecules respectively) have opposite polarities in opposite halves of the same sarcomere ( 4 9 ) . The substructure of thick filaments of heart muscle is probably similar

Figure 18. Transverse section through an A band showing both thick and thin filaments. A portion of a mitochondrion (M) which demonstrates continuity between cristae and the inner membrane of the double peripheral membrane complex is seen. Mag. X 90,000.

34 THE MYOCARDIAL CELL in many ways to those of skeletal muscle despite some known chemical differences between the myosins of certain types of skeletal muscle and heart muscle (63, 64, 65, 66, 67). The location of tropomyosin, (68) another myofibrillar protein is not certain. Huxley has presented evidence that tropomyosin is in part located within the Z lines (49). The sliding filament hypothesis as a mechanism for muscular contraction was proposed in 1954. This hypothesis envisages mus-

Figure 19. Similar to Fig. 18 but at higher magnification. The primary hexagonal array of thick filaments plus the interposed secondary set of thin filaments is shown (hexagon). Note that six thin filaments, located at the trigonal points of the primary lattice, surround each thick filament (circles) and that the thin:thick filament ration is 2 : 1. Mag. X 148,000.

THE MYOCARDIAL

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35

cular contraction as a sliding of the thin actin filaments relative to the thick myosin filaments, the lengths of the filaments themselves remaining constant. H. E. Huxley and Hanson and A. F. Huxley and Niedergerke posed the sliding filament hypothesis simultaneously on the basis of the filamentous fine structure of the sarcomere, the protein nature of the two types of filaments and changes in b a n d

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36

THE MYOCARDIAL

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patterns observed under the light microscope at varying muscle lengths (31, 46, 55, 69, 70, 71). The initial direct support for this sliding mechanism stemmed from the fact that in skeletal muscle A band length remains constant while I band length is a direct function of muscle and sarcomere length ( 69, 71). In addition an expanding lighter zone termed the H zone, is seen on either side of the M-L complex, with progressive increments in muscle length (55, 72). The H zone results from the withdrawal of thin filaments from the A bands as the I

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THE MYOCARDIAL band widens.

CELL

37

The successive interactions between thick and thin

filaments resulting in filament sliding is presumably mediated by

Figure 22. Frog sartorius muscle fixed at the bottom of the ascending limb of the length-tension curve (Lo). No I band is seen. The sarcomere, which consists only of the A band and its central M line-L line complex, measures 1.5/x in length. Note the A contraction bands (vertical arrows) flanking the L lines. The horizontal arrows extend from the Z line to the far edge of the A contraction band in the opposite half of the sarcomere and measure 1 //. in length. (See text). Mag. X 20,000.

38

THE MYOCARDIAL

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the A band cross bridges composed of H meromyosin (46, 4 9 ) . The sliding filament model has been analyzed and is sound from a thermodynamic point of view ( 7 2 ) .

Figure 23. Heart papillary muscle fixed at Lo. The sarcomeres measure about 1.6//. in length and include narrow I bands (I) adjacent to the Z lines. The A contraction bands (vertical arrows) are shorter than those in Fig. 22 in proportion to the increment in sarcomere length. Horizontal arrow from Z line to far edge of A contraction band is again 1/x. Mag. X 20,000.

THE MYOCARDIAL

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39

A fundamental property of muscle is the relationship between muscle length and active or developed tension. This relationship defines the well known length-tension or L T curve. In Figure 21 the length tension curves for cat heart papillary muscle and for frog sartorius skeletal muscle are shown. Active tension increases with increments in muscle length from zero (at a starting muscle length termed L o ) to a maximal value (at length L m a x ) which is about 4 5 % greater than Lo. Sarcomeres measure about 1.5/x at Lo and 2 . 2 n at Lmax ( 4 4 ) . The portion of the curve from Lo to Lomax is known as the ascending limb. Further stretching of the muscle beyond Lmax is associated with sarcomeres which are longer than 2.2u and with a progressive decline in active tension along the descending portion of the L T curve. T h e shapes of the active tension curves are similar for many types of muscle (48, 73, 7 4 ) . However, the maximal tension developed by heart muscle is 3 or 4 fold less than that of frog sartorius muscle. This can partially be accounted for by the relatively larger volume occupied by mitochondria in heart muscle in comparison to skeletal muscle which possesses fewer mitochondria (48, 7 5 ) . T h e difference in developed tension between the two types of muscle may also be related to the fact that cardiac myosin has a lower A T P ' a s e activity than certain types of skeletal myosin (63, 64, 65, 66, 6 7 ) . T h e resting tension curves of heart and skeletal muscle also differ. In heart muscle resting tension is positive along the entire ascending limb of the active tension curve and is quite appreciable at Lmax, while in skeletal muscle resting tension is absent or negligible along most of this portion of the curve (76, 7 7 ) . The higher resting stiffness of heart muscle relative to skeletal muscle may correspond to the larger quantities of collagen found in the former ( 7 8 ) . The descending limb of the length-tension curve in skeletal muscle can be adequately explained by the sliding model. Linear increments in I band width and H zone width due to the withdrawal of thin filaments from the A band as skeletal muscle is extended occurs along the descending limb of the length tension curve ( 4 8 , 72, 7 9 ) . Thus the fall in active tension here is most likely due to the decreasing degree of overlap and therefore of interaction between the thick myosin and thin actin filaments (55, 72, 8 0 ) . T h e fact that active tension falls to zero when skeletal muscle is extended to

40

THE MYOCARDIAL

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Figure 24. Transverse section through an A contraction band of papillary muscle. Approximately double the number of thin filaments are present. The thin : thick filament ratio is 3.5 : 1 rather than 2 : 1 . (See Fig. 19). Mag. X 100,000. the point where the two sets of filaments are completely disengaged offers firm support for this suggestion ( 8 1 ) . Although Huxley had presented some evidence that the thin filaments passed across the center of the sarcomere at short muscle lengths (75, 82), the purely sliding mechanism was recently questioned for heart and skeletal muscle operating on the ascending limb of the active L T curve since a changing H zone is not seen

THE MYOCARDIAL

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41

Figure 25a, b. Similar to Fig. 24. The thick filaments in these A contraction bands are surrounded by thin filaments which are more than 6 in number. In areas as many as 10 or 11 thin filaments may be counted around individual thick filaments (Asterisks over thick filaments—compare to Figure 19). Fig. 25a Mag. X 130,000. Fig. 25b Mag. X 170,000.

along this portion of the curve (44, 8 3 ) . Moreover the fall in active tension with decreasing muscle lengths along this part of the curve was not adequately explained since thick, and thin filament overlap is maximal at or near Lmax. These facts are of particular

42

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importance with regard to heart muscle which normally functions only on the ascending limb of the active length tension curve. T h e upper limit of normal end diastolic pressure is about 12 m m of mercury and at this pressure sarcomeres measure about 2.2u, the sarcomere length at which active developed tension is maximal ( L m a x ) ( 4 2 , 8 4 ) . This indicates that heart muscle operates along the

Figure 26. Heart papillary muscle fixed in an unloaded state. Muscle is marketdly contracted, the sarcomeres measuring less than 1.5/j. in length. A contraction bands (delimited by arrows) are correspondingly wider than those shown in Figures 22 and 23. The broadening in the regions of the Z lines (Z) is due to the presence of Z contraction bands. Mag. X 20,000.

THE

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43

ascending limb of the curve under normal conditions with initial sarcomere lengths of 2.2« or less. In the intact heart the fact that increasing muscle length may engender an increased force of contraction forms the basis of the Frank-Starling Law (85, 8 6 ) . Significantly this law probably pertains only to the ascending limb of the active length-tension curve

Figure 27. Frog sartorius muscle fixed along the ascending limb of the active length-tension curve. Sarcomeres measure approximately 1.8fi and have broader I bands than those previously shown. The A band with its central M line-L line complex is unchanged. No A contraction bands are visible. A prominent N line (arrow) is seen within the confines of the I band. Mag. X 20,000.

44

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normally since inordinate filling pressures would be required to attain the descending limb of the curve ( 8 7 , 8 8 ) . Recently the active length-tension curves of heart ( 4 8 ) and skeletal ( 2 6 , 4 8 ) muscle have been explored in some detail relative to sarcomere lengths and fine structure. These studies were greatly facilitated by the employment of glutaraldehyde as the initial fixative inasmuch as it has been shown that this method of fixation does not result per se in significant artefactual changes in tension (44). Along the ascending limb of the active L T curve, heart and skeletal muscle sarcomere structure is similar ( 4 8 ) . Thus at L o where active tension is O sarcomeres measure about 1.5M to 1.6M in length (Figs. 2 2 and 2 3 ) . At sarcomere lengths of 1.5M no I bands are present, the entire sarcomere consisting only of A band ( F i g . 2 2 ) . In sarcomeres measuring 1.6M narrow I bands have appeared which measure .05M in width ( F i g . 2 3 ) . At these sarcomere lengths additional dark bands are present toward the center of the A band flanking the M - L complex ( 2 6 , 4 8 ) (Figs. 2 2 and 2 3 ) . These bands, which are termed A contraction bands, have widths which are inversely proportional to muscle and sarcomere lengths. Furthermore, the distance from the Z line to the far edge of the A contraction band in the opposite half of the sarcomere is constant regardless of sarcomere lengths and measures 1 .0M which is also the length of the thin filaments ( 2 6 , 4 8 , 8 9 ) (Figs. 2 2 and 2 3 ) . T h e A contraction bands are produced by the thin filaments penetrating into the opposite half of the sarcomere at short muscle lengths to create a double overlap of these elements. Thus in transverse sections through A contraction bands approximately double the number of thin filaments are present with a thin to thick filament ration of about 3.5 : 1 instead of the usual A band ratio of 2 : 1 ( 2 6 , 9 0 ) ( F i g . 2 4 ) and individual thick filaments within A contraction bands are surrounded by as many as 10 to 11 thin filaments rather than 6 ( F i g . 2 5 ) . These A contraction bands should be distinguished from Z contraction bands which are contiguous with the Z lines and which appear in sarcomeres measuring less than 1.5n ( F i g . 2 6 ) . T h e Z contraction bands probably result from the deformation of the ends of the thick filaments when sarcomere lengths are less than those of the thick filaments ( 3 1 . 5 5 ) . A contraction bands which

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45

Figure 28. Cat papillary muscle fixed along the ascending limb of the curve with sarcomeres measuring about 2.0/i. The increment in sarcomere length relative to previous figures is accounted for by increments in I-band length, the A band remaining unchanged. Mag. X 20,000. are proportionally widened c a n also be d e m o n s t r a t e d in these very short sarcomeres (Fig. 2 6 ) . L i n e a r increments in muscle length a n d I b a n d lengths are noted along the ascending limb of the active length tension curve, but the width of the A b a n d remains constant ( a t 1.5M) at all sarcomere lengths greater t h a n 1.5w (Figs. 22, 2 3 and 2 7 - 3 0 ) . T h e width of the M - L complex also remains constant at all muscle lengths. However, the L lines are m o r e clearly defined a n d a p p e a r to be of lower density in sarcomeres which m e a s u r e 2.2u o r m o r e d u e to the withdrawal of the thin filaments f r o m the

46

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Figure 29. Cat papillary muscle fixed at Lmax. Sarcomeres are 2.2/x; lip. is probably a lipid droplet. Mag. X 20,000. center of the s a r c o m e r e as the muscle is extended. A t the apex of the length tension curve sarcomeres m e a s u r e a b o u t 2 . 2 f i as previously m e n t i o n e d ( F i g s . 2 9 and 3 0 ) . In s a r c o m e r e s w h i c h m e a s u r e 2.2/j., i.e. at L m a x or m a x i m a l physiological diastolic length, the 1 fi in length thin filaments termin a t e at the m a r g i n s of the M - L complex. T h e fall in active tension along the ascending p o r t i o n of the length tension c u r v e is associated with the progressive p e n e t r a t i o n of thin filaments first into the M - L c o m p l e x and then into the opposite half of the s a r c o m e r e with the f o r m a t i o n of A c o n t r a c t i o n b a n d s as the muscle is allowed to shorten (See Fig. 3 1 ) . This decline in active tension is p r o b a b l y related to steric interference of

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Figure 30. Frog sartorius muscle fixed at the apex of the length-tension curve (Lmax). Sarcomeres are about 2 J / i and I band widths are proportional. The length of the A band has remained constant at 1 . 5 A narrow H zone (see Figs. 34 and 35) is barely apparent adjacent to L lines. Note transverse bridges within the M lines. Mag. X 20,000.

thick and thin filament interaction due to the presence of the additional set of thin filaments in the A contraction b a n d s which enlarge as he sarcomeres shorten. H. E. Huxley has suggested that this interference stems f r o m the fact that the polarity of the second set of thin filaments is opposite to that of the original set of thin filaments and that of the thick filaments ( 2 6 , 4 9 ) . T h e above results are entirely in accord with those predicted by A. F. Huxley in his elegant studies correlating skeletal muscle

Lmax

2 . 2 fj.

2 . 0 fi

1.8/1

Lo

1.5/x '-AC

ml 2 .4/1 H Figure 31. Schematic diagram which demonstrates the penetration of thin filaments into the opposite half of the sarcomere at short sarcomere lengths to produce the A contraction bands (AC). At 2.2/x (Lmax) thin filaments terminate at the edge of the M-L complex (ml); at 2.0/jl the thin filaments meet head on in the center of the sarcomere. At sarcomere lengths of 1.8/u. the thin filaments have started to by-pass one another largely within the confines of the M-L complex. At still shorter sarcomere lengths the zone containing twice as many thin filaments is proportionally longer creating the A contraction bands. Note that in over-extended muscle (2.4fi) the withdrawal of thin filaments from the A band into the I band creates an expanding H zone (which includes the central M-L complex). At all muscle lengths depicted the lengths of thick and thin filaments remain constant at 1.5/x and l.Oju respectively.

THE

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49

1.7 r A H-

AAA

1.5

A A A A A A AA A A

AAA

M-L ft

0.5 -

2.5

-max SARCOMERE

L E N G T H /x

Figure 32. Lengths of sarcomere bands of papillary muscle relative to sarcomere lengths are depicted. A band (A) length remains constant, while I band (I) length is a direct function of sarcomere length. The M line-L line complex (M-L) is also constant, and no H zone is apparent.

50

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SARCOMERE BANDS AT VARYING MUSCLE LENGTHS (FROG SARTOR I US)

SARCOMERE2 4 LENGTH LL Q Q 2.2 2.0

DEVELOPED TENSION

1.8

1.6 1.4

-40

LMAX MUSCLE

LENGTH

+10 (%)

Figure 33. Lengths o ! the various sarcomere bands relative to muscle length and the active-tension curve are graphically depicted for frog sartorius muscle. The A band (A) length does not vary, while sarcomere and I band (I) lengths are linear functions of muscle length. At sarcomere length greater than 2.2jk the H zone appears and widens in proportion to increments in muscle length. H zone measurements include the width of the M line-L line complex which remains constant in width at all muscle lengths.

THE

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51

length-tension mechanics with sarcomere lengths ( 9 1 ) . Bands similar to A contraction bands were previously observed in shortened skeletal muscle sarcomeres by Gilev who concluded, however, that they resulted from filament folding ( 9 2 ) . The lengths of the major sarcomere bands are summarized graphically as a function of sarco-

Figure 34. Frog sartorius muscle fixed on the descending limb of the active length-tension curve. The sarcomeres measure 2.3 to 2,4/i. The increments in sarcomere lengths are accounted for by the broadening of the H zones (delimited by arrows), in addition to increments in I band length along the descending portion of the curve. The M line-L line complex in the center of the H zone is unchanged. Total width of A band (A band proper and H zone) is 1.5^. Mag. X 20,000.

52 THE MYOCARDIAL CELL mere length or muscle length for heart and skeletal muscle respectively in Figures 32 and 33. Along the descending limb of the length-tension curve of skeletal muscle the decline in active tension is as previously noted adequately accounted for by the gradual withdrawal of thin filaments from the A band (26, 48, 72, 79) (Figs. 31, 34 and 35). This is reflected by a direct relationship between muscle length and sarcomere length and the presence of linearly expanding I bands and H zones (Fig. 33).

Figure 35. Frog sartorius muscle which is fixed further along the descending limb of the curve than that shown in Fig. 34. Note proportional increments in lengths of sarcomeres (which measure about 3.0/*) and of the H zones (delimited by arrows) and I bands relative to Fig. 34. Total length of A band does not change. Mag. X 20,000.

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The explanation for the descending limb of the length tension curve in heart muscle is not as clear (44, 4 8 ) . In heart as in skeletal muscle H zones may be present in sarcomeres measuring greater than 2 . 2 ^ (42, 9 3 ) . H zones are not always present in sarcomeres measuring from 2.2-2.4/x (Fig. 36) but are almost always noted if sarcomere length exceeds 2.4/x (Fig. 3 7 ) . However, unlike skeletal muscle, no linear relationship exists between overall heart muscle

Figure 36. Heart papillary muscle fixed along (he descending limb of the length-tension curve at a muscle length approximately 20% greater than that at Lmax. In contrast with Figs. 34 and 35, the lengths of the sarcomeres, which measure about 2.3/x do not correspond to over-all increments in muscle lengths, nor are H zones apparent. The M line-L line complex is unchanged. Mag. X 20,000.

54

THE MYOCARDIAL

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length and sarcomere length on this part of the curve (Fig. 36). The maximal sarcomere lengths which can be attained are about 2.6/* for heart muscle as opposed to 3.65 or even greater for skeletal muscle (44, 48, 81). Inasmuch as intercalated disc structure appears unchanged in extended heart muscle, (Fig. 36) relative slippage of columns of muscle cells may account for the increments of overall muscle length in the absence of corresponding increments of sarcomere length. The differences in the behavior of heart and skeletal muscle on the descending limb of the curve are probably related to the higher resting stiffness of the former type of muscle. However, since heart muscle under physiological conditions func-

Figure 37. Overdistended left ventricular muscle exhibiting sarcomeres measuring about 2.4/z in length. Irregular H zones (delimited by arrows) are present. Mag. X 20,000.

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tions on the ascending limb of its length tension curve, the normal contractile process of myocardium is adequately accounted for by the sliding filament hypothesis. Further work correlating fine structural and mechanical parameters may disclose whether the pathological or failing myocardium is operating on the descending limb of the active length tension curve with elongated sarcomeres in which the zone of thick and thin filament overlap is diminished.

REFERENCES 1. Stenger, R. J. and Spiro, D.: Amer. J. Med., 30:653 ( 1961). 2. Spiro, D. and Sonnenblick, E. H. Cyclopedia of Med., 3 (9) 64. F. A. Davis, Phila. 3. Spiro, D., and Sonnenblick, E. H. Prog. Cardiovasc. Dis. 7, 295 (1965). 4. Robertson, J. D. : Proc. Fourth Inter. Cong. Electron Microscopy. 2:159 (1960). Springer-Verlag, Berlin. 5. Davison, H. and Danielli, J. F.: The Permeability of Natural Membranes. Cambridge University Press, Cambridge, 1943. 6. The Plasma Membrane, Symposium. A. P. Fishman, Ed. Circulation, 26:983 (1962). 7. Porter, K. R.: The Cell. Brächet, J. and Mirsky, A. E., Eds. Academic Press, New York, 1961, Vol. 2, p. 621. 8. Shanes, A. M.: Pharmacol. Rev. 70:59 and 165 (1958). 9. Woodbury, J. W.: In Handbook of Physiology, Hamilton, W. F., Ed. Amer. Physiol. Soc. Washington, Circulation, Vol. 7:237 (1962). 10. Hoffman, B. F. and Cranefield, P. F.: Electrophysiology of the Heart. McGraw-Hill, New York, 1960. 11. Hutter, O. F. and Trautwein, W.: J. Gen. Physiol. 59:715 (1956). 12. Hajdu, S. and Leonard, E.: Pharmacol. Rev. 77:173 (1959). 13. Fawcett, D. W. and Selby, C. C.: J. Biophys. Biochem. Cytol. 4:63 (1958). 14. Moore, D. H. and Ruska, H.: J. Biophys. Biochem. Cytol. 3:261 (1957). 57

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15. Sjostrand, F. S., Anderson-Cedergren, E., and Dewey, M. M.: J. Ultrastruct. Res. 7:271 (1958). 16. Porter, K. R. and Palade, G. E.: J. Biophys. Biochem. Cytol. 3:269 (1957). 17. Stenger, R. J. and Spiro, D.: J. Biophys. Biochem. Cytol 9:325 (1961). 18. Cottrell, T. S., Wiener, J. and Spiro, D.: Unpublished observations. 19. Palade, G. E.: J. Histochem. Cytochem. 7:188 (1953). 20. Green, D. E. and Goldberger, R. F.: Amer. J. Med. 30:666 (1961). 21. Lehninger, A. L.: The Mitochondrion. Benjamin, New York, 1964. 22. Porter, K. R.: J. Biophys. Biochem. Cytol. 70:219 (1961). 23. Fawcett, D. W.: Circulation 24:336 (1961). 24. Smith, D. S.: Proc. 5th Inter. Conf. of Electron Microscopy, 2, 1962. Acad. Press, New York. 25. Franzini-Armstrong, C.: J. Cell Biol. 22:675 (1964). 26. Huxley, H. E.: Univ. of Alberta Symposium on Muscle Structure and Function. Pergamon Press (in press). 27. Nelson, D. A. and Benson, E. S.: J. Cell Biol. 76:297 (1963). 28. Huxley, A. F. and Taylor, R. E.: J. Physiol. 144-426 ( 1958). 29. Bendall, J. R.: Nature (London) 770:1058 (1952). 30. Marsh, B. B.: Nature. 767:1065 (1951). 31. Huxley, H. E. : The Cell. Brächet and Mirsky, Eds. Academic Press, New York, 1960, Chap. 7. 32. Muscatello, V., Anderson-Cedergren, E., Azzone, G. F., and von der Becken, A.: J. Biophys. Biochem. Cytol. 70:201 (1961). 33. Tice, L. W. and Engel, A. G.: J. Cell. Biol. 23:97A (1964). 34. Essner, E. and Quintana, N.: J. Cell. Biol. 79:22A (1963). 35. Bianchi, P. and Shanes, A. M.: J. Gen. Physiol. 42:803 (1959). 36. Podolsky, R. J.: J. Gen. Physiol. 45:613A (1962). 37. Weinegrad, S.: Circulation. 24:523 (1961). 38. Gergely, J.: J. Biol. Chem. 200-543 (1953). 39. Hasselbach, W. and Makinose, M.: Conf. the Biochemistry of Muscle Contraction, Dedham, Mass., 1962.

THE 40. 41. 42.

43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

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Weber, A. M„ Hertz, R„ and Reiss, J.: J. Gen. Physiol. 46:679 ( 1 9 6 3 ) . Ebashi, S. and Lipmann, F.: J. Cell Biol. 14:389 ( 1 9 6 2 ) . Sonnenblick, E. H., Spotnitz, H. and Spiro, D.: Symp. on Structure and Function of Heart Muscle. Circ. Res. Suppl. 11:70 ( 1 9 6 4 ) . Jamieson, J. D. and Palade, G. E.: J. Cell Biol. 25:151 (1964). Sonnenblick, E. H., Spiro, D., and Cottrell, T. S.: Proc. Nat. Acad. Sci., U. S. A. 49:193 ( 1 9 6 3 ) . Sjostrand, F. S. and Anderson-Cedergren, E.: J. Ultrastr. Res. 1:74 ( 1 9 5 7 ) . Huxley, H. E.: J. Biophys. Biochem. Cytol. 5:631 ( 1 9 5 7 ) . Spiro, D.: Trans. N. Y. Acad. Sci. Sec. II. 24:879 ( 1 9 6 2 ) . Spiro, D. and Sonnenblick, E. H.: Symp. on Structure and Function of Heart Muscle. Circ. Res. Suppl. II, 14 ( 1 9 6 4 ) . Huxley, H. E.: J. Mole. Biol. 7:281 ( 1 9 6 3 ) . Huxley, H. E.: Discussions Faraday Soc. 77:148 ( 1 9 5 1 ) . Knappeis, G. G. and Carlson, F.: J. Cell Biol. 75:323 (1962). Szent-Gyorgi, A.: Chemical Physiology of Contraction in Body and Heart Muscle. Academic Press, New York, 1953. Hasselbach, W. and Weber, A.: Pharmacol. Rev. 7:97 (1955). Weber, H. H. and Portzehl, H.: Prog. Biophys. 4:60 ( 1 9 5 4 ) . Hanson, J. and Huxley, H. E.: Sympos. Soc. Exp. Biol. 9:228 ( 1 9 5 5 ) . Hasselbach, W.: Naturforsch. 86-449 ( 1 9 5 3 ) . Finck, H., Holtzer, H., and Marshall, J. M.: J. Biophys. Biochem. Cytol. 2 (Suppl. 4:175 ( 1 9 5 8 ) . Tice, L. W. and Barrnett, R. J.: J. Cell Biol. 75: 401 ( 1 9 6 2 ) . Hanson J., and Lowy, J.: J. Mol. Biol. 6:46 ( 1 9 6 3 ) .

60. Hanson, J. and Lowy, J.: Structure and Function of Heart Muscle. Circ. Res. Suppl. II, 4, 1964. 61. Szent-Gyorgi, A. G.: Advances Enzym. 7(5:313 ( 1 9 5 5 ) . 62. Rice, R. V.: Biochim et Biophys. Acta. 7:281 ( 1 9 6 3 ) .

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63. Perry, S. V.: Comparative Biochemistry, A Comprehensive Treatise. Florkin, M. and Mason, H. S., Eds. Academic Press, N. Y„ 1960, pp. 245-340. 64. Brahms, J. and Kay, C. M.: J. Biol. Chem. 238:198 (1963). 65. Gergely, J.: Ann. New York Acad. Sei. 72:538 (1959). 66. Kay, D.: Symp. on Structure and Function of Heart Muscle. Circ. Res. Supp. II, 38 (1964). 67. Mueller, H., Franzen, J., Rice, R. V. and Olson, R. E.: J. Biol. Chem. 239, 1447 (1964). 68. Bailey, K.: The Proteins, Academic Press, New York, 1954, Vol. 2B, p. 951. 69. Huxley, H. E. and Hanson, J.: Nature (London). 775:973 (1954). 70. Huxley, H. E. and Hanson, J.: In the Structure and Function of Muscle. Bourne, G. H., Ed. Academic Press, New York, 1960. 71. Huxley, A. F. and Niedergerke, R.: Nature (London). 173:971(1954). 72. Huxley, A. F.: In Progress in Biophysics. Butler, J. A. V. and Katz, B., Eds. Pergamon Press, London, 1957 Vol. 7, pp. 255-318. 73. Ramsey, R. W. and Street, S. F.: J. Cell Comp. Physiol. 15:11 (1940). 74. Csapo, A.: In the Structure and Function of Muscle. Bourne, G. H., Ed. Academic Press, New York, 1960, Vol. 1, p. 229. 75. Huxley, H . E . : Circ. 24, 328 (1961). 76. Abbott, B. C. and Mommaerts, W. F. H. M.: J. Gen. Physiol. 42-533 (1959). 77. Sonnenblick, E. H.: Amer. J. Physiol. 202:931 (1962). 78. Personal Communication. Dr. M. Barany, Institute for Muscle Disease. 79. Carlsen F., Knappeis, G. G., and Bachthal, F.: J. Biophys. Biochem. Cytol. 77:95 (1961). 80. Huxley, A. F.: Physiol. Rev. 26, 131 (1964). 81. Huxley, A. F. and Peachey, L.: J. Physiol. 156:150 (1961). 82. Huxley, H. E.: Muscle As a Tissue, Ed. by K. Rodahal and S. M. Horvath. (p. 153 discussion). McGraw-Hill, 1962.

