The Dynamic State of Muscle Fibers: Proceedings of the International Symposium. October 1–6, 1989, Konstanz, Federal Republic of Germany [Reprint 2019 ed.] 9783110884784, 9783110121681

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The Dynamic State of Muscle Fibers

The Dynamic State of Muscle Fibers Proceedings of the International Symposium October 1-6,1989 Konstanz, Federal Republic of Germany

Editor

Dirk Pette

W DE _G Walter de Gruyter • Berlin • New York 1990

Editor Dirk Pette, Dr. med. Professor of Biochemistry Department of Biology University of Konstanz D-7750 Konstanz 1 Federal Republic of Germany

Library of Congress Cataloging in Publication Data The dynamic state of muscle fibers : proceedings of the international symposium, October 1-6,1989, Federal Republic of Germany / editor, Dirk Pette. Complication of work presented at the Symposium "The Dynamic State of Muscle Fibers", held at the University of Konstanz. Includes bibliographical references. Includes indexes. ISBN 3-11-012168-9.--ISBN 0-89925-603-1 (New York) : I. Muscles—Congresses. 2. Muscle proteins—Congresses. 3. Muscle contraction-contraction-Congresses. I. Pette, Dirk. 1933- . II. mimposium "The Dynamic State of Muscle Fibers" (1989 : University of Konstanz). [DNLM: 1. Gene Expression, Regulation-congresses. 2. Muscle Proteinsmetabolism—congresses. 3. Muscles-metabolism-congresses. 4. Muscles—physiology-congresses. WE 500 D997 1989] QP321.D96 1990 599'.01852-dc20 DNLM/DLC 90-3346 for Library of Congresses CIP

CIP-Kurztitelaufnahme der Deutschen Bibliothek The dynamic state of muscle fibers : proceedings of the international symposium, October 1-6,1989, Konstanz, Federal Republic of Germany / ed. Dirk Pette. - Berlin ; New York : de Gruyter, 1990 ISBN 3-11-012168-9 NE: Pette, Dirk [Hrsg.]

© Printed on acid free paper. © Copyright 1990 by Walter de Gruyter& Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in Germany. Printing: Gerike GmbH, Berlin. - Binding: Lüderitz & Bauer-GmbH, Berlin.

PREFACE

Ten years ago, a symposium entitled "Plasticity of Muscle" (1) was held in Konstanz. The intention of that conference was to bring together investigators who, although being committed to various relevant and trend-setting developments in muscle research, had not, or had only insufficiently, been in contact with each other at that time. The face-to-face exchange of facts, ideas, and speculations proved to be extremely pleasant and stimulating. Since that time, I have been asked by several participants of the symposium and other colleagues if a follow-up symposium would some day be organized. Obviously, after a 10-year intermission, the time had come for a "Plasticity of Muscle Revisited" Symposium. I have to admit that it was a pleasure to organize another conference in Konstanz. The enthusiastic responses to my invitations for participation indicated that the follow-up symposium which was scheduled for the tenth anniversary of its predecessor, was timely. I wish to express my gratitude to my collaborator Dr. Sabine Düsterhöft for her valuable assistance in organizing the conference. Also, I would like to thank the generous financial contributions of the Deutsche Forschungsgemeinschaft and the Ministerium für Kunst und Wissenschaft des Landes Baden-Württemberg without which this conference could not have taken place. Additional financial support was given by the Gesellschaft der Freunde und Förderer der Universität Konstanz and the following companies: ASTA Pharma AG Frankfurt, Bakken Research Center Maastricht, Byk Gulden Lomberg Chemische Fabrik GmbH Konstanz, Dr. Falk Pharma GmbH Freiburg, Schering AG Berlin and SYNCOTEC GmbH Aßlar. The present volume is a compilation of work presented at the Symposium "The Dynamic State of Muscle Fibers" which was held

VI

at the University of Konstanz on October 1-6, 1989. It took place under the honorary chairmanship of two distinguished pioneers in the field, Theodor Bucher and Lloyd Guth. Theodor Biicher, my academic teacher, initiated a series of comparative enzymological studies thirty years ago (2,3), which opened the new field of comparative muscle biochemistry and directed my scientific interest in this direction. In the late sixties, Lloyd Guth contributed to our understanding of the phenotypic differences of muscle fiber types and their neural control (46). His foresighted article in 1971 "The Dynamic Nature of the So-Called Fiber Types of Mammalian Skeletal Muscle" (7) predetermined him to act as the honorary chairman of this Symposium. The major purpose of the present Symposium was to elaborate the dynamic state in which muscle fibers exist. This plasticity is the result of diverse developmental origins and phenotypic modulations in the adult which are under the influence of exogenous factors. The combined approaches of molecular and cellular biology, protein chemistry, enzymology and microbiochemistry at the single fiber level, ultrastructural research, biomechanics, and neurophysiology have provided new insights and opened new horizons for future research and collaboration. I wish to thank the participants of the Symposium for their contributions and discussions which were essential for setting a high scientific level and creating an extremely stimulating atmosphere. After having put together the proceedings of this Symposium in the form of the present book, I hope that it will have a similar illuminating effect upon its readers.

Konstanz, January 1990

Dirk Pette

VII

References 1. Plasticity of Muscle, D. Pette ed., Walter de Gruyter, Berlin New York, 1980. 2. Vogell, W., F.R. Bishai, Th. Bücher, M. Klingenberg, D. Pette, E. Zebe: Über strukturelle und enzymatische Muster in Muskeln von Locusta migratoria. 1959. Biochem. Z. 332. 81-117. 3. Pette, D., Th. Bücher: Proportionskonstante Gruppen in Beziehung zur Differenzierung der Enzymaktivitätsmuster von Skelett-Muskeln des Kaninchens. 1963. Hoppe-Seyler 1 s Z. Physiol. Chemie. 331. 180-195. 4. Guth, L., F.J. Samaha: Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle. 1969. Exp. Neurol. 25, 138-152. 5. Samaha, F.J., L. Guth, R.W. Albers: Phenotypic differences between the actomyosin ATPase of the three fiber types of mammalian skeletal muscle. 1970. Exp. Neurol. 26, 120-125. 6. Guth, L., F.J. Samaha: Procedure for the histochemical demonstration of actomyosin ATPase. 1970. Exp. Neurol. 28. 365-367. 7. Guth, L., H. Yellin: The dynamic nature of the so-called "fiber types" of mammalian skeletal muscle. 1971. Exp. Neurol. 31, 277-300.

CONTENTS

An Historical Perspective by Lloyd Guth Section I.

XXV

Patterns and Regulation of Gene Expression in Muscle

Myosin Heavy Chain Gene Expression: Interplay of cis and trans Factors Determines Hormonal and Tissue Specificity by J.N. Rottman, W.R. Thompson, B. Nadal-Ginard, V. Mahdavi

3

Isolation and Characterization of Human Myogenic Factors Involved in Lineage Determination and Regulation of Gene Expression by H.H. Arnold,T. Braun, E. Bober, B. Winter, G. Buschhausen-Denker

17

Regulatory Elements Involved in Chicken Myosin Alkali Light Chain Gene Expression by Y.-i. Nabeshima,T. Uetsuki, T. Komiya, Y. Nabeshima, A. Fujisawa-Sehara

33

Regulation of the Human ß-Myosin Heavy Chain Gene and an Approach to a Functional Analysis of Recombinant Protein Subregions of the ß-Chain by H.-P. Vosberg, U. Horstmann-Herold, T. Jaenicke, I. Morano, J.C. Riiegg, P. Eldin, J.J. Leger

45

Regulation of the Myosin Heavy Chain & Promoter in Skeletal and Cardiac Myocytes by P.K. Umeda, R.L. Carter, R.S. Hall, J.M. Welborn, L.B. Bugaisky

61

Developmental Regulation of Muscle Gene Transcripts in Embryonic Mouse Muscle by G.E. Lyons, D. Sassoon, M.-O. ott, M.E. Buckingham, M. Ontell

75

Regulation of a-Tropomyosin Expression in Embryonic Stem Cells by D.F. Wieczorek, P. Howies, T. Doetschman

91

The Insertion and Release of Contractile Proteins from Myofibrils by S.M. Goldfine, J. Peng, D.A. Fischman

103

X

Section II.

Muscle Fiber Development and Origins of Diversity

Muscle Fiber Development and Origins of Fiber Diversity by F.E. Stockdale

121

Regulation of Myosin Heavy Chain Expression during Development, Maturation, and Regeneration in Avian Muscles: The Role of Myogenic and Non-Myogenic Factors by E. Bandman, D.L. Bourke, M. Wick

127

Effects of Induced Contractile Activity on Cultured Chick Breast Muscle Cells by S. Diisterhoft, S. Hofmann, D. Pette

139

Mechanically-Induced Alterations in Cultured Skeletal Myotube Growth by H.H. Vandenburgh, S. Hatfaludy,P. Karlisch, J. Shansky

151

Isoforms of Troponin Components in Developing Muscle Fibres by G.K. Dhoot

165

Biochemical Properties of the Diaphragm during Development of Respiratory Function in the Rat by A.M. Kelly, B.W.C. Rosser, N.A. Rubinstein, P.M. Nemeth

181

Cultured Adult Rat Cardiomyocytes as a Model for Differentiation by H.M. Eppenberger, M. Messerli, M. Miiller, P. Schwarb, M.E. Eppenberger-Eberhardt

193

Motor Activity Dependant Muscle Fibre Transformation of the Rat Soleus by G. Vrbova, M.B. Lowrie, A.L. Connold

205

Motoneurons - Muscle Fiber Connectivity and Interdependence by V.R. Edgerton, R.R. Roy, S.C. Bodine-Fowler, D.J. Pierotti, G.A. Unguez, T.P. Martin, B. Jiang, G.R. Chalmers

217

Metabolic Uniformity of the Motor Unit by P.M. Nemeth, R.S. Wilkinson

233

Myosin Expression in Denervated Fast- and SlowTwitch Muscles: Fiber Modulation and Substitution by U. Carraro, C. Catani, A. Degani, C. Rizzi

247

XI Section III.

Cellular and Molecular Diversity of Muscle Fibers

Regulation of Regional Specialization in Muscle Fibres by S.M. Hughes, ü.M. Blau

265

Expression of Fast Thin Filament Proteins. Defining Fiber Archetypes in a Molecular Continuum by F.H. Schachat, M.M. Briggs, E.K. Williamson, H. McGinnis, M.S. Diamond, P. W. Brandt

279

Correlations between Troponin-T and Myosin Heavy Chain Isoforms in Normal and Transforming Rabbit Muscle Fibers by T. Schmitt, D. Pette

293

Complexity of Sarcomeric Myosin Species at the Protein Level by J.I. Rushbrook, C. Weiss, T.-T. Yao

303

The Multiplicity of Myosin Light and Heavy Chain Combinations in Muscle Fibers by R.S. Staron, D. Pette

315

Muscle Fiber Types Expressing Different Myosin Heavy Chain Isoforms. Their Functional Properties and Adaptive Capacity by S. Schiaffino, L. Gorza, S. Ausoni, R. Bottinelli, C. Reggiani, L. Larson, L. Edstróm, K. Gundersen, T. Lomo

329

Characterization of IIx Fibres in Mouse Muscles by D.J. Parry, D. Zardini

343

Varied Expression of Myosin Alkali Light Chains is Associated with Altered Speed of Contraction in Rabbit Fast-Twitch Skeletal Muscles by R.L. Moss, P.J. Reiser, M.L. Greaser, T.J. Eddinger

355

M-Band Structure and Composition in Relation to Fiber Types by L.-E. Thornell, E. Carlsson, F. Pedrosa

369

Fibre Type-Specific Enzyme Activity Profiles. A Single Fibre Study of the Effects of Chronic Stimulation on the Rabbit Fast-Twitch Tibialis Anterior Muscle by J. Henriksson, P.M. Nemeth, K. Borg, S. Salmons, O.H. Lowry

385

XII

Section IV.