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83. Spiro, D., Sonnenblick, E. H., and Ccttrell, T. S.: Fed. Proc. 22 ( 1 9 6 3 ) . 84. Sonnenblick, E. H., Spiro, D. and Spotnitz, H.: Am. Ht. J. 68, 336 ( 1 9 6 4 ) . 85. Frank, O. Z.: Zur Dynamik Der Merzmuckles Z. Biol. 32-370 ( 1 8 9 5 ) . Translated by Chapman, C. B. and Wassenman, E., Amer. Heart J. 58-282, 467 ( 1 9 5 9 ) . 86. Starling, E. H.: Linacre Lecture on the Law of the Heart. Longmans, Green, London, 1918. 87. Monroe, R. G. and French, G. N.: Circ. Res. 9:362 ( 1 9 6 1 ) . 88. Ulrich, K. J., Riecker, G., and Kramer, K.: Pfueger Arch. Ges. Physiol., 259, 481 ( 1 9 5 4 ) . 89. Page, S. G. and Huxley, H. E.: J. Cell Biol. 79:369 ( 1 9 6 3 ) . 90. Spiro, D., Sonnenblick, E. H., and Spotnitz, H.: J. Cell Biol. 23:88A ( 1 9 6 4 ) . 91. Huxley, A. F.: Muscle Cells Under the Microscope. Columbia Univ. Press (in press). 92. Gilev, V. P.: J. Cell Biol. 72:135 ( 1 9 6 2 ) . 93. Huxley, H. E.: 2nd Inter. Meeting on Pharmocology. Pergamon Press (in press).

The Function of the Cell Membrane DR. R. D.

KEYNES

Institute of Animal Physiology Babraham, Cambridge, England BEFORE W E DECIDE ON THE FUNCTION OF THE CELL

MEMBRANE,

we have to be clear in our minds that there is such a thing. I expect that I am preaching to the converted, but this meeting is being held, so to speak, on the home ground of Ling ( 1 ) who— and I do not wish to give offense—is responsible for a major heresy in this field. Therefore, I thought that I might start by explaining very briefly why it seems to me that we do have to suppose that there is such a thing as the cell membrane; and by explaining why I reject the idea that the asymmetrical distribution of ions between the insides of cells—and particularly muscle cells—and the external medium arises from the special properties of the cytoplasm rather than special properties of the surface boundary layer which we call the membrane. The first thing that is difficult to swallow is that it is hard to see how any mechanism by which sodium and potassium ions are specifically adsorbed on to the proteins of the cytoplasm can operate without affecting the electrochemical activity of the ions inside the cells. Hodgkin and 1 ( 2 ) did an experiment which is relevant to this point, which was to demonstrate that in Sepia giant 63

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axons the mobility of labelled potassium ions was the same inside the living, functioning nerve fibre as it was outside. We also found ( 3 ) that sodium had about the same diffusion constant for radial movements inside the squid axon as in free solution, while calcium —which most of us would expect to be intracellular^ bound— scarcely moved at all in a longitudinal electric field ( 4 ) . This kind of experiment has not. as far as I know, been done on heart muscle, but it has been done on frog muscle by Harris ( 5 ) , who again found that K42 moved freely along the fibres in an electric field. Recently Lev ( 6 ) has reported measurements of the activity of sodium and potassium ions inside frog muscle fibres which were made with the help of selective glass electrodes. He seems to find no evidence for any appreciable binding of potassium, but does get very low values for the intracellular activity coefficient of sodium. One of the principal arguments used by Ling against the concept of the cell membrane is concerned with the energy requirement for extrusion of sodium from frog muscle fibres. He measured the rate at which labelled sodium emerged from frog muscles at 0 ° C after they had been poisoned to block both glycolysis and oxidative phosphorylation, and calculated the amount of work that would have been necessary to transport sodium actively at this rate. He then showed that this rate of consumption of energy was much greater than the actual rate at which the reserves of phosphate-bond energy in the muscle decreased, and concluded that the postulate of a sodium-pumping mechanism in the membrane was untenable. This is not, however, a legitimate argument, because no proponent of membranes would claim that the exchange of labelled ions observed in a muscle at 0 ° C results wholly from the operation of an energy-consuming process; it seems much more likely that under these conditions, labelled sodium mainly crosses the membrane through the type of mechanism that has been termed "exchange diffusion." I have to admit that there is rather little direct experimental evidence on the validity of the exchange diffusion idea, but I am nevertheless sure that Ling's argument does not rest on a satisfactory basis. Recently, Fugelli examined in my laboratory the action of ouabain on the influx of labelled potassium into a freshly dissected frog muscle, and found no detectable change, al-

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though in similar muscles the efflux of labelled sodium was roughly halved by ouabain, while in sodium-loaded muscles ouabain blocks very effectively the uptake of potassium from a solution containing 10 mM—K. This observation is consistent with the view that when the sodium content of the muscle is low, most of the sodium efflux is not coupled to potassium influx, and represents only an 'idling' of the sodium p u m p mechanism which does not involve the performance of osmotic work. Those who object to membranes on the grounds that altogether unreasonable properties have to be attributed to them should also remember that there are many types of epithelium which are capable of precisely similar feats of ionic transport f r o m one aqueous solution to another where there can be no question of any binding of ions. Since it must be accepted that layers of cells can transport ions and water, it seems hardly logical to deny similar capabilities to the cell membrane. Perhaps the most dramatic piece of evidence for the functional existence of cell membranes is to be found in the recent work of Baker, Hodgkin & Shaw ( 7 ) and others on the perfused squid axon. F o r years, those of us who have been working on the squid giant axon have been obtaining samples of axoplasm u n c o n t a m i n a t e d by extra-cellular material by a simple process of squeezing out the contents of axons, in the kind of way that toothpaste is squeezed f r o m its tube. W h e n this has been done, the sheath remains behind as a flat squashed-out ribbon, and it never occurred to me that this ribbon could still be excitable. However, Baker and Shaw were brave enough to put the matter to test, and it turned out not only that the sheath was still excitable but also that it could be reinflated with a variety of artificial solutions without impairing its ability to generate a propagated action potential. O n one occasion when I happened to be present an axon whose cytoplasm had been wholly replaced by an isotonic solution of potassium sulphate successfully conducted over four h u n d r e d thousand impulses; after witnessing this kind of experiment one must be forgiven for being impatient with those who attribute too m u c h importance to special properties of intracellular proteins. But of course the main use of the perfused squid axon preparation has been to examine the effect of changing the internal ionic environ-

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m e n t in a way which was previously applicable only to the external solution. A somewhat unexpected development has been the discovery (8, 9, 10) that axons perfused with salt-free sugar solutions can produce large and very much prolonged action potentials although their resting potentials are then close to zero. Baker, Hodgkin & Meves ( 8 ) have suggested that the maintenance of excitability in axons perfused with solutions whose ionic strength is low may result from the presence of a layer of fixed negative charges on the inner face of the m e m b r a n e which are normally masked by the high internal concentration of mobile counter-ions ( K + ) , but which maintain an appropriate potential gradient across part of the m e m b r a n e when the counter-ions are absent. In concluding this part of my talk, I would like to suggest that some of Ling's highly ingenious arguments about the behaviour of fixedcharge systems might profitably be deployed to treat this very interesting situation. If, then, we accept the existence of the m e m b r a n e , what is its function in the particular context of the cardiac muscle cell? T h e obvious answer to this question is control. T h e function of the cell membrane is to provide a means by which the contractile machinery of the cell can be switched on, by which it can be coordinated in a purposeful fashion, and by which the strength of the contractions can be graded in c o n f o r m a n c e with the commands of the homeostatic vasomotor center. Perhaps I might amplify this statement slightly, if only because I have all too few remarks to m a k e that are really relevant to the title of my talk. In the first place, the m e m b r a n e clearly has the function of providing a system for conducting impulses in an orderly fashion through a complicated network of cells; Spiro has shown us just how complicated the network is, and it must be remembered that the impulses which activate its contraction have to be conducted through it in such a way that all of its parts are brought into operation at precisely the right moment, in order to ensure an efficient p u m p i n g action. Most of you know much better than I what h a p p e n s when the conduction mechanism develops a fault, and the heart ceases to p u m p in a fully effective manner. In the second place, the m e m b r a n e system has to see to it that immediately on the arrival of the conducted action potential at each point, the myofibrils contract si-

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multaneously right across the cell. In saying this, I am intending to include with the surface membrane of the cell the elaborate sarcoplasmic reticulum which has already been described by Spiro. N o doubt, others will deal with the role of the sarcoplasmic reticulum in providing a link between the propagated spike and the contraction of the myofibrils. A n d in the third place, both the frequency and the strength of the cardiac contraction cycle have to be constantly adjusted to suit the needs of the circulatory system, and the m e m b r a n e plays an important part in enabling these adjustments to be made. In what ways does the membrane of cardiac muscle cells differ f r o m that of other excitable cells? O n e property of cardiac muscle which is obviously important in its functioning is its innate rhythmicity, and in particular the ability of the specialized pacem a k e r regions to fire spontaneously at a low but regular and well controlled frequency. Another special feature of cardiac muscle is its sensitivity to neurohumoral transmitters, such as acetylcholine and adrenaline, which do not affect the membranes of striated muscle fibres except at the neuromuscular junction. Lastly, the duration of the action potential in cardiac muscle fibres is characteristically much greater than it is in nerve fibres or even in striated muscle fibres, not because the upstroke is slow but because of its long plateau. Presumably, if I m a y use a teleological argument, the purpose of this long plateau is to prolong the refractory period of the tissue and so to make it incapable of giving summated contractions like striated muscle, since contractile activity of this kind would not be appropriate to its function. I will next try to summarise the points in the cardiac muscle system at which drugs might be expected to act: ( 1 ) O n excitation and conduction in the membrane. A s examples under this heading I would cite first the effect of changing the external concentration of calcium. T h e stabilizing action that calcium has on excitable membranes is well known, and one of the first quantitative studies of the basis of this action was m a d e by W e i d m a n n ( 1 1 ) , who applied a simplified voltage clamp technique to examine the properties of the Purkinje fibres

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from the sheep's heart. He showed that the effect of [Ca 2 + ] on the critical (threshold) potential for excitation was brought about by a shift along the voltage axis of the curve relating peak sodium conductance to membrane potential, without any marked change in the shape of the curve. In the pacemaker region, such a shift in the critical potential has an obvious influence on the interval between spikes. Weidmann ( 1 1 ) also showed that drugs like procaine may act in a somewhat similar way. Another example of a drug which acts on the mechanism of excitation in the membrane is, of course, acetylcholine. It appears from the work of Burgen & Terroux ( 1 2 ) , Hutter ( 1 3 ) and others that in cardiac muscle acetylcholine produces its effect by increasing the potassium permeability of the membrane; in the pacemaker, the spike interval is lengthened, while in other cardiac muscle fibres the spike itself is shortened and the mechanical force of the contraction is reduced. Yet another drug which must act on excitation (and which may, as we shall see, act elsewhere as well) is adrenaline; but I do not know of any evidence as to exactly what specific permeability changes underlie its action. And for the sake of completeness I must also mention drugs like quinidine, which certainly act on excitation and conduction through the heart, and are used clinically for this reason. ( 2 ) On active transport through the membrane. The drug actions in category ( 1 ) are all ones which affect the passive permeability of the cell membrane, and alter the ease with which various ions are permitted to flow across the membrane down the pre-existing concentration gradients. However, in parallel with the passive permeability channels there are also active transport channels through which ions can be transferred uphill, against the concentration gradients, and we have therefore to consider a second category of drugs which may affect active transport. Many years after the discovery and clinical application of the cardiotonic effects of the cardiac glycosides, it was found by Schatzmann ( 1 4 ) that these substances are potent inhibitors of the sodium pump, and they have now become a favourite tool of all those engaged in studying active transport mechanisms. You have already had

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an example of this in my remarks about the action of ouabain on the sodium and potassium fluxes in frog muscle. A question of particular interest for this symposium is whether or not the cardiotonic action of cardiac glycosides can be accounted for in terms of their interference with active transport. I wish to draw your attention to two points connected with the inhibition of active transport. One is that in its prime effect on the coupled pumping of sodium and potassium ions, ouabain appears to act only at the outer face of the m e m b r a n e , on the side towards which sodium and away f r o m which potassium is being moved; this statement rests on the lack of effect of ouabain when it is injected into squid axons ( 1 5 ) and on the asymmetry of its effect on ion transport through epithelia like frog skin. T h e second point is that if the transport of sodium through the membrane is an electrogenic process, then its inhibition m a y have a more immediate effect on the m e m b r a n e potential than it will if it is a tightly coupled neutral process. My reason for introducing this somewhat controversial issue is that the recent experiments of Kernan ( 1 6 ) and of R y b o v a and myself ( 1 7 ) suggest that at least under some conditions the sodium p u m p in frog muscle is electrogenic. In erythrocytes there c a n be no doubt that the movements of sodium and potassium are chemically coupled, and I have evidence that under certain circumstances this is also true for frog muscle. While the question has not yet been critically examined in cardiac muscle, the possibility that the sodium p u m p is electrogenic has been suggested by Conn & W o o d ( 1 8 ) and should not be overlooked. In this category, adrenaline should again be included, because Fleckenstein and his co-workers ( 1 9 ) believe that when adrenaline counter-acts the depolarization of cardiac muscle fibres produced by high concentrations of potassium, it does so by stimulating the sodium p u m p . ( 3 ) On excitation-contraction coupling. As noted by Winegrad in this symposium, it is now believed that a release of calcium f r o m some element of the sarcoplasmic reticulum plays an important role in linking contraction with the propagated depolarization of the surface m e m b r a n e of muscle

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fibres. If I may again regard calcium as a drug, it is obvious that changes in [Ca 2 *] may have effects at this point in the system which will be quite distinct from their effects on excitability that were included in category ( 1 ) . Another drug which probably acts on this part of the machine is caffeine, and I would also tentatively add eserine (physostigmine) and cocaine to the list because of the interesting work of Novotny, Vyskocil, Vyklicky & Beranek ( 2 0 ) on the effect of eserine and cocaine in suppressing the extra consumption of oxygen by frog muscles exposed to 20 mM-K. ( 4 ) On contractility. Under this heading I mean to include an action on the contractile machinery beyond the stage of calcium release. I do not know of any drugs that definitely affect contractility in this manner, but it should be remembered that the cardiotonic effect of the cardiac glycosides has sometimes been attributed to a direct action on actin ( 2 1 ) . After studying the effects on ion transport in erythrocytes of various structural modifications in the cardiac glycoside molecule, Glynn ( 2 2 ) concluded that the structural requirements for the cardiotonic action were closer to those for interference with active transport than to those for affecting actomyosin. But it would perhaps be unwise to dismiss this possibility altogether before a really satisfactory explanation has been found for the way in which cardiac muscle is affected by very low concentrations of the cardiac glycosides. ( 5 ) On cellular metabolism. Several of these processes require supplies of phosphate-bond energy for their operation. Certainly contraction cannot take place without ATP, and there is also good evidence that the sodium pump is driven by phosphate-bond energy. In addition, it may well be that excitation-contraction coupling needs A T P in order to be kept fully primed or, to be more specific, to pump calcium back into the appropriate part of the sarcoplasmic reticulum. In order to complete my list, therefore, I must add any drug which interferes with the regeneration of energy-rich phosphate bonds by blocking the glycolytic pathway or oxidative phosphorylation.

REFERENCES 1. Ling, G. N.: A Physical Theory of the Living State. Blaisdell, New York, 1962. 2. Hodgkin, A. L. and Keynes, R. D.: J. Physiol. 779:513 (1953). 3. Hodgkin, A. L. and Keynes. R. D.: J. Physiol. 737:592 (1956). 4. Hodgkin, A. L. and Keynes, R. D.: J. Physiol. 738:253 (1957). 5. Harris, E. J.: J. Physiol. 724:248 (1954). 6. Lev, A. A.: Nature (London) 201:1132 (1964). 7. Baker, P. F., Hodgkin, A. L. and Shaw, T. I.: J. Physiol. 164:330 (1962). 8. Baker, P. F., Hodgkin, A. L. and Meves, H.: J. Physiol 170:541 (1964). 9. Narahashi, T.: J. Physiol. 769:91 (1963). 10. Tasaki, I. and Shimamura, M.: Proc. Nat. Acad. Sei., Wash. 48:1571 (1962). 11. Weidmann, S.: J. Physiol. 729:568 (1958). 12. Burgen, A. S. V. and Terroux, K. G.: J. Physiol. 720:449 (1953). 13. Hutter, O. F.: In Nervous Inhibitions. Pergamon Press, London, 1961, p. 114. 14. Schatzmann, H. J.: Helv. Physiol. Acta 77:346 (1953). 15. Caldwell, P. C. and Keynes, R. D.: J. Physiol. 148:% (1959). 16. Kernan, R. P.: Nature (London) 793:986 (1962). 71

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Keynes, R. D. and Rybova, Renata: J . Physiol., 7 6 S : 5 8 P (1963). 18. Conn, H. L. and Wood, J. C.: Am. J. Physiol. 797:631 (1959). 19. Antoni, H., Engstfeld, G. and Fleckenstein, A.: Pfliigers Archiv. 2 7 2 : 9 1 ( 1 9 6 0 ) . 20. Novotny, I., Vyskocil, F., Vyklicky, L. and Beranek, R.: Physiol. Bohemeslov. 7 7:277 ( 1 9 6 2 ) . 21. Horvath, I., Kiraly, C. and Szerb, J . : Nature (London) 764:792 (1949). 22. Glynn, I. M., J. Physiol. 136:148 (1957).

Transmembrane Potentials of Cardiac Cells and Their Ionic Basis D O N A L D H. S I N G E R , R A L P H L A Z Z A R A , and B R I A N F. H O F F M A N

Columbia

Department of Pharmacology University College of Physicians & Surgeons New York, N. Y.

I. T R A N S M E M B R A N E P O T E N T I A L S O F T H E H E A R T Interest in the electrical activity of the heart dates back many years. By the end of the 19th century the excitability characteristics of the heart had been generally defined through the efforts of such investigators as Bowditch ( 1 ) , Englemann ( 2 ) , and Marey ( 3 ) . By the 1930's many studies had already been directed toward the elucidation of the special characteristics of the pacemaker potential. The typical spontaneous diastolic depolarization exhibited by these cells was suggested by Bozler in 1943 ( 4 ) , on the basis of his observations of diastolic prepotenThis investigation was supported in part by a Public Health Service G e n eral Research Support Grant, Public Health Service fellowship no. 2-F2HE-13, 3 5 4 - 0 4 from the National Heart Institute and a fellowship from the John Polachek Foundation for Medical Research.

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tials in the vicinity of a pacemaker. Despite these advances, the inability to measure changes in transmembrane potentials directly and to correlate them with surface records of electrical activity made systematic interpretation of the latter difficult at best. T h e introduction of the glass microelectrode by Ling and Gerard in 1 9 4 9 ( 5 ) , and its subsequent adaptation to the study of cardiac tissue by Draper and Weidman ( 6 ) and Woodbury et al ( 7 ) , made it possible to record the electrical activity of single cardiac cells and thereby opened the way to a better understanding of the electrical events of the heart and its specialized conduction system. T h e Ling-Gerard microelectrode is a fine glass capillary tube drawn to a tip diameter of less than .5 micron and filled with a concentrated electrolyte solution. Usually 3 M KC1 is used to minimize junctional potentials between the electrode and the cytoplasm ( 8 ) . When the intracellular microelectrode is paired with a suitable extracellular indifferent electrode and led into a cathode follower and appropriate D C coupled amplifier, the potential differences across the cell membranes can be recorded satisfactorily. T h e considerations involved in the selection of the appropriate tip diameter, as well as the actual details of the recording techniques used, have been described elsewhere ( 9 ) . The transmembrane potentials recorded from a canine ventricular muscle cell together with a simultaneously recorded surface electrogram are illustrated in Figure 3 8 . In a resting fiber, the cell interior is negative with respect to the extracellular fluid by about 9 0 m V . This is the so-called resting potential. Resting potentials ranging from — 5 0 — 9 5 mV have been reported as occurring in cardiac cells from different species as well as from cells in different parts of the heart. T h e lower values are usually recorded from S A and A V nodal fibers. In all types of cardiac fibers except those exhibiting pacemaker characteristics the resting potential remains constant until excitation occurs. This period of electrical quiescense is designated as phase 4. Following excitation, the transmembrane potentials undergo a characteristic sequence of changes resulting in the inscription of the action potential. It may be seen from Figure 38 that the action potential bears a striking resemblance to the monophasic current of injury which was first described by Burdon-Sanderson and Page in 1 8 8 4 ( 1 0 ) .

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Figure 38. Diagrammatic representation of the transmembrane action potentials and unipolar electrogram recorded from an isolated preparation of canine ventricular muscle. The zero or reference line, (a) is recorded when the tip of the microelectrode is extracellular in position. When the tip of the microelectrode penetrates the cell, the transmembrane resting potential (b) is recorded. Inscription of the action potential commences at (c). The phases of the action potential are designated by the symbols 0, 1, 2 3. Diastole is designated as phase 4. The bottom trace shows a unipolar electrogram recorded in the immediate vicinity of the microelectrode tip. The intrinsic deflection of the R wave is synchronous with phase 0 of the action potential. The ST-T segment is inscribed during phases 2 and 3. (Modified after Hoffman and Singer Progress in cariovascular disease 7:227 (1964)

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The initial portion of the action potential, the upstroke or phase 0, is inscribed during depolarization of the cell. The rate of depolarization varies considerably in different cell types, being highest for Purkinje fibres. For these fibres, values of up to 670 Volts/sec have been reported ( 6 ) . This is comparable to the fastest rising velocities reported for noncardiac excitable tissue. The slowest rates of depolarization occur in fibers exhibiting spontaneous pacemaker activity and in fibers of the AV node. By the end of phase 0 a reversal of cell polarity, the overshoot, has occurred, the interior becoming 20-30 mV positive with respect to the extracellular fluid. This reversal is more or less prominent in all cardiac fibers except those exhibiting pacemaker activity and A V nodal fibers. Phase 0 corresponds to the R wave of the simultaneously recorded surface electrogram. Depolarization is followed by a prolonged period of repolarization during which the membrane potential is restored to the resting level. Repolarization may be divided into 3 phases. An initial brief period of rapid depolarization (phase 1) is followed in some fibers by a period during which the recovery of membrane potential proceeds very slowly. This period is phase 2 or the plateau. It is much more prominent in ventricular muscle and His-Purkinje cells than in ordinary atrial or nodal fibers and is responsible for the greater duration of the action potential in the former. Recent studies of the transmembrane potentials of specialized conducting fibers located in the interatrial band and the crista terminales have shown them to be similar to those recorded from Purkinje fibers (11, 12), in that, among other shared characteristics, they also exhibit a prominent plateau. Phase 2 is followed by a period of more rapid repolarization, phase 3, during which the resting level of membrane potential is restored. Phases 2 and 3 correspond to the ST and T deflections of the simultaneously recorded surface electrogram. A more detailed description of the transmembrane potentials of cardiac cells may be found elsewhere ( 9 ) . Figure 39 illustrates transmembrane potential recordings from different portions of the canine heart and the time sequence of the activation of these fibers. Excitation of most cardiac cells occurs as the result of the arrival of a propagated action potential. When the level of membrane potential is decreased to a critical level, the threshold po-

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100

potentials from different parts of the canine heart illustrating the differences in their amplitude, configuration and duration as well as the sequence of activation at the various sites. From above down the action potentials are from: sinoatrial node, specialized atrial fiber (e.g. interatrial band), atrium, atrioventricular node, bundle of His, Purkinje fiber in a false tendon, terminal Purkinje fiber, and ventricular muscle fiber. (Modified from Hoffman and Cranefield (9))

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tential, the response becomes regenerative and the upstroke of an action potential is inscribed. Certain cardiac cells, called automatic cells, are capable of undergoing spontaneous, repetitive self-excitation. These cells are clustered in the sinus node and adjacent venous tissue, in certain other specialized atrial fibers, and in the His-Purkinje system. The widespread view that automatic cells also occur in the AV node has been disputed recently on the ground that the characteristic feature of automatic fibers could not be found in AV nodal tissues ( 1 3 ) . Automatic cells are characterized by the fact that the level of membrane potential does not remain constant during electrical diastole (phase 4 ) . Once repolarization has been completed, the level of membrane potential begins to decline. If this results in a decrease in the membrane potential to the level of the threshold potential, an action potential will ensue. If this action potential can propagate and excite the heart, it will serve as the pacemaker for at least that beat. In most instances, before spontaneous depolarization can proceed to the level of the threshold potential, excitation occurs due to the arrival of a propagated action potential. Automatic cells brought to threshold in this manner are the so-called latent pacemakers. Although many factors are undoubtedly involved in the determination of which cell will initiate the cardiac impulse, it seems reasonable to believe that the rate at which diastolic depolarization proceeds is the dominant consideration. The speed of spontaneous diastolic depolarization varies considerably for automatic cells located in different portions of the heart. The fastest rates occur in the automatic cells of the sinus node and adjacent venous tissue, which serve as the primary pacemakers of the heart. The rates are generally slower for automatic cells of the His-Purkinje system, which ordinarily function as reserve pacemakers. Although changes in the repetition rate of the pacemaker are probably most frequently caused by changes in the rate of phase 4 depolarization, they also can be caused by changes in the threshold potential and in the level of maximum diastolic potential. Changes in more than one of these variables may occur at any given time, the net effect on rate being the resultant of these changes. Transmembrane potentials from automatic cells of the

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Figure 40. Diagrammatic representation of the transmembrane action potentials recorded from a fiber of the sinoatrial node (SA) and from an ordinary atrial muscle fiber (A) illustrating their differences in amplitude, rising velocity and general configuration as well in the constancy of the level of membrane potential during phase 4. The spontaneous decrease in the level of diastolic potential during phase 4 observed in the recording from the S-A node is the hallmark of automatic cells. If phase 4 depolarization decreases the membrane potential to a critical value, the threshold potential (TP), spontaneous firing will occur. (Modified after Hoffman and Singer: Progress in Cardiovascular Disease, p 229.)

sinus node are compared with potentials from ordinary atrial fibers in Figure 4 0 . The ability of most cardiac fibers to generate an action potential in response to stimulation, i.e., excitability, is related to the level of membrane potential at the time the stimulus is applied. This was clearly shown for Purkinje fibers by Weidman in 1 9 5 5 ( 1 4 ) and by Hoffman et al in 1957 ( 1 5 ) . During repolarization,

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stimuli applied before the membrane potential has been restored to approximately — 5 0 mV will not result in a response. At higher levels of membrane potential a response usually is generated. The amplitude and rising velocity of the resultant action potentials increase progressively with further increases in the level of membrane potential, a normal action potential occurring at approximately — 9 0 mV. Since the amplitude and rising velocity of an action potential affect its ability to excite adjacent fibers and therefore its ability to propagate, action potentials elicited at higher levels of membrane potential will propagate more effectively and more quickly than those arising at lower potentials. Action potentials elicited at levels of membrane potential of — 5 0 to — 5 5 mV are so small and slow rising that they are ineffective in exciting adjacent fibers and thus usually result in a local response or decrement. The relationship between excitability and level of membrane potential is also reflected in the changing stimulus requirements needed to elicit a response, the necessary stimulus generally decreasing with increasing levels of membrane potential. Just prior to complete repolarization, the stimulus requirement is somewhat less than that needed upon full restoration of the membrane potential to resting levels. This is the supernormal phase of excitability ( 1 6 ) . However, the action potentials occurring during this period are not normal. No evidence has been found to date for a true supernormal phase of conduction, i.e., potentials elicited prior to completion of recovery which have an amplitude and rising velocity greater than that of potentials occurring at higher levels of membrane potential. The interrelationship between excitability and level of membrane potential during repolarization is illustrated in Figure 41. Stimulation of Purkinje fibers exhibiting loss of membrane potential during diastole due to the development of spontaneous phase 4 depolarization results in much the same spectrum of responses as occurs during stimulation of incompletely repolarized fibers ( 1 4 ) . If the loss of membrane potential is sufficient, the initiation and propagation of the impulse in these fibers will be depressed. Propagation of impulses into areas of enhanced automaticity, e.g. the His-Purkinje system in instances when the sinus is markedly slowed, may well be an important cause of conduction disturbances in the intact heart.

A 40

V

20

+

20 40

60 80

ous times during repolarization. The amplitude and rising velocity of the responses are related to the level of membrane potential at the time of stimulation. The earliest responses, " a " and " b " , arise at such low levels of membrane potential and are so small and slow rising that they do not propagate (graded or local responses). The first propagated response, " c " , defines the end of the effective refractory period. Although response " d " arises at a time when the membrane potential approximates the threshold potential ( T P ) , i.e., during the supernormal period of excitability, it is still smaller and more slowly rising than response " e " which occurs after repolarization is complete. The first normal response (e) defines the end of the full recovery time. B. Schematic representation of the usual relationships between transmembrane potentials and cathodal excitability. The changes in threshold are related to an arbitrary scale of current strength. The fiber becomes inexcitable coincident with the inscription of phase 0 of the action potential. Recovery of excitability, as indicated by changes in threshold, progresses slowly during phase 3. The terminal portion of phase 3 is associated with a period of supernormal excitability. The diagram also illustrates the approximate duration of the absolute refractory period ( A R P ) , the effective refractory period (ERP), relative refractory period ( R R P ) , total refractory period ( T R P ) , full recovery time ( F R T ) , and the period of supernormal excitability (SNP). (Modified from Singer and Hoffman: Progress in Cardiovascular Disease, p. 231.)

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This may also be one of the explanations for such diverse phenomena as abberrant intraventricular conduction of supraventricular escape beats ( 1 7 ) , bundle branch block occurring only at slow heart rates and the exit and entry blocks said to exist about parasystolic foci. The instability of the His-Purkinje pacemakers

Dr.

E Figure 42. A. Schematic diagram of a free running strand of canine Purkinje fibers with attached ventricular muscle. The preparation is stimulated at one end by bipolar silver wire electrodes (Dr) inserted into the muscle. Simultaneous transmembrane potentials are recorded through glass microelectrodes inserted near (P) to and distant from (D) the drive site. A simultaneous bipolar surface electrogram is recorded in the immediate vicinity of the distant microelectrode.

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which take control of the heart in instances of sinus failure or advanced atrioventricular block may be due to variations in the membrane potential of surrounding fibers caused by variations in the rate of phase 4 depolarization. A sufficient decrease in membrane potential of the fibers surrounding the ectopic pacemaker could block propagation of the impulse resulting in an apparent cessation of activity of the pacemaker. The development of phase 4 depolarization may also be one of the causes of so-called supernormal conduction since impulses arising later in the cycle because of the progressive decline in membrane potential. Figure 42 illustrates some of the effects of the development of phase 4 depolarization in Purkinje fibers. T h e relationship between excitability and responsiveness and the level of membrane potential does not seem to hold strictly for fibers of the SA and A V nodes (9, 18). For example, the amplitude and rising velocity of action potentials resulting from stimulation of SA nodal fibers during phase 4 may increase as the level of resting potential falls, suggesting that the duration of refractoriness outlasts the recovery of resting levels of membrane potential. The rate induced changes in action potential duration are also much less marked in these fibers than in ordinary atrial or ventricular muscle. In these fibers conduction disturbances, including decrement, probably result most frequently from increases in the rate of stimulation whereas in Purkinje fibers they usually result from decreases in the level of diastolic membrane potential. Conduction is ordinarily decremental in these fibers. The cause of this anomalous behavior is unknown but may relate to a recent proposal that the mechanisms involved in the depolarization of these fibers may differ from those of other cardiac cells ( 1 9 ) .

II. T H E I O N I C B A S I S O F C A R D I A C ACTIVITY

ELECTRICAL

T h e electrical phenomena in excitable tissue must originate from the movements of the ions present in the tissue. The cell membrane separates media of different ionic composition. In the intracellular fluid, the most abundant cation is potassium and the anions are mostly organic ions which probably do not cross the membrane.

20 msec

surface electrograms from a canine Purkinje fiber preparation similar to that described in fig. 42 A. Recordings were made at a fast sweep velocity (20 mSec./cm.) The electrogram is displayed on the bottom trace of each frame. In frame "A" the preparation was stimulated at a frequency of 100/min. At this frequency significant diastolic depolarization was not present. The driving frequency was then gradually slowed to 30/min in order to facilitate the development of phase 4 depolarization. As phase 4 depolarization developed (frames "B"-"E") the amplitude and rising velocity of the action potentials recorded from both sides diminished progressively. In many instances the upstrokes of the action potentials were preceded by slow prepotentials. The action potentials became similar in many respects to those recorded from AV nodal fibers (18). These changes were associated with the development of progressive slowing of conduction along the fiber as manifested by the increasing latency between the stimulus artifact and the upstroke of the proximal action potential and by the increasing separation between the upstrokes of the proximal and distol action potentials. With the most marked degrees of phase 4 depolarization, conduction becomes decremental; local block developed and frequently the sequence of activation of the fiber was altered. These changes were also reflected in the alterations of the surface electrogram.

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T h e predominant extracellular cation is sodium whereas the m a j o r anion is chloride. T h e proposed mechanisms for the maintenance of the ionic gradients across the m e m b r a n e will be discussed below. Notwithstanding the mechanisms producing the gradients, it is proposed that the concentration gradients of ions provide both the potential electrical energy manifest in the resting potential and the driving force for flow of current during activity. F o r many years it was postulated that the resting potential across cell membranes is a diffusion potential, resulting f r o m free permeability of the m e m b r a n e to potassium and impermeability to organic intracellular anions ( 2 0 ) . The representation of the resting m e m b r a n e potential as a potassium equilibrium potential has been f o u n d to be an accurate approximation for most tissues except at low concentrations of potassium where the potential is less than predicted. The potential at equilibrium for any monovalent ion is given by the well known Nernst equation: vV e q

R T - —— .In =

F

fC]

-

[C]i

where Veq is the electrical potential at equilibrium inside relative to outside, [C]f and [C]i are the activities in the extracellular and intracellular fluids respectively, R is the gas constant, T the absolute temperature, and F is Faraday's number. F o r the mammalian heart the value of Veq for potassium is about - 9 0 mV., for sodium about + 4 0 mV., and for chloride, it is close to the value for potassium. T h e measured m e m b r a n e potentials at different external potassium concentrations have been compared with the predicted potassium equilibrium potentials for m a n y types of cells ( 9 ) . In general, the relationships f o u n d are depicted in Figure 43. At low external concentrations of potassium the observed potentials deviate increasingly f r o m the predicted values and in some cells, the m e m b r a n e potential actually decreases at very low concentrations. T h e curved portion of the graph of observed potential has usually been f o u n d in the physiologic range of potassium concentrations, whereas the linear portion closely approximating the theoretical line usually is f o u n d with higher than physiologic concentrations of potassium. There is some evidence that the resting m e m b r a n e potential of certain specialized tissues of the

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L o g (K ) e Figure 43. Relationship between theoretical potassium equilibrium potential (dashed line) and observed membrane potential (solid line). Ordinate is electrical potential, V, and abscissa is the logarithm of the external potassium concentration. The dotted extension of the solid line represents the fall in potential observed in some cells at very low concentrations of potassium. heart, for example nodal tissues, may be less dependent on extracellular concentration of potassium ( 2 1 ) . Just as the development of the ultramicroelectrode supplied a fresh impetus f o r the study of the cellular electrophysiology of the heart, the ionic hypothesis developed by Hodgkin and Huxley and

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their associates has provided the m a j o r theoretical basis for the interpretation of the changes in t r a n s m e m b r a n e potential occurring with activity. In a series of now classic studies ( 2 2 - 2 6 ) on the giant axon of the squid, Hodgkin and Huxley were able to control the m e m b r a n e potential and accurately measure the current crossing the membrane. F u r t h e r m o r e , by using sodium-poor solutions and comparing the currents with those recorded in normal solutions, they were able to achieve an approximate separation of the current into components carried by sodium and potassium. A number of important observations were made. At small displacements of the m e m b r a n e potential f r o m the resting value, the current (equivalent of net flow of positive ions) was inward with a more negative potential (hyperpolarization) and outward with less negative potential (depolarization). However, maintained depolarizations beyond a certain level, the threshold potential, resulted in a reversal f r o m outward current to a large transient inward current of sodium ions, followed by a delayed, maintained o u t w a r d flow of potassium ions. Restoration of the m e m b r a n e potential to the resting state restored the potassium current to near zero. T h e anionic current was considered to remain small and constant. T h e plot of the peak of the early (largely s o d i u m ) m e m b r a n e current against voltage was non-linear and showed a limb of negative resistance. The form of the current voltage relation is shown in Figure 44. In this curve there are two "stable" points at which the current is zero, one of which corresponds to the potassium equilibrium potential ( A ) and the other to the sodium equilibrium potential ( B ) . T h e threshold potential ( C ) also represents a point of zero current, since at this point the current changes f r o m o u t w a r d to inward, but it is "unstable," since this point is on the limb of negative resistance ( D - E ) . If the m e m b r a n e potential is brought to any point between C and E , it will move regeneratively along the curve to the sodium equilibrium potential. Using these and other observations and drawing heavily f r o m the G o l d m a n treatment of classical liquid junction theory ( 2 7 ) , Hodgkin and Huxley proposed that alteration of the specific permeability of the m e m b r a n e with respect to ions was responsible for the transition f r o m the resting to the active state. A general equation for m e m b r a n e potential at equilibrium, taking into account the

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I

Figure 44. Form of the current-voltage relation observed in excitable membranes. The ordinate is the peak value of the early current observed when the membrane potential, V, is held at the abscissal valve. The portion of the ordinate above zero represents the equivalent of net flow of positive ions out of the cell, whereas the portion of the ordinate below zero represents the equivalent of net flow of positive ions intp the cell. The fine portion of the curve between points D and E is the region of negative resistance. See text for further discussion.

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concentrations of the m a j o r ions ( N a , K. and C I ) and the permeability of the m e m b r a n e to them was derived by Hodgkin and Katz ( 2 8 ) from the constant field theory of G o l d m a n :

V,(1 = R T / F In

P • [K]e+ — ( N a ) o + ^ [K], +

r K

(Na): +

P (CI); ^ rn

(Cl)o

where V e(| is the m e m b r a n e potential at zero net flux; ( K ) e , ( N a ) e and ( C I ) e are the activities of K, Na, and CI respectively at the outside of the m e m b r a n e ; (K)¡, ( N a ) i and (CI)• are the respective activities at the inside of the membrane; and Pk, P.va and PA are the respective permeabilities. This equation shows the importance of the ratios of the permeabilities of the m a j o r ions in determining the m e m b r a n e potential. Hodgkin and Huxley postulated that at rest the m e m b r a n e of squid giant axon is far more permeable to potassium than to sodium or chloride. Therefore the potassium concentration ratio largely determines the potential, whereas during activity a large but transient specific increase in sodium permeability causes the potential to approach (not necessarily a t t a i n ) the sodium equilibrium potential. A delayed rise in potassium permeability, coupled with the fall in sodium permeability repolarizes the membrane. However, the analysis of the electrical data was not made in terms of permeability. A new parameter, the ionic chord conductance, gs, was defined by the following equation: Is =

gs

(V-Vs)

where Is is the current carried by a given ion, gs is the chord conductance of the ion, V is the membrane potential, and V» is the equilibrium potential for the ion. Intuitively this parameter is similar to the permeability, P, as a measure of the ease of movement of an ion through the m e m b r a n e . Measured "instantaneously" after a change in voltage, gs appeared to be constant ( 2 4 ) . However, after a voltage step, g s underwent changes with time which were dependent on the m e m b r a n e potential. The changes in gs for each ion were in the same direction as the postulated changes in permeability discussed above, i.e. depolarization of the m e m b r a n e beyond

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threshold produced a large transient increase in gN>, followed by a delayed increase in gK. T h e concept of the chord conductance has resulted in some confusion. It is necessary to distinguish gs from the more familiar slope conductance (1/ V ) of the membrane. The former is not a derivative, whereas the latter is, and the former is defined in terms of specific ionic currents, whereas the latter is defined in terms of the entire current. Woodbury ( 2 9 ) has formulated the relationship between the chord and slope conductances in terms of the major ions. O f course the common method of measuring membrane conductance, by noting the change in potential for a given current through the membrane, actually measures neither the chord nor the slope conductance, but approximates the latter. Also it is necessary to distinguish gs from the permeability P of the membrane to ions. Although both represent a measure of the ease of movement of ions across the membrane, they are not formally or experimentally the same. Using the constant field assumptions, Hodgkin and Horowitz ( 3 0 ) have derived the relationship between gs and P for the steady state. It was shown that gs depends on P, the membrane potential, and the concentration of ions on either side of the membrane. Furthermore, it has been shown that gs must depend on the concentration of ions within the membrane as well as their respective mobilities ( 3 1 ) . Deviations from equilibrium in which ionic currents flow through the membrane would be expected to result in changes in gs even if the intrinsic characteristics of the membrane do not change. The constant field theory predicts nonlinear current-voltage relationships and variable gs ( 2 7 ) . However, Hodgkin and Huxley found a constant gs on "instantaneous" measurement. N o explanation was given for this experimental discrepancy. It may be related to time dependent changes, since the equations are formulated for the steady state, which is not established instantaneously. Frankenhaeuser and Hodgkin ( 3 2 ) have attempted to explain the discrepancy by assuming that the fixed charges are localized at one site in the membrane. Until more is known about the behavior of the parameter, gs. in different types of membrances, it is hazardous to deduce that changes in g s reflect alterations in intrinsic characteristics of the membrane such as pore size, fixed charges, carrier transport, etc.

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Figure 45. Electrical model of the excitable membrane. The equilibrium potentials of K, Na, and CI are represented as the EMF's, VK, \NO, and Vci and the resistance of the membrane to the net flux of Ka, Na, and CI is shown as the variable resistances Rk, R\N, and RCi. The membrane capacity is in parallel. The membrane potential is the potential between points X and Y. An understanding of the ionic hypothesis is facilitated by the equivalent circuit model in Figure 45. The equilibrium potential for each ion is represented by an electromotive force, ( E M F ) , in series with a resistance (or conductance) which represents the permeability of the membrane, and the capacitance of the membrane is in parallel. In this circuit, variations in the relative values of the

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resistances would alter the net potential across the whole parallel circuit (the membrane). Specifically, at rest the resistance in the sodium line is far greater than that in the potassium line, so that the net potential is close to that of the E M F in the K circuit, whereas during activity the resistance of the sodium circuit is transiently but markedly lowered, allowing the E M F in the sodium circuit to predominate. In cardiac cells of various types in various species the overshoot of the action potential and maximum rate of rise of the upstroke have been found to vary in a roughly linear relationship with the sodium concentration (9, 2 9 ) . An exception is the cells of the guinea pig ventricle which generate good action potentials at very low sodium concentrations ( 3 3 ) . The dependence of overshoot and maximum rate of rise on external sodium concentration is predictable from the ionic hypothesis since the overshoot represents the approach to the sodium equilibrium potential, whereas the maximum rate of rise of the action potential is a first approximation of the inward sodium current (14, 2 8 ) . It has also been shown that the maximum rate of rise is related to the level of membrane potential at the time of excitation by an S-shaped function, much like the Hodgkin-Huxley curve of maximum inward sodium current plotted against the membrane potential preceding a large depolarization ( 1 4 ) . Prior hyperpolarization of the membrane results in larger inward sodium current and maximum d V / d t upon excitation, whereas prior partial depolarization results in a smaller inward sodium current and maximum d V / d t upon excitation. Hodgkin and Huxley postulated that the amount of a hypothetical "carrier" available to transport sodium inward was dependent on the membrane potential, and the concept of "available sodium carrier" appears often in the literature. However, a carrier for the current flowing during activity has not been demonstrated to date and is not a necessary postulate since the electro-chemical gradient could supply the driving force for the inward current flow, if the permeability of the membrane to free sodium ions were transiently increased. Action potentials of nodal fibers showing very slow rates of depolarization and no overshoot respond to acetylcholine and changes in extracellular K concentration differently from other car-

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diac fibers. It has been postulated that such cells may generate action potentials solely by a decrease in permeability to K, and not by an increase in permeability to N a ( 1 9 ) . Because of the technical difficulties involved in studying these cells, direct experimental verification of this postulate is lacking to date. T h e process of repolarization in cardiac cells has engendered a great deal of speculation in terms of the ionic hypothesis. T h e duration of repolarization in the heart is a thousand times as long as that in nerve. Furthermore, it is a very labile process, quite sensitive to rate, temperature, drugs, alterations in ionic concentrations and other influences. A m a j o r issue has been whether relatively simple modifications of the Hodgkin-Huxley hypothesis will suffice to explain cardiac repolarization. Intuitively a delay in the fall of gsa or the rise of gx or both would delay the repolarization of the membrane. Because of the inability to carry out technically adequate voltage clamping, reliance has been placed on indirect data. Important observations have been the finding for Purkinje fibers of lower slope conductance during repolarization than during diastole ( 3 4 ) ; the finding of a threshold for regenerative repolarization ( 3 4 , 3 5 ) ; and the finding of a nonlinear current-voltage relation in Purkenje fibers in Na-free Tyrode solution ( 3 6 ) . These findings indicate that the chord conductances are dependent on voltage as well as time during repolarization and that they probably are low. Noble has m a d e modifications of the Hodgkin-Huxley hypothesis based largely on the above observations ( 3 7 ) . T h e result of his assumptions is that the calculated g.\a is very high during the upstroke and then rapidly decreases to a level somewhat above the resting value where it remains for most of the action potential. On the other hand, gx falls below the resting value during the action potential. Recently published experiments by Deck and Trautwein, ( 3 8 , 3 9 ) in which the voltage clamp m e t h o d was applied to ligated Purkinje fibers, have show than gK is actually less than the resting value 12.5 msec after a large depolarization and declines with time if the depolarization is maintained. However, gNa is quite high at 12.5 msec and also declines with time. Some authors have postulated that the conductance changes during repolarization are dependent on time, and not voltage, because of failure to ob-

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serve a negative slope c o n d u c t a n c e during repolarization and the failure by some investigators ( 4 0 , 4 1 ) to find non-linear current voltage curves or regenerative repolarization in ventricular muscle. T h e work of Trautwein and D e c k , however, establishes the voltage dependence as well as the time dependence of g.sa and gK.

Noble

( 4 2 ) has suggested that failure to find non-linear current voltage relationships m a y be attributed to the experimental method and the preparation.

A summary of the various hypotheses for the m e c h -

anism of repolarization may be found in W o o d b u r y ' s recent review (29).

O f course, the establishment of the b e h a v i o r of gNa and gK

does not constitute an " e x p l a n a t i o n " for repolarization. A n observation made by W e i d m a n n ( 4 3 ) has produced some interesting speculation about the mechanism for a rise in gK and consequent repolarization of the cardiac fiber. It was observed that an injection of high K solution during the prolonged action potential of the cooled turtle heart produced shortening of the action potential.

W i e d m a n n considered the possibility that a c c u m u l a t i o n of

K ions outside the m e m b r a n e during the action potential might b e the normal mechanism for repolarization.

However, he

rejected

this idea because calculations indicated that the amount of K which could accumulate was insufficient.

Hoffman and Cranefield

(9)

have proposed the more general hypothesis that gK ( o r P k ) is inversely proportional

to the gradient of e l e c t r o c h e m i c a l

potential

acting on K . T h e r e f o r e , a high ( K ) < would result in a less negative equilibrium potential for K , which in turn would result in a decrease of the difference between the actual m e m b r a n e potential during repolarization ( n e a r z e r o ) and the potassium equilibrium potential.

Since this difference is a measure of the driving force on

K , a decrease of this difference would result in an increase in PK and

gK.

T h e chloride ion may be more important as a carrier of current in c a r d i a c cells than in the squid axon ( 3 8 , 3 9 , 4 4 , 4 5 ) .

The

chloride c o n d u c t a n c e has been estimated to be close to 3 0 per cent of the total c o n d u c t a n c e of the m e m b r a n e at voltages close to the resting value.

With depolarization of the m e m b r a n e , gs p r o b a b l y

increases. T h e slow diastolic depolarization that o c c u r s in

pacemaker

cells could be associated with a decrease in gK or an increase in

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gNa or both. The progressively decreasing slope conductance during diastolic depolarization suggests that there is a decrease in gK. The separation of membrane current into the individual ionic currents has generally required the use of unphysiologic solutions and inferences from indirect data. T o date, direct verification of the magnitude and time course of the bidirectional fluxes of the ions by tracer techniques has not been possible. The important studies of Keynes ( 4 6 , 4 7 ) showed that activity in nerve was accompanied by increased exchange of sodium and potassium. Furthermore, the amount exchanged per impulse was fairly close to that predicted by the ionic hypothesis. T h e data regarding the changes in exchange of N a and K with activity of cardiac muscle are far less definite and quantitative ( 4 8 - 5 0 ) but there does appear to be an increased exchange of both sodium and potassium with activity. The details of the magnitude, direction, and time course of the exchange are not known. The well known studies of Wilde and O'Brien ( 5 1 ) showed a pulsatile release of K' 2 from the perfused coronary system of the turtle heart. Lorber and associates ( 5 2 ) showed a similar phenomenon with frog ventricular strips. It was thought that the greatest efflux of K 42 was during the action potential. Lorber and associates calculated that the amount of efflux was less than predicted f r o m the Ussing equation for flux ratios, indicating a low permeability to potassium during repolarization. However, recent studies by Sjostrand ( 5 3 ) with the perfused sinus venosus of the frog have cast doubt on the interpretation of the pulsatile release. At low rates of flow of the perfusate, the concentration of K 42 in he perfusate showed a periodic variation consonant with the rate of contraction. However, at high rates of perfusion this periodicity was lost. Sjostrand postulated that the pulsatile release of tracer from the tissue may be related to the mechanical effect of contraction on unstirred layers. There was some indication f r o m his studies that the uptake of Na was periodic. Clearly, available data are not adequate.

III. ION M O V E M E N T S The unequal distribution of ions between the cell and interstitial fluid is generally believed to be due to the properties of the

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cell membrane. How it functions in this regard is largely unknown. In line with early concepts of the m e m b r a n e as an inert structure which did not participate actively in ion movements, it was initially believed that the unequal distribution of sodum and potassium reflected the impermeability of the membrane to cations ( 5 4 ) . T h e concept that the transmembrane gradients and potentials reflected the operation of a semi-permeable rather than an impermeable m e m b r a n e was developed at the turn of the century by several investigators, notably Bernstein. He proposed that the resting membrane was selectively permeable to potassium and that the resting potential was a potassium equilibrium potential ( 5 5 ) . Given a m e m b r a n e permeable to potassium, and assuming that the intracellular potassium exists in dissociated form, the greater intracellular concentration of this ion suggested the operation of a D o n n a n type equilibrium or of an active cation transport system capable of moving potassium against the concentration gradients. Since the D o n n a n equilibrium hypothesis, as outlined by Boyle and Conway ( 5 6 ) , was based, at least in part, on data suggesting membrane impermeability to sodium, the demonstration that sodium was also permeant ( 5 7 ) necessitated some modifications of this view. It led Dean in 1941 ( 5 8 ) to suggest the operation of a cation p u m p which could continuously extrude sodium from the cell or p u m p potassium in, or both. At about the same time several investigators noted that cold storage of erythrocytes caused the cells to lose potassium and to gain sodium ( 5 9 , 6 0 ) . These movements of sodium and potassium down their concentration gradients could be explained in terms of the Boyle-Conway theory by assuming that the cold had damaged the m e m b r a n e with resultant increase in ionic permeance. However, findings that the gradients could be restored by replacing the cells into the circulation or by rewarming them in the presence of glucose were more in line with the operation of a metabolically driven active cation transport system. Subsequent studies of transmembrane ion movements, such as those by Hodgkins and Keynes in squid giant axones ( 6 1 , 6 2 ) and Glynn in h u m a n erythrocytes ( 6 3 ) have provided strong support for this concept. They found that sodium efflux and potassium influx were markedly inhibited by depletion of adequate substrates or by application of metabolic inhibitors, e.g., fluoride to mam-

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m a l i a n erythrocytes, dinitrophenol to nerve, suggesting that these ion m o v e m e n t s were active in the sense of being dependant on the availability o f cellular energy supplies.

T h i s was also suggested by

the m a r k e d temperature sensitivity of these fluxes ( Q l O ' s 3 - 4 ) . O n the other h a n d , inward sodium and outward potassium movements, which o c c u r down their e l e c t r o c h e m i c a l

gradients were not sig-

nificantly affected by m e t a b o l i c ihibitors and had a low Q 1 0 .

The

active ion fluxes seemed to be related in other ways as well. Sodium efflux was not only dependent on the c o n c e n t r a t i o n of sodium inside the cell, but o n the external potassium c o n c e n t r a t i o n as well.

In

the a b s e n c e of e x t r a c e l l u l a r potassium, outward sodium m o v e m e n t decreased by a b o u t two-thirds.

T h e potassium dependant c o m p o -

nent of sodium efflux o c c u r r e d only in the presence o f adequate substrates a n d was a p p r o x i m a t e l y c o m p a r a b l e in m a g n i t u d e to the d e c r e a s e in potassium influx c a u s e d by m e t a b o l i c inhibitors. inter-relationships between these

fluxes

The

were interpreted to m e a n

that sodium extrusion o c c u r s in e x c h a n g e f o r potassium and that this process is linked to cellular energy supplies.

This

sodium-

potassium e x c h a n g e process has been designated as the sodiumpotassium e x c h a n g e pump o r simply the sodium p u m p . T h e extent to which these fluxes are c o u p l e d is still unsettled ( 6 4 ,

65).

Studies such as these have led to a general a c c e p t a n c e of the m e m b r a n e t h e o r y of ion transport including the c o n c e p t of an a c tive c a t i o n " p u m p , " although dissenting opinions are still heard (66).

A s s u m m a r i z e d by Hodgkin ( 6 7 ) this viewpoint holds that

the passive m o v e m e n t s of sodium into cells and potassium out of cells are c o u n t e r e d by a m e t a b o l i c a l l y driven " p u m p " which actively extrudes sodium in e x c h a n g e for potassium, although the relationship is not necessarily 1 : 1 .

T h e ionic c o m p o s i t i o n of cells repre-

sents a d y n a m i c equilibrium between these opposing a semipermeable membrane.

fluxes

across

In resting cells these fluxes just count-

e r each o t h e r .

I n e x c i t a b l e cells this b a l a n c e is altered at the time

of excitation.

T h e c h a n g e s in m e m b r a n e permeability during ex-

citation results in an increase in intracellular sodium and a loss of potassium.

F o r the cells to c o n t i n u e to f u n c t i o n , this process must

b e reversed during recovery b y e n h a n c e d activity of the sodium potassium e x c h a n g e m e c h a n i s m .