Adaptive and Induced Fiber Transformations: Myofibrillar and Ca-Sequestering Proteins

On the Reversibility of Stimulation-Induced Muscle Transformation by S. Salmons

401

Dynamics of Stimulation-Induced Fast-to-Slow Transitions in Protein Isoforms of the Thick and Thin Filament by D. Pette

415

Muscle Fibre Transformations in Myotonic Mouse Mutants by H. Jockusch

429

Myosin and Sarcoplasmic Reticulum Ca2+-ATPase Isoforms in Electrically Stimulated Rabbit Fast Muscle by K. Mabuchi, F.A. Sreter, J. Gergely, A.O. Jorgensen

445

Myosin Heavy Chain Isoforms in Single Fibers of Transforming Rat Muscle by A. Termin, R.S. Staron, D. Pette

463

Effects of Increased Neuromuscular Activity at Altered Thyroid Hormone Levels on Myosin Expression by B.J. Kirschbaum, H.-B. Kucher, A.M. Kelly, D. Pette 473 Change in Protein Synthesis During Induced Muscle Fiber Transformation by J. Gagnon, P. Gregory, T. Kurowski, R. Zak

481

Influence of Neuromuscular Activity on the Expression of Parvalbumin in Mammalian Skeletal Muscle by E. Leberer, D. Pette 497 Conformational States of the Sarcoplasmic Reticulum Ca 2+ -ATPase in Normal and Transforming Rabbit Fast-Twitch Muscle by L. Dux, D. Pette

509

The Mechanogenic Transduction of the Mammalian Myocardium by K. Schwartz, A.M. Lompre, D. de la Bastie, P. Due, K.R. Boheler, J.L. Samuel, L. Rappaport, J.J. Mercadier

521

Adaptive Changes in Sarcomeric Proteins of Heart Muscle by M.C. Schaub, U.T. Brunner, P.A.J. Huber

533

XIII

Section V.

Adaptive and Induced Fiber Transformations: Metabolic Changes

Adaptive Changes in Myosin Isoforms and in Energy Metabolism in Muscles Containing Analogues of Creatine as the Phosphagen by M.J. Kushmerick, T.S. Moerland, N.G. Wolf

551

The Range of Mitochondrial Adaptation in Muscle Fibers by H. Hoppeler

567

Species-Specific Ranges of Metabolic Adaptations in Skeletal Muscle by J.-A. Simoneau

587

Intracellular Signals Mediating Contraction-Induced Changes in the Oxidative Capacity of Skeletal Muscle by W.E. Kraus, R.S. Williams

601

Time Dependent Changes in Metabolites of Energy Metabolism in Low-Freguency Stimulated Rabbit Fast-Twitch Muscle by H.J. Green, S. Düsterhöft, L. Dux, D. Pette

.... 617

Muscle Structure and Function after Exposure to High Altitude Hypoxia by H. Howald, H. Hoppeler, H. Ciaassen, S. Kayar, E. Kleinert, C. Schlegel, R. Winterhaider, M. Martineiii, D. Pette, J.-A. Simoneau, A. Uber, P. Cerretelli, P.E. di Prampero, G. Ferretti

629

XIV

Section VI.

Adaptive and Induced Fiber Transformations: The Role of satellite Cells

Satellite Cells and the Concept of Cell Lineage in Avian Myogenesis by F.E. Stockdale, J.L. Feldman

641

Transformation and Cloning of Different Types of Myoblasts during Avian Development by V. Mouly, M. Lemonnier, D. Libri, F. Gros, M.Y. Fiszman

651

The Role of Satellite Cells in Adaptive or Induced Fiber Transformations by E. Schultz, K.C. Darr

667

Are Satellite Cells Essential for Isomyosin Switching? by B.R. Eisenberg, J. Jacobs-El

681

Proliferation and Differentiation of Myoblasts: The Role of Platelet-Derived Growth Factor and the Basement Membrane by Z. Yablonka-Reuveni, D.F. Bowen-Pope, R.S. Hartley

693

The Control of Satellite Cell Growth in Skeletal Muscle during Hypertrophy and Regeneration by R.C. Strohman, J. DiMario, N. Buffinger, S. Yamada

707

Index of Contributors

719

Subject Index

723

XV

LIST OF CONTRIBUTORS H.H. Arnold, Department of Toxicology, Medical School, University of Hamburg, D-2000 Hamburg 13, Germany 8. Ausoni, CNR Unit for Muscle Biology and Physiopathology, Institute of General Pathology, 1-35100 Padova, Italy E. Bandman, Department of Food Science and Technology, University of California, Davis, CA 95616, USA D. de la Bastie, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France H.M. Blau, Department of Pharmacology, Stanford University, Medical Center, Stanford, CA 94305-5332, USA E. Bober, Department of Toxicology, Medical School, University of Hamburg, D-2000 Hamburg 13, Germany B.C. Bodine-Fowler, Department"of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA K.R. Boheler, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France K. Borg, Department of Neurology, Karolinska Sjukhuset, S-104 01 Stockholm, Sweden R. Bottinelli, Institute of Human Physiology, University of Pavia, 1-27100 Pavia, Italy D.L. Bourke, Department of Food Science and Technology, University of California, Davis, CA 95616, USA D.F. Bowen-pope, Department of Pathology, School of Medicine, University of Washington, Seattle, WA 98195, USA P.W. Brandt, Department of Anatomy and Cell Biology, Columbia University Medical School, New York, NY 10032, USA T. Braun, Department of Toxicology, Medical School, University of Hamburg, D-2000 Hamburg 13, Germany M.M. Briggs, Department of Cell Biology, Duke University, Medical School, Durham, NC 27710, USA U.T. Brunner, Institute of Pharmacology, Medical Faculty, University of Zürich, CH-8006 Zürich, Switzerland M.E. Buckingham, Department of Molecular Biology, Pasteur Institute, F-75724 Paris Cedex 15, France

XVI

N. Buffinger, Department of Cell and Molecular Biology, University of California, Berkeley, CA 94720, USA L.B. Bugaisky, Departments of Medicine, Pathology, Cell Biology and Anatomy, University of Alabama, Birmingham, AL 35294, USA 6. Buschhausen-Denker, Department of Toxicology, Medical School, University of Hamburg, D-2000 Hamburg 13, Germany E. Carlsson, Department of Anatomy, University of Umea, S-901 87 Umea, Sweden U. Carraro, C.N.R. Unit for Muscle Biology and Physiopathology, Institute of General Pathology, Padova, Italy R.L. Carter, Departments of Medicine, Pathology, Cell Biology and Anatomy, University of Alabama, Birmingham, AL 35294, USA C. Catani, C.N.R. Unit for Muscle Biology and Physiopathology, Institute of General Pathology, Padova, Italy P. Cerretelli, Department of Physiology, University of Geneva, Medical School, CH-1211 Geneva 4, Switzerland 6.R. Chalmers, Department of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA H. Claassen, Department of Anatomy, University of Bern, CH-3 000 Bern 9, Switzerland A.L. Connold, Department of Anatomy and Developmental Biology, Centre for Neuroscience, University College London, London WC1E 6BT, Great Britain K.C. Darr, Department of Anatomy, University of Wisconsin, Madison, WI 53706, USA A. Degani, C.N.R. Unit for Muscle Biology and Physiopathology, Institute of General Pathology, Padova, Italy G.K. Dhoot, Department of Basic Sciences, The Royal Veterinary College, University of London, London, Great Britain M.S. Diamond, Department of Anatomy and Cell Biology, Columbia University Medical School, New York, NY 10032, USA J. DiMario, Department of Cell and Molecular Biology, University of California, Berkeley, CA 94720, USA T. Doetschman, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0524, USA

XVII

P. Duc, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France 5. Düsterhöft, Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany L. Dux, Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany T.J. Eddinger, Department of Biology, Marquette University, Milwaukee, WI 53233, USA V.R. Edgerton, Department of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA L. Edström, Departments of Clinical Neurophysiology and Neurology, Karolinska Hospital, Karolinska Institute, S-104 01 Stockholm, Sweden B.R. Eisenberg, Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60680, USA P. Eldin, I.N.S.E.R.M. Unité 300, Faculté de Pharmacie, Université de Montpellier, Montpellier, France H.M. Eppenberger, Institute for Cell Biology, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland M.E. Eppenberger-Eberhardt, Institute for Cell Biology, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland J.L. Feldman, Stanford University School of Medicine, Stanford, CA 94305-5306, USA 6. Ferretti, Department of Physiology, University of Geneva, Medical School, CH-1211 Geneva 4, Switzerland D.A. Fischman, Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021, USA M.Y. Fiszman, Unité de Biochimie, Institut Pasteur, F-75724 Paris Cedex 15, France A. Fujisawa-Sehara, Division of Molecular Biology, National Institute of Neuroscience, Tokyo 187, Japan J. Gagnon, Departments of Medicine, Pharmacology, Anatomy, The University of Chicago, Chicago, IL 60637, USA J. Gergely, Department of Muscle Research, Boston Biomedical Research Institute, Boston, MA 02114, USA

XVIII S.M. Goldfine, Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021,

USA

L. Gorza, C.N.R Unit for Muscle Biology and Physiopathology, Institute of General Pathology, 1-35100 Padova, Italy M . L . Greaser, M u s c l e Biology Laboratory, University Wisconsin, Madison, WI 53706, USA

of

H . J . Green, Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 P. Gregory, Departments of Medicine, Pharmacology, The University of Chicago, Chicago, IL 60637, USA

Anatomy,

F. Gros, Unité de Biochimie, Institut Pasteur, F-75724 Cedex 15, France

Paris

K. Guildersen, Institute of Neurophysiology, University Oslo, N-0162 Oslo 1, Norway

of

L. Guth, Department of Anatomy, University of Maryland, School of Medicine, Baltimore, M D 21201, USA R.S. Hall, Departments of Medicine, Pathology, Cell Biology and Anatomy, University of Alabama, Birmingham, A L 35294, USA R.S. Hartley, Department of Biological Structure, School of Medicine, University of Washington, Seattle, W A 98195, USA S. H a t f a l u d y , Department of Pathology, Brown University The M i r i a m Hospital, Providence, RI 02906, USA

and

J. Henriksson, Department of Physiology III, Karolinska Hospital, Karolinska Institute, S-104 01 Stockholm, Sweden S. Hofmann, Fakultät für Biologie, Universität D-7750 Konstanz, Germany

Konstanz,

H. Hoppeler, Department of Anatomy, University of Bern, CH-3 000 Bern 9, Switzerland U. H o r s t m a n n - H e r o l d , Max-Planck-Institut Forschung, D-6900 Heidelberg, Germany

für

medizinische

H. Howald, Human Pharmacology, Pharmaceuticals Ciba-Geigy Ltd., CH-4002 Basel, Switzerland

Division,

P. Howies, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0524, USA P.A.J. Huber, Institute of Pharmacology, Medical Faculty, University of Zürich, CH-8006 Zürich, Switzerland