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T h e metabolic energy to drive the ionic p u m p is believed to come f r o m oxidative phosphorylation, a view based on experiments such as those by Caldwell and associates on squid giant axons ( 6 8 - 9 ) . T h e y showed that the depression of active ionic fluxes by dinitrophenol is accompanied by a decrease in the intra-axonal content of high energy phosphates such as adenosine triphosphate ( A T P ) , and that the effects of metabolic inhibitors could be countered by the intracellular injection of A T P . Since A T P applied to the outside of the m e m b r a n e was ineffective in this regard, it was f u r t h e r concluded that splitting of the phosphates probably occurred intracellularly. Observations in erythrocytes as well as in other tissues ( 7 0 - 7 4 ) have corroborated the importance of the b r e a k d o w n of high energy phosphates to active transport. A l t h o u g h the mechanisms by which ions actually penetrate and traverse the cell m e m b r a n e have been the subject of intensive study ( 7 5 - 7 9 ) , they are still largely unknown. Conceptually, the simplest m e a n s by which such movements of ions could occur would be by simple diffusion, for example, through aqueus pores penetrating the lipidprotein layers of the m e m b r a n e ( 8 0 - 8 3 ) . Estimates of the pore radii in h u m a n erythrocytes (3.5-4, 5 A ) by A . K. Solomon ( 8 2 ) , when c o m p a r e d with the estimates of the radii of hydrated ions (sodium, 2 . 5 6 A ; potassium 1.98A, and chloride 1 . 9 3 A ) suggest that such ion movements are theoretically possible. T h e difference in size a m o n g these ions could also explain the greater permeability of the resting m e m b r a n e to potassium c o m p a r e d to sodium but not the greater permeance of chloride c o m p a r e d to potassium. In order to explain this, some additional assumption is required, for example, the occurrence of an assemblage of positive charges within the pores. Since simple diffusional movements are directly dependent on the t r a n s m e m b r a n e electrochemical gradients, they cannot explain the active fluxes. Moreover, it is becoming increasingly unlikely that even the passive components of the sodium and potassium fluxes occur solely or even predominantly in response to electrical and diffusional forces. Kinetic analyses of these fluxes by Hodgkin and Keynes in squid axons ( 6 1 , 6 2 ) , by Glynn ( 6 3 ) and Solomon ( 8 4 ) in erythrocytes and by Keynes and Swan ( 8 5 ) and A d r i a n ( 8 6 ) in skeletal muscle

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suggest that these fluxes d o not meet the criteria of independance proposed by Ussing 1 ( 8 7 ) for simple diffusional systems. In instances where simple diffusion cannot explain transmemb r a n e ion movements, for example, movements against a concentration gradient, alternate hypotheses have been proposed involving more active participation of the m e m b r a n e in the transport process. T h e proposition that has gained the widest acceptance stems from suggestions by Osterhaut ( 8 8 ) and L u n d e r g a r d h ( 8 9 ) that for substances to cross the cell m e m b r a n e they must enter into a temporary combination with some m e m b r a n e c o m p o n e n t . C o m binations with both fixed and mobile m e m b r a n e constituents have been proposed ( 9 0 ) . Stein's and Danielli's suggestion ( 8 1 ) that the aqueous channels through the m e m b r a n e are lined by polar binding sites and that transmembrane passage depends on bond formation with these sites, so-called facilitated diffusion, is an example of transport in combination with fixed m e m b r a n e components. Alternately, the formation of the complex between substrate and m e m b r a n e component could result in the alteration of the characteristics of the substrate or m e m b r a n e so that on dissociation t r a n s m e m b r a n e passage substrate would be facilitated. F o r example, the combination could result in an enhanced kinetic energy or an altered spatial configuration of the subtrate or in alterations of pore size or configuration. The concept of transport in combination with a mobile membrane component or carrier is the more generally accepted of the two proposals. T h e carrier system that is most frequently cited is 1 If ions move freely across a membrane solely under the influence of their own kinetic energy then, assuming that the chance of any one ion crossing the membrane is uninfluenced by the presence of the other ions, the ratio of the outward movement ( M 0 ) to the inward movement ( M , ) of the ion may be described as follows:

_ ZEF/RT M, C„ C| and C„ are the internal and external concentrations, Z the valency of the ion. E the membrane potential. a n d £ , F, R. & T have their usual meanings. If the flux of any ion species does not satisfy this equation, i.e., if the ions do not cross the membrane independantly of each other, the flux cannot be attributed to a simple diffusional system.

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based on Osterhaut's guaicol model ( 8 8 ) . The substrate is visualized as forming a complex which diffuses across the m e m b r a n e to the opposite surface where it dissociates, discharging the unchanged substrate. T h e movement of the substrate-carrier complex is always downhill even though substrate itself may be moving against its electrochemical gradient. The system is energy dependent. Presumably energy is involved in the formation and dissociation of the carrier-substrate complexes. A carrier system which would explain the maintenance of transmembrane concentration gradients must allow for net movements of ions f r o m one side of the m e m b r a n e to the other. Shaw proposed such a system to explain cation movements in horse erythrocytes ( 9 1 ) . He suggested a single cyclical carrier f o r both sodium and potassium which could only move through the membrane in combination with one or the other of these ions. At the outer surface of the membrane it could complex only with potassium. T h e potassium-carrier complex would then cross the membrane. O n reaching the inner surface, potassium would be released and the molecule would be converted to a sodium carrier. T h e sodium-carrier complex would then cross to the outer surface where sodium would be discharged and the carrier would again become available for potassium transport. One round trip would result in the extrusion of a sodium ion in exchange for a potassium ion, thus accounting for the coupling between these fluxes, as well as for active transport of sodium and potassium. The carrier concept can also be adapted to explain other modes of ion exchange, such as Ussing's exchange diffusion ( 9 2 ) in which both the influx and efflux of an ion species are increased equally without any net change in electrochemical gradient. A carrier which could only cross the membrane in association with a particular ion species would, during the course of one round trip, simply result in the exchange of an intracellular for an extracellular ion of that species. Although these as well as most of the other proposed carrier models have been designed to account for the movements of ions against their electrochemical gradients, no specific evidence has been found to deny the participation of carriers in passive ion movements as well. In their recent detailed analysis of carrier transport mechanisms Wilbrandt and Rosenberg ( 7 8 ) stressed that there is no unequivocal way of

THE

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proving that a flux does not involve carriers. They also noted that under conditions of low substrate concentration, the kinetics of all transport systems, including simple diffusional systems, become virtually indistinguishable from each other. In light of these observations and in light of findings that passive ion fluxes probably are not due to simple diffusion, one may speculate that carriers are involved in passive fluxes as well. If this is so, a unitary carrier hypothesis might be formulated to explain both active and passive ion movements. All of the proposed models are speculative since the actual membrane components have not been isolated. Of the many substances that have been said to possess carrier properties, ATPase is currently receiving the most attention. Interest in this substance stems from the apparent importance of the hydrolysis of energy rich phosphates, particularly A T P , to the operation of the sodium potassium pump. Observations by Skou in 1957 ( 9 3 ) that the magnesium dependent ATPase in crab nerve required sodium and potassium for maximum activation suggested parallels between ATPase and the ion pump. The dependence of ATPase on the simultaneous presence of sodium and potassium has been subsequently confirmed ( 9 4 - 9 7 ) and there is suggestive evidence that potassium must be on the outside and sodium on the inside of the membrane ( 9 6 - 9 8 ) , circumstances reminiscent of the coupled movements of these ions across the membrane. Some investigators have found that the linkage of sodium and potassium with ATPase occurs at specific sites ( 9 4 - 9 6 ) . Other important shared characteristics have also been described including the glycoside sensitivity of ATPase (95-97, 9 9 ) . The concentrations of glycoside needed for ATPase inhibition are of the same magnitude as those required to inhibit the sodium-potassium pumping mechanism. Digitalis inhibition of both systems could be blocked by increasing potassium concentrations. The parallels between the two systems suggest that they are somehow linked in their operation. Some authors have speculated that ATPase may in fact be the carrier molecule (97, 1 0 0 ) . In 1956 Stein and Danielli commented that the "urgent requirement in this field is the isolation of the effective membrane component" ( 8 1 ) . The needs in this field remain the same today.

REFERENCES 1. Bowditch, H. P.: Ueber die Eigentümlichkeiten der Reizbarkeit, welche die Muskelfasern der Herzen zeigen: Arbeit aus der physiologischen Anstalt, zu Leipzig, 6:139 (1871). 2. Engelmann, T. W.: Refractare Phase und compensatorische Ruhe in ihrer Bedeutung für den Herzrhythmus. Pflug. Arch, ges. Physiol. 59:309-349 (1895). 3. Marey, E. J.: Des excitations electriques du coeur. Physiologie Experimentale. Travaux du Laboratoire de M. Marey, Paris, Masson, Volume 11:63 (1876). 4. Bozler, E.: The initiation of impulses in cardiac muscle. Am. J. Physiol. 755:273-282 (1943). 5. Ling, G., and R. W. Gerard: The normal membrane potential of frog sartorius fibers. J. Cellular Comp. Physiol. 34:383-396 (1949). 6. Draper, M. H., and S. Weidmann: Cardiac resting and action potentials recorded with an intracellular electrode. J. Physiol. 775:74-94 (1951). 7. Woodbury, L. A., H. H. Hecht, and A. R. Christopherson: Membrane resting and action potentials of single cardiac muscle fibers of the frog ventricle. Am. J. Physiol. 164:307318 (1951). 8. Nastuk, W. L., and A. L. Hodgkin: The electrical activity of single muscle fibers. J. Cellular Comp. Physiol. 35:39-73 (1950). 9. Hoffman, B. F., and P. F. Cranefield: Electrophysiology of the Heart. McGraw-Hill, New York, 1960. 103

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10. B u r d o n - S a n d e r s o n , J . S., a n d F . J . M . P a g e : O n the electrical p h e n o m e n a of the e x c i t a t o r y process in the h e a r t of the f r o g a n d of the tortoise, as investigated p h o t o g r a p h i c a l l y . J . Physiol. 4 : 3 2 7 - 3 3 8 ( 1 8 8 4 ) . 11.

H o r i b e , H . : Studies o n the s p r e a d of the right atrial activation b y m e a n s of i n t r a c e l l u l a r m i c r o e l e c t r o d e .

J a p a n Circu-

lation J o u r n a l 2 5 : 5 8 3 - 5 9 3 ( 1 9 6 1 ) . 12.

Horiba, Maretsugu:

S t i m u l u s c o n d u c t i o n in atria studied by

m e a n s of i n t r a c e l l u l a r m i c r o e l e c t r o d e s . P a r t I. T h a t in B a c h mann's bundle. 13.

Japanese Heart Journal 4:333-345

H o f f m a n , B. F . a n d basis of c a r d i a c

P. F . C r a n e f i e l d : T h e

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W e i d m a n n , S.: T h e effect of the c a r d i a c m e m b r a n e p o t e n tial o n the r a p i d availability of the s o d i u m c a r r y i n g system. J. Physiol. 7 2 7 : 2 1 3 - 2 2 4 ( 1 9 5 5 ) .

15.

H o f f m a n , B. F . , C . Y . K a o , a n d E . E . S u c k l i n g : R e f r a c t o r i ness

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E f f e c t s of c a l c i u m ions a n d local anesthetics

o n electrical p r o p e r t i e s of P u r k i n j e

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568-582 (1955). 17

Singer, D . H., B. K. Y e h , a n d B. F. H o f f m a n : A b e r r a t i o n of s u p r a v e n t r i c u l a r e s c a p e beats. F e d . Proc. 2 3 : 1 5 8 ( 1 9 6 4 ) .

18.

H o f f m a n , B. F . : E l e c t r i c a l Activity of the A t r i o - V e n t r i c u l a r N o d e : Specialized T i s s u e of the H e a r t .

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C o . , A m s t e r d a m , T h e N e t h e r l a n d s , 1 9 6 1 , pp. 1 4 3 - 1 5 8 . 19.

Paes de Carvalho, Antonio: Personal communication. B e r n s t e i n , J . : E l e c t r o b i o l o g i e ; die L e h r e von d e n electrischen

20

V o r g a n g e n im Organismus auf m o d e r n e r G r u n d l a g e dargestellt. F . V i e w e g & Son, B r a u n s c h w e i g , 1 9 1 2 . d e M e l l o , W . C., a n d B. F. H o f f m a n :

21

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Physiol 7 9 9 : 1 1 2 5 - 1 1 3 0 ( 1 9 6 0 ) . H o d g k i n , A . L., A . F . H u x l e y , a n d B. K a t z : M e a s u r e m e n t s of c u r r e n t - v o l t a g e r e l a t i o n s in the m e m b r a n e of the giant a x o n of L o l i g o . J. Physiol. 7 7 6 : 4 2 4 - 4 4 8 ( 1 9 5 2 ) .

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Hodgkin, A . L., and A. F. Huxley: C u r r e n t s carried by sodium and potassium ions through the m e m b r a n e of the giant axon of Loligo. J. Physiol. 7 7 6 : 4 4 9 - 4 7 2 ( 1 9 5 2 ) . Hodgkin, A . L., and A. F. Huxley: T h e components of membrane conductance in the giant axon of Loligo. J. Physiol. 776:473-496 (1952). Hodgkin, A. L., and A. F. Huxley: T h e dual effect of membrane potential on sodium conductance in the giant axon of Loligo. J. Physiol. 7 7 6 : 4 9 7 - 5 0 6 ( 1 9 5 2 ) . Hodgkin, A . L., and A. F. Huxley: A quantitative description of m e m b r a n e current and its application to conduction and excitation in nerve. J. Physiol. 7 7 7 : 5 0 0 - 5 4 4 ( 1 9 5 2 ) . Goldman, D. E . : Potential, impedance, and rectification in membranes. J. Gen. Physiol. 2 7 : 3 7 - 6 0 ( 1 9 4 3 ) . Hodgkin. A . L., and B. Katz: T h e effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 7 0 S : 3 7 - 7 7 ( 1 9 4 9 ) . Woodbury, J. W.: Cellular electrophysiology of the heart: Handbook of Physiology, Section II Circulation, Vol. 7 : 2 3 7 286. The A m e r i c a n Physiological Society, Washington, D. C „ 1962. Hodgkin, A . L., and P. Horowitz: T h e influence of potassium and chloride ions on the m e m b r a n e potential of single muscle fibers. J. Physiol. 7 4 5 : 1 2 7 - 1 6 0 ( 1 9 5 9 ) . Finkelstein, A., and A . M a u r o : Equivalent circuits as related to ionic systems. Biophys. J. 5 : 2 1 5 - 2 3 7 ( 1 9 6 3 ) . Frankenhaeuser, B.: Sodium permeability in toad nerve and in squid nerve. J. Physiol. 7 5 2 : 1 5 9 - 1 6 6 ( 1 9 6 0 ) . Coraboeuf, E., et M. O t s u k a : L ' A c t i o n des solutions hyposodiques sur les potentiels cellulaires de tissu cardiaque de Mammifères. C o m p t . Rend. A c a d . Sci. 243 \ 4 4 1 - 4 4 4 (1956). Weidman, S.: Effect of current flow on the m e m b r a n e potential of cardiac muscle. J. Physiol. 7 7 5 : 2 2 7 - 2 3 6 ( 1 9 5 1 ) . Cranefield, P. F., and B. F. H o f f m a n : Propagated repolarization in heart muscle. J. Gen. Physiol. 4 7 : 6 3 3 - 6 4 9 ( 1 9 5 8 ) . Hutter, O. F., and D. N o b l e : Rectifying properties of heart muscle. N a t u r e 7 8 5 : 4 9 5 ( 1 9 6 0 ) .

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37. Noble, D.: A modification of the Hodgkin-Huxley equations applicable to Purkinje fiber action and pacemaker potentials. J. Physiol. 760:317-352 ( 1 9 6 2 ) . 38. Deck, K. A., R. Kern, and W. Trautwein: Voltage clamp technique in mamalian cardiac fibers. Pflugers Arch. 280: 50-62 ( 1 9 6 4 ) . 39. Deck, K. A., and W. Trautwein: Ionic currents in cardiac excitation. Pflugers Arch. 280:63-80 (1964). 40. Johnson, E. A., and J. Tille: Evidence for independance for voltage of the membrane conductance of rabbit ventricular fibers. Nature 792:663-664 ( 1 9 6 1 ) . 41. Johnson, E. A., and J. Tille: Changes in polarization resistance during the repolarization phase of the rabbit ventricular action potential. Australian J. Exp. Biol. Med. Sci. 58:509513 ( 1 9 6 0 ) . 42. Noble, D.: The voltage dependance of the cardiac membrane conductance. Biophys. J. 2:381-393 ( 1 9 6 1 ) . 43. Weidmann, S.: Shortening of the cardiac action potential due to a brief injection of KC1 following the onset of activity. J. Physiol. 732:157-163 ( 1 9 5 6 ) . 44. Hutter, O. F., and D. Noble: Anion conductance of cardiac muscle. J. Physiol. 757:335-350 ( 1 9 6 1 ) . 45. Carmeliet, E. E., Chloride ions and the membrane potential of Purkinje fibers. J. Physiol. 756:375-388 ( 1 9 6 1 ) . 46. Keynes, R. D.: The leakage of radioactive potassium from stimulated nerve. J. Physiol. 775:99-114 ( 1 9 5 1 ) . 47. Keynes, R. D.: The ionic movements during nervous activity. J.Physiol. 774:119-150 ( 1 9 5 1 ) . 48. Humphrey, E. W., and J. A. Johnson: Potassium flux in the isolated perfused rabbit heart. Am. J. Physiol. 198:12171222(1960). 49. Conn, H. L. Jr., and J. C. Wood: Sodium exchange and distribution in the isolated heart of the normal dog. Am. J. Physiol. 797:631-636 ( 1 9 5 9 ) . 50. Grupp, G.: Potassium exchange in the dog heart in situ. Circulation Res. 73:279-289 ( 1 9 6 3 ) .

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Wilde, W. S.: The pulsatile nature of the release of potasium from heart muscle during the systole. Ann. N. Y. Acad. Sei. 65:693-699 ( 1 9 5 7 ) . Lorber, V., J. L. Walker. E. A. Greene, M. H. Minarik, and M. J. Pak: Phase efflux of potassium from frog ventricles. Am. J. Physiol. 203:253-257 (1962). Sjostrand, U.: Analysis of ionic tracer movements during single heart cycles. Acta Physiol. Scand. 61: Suppl. 227: 1-139 ( 1 9 6 4 ) . Hober, R.: Physikalische Chemie der Zelle und der Gewebe. 2nd Ed. W. Engelmann, Leipzig, 1906. Bernstein, J.: Untersuchungen zur Thermodynamik der bioelektrischen Strome. Erster Theil., Pflug. Arch. ges. Physiol. 92:521-562 ( 1 9 0 2 ) . Boyle, P. J., and E. J. Conway. Potassium accumulation in muscle and associated changes. J. Physiol. 700:1-63 (1941). Cohn, W. E., and E. T. Cohn: Permeability of red corpuscles of the dog to sodium ion. Proc. Soc. Exp. Biol. N. Y. 47:445-449 ( 1 9 3 9 ) . Dean, R. B.: Theories of electrolyte equilibrium in muscle. Biol. Sym. 3:331-348 ( 1 9 4 1 ) . Harris, J. E.: The influence of the metabolism of human erythrocytes on their potassium content. J. Biol. Chem. 141:519-595 (1941). Danowski, T. S.: The transfer of potassium across the human blood cell membrane. J. Biol. Chem. 739:693-705 (1941). Hodgkin, A. L., and R. D. Keynes: Active transport of cations in giant axons from Sepia and Loligo. J. Physiol. 728:28-60 ( 1 9 5 5 ) . Hodgkin, A. L., and R. D. Keynes: The potassium permeability of a giant nerve fibre. J. Physiol. 728:61-88 ( 1 9 5 5 ) . Glynn, I. M.: Sodium and potassium movements in human red cells. J.Physiol. 734:278-310 ( 1 9 5 6 ) . Conway, E. J.: The nature and significance of concentration relations of potassium and sodium ions in skeletal muscle. Physiol. Rev. 37:84-132 ( 1 9 5 7 ) .

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Conway, E. J.: Principles underlying the exchanges of K and Na ions across cell membranes. J. Gen. Physiol., Suppl. 45:17-41 ( 1 9 6 0 ) . 66. Ling, G. N.: A Physical Theory of the Living State. Blaoisdell Publishing Co., New York, 1962. 67. Hodgkin, A. L.: Ionic movements and electrical activity in giant nerve fibers. Proceedings of the Royal Society of London. Series B 745:1-37 ( 1 9 5 8 ) . 68. Caldwell, P. C.: The phosphorous metabolism of squid axons and its relationship to the active transport of sodium. J. Physiol. 752:545-560 ( 1 9 6 0 ) . 69. Caldwell, P. C., A. L. Hodgkin, R. D. Keynes, and T. I. Shaw: The effects of injecting "energy-rich" phosphate compounds on the active transport of ions in the giant axons of Loligo. J. Physiol. 752:561-590 ( 1 9 6 0 ) . 70. Gardos, G.: Akkumulation der Kaliumionen durch menschliche Blutkörperchen. Acta Physiol. Hung. 6:191-199,. 1954. 71. Dunham, E. T.: Linkage of active cation transport to A T P utilization. Physiologist 7:23 ( 1 9 5 7 ) . 72. Hoffman, J. F.: The link between metabolism and the active transport of sodium in human red cell ghosts. Fed. Proc. 79:127 ( 1 9 6 0 ) . 73. Hoffman, J. F.: The active transport of sodium by ghosts of human red blood cells. J. Gen. Physiol. 45:837-859 (1962). 74. Wittam, R.: Potassium movements and A T P in human red cells. J.Physiol. 740:479-497 ( 1 9 5 8 ) . 75. Symposium on Active Transport and Secretion. Symposia of the Soc. Exp. Biol. m £ cn m

.80

1.0 LENGTH

1.2 L'/Lo

Figure 57. A plot of PC hydrolysis and tension developed in a 20 second isometric tetanus (10 stimuli/sec.) as a function of the length of the muscle referred to resting length, Lo. J PC hydrolysed at length L' referred to PC hydrolysed at Lo. J Maximum tension developed at L' referred to tension developed at Lo. (2) there is a hydrolysis of phosphorylcreatine for both short and long lengths when there is no tension developed. In fact, there appears to be both a tension independent and a tension dependent component of phosphorylcreatine hydrolysis. While this is not an unexpected result, its full significance is not apparent at this time. But ultimately we expect to derive a picure of the chemical events of contraction that can be related to the structural changes associated with the mechanical interdigitating of action and myosin filaments.

REFERENCES 1. Carlson, F. D.: Abstr. Biophys. Soc. TC7 (1961). 2. Sandberg, J. and Carlson, F. D.: Abstr. Biophys. Soc. FE4 (1964).

155

The Role of ATP in Contraction R. E. DA VIES Department of Animal Biology School of Veterinary Medicine The University of Pennsylvania Philadelphia, Pennsylvania THE E X P E R I M E N T S I SHALL DESCRIBE WERE IN

COLLABORATION

with Drs. D. F. Cain, A. M. Delluva, A. A. Infante and M. J. Kushmerick. The problem of muscle contraction has interested people since time immemorial. For example, the ancient Greeks had a quite nice theory of muscle contraction which is the one that lasted longest, i.e. about eighteen hundred years. Nowadays theories of contraction rarely last more than a few months. Albert Szent-Gyorgyi, many years ago, said that life was a very similar process in cabbages and kings, but since cabbages were cheaper and easier to come by, he worked on cabbages. Now biochemists feel that the mechanism of contraction is similar in skeletal muscle and heart muscle at the biochemical level, and so they work on skeletal muscle because it's much easier. In fact, most of biochemistry has been developed with muscle. The basic mechanism of anaerobic glycolysis was worked out mainly with pressed muscle juice and the basic mechanism of oxidation, the tricarboxylic acid, or citric acid, or Krebs' cycle was worked out on pigeon breast muscle. About a hundred years ago, it was thought that muscle con-

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tained a vast molecule, myogen or inogen, which contracted when it took up oxygen and gave of! carbon dioxide. This was disproved nearly 60 years ago by Fletcher and Hopkins (1), who showed that muscles could contract in the absence of oxygen and that lactic acid was formed. In the next 10 or 20 years, work by Parnas ( 2 ) , Meyerhof (3) and Hill (4) showed that the energy for anaerobic contractions of muscles could be accounted for by the formation of lactic acid from glycogen and, in fact, muscle contraction seemed to be a completed story. There was no problem as to the energy source until Lundsgaard (5), using a drug, iodoacetic acid, found that animals seemed to go into rigor mortis whilst they were still alive. He investigated this material on isolated muscles and found that he could observe contractions of the muscles without the formation of any lactic acid, so the lactic acid theory of muscle contraction went down the drain along with a very large number of other theories that have also been consigned to the graveyard. However, creatine phosphate was broken down in the intact iodoacetate-treated muscle but could only break down in muscle extracts in the presence of a co-factor. Lohmann (6) found that this co-factor was adenosine diphosphate ( A D P ) . Hence it seemed that the role of creatine phosphate (or phosphylcreatine as it should be called nowadays) was to reconstitute ATP (adenosine triphosphate) from ADP. Next Engelhardt and Ljubimowa ( 7 ) , found that the main structural protein in muscle, myosin, was also an enzyme and, in fact, was an enzyme that broke down ATP. Then Szent-Gyorgyi (8) and Weber (9) and Needham (10) and many others investigated the quite fascinating contraction of actomyosin threads and reconstituted glycerinated muscle models which contract in the presence of ATP, so everything seemed fine, and all the textbooks said that ATP really was the energy source for muscle contraction. Fifteen years ago only A. V. Hill ( 1 1 ) seemed doubtful and he kept issuing warnings saying, nobody has ever proved that ATP does anything in living muscle except prevent rigor mortis, and the only time it ever changes is when the muscle is almost completely fatigued. Thus there was the problem of trying to find out what changes, if any, in ATP actually occurred.

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Some earlier experiments were made about 12 years ago to try to find changes in A T P in isolated muscles during single contractions, and these failed ( 1 2 ) . There seemed to be no measurable changes in A T P , A D P . creatine and phosphorylcreatine, or in m a n y other compounds tried, so this was a real problem. It resulted in 10 years of frustration and failure before anything even slightly significant began to appear. N o w you might think that these experiments would be very primitive and easy. All you need to do is get a large amount of muscle, analyze it, make it contract, analyze it again and see what the difference is. However, you just can't do that. F o r m a n y years all attempts to find out changes in single contractions failed because it was impossible ever to get a control. This was because of the methods used for extracting the muscle, which were grinding it with trichloroacetic acid or alcohol, or boiling it. These always caused both the experimental and control muscles to contract. It was only with the use of thin muscles and the advent of rapid freezing techniques that it became possible to have a control muscle that hadn't contracted or done any work. T h e principle was simple. W e went down to the Physiology D e p a r t m e n t to get a primitive kymograph that they had thrown in the junk store because it was too primitive for use by medical students. We hung the muscle on a little lever and cooled it down to 0 ° C . We hoped to keep the reactions sufficiently slow so that we could dissect the time course of a single contraction and at the appropriate moment stop the reaction with a freezing mixture. W e n o w use a mixture of Freons cooled by liquid nitrogen to about ~ 1 7 0 ° C . , and this is lifted up rather quickly by a simple device made f r o m a motor car tire p u m p and an old washing machine relay. T h e whole apparatus cost us only $2. Most of the early work was done on the rectus abdominis of female frogs because 14 years ago I fell among pharmacologists, and pharmacologists, as you know, like the rectus abdominis. At 20°C., it doesn't do much in the way of work if you stimulate it once, but at 0 ° C . if you stimulate it electrically about 12 times per second, it responds with a beautiful nice single tetanic contraction. It can do 120 gm. cm. of work per gm., and you can freeze it at any point. If you stop the stimulation, it will relax very nicely. N o w if the work is sup-

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plied by the breakdown of a molecule liberating around 10,000 calories per mole at an efficiency guessed at say 50 per cent, the expected breakdown for 120 gm.-cm. per gm. is in the range of 0.5 or 0.6 micromoles per gram muscle. This then is what you have to measure. In order to freeze the muscle quickly enough it has to be very small and thin. You can manage with a sartorius or a rectus abdominus which are usually not more than 1 0 0 - 2 0 0 mg. but what with duplicates and multiple assays the bit of it you actually analyze is not more than 10 or 20 mg., so you have to measure changes of around a few thousand-millionths of a mole of material which is fairly small. Once the muscle has been frozen solid, it is removed, weighed while it is still frozen and then, either ground up in a stainless steel centrifuge tube or put in a percussion mortar and hit with a heavy hammer. This is much better for your repressions. You can swing the hammer, bash at it and get rid of all your annoyance at the way the experiments are going. As I said, for the first 10 years everything went wrong. We couldn't find a change in anything, until eventually a very delicate new method for measuring inorganic phosphate was developed by Wahler and Wollenberger (13) in East Berlin. We modified this technique and with it found that inorganic phosphate was, in fact, released during the working part of a single contraction. By the way, we tried to keep all our experiments down to one contraction in less than a second because we didn't know what happened during the recovery processes. It had been shown by Lundsgaard that for a long series of contractions phosphorylcreatine is the energy source, and this finding has been extended considerably by Carlson and Siger (14) and by Mommaerts, Seraydarian and Marechal (15) over the last few years. What happened in a single contraction was still a mystery. During the first 10 years virtually all the soluble and insoluble phosphorus compounds were investigated until about 1960—all of the possible sources of inorganic phosphorus in the muscle had been analyzed and all of them were found not to change on contraction. Yet inorganic phosphate appeared and this raised several problems, of which the most likely was that someone had

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done some bad experiments. We started repeating some of the earlier ones with the better methods that had been developed and found that the classical reaction did in fact appear to be working in muscles during a single contraction. This classical LundsgaardLohmann scheme is that ATP is broken down by the myofibrillar ATP to make inorganic phosphate and ADP. This ADP is rapidly regenerated to ATP by phosphorylcreatine even during the time that the contraction is occurring and the overall process is thus the formation of inorganic phosphate and creatine from phosphorylcreatine. Now the amount of creatine phosphate in muscles is fairly large and we wanted to reduce this, which was done by pretreatment with 2, 4-dinitrophenol. Under these conditions, we could find changes during single contractions. Of course, lots of things happen in a contraction as shown by the whole massive edifice of Professor Hill's work over the last 50 years or more. There are major heat changes associated with activation, with shortening and with recovery, and we wanted to see if there were biochemical changes associated with these. By the way he used mainly sartorius, but with the rectus abdominis we couldn't measure any work associated with activation in this muscle even though we stimulated it 36 times in 3 seconds. The change was not significantly different from zero nor from 0.1 /xmole/gm. either. I should stress that these muscles were frozen rapidly immediately after the stimulation. You'll see later that there is an observable chemical concomitant of the activation process but this occurs during the recovery process after the physically observed activation is over. A series of experiments were then carried out in which a muscle was loaded and allowed to shorten various distances, so that a varying amount of work was done. There is a rather nice straight relation between work and phosphorylcreatine breakdown which passes through the origin (16). This was a surprise because Hill has said that there is a heat change associated with shortening rather than with work. We then separated work and shortening. The total external work was kept constant but the load was varied so some muscles

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lifted a heavy load a short distance and some a light load a long distance. The percentage shortening varied by a factor of three times but, the breakdown of phosphorylcreatine remained constant. In another series of experiments, the shortening was kept constant and the work was changed by allowing different muscles to lift different loads through a constant distance. It was found that the breakdown of phosphorylcreatine varied directly with the work done. The length-tension relationship shows that muscles at great lengths and at short lengths don't develop tension, whereas, around their natural resting length, they show maximum tension during isometric contraction. It turns out that this diagram for the rectus abdominis is approximately superposable on the curve giving the phosphorylcreatine breakdown during a 5-second tetanus at various lengths. Thus, if tension isn't developed, there isn't a breakdown of phosphorylcreatine. When the developed tension is maximal so is the breakdown. (17) It is obvious and has been stated many times in the literature that the observation of a breakdown of phosphorylcreatine does not prove that ATP is the energy source. Chance and Connelly (18) by very delicate spectrophotometric means have found a change of ADP in contraction that was only 3 per cent of the amount required. This is not overwhelming evidence that the real breakdown is 100 per cent, and that 97 per cent of it is being reconstituted enzymically. Better or firmer evidence was clearly needed, and many people have tried unsuccessfully to find an inhibitor that could block the creatinephosphokinase in muscle and leave the immediate energy reactions to continue. It's very interesting commentary on the progress of science that Kuby and Mahowald (19), in 1959, at the Federation Meeting, published an abstract showing that a compound, 1-fluoro, 2, 4-dinitrobenzene, which is widely known as the Sanger reagent and is used for getting at the structure of proteins, would inhibit crystalline creatinephosphokinase and also myokinase. But nobody did anything about it, largely because this is a very aggressive compound and it seemed reasonable that it would also destroy all enzymic activity in the muscle. However, when we saw the abstract it seemed the sort of

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thing we would only try when we were desperate, and sure enough two years later we were desperate, so we tried it. Dr. Cain found to his astonishment that if you incubate the muscle at 0 ° C . in properly defined conditions, then the enzyme, creatinephosphokinase is completely and irreversibly inhibited, and the muscle only contracts and does work to an amount equivalent to the A T P content ( 2 0 ) . When a normal sartorius muscle works there is an appearance of inorganic phosphate and of creatine. After pre-treatment with fluorodinitrobenzene. the inorganic phosphate production was virtually unchanged but no significant change was noted in creatine though the work done was quite similar. It was the same with the rectus abdominis. This was the next step past Lundsgaard's lactic acid contraction. It was an aphosphorylcreatine contraction, and it was immediately obvious that the A T P content in the muscle had to be looked at. It was looked at by two techniques in the rectus abdominis. O n e was the a m o u n t of light given off by an extract of firefly tails when you add an extract of muscle to it. In this case the flash of light f r o m the extract of firefly tails is related to the amount of A T P present. T h e other was the more commonly used change of the fluorescence of reduced N A D , or D P N as it used to be called, in the 3-phosphoglyceraldehyde dehydrogenase reaction. There was a clear breakdown of A T P in a single contraction and an a p p e a r a n c e of A D P , but the results looked bad. In two consecutive contractions, there was by no means a doubling of the A T P change, and the change in A D P was tiny. We then measured A M P to find out what had happened. It turned out that in this particular muscle which has a very low initial content of A T P there was a formation of A M P even during a single contraction. The results gave a good fit with those expected in muscles in which the actomyosin A T P a s e and myokinase were operative ( 2 0 ) . It was then f o u n d that myokinase was not completely inhibited. T h e r e was enough left to keep the reactions going. It was clear that A T P was breaking down and myokinase was active during a single contraction. This result immediately excludes several theories of muscle contraction, including those in which the A T P is b o u n d at the start of contraction, remains there during

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the whole process, and comes off again as A D P and inorganic phosphate at the end. It was quite clearly being regenerated during the actual contractile process. Most of this work was done during contractions of frog rectus abdominis, but the classic basic phenomenon much investigated by muscle physiologists is the single twitch of frog sartorius and this is not an easy thing for us to work on because under normal conditions the work done in a single twitch is much less than in a full tetanic contraction. Under our particular conditions of loading, the sartorius was doing somewhat less than 2 0 gm. - cm. of work per gm. Even so we were able to show that a breakdown of A T P occurred when the muscle was frozen at the peak of the twitch. T o our great surprise we found that an extra breakdown occurred if it was frozen during relaxation ( 2 1 ) . This was a surprise because heat measurements have shown that there is no heat change during relaxation except for what used to be called the relaxation heat, which is purely a mechanical effect of lowering the load. If a muscle lifts a load and then during relaxation the load stretches the muscle, the work done on the muscle by the loss of gravitational potential appears in the musicle as heat. If, however, you let the muscle lift the load and then hold the load up so it doesn't pull the muscle down, this so-called relaxation heat doesn't appear anymore. It is interesting that the observed extra breakdown is of the right order of magnitude to account for the pumping back of calcium into the sarcoplasmic reticulum described by Dr. Annemarie Weber ( 2 6 ) . It is quite likely the biochemical equivalent of the activation process, that is, if activation involves a process of spontaneous ionic changes that get the calcium to the part of the muscle where it's needed, a biochemical mechanism is then needed to pump all the calcium back again and this is what causes this extra breakdown of A T P during the decay of the active state. One of the most dramatic discoveries in muscle physiology in the last 10 years has followed from work in Professor A. V. Hill's lab done by himself, Howarth, Aubert and Abbott ( 2 2 ) . This discovery is that if you take a muscle, stimulate and then stretch it, the heat changes are such that there is an apparent disappearance of energy. Hill has repeatedly said that the results

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can be interpreted as showing that when you do work on such a stimulated muscle, some of this work is used to reverse the immediate chemical processes of muscle contraction. Thus it was important to try experiments like this to see if there was a resynthesis of adenosine triphosphate. More than a thousand measurements were made and the results show clearly that in all cases, stretching reduced the breakdown of ATP but did not cause a resynthesis. A difficulty was that while we were doing these experiments, Marechal and Aubert (23) published a paper saying that stretching an activated muscle caused an increased breakdown of phosphorylcreatine. There was also a statement at the Royal Society meeting last year by Dr. Wilkie that Dr. Abbott had done unpublished experiments which showed an increased breakdown, and so we were on our own with two very efficient workers on the wrong side, or maybe we were on the wrong side. Well, I wrote to Dr. Aubert about this, and found that they've since done some more experiments. Those with increased phosphorylcreatine usage were done at quite rapid rates of stretch. When they stretched at the rate we and A. V. Hill used, they also got a decreased usage and no resynthesis. I also talked to Dr. Abbott who said Dr. Wilkie must have misunderstood him and certainly had misquoted him. Dr. Abbott most definitely did not get an increased usage and said that his results were in the range showing a decrease. Now if A. V. Hill's conclusions are correct and apply to American frogs treated with 1 fluoro 2, 4-dinitrobenzene, this means that ATP is not the immediate energy source for muscle contraction. This result created problems so we need to consider the microanatomy of muscle. A recent paper of Tice and Barrnett (24) shows that there is an ATPase in muscles in the region of overlap of the thick and thin filaments. There is also the biochemical finding that the H-meromyosins, which form the cross bridges sticking out from the myosin rods, are the sites of the ATPase and also can bind to actin. Most people in the muscle field believe that contraction involves some interaction between the cross bridges of the myosin

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filaments and the actin in the thin filaments and this causes a relative movement so that one filament moves over the other. Whether they remain as straight sliding filaments or whether there is also folding as the ends get to the middle is still a matter for discussion, but it seems most likely that this region is the site of interaction. Now there isn't occasion, need or time to go into a possible theory as to how this might happen, but I'll present a very broad outline of it. Anyone who might possibly be interested can read it in the September 14th, 1963 issue of Nature ( 2 5 ) . It is based on the assumption that these side-pieces interact and cause movement because of a change that's well known in biochemistry:— the change in protein conformation from an alpha-helix to a mixture of a fully extended beta configuration and a randomly coiling form. Although the term random coil is widely used it is important to realize that this term "random coil" is not a good one. It should be randomly coiling. The polypeptide chains are in fact wriggling around like a bucketful of worms. It seems likely that sarcomeres move 100A for a usage of one A T P per interaction site and if you assume, and this is a complete assumption, that there is a 50 - unit flexible polypeptide in the head of the Hmeromyosin, it will move by 100A during the transition from the beta form to the alpha helix. This would occur during a sequence like this which has been worked out in some detail in the paper in Nature with some quantitative conclusions from it. The basic idea is that in resting muscle the A T P known to be bound to each myosin is assumed to be bound on the moving end of the postulated flexible polypeptide and that there is a negative charge near the fixed end. Thus charge repulsion will prevent the formation of an alpha helix and the polypeptide will continuously sweep out the space available to it. The ATPase is known to be in the head of the H meromyosin and there is good evidence that the binding site for A T P is different from the enzymically active site. In the resting muscle actin and myosin are separate and presumably repel each other. It's known, by the way, that there is a firmly bound A D P in the filaments of fibrous F-actin and this would repel the A T P of the myosin. However, activation of mus-

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cle seems to cause a liberation of calcium into the sarcoplasm and this ion would form strong links between the myosin-bound A T P and the actin-bound A D P . When these are linked the original charge repulsion will be obliterated so that the peptide can now contract to form an alpha-helix, the contraction actually being due to the formation of hydrogen bonds. This contraction should bring the A T P to the place where it can be cleaved by the A T P a s e which splits the link. It is clear that any theory of muscle contraction involving cyclic interaction between the actin and these side pieces of the myosin has to have a mechanism to develop the link, develop tension, cause movement and then break the link. All these three are included here. T h e formation of the link is by the electrostatic charge, of the calcium and the bound A T P and A D P ; the contraction is by the formation of hydrogen bonds; and the link breaking by the splitting of the A T P by the A T P a s e . The A D P formed is now rephosphorylated by the cytoplasmic A T P , or possibly the A D P physically exchanges for an A T P and then is reconstituted by glycolysis or by oxidative phosphorylation. The recreation of the myosin-bound A T P immediately breaks the alpha helix which rapidly re-extends and can reconnect with another site further along the thin actin filament. Thus the filaments will continue to interdigitate until the calcium ions are returned to the sarcoplasmic recticulum by the action of the ATP-dependent pump. This theory can explain the effects of calcium, and states that A T P is needed for micro-extensions of part of the myosin side pieces during this ratchet-like process of micro contractions and re-extensions but that the actual final immediate energy source for muscle contraction is the formation of hydrogen bonds. Thus the stretch experiments should break hydrogen bonds and not cause a resynthesis of A T P . However, they should cause a reduction of A T P breakdown because during stretch the actin is pulled backwards physically, so the A T P will be pulled away from the A T P a s e site and will not get split. Further pulling will cause a rise in tension until the electrostatic link is pulled apart allowing a new link to be formed further down the actin. The A T P will thus have its rate of breakdown reduced.

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The experiments were done to test this theory because if A T P was resynthesized the theory would have to be wrong. The increased rate of breakdown at high rates of stretch and many other observations on muscle are also explicable. This then is my view of the role of A T P in contraction.

REFERENCES 1. Fletcher, W. M. and Hopkins, F. G.: Lactic acid in amphibian muscle. J. Physiol. (London) 35:241 ( 1 9 0 7 ) . 2. Parnas, J. and Wagner, R.: Uber den Kohlenhydratumsatz isolierter Amphibienmuskeln und über die Beziehungen zwschen Kohlenhydratschwund und Milchsaurebildung im Muskel. Biochem, Z. 67:387 ( 1 9 1 4 ) . 3. Meyerhof, O.: Die Energieumwandlungen in Muskel. 1. über die Beziehungen der Milchsaure zur Warmebildung und arbeitsleistung des Muskels in der anaerobiose. Arch. ges. Physiol. Pflügers. 752:232 ( 1 9 2 0 ) . 4. Hill, A. V.: The recovery heat-production in oxygen after a series of muscle twitches. Proc. Roy. Soc. B 703:183 ( 1 9 2 8 ) . 5. Lundsgaard, E.: Untersuchungen über muskelkontraktionen ohne Milchsaurebildung. Biochem Z. 27 7:162 ( 1 9 3 0 ) . 6. Lohmann, K.: Uber die enzymatische aufspaltung der Kreatinphosphorsaure; zugleich ein Beitrag zum Chemismus der Muskelkontraktion. Biochem Z. 2 7 7 : 2 6 4 ( 1 9 3 4 ) . 7. Engelhardt, W. A. and Ljubimowa, M. N.: Myosine and adenosine-triphosphatase. Nature (London) 144:66% (1939). 8. Szent-Gyorgyi, A.: Chemistry of Muscular Contraction. Academic Press, New York, 1953. 9. Weber, H. H.: The Motility of Muscle and Cells. Harvard Univ. Press, Cambridge, Mass., 1958. 10. Needham, D. M.: Energy production in muscle. Brit. Med. Bull. 72:194 ( 1 9 5 6 ) . 169

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11. Hill, A. V.: A discussion on muscular contraction and relaxation: their physical and chemical basis. Introduction. Proc. Roy. Soc. B. 757:40 (1950). 12. Fleckenstein, A., Janke, J., Davies, R. E., Krebs, H. A. and Mommaerts, W. F. H. M.: Chemistry of muscle contraction. Nature (London) 774:1081 (1954). 13. Wahler, B. E. and Wollenberger, A.: Zur Bestimmung des Othophosphats neben sauremolybdat labilen Phosphorsauteverbindungen. Biochem. Z. J29:508 (1958). 14. Carlson, F. D. and Siger, A.: The mechanochemistry of muscular contraction. 1. The isometric twitch. J. Gen. Physiol. 44:33 (1960). 15. Mommaerts, W. F. H. M., Seraydarian, K. and Marechal, G.: Work and chemical change in isotonic muscular contractions. Biochem. Biophys. Acta. 57:1 (1962). 16. Cain, D. F., Infante, A. A. and Davies, R. E.: Adenosine triphosphate and phosphorylcreatine as energy supplies for single contractions of working muscle. Nature (London) 796:214 (1962). 17. Infante, A. A., Klaupiks, D. and Davies, R. E.: Relation between length of muscle and breakdown of phosphorylcreatine in isometric tetanic contractions. Nature (London) 207:620 (1964). 18. Chance B. and Connelly, C. M.: A method for the estimation of the increase in concentration of adenosine diphosphate in muscle sarcosomes following a contraction. Nature (London) 779:1235 (1957). 19. Kuby, S. A. and Mahowald, T. A.: Studies on ATP-transphosphorylases. Fed. Proc. 78:267 (1959). 20. Cain, D. F. and Davies, R. E.: Breakdown of adenosine triphosphate during a single contraction of working muscle. Biochem. Biophys. Res. Commun. 8:361 (1962). 21. Infante, A. A. and Davies, R. E.: Adenosine triphosphate breakdown during a single isotonic twitch of frog sartorius muscle. Biochem. Biophys. Res. Commun. 9:410 (1962). 22. See Hill, A. V.: Production and absorption of work by muscle. Science 737:897 (1960).

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Aubert, X. and Maréchal, G.: Le bilan énergétique des contractions muscularies avec travail positif ou négatif. J. Physiol. (Paris) 5 5 : 1 8 6 ( 1 9 6 3 ) . 24. Tice, L. W. and Barrnett, R. J.: Fine structual localization of adenosine triphosphatase activity in heart muscle myofibrils. J. Cell. Biol. 75:401 ( 1 9 6 2 ) . 25. Davies, R. E.: A molecular theory of muscle contraction: calcium-dependent contractions with hydrogen bond formation plus ATP-dependent extensions of part of the myosinactincross-bridges. Nature ( L o n d o n ) 7 9 9 : 1 0 6 8 ( 1 9 6 3 ) . 26. Weber, A.: The role of calcium in the regulation of muscular activity. In S. A. Briller and H. L. Conn, Jr., Eds., The Myocardial Cell. Univ. of Pennsylvania Press, Philadelphia, 1965.

The Mechanics of Myocardial Contraction E D M U N D H. S O N N E N B L I C K ,

M.D.

Senior Investigator Cardiology Branch National Heart Institute Bethesda, Maryland THE PURPOSE OF THIS REVIEW IS TO PRESENT SOME IDEAS CONCERN-

ING the intrinsic mechanical properties of heart muscle. Consideration of the heart as a muscle is not exactly new. About three hundred years ago, Niels Stensen ( 1 ) , noted that "the heart merits the name of muscle because it has tendons and flesh and nerves . . . thus from the fibers proceeds all movement of the heart." This view prevailed until the latter part of the nineteenth century when Otto Frank ( 2 ) , following studies on skeletal muscle (3, 4 ) , demonstrated that the mechanical analysis of muscular contraction, ordinarily characterized by the relations of muscle lengths, tensions and shortening, could also be applied to the beating heart if transposed into analogous terms of volumes, pressures and flows. Thereafter, the relative ease of measuring pressure and flow tended to solidfy the conception of the heart as a pump rather than a muscle. This approach has provided an important basis for our understanding of cardiac function especially when considering the heart relative to its role in the total organism. Indeed, so readily ap-

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plicable have been these views ( 5 - 8 ) that the intrinsic muscle properties of the myocardium have been largely neglected, and the analysis of cardiac function has been primarily concerned with the hydrodynamics of a compression pump. However, with the renaissance of our knowledge of the properties of skeletal muscle during the past twenty-five years ( 9 - 1 5 ) it is now appropriate to analyze the performance of the heart in terms of its properties as muscle. It may be expected that this approach will permit more basic definitions and further understanding of such phenomena as Starling's Law of the Heart ( 4 A ) or the poorly defined term, "myocardial contractility." As Hill ( 1 6 ) has suggested, the underlying proposition to this approach is that "all the dimensional relations referred to in voluntary muscle have their counterpart in the heart. T h e principles of similarity may not be applicable in all detail but they provide a general guide to the comparative physiology of the heart which could scarcely be found elsewhere." This account, of course, will of necessity be selective and brief, and is thus subject to much oversimplification. N o attempt will be made to review the literature completely. Further, our knowledge of myocardial mechanics and energetics remains far from complete. Following a description of some of the mechanics of heart muscle, certain structural aspects of the myocardium will be related to mechanics in order to demonstrate how ultrastructural properties may provide insight into such seemingly distinct properties as heart size and cardiac output ( 2 5 ) . For more detailed accounts of mechanics, the reader is referred to several recent reviews relative to skeletal muscle ( 1 7 - 2 2 ) as well as to a recent symposium ( 2 3 ) and review ( 2 4 ) in which some of the mechanics of muscle have been considered in relation to the heart. I. T H E M E C H A N I C S O F H E A R T A. The Two Component

Model for A dive

MUSCLE

Muscle

A. V. Hill ( 1 1 ) has provided a useful model for muscular contraction which serves as a framework for viewing experimental results, a background for theoretical speculation, and a basis for planning future experiments. As long as the model serves these pur-

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poses it is of value and therefore retained, although further advances may require alternative models or modifications of the present one

CONTRACTILE ELEMENT(CE) PARALLEL ELASTIC ELEMENT

SERIES ELASTIC ELEMENT ( S E )

A.V. Hill

1938

Figure 58. Hill's Three Component Model for Muscle (9).

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( 1 9 ) . On the basis of a series of elegant experiments, Hill ( 9 ) suggested that active muscle behaved as if it were composed of two components or elements arranged in series (Fig. 5 8 ) : ( 1 ) a contractile element ( C E ) , which at rest is freely extensible but when activated is capable of shortening and developing force; and ( 2 ) a passive elastic element arranged in series with the contractile element, termed the series elastic component ( S E ) . The contractile element is connected to the external environment only through this series elastic component. The series elastic component, in effect a passive spring that is stretched by shortening of the CE, is functionally, but not necessarily structurally, separate from the contractile element. Arranged in parallel with these two components is another elastic component, the parallel elastic element, which plays no role during contraction but is thought to support resting tension so that little force exists across the resting CE-SE complex. Again, the anatomical site of this parallel elastic component is not defined but may reside either in the contractile substance itself, in the sarcolemma (surface membrane), or to some extent in both. B. The Active

State

The mechanical properties of the contractile system as well as the series elastic component are manifest only during the presence of the active stale (18, 26-28). The active state of the C E is said to be present when those chemical processes which generate force or shortening at contractile sites in the C E are operative. Expressed in quantitative terms the active state in a single contraction has an intensity and a duration (26, 2 8 ) . The intensity of the active state relates to the magnitude of the force generated and the velocity with which the C E is shortening relative to force ( 1 4 ) . It has been observed experimentally, that an inverse relation exists between the velocity with which the contractile element can shorten and the load it is made to carry. This experimental fact forms the basis of the force-velocity relation of Hill ( 9 ) which characterizes the active CE. Maximum force (Po) in the C E occurs when velocity is zero, i.e. maximum isometric contraction. The maximum velocity of shortening of the CE (Vmax) is achieved when the load is zero, i.e. with an unloaded isotonic contraction. Velocity of shortening

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of the C E decreases as force increases. W h e n force is so large that s h o r t e n i n g c a n n o t o c c u r C E velocity falls to zero and isometric f o r c e ( P o ) is manifest. T h u s the active state is a mechanical reflection of those chemical processes in the contractile element which g e n e r a t e force o r shortening and the intensity of the active state m a y be expressed quantitatively by the force-velocity relation at any instant in time ( 1 4 , 2 9 ) . This definition of active state which includes the p a r a m e t e r s of both force and velocity is more inclusive t h a n Hill's original definition in terms of force ( o r active stiffness) alone ( 3 0 ) . This is especially true f o r heart muscle where the forcevelocity relations m a y be altered by multiple inotropic interventions such as n o r e p i n e p h r i n e or calcium, a n d where the muscle c a n n o t be tetanized. T h e i n s t a n t a n e o u s relation between force and velocity will be dealt with in greater detail when the specific properties of the active C E are explored. T h e duration of the active state also requires consideration in the twitch type c o n t r a c t i o n of heart muscle. ( 2 8 ) . T h e m a n n e r in which the active state in heart muscle is turned on and off is not entirely u n d e r s t o o d , but it would not a p p e a r to correspond directly to w h a t is k n o w n of the time course of the active state in skeletal muscle ( 2 6 - 3 0 ) . Recently the course of the active state has been studied in the cat papillary muscle p r e p a r a t i o n using a modified quick release m e t h o d ( 3 1 , 3 1 a , 3 1 b ) . By determining contractile element velocity as a f u n c t i o n of time with constant load and muscle length the entire course of the active state as well as the instantaneous force-velocity curve has been delineated ( 3 1 a ) . These studies have shown that the m a x i m u m intensity of the active state of heart muscle does not have an a b r u p t onset as has been demonstrated in skeletal muscle ( 2 6 ) but requires 100 to 150 msec, to develop (at 2 5 ° C ; 12 c o n t r a c t i o n s / m i n . ) . This m a x i m u m intensity of active state is then m a i n t a i n e d f o r 150 to 2 5 0 msec., following which the active state declines as peak isometric tension is reached and relaxation ensues. T h u s the active state in heart muscle does not switch on a b r u p t l y but develops with time. F u r t h e r the d u r a t i o n of maxim u m active state is directly p r o p o r t i o n a l to the time required to develop peak isometric tension. This course of events is consistent with the view that with electrical activation a substance, most likely calcium, is released f r o m a site external to the contractile

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sites in the sarcomere ( 3 2 ) . This activator then requires time to diffuse f r o m this external site of release to the sites of force generation in the sarcomere ( 3 3 ) . Such a suggestion is also consonant with the observations that the papillary muscle can shorten with maximum speed ( V m a x ) prior to the development of maximum active stiffness as seen in quick stretch experiments ( 3 4 ) . These findings could result if the establishment of Vmax required only superficial activation of the sarcomere while Po required activation of the entire contractile mass. Other problems inherent in describing the active state will be dealt with subsequently. In describing contraction of the heart in mechanical terms it is first necessary to define the properties of the C E and SE. In order to examine contractile and series elastic components separately, muscular contraction is analyzed under different mechanical arrangements. In Figure 59, a muscle has been arranged for an isometric contraction, i.e. its external ends have been fixed. T h e implications of such isometric measurements are of special interest since this is the m a n n e r in which heart muscle strips have usually been studied in the past. On the left in Figure 59, is the model; on the right, the mechanical event, with recorded isometric force depicted as a function of time. Following activation, the C E rapidly begins to shorten at its maximum velocity ( V m a x ) . In the process of shortening, the C E stretches the SE. This builds up force in the SE, and the force in turn is delivered to the external attachments of the muscle (solid line). As the force is generated in the SE by the shortening of the C E , the velocity of shortening of the C E falls in accordance with the force-velocity relation. The manner in which force builds up externally at the attachments of the systems depends primarily on three factors: ( 1 ) the shortening properties of the C E which are defined by the force-velocity relation at any instant during contraction under the given set of conditions: ( 2 ) the elastic properties of the SE, as defined by the stress-strain relations of this internal spring; and ( 3 ) the time allowed for the interaction between the C E and SE, i.e. the duration of the active state. Included in this last item are considerations of the rapidity with which the active state turns on and off. In Figure 59, on the right, the course an isometric contraction is portrayed between points ( A ) and ( B ) ; the broken line represents the hypothetical curve for the active state

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ISOMETRIC

Figure 59. Isometric contraction. On the left, the model; on the right, isometric force (solid line), and the hypothetical maximum force potential or active state of the contractile element (dashed line) are shown as functions of time after stimulation. In both panels, Point A is the initial resting state; Point B, some time during the active contraction. in the C E expressed as the maximum force which it could develop ( P o ) if no series elastic attachments existed. Expressing the active state in this manner is of course incomplete since velocity of shortening is not considered. T h e isometric force development (Fig. 59, solid line) is found to lag behind the relatively rapid onset of the active state (dashed line). This lag in force development results

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largely from the time necessary for the C E to stretch the SE ( 9 ) . A s force is built up in the SE due to the shortening of the C E , the velocity of shortening of the C E declines, following a course prescribed by the force-velocity relation. When the force generated in the SE by the shortening C E equals maximum isometric force, C E velocity reaches zero and force then remains constant. This force represents tetanus tension or true Po. However, since a finite time is necessary for the C E to stretch the SE, tetanus is reached only if the duration of the active state is sufficiently long. In the "twitch" resulting f r o m a single action potential, maximum or tetanic force may not be reached since the duration of the active state is generally limited, especially at physiologic temperatures. Thus, at no point during the single isometric twitch does a steady state exist. This is the general case for heart muscle which cannot be tetanized. The isometric force and the manner in which it is developed in the twitch will depend on the intensity of the active state, as characterized by the force-velocity curve at any instant in time, and the duration of the active state. Increased force could result from an increase in the duration of the active state with a constant intensity; ordinarily this does not occur in mammalian heart muscle as it does in skeletal muscle except with hypothermia. O n the other hand, an increase in force could result from increasing the intensity of the active state without a change in its duration. Such a change results in an increase in the rate of force development (given the same SE c o m p o n e n t ) with no change in the time to maximum force. This is illustrated in the change from A to B in Figure 60. T h e manner in which the force velocity curve of the C E , which characterizes the active state intensity, is shifted is not defined. It is also possible to change the intensity of the active state with no change in developed force. Thus, as in B to C in Figure 60, the duration of the active state is abbreviated while the intensity of the active state is augmented proportionately. This result is an increase in the rate of force development, while the developed force remains unchanged. Despite the fact that peak developed force is unchanged, the contractile state of the muscle has been profoundly altered. Such a change in active state is important for heart muscle ( 3 7 . 3 8 ) . The manner in which such changes may occur requires more detailed knowledge of

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both the C E and SE. Mechanical analysis of the isometric contraction is thus a complex affair, determined by an interaction

T T I M E Stimulation Figure 60. Isometric force development. Hypothetical isometric contractions are demonstrated. The change from curve A to B would result from a change in initial muscle length. The change from B to C illustrates how a profound change in contractile activity as may occur with changing frequency of contraction may not be reflected in increased force development but rather by an increase in the rate of force development.