XIX

S.M. Hughes, Department of Pharmacology, Stanford University,, Medical Center, Stanford, CA 94305-5332, USA J. Jacobs-El, Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60680, USA T. Jaenicke, Max-Planck-Institut für medizinische Forschung, D-6900 Heidelberg, Germany B. Jiang, Department of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA H. Jockusch, Developmental Biology Unit, University of Bielefeld, D-4800 Bielefeld 1, Germany A.O. Jorgensen, Department of Anatomy, University of Toronto, Toronto, Ontario, Canada M5S 1A8 P. Karlisch, Department of Pathology, Brown University and The Miriam Hospital, Providence, RI 02906, USA S. Kayar, Department of Anatomy, University of Bern, CH-3000 Bern 9, Switzerland A.M. Kelly, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA B.J. Kirschbaum, Laboratory of Biochemical and Molecular Biology, The Rockefeller University, New York, NY 10021, USA E. Kleinert, Department of Anatomy, University of Bern, CH-3000 Bern 9, Switzerland T. Komiya, Division of Molecular Biology, National Institute of Neuroscience, Tokyo 187, Japan W.E. Kraus, Department of Medicine, Duke University, School of Medicine, Durham, NC 27710, USA H.-B. Kucher, Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany T. Kurowski, Departments of Medicine, Pharmacology, Anatomy, The University of Chicago, Chicago, IL 60637, USA M.J. Kushmerick, Departments of Radiology and Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA L. Larson, Departments of Clinical Neurophysiology and Neurology, Karolinska Hospital, Karolinska Institute, S-104 01 Stockholm, Sweden

XX

E. Leberer, NRC Biotechnology Research Institute, Genetic Engineering Section, Montreal, Quebec, Canada H4P 2R2 J.J. Leger, I.N.S.E.R.M. Unité 300, Faculté de Pharmacie, Université de Montpellier, Montpellier, France M. Lemonnier, Unité de Biochimie, Institut Pasteur, F-75724 Paris Cedex 15, France D. Libri, Unité de Biochimie, Institut Pasteur, F-75724 Paris Cedex 15, France T. Lömo, Institute of Neurophysiology, University of Oslo, N-0162 Oslo 1, Norway A.M. Lompré, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France M.B. Lowrie, Department of Anatomy and Developmental Biology, Centre for Neuroscience, University College London, London WC1E 6BT, Great Britain O.H. Lowry, Department of Pharmacology, Washington University, School of Medicine, St. Louis, MO 63124, USA 6.E. Lyons, Department of Molecular Biology, Pasteur Institute, F-75724 Paris Cedex 15, France K. Mabuchi, Department of Muscle Research, Boston Biomedical Research Institute, Boston, MA 02114, USA V. Mahdavi, Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA T.P. Martin, Department of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA M. Martinelli, Department of Anatomy, University of Bern, CH-3000 Bern 9, Switzerland H. McGinnis, Department of Cell Biology, Duke University, Medical School, Durham, NC 27710, USA J.J. Mercadier, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France M. Messerli, Institute for Cell Biology, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland T.S. Moerland, Department of Biological Sciences, Florida State University, Tallahassee, FL, USA

XXI

I. Morano, Institut für Physiologie II der Universität Heidelberg, D-6900 Heidelberg, Germany R.L. Moss, Department of Physiology, University of Wisconsin, Madison, WI 53706, USA V. Mouly, Unité de Biochimie, Institut Pasteur, F-75724 Paris Cedex 15, France M. Müller, Institute for Cell Biology, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland Y. Nabesbima, Division of Molecular Biology, National Institute of Neuroscience, Tokyo 187, Japan Y.-i. Nabeshima, Division of Molecular Biology, National Institute of Neuroscience, Tokyo 187, Japan B. Nadal-Ginard, Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA P.M. ttemeth, Department of Neurology, Washington University, School of Medicine, St. Louis, MO 63124, USA M. Ontell, Department of Anatomy, University of Pittsburgh, Pittsburgh, PA, USA M.-O. Ott, Department of Molecular Biology, Pasteur Institute, F-75724 Paris Cedex 15, France D.J. Parry, Department of Physiology, University of Ottawa, Ottawa, Canada K1H 8M5 P. Pedrosa, Department of Anatomy, University of Umea, S-901 87 Umea, Sweden I. Peng, Department of Cell Biology and Anatomy, Cornell University, Medical College, New York, NY 10021, and Department of Anatomy, U.M.D.N.J. - Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA D. Pette, Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany D.J. Pierotti, Department of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA P.E. di Prampero, Department of Physiology, University of Geneva Medical School, CH-1211 Geneva 4, Switzerland L. Rappaport, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France

XXII

C. Reggiani, Institute of Human Physiology, University of Pavia, 1-27100 Pavia, Italy P.J. Reiser, Department of Physiology and Biophysics, University of Illinois, Chicago, IL 60680, USA C. Rizzi, C.N.R. Unit for Muscle Biology and Physiopathology, Institute of General Pathology, Padova, Italy B.W.C. Rosser, Department of Neurology, Washington University, School of Medicine, St. Louis, MO 63124, USA J.N. Rottman, Laboratory of Molecular and Cellular Cardiology, Howard Hughes Medical Institute, Boston, MA 02115, USA R.R. Roy, Department of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA N.Ä. Rubinstein, Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA J.C. Rüegg, Institut für Physiologie II der Universität Heidelberg, D-6900 Heidelberg, Germany J.I. Rushbrook, Department of Biochemistry, SUNY Health Science Center at Brooklyn, Brooklyn, NY 11203, USA S. Salmons, Department of Human Anatomy and Cell Biology and The Muscle Research Centre, University of Liverpool, Liverpool L69 3BX, Great Britain J.L. Samuel, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France D. Sassoon, Department of Molecular Biology, Pasteur Institute, F-75724 Paris Cedex 15, France F. Schachat, Department of Cell Biology, Duke University, Medical School, Durham, NC 27710, USA M.C. Schaub, Institute of Pharmacology, Medical Faculty, University of Zürich, CH-8006 Zürich, Switzerland S. Schiaffino, C.N.R. Unit for Muscle Biology and Physiopathology, Institute of General Pathology, 1-35100 Padova, Italy C. Schlegel, Department of Anatomy, University of Bern, CH-3 000 Bern 9, Switzerland T.L. Schmitt, Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany

XXIII

E. Schultz, Department of Anatomy, University of Wisconsin, Madison, WI 53706, USA P. Schwarb, Institute for Cell Biology, Swiss Federal Institute of Technology (ETH), CH-8093 Zürich, Switzerland K. Schwartz, I.N.S.E.R.M. Unité 127, Hôpital Lariboisière, F-75010 Paris, France J. Shansky, Department of Pathology, Brown University and The Miriam Hospital, Providence, RI 02906, USA J.-A. Simoneau, Physical Activity Sciences Laboratory, Laval University, Ste-Foy, Québec, Canada G1K 7P4 F.A. Sréter, Department of Muscle Research, Boston Biomedical Research Institute, Boston, MA 02114, USA R.S. Staron, Department of Zoological & Biomedical Sciences, College of Osteopathic Medicine, Athens, Ohio 45701, USA P.E. Stockdale, Stanford University, School of Medicine, Stanford, CA 94305-5306, USA R.C. Strohman, Department of Cell and Molecular Biology, University of California, Berkeley, CA 94720, USA A. Termin, Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany W.R. Thompson, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA L.-E. Thornell, Department of Anatomy, University of Umea, S-901 87 Umea, Sweden A. Uber, Fakultät für Biologie, Universität Konstanz, D-7750 Konstanz, Germany T. Uetsuki, Division of Molecular Biology, National Institute of Neuroscience, Tokyo 187, Japan P.K. Umeda, Departments of Medicine, Pathology, Cell Biology and Anatomy, University of Alabama, Birmingham, AL 35294, USA G.A. Unguez, Department of Kinesiology and Brain Research Institute, University of California, Los Angeles, CA 90024-1568, USA H.H. Vandenburgh, Department of Pathology, Brown University and The Miriam Hospital, Providence, RI 02906, USA H.-P. Vosberg, Max-Planck-Institut für medizinische Forschung, D-6900 Heidelberg, Germany

XXIV

G. Vrbova, Department of Anatomy and Developmental Biology, Centre for Neuroscience, University College London, London WC1E 6BT, Great Britain C. Weiss, Department of Biochemistry, SUNY Health Science Center at Brooklyn, Brooklyn, NY 11203, USA J.M. Welborn, Departments of Medicine, Pathology, Cell Biology and Anatomy, University of Alabama, Birmingham, AL 35294, USA M. Wick, Department of Food Science and Technology, University of California, Davis, CA 95616, USA D.F. Wieczorek, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 452 67-0524, USA R.S. Wilkinson, Department of Anatomy and Neurobiology, Washington University, School of Medicine, St. Louis, MO 63110, USA R.S. Williams, Department of Microbiology, Duke University, Medical School, Durham, NC 27710, USA E.K. Williamson, Department of Cell Biology, Duke University, Medical School, Durham, NC 27710, USA B. Winter, Department of Toxicology, Medical School, University of Hamburg, D-2000 Hamburg 13, Germany R. Winterhaider, Department of Anatomy, University of Bern, CH-3000 Bern 9, Switzerland N.6. Wolf, Harvard Medical School, Boston, MA, USA Z. Yablonka-Reuveni, Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA 98195, USA S. Yamada, Department of Sports Science, College of Arts and Science, The University of Tokyo, Tokyo 153, Japan T.-T. Yao, Department of Biochemistry, SUNY Health Science Center at Brooklyn, Brooklyn, NY 112 03, USA R. Zak, Departments of Medicine, Pharmacology, Anatomy, The University of Chicago, Chicago, IL 60637, USA D. Zardini, Department of Physiology, University of Ottawa, Ottawa, Canada K1H 8M5

THE DYNAMIC STATE OF MUSCLE FIBERS An Historical Perspective LLOYD GUTH Department of Anatomy, University of Maryland School of Medicine, Baltimore, MD 21201, USA Although scientific progress represents a continuum of discovery, a single dramatic observation often exerts a seminal influence on the direction of scientific investigations for succeeding decades. Such was certainly the case for the field of skeletal muscle plasticity, where the landmark work of Buller, Eccles and Eccles (1) revealed a degree of plasticity that was quite unanticipated. In the course of physiological studies on the motor unit, Eccles (2) had observed that the contractile properties specific to slow and fast muscles were appropriately matched to the physiological properties of the motor neurons that innervated them; he pointed out that this physiological matching served to maximize work output and minimize energy expenditure for the tonically-active slow muscles and the phasically-active fast ones. Eccles and Buller therefore set out to clarify the developmental mechanism by which so appropriate a neuromuscular relationship became established. Because of the intimate dependence of muscle development on its nerve supply, they suspected that this might be a neurally regulated, developmental phenomenon, and they tested this hypothesis by cross-reinnervating slow and fast muscles in neonatal cats (at which age most muscles are still immature, being composed in large measure of myoblasts and myotubes). They observed that the contraction time of the cross-reinnervated slow muscle became shortened and that of the fast muscle became prolonged (2). The success of this experiment raised the question whether this neural regulation was necessarily restricted to developmental stages of muscle formation. They therefore repeated the cross-reinnervation procedure on adult cats and obtained identical results.

XXVI

These findings led to two inescapable conclusions: that the contraction time of muscles is neurally regulated and that this regulation is not temporally restricted to immature muscle but is subject to change throughout life. As with all important experimental discoveries, the observations raised several critical questions requiring further experimental analysis: (a) Are the specific biochemical and anatomical properties of slow and fast muscles also neurally regulated? (b) What cellular changes are responsible for the transformation of contraction time of cross-reinnervated fast and slow muscles? (c) Does this capacity for plasticity, that can be brought about by experimental surgical means, reflect ongoing dynamic transformations throughout life? (d) What are the molecular mechanisms by which the nerve regulates the physiological properties of the muscle it innervates? The Regulation of Other Properties of Slow and Fast Muscles The first question, whether the specific biochemical and anatomical properties of slow and fast muscles are also neurally regulated, was addressed in short order. Within a few years after Buller and Eccles 1 initial reports, it was apparent that virtually every physiological, biochemical and morphological property specific to fast and slow muscles could be transformed by surgical cross-reinnervation (3). However, in every case the transformations were incomplete; it was simply not possible to obtain a complete quantitative or qualitative transformation of any of these parameters by cross-reinnervation. This finding puzzled investigators in the field, raising interpretive complexities that they discussed at length but have never been able to explain satisfactorily. Indeed, this issue remains so enigmatic that it has been the subject of considerable discussion at the present conference.

XXVII

The Cellular Basis of Muscle Fiber Transformations The second of our questions was initially approached histochemically rather than physiologically because, in the 1960s, the techniques for stimulating and recording from single motor units were still rather primitive. Enzyme histochemical studies on biochemical-physiological relationships revealed that slow and fast muscles consisted of a mosaic of fiber types having either high or low activities of the various glycolytic, oxidative and ATPase enzymes. Thus the proportion of high-activity to low-activity fibers determined the overall enzyme activity of the muscle. By examining serial transverse sections of muscle histochemically it was possible to group muscle fibers into a small number of classes which became known as "fiber types". Dubowitz (4) had initiated this concept by showing that most muscle fibers were either thigh-oxidative and low-glycolytic] or flow-oxidative and high-glycolytic] in activity. This observation formed the basis for E n g e l 1 s classification (5) which included myosin ATPase activity as well. He described TYPE I fibers as being [low-ATPase, high-oxidative and low-glycolytic] and TYPE II fibers as being fhigh-ATPase, low-oxidative, and highglycolytic] in activity. Unfortunately ignored in this classification were the conclusions of Stein and Padykula (6) that three distinct fiber types could be discerned by staining for oxidative enzymes alone and of Yellin and Guth (7) that at least three fiber types could be discerned with the myofibrillar ATPase procedure alone. Eventually the dispute over the number of the fiber types was resolved, for the time being, with the acceptance of a modified classification scheme in which fibers were described as TYPE I, IIA and IIB (8). However, we now know that even this tripartite classification fails to recognize subtle but critical distinctions among the fibers in each class. Indeed the inadequacies of this classification could have been predicted from the early studies of Romanul (9), who used a battery of histochemical procedures to reveal at least eight histochemical fiber types.

XXVIII

Armed with the knowledge that the fiber composition of most muscles could be categorized into a relatively restricted number of histochemically-distinct, fiber-type classes and that fast and slow muscles differed primarily in the relative proportion of these fiber types, muscle biologists now turned their attention to elucidating whether each type of muscle fiber is a rigidly specified, relatively immutable cell type or whether it is a cell capable of a variety of phenotypic expressions. This issue was soon resolved by experiments demonstrating that the characteristic histochemical fiber types of slow and fast muscle were transformed by crossreinnervation (3) and there was only a transitory appearance of fiber types having histochemical activities intermediate between the Type I and Type II categories. This finding had implications whose importance transcended the mere issue of the nature of muscle fiber types. First of all, it meant that the quantitative changes in ATPase activity that had been observed in myosin preparations from homogenates of crossreinnervated slow and fast muscles was brought about by a change in the relative proportions of high and low ATPase muscle fibers. Secondly, it indicated that slow and fast muscle transformation is brought about by the metamorphosis of the high-activity fibers to low-activity ones (and vice versa) rather than by the appearance de novo of new classes of fibers having intermediate levels of enzyme activity. These conclusions implied that a comparable cellular mechanism was responsible for the transformation of the contractile properties of cross-reinnervated muscles, an interpretation that was indeed eventually verified by physiological experiments.

Muscle Plasticity as a Physiological Adaptation The resolution of these issues enabled scientists to address the third question, viz., whether muscle fiber plasticity, as demonstrated experimentally by neurosurgical manipulations, might actually represent a functionally-important adaptive

XXIX

mechanism in the unoperated animal. Before discussing the experiments that gave rise to this concept, it is appropriate to review the state of our understanding of certain relevant aspects of neurobiology at the beginning of 1960s. First of all, the principle of axonal transport, although enunciated by Weiss (10) only twelve years previously, had by 1960 gained universal acceptance. This principle, which is fundamental to our understanding of regeneration and trophic function, was even then accepted by most developmental neuroscientists as the single most important neurobiological discovery since the neuron doctrine of Cajal. Less well appreciated in this context is a second major discovery that came from Weiss' laboratory at that time and which concerned the subject of collateral nerve sprouting in partially denervated muscle (11). In contrast to the progressive atrophy that occurs after total denervation, atrophy lasts for only several weeks in partially denervated muscles. The muscles then begin to recover, and they ultimately regain much of their original weight and strength. Edds, a postdoctoral fellow in Weiss' laboratory, provided the explanation for this phenomenon by showing that axonal sprouts emerged from the residual axons of the partially denervated muscle and reinnervated previously denervated muscle fibers (11). This demonstration of the expansion of the motor unit under pathological conditions prepared neuroscientists to accept morphological remodeling of the motor unit as an adaptive physiological process. The elucidation of collateral sprouting as a fundamental neurobiological principle enabled experimental scientists to address the issue of motor unit homogeneity and its relationship to muscle fiber plasticity. Histochemical studies of cross-sections of normal muscles had revealed that the various fiber types are distributed in an apparently random or mosaic pattern. However, after reinnervation, cross-reinnervation, or collateral reinnervation, the fiber types become arranged in groups or clusters (3). The definitive explanation for this change in distribution of fiber types was provided by the studies of Edstrom and

XXX

Kugelberg (12,13). They found that the motor unit is histochemically homogeneous in normally-innervated muscle — i.e., every muscle fiber supplied by axonal branches of a single neuron is histochemically identical. Consequently, the appearance of fiber type groups in reinnervated muscle results from the reinnervation of adjacent muscle fibers by collateral branches of axons of a single motor unit. Many years later the motor unit was shown to be physiologically homogeneous (14), thus providing explanation for the alterations in contractile properties of cross-reinnervated muscles. These findings dramatically advanced our understanding of the neural regulation of muscle fiber plasticity. If the innervation of each mammalian muscle fiber is rigidly restricted to one motor axon, and _if the properties of each muscle fiber are determined by its innervation, and _if these properties are capable of adaptive change, then the transformations in biochemical, physiological and morphological properties of a given muscle fiber must be accompanied by commensurate changes not only in its neuron but in all other muscle fibers of that motor unit as well. Experimental Evidence for the Dynamic State of Muscle Fibers Thus, by 1970, we had come to understand (a) that the nervous system exerted a comprehensive control over the physiological, metabolic and structural properties specific to slow and fast muscles, (b) that this control was achieved by a regulation of the properties of individual motor unit, and (c) that the size of the motor unit was not rigidly specified but could expand or contract. Clearly the key is sue that remained to be resolved was to determine whether such muscle fiber plasticity reflected an ongoing, adaptively-useful, physiological process throughout the lifetime of the animal. The resolution of this question required experiments to elucidate whether comparable transformations in muscle properties could be achieved without surgical disruption of normal neuromuscular relationships.

XXXI T h e first clue came

f r o m e x p e r i m e n t s by V r b o v a

showed that t e n o t o m y p r o d u c e d both a l t e r e d activity patterns and altered contractile hypothesized that

p r o p e r t i e s of t h e m u s c l e hypothesis was

properties.

t h e p a t t e r n of a c t i v i t y

m u s c l e by i t s m o t o r n e r v e d e t e r m i n e s (16).

recognized

the

(15), w h o

electromyographic She

imposed on

the

contractile

The importance

of

this

i m m e d i a t e l y , and its v a l i d i t y

was

e x a m i n e d by a g r e a t m a n y s c i e n t i s t s w h o u t i l i z e d a v a r i e t y experimental

test procedures.

experimental

m o d e l s were the a p p l i c a t i o n

electrical The

Principal

these

patterned

s t i m u l a t i o n of m u s c l e s v i a i m p l a n t e d

electrodes.

r e s u l t s of s u c h s t u d i e s c o n f i r m e d t h a t a c t i v a t i o n

m u s c l e v i a d i r e c t or activity patterns

indirect

resulted

specific physiological fast muscles.

Among

substantiated

and biochemical

the first

r e p o r t s of t h i s p h e n o m e n o n

was

(17) a n d t h e i r

(18-22).

in s h o w i n g

that muscle transformation

or by d i r e c t s t i m u l a t i o n muscles.

The

s t i m u l a t i o n of

innervated

studies utilizing

and compensatory hypertrophy

stimulation

programmed

to e v a l u a t e

of u s e a n d d i s u s e to the a l t e r a t i o n

phenotypes.

The

studies

about muscle

denervated

contribution

r e s u l t s of t h e s e

important

c o u l d be b r o u g h t

of i n n e r v a t e d or of

by o t h e r

were

investigators

studies using patterned electrical

complemented

exercise

results

investigations were also

(neural)

phasic

the and

thereafter

e i t h e r by i n d i r e c t

of m a n y of

p r o p e r t i e s of slow

a n d e x t e n d e d by n u m e r o u s These

of

s t i m u l a t i o n w i t h t o n i c or

in c o n v e r s i o n

t h a t of S a l m o n s a n d V r b o v a

were

among of

of

(23-28)

the of

muscle

confirmed

t h a t c h a n g e s c o m p a r a b l e to t h o s e of c r o s s - r e i n n e r v a t i o n patterned

s t i m u l a t i o n c o u l d be a c h i e v e d

programs

or by p r o c e d u r e s w h i c h e l i c i t e d

by s p e c i f i c

and

exercise

compensatory

hypertrophy. Thus, scientific advances b e t w e e n 1960 and 1976 had

brought

about the following

the

changes

b i o l o g y of t h e m u s c l e

in our u n d e r s t a n d i n g

fiber,

(a) M u s c l e p l a s t i c i t y w a s

to represent d y n a m i c t r a n s f o r m a t i o n s occurring

throughout

life,

of

of m u s c l e

(b) C h a n g e s

properties

in use a n d

p r o v e d t o be a m a j o r m e c h a n i s m u n d e r l y i n g

the

shown

disuse

neural

XXXII

r e g u l a t i o n of m u s c l e p l a s t i c i t y ,

(c) B e c a u s e

physiological, biochemical and morphological

so many p r o p e r t i e s of the

m u s c l e cell are c a p a b l e of e x t e n s i v e t r a n s f o r m a t i o n life, m u s c l e "fiber t y p e s " immutable.

c o u l d no longer be

throughout

considered

(d) Since neural i n f l u e n c e s of v a r i o u s s o r t s are

r e s p o n s i b l e for t r a n s f o r m a t i o n s

in the m u s c l e

fiber's

p h e n o t y p i c a p p e a r a n c e , t h e s e c h a n g e s in m u s c l e

phenotype

r e p r e s e n t a n e u r a l r e g u l a t i o n of gene e x p r e s s i o n

(29).

(e) In

m a n y r e s p e c t s , m u s c l e d i f f e r s but little from other o r g a n s the body

(such as l i v e r , kidney and even b o n e ) , w h o s e

are c a p a b l e of e x t e n s i v e b i o c h e m i c a l adaptations.

and

physiological

H o w e v e r , b e c a u s e of the m u s c l e f i b e r ' s

intimate

r e l a t i o n to the n e r v o u s s y s t e m , a d a p t i v e t r a n s f o r m a t i o n s disseminated throughout each motor M u s c l e F i b e r P l a s t i c i t y as

in

cells

are

unit.