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ISOTONIC A

B

C

CE\

A I ofSE

P(load)

\ A! of toad 1 Stimulation

Time •

Figure 61. Afterloaded isotonic contraction. On the left, changes in the model with time: on the right, force and shortening as functions of time after stimulation. Between A and B, the CE shortens at the expense of the SE. This represents the isometric portion of the contraction. At B, the force generated in the SE equals the load, P, and external shortening begins. From B to C, the SE remains constant in length and the shortening of the muscle reflects the shortening of the CE alone.

between the C E and SE in which both components are altered as force is generated. This is further complicated by a potential limitation of the time available for forcc development. In contrast to the isometric contraction, the isotonic contraction provides a

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m o r e steady state so that the contractile and series elastic components may be dissociated and analyzed separately. II. T H E C O N T R A C T I L E

ELEMENT

In Figure 61, the same contractile components are shown as in Figure 59 but with the analogue for an afterloaded isotonic contraction. On the left is the model; on the right, force and shortening are recorded as functions of time. Unlike the isometric system, the lower end of the model is not fixed but attached to a load ( P ) which is supported when the muscle is at rest (point A ) . This type of load ( P ) which is encountered by the C E only when it attempts to shorten, is termed the ajterload while the small load used to stretch the system to its initial length while at rest is termed the preload. With stimulation of the system, the C E begins to shorten at m a x i m u m speed ( V m a x ) . As the C E shortens and stretches the SE, between A and B, force is generated. As the force builds up, the velocity with which the C E shortens decreases. At B, the force in the SE matches the load ( P ) , and the load starts to move. Once the load moves, the SE, which was being stretched until this point, now remains constant in length as a function of the load ( P ) . Hence, subsequent to the point B, shortening of the system reflects the shortening of the C E alone apart f r o m any changes in the length of the SE. Thus, if the shortening of the muscle between B and C is analyzed, the C E can be studied independent of the S E component. In Figure 62, the experimental arrangement for the study of an afterloaded isotonic contraction is shown diagrammatically. In such a manner, the characteristics of the activated C E may be analyzed relative to load and time independent of the properties of the SE.

A. The Force Velocity Relation of the CE By analyzing the afterloaded contraction, the mechanical properties of the contractile systems have been analyzed in many different muscles ( 9 - 2 0 , 3 6 - 4 4 ) , including heart muscle ( 3 4 , 37, 38, 45, 4 7 ) . In studying the effects of increasing load on the initial velocity of shortening, what is perhaps the most characteristic property of the contractile element, namely the force-velocity relation,

0

500 T I M E (msec)

Figure 62. Experimental arrangement for study of the afterloaded isotonic contraction of the papillary muscle (diagrammatic) (37). The muscle, held in a bath (not shown), is attached below to a force transducer and above to a lever for the measurement of displacement. The initial length of the muscle is set by a preload, and is held constant by a stop above the lever. Added loads, afterloads, are not encountered by the muscle until it attempts to shorten. The total load comprises the preload plus the afterload. Below, a typical afterloaded contraction is shown. Time zero is at stimulation. Tension and shortening are illustrated. The tangent to the shortening trace denotes the initial velocity of shortening under the given condition.

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I N I T I A L L E N G T H CONSTANT (llmm) (preload 0.4g) INCREASING AFTERLOAD

/

Figure 63. Superimposed afterloaded isotonic contractions. Tension and shortening are shown as functions of time after stimulation. Initial muscle length is constant but load has been increased progressively. The dashed lines on the shortening portion of the trace represent the initial velocity of shortening. Note that with increasing loads, the velocity of shortening decreases. Further, the isometric phase of relaxation is prolonged as load is increased.

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may be defined. When a muscle contracts against increasing loads (Fig. 63) from a constant initial muscle length, characteristic findings are recorded. In Figure 63, superimposed recordings from such an experiment have been shown for the cat papillary muscle. 1 Force is recorded below and shortening above. Both are shown as functions of time after stimulation and it may be noted that as the afterload is increased the initial velocity of shortening declines. With increasing load, the time for the stimulus to the onset of shortening is prolonged but the time to maximum shortening is unchanged. When the initial velocity of isotonic shortening, i.e. the tangent to the initial shortening slope, is plotted as a function of load, the characteristic force-velocity curve is obtained. In each case, initial muscle length is constant. In Figure 64, this relation has been plotted on the left. As in other muscles, the initial velocity of the shortening muscle is determined by the load. When the load is extrapolated to zero, velocity is maximal (Vmax or intrinsic velocity); when the load is increased to the point that shortening cannot occur, maximum force is manifest (Po or intrinsic force). Thus, force and velocity of shortening of the CE are intimately related. One criticism of obtaining the force-velocity relation in this manner is that with increasing loads, increasing time is spent in force development, and the initial velocity of shortening is measured at progressively later periods in time after stimulation. If it were not for the fact that the maximum active state is maintained for a considerable portion of the contraction time ( 3 1 ) , this factor would be a limiting experimental difficulty. However, when the velocity of shortening with increasing loads is measured at a constant time after stimulation, as in Figure 65, using the methods of Jewell and Wilkie ( 1 4 ) , the inverse relation between velocity of shortening and load still pertains. Thus, while the dependence of the instantaneous force-velocity curve on time has been recognized ( 3 1 a ) , it has been found that the instantaneous force-velocity curve is relatively stable over a large extent of the rising portion of 'In all subsequent experiments to be described the cat papillary muscle has been used. It serves as a most useful experimental tool since it is a linear sample of heart muscle with its fibers arranged in parallel, without the geometric complexities of the intact ventricle.

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Cat Papillary Muscle l 0 = 13mm r -

i

Vmax \ \ \ \

Hill

'38

(P + a)(V

+ b)=(P„ + a)b

\°Sp*0.45 1 b = 5.5 mm/sec.or when

.42

lo/sec.

P - 0 Vmax * (P0

I

/a)b

1

5

10 LOAD (gm)

LOAD ( g m )

Figure 64. Force-velocity relation of the cat papillary muscle. The Hill equation is given in the insert with the derived constants a and b for the muscle. On the right, power (force x velocity of shortening), and work [force (or load) x displacement (AL)] are given as functions of load. the isometric force curve, the maximum intensity of active state being maintained for approximately 70 per cent of this period of time ( 3 1 ) . Accordingly, the absolute values derived for Vmax are somewhat underestimated but the general principles and relative changes remain pertinent. T h e force-velocity relation is of especial interest because it not only describes the mechanical limits of C E at any moment,

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but also provides some insight into the energetics of the active C E . By studying heat and energy flux relative to load, Hill ( 9 ) , following the lead provided by the studies of Fenn ( 4 3 , 4 9 ) , clearly

INSTANTANEOUS FORCE - V E L O C I T Y R E L A T I O N AT 2 7 0 MSEC A F T E R S T I M U L A T I O N (QUICK R E L E A S E )

LOAD-GRAMS Figure 6 5 . The instantaneous force-velocity relation of the cat papillary muscle, determined by quick release 2 7 0 msec, following stimulation. On the ordinant is velocity of shortening following quick release; on the abscissa is load. Initial length 9.5 mm. Cross-sectional area .77 mm 2 . Temperature 2 3 ° C . Frequency 1 2 / m i n .

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established the fact that the rate of energy liberation by the CE is controlled by the mechanical conditions during contraction. With increasing demands for work, extra energy is mobilized by the contractile system (Fenn effect). In addition to proving that the mechanical behavior of the CE could not be explained by viscous properties these experiments serve as the basis for assuming a tight linkage between mechanics and the chemical reactions during contraction. As Podolsky has stressed (22, 29) the energy produced during contraction results from "the driving chemical reaction" and the chemistry in turn is controlled by mechanical demands. The hyperbolic curve of the force-velocity relation has been characterized by Hill's well known equation (9), as given in Figure 64, where P is the load, V the velocity, and a and b, constants which fit the shape of the curve when it is considered to be a displaced hyperbola. While other equations, e.g., Aubert (12) or Fenn and Marsh ( 3 9 ) , also may be made to fit the mechanical data, Hill's equation has the added virtue that it may also be obtained from experiments involving the measurement of heat and work. The initial work of Hill ( 9 ) held that the constants a and b are the same whether obtained from mechanical or heat experiments. This identity supports the concept of a tight mechanochemical link ( 2 9 ) . However, this equality of mechanical and heat constants has been derived only from the frog sartorius. Further, more recent work of A. V. Hill (15) would suggest that certain modifications of the initial formulations are required in order to equate the mechanical and heat constants a and b. Nevertheless, the general principles appear to remain valid. The constants of the Hill equation can be derived from the mechanical experiment if the curve is considered to be a displaced hyperbola. This is the form in which the equation is written in Figure 64. Po, a and b determine the shape of the curve. By heat measurements, Hill demonstrated a to be the extra heat liberated by the muscle with shortening, while b was shown to be constant for the rate at which chemical energy was converted to mechanical energy, i.e. the rate constant of the driving chemical reaction in contraction ( 9 ) . Since when the load is zero, velocity is maximum (Vmax), the equation may be simplified to Vmax = ( P o / a ) b . In heart muscle, experiments would indicate that Po/a is a rather

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constant value ( 3 8 ) . Vmax then becomes a direct function of b; the constant b in turn is a measure of the rate with which the chemical reactions occur at the unloaded contractile sites ( 2 9 ) . Therefore, measurement of Vmax provides meaningful insight into the rate of mechanochemistry in the contractile sites of the muscle. In the particular instance depicted in Figure 64, these constants have been calculated from the mechanics of the cat papillary muscle. The ability to transpose these mechanical constants into energetic constants remains totally speculative although Hill has provided a precedent for this with his work on the frog sartorius ( 9 ) . 2 Moreover, not all experiments with the cat papillary muscle allow for the mechanical derivations of the constants in Hill's equation since a hyperbolic force-velocity relation is necessary. In instances where the active state is abbreviated, Po is not reached and the curve diverges from an hyperbolic form at higher loads. B. The Contractile State of the Heart Since the force-velocity relation of the C E at any instant in time is unique for that given functional state and muscle length, it can be used to define the contractile state of the contractile element, i.e. the manner in which the C E converts chemical energy into mechanical energy. If the character of the force-velocity relation at any instant is defined and the duration of active state determined, the limits of performance of the contractile elements are also defined. This view offers insight into the two major functional properties of heart muscle: ( 1 ) changing muscle length which is the basis of the Frank-Starling principle; and ( 2 ) changing contractility or the inotropic state of the muscle. 1. CHANGING MUSCLE LENGTH ( T H E FRANK-STARLING PRINCIPLE) AND THE INSTANTANEOUS FORCE-VELOCITY-LENGTH RELATION

When the length of either heart or skeletal muscle is increased prior to contraction, the ensuing contraction produces an increased 2 Hill ( 1 5 ) has recently presented evidence that the constant a obtained by heat measurements is load dependent. However, with the proper correction factor, the previous relation between the heat measurement and the constant a obtained from mechanical experiments still pertains.

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VELOCITY OF SHORTENING OF THE C E : INCREASING MUSCLE LENGTH

Figure 66. The effects of increasing initial muscle length on the forcevelocity relation. Initial velocity of shortening is plotted as a function of load. The insert indicates the muscle lengths corresponding to the increasing preload. Note that with increased initial muscle length, Po is increased with no change in Vmax.

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force. When the muscle is stretched beyond a critical length, the developed force falls. This dependence of the developed force on the initial muscle length is the basis of the well known principle espoused by Frank ( 2 ) and Starling ( 4 a ) , by which an increase in the initial volume of the heart allows for a more forceful contraction, and thus the ejection of a greater stroke volume. When the initial muscle length of the isolated papillary muscle is increased, characteristic shifts in the force-velocity curve occur as shown in Figure 66 (37, 3 8 ) . Here initial velocity of shortening has been plotted as a function of increasing afterload. The initial muscle length has been increased by augmenting the preload as noted on the graph and each of the curves portrayed is derived from one muscle length. With increased initial length, Po (intrinsic force) is increased with little or no change in Vmax (intrinsic speed) as indicated by the convergence of the curves towards a common intercept on the vertical axis at zero load. In this circumstance the muscle performance has been moved along a given length-tension curve, as shown in the insert of Figure 66. Such a shift in the force-velocity curve in which Po changes as a functional muscle length while Vmax remains unchanged connotes no change in the contractile state of the muscle. The relation of force and velocity not only characterizes the initial velocity of shortening of the muscle at a given length, but, as recent studies have shown ( 4 6 ) , the instantaneous velocity of shortening remains a function of instantaneous muscle length and afterload throughout contraction. This view is also consonant with findings in skeletal muscle ( 5 0 ) . The presentation of such data lends itself to a three dimensional plot of force (load), velocity and instantaneous length, as suggested by Fry ( 5 1 ) . In Figure 67, a set of curves has been derived from single contractions with increasing afterloads by plotting the instantaneous velocities of shortening during each contraction as a function of instantaneous muscle length. The velocity of shortening of the contractile element at any instant depends not only on the initial muscle length, but during contraction depends on the force-velocity curve appropriate to the muscle length at that instant in time ( 4 6 ) . The similarity of such a set of curves in Figure 67, to those derived from multiple initial muscle lengths in Figure 66 is apparent. Thus

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the entire course of an individual afterloaded contraction can be described with this myriad of curves relating instantaneous velocity

Figure 67. A three-dimensional representation of the relations of force (load), velocity, and muscle length (46). On the base is shown load (force) and muscle length. On the vertical axis is velocity of shortening. Thus the basal plane shows the length-tension relation; the vertical plane to the right, the force-velocity relation; and the plane to the left, the velocity length relation. The superimposed dashed line shows the course of a single afterloaded contraction plotted relative to this construct. See text.

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of shortening load and muscle length. The shortening muscle moves across the surface formed by these curves. For example, the muscle in Figure 67 is activated at point A. The rapid development of the maximum active state allows the contractile elements to shorten at maximal velocity (Vmax), placing the muscle on the force-velocity curve at point B. The delay which is involved in reaching point B has already been discussed relative to active state (vide supra). As the CE shortens internally between B and C, force is built up as it stretches the SE. With this build-up of force during the isometric phase of contraction, velocity of shortening of the C E declines as required by the force-velocity relation. At point C, the generated tension in the muscle equals the imposed afterload and shortening of the muscle begins. Velocity does not remain constant as the muscle shortens, but declines largely as a function of length (C to H ) , the load remaining constant. In a given functional state such as in Figure 67, the muscle starts on one curve, and its velocity changes as it moves to another curve with shortening. Further, should the active state begin to decline during shortening, i.e. the activity of the muscle begin to turn off, the muscle's velocity will fall away from these curves as relaxation ensues. In that case the muscle would follow the course from F to K. This representation of the instantaneous force-velocity-length relation comprises the limits of one contractile state of the muscle. 2 . CHANGING THE CONTRACITLE STATE OF THE MUSCLE

Not only can the force-velocity relation be displaced by altering initial muscle length, but it also can be shifted at any one muscle length by various inotropic interventions which alter the basic contractile state of the muscle (34, 37, 38). Two such inotropic interventions are illustrated in Figure 68: on the left is shown the effect of increasing frequency of contraction; and on the right, are shown the effects of norepinephrine. As initially demonstrated by Abbott and Mommaerts for heart rate ( 3 4 ) , and further characterized by Sonnenblick (37, 38, 46), the hallmark of a change in the contractile state of a muscle is an increase in Vmax, with or without a change in Po. A usual accompaniment of this increase in Vmax is a decrease in the duration of the active

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HEART RATE

NOREPINEPHRINE

L 0 A 0

loml

Figure 68. The effect of changing heart rate and the addition of norepinephrine on the force-velocity relation. Initial velocity of shortening is plotted as a function of load. The time from stimulation to maximal shortening ( A L ) , time to maximal force, and the time to maximum rate of force development (in g/sec.) are also shown. Note that with the augmentation of Vmax, all of these time parameters are abbreviated. Thus a change in the contractile state is represented by a change in the coordinate of velocity (Vmax) whether or not a change in Po occurs.

state ( 3 7 , 3 8 ) . This decrease in the duration of active state is reflected by a decrease in the time required to reach maximum force or shortening, as noted in the insert in Figure 6 8 . Indeed, a general, but not necessarily linear, inverse relation holds between intrinsic velocity and the duration of the active state ( 3 8 , 5 2 ) . Supporting this argument is the fact that changing initial muscle

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length, which does not change Vmax, does not alter the duration of the active state (37, 38, 52, 53, 5 4 ) . Thus, in mammalian heart muscle, an increase in force which may accompany an inotropic intervention cannot be attributed to prolongation of the active state but must evolve from an increase in the intensity of the active state, characterized by a shift in the force-velocity curve with an increase in Vmax. This is opposed to what is observed in the skeletal muscle twitch, where force may be augmented by prolonging the duration of the active state without changing Vmax (21, 5 5 ) . However, some of the responses of amphiban heart muscle more closely resemble the findings in skeletal muscle by demonstrating a prolongation of active state (56, 5 7 ) . A measured increase in C E force might occur in a twitch without a real change in Po if Vmax alone were augmented in the face of a limited duration of the active state, since the C E might shorten further during the same limited period of time and thus develop a force more nearly approaching Po. Although this possibility exists, recent work indicates that a true increase in Po may also accompany an augmentation of Vmax following inotropic interventions such as cardiac glycosides, calcium and norepinephrine ( 3 8 ) . This is especially evident when heart muscle is studied at lower temperatures. Therefore a change in contractility may be defined by a shift in the force-velocity-muscle length relation which establishes a new Vmax. In this new state, the muscle is still subject to the same operational limits of instantaneous length as in Figure 67, but the curves themselves are established between new limits. The definition of a change in contractile activity by a shift in the force-velocity curve with a change in Vmax may be generalized further with the use of instantaneous force-velocity-length relations in the manner similar to that in Figure 67. In Figure 69, when Vmax is increased, the curve at any instantaneous length is augmented and the course of contraction is shifted upward on to a new set of force-velocity-length curves. With any constant load, the instantaneous velocity will be increased (for example from D to D 1 ) . This second set of curves (light grey line) thus defines a change in the contractile state of the muscle, both at the initial muscle length and at any instant during contraction ( 4 6 ) .

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C. Work and Power Implicit in the force-velocity curve are the relations of power and work. Power, the rate of doing work, is the product of the force ( P ) and the velocity ( P • d l / d t ) , and the work performed is the power integrated as a function of time ( P • dl/dt or P A L.). 3 Both work and power as functions of load are shown in Figure 64. These relations of work and power are of some practical interest since they have been used widely in evaluating the performance of the intact ventricle ( 8 ) . Some of the limitations involved in using work and power to define a contractile state are seen in Figure 70 as well as Figure 64. From Figure 64 it is apparent that at any one muscle length both work and power vary with load reaching a maximum where the load ( P ) is approximately 509? of the maximum force the muscle could develop at the given muscle length ( P o ) . Either an increase or decrease in P / P o results in a fall in work and power. In Figure 70, velocity of shortening, extent of shortening ( A L ) , power, and work are given for three different frequencies of contraction. With the initial increment in frequency of contraction (20 to 3 0 / m i n ) , both power and work are augmented at any load. However, with a further increase in the frequency of contraction from 30 to 50/min., the intrinsic speed of the muscle shortening is increased but without further increase in either the extent of shortening or developed force. Hence, in this instance, power increases with no change in work. Concommitant with the increase in Vmax, an increase in the rate of force development ( d p / d t ) and an abbreviation of the duration of the active state occurs, but without a change in developed force ( 3 8 ) . Thus alterations in work and power may not correspond to changes in the contractile state of the muscle. Further, if load were to decrease considerably while Vmax increased, calculated power might also be misleading. 3

Power — W; work = W; P = load (or its equivalent, force); dl/dt velocity and A L = displacement. W = P dl/dt

=

W = Jf

. dt = Jf Pdl = P • A L dt The kinetic component or the acceleration component of work equals Wmv2, Since it is quite small it has been ignored.

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Figure 69. The effects of augmenting the contractile state of heart muscle on the instantaneous force-velocity-length relation. The diagram is similar to fig. 67. However, Vmax has been increased with the result that the afterloaded muscle now follows a path along this new set of curves. D to D 1 shows the shift in the instantaneous velocity during the contraction at comparable muscle length and load. The change in the contractile state is defined by the change in the coordinate of velocity.

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L O A D (g) Figure 70. The effects of increasing frequency of contraction in the cat papillary muscle (33). Velocity of shortening, extent of shortening (AL), power, and work are all given as functions of load. See text.

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Before proceeding to more speculative considerations, these mechanical properties of the contractile elements of heart muscle may be summarized. In Figure 71, hypothetical shifts of the forcevelocity curve are shown with their derived power curves. It is apparent that considerable modification of myocardial performance can occur within this framework of shifting power curves. Increasing muscle length, the basis of the Frank-Starling prin-

Reaulotion

of Conlroclile

Response

Figure 71. Changes in the force-velocity relation and derived powerload (force) curves of heart muscle following various interventions.

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ciple, alters the force-velocity curve by increasing Po without a change in Vmax or alteration in the duration of the active state (the dashed curve in Fig. 71). Changing frequency of contraction (dotted line) the first inotropic change, shifts the force-velocity curve by augmenting Vmax without a change in Po. Both these changes are intrinsic to the heart muscle: muscle length modulating primarily force; heart rate modulating speed. Norepinephrine and strophanthidin also increase Vmax but with more variable changes in Po. The shifts in the force-velocity curve also have their counterpart in the mechanically derived constants of the Hill Equation, since a, b and Po determine the shape of the curve (Fig. 72). When these shifts in the force-velocity curves are related to the entire course of contraction, the force-velocity-length relations of Figure 69 are obtained and within this framework contractile state of heart muscle is defined. A generalized view of power in these terms also can be formulated: ( 1 ) power, and hence work, depend on the limits of the force-velocity relation and are thus load dependent; and ( 2 ) at any given load, power (but not necessarily work) can be increased

Muscle Length Inotropism

Vmax

b

a/Po

No Change

No Change

No Change

t

No Change

t

(velocity)

Po (Force)

Figure 72. Effects of changing initial muscle length and the contractile state of the muscle (inotropism) on the constants of the Hill equation (38).

t i

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by increasing muscle length or by increasing intrinsic velocity (inot r o p i s m ) . T h e duration of the active state plays little direct role, decreasing inversely with V m a x , but not contributing per se to an increase in contractile force. F u r t h e r these generalities apply to the f u n c t i o n of the contractile element both at the onset and throughout the entire course of contraction. In contrast to heart muscle, skeletal muscle does not have the ability to shift its force-velocity curve u n d e r physiologic conditions. T h e force developed by skeletal muscle is modified primarily by neural innervation which changes the n u m b e r of contracting fibers while each muscle fiber operates at that muscle length at which the force of contraction is near maximal. O n the other hand, the heart cannot alter the total n u m b e r of its contracting fibers by neural mechanisms. However, myocardial force can be modified by allowing the muscle to operate along the ascending portion of the length-tension curve. In effect, changing cardiac muscle length along the length-tension curve accomplishes what neural control does f o r skeletal muscle, i.e. it permits a change in the n u m b e r of units contracting effectively in parallel. Coupled with this intrinsic ability to modulate force by changing length is the potential for altering the velocity and force of contraction through sympathetic nerve activity allowing for a remarkable degree of functional adaptation.

III. T H E S E R I E S E L A S T I C

COMPONENT

T h e foregoing considerations have dealt largely with the contractile element of heart muscle with only passing mention of the series elastic c o m p o n e n t ( S E ) . However, all external force generation must ultimately derive f r o m the interaction of the C E with the SE. T h u s the role of the S E in such p h e n o m e n a as changing muscle length and contractile state must also be considered.

A. The Load-Extension

Curve of the SE

T h e characteristics of the series elastic c o m p o n e n t have been defined mechanically in skeletal muscle in several ways (9, 26,

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3 6 ) , all of which depend on the functional independence of the properties of the SE and C E . In skeletal muscle, the series elastic c o m p o n e n t has generally been studied by measuring the decrease in force which results from a very rapid decrease in length (quick release) ( 2 6 , 3 6 ) . If the properties of the contractile elements a n d series elastic components are considered to be identical in the transition from isometric to isotonic contraction under afterloaded conditions, which is a basic assumption of the Hill two c o m p o n e n t model, the properties of the SE may also be derived f r o m afterloaded contractions ( 2 6 , 29, 4 7 ) . 4 In this m a n n e r the stress-strain curve for the series elastic c o m p o n e n t of heart muscle has been obtained (Fig. 7 3 ) . Since it has been shown that the stiffness ( d P / d l ) o f the series elastic component is a linear function of load, the load-extension curve of the series elastic component will be exponential in form ( 2 9 , 4 7 ) . This makes the properties of the SE distinctly different f r o m a simple Hookian spring where the relation of load and extension is linear such that d P / d l is a constant. T h e load-extension curve of the SE has also been obtained by plotting the initial velocity of isotonic shortening as a function

'In the two component model, force ( P ) equals the product of the displacement or shortening of the contractile element, ( A L ) , and the stiffness of the (dP) series spring Hence: P = A L • d P / d l Differentiating this equation relative to time: dP _ _dj_ _dP_ dt — dt dl By determining the rate of force development ( d P dt) just prior to the onset of shortening and the velocity of shortening (dl dt) just after shortening begins, d P / d l , the stiffness of the series elastic component can be determined for the given load since: dP/dl =

d P / d t

at load P. dl/dt Since d P / d l has been shown to be a linear function of the load ( P ) the length-extension curve of the series elastic component can be derived ( 3 8 , 47). Hence: d P / d l = k P where P is the load and k is a constant, and P 2 = P , e ~ k ( I 1 - I 2 ) where Po is the developed force; Pi the initial force extending the SE; l i the initial SE length produced by P,; and 1, the SE length when the load is P2

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Load-extension curve of the series heart muscle (normalized)

elastic

element

CELL

of

Figure 73. Load-extension relation of the series elastic component (SE) of the cat papillary muscle. Normalized for muscle length (47). Note that the SE is extended 8% for a developed tension of 5g/ mm2. of the time from stimulation to the onset of shortening (Fig. 7 4 ) . During isometric contraction, shown as the dashed line in Figure 74A, the velocity of the C E (solid line) must be equal to the velocity with which the SE is lengthening. With increasing load, the time from stimulation to the onset of shortening is prolonged

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since the SE must be stretched before external shortening occurs. Further, as increased force is generated. C E velocity falls. If the area under the velocity curve (solid line) is integrated as a f u n c tion of the time [ ( ^ - L of SE = V d V / d t • d t . ) , t, being the time of onset of C E shortening and t 2 being the time to onset of C E shortening] and A L is then plotted relative to load, the load-extension or stress-strain curve of the SE is obtained. This is shown in Figure

74B.

Figure 74. A. Contractile element velocity and isometric force as functions of time after stimulation in the cat papillary muscle. Obtained from isotonic contractions with increasing afterloads (47). B. Load extension curve for the series elastic component obtained from A. See text.