Illustrative

of the D y n a m i c State of B o d y

Constituents

T h a t the m a c r o m o l e c u l a r c o n s t i t u e n t s of all cells e x i s t in a "dynamic s t a t e " w a s a c o n c e p t first e n u n c i a t e d S c h o e n h e i m e r ' s 1942 m o n o g r a p h , Constituents"

in

"The D y n a m i c S t a t e of B o d y

(30), and u p d a t e d in R a t n e r ' s 1979 r e v i e w ,

D y n a m i c S t a t e of B o d y P r o t e i n s "

(31).

c o l l e a g u e s fed rats a diet c o n t a i n i n g a m i n o acids and found, for e x a m p l e ,

"The

S c h o e n h e i m e r a n d his isotopically-labeled t h a t the l a b e l e d

amino

a c i d s r e p l a c e d u n l a b e l e d amino a c i d s in t i s s u e p r o t e i n s . c o n c l u d e d that tissue p r o t e i n s are not inert, as had t h o u g h t , but are c o n t i n u a l l y being d e g r a d e d and

replaced.

S c h o e n h e i m e r d e v e l o p e d the c o n c e p t of t u r n o v e r and t i m e s to d e s c r i b e the c o n t i n u a l macromolecules.

half-life

s y n t h e s i s and d e s t r u c t i o n

The a c c e p t a n c e of these n o t i o n s came

b e c a u s e s c i e n t i s t s f o u n d it d i f f i c u l t to a t t r i b u t e significance

to so e n e r g y - e x p e n s i v e

a process.

They

been

of

slowly

functional

The

e x p l a n a t i o n w a s u l t i m a t e l y p r o v i d e d by S c h i m k e and h i s colleagues

(32) who

showed t h a t the e x t r e m e l y rapid

turnover

of r a t e - l i m i t i n g e n z y m e s p e r m i t s a n i m a l s to adapt p r o m p t l y environmental viscisitudes

(such as a l t e r a t i o n s

n e e d s , h o r m o n e levels or s u b s t r a t e

to

in n u t r i t i o n a l

availability).

XXXIII

Certainly, by 1970 it was widely accepted that cellular proteins of adult animals are being continually degraded and replaced by synthesis at rates characteristic of specific proteins. This concept forms the basis for understanding muscle plasticity, since transformations of muscle fiber phenotype result from changes in the rate of breakdown and synthesis of the cell's macromolecular constituents. Insofar as the kinetics of these processes are neurally determined, such transformations of phenotype reflect a neural regulation of gene expression in the muscle cell (29). This, in essence, is what we mean by the Dynamic State of the Muscle Fiber. During the 1940s and 1950s when the rules governing the dynamic state of proteins and other macromolecules were being elucidated, comparable studies were being carried on at the cellular level by Leblond (33). His studies on the kinetics of renewing cell populations, led to an investigation of muscle and to the discovery of the satellite cell (34,35) and its role in reactive myogenesis in the adult animal. This discovery led to a modification of our views on muscle dynamics. We came to recognize that under normal, steadystate conditions, the ongoing turnover of the muscle cell's constituent proteins is regulated qualitatively by neural influences, whereas under conditions of rapid growth, traumatic injury, and severe mechanical stress, muscle cell modification can also be effected by incorporation of satellite cells. Molecular Mechanisms Regulating the Dynamic State of Muscle Fibers This brings us to the last of the four questions with which we began this review, viz., how do changes in pattern of activation regulate the physiological and biochemical properties of a muscle? At issue is the elucidation of the molecular mechanisms by which changes in transmembrane potential alter gene expression in the muscle cell. In general, changes in membrane potential induce conformational

XXXIV

or other changes in certain transmembrane proteins that alter their specific functions (e.g., ion transport, receptor binding, second messenger activation, or enzymatic activity). The end result is the regulation of the intracellular concentrations of particular ions, hormones, nutrients and various specific metabolites. Precisely how the changes in concentration of these molecules control the rate of transcription of specific genes is a subject that will concern us for many years to come. But we can be confident that we shall succeed in determining how patterned alterations of transmembrane potential can influence the expression of the muscle cell's varied menu of genetic capabilities. From this brief historical review, we have seen that what started out in the 1960s as a seemingly straightforward issue of physiological regulation has proved to be an exceedingly complex problem of molecular biology. Moreover, the issue of the regulation of the dynamic state of muscle fibers cannot be approached, much less resolved, without first addressing the fundamental cell biological question of how changes in transmembrane potential regulate transcriptional, translational and post-translational aspects of gene expression. Perhaps the most important lesson to be learned from this review of the adventures and misadventures of a past generation of scientists is that we would do well in the future to heed the sage advice of R. A. Fisher (36) when designing experiments to elucidate such complex regulatory mechanisms: "No aphorism is more frequently repeated ... than that we must ask Nature few questions, or, ideally, one question, at a time. I am convinced that this view is wholly mistaken. Nature... will best respond to a logically thought out questionnaire; indeed, if we ask her a single question, she will often refuse to answer until some other topic has been discussed."

XXXV

References 1. Buller, A.J., J.C. Eccles and R.M. Eccles. 1960. J. Physiol. 159, 417-439. 2. Eccles, J.C. 1963. In: The Effect of Use and Disuse on Neuromuscular Functions (E. Gutmann and P. Hnik, eds.). Czech. Acad, of Sci., Prague, pp. 111-128. 3. Guth, L. 1968. Physiol. Rev. 48r

645-687.

4. Dubowitz, V. and A.G.E. Pearse. 1960. Histochemie 2, 105-117. 5. Engel, W.K. 1962. Neurology 12, 778-794. 6. Stein, J.M. and H.A. Padykula. 1962. Am. J. Anat. 110, 103-124. 7. Yellin, H. and L. Guth. 1970. Exp. Neurol. 26, 424-432. 8. Brooke, M.H. and K.K. Kaiser. 1970. Arch. Neurol. 23, 369379. 9. Romanul, F.C.A. 1964. Arch. Neurol. 11, 355-368. 10. Weiss, P. and H.B. Hiscoe. 1948. J. Exp. Zool. 107, 315-396. 11. Edds, M.V.,Jr. 1953. Quart. Rev. Biol. 28, 260-276. 12. Edstrom, L. and Kugelberg, E. 1968. J. Neurol., Neurosurg., Psychiat. 3J,, 415-423. 13. Edstrom, L. and Kugelberg, E. 1968. J. Neurol., Neurosurg., Psychiat. 31., 424-433. 14. Burke, R.E., D.N. Levine, P. Tsairis, P. and F.E. Zajac, III. J. Physiol. 1971. 234, 723-748. 15. Vrbova, G. 1963. J. Physiol. 16.6/ 241-250. 16. Vrbova, G. 1963. J. Physiol. 169- 513-526. 17. Salmons, S. and G. Vrbova. 1969. J. Physiol. 201, 535-549. 18. Pette, D., M.E.Smith, H.W.Staudte and G. Vrbova. Pflugers Arch. 338, 257-272.

1973.

19. Lomo, T., R.H. Westgaard and H.A. Dahl. 1974. Proc. Roy. Soc. B. 187, 99-103.

XXXVI

20. L o m o , T. a n d R.H. W e s t g a a r d . 1975. J. P h y s i o l . 603-626.

252,

21. S a l m o n s , S. and F . A . S r e t e r . 1976. N a t u r e 263,

30-34.

22. P e t t e , D . , W . M u l l e r , E. L e i s n e r a n d G. V r b o v a . P f l u g e r s A r c h . 364, 1 0 3 - 1 1 2 .

1976.

23. E d g e r t o n , V . R . , L. G e r c h m a n and R. C a r r o w . 1969. N e u r o l . 24/ 1 1 0 - 1 2 3 .

Exp.

24. G u t m a n n , E., I. Hajek and P. H o r s k y . 1 9 6 9 . J P h y s i o l . 46P-47P. 25. G u t m a n n , E., S. S c h i a f f i n o , a n d V. H a n z l i k o v a . 1 9 7 1 . N e u r o l . 31, 4 5 1 - 4 6 4 .

203, Exp.

26. L e s c h , M . , W . W . P a r m l e y , M. H a m o s h , S. K a u f m a n , a n d E.J. S o n n e n b l i c k . 1969. A m . J. P h y s i o l . 2 1 4 , 6 8 5 - 6 9 0 . 27. G u t h , L. and H. Y e l l i n . 1971. Exp. N e u r o l . 31,

277-300.

28. Guth, L. a n d J.B. W e l l s . 1972. Exp. N e u r o l . 36»

463-471.

29. S a m a h a , F . J . , L. G u t h and R.W. A l b e r s . 1970. E x p . 27, 2 7 6 - 2 8 2 . 30. S c h o e n h e i m e r , R. 1 9 4 2 . The D y n a m i c S t a t e of B o d y Constituents.Harvard University Press, Cambridge, 31. R a t n e r , S. 1979. A n n a l s N . Y . A c a d . S e i . 325,

Neurol.

Mass.

189-209.

32. S c h i m k e , R.T. and D. D o y l e . 1970. A n n . Rev. B i o c h e m . 929-976. 33. L e b l o n d , C . P . , W a l k e r , B.E. 1 9 5 6 . P h y s i o l . Rev. 255-276. 34. M a u r o , A. 1961. J. B i o p h y s . B i o c h e m . A c t a . 9,

30,

493-495.

35. M o s s , F . P . , L e b l o n d , C.P. 1970. J. Cell B i o l . 44/ 36. F i s h e r , R.A. 1926. J. Min. A g r . Gt. B r i t .

39,

459-462.

¿3_, 503.

Section I.

Patterns and Regulation of Gene Expression in Muscle

Myosin Heavy Chain Gene Expression: Interplay of cis and trans factors determines hormonal and tissue specificity Jeffrey N. Rottman, W. Reid Thompson, Bernardo Nadal-Ginard, Vijak Mahdavi Laboratory of Molecular and Cellular Cardiology, Howard Hughes Medical Institute; Department of Cardiology, Children's Hospital; and Department of Pediatrics, Harvard Medical School 300 Longwood Avenue, Boston, Massachusetts 02115, USA

Abstract The myosin heavy chain genes are particularly well-suited as a model system for understanding gene regulation because they show a clearly defined and often reciprocal pattern of developmental, hormonal, and physiological modulation. Gene regulation involves cis and trans factors. The antithetic expression of different myosin heavy chain isoforms in the same cell at the same time clearly implies differences between the genes in cis regulation. The differential pattern of expression of a single gene during development or in response to hormonal or physiological stimuli necessitates a complementary set of trans regulatory factors. We describe a set of cis and trans factors and their interaction in the regulation of myosin heavy chain gene expression. In the initial section we briefly describe the myosin heavy chain genes and their biological regulation. Then by focusing on the gene itself a variety of cis regulatory sequences are described. Most importantly a thyroid responsive element or TRE is characterized; since the nuclear receptors for thyroid hormone have been cloned this regulatory interaction can be examined in detail. The effects of different thyroid receptor isoforms, and of the receptors for the morphogen retinoic acid, which are highly similar to the thyroid receptors, can be evaluated. Finally we turn to inter-species differences in a and 6 MHC gene regulation as another illustration of the interaction of cis and trans regulatory factors. The pattern of MHC expression differs in small and large animals, and this divergence can be analyzed in terms of cis and trans influences. Introduction The contractile state of the myocardium changes in response to developmental, hormonal, physiological, and pathological stimuli. The major contractile proteins, the sarcomeric myosin heavy chains, are encoded by a multigene family, with different protein isoforms manifesting different ATP'ase activity (1). The predominant cardiac isoforms, VI and V3, are homodimers of the a and & myosin heavy chain types, respectively. a-MHC has high ATP'ase activity, fast shortening velocity, and low efficiency of force production; 6-MHC has the opposite attributes. The changes in the contractile state of the heart are mirrored by changes in myosin heavy chain isoform composition.

The Dynamic State of Muscle Fibers © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in Germany

4

Myosin heavy chain expression has a clear developmental pattern (2,3). In the ventricle of small animals fi MHC is the predominant isoform in late fetal life, but is almost completely replaced by a MHC following birth. In large animals, including man, 6 MHC is the predominant isoform in the ventricle in all developmental stages. In both, a MHC is predominantly expressed in the atrium throughout development. Thyroid hormone insufficiency or excess affects MHC isoform composition (2,4). In hyperthyroid rats a MHC expression in the ventricle remains predominant; in hypothyroid rats ventricular a MHC is almost entirely replaced by B MHC. In hyperthyroid humans the situation is less clear, since thyroid excess or deficiency is usually promptly remedied once recognized. However, most likely a MHC expression increases in the ventricle as R MHC expression, normally high, reciprocally decreases. MHC expression in the atria of both species appears less sensitive to thyroid hormone levels. In rats exposed to excessive cardiac afterload, cardiac hypertrophy is associated with a transition from a MHC to B MHC in the ventricle. In the human heart exposed to the comparable stimulus the predominant ventricular MHC isoform remains 6 MHC, and the predominant atrial isoform changes from a to 6 also (1,5). The genes encoding a and & MHC are organized in tandem, spanning 50 kb of the genome in both humans and rats, are highly similar in primary structure, and probably arose by duplication (6). The changes in isoform expression can be completely accounted for by changes in corresponding mRNA transcription. The complex pattern of regulation of the MHC isoforms suggests an interesting interaction of cis and trans acting factors. Results The 5' flanking sequence of the cardiac a MHC gene contains a positive thyroid responsive element Functional analyses of the promoters and 5' upstream sequences were performed for the rat a and 6 MHC genes to characterize the cis regulatory sequences. Reporter construct plasmids bearing different portions of the 5' regulatory region were linked to the CAT (chloramphenicol acetyl transferase) gene and tested for transcriptional activity in transient expression assays. Transfections were performed in primary cardiocytes, skeletal muscle cell lines (fast and slow twitch), and non-muscle cell lines (CV-1 and HeLa). The endogenous a and & MHC genes are expressed and are thyroid responsive in cardiocytes, and the endogenous 6 MHC gene is expressed in the slow twitch (sol8) myogenic cell line. The presence or absence of thyroid responsiveness and of celltype specificity can therefore also be assessed in mapping the functional role of the cis elements which must interact with the responsible trans factors. The nuclear receptors for thyroid hormone have been identified and cloned as the cellular analogues of the viral oncogene v-erb-A, and belong the to "steroidthyroid superfamily" of nuclear regulatory proteins (7-12). At least two classes of

5 these receptors, a and fi, are widely distributed. Expression plasmids for these receptors w e r e co-transfected with the M H C reporter constructs w h e n appropriate, and cells w e r e grown in m e d i u m containing, or depleted of, T 3 (30nM).

Induction of a MHC gene expression by T3 600

500

400

200

100

>

>

is

Construct

Figure 1: Transient expression of a MHC 5' regulatory region constructs in soi8 myotubes. CAT activity expressed as percent of positive control (PSV2-CAT), normalized by internal ßgalactosidase controls for differences in transfection efficiency. Co-transfection are with a null vector in T , depleted serum, or TR a 1 in the presence of 30nM T«.Constructs are named by the number of Base pairs of the 5' regulatory region (preceeding the transcription start site) included, with specific internal elements deleted as noted. Significant induction in the presence of thyroid receptor indicates the presence of a TRE in that construct. Changes in relative level with sequential deletions indicate the presence of positive or negative regulatory elements as described in the text.

6 A panel of sequential deletions showing the basic pattern of expression of the a MHC constructs is illustrated in Figure 1. It is worth examining this figure in detail: a) The construct containing the entire 5' regulatory region to -674 is expressed in sol8 myocytes, but is not expressed in non-myogenic cells (not shown). Thus a sequence responsible for muscle specificity is probably present. The construct also shows several-fold induction on cotransfection with thyroid hormone receptor and T3, suggesting the presence of some sequence also responsible for this. b) When the region from -674 to -369 is deleted the overall level of expression increases about 3-fold, implying that a negative regulatory sequence element has been fully or partially deleted. Thyroid hormone responsiveness is maintained. Similarly when the region from -369 to -194 is deleted expression once again decreases 3-fold, consistent with a positive regulatory element in the deleted region. Another negative element can be seen to lie between -194 and -161. Deletion of this element increases expression, but also allows expression in non-muscle cells and levels comparable to that in muscle cells (not shown). Thus the sequence between -194 and -161 must mediate interaction with some of the trans factors determining tissue specificity. Furthermore deletion of this negative element appears to dramatically enhance the thyroid responsiveness of the remaining construct (see the -674delNRE construct, figure 1). c) Very dramatic thyroid responsiveness is still maintained in the -161 construct. However, this responsiveness is ablated by deletion of the region from -161 to -79. In fact this thyroid responsive element (TRE) can be localized to a 29 BP sequence: 5' GCTGTCCTCCTGTCACCTCCAGAGCCAAG 3' This sequence, which is relatively homologous to the growth hormone TRE, has a striking number of symmetries, near palindromes, and repeats. Such motifs may be important for interaction with the thyroid receptors, which appear to bind as homodimers or heterodimers. When this sequence element is deleted from the 5' regulatory region the resulting constructs are not thyroid responsive (construct -674delTRE in figure 1).

7

Thyroid responsiveness can transferred to be heterologous promoters by this 29 BP sequence, in both muscle and nonmuscle cells. Figure 2 shows co-transfection data with this T R E in the context of the SV-40 alO promoter. The promoter construct itself is not thyroid-responsive, but when the a M H C element is placed immediately before the promoter the resulting construct is upregulated 8-10 fold on cotransfection with thyroid receptor a or thyroid receptor R and T3. This is seen in both muscle and non-muscle cells. The degree of up-regulation produced by this element is quantitatively much greater than that conferred by the native T R E from the growth hormone gene.

The thyroid hormone receptor can also be shown to bind to this region of D N A binding by gel retardation and by avidin-biotin D N A binding (Figure 3). In concordance with the stronger functional effect the avidin-biotin D N A binding assay 5 demonstrates an apparently higher affinity of thyroid receptor a for the a M H C T R E than for other thyroid responsive elements, such as that from the growth hormone gene (13).

The a MHC T R E confers T3 responsiveness to heterologous promoters

• gj

a10-CAT TRE-a10-CAT

Figure 2: a M H C TRE in heterologous promoter context. Co-transfections with a null vector, TR a ^ and TR a ? in so/8 and HeLa cells. Control construct consists o f l h e a10 promoter linked to the CAT reporter. TRE-a10-CAT contains a single copy of the a M H C TRE in front of the a10 promoter. TR a . , but not TR a 2 , boosts transcription of this construct by 10-fold.

Avidin-Biotin Complex DNA Binding Assay

aMHC

GH

Ct MHC

GH

C

Figure 3: A. Tg binding of in vitro translated product, and nuclear extracts from transiently transfected cos cells, by avidin-biotin DNA binding assay, a M H C TRE binds more avidly than growth hormone (GH) TRE; both are significantly above control (C).

8

The 5' regulatory region of the 0 MHC contains a negative TRE Similar regulatory region-CAT reporter constructs were constructed for the rat 6 MHC gene 5' regulatory region (Figure 4). In contrast to a MHC, & exhibits significant down-regulation on co-transfection with either the a and 6 thyroid receptors. Such down-regulation is apparent in both the 667 and 348 6 MHC constructs. Deletions beyond 348 result in insufficient levels of baseline expression to assess whether down-regulation by thyroid hormone receptor is still present. The exact sequence(s) and mechanism responsible for the S MHC thyroidresponsiveness are currently being characterized. Inhibition of

p MHC expression by T3

400 -l

Construct

Figure 4: Transient expression of R MHC reporter constructs in sol8 myotubes. CAT activity expressed as % of positive control, normalized for differences in transfection efficiency by 6-gal internal controls. Constructs are named by number of base pairs in the 5' regulatory region included. Co-transfections are with null vector in T , depleted serum, and TR 6 with Tg. Downregulation with thyroid hormone receptor and T- is seen with -667 and -348 constructs. Level of expression is too low to assess subsequent deletions for thyroid responsiveness.

An alternatively spliced isoform of thyroid receptor a does not bind Tj, and has no transcriptional effects on MHC expression The regulatory effects of the different thyroid receptor isoforms TR a and TR B on the MHC isoforms (MHC a and MHC 6) were evaluated. Both TR a and TR B up-regulate a MHC gene expression; both thyroid receptors also downregulate 6 MHC gene expression. TR a appears to be slightly more effective at upregulation than is TR B, but TR 6 appears to be slightly more potent at down-

9

regulation. These differences are not particularly marked when receptors from the same species are used. A clear functional difference underlying the existence of multiple thyroid receptor isoforms still remains to be defined. A much more dramatic difference is seen between two variants of the thyroid receptor a produced by alternative splicing. A lambda gtll cDNA library from adult rat ventricular RNA was screened using a fragment of the v-erb-A gene, and three positive clones were sequenced. These clones represented three distinct mRNA's with the coding capacity for two different proteins (13,14). One protein essentially corresponded to the previously described TR a receptor, and for greater specificity will be denoted TR a j in the remainder of this section. The other protein, of 492 amino acids, was identical in the first 370 amino acids to TR ai, including the DNA binding domain, but then diverged. This clone was denoted TR 0=2. Genomic hybridization to a single locus was consistent with alternative splicing as the generative mechanism. The in vitro reticulocyte lysate translation product of TR a^ did not bind T3. Transient transfection of cells with an expression plasmid for TR 02 also did not result in the 40- to 80- fold (corrected) increase in T j binding that was produced by TR a (see figure 3). Co-transfection of the expression plasmid for TR 02 with a MHC regulatory constructs containing the TRE also failed to produce significant additional T3 dependent or independent transcriptional stimulation (see figure 2). These observations are one of the few known examples of dramatic differences in the functional properties of a transcriptional factor produced by alternative splicing. Such modification of trans factors exponentially increases the capabilities and complexities of such a system of gene regulation. Retinoic acid also affects the expression of a and fl MHC regulatory region expression constructs. The nuclear receptors for the morphogen retinoic acid have been identified and cloned (15-17) and belong to the same steroid/thyroid receptor superfamily as the receptors for thyroid hormone. Receptor isoforms a and 6 appear to be widely distributed, and a receptor isoform t is localized to the skin. The DNA binding domain of the retinoic acid receptors (RAR's) is considerably homologous to the corresponding domain of the thyroid receptors (for example, 62% amino acid homology between rat retinoic acid receptor a and rat thyroid receptor 6). A triple modified (palindromic) copy of the growth hormone TRE is capable of conferring up-regulation by the retinoic acid receptor a (18,19). We examined whether the a MHC TRE can also function as a retinoic acid responsive element. The heart contains mRNA for the RAR receptors (Figure 5), and the important cardiac teratogenicity of excess retinoic acid suggests a biologically important role for these receptors.

10 Figure 5: Northern blot of polyA cardiac tissue for thyroid and retinoic acid receptors. Specific thyroid probes, non-specific retinoic acid probes; high stringency washes. Both classes of receptors appear to be present in atria and ventricles.