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Using isotonic quick releases, Abbott and M o m m a e r t s ( 3 4 ) have demonstrated a curve similar to Figures 73 and 74 for the series elastic c o m p o n e n t in heart muscle. This load-extension curve of the series elastic c o m p o n e n t in heart muscle resembles that of skeletal muscle, although the S E of heart muscle is only approximately one third as stiff ( 3 4 , 4 7 ) . Since the rate of development of force ( d P / d t ) , depends on the velocity of shortening of the C E and the stiffness of SE ( d P / d t = dl dt x d P / d l ) , the rate of force generation in heart muscle would be one third of that in skeletal muscle at identical velocities of the C E . This is generally observed. T h e anatomical localization of the passive series elastic component is not known. Some elasticity undoubtedly resides in tendinous attachments of the muscle. Further, some of the series elasticity is thought to reside in the contractile substance itself, either in the contractile proteins or in the links between actin and myosin. T h e extent to which a synchronous excitation may allow one portion of the muscle to use another portion as elasticity is unclear. Further, the three-fold greater distensibility of the series elasticity in heart muscle as c o m p a r e d to skeletal muscle remains unexplained. ( 4 7 )

B. The Effect of Changing Muscle Length on the SE T h e properties of the series elastic component in heart muscle have also been studied at various muscle lengths ( 4 7 ) and contractile states of the muscle ( 3 4 , 38, 59, 6 0 ) . When the stiffness of the series elastic c o m p o n e n t ( d P / d l ) is expressed as a function of dP/dl load ( P ) , — - — is not only linear, but independent of initial muscle length (Fig. 7 5 ) ( 4 7 ) . In Figure 76 velocity of shortening has been plotted as a function of time for the several initial muscle lengths in the same m a n n e r as shown in Figure 74. Developed force has also been plotted relative to time for these muscle lengths. Significantly, all of the initial velocities of shortening in Figure 76 fall on the same curve, independent of initial muscle length. Since the area under this curve, which can be equated with series elastic extension, is the same for all of the force curves,

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M O D U L U S OF E L A S T I C I T Y OF T H E S E : INCREASING MUSCLE LENGTH 20

15

r

io 2 46

Preload (g)

A O A

• •

0

5

.1 .2 .4 .6 .8

Muscle Lengfh(mm) 11.7 12.4 13.0 13.3 13.5

10

LOAD ( g ) Figure 75. The relation of stiffness of the series elastic component (dP/dl) to load for contractions initiating from several initial muscle lengths. Legend as on fig. 66. Note that the relation of dP/dl to load (P) is independent of initial muscle length.

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CONTRACTILE ELEMENT V E L O C I T Y AND FORCE AS F U N C T I O N OF T I M E : I N C R E A S I N G M U S C L E L E N G T H

I0 r

-110 Muscle Preload Length (g) (mm/ A 0 A

.1 2 4

m •

6 .8

II 7 12.4 13.0 13.3 135

o

5

0

100

200

300

TIME AFTER STIMULATION

33

o

400 (msec)

Figure 76. The effect of increasing initial muscle length on contractile element velocity (solid line) and isometric force (dashed lines) as functions of time after the stimulation of the muscle. See text.

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209

it may be concluded that the extent to which the series elastic c o m p o n e n t is stretched remains constant independent of initial muscle length and thus, in turn, of maximal developed force. As muscle length increases therefore, SE stiffness increases pari passu with the ability to generate increased force (Figure 7 7 ) . Thus, for every C E added in parallel by increasing muscle length, an equivalent increase in SE is noted. Such a finding has been used to support the view that some of the SE must reside at contractile sites in the C E ( 4 7 ) .

C. Effect of Changing

Contractile

State on the SE:

Strophanthidin

The cardiac glycoside, strophanthidin, was used to examine the effects of an inotropic agent on the series elastic component. T h e effects of glycosides on muscle mechanics have only recently been described (59, 6 0 ) . In Figure 78, the effects of strophanthidin on the force velocity relation, the stiffness of the elastic component ( d P / d l ) , and the load-extension curve of the series elastic component are illustrated. Although strophanthidin shifts the force-velocity curve with an increase of both Vmax and Po, no change in SE stiffness (Fig. 7 8 B ) or the SE load-extension curve occurred (Fig. 7 8 C ) . On the left in Figure 79, C E velocity (solid line) and force (dashed line) are both plotted as functions of time after stimulation in a m a n n e r similar to Figure 71. Following the addition of strophanthidin, the C E shortens more rapidly than in the control state. However, the velocity of shortening declines more rapidly than in the control state. This increase in the velocity of shortening ( d l / d t ) following strophanthidin is reflected in an increase in the rate of force development ( d P / d t ) and a decrease in the time to peak isometric tension (Fig. 79, left, dashed line). Since d P / d t = d l / d t • d P / d l , an increase in d P / d t would be expected to occur following an increase in d l / d t . This relation, however, is not a simple one since d P / d l and d l / d t (Fig. 7 8 A ) are both functions of load ( P ) . When the series elastic component extension is derived from the contractile element velocitytime relation (Fig. 79, left), and plotted as a function of developed force, the load-extension curves for the series elastic component are identical in both the control state and following the addition

210

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LOAD ( S T R E S S ) - E X T E N S I O N (STRAIN) R E L A T I O N OF T H E I N C R E A S I N G MUSCLE

SE : LENGTH

Figure 77. The effect of increasing initial muscle length on the loadextension curve of the series elastic component. Calculated from fig. 76. As muscle length is increased, the stiffness of the SE is augmented.

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211

of strophanthidin (see Fig. 79, left). This constancy of the stiffness of the series elastic component is also noted in Figure 80. In this experiment the contractile state of the papillary muscle was depressed by the addition of pentabarbital. Velocity of shortening, the rate of force development and the extent of shortening were all depressed but the series elastic component stiffness (dP/'dl) remained unaffected. Subsequently, strophanthidin was added to the bath with a resultant improvement of the contractile state shown

A.

FORCE V E L O C I T Y R E L A T I O N OF C E : o

Control

• Strophanthidin t/i/cc Lo 9mm HR

3

30/mm

VELOCITY OF SHORTENING (mm/sec)

dp/dl (gm/mm)

2

2

5

LOAD In I

Figure 78. The effect of the addition of strophanthidin: A. The force velocity relation; B. the relation of series elastic stiffness (dP/dl) to load and C. the load extension curve of the series elastic component. The curve in C is calculated from the equation ^ p ^ *

= 4.1 as obtained in

B. Note that the same curve in B is obtained before and after the addition of strophanthidin, resulting in a single load extension curve for the SE (C). Thus strophanthidin shifts the force-velocity curve of the CE with an augmentation of both Vmax and Po, but does not alter the SE component stiffness.

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by a shift to the right of the force-velocity curve. This improvement in the contractile state of the muscle occurred without a

CE VELOCITY AND FORCE AS FUNCTIONS OF TIME:

L O A D - E X T E N S I O N CURVE OF SE

- 4

o

^o—o-.0

-d o

TO

-23 o Control • Strophanthidin 1/i/mt

200

400

TIME (msec)

1

2

LOAD (gm)

3

Figure 79. The effects of strophanthidin on the load-extension curve of the series elastic component (right), derived from contractile element velocity and isometric force as functions of time after stimulation (left). Note that following strophanthidin, CE velocity is higher earlier in the contraction but falls more rapidly with time during contraction. These changes are accompanied by an increase in the rate of isometric force development (dp/dt), and an increase in the force developed while the time required to reach maximum force is abbreviated.

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213

change in the stiffness of the series elastic c o m p o n e n t since d P dl remained constant throughout. Similar findings have been obtained by altering frequency of contraction ( 3 4 ) or adding norepinephrine ( 3 8 ) . Thus, alterations of the contractile state of the heart induced by various inotropic interventions, such as changes in heart rate or drugs such as pentobarbital, norepinephrine and glycosides must be attributed entirely to alterations in the activity of the contractile elements as shown by shifts in the force-velocity relation and are not contributed to by changes in the series elastic component. IV. T H E F R A N K - S T A R L I N G

MECHANISM

If the muscle is considered to contain multiple contractile and series elastic components arranged both in series and parallel, the results of mechanical experiments allow certain suggestions to be made relative to mechanisms involved in the change of force of contraction observed with changing muscle length. T h e fact that increasing initial muscle length increases m a x i m u m force without a change in m a x i m u m velocity of shortening ( V m a x ) would support the view that an increase in muscle length brings about an increase in the number of contractile elements acting in parallel without changing their qualitative activity ( 2 9 , 3 8 ) . A simple analogy would be provided by a n u m b e r of horses arranged in parallel: T h e total load they can haul depends on their n u m b e r ; without a load, their speed is not dependent on how m a n y there are in tandem, but only on how fast any one of them can move. The constancy of Vmax and the Hill constant b suggests that increasing muscle length induces a change in the n u m b e r of similar force generating sites rather than a change in the nature of the mechanochemical coupling at the contractile site ( 2 9 ) . Further, the demonstration that increased muscle length brings about an increase in the stiffness of the SE pari passu with increased force has been used to suggest that f o r every additional C E brought into play by increased length, a new SE c o m p o n e n t is added ( 4 7 ) . How then might the muscle length increase the n u m b e r of activated C E operating effectively in parallel? Starling ( 4 a ) suggested that increasing muscle length "increases the extent of active sur-

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Na Pentobarbital Induced Deiwession Reversed by Strophanthidin

0.4 f | HR 20/mm T 23'C MkrM 1.2 gConstant PENTOBARB l/i/cc) STROPHANTHIDIN T I M E TO PEAK AL (O)

Wml)

700

• Pentobarbital 140/1Ac O Strophanthidin !.3p/cc

600

VELOCITY OF SHORTENING mm/sec (A) RATE OF FORCE DEVELOPMENT g/sec

(A) 6L

ELASTICITY MODULUS dp/dl (•'

40

3.0 2

10 m

3 L O A D (gm)

Figure 80. Depression of the contractile state of the cat papillary muscle and its reversal by strophanthidin. On the left the muscle has been arranged for afterloaded contractions with a constant load. The time from stimulation to maximal shortening, extent of shortening, velocity of shortening, rate of isometric development and the "elasticity modulus" (stiffness) of the series elastic component are given as functions of time. The effects of sodium pentobarbital added to the bath are shown, followed by the effects of the addition of strophanthidin. On the right, the improvement of the contractile state resulting from the addition of strophanthidin is demonstrated by the shift to the right of the forcevelocity curve.

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face". This would mean that each myofibril is altered. A proposal based on activation of the myofibril has been put forward by Podolsky to explain the length-tension phenomenon ( 2 9 ) . He has suggested that heart muscle, unlike skeletal muscle, might not be activated completely with each action potential and that an increase in initial muscle length might engender an increased degree of activation. Podolsky has postulated that if a given limited amount of an activating substance were released with each action potential and were to diffuse a given depth into the fiber, activation might not reach the center of the fiber. When the length of a fiber were increased (assuming that volume remains constant) its radius would decrease and the same depth of activation would activate a larger portion of the fiber. Although there is no direct evidence for this view, the constancy of the ratio a / P o is consistent with such a mechanism as is the finding in heart muscle that in the presence of a small load Vmax is established before the attainment of maximum stiffness. The attainment of Vmax requires only superficial activation of the fibers while force depends on the crosssectional area of myofibril that is activated. T h e observed delay in the onset of maximum active state in heart muscle ( 3 1 a , 3 1 b ) is also consonant with progressive recruitment of myofibrils as an activator penetrates the fiber. There are, however, certain basic criticisms of this depth of activation hypothesis. First, a 30 per cent decrease in muscle length theoretically decreases force by 80 per cent while increasing the radius by only 16 per cent. Further, there is no assurance that sarcomere volume is independent of muscle length. Second, the depth of activation hypothesis does not consider known changes in the ultrastructure of heart muscle which occur with changing length (see Spiro, 6 1 - 6 3 ) . These changes in sarcomere length alone are adequate to explain the length tension curve. A n alternative explanation for the change in developed force with changing muscle length derives f r o m the ultrastructure of muscle. Electron microscopic studies have shown that the sarcomere of both skeletal ( 2 0 , 64) and heart ( 6 5 ) muscle is composed of an overlapping array of functionally rigid protein filaments, actin (1.0/ti in length) and myosin ( 1 . 5 ^ in length). In the individual sarcomere, which is the f u n d a m e n t a l unit of contraction, actin fila-

216

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ments extend toward one another from the two ends of the sarcomere, overlapping with myosin filaments in the central portion of the sarcomere. Evidence is accumulating to support the view that with shortening of skeletal muscle actin filaments interact with the myosin filaments at multiple specific reaction sites and thus the actin filaments slide into the center of the sarcomere without a change in overall myofilament length (64, 66-68). Similar evidence has now been adduced for heart muscle ( 6 3 ) . The optimal overlap of thick and thin filaments for maximum interaction would be expected to occur at a sarcomere length of 2.2n, granting a central zone on the myosin filaments devoid of contractile sites. The maximum apposition of contractile sites on thick (myosin) and thin (actin) filaments would thus be expected at a sarcomere length of 2.2 microns for both skeletal and heart muscle. If generated force depends on the number of force generating sites, maximum force also should be seen with a sarcomere length of 2.2/x. This has been observed for both skeletal (61, 63, 69) and heart muscle ( 6 2 ) . As the sarcomere shortens, thin actin filaments appear to pass into and through the center of the sarcomere ( 6 8 ) . It has been suggested that the thin actin filaments, forming a double overlap of thin filaments in opposite halves of the sarcomere, interfere with force-generating sites ( 6 9 ) and force decreases accordingly. Since there is suggestive evidence that thin filaments also penetrate the center of the sarcomere in heart muscle ( 6 3 ) , similar considerations might also apply to the myocardium. This alters the question from what increases force as the muscle length is increased to what decreases it as the muscle shortens? Such a mechanism would be consistent with the suggestion that the series elastic component resides largely at or near the contractile sites ( 4 7 ) . Fewer active sites would produce a parallel decrease in the stiffness of the SE. Thus, the sliding filament model for contraction readily provides a framework for understanding how a change in muscle length brings about a change in the number of the force generating sites without changing the force generating process at any individual site. An inotropic agent such as norepinephrine or strophanthidin, in altering Vmax, the intrinsic speed of the muscle, as well as Hill's velocity constant b, may be presumed to change the qualitative

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217

nature of the reaction at the contractile sites with an increase in the maximal rate of mechanochemical interaction. Whether this results from an increased rate at which an activator is made available to the contractile site or a change in the enzymatic characteristics of the force-generating sites themselves is undefined. In simplest form then it may be suggested that an increased muscle length engenders a quantitative increase in the number of active force generating sites working effectively in parallel, while a change in the contractile state of the muscle qualitatively alters these sites with or without a change in their number.

V. E N E R G E T I C

SPECULATIONS:

EFFICIENCY

The strict theoretical concept of efficiency as employed in the thermodynamics of heat engines is not readily applicable to physiological performance ( 7 0 ) , as recently emphasized by Mommaerts (71).

Stated in simplest form, the efficiency of the machine being

analyzed is the ratio of the useful energy output

(work)

to the

total energy expenditure, i.e. the heat produced by the system plus the work performed.

Efficiency can also be evaluated in terms of

the force-generator itself.

In this instance the contractile element

is considered apart from the transmission system which connects the contractile elements to the external world.

The latter situation

involves the interaction of the contractile element, the generator of force, with the series elastic component, the transmission system. Although the determinants of C E efficiency and the implications of the transmission of C E force to the external world through a series elastic component are quite speculative, insight into some determinants of C E efficiency may be obtained from a consideration of the constants of Hill's equation studied under various experimental conditions. has two components:

Total energy released by the active muscle work ( W ) and heat ( H ) .

In skeletal muscle

( 9 ) , the heat ( H ) is composed of two portions:

a shortening heat,

which is the heat of shortening (a)

times the extent of shortening

( A L ) , and the product of the heat concerned with initiating and maintaining the active state ( k ) and the time ( t ) during which the system is active. T h u s : H = a • A L + k • t

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The constant k has not been directly evaluated for heart muscle but on the basis of Hill's experiments ( 7 2 ) it ought to approximate the product of a and b where a and b are constants of the Hill equation for the force velocity curve. Hence: Contractile Element Efficiency (Ece) Since, and

W -f H W = A L P H = a ' AL+kt P A L ECE = ( P - A L + a - A L + kt =

The C E efficiency then is dependent on two primary mechanical relations: the ratio a / P o and the ratio of load to maximum force ( P / P o ) . These relations are plotted in Figure 81. On

B Efficiency os o Function CE efficiency f£l • of Lood where W • worh -P e Htneot} -- mmtennnce IttelWH shortening heot tot)

Efficiency as a Function of Velocity o/P„ -- 45 b -- 4 ty'sec

.,. P> C ~ (01*111+ PI PI

' (Pto)X-Hll

50 EFFICIENCY PERCENT

0

0.5 P/P„

1.0

0

25

50 V E L O C I T Y OF SHORTENING PERCENT OF Vina.

Figure 81. Contractile element efficiency. A. as a function of a/Po, the ratio of the shortening heat from Hill's equation to the maximum force of contraction. B. as a function of the P/Po, the ratio of the force actually developed (load) to the maximum force. C. as a function of the velocity of shortening.

7

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219

the left in Figure 81, one finds that efficiency is an inverse function of a ratio of a/Po, i.e. the more heat produced in the process of muscle shortening, the lower the efficiency. In the center one notes that for a given a/Po, obtained from the previously illustrated experiment ( 3 8 ) , efficiency, like work performed, is greatest when the load P is about 50 per cent of isometric force. Since a / P o is independent of muscle length ( 3 8 ) , C E efficiency should also be independent of muscle length. This is in accord with the findings of Aubert (12) and Hill ( 7 3 ) in skeletal muscle, assuming that the maintenance heat changes pari passu with a as the muscle shortens. Further, the constancy of b and a / P o with changing muscle length is consistent with an increase in the number of myofilaments contracting in parallel without a change in the maximum rate of the process generating force, a view which requires a constant C E efficiency, with changing muscle length. Efficiency of the C E can also be expressed as a function of velocity. It is found (Fig. 81) that maximum efficiency occurs when the velocity is about one-fourth of maximum velocity (Vmax). With either an increase or decrease in velocity contractile element efficiency falls. Thus, the efficiency depends on the loading conditions, given the force-velocity relations of the C E which limit performance. In contrast to changing muscle length, interventions such as changing the frequency of contraction, the addition of norepinephrine, or strophanthidin, appear to change the rate of mechanochemistry at the contractile sites in the myocardium as signified by an increase in Vmax and a concomitant rise in the constant b. As far as it has been assessed the relation a / P o remains unchanged ( 3 8 ) . Thus, in the presence of an inotropic intervention, CE efficiency is unchanged relative to load. Maximum efficiency occurs at the same per cent of Vmax as before but in absolute terms the velocity of shortening for maximum efficiency is augmented since Vmax is increased. It is thus apparent that C E efficiency is dependent on load and velocity but independent of changing muscle length. The suggested importance of velocity in determining efficiency in the intact heart awaits further definition. There are certain important limitations to such an analysis of CE efficiency. First, the constant k may be dependent on both

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THE MYOCARDIAL

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muscle length and time (12, 7 1 ) . Further, the duration of the active state and its dissapation may also be dependent on load ( 7 4 ) . In heart muscle, the importance of muscle length is further emphasized by the fact that heart muscle normally functions along the ascending portion of the active length-tension curve where relatively small changes in muscle length may produce large changes in developed force. Second, as the muscle shortens with constant load ( P ) , the maximum force ( P o ) is decreasing as the muscle moves down the length tension curve such that the ratio of load ( P ) to maximum force (Po) is constantly increasing. This change in the ratio of P / P o might serve to change the contractile element efficiency during any given contraction. Third, the expression for efficiency as developed here applies to the C E alone. However, in the intact muscle consideration must be taken of the work which the C E performs in stretching the series elastic component during the isometric phase of contraction as well as the external work being performed. This consideration is paramount in heart muscle since the C E component is quite extensible while in skeletal muscle, with a relatively stiff SE this problem is of relatively minor importance. Further, in the intact heart factors which contribute to the effective SE such as the mitral valve and asynchronous contraction would increase the effective series elastic component of the system by leaving incompletely activated portions of the ventricle stretched by more completely activated segments of the myocardium. Thus C E efficiency depends on the constants which determine the shape of the force-velocity curve as well as loading conditions which pertain to this curve (Fig. 8 1 ) . Generally when the efficiency of the intact muscle is evaluated, only the external displacement of load is considered since this is the useful work. However, in order to assess C E efficiency total C E work must be evaluated. This includes the work that the CE performs in stretching the SE during the isometric phase of contraction as well as the isotonic phase. Thus in Figure 82, both internal and external work have been calculated ( 4 7 ) . The extent of external muscle shortening and series elastic component extension have both been portrayed as functions of load under the conditions of afterloaded contraction. Below, external work (load x external muscle shortening), and internal or isometric work (load

THE

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221

(Isotonic) Shortening

Total Contractile Element Work (Isometric + Isotonic)

V

ISOMETRIC SE

WORK(W)

Extension

dwP-dt Since

dw -

P -

-

(AL)

• a v.

force

dp/d-l

dp/d-l

ne -

dp

T

Integrating

W-A

LOAD(g) Figure 82. Contractile element work for the cat papillary muscle contracting from a given muscle length with increasing afterloads. The external shortening of the muscle (AL), ¿he extension of the series elastic component, and work (total contractile element, isotonic, and isometric) are given as functions of load. Total contractile element work in the lower panel equals the isometric plus the isotonic work. The isometric or "internal" work which is performed by the CE in stretching the SE during the isometric power of contraction is derived on the right on the figure. P=the load and K, the constant for the stiffness of the SE (47).

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x series elastic extension) are shown. The total contractile element work is the sum of the internal (isometric) and external (isotonic) work. From this it is apparent that a considerable amount of contractile element effort is expended during the isometric phase of contraction, which, by classical methods of calculating work and efficiency, is ignored. The calculation of total contractile element work may prove to be a useful concept (Fig. 82). For example, Britman and Levine (75) have recently suggested a correlation between oxygen consumption and contractile element work. Although this is an interesting correlation, one suspects that it may be incomplete since other considerations such as time and velocity are ignored. From considerations of contractile element efficiency, it would seem that velocity parameters should ultimately play an important role in the energy balance of heart muscle, since it is not only the work relations of the contractile elements which must be defined but also the parameters which alter the efficiency of the mechanochemical coupling process. (76a) The analysis of overall efficiency of the muscle thus requires a consideration of two independent systems: (1) the mechanochemical coupling of the CE and (2) the mechanical coupling of the CE and SE. The derivation of these two aspects of efficiency is shown in Figure 83.

VI. M E C H A N I C S A N D U L T R A S T R U C T U R E The ultrastructure of heart muscle has recently been related to mechanics in terms of the length-tension curve (62). It has been demonstrated that along the ascending portion of the length-tension curve, the portion of the curve which is physiologically pertinent to the intact heart, sarcomere length is directly proportional to muscle length. Accordingly, the length-tension curve of the muscle can be generalized to the length-tension curve of the sarcomere (Fig. 84). This sarcomere length-tension curve forms the ultrastructural basis of Starling's law of the heart (25, 76). Some of the previously described mechanics can now be considered in terms of the sarcomere, the fundamental ultrastructural unit of contraction. It has been found that in heart muscle actively developed ten-

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223

E F F I C I E N C Y = E c t x E mech 1) M e c h a n o c h e m i c a l ( c e )

E cc

"[P-AL

^

+a.L]+kr»

where P = load AL a

= external displacment = h e a t of s h o r t e n i n g

k ] = a c t i v a t i o n or m a i n t a n c e h e a t t 2)

=time

Mechanical P'AL ^mech ~

. dp/dl , w h e r e k 2 = — = — , t h e spring

p '

c o n s t a n t of

+

k2

series elastic component

- — = i s o m e t r i c ( i n t e r n a l ) w o r k , s i n c e dw = — k2 k2 a n d dw =

.dl

k2

P ' A L = i s o t o n i c ( e x t e r n a l ) work

Figure 83. Overall efficiency of the muscle. Total efficiency is the product of the efficiency of the contractile element (ECE) with its mechanochemical coupling and the efficiency of the mechanical coupling system (E mech.). The mechanical system requires the expenditure of energy in performing non-useful work while stretching the elastic connections of the muscle (SE). These two considerations are shown.

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SARCOMERE TENSION

CELL

LENGTH RELATION

m S
A - > B - > C ~ > n ~ > response, in which

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333

H symbolizes the neurohumoral agent or drug and R the receptor. HR represents the complex of humoral agent and receptor which, itself or a reaction product, starts the sequential reaction depicted by A, B, C, and n leading to the measurable response, such as change of heart rate, force of contraction, transmembrane potential, oxygen consumption or altered enzyme activity. Specificity of receptors ideally would be determined by isolation and chemical identification. This step has not been accomplished as yet. Consequently, functional identification is necessary. Specificity of receptors can only be deduced at the present time from an analysis of the relationships between pharmacological activity and chemical structure of chemical compounds and by the actions of selective antagonists. For instance, acetylcholine and norepinephrine are chemically dissimilar and their actions on the heart are distinctly different. Furthermore, the actions of each is selectively antagonized by different drugs, that of acetylcholine by atropine and not by dichloroisoproterenol and that of norepinephrine by dichloroisoproterenol and not by atropine. Thus, in the heart the distinction between the adrenergic and cholinergic receptors seems clear. Receptor dilineation becomes more difficult when a tissue responds in a similar manner to two different types of compounds. Selective blockade is necessary to differentiate receptor types in the absence of detailed knowledge of the precise sites of action of the compounds. Also, if the responses of several tissues or several different responses in one tissue all appear to be mediated by one type of receptor on the basis of the effectiveness of one type of agent, for instance the actions of acetylcholine on smooth muscle, heart and glands, on the motor end plate and on autonomic ganglia, separation into subclasses is more difficult. This separation requires the comparison of orders of potency of a chemically related series of compounds on the different tissues and also comparison of antagonists. Adrenergic receptors. As a general class of drugs, sympathomimetic amines elicit a wide variety of physiological and metabolic responses. However, in the past it has been difficult to conceive of these agents acting on a single receptor type because of 1) some apparently opposite types of response (vasoconstriction and vaso-

334

THE

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dilation), 2 ) differing orders of potency on different systems and 3 ) the failure of "conventional" sympathetic blocking drugs, such as the ergot alkaloids and phenoxybenzamine (Dibenzyline), to antagonize all of the actions of sympathomimetic amines. The proposal of Cannon and Rosenblueth ( 1 ) of two sympathins left unexplained contradictions (e.g., the "excitatory" responses of the heart were not antagonized by the then known adrenergic blocking drugs whereas the "excitatory" response of vasoconstriction was blocked). In an attempt to produce a more rational classification of the actions of sympathomimetic amines Ahlquist (2, 3) compared the orders of potency of a series of closely related phenylethylamines on several systems. He proposed two types of adrenotropic receptors to be called by the Greek letters, alpha and beta instead of two types of transmitters. In Ahlquist's classification the alpha adrenergic receptors, in terms of the circulatory system, subserve only vasoconstriction whereas the beta adrenergic receptors subserve vasodilator and cardiac responses. This classification was compatible with the actions of the then known adrenergic blocking compounds, that is they could antagonize adrenergically-induced vasoconstruction but could antagonize neither vasodilation nor cardiac stimulation. At the time Ahlquist formulated his classification no drugs were known which blocked the beta adrenergic receptors (4). In 1958 Powell and Slater ( 5 ) described some unique adrenergic blocking actions of a new compound, dichloroisoproterenol ( D C I ) , a chlorinated analogue of the catecholamine, isoproterenol. They and subsequent workers found DCI to antagonize those cardiovascular actions of catecholamines subserved by beta adrenergic receptors (6, 7, 8, 9, 10). The following discussion is based on the use of DCI and a newer related drug, pronethalol (Nethalide) (11). An analysis of the actions of catecholamines on myocardial contractility by the use of adrenergic blocking drugs. If one considers the relative cardiac positive inotropic activities of four sympathomimetic amines, isoproterenol, epinephrine, norepinephrine and methoxamine, the order of potency is as follows: isoproterenol > epinephrine = norepinephrine > > > methoxamine. In the dog

THE MYOCARDIAL

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CELL

N

140

Ca

D

• ^

Control After DCI

(3)

(6) (7)

«6 E 120 Ol I Id

o 100 o

Ll y %

so

2

60

i

40 20

É

0 -10 0)

(9)

(9)

(5)

(5) (5)

Figure 104. Selective cardiac adrenergic blockade by dichloroisoproterenol (DCI). Vertical bars represent mean change in right ventricular contractile force in anesthetized, vagotomized, open-chest dogs in response to i.v. injections of various cardiac stimulant drugs or to nerve stimulation. Open bars, control responses; hatched bars, responses after 15 mg/kg of DCI. E—epinephrine, 1 /xg/kg; N—norepinephrine, 1 /¿g/kg; I—isoproterenol, 0.5 fj.g/kg; S—right cardiac sympathetic nerve stimulation; CA—calcium chloride, 20 mg/kg; D—digoxin, 0.15 mg/kg; T— theophylline, 10 mg/kg. Numbers in parentheses—number of dogs.