Accumulation of TR and RAR m R N A s in Human Myocardium ^ • ^ V

/

V

•

. •*>». -18S

TRii

TR|3

RARa

R A R (5

Retinoic acid receptor a, in the presence of retinoic acid (5xlO"^M to H T M ) , can up-regulate reporter constructs containing the a MHC regulatory region. This effect is concordant in direction with that produced by either TR a or TR 6, but smaller in magnitude (figure 6a). When the a MHC T R E is placed in the viral alO or thymidine kinase (TK) contexts the resulting constructs are also upregulated by retinoic acid receptor a in both myogenic (figure 7a) and nonmyogenic (figure 7b) cells. The up-regulation conferred by retinoic acid receptor a on the a MHC constructs is also ablated if the TRE is deleted (figure 6b). Thus the a M H C TRE can also function as a retinoic acid responsive element in muscle and non-muscle cells. a MHC -367

a MHC •367

c o

o

Ol > o c o o T3

< >5 o c o o

A TRE

•

Control + RA R A R a + RA

m

RAR (3 + RA TR a + T3

3 T3 C

o u.

Di

m

Li

Figure 6: Transient co-transfections in sol8 myotubes of a MHC constructs with thyroid and retinoic acid receptors. A. -367 a MHC. B. -367 a MHC with TRE deleted. Average of multiple experiments, corrected for transfection efficiency and normalized for fold induction over control (null) co-transfection. RAR a and TR a can both up-regulate an a M H C construct with intact TRE; this up-regulation is lost with deletion of the TRE. Retinoic acid alone (+RA) or with RAR 6 results in down-regulation of constructs with and without the TRE. RAR a and TR a also downregulate the TRE-deleted construct.

11 So!8

myogenic cells

(xTRE-TK

non-myogenic cells

CV-1

(X TRE - pal0

aTRE-TK

aTRE-palO

C

o o 3 T3 C

o

I

|

Control RAR a + RA

2 3 RARß + RA rimi TR a + T3

Figure 7: Transient co-transfections of the a MHC TRE in heterologous promoter (TK and a10) contexts with thyroid and retinoic acid receptors. A. so/8 myotubes. B. CV-1 cells. Average of multiple experiments, normalized and corrected for transfection efficiency. RAR a and TR a, but not RAR 6 , result in noticeable up-regulation of TRE constructs in sol8 and CV-1 cells.

The retinoic acid receptors a and 6 in the presence of retinoic acid can also down-regulate B MHC constructs (figure 8). Determination of the particular cis sequences essential for this downregulation awaits the definition of the 6 MHC thyroid responsive element.

ß MHC expression in sol8 cells 100%

•

Control

•

+RA

§ •

RAR a + RA RAR ß + RA

•

TR a + T3

50%

Q

CL 0% Figure 8: Transient expression 6 MHC construct (-667) in sol8 cells, co-transfected with retinoic acid and thyroid receptors. RAR a, RAR B, and TR 6 all result in down-regulation.

In sol8 but not CV-1 cells down-regulation of reporter constructs bearing the a MHC promoter and 5' regulatory region is produced by RA alone and by RAR 6 (with RA, figure 6b). This effect is not transferred by the isolated TRE, and is still present in those constructs in which the TRE has been eliminated (figures 6b,7a,7b). We are currently determining the sequence element responsible for this effect. Such an effect could result directly from an interaction of a retinoic acid receptor with the promoter, some other sequence element, or, for example, by regulation of, or dimer formation with, another regulatory factor. The presence of

12

this effect in a MHC constructs without the TRE suggests that this effect is not simply due to interference with endogenous thyroid receptors. Comparison of human and rat a and B MHC regulatory regions We have also investigated the differences between the human and rat a and 6 MHC genes to better elucidate the importance of cis and trans factors. If corresponding reporter constructs from the two species contain an important cis difference then they may show different patterns of expression or regulation when transfected into the same cell types. These differences would not be present if the divergent pattern of regulation of the a and 15 MHC genes between rat and man (for example, the replacement of B MHC by a MHC as the predominant ventricular species in small but not large animals) is instead due to differences in trans factors. Human a MHC

Rati MHC 15

Human (MHC

Rat ¡MHC

Carfa|1es

Cardiocyies

10

li

II • • •I

Ml

Soli

15 10

5

tf V

CP

IV

Regulatory construct (from initiation site)

Figure 9: Comparison of transient expression assays, human and rat a and 6 MHC constructs in cardiocytes and myotubes. A. a MHC. B. 8 MHC. Ordinate gives number of base pairs of regulatory region in construct. Corrected for transfection efficiency and normalized to a positive control. The general pattern of positive and negative elements is similar, although the parallel constructs differ in exact number of base pairs.

13

Using the first coding exon of the rat a and B MHC genes the corresponding human a and 0 genomic clones were isolated (20). The 5' regulatory regions of the rat a and rat & MHC genes are not significantly homologous. However the 5' regulatory regions of the rat a MHC gene and 5' regulatory region of the human a MHC gene show 80% nucleotide identity, and the corresponding rat and human regulatory regions of the & MHC gene show 82% regulatory identity. Expression plasmids for the human a and 6 MHC gene regulatory regions were constructed, and functional studies paralleling those for the rat constructs were performed. The pattern of expression between the two species is very similar, with positive elements in the human a sequence at positions -536 to -379 and -199 to -74 (figure 9a). For the 6 human regulatory region a positive element occurs between -357 and -248 (figure 9b).

Rat B MHC

Human B MHC < o c\j >

300

• •

T-/TRT+/TR+

300

• •

T-/TRT+/TR+

CO

Q.

200

200

100

100

>

o

CO




MCA-Myf-5/2

c

>

( +) + +

n.d.

a

) conversion w i t h b) conversion w i t h conversion w i t h d) conversion with

pEMSV-Myf-4/Sst pEMSV-Myf-5/18 pEMSV-Myf-5 pEMSV-MyoDl

+ indicates abundant or moderate mRNA levels, (+) barely detectable mRNA levels and n.d. was not determined Mvf

5 fusion

protein

produced

in E.coli

binds

to

regulatory

sequence elements present in muscle specific genes. The

observation

nucleus

and

typical

for

capacity specific Myf

cDNAs

proteins for

that

contain DNA

for

a

proteins proteins,

autoactivation

were

binding

mobility-shift

to

assay. were

sequence

together

prompted

as

us

to

in

the

which

with

is

their

search

glutathione-transferase

and purified

synthetic

oligo-nucleotides

localized

for

of Myf proteins w i t h DNA. To this end,

expressed

E.coli

are

helix-loop-helix

binding

interactions in

Myf

The

DNA

hybrid

from

sequences

muscle

fusion

were

oligonucleotides

nucleotide

selected

proteins

tested

in

a of

specific

gel the genes

28 according to a consensus sequence which was

first

for

enhancer

factor

binding

to

the

muscle-specific

postulated of

mouse M - C P K gene (13). More recently, this sequence was to

bind

MyoDl

computer MLClemb which

which

assisted genes

are

enhancer

was

revealed

located

sequence

synthesized

search

the bin-

the

human

and

in

in

bacteria

human

MyoDl

in (15)

of

ding

the

(14).

MLC1/3

and

consensus

MLC1/3 MLClem^

the

shown A the

sequences

muscle-specific promoter

region

(unpublished). As shown in fig.7, MY! 4 !ngx10> Myl 5 (ugxio: ' 1 3 5 8 13 5 8*13 5 8 ! 3 5 ti MLC-enh MLC-Mut ' MlC-enh " MlC-Mut

1 2 3 4 5 6 7 8 9 » H B U 14 K IK Figure 7: Gel mobility shift assay with human Myf-4 and Myf-5 glutathione transferase (GS) fusion proteins. The synthetic MLC1/3 enhancer sequence MLC-enh (AAGTAACAGCAGGTGCAAAATAAAGT) or the mutated version MLC-Mut (AAGTAAGTAACTGTGCAAAATAAAGT) were radiolabelled and the ds oligonucleotides were incubated w i t h the indicated amounts of affinity purified GS-fusion protein (17). the forms

oligonucleotide complexes

complex Myf

protein

with

formation

proteins

representing

but

its

concentration.

cooperativity

for

Myf-4

appears

and

Myf-5

of

the

fusion

MLC-ei|hancer proteins.

The

to be dependent on the presence of

increase This

binding

part

is

not

observation

exists,

colinear could

probably

due

with

suggest to

the that

dimeri-

29 zation of for tide was

the Myf

binding

MLC-Mut, used

for

binding. complex

mutant

(MyoDl,

ces,

however,

complexes

data

the

the

exhibit

results

all,

sequence,

only

a

weak

fold)

with

obtained

with

10

were

unrelated

binding

at

oligonucleo-

consensus

Completely

API

proteins

selectivity

the

(approximately

shown).

as

Myf

of

and Myf-4

Similar

not

such

with

version

Myf-5

moderate

obvious w h e n

formation

substrate.

Myf-3

The merely

becomes

a mutated

reduction of the

proteins.

sequences

site

even

did

when

sequennot

form

very

high

concentrations of proteins were used (data not shown). Transient

expression

of Mvf

proteins

in non-muscle cells

can

activate cotransfected muscle specific promoters. Lassar

et

cardiac into

al.

actin

10T1/2

have

previously

promoter

is

fibroblasts

5-azacytidine-derived

shown

not

but

that

the chicken

transcribed is

myoblasts

when

expressed (4).

To

alpha

transfected

and

regulated

in

whether

the

assess

activation of this muscle-specific promoter could be mediated by the myogenic determination factors, cotransfection

of

the

cardiac

actin

chloramphenicol-acetyltransferase with

pEMSV

promoter

gene

(16)

Myf-3,

transient

joined in

to

the

combination Myf-5 alpha

transfected

for

the

and

gene was

vehicles

used

into 10T1/2 cells. As shown in fig.8, w h e n the reporter actin CAT

expression

we

alone

or

Myf-4,

in combination

with

RSV-neo or pEMSV alpha-scribe used as control plasmids, no or very

low CAT

activity was obtained.

In contrast, w h e n

either

of the 3 Myf expression vehicles was

contransfected

with

reporter

activity

observed.

Similar hancer

plasmid, activation CAT

constructs the

considerable

SV40,

was

CAT

achieved

with

the

chicken

MLC

(data

not

shown).

Non-muscle

the TK,

or

2A

the

and

was

human

promoter

MLC1/3

CAT

promoters,

the fi-actin promoters,

en-

reporter such

were either

activated at all or only very moderately. We have

the

not

as not

obser-

ved any substantial differences

in the transactivation of the

various

genes.

muscle-specific

target

A

similar

degree

of

30

transactivation three Myf

was

achieved

factors was

irrespective

supplied

of

which

of

the

in trans. The activation of

muscle specific promoters by the Myf proteins was not restricted

to

10T1/2 fibroblast but was also seen in CV1, NIH 3T3

and primary chicken fibroblast cells. We conclude from these results that Myf proteins, presumably by direct with

specific

transcription

sequences

in

muscle

genes,

interaction

can

activate

of a selected set of promoters thereby adding

to the establishment of the myogenic phenotype

in committed

cells.

I t I

; > H s

I 5 » S

I ? 6 S

I I £ n a

5 a £

« actmCAT

Ac CM

A ™

#

(gk

4fc

Figure 8: Muscle-specific promoter activation by transient expression of pEMSV-Myf proteins in 10T1/2 fibroblasts. The chicken cardiac alpha-actin CAT reporter construct (16) was contransfected with control plasmids RSVneo (lane 1) and pEMSV scribe (lane 5) or with pEMSV expression vehicles for Myf-3 (lane 2), Myf-4 (lane 3), and Myf-5 (lane 4). The B-actin CAT construct (18) was used to control the transfection efficiency.