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CELL

isoproterenol is 4 to 5 times more potent than epinephrine and norepinephrine, which are equipotent, ( 1 2 ) and methoxamine has no positive inotropic action (13, 14). This order of potency holds for adrenergic vasodilator activity except for the fact that norepinephrine is much less active than is epinephrine.

EPI I jig/kg

Cardioc

\

NOREPI Ipg/kg

{

ISO .05yj/kfl

J

NERVE STIM

l

CoCI2 20 mfl/kg

i

Contractu«

FOfC«

AFTER PRONETHALOL

15

mg/kg

Figure 105. Selective cardiac adrenergic blockade by pronethalol. Selected polygraph tracings from experiment on anesthetized, vagotomized, open-chest dog. At arrows drugs were injected i.v. at doses indicated (EPI—epinephrine; NOREPI—norepinephrine; ISO—isoproterenol; CAC12—calcium chloride) or the cardiac sympathetic nerve was stimulated. Upper pairs of tracings before administration of pronethalol; lower pairs of tracings after pronethalol, 15 mg/kg.

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337

CUMULATIVE-mg/kg Figure 106. Comparison of the effects of dkhloroisoproterenol (DCI) and pronethalol on the heart. Each point represents the mean change in contractile force of the dog heart in response to norepinephrine (1 /¿g/kg, i.v.) and to stimulation of the cardiac sympathetic nerves. The abscissae represent cumulative doses of DCI and pronethalol. DCI and pronethalol are of approximately equivalent potency.

338

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DCI is capable of blocking selectively the positive inotropic effects of sympathomimetic amines. Figure 104 shows the antagonism by DCI of adrenergically-induced augmented contractile force in the anesthetized dog but no blockade of the positive inotropic effects of digoxin, calcium chloride and theophylline. A similar selective blockade can be produced with pronethalol as shown in Figure 105. The blockade produced by both pronethalol and DCI is dose dependent and surmountable. Both agents are of approximately the same potency (Fig. 106). Specificity of blockade can be demonstrated in another way by comparing the actions of DCI, a beta adrenergic blocking drug, with those of phenoxybenzamine and phentolamine, alpha adrenergic blocking compounds. Whereas DCI antagonizes the positive inotropic effect of catecholamines in the dog heart in situ and in the isolated rabbit heart ( 6 ) and in the cat papillary muscle (9, 10) neither phenoxybenzamine nor phentolamine antagonizes these effects selectively (7, 10). *In the isolated rabbit heart phenoxybenzamine and phentolamine depress contractility and the responses to both catecholamine and calcium chloride, whereas DCI selectively antagonizes only the effect of the catecholamine. Figure 107 demonstrates the nonspecific action of phenoxybenzamine on the rabbit heart. Cardiac rate responses to catecholamines evaluated by means of adrenergic blocking drugs. The same order of potency of the four sympathomimetic amines, e.g., isoproterenol > epinephrine _ norepinephrine > > > methoxamine, is operative in the positive chronotropic actions as in the positive inotropic effects. Heart rate responses to these amines are also selectively antagonized by the beta adrenergic blocking drugs ( 6 ) and not by alpha adrenergic blocking drugs. Figure 108 depicts the selective blockade, by DCI and pronethalol, of the positive chronotropic action of isoproterenol on the dog heart. Adrenergically-induced cardiac ectopic activity evaluated by adrenergic blockade. In Ahlquist's original classification adrenergically-induced cardiac arrhythmias were considered to be subserved by alpha adrenergic receptors because of the ability of dibenamine to prevent arrhythmias induced by epinephrine and cyclopropane in dogs ( 1 5 ) . Moe et al. (16) had challenged the concept that the

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PHENOXYBENZAMINE

DCI

m

Ca

Ca

Ca

Figure 107. Lack of selective cardiac adrenergic blocking effect of phenoxybenzamine. Isolated perfused rabbit heart. Each open bar represents the control contractile amplitude prior to the administration of either 1 ¡j.g of epinephrine ( E ) or 25 mg of calcium chloride (Ca). T h e hatched upper part of each bar shows the response to these agents. Phenoxybenzamine (10 /¿g/ml) was infused and effects of E and Ca were determined. Control perfusion fluid was re-instituted and later a single injection of D C I (0.2 m g ) was followed by E and Ca. Phenoxybenzamine depressed contractility and the responsiveness of the heart to both E and Ca. Subsequently D C I selectively antagonized the effect of E.

Ca

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PR0NETHAL0L CU) Control response After blocking drug

100 80

60 40

i

20

X

V/ 0

ISO

THEO

äEL

ISO

THEO

Figure 108. Comparison of the effects of DCI and pronethalol on heart rate. Each open bar represents the mean change in heart rate in anethetized, open chest dogs with denervated hearts before administration of a blocking drug, the hatched bar after administration of the blocking drug. ISO—isoproterenol, 0.5 /ig/kg, i.v.; THEO—theophylline, 10 mg/kg, i.v. Dose of DCI, 7 mg/kg; of pronethalol, 15 mg/kg. Demonstrates selective adrenergic blockade of heart rate responses.

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BEFORE LIGATION "t~~I—n—i—i i i—r

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FOURTH DAY AFTER

LIGATION

i I i i i i—I I I I I I

E S I SINUS R A T E ECTOPIC RATE

0

BEFORE DCI

+1

+2

+3

AFTER DCI-I6M«/K«

Figure 109. The effect of DCI on ventricular arrhythmias induced by norepinephrine in conscious dog. First block shows sinus heart rate (stippled bars) and ectopic rate (black bars) for 3 0 second periods before and after i.v. injection of norepinephrine, 10 /¿g/kg. Selected electrocardiograms below. Second block shows exaggerated ectopic response to same dose of norepinephrine in the same dog four days after ligation of the anterior descending coronary artery. The last block shows the lack of ectopic response to norepinephrine in the same dog following administration of 16 mg/kg of DCI. (Reproduced with permission of The Journal of Pharmacology and Experimental Therapeutics and The Williams and Wilkins Company from Moran et al., 1962).

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dibenamine antagonism was due to a direct cardiac effect by presenting evidence that these arrhythmias could largely be circumvented by preventing the epinephrine-induced rise in blood pressure. In a study of the ventricular arrhythmias which norepinephrine and epinephrine produce in conscious dogs four to five days after ligation of the anterior descending coronary artery ( 1 7 ) Moran et al ( 1 8 ) demonstrated an antiarrhythmic action of DCI despite marked hypertensive and reflex bradycardic responses to the catecholamines (Fig. 109). In contrast, phenoxybenzamine antagonized the catecholamine-induced arrhythmias only when the vasopressor effects of the amines were obtunded (Fig. 110). It was concluded that the adrenergically-induced ventricular arrhythmias are not subserved by alpha receptors. However, it is not possible to conclude that the antagonism by DCI is due entirely to beta adrenergic blockade since Lucchesi and Hardman ( 1 9 ) demonstrated a nonspecific antiarrhythmic action for DCI and Vaughan Williams and Sekiya ( 2 0 ) have been able to block ouabain-induced arrhythmias with pronethalol. Activation of myocardial glycogen phosphorylase evaluated with adrenergic blocking drugs. Catecholamines are known to induce glycogenolysis in several tissues, the heart included. The details of the mechanism of this reaction have been clarified by the demonstration of the epinephrine-induced activation of the enzyme glycogen phosphorylase by Sutherland (21, 2 2 ) and Sutherland and Cori ( 2 3 ) and more recently by the demonstration by Sutherland and Rail ( 2 4 ) of the key role of a cyclic adenosine monophosphate (cyclic 3', 5'-AMP) in this reaction. Briefly, catecholamines increase the synthesis of cyclic 3', 5'-AMP from A T P in liver and muscle. The cyclic nucleotide then accelerates the conversion of phosphorylase b kinase from an inactive to an active form. The latter catalyzes the conversion of inactive phosphorylase b to active phosphorylase a. Details of these reactions can be found in the review by Sutherland and Rail ( 2 4 ) . Hess and Haugaard ( 2 5 ) and Kukovetz et al ( 2 6 ) demonstrated a correlation between the positive inotropic effect and phosphorylase activating effect of sympathomimetic amines in the perfused rat heart. Kukovetz et al. ( 2 6 ) showed that only sympa-

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TIME IN MINUTES

Figure 110. Effect of phenoxybenzamine on ventricular arrhythmias induced by norepinephrine in the conscious dog. Upper graphs depict total heart rate (X) and ectopic rate (O) in response to i.v. injection of 10 /ig/kg of norepinephrine. Lower graphs show changes in diastolic blood pressure. Figure demonstrates the exaggerated ectopic response to norepinephrine as shown in figure 109 four days after coronary artery ligation and the effect of increasing doses of phenoxybenzamine on the response. Even with large dose of phenoxybenzamine, norepinephrine still produced a significant, but diminished, ectopic response. The magnitude of the ectopic response appears to be related in part to the magnitude of the vasopressor response.

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thomimetic amines which augment cardiac contractility activate the enzyme. Mayer and Moran (27) subsequently confirmed and extended the observations of Hess and Haugaard and Kukovetz et al. but used the dog heart in situ instead of the isolated rat heart. They showed a direct correlation between the positive inotropic effects of adrenergic stimuli and activation of myocardial glycogen Phosphorylase. Only adrenergic stimuli which increased contractile force activated phosphorylase (e.g., cardiac sympathetic nerve stimulation, epinephrine, norepinephrine, ephedrine) whereas the vasoconstrictor sympathomimetic amine, methoxamine, which lacks cardiac stimulant activity (13, 14) also shows no effect on phosphorylase. Non-adrenergic cardiac stimulants such as theophylline, ouabain and serotonin produced no activation of the enzyme. Calcium chloride produced a significant activation of phosphorylase only at an excessive dose (80 mg/kg or 28.8 mg/kg of calcium cation), a dose which might have released endogenous catecholamines. No correlation was observed between positive chronotropic actions and activation of phosphorylase. For example, the largest mean increase in heart rate was observed with administration of theophylline which produced no activation of the enzyme. Submaximal electrical stimulation of the cardiac sympathetic nerve produced a positive chronotropic effect only one-fourth as large as that produced by theophylline, but elicited a positive inotropic effect greater than that produced by theophylline and a highly significant activation of phosphorylase as well. Similar experiments in which DCI or phenoxybenzamine were used further demonstrated a correlation between blockade of the effects of adrenergic stimuli on contractility and enzyme activation. That is, DCI blocked both the adrenergically-induced inotropism and activation of phosphorylase whereas phenoxybenzamine had no antagonistic effect (Figure 111). On the basis of these results it may be concluded that the sympathetically-induced activation of myocardial glycogen phosphorylase is mediated through the beta adrenergic receptors of the heart. However, these data on phosphorylase activation do not permit the conclusion that the enzyme activation and ensuing glycog e n o s i s is the cause of the positive inotropic effect of catechola-

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CHANGE N CONTRACTILE FORCE IN GRAMS

too

è

50

PERCENT ACTIVE PHOSPHORYLASE 100 50

n

CON

ri—j

Hfc-

E

SUM

mn DCI

DCI

PBZ

E

STIM

E

•

+

Figure 111. Selective blocking effect of DC I on adrenergically-induced activation of myocardial glycogen phosphoryiase. Results from experiments on anesthetized, open-chest dogs in which myocardial biopsies were obtained for assay of phosphoryiase. Upper bars represent mean changes in contractile force and the lower the amount of phosphoryiase in the active form as percent of total. Strippling represents significant change from control. CON—control; E—epinephrine, 1 /xg/kg, i.v.; STIM—cardiac sympathetic nerve stimulation; DCI—-dichloroisoproterenol, 7 mg/kg; PBZ—phenoxybenzamine, 15 mg/kg. (Taken from Mayer and Moran, 1960.)

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mines. In fact, Mayer et al. ( 2 8 ) subsequently demonstrated a distinct dissociation of the two actions under several circumstances. One, small single doses of epinephrine augmented contractile force in dogs with no change in the level of active phosphorylase. Two, intravenous infusions of epinephrine for 15 minutes produced a sustained increase in contractile force whereas the percent active phosphorylase, after an initial increase, declined in spite of continuous administration of the amine. Three, under the specific conditions of hypothermia and prior administration of ouabain, norepinephrine produced a negative inotropic effect in the dog ( 2 9 ) but still activated phosphorylase. Figure 112 presents a schematic representation of possible ways in which catecholamines alter myocardial contractility and metabolism. First, it is firmly established that these amines augment glycogenolysis through the activation of phosphorylase. As a result of augmented glycogenolysis, and subsequent oxidation of pyruvate to C 0 2 and water, A T P is generated, some of which may become involved in the contractile machinery. However, it is unlikely that this route is of direct importance to the rapid increase in contractile force which results from the actions of catecholamines. Other possible routes include ( 1 ) the increased generation of hexose phosphates through glycogenolysis, these compounds in some way augmenting contraction (30, 31, 3 2 ) , ( 2 ) an effect of cyclic 3'-5', A M P on the contractile machinery independent of the phosphorylase activating reactions and ( 3 ) an action of catecholamines independent of the action on the adenyl cyclase system. Our present limited knowledge does not permit specification of the exact site of the positive intropic action of these amines. It is tempting to invoke a unitary hypothesis of the basis of the known effects of catecholamines on generation of cyclic 3', 5'-AMP and the antagonism of this effect by DCI ( 3 3 ) . This hypothesis would place the adrenergic receptor of heart muscle in the adenyl cyclase system implying that all of the effects of catecholamines on the heart result from this specific action. However, it must be emphasized that this proposal is presently not supported by experimental evidence. One important aspect of the actions of neurohumoral transmitters and their mimetic drugs upon the heart is the possible interrelationships with the actions of hormones such as thyroxine and

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GLYCOGEN

CARDIAC CONTRACTION Figure 112. Schematic representation of possible pathways of action of catecholamines on the heart. Glycogenolysis is mediated through activation of glycogen phosphorylase secondary to augmented synthesis of adenosine^', 5'-phosphate (3' 5' AMP) from ATP. Possible mechanisms for catecholamine-induced positive inotropic effect are indicated. glucocorticoids. Investigations into these interrelationships have not confirmed the commonly held concept that excess thyroid hormone increases the sensitivity of the cardiovascular system to catecholamines. Daily administration of thyroxine to animals, sufficient to produce hypermetabolism, augmented neither the positive ino-

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tropic nor the positive chronotropic effects of catecholamines in the dog heart in situ or in the rat or rabbit heart in vitro (34). Similar results have been obtained by Margolius and Gaffney (35). These findings cast serious doubt on the concept that thyrotoxicosis increases the sensitivity of the heart to sympathetic stimulation. We have also investigated the influence of glucocorticoids on the circulatory system by the use of chronic administration to dogs of dichlorodiphenyldichloroethane (o,p' DDD) an analogue of the insecticide, DDT. This compound has a selective toxic effect on the inner two zones of the adrenal cortex of the dog resulting in an abolition of the ability of the gland to synthesize glucocorticoids. Utilizing this method of depriving the animal of its adrenal hormones, we found that these dogs responded as well as normal dogs to the positive chronotropic and vasopressor effects of epinephrine and norepinephrine but the positive inotropic actions of the two amines were diminished. Furthermore, the animals were very intolerant of the stresses of anesthesia, surgery and repeated administration of catecholamines, showing a marked loss of plasma volume and reduction in blood pressure during the acute experimental procedure. These effects in o,p'-DDD-treated dogs were prevented by pretreatment of the animals with a glucocorticoid (Cortisol or prednisilone) for several days prior to experiment (36). These brief descriptions of the influence of hormones on catecholamine action emphasize the need for careful and rigorous examination of possible interactions between hormones and neurohumoral substances. Conclusions. Descriptive information concerning the effects of neurohumoral substances on the heart can be obtained by pharmacological analyses involving the use of synthetic compounds which are chemically identical with or related to the known neurohumoral transmitter compounds and by the use of selective antagonists. For example, certain phenylethylamine derivatives (including the naturally-occurring compounds epinephrine and norepinephrine) mimic the actions of electrical stimulation of the cardiac sympathetic nerves in terms of heart rate, force of contraction and activation of glycogen phosphorylase. The order of potency of these compounds and the selective antagonism of their cardiac actions by dichloroisoproterenol and pronethalol support the viewpoint

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that the mammalian cardiac adrenergic receptor is of the beta type in Ahlquist's classification. The failure of hypermetabolism produced by administration of thyroxine to alter the dose-response characteristics of epinephrine and norepinephrine action on the heart and blood vessels challenges the concept that thyroid hormone increases the sensitivity of the adrenergic receptors to catecholamines. On the other hand induced, selective reduction in endogenous adrenal glucocorticoid synthesis by administration of DDD to dogs depressed the responsiveness of the ventricular myocardium to the positive inotropic effects of catecholamines, supporting the view that glucocorticoids modify some of the cardiac actions of these neurohumors.

REFERENCES 1. Canon, W. B. and Rosenblueth, A.: Autonomic NeuroEffector Systems. The Macmillan Company, New York, 1937. 2. Ahlquist, R. P.: A study of the adrenotropic receptors. Amer. J. Physiol. 753:586-599 ( 1 9 4 8 ) . 3. Ahlquist, R. P.: Chapter 27 in Pharmacology in Medicine, 2nd ed., Drill, V. A. ed., McGraw-Hill Book Company, Inc., New York, 1958. 4. Nickerson, M.: The pharmacology of adrenergic blockade. Pharmacol. Rev. 7:27 ( 1 9 4 9 ) . 5. Powell, C. E. and Slater, I. H.: Blocking of inhibitory adrenergic receptors by a dichloro analogue of isoproterenol. J. Pharmacol. Exptl. Therap. 722:480-488 ( 1 9 5 8 ) . 6. Moran, N. C. and Perkins, M. E.: Adrenergic blockade of the mammalian heart by a dichloro analogue of isoproterenol. J. Pharmacol. Exptl. Therap. 724:223-231 ( 1 9 5 8 ) . 7. Moran, N. C. and Perkins, M. E.: An evaluation of adrenergic blockade of the mammalian heart. J. Pharmacol. Exptl. Therap. 133:192-210 (1961). 8. Furchgott, R. F.: The receptors for epinephrine and norepinephrine (adrenergic receptors). Pharmacol. Rev. 77:429441 ( 1 9 5 9 ) . 9. Dresel, P. E.: Blockade of some cardiac actions of adrenaline by dichloroisoproterenol. Canad. J. Biochem. Physiol. 38: 375-381 ( 1 9 6 0 ) . 10. Nickerson, M. and Chan, G.C-m.: Blockade of the responses of isolated myocardium to epinephrine. J. Pharmacol. Exptl. Therap. 735:186-191 ( 1 9 6 1 ) .

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11. Black, J. W., and Stephenson, J. S.: Pharmacology of a new adrenergic beta-receptor blocking compound (nethalide). Lancet 2:311 ( 1 9 6 2 ) . 12. Cotten, M. deV., and Pincus, S.: Comparative effects of a wide range of doses of 7-epinephrine and /-norepinephrine on the contractile force of the heart in situ. J. Pharmacol. Exp. Therap. 774:110-118 ( 1 9 5 5 ) . 13. Melville, K. I., and Lu, F. C.: Effects of ephedrine, phenylephrine, isopropylarterenol and methoxamine on coronary flow and heart activity as recorded concurrently. Arch. int. pharmacodyn., 92:108-118 ( 1 9 5 2 ) . 14. Goldberg, L. I., Cotton, M. deV., Darby, T. D. and Howell, E. V.: Comparative heart contractile force effects of equipressor doses of several sympathomimetic amines. J. Pharmacol. 70S: 177-185 ( 1 9 5 3 ) . 15. Nickerson, M. and Smith, S. M.: Protection against cyclopropane-epinephrine arrhythmias by dibenamine and other agents. Anesthesiology 70:562-576 ( 1 9 4 9 ) . 16. Moe, G. K., Malton, S. E., Rennick, B. R., and Freyburger, W. A.: The role of arterial pressure in the induction of idioventricular rhythms under cyclopropane anesthesia. J. Pharmacol. Exptl. Therap. 94:319-327 ( 1 9 4 8 ) . 17. Maling, H. M. and Moran, N. C.: Ventricular arrhythmias induced by sympathomimetic amines in unanesthetized dogs following coronary artery occlusion. Circulation Research 5: 409-413 ( 1 9 5 7 ) . 18. Moran, N. C., Moore, J. I., Holcomb, A. K., and Mushet, G. : Antagonism of adrenergically-induced cardiac arrhythmias by dichloroisoproterenol. J. Pharmacol. Exptl. Therap. 736:327335(1962). 19. Lucchesi, B. R. and Hardman, H. F.: The influence of dichloroisoproterenol and related compounds upon ouabain and acetylstrophanthidin induced cardiac arrhythmias. J. Pharmacol. Exptl. Therap. 732:372-381 ( 1 9 6 1 ) . 20.

Vaughan Williams, E. M. and Sekiya, A.: Prevention of arrhythmias due to cardiac glycosides by block of sympathetic beta receptors. Lancet 7:420-421 ( 1 9 6 3 ) .

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Sutherland, E. W.: The effect of the hyperglycemic factor of the pancreas and of epinephrine on glycogenolysis. In Recent Progress in Hormone Research. Proceedings of the Laurentian Hormone Conference, edited by G. Pincus, Vol. 5, pp. 441-463. Academic Press, Inc., New York, 1950. Sutherland, E. W.: The effect of the hyperglycemic factor and epinephrine on liver and muscle phosphorylase. In Phosphorus Metabolism, edited by W. D. McElroy and B. Glass, vol. 1, pp. 53-66. The Johns Hopkins Press, Baltimore, 1951. Sutherland, E. W. and Cori, C. F.: Effect of hyperglycemicglycogenolytic factor and epinephrine on liver phosphorylase. J. Biol. Chem. 755:531-543 ( 1951). Sutherland, E. W. and Rail, T. W.: The relation of adenosine-3', 5'-phosphate and phosphorylase to the actions of catecholamines and other hormones. Pharmacological Reviews 72:265-300 ( 1 9 6 0 ) . Hess, M. E. and Haugaard, N.: The effect of epinephrine and aminophylline on the phosphorylase activity of perfused contracting heart muscle. J. Pharmacol. Exptl. Therap. 722:169-175 ( 1 9 5 8 ) . Kukovetz, W. R., Hess, M. E., Shanfeld, J. and Haugaard, N.: The action of sympathomimetic amines on isometric contraction and phosphorylase activity of the isolated rat heart. J. Pharmacol. Exptl. Therap. 727:122-127 ( 1 9 5 9 ) . Mayer, S. E., and Moran, N. C.: Relation between pharmacologic augmentation of cardiac contractile force and the activation of myocardial glycogen phosphorylase. J. Pharmacol. Exper. Therap. 729:271 ( 1 9 6 0 ) . Mayer, S. E., Cotten, M. deV. and Moran, N. C.: Dissociation of the augmentation of cardiac contractile force from the activation of myocardial phosphorylase by catecholamines. J. Pharmacol. Exptl. Therap. 139:275-282 ( 1 9 6 3 ) . Cotton, M. deV. and Cooper, T.: Blockade of the positive inotropic action of norepinephrine by cardiac glycosides in the dog during hypothermia. J. Pharmacol Exptl. Therap. 756:97-106 (1962). Ellis, S.: Increased hexosemonophosphate, a common factor in muscular contraction potentiated by treppe, a short tetanus,

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epinephrine, or insulin. Amer. J. Med. Sci. 229:218-219 (1955). Ellis, S.: The metabolic effects of epinephrine and related amines. Pharmacol. Rev. 8:485-562 ( 1 9 5 6 ) . Ellis, S.: Relation of biochemical effects of epinephrine to its muscular effects. Pharmacol. Rev. 77:469-479 (1959). Murad, F „ Chi, Y. M., Rail, T. W. and Sutherland, E. W.: Adenyl Cyclase. III. The effect of catecholamines and choline esters on the formation of adenosine 3',5'-phosphate by preparations from cardiac muscle and liver. J. Biol. Chem. 237: 1233-1238 ( 1 9 6 2 ) . van der Schoot, J. B., and Moran, N. C.: An experimental evaluation of the reputed influence of thyroxine on the cardiovascular effects of catecholamines. J. Pharmacol. Exptl. Therap. (In Press.) Margolius, H. S. and Gaffney, T. E.: The effects of injected norepinephrine and sympathetic nerve stimulation in hypothyroid and hyperthyroid dogs. J. Pharmacol. Exptl. Therap. (In Press.) Cueto, C. Jr.: Glucocorticoid deficiency and circulatory responses to catecholamines. A Dissertation for Ph.D. degree, Emory University, Atlanta, 1964.

Agents Modifying Myocardial Blood ROBERT M. BERNE, M.D.2 Department of Physiology Western Reserve University School of Medicine Cleveland, Ohio THE CORONARY BLOOD F L O W ( C B F )

IS I N F L U E N C E D BY SEVERAL

physiological factors; the action of drugs on coronary resistance must be considered in terms of their effects on these physiological factors as well as by a direct effect on the vascular smooth muscle. For simplicity, the determinants of CBF can be divided into physical, neural and neurohumoral, and metabolic factors. Physical determinants of CBF are effective perfusion pressure (coronary arterial pressure minus venous pressure), extravascular compression (intramyocardial pressure) and blood viscosity. Neural and neurohumoral factors refer to the influence of the vagi and accelerator nerves and their transmitter substances. Metabolic factors encompass the local adjustments in coronary resistance associated with changes in the metabolic state of the myocardium and its oxygen supply. With the exception of blood viscosity, all the de1

Supported in part by U.S.P.H.S. Grants No. HE 06031-03 and HE 0630403. Research Career Awardee U.S.P.H.S. No. HE-K6-9593.

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terminants of C B F act to some measure, by altering the caliber of the coronary vessels. Few substances elicit a reduction in C B F whereas many compounds produce an increase in C B F when administered orally, intravenously or directly into a coronary artery. Since a complete survey of the numerous coronary vasodilators is available ( 1 ) , the present report will be limited to discussion of three substances which serve as examples of known or postulated mechanisms whereby drug-induced increases in C B F may occur. The first compounds to be considered are the catecholamines, epinephrine and norepinephrine. The mechanism of action of these substances on the coronary vessels is complex, and has been the subject of considerable study and controversy. Perhaps the principle reason for the lack of agreement on the effect of the catecholamines on coronary resistance can be traced to the fact that they may alter C B F in several ways, e.g. elevate aortic pressure (coronary perfusion pressure), increase extravascular compression, enhance myocardial metabolic activity, change the ratio of the duration of systole to diastole and directly effect the smooth muscle of the resistance vessels. When administered intravenously to the intact animal the observed effect on C B F is the algebraic sum of the several actions of the drugs and provides little information about the primary effect on the coronary resistance vessels. Elevation of arterial blood pressure increases C B F directly by virtue of a greater perfusion pressure and indirectly by increasing cardiac work. The reduction in coronary resistance associated with the increase in cardiac work is presumably mediated by vasoactive "metabolites" released from the myocardium. Opposing the effect of a rise in arterial pressure is an increase in intramyocardial pressure, due to the more forceful cardiac contractions, and an increase in the total duration of systole per unit of time, due to the tachycardia. The increase in contractile force and the tachycardia result in a significant increment in myocardial oxygen consumption, which, in the presence of a normally low coronary venous blood oxygen level, can only be achieved by a reduction in coronary resistance. Any direct effect of epinephrine or norepinephrine on coronary arteriolar smooth muscle is masked when the drugs are administered intravenously and the overall effect observed is one of increased CBF.

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In order to determine the primary effect of the catecholamines on coronary resistance we injected the drugs directly into the coronary arteries of open-chest dogs and fibrillating dog heart preparations in which the interfering factors could be controlled ( 2 ) . In the fibrillating heart experiments arterial blood f r o m a donor dog

•HfsniH

120 r

Figure 113. Intracoronary injection (arrows) of 0.5 fig. of epinephrine (upper record) and norepinephrine (lower record) in the fibrillating dog heart; P.P. = perfusion pressure in mm. Hg; CBF = mean coronary blood flow in ml./min.; pOo=coronary sinus blood oxygen tension in mm. Hg.

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was used to perfuse the cannulated left coronary artery at a constant pressure.

C B F , coronary perfusion pressure and coronary sinus

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