CM

Conclusions Several myogenic factor genes related to the myc super-gene family are expressed in human skeletal muscle. All Myf proteins contain the amphipathic helix-loop-helix structure which presumably is important for DNA binding and/or gene

31

activation. in

Constitutive

mesodermally

germ-layers cells. specific genous however,

derived

results

During Myf

such

genes

is

in

this

genes

expression cells the

transition

become

criptional

A

possible

activation

direct

regulatory binding

DNA

sequences

in cells

from

not

only

and actin

Their

controlled

in

mode

of

action

also

Myf

other muscle

muscle-

the

endo-

autoregulation, cellular

different involves

muscle-specific

of

cDNAs

to

typical

but

activated.

of

interaction

Myf

conversion

suggested by co-transfection experiments The

individual

also

phenotypic

as myosin

differentially

backgrounds.

but

of

the

trans-

promoters

in non-muscle

proteins

with

can be demonstrated

by

as

cells.

specific

"in

vitro"-

assays.

Acknowledgements We

like to thank C. Mink, without her help it w o u l d not have

been

possible

Paterson

for

to produce

this

kindly

providing

struct. The w o r k was

supported

manuscript. the

We

cardiac

by Deutsche

also actin

thank CAT

B.

con-

Forschungsgemein-

schaft and Deutsche Muskelschwundhilfe e.V.

References 1.

Taylor, S.M., and Jones, P.A. 1979. Cell 17, 771-779

2.

Jones, P.A. and Taylor, S.M. 1980. Cell 20, 85-93

3.

Konieczny, S.F., and Emerson, C.P.J. 1984. Cell, 38. 791-800.

4.

Lassar, A.B., Paterson, B.M., and Weintraub, H. 1986. Cell £2, 179-184

5.

Pinney, D.F., Pearson-White, S.M., Konieczny, S.F., Latham, K.E., and Emerson, Jr., C.P. 1988. Cell 51, 781-793

32 6.

Davis, R.L., Weintraub, H., and Lassar, A.B. 1987. Cell 51, 987-1000

7.

Weintraub, H., Tapscott, S., Davis, R., Thayer, M., Adam, M., Lassar, A.B., and Miller, A.D. 1989. Proc.Natl.Acad. Sei.USA 86, 5434-5438

8.

Wright, W.E., Sassoon, D.A., and Lin, V.K. 1989. Cell 56. 607-617

9.

Edmondson, D.G., and Olson, E.N. 1989. Genes & Develop. 3, 628-640

10. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E., and Arnold, H.H. 1989. EMBO J. 701-709 11. Tapscott, S.J., Davis, R.L., Thayer, M.J., Cheng, P.F., Weintraub, H., and Lassar, A.B. 1988. Science 242. 405-411 12. Braun, T., Bober, E., Buschhausen-Denker, G., and Arnold, H.H. 1989. EMBO J. 8, 3617-3625 13. Buskin, J.N., and Hauschka, S.D. 1989. Mol.Cell.Biol. 9, 2627-2640 14. Lassar, A.B., Buskin, J.N., Lockshon, D., Davis, R.L., Apone, S., Hauschka, S.D., and Weintraub, H. 1989. Cell M , 823-831 15. Seidel, U. and Arnold, H.H. 1989. J.Biol.Chem. 264. 1610916118 16. Quitschke, W.W., dePonti-Zilli, L., Lin, Z.-Y., and Paterson, B.M. 1989. Mol.Cell.Biol. 9, 3218-3230 17. Smith, D.B., and Johnson, K.S. 1988. Gene 67, 31-40 18. Lohse, P. and Arnold, H.H. 1988. Nucleic Acids Res. 16, 2787-2803 19. Thisse, B., Stoetzel, C., Garostiza-Thisse, C., and Perrin-Schmitt, F. 1988. EMBO J. 7, 2175-2185 20. Villares, R., and Cabrera, C.V. 1987. Cell 50, 4151-424 21. Cronmiller, C.R., Schedl, P., and Cline, T.W. 1988. Genes and Dev. 2, 1666-1676 22. Murre, C., Schonleber-McCaw, P., and Baltimore, D. 1989. Cell 56, 777-783 23. Caudy, M., Vaessin, H., Brand, M., Tuma, R., Yan, L.H., and Yan, Y.N. 1988. Cell 55, 1061-1067

Regulatory elements involved in chicken myosin alkali light chain gene expression

Yo-ichi Nabeshima, Taichi Uetsuki, Thorn Komiya, Yoko Nabeshima and Atsuko Fujisawa-Sehara Division of Molecular Biology, National Institute of Neuroscience Ogawahiashi-cho 4-1-1, Kodaira-shi, Tokyo 187, Japan Introduction Myosin alkali light chain (MLC) isoforms are encoded in a multigene family, the expression of which is developmentally regulated in a tissue specific manner. To study how the transcription of skeletal muscle MLC gene is dramatically increased and continuously expressed in skeletal muscle myotubes, and how cardiac muscle MLC and embryonic MLC (L23) genes are transiently expressed during myogenesis (1), we have analyzed the functions of 5' upstream regions of the three genes mentioned above, using transfection methods of fusing genes into primary cultured muscle cells. Here, we have identified three cis-acting regions, one of which is an enhancer sequence located about 2kb upstream from the transcriptional initiation site of the skeletal muscle MLClf gene and second of which is a common element essential for the expression of MLC genes at approximately 100 bp upstream from mRNA start sites of the above three MLC genes and the third is suppressor like elements which may correspond to the transient expression of cardiac MLC1 and L23 genes. The common cis-acting element is found to be highly conserved in the promoter region of MLC genes of chicken, mouse and rat. Therefore, we designate this element as "MLC box". We have also analyzed the nuclear protein(s) binding to the MLC box and the enhancer sequence and have revealed that the binding proteins of MLC box, CArG box and SRE of c-fos are indistinguishable. Results MLC box was identified by 5' deletion mutation analysis of L23 gene To analyze the cis-acting elements of L23 gene (1), we have constructed a fusion gene containing the 3.7

kb 5' upstream region of the L23 gene linked to bacterial

chloramphenicol acetyltransferase (CAT) gene (pCD3.7) and introduced it into the primary cultured cells prepared from the breast muscles of 11 day chick embryos. We detected sufficient CAT activity from pCD3.7 compared with the pMLCAT which lacks

The Dynamic State of Muscle Fibers © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in Germany

34

Fig. 1 Deletion analysis of 5' upstream region of L23 gene (a)

mRNA -3.7kb

t-20bp

1.23 Gene

C A T gene

+1

(b)

Primary cultured muscle cells

pSV2CAT pMLCAT

-3.7kb

-2.7kb

-l.5kb

-375bp

-80bp

Primary cultured fibroblast cells

pSV2CAT

pMLCAT

-3.7kb

-2.7kb

1.5kb

-375bp

-80bp

the promoter Jinked to CAT gene and then we also confirmed by the primer extension analysis that the start site originating from pCD3.7 was identical to the initiation site of L23 mRNA in vivo. To define the cis-acting element in this region which regulates the expression of L23 gene, we prepared a series of deletion mutants and introduced them into primary cultured muscle cells. As shown in Fig.l, the rate of transcription gradually increased as the 5' upstream region became shorter. Furthermore, we confirmed that the increased expression of fusion gene was not the effects of read through transcripts by primer extension analysis. Therefore, there may be two cis-acting regulatory elements, one of which is a suppressor -like element between -3.7 kb and -375 bp (Fig.2) Fig.2

-3.7kb

Upstream

1 kb region suppresses

the activity

of L23

promoter

(c)

-2.7kb

(d)

CAT

L23 promoter

SV40 spHcefeplice

(-128bp)

* if JC pCD-128

m

O

o Z

q;

LJ Q_

2 0

--

I

O UJ

cc in -20 -L

SERUM + MEDIUM

S E R U M - F R E E MEDIUM

Figure 8. Effects of medium on stretch-induced cell growth. Muscle cultures 6 to 7 days postplating are rinsed 3 times rapidly and then incubated for 2 h at 3 7°C with serum-free medium. Fresh medium is added ± 10% serum, 5% embryo extract and mechanical stimulation (TRIAL39.PGM) initiated for 2 to 4 days with medium changes every 24 h. Percent stretch-induced increase or decrease are relative to time-matched static controls. (Adapted from references 31,32).

162 stretch-induced

hypertrophy

in u n s u p p l e m e n t e d

medium

is

due

t o a 55% r e d u c t i o n in s t r e t c h - s t i m u l a t e d p r o t e i n s y n t h e s i s (31) and a

stretch-induced

increase

in t o t a l

protein

degradation

r a t e s (Figure 8) . M e c h a n i c a l l y - s t i m u l a t e d m y o t u b e h y p e r t r o p h y in

vitro

is

thus

independent

tetrodotoxin-insensitive)

of

but

electrical

dependent

activity

on

medium

(i.e. growth

f a c t o r s p r e s e n t in s e r u m a n d / o r e m b r y o e x t r a c t . S t r e t c h - i n d u c e d muscle

growth

hormones

in v i v o

(36).

The

can also

medium

be modulated

growth

factors

by

circulating

which

apparently

interact synergistically with mechanical activity to stimulate m u s c l e c e l l g r o w t h in v i t r o a r e n o t k n o w b u t i n s u l i n , like

growth

current In

factors,

and

testosterone

are

insulin-

candidates

summary,

mechanically

two

models

stimulating

systems cultured

are

now

skeletal

available muscle

cells

t e n s i l e f o r c e s w h i c h c a n s i m u l a t e s o m e of t h e m e c h a n i c a l found cells,

under

investigation.

in

vivo.

can

These

induce

forces, muscle

when cell

adaptations, and muscle hypertrophy. supplement

other

model

systems

in

applied

to

the

organogenesis,

for by

forces

cultured metabolic

The model systems should understanding

transduction processes at the molecular

mechanical

level.

T h i s w o r k is s u p p o r t e d b y g r a n t s from N I H a n d N A S A .

References 1.

S t e w a r t , D.M. 1972. In: R e g u l a t i o n o f O r g a n a n d T i s s u e G r o w t h . (R.J. G o s s , ed). A . P . , N e w Y o r k p p . 7 7 - 1 0 0 .

2.

W e i s s , P. 1933. A m e r . N a t u r a l i s t 118,

3.

W i l l i a m s , P.E. a n d G. G o l d s p i n k . 751-767.

389-407.

1971. J. Cell S c i . 9,

163

4.

Goldberg, A.L., J.D. Etlinger, D.F. Goldspink, and C. Jablecki. 1975. Med. Sci. Sports 2, 248-261.

5.

Vandenburgh, H.H. 1987. Med. Sci. Sports Exer. 19, S142 S149.

6.

Jolesz, F. and F.A. Sreter. 1981. Ann. Rev. Physiol. 43, 31-52.

7.

Felig, P. 1983. In: Frontiers of Exercise Biology. ( K.T. Borer, D.W. Edington, and T.P. White, eds.). Human Kinetics, IL.

8.

Barnes, W.S. 1987. Comp. Biochem. Physiol. 86, A229-232.

9.

Feng, T.P. 1932. J. Physiol.

(Lond.) 24, 441-454.

10. Ivy, J.L. and J.O. Holloszy. 1981. Am. J. Physiol. 241. C200-C203. 11. Palmer, R.M., P.J. Reeds, G.E. Lobby, and R.H. Smith. 1981. Biol. J. 198, 491-498. 12. Periasamy, M., P. Gregory, B.J. Martin, and W.S. Stirewalt. 1989. Biochem J 252, 691-698. 13. Florini, J.R. and K.A. Magri. 1989. Am. J. Physiol. 256. C701-C711. 14. Vandenburgh, H.H. 1988. In Vitro 24, 609-619. 15. Vandenburgh, H.H. and P. Karlisch. 1989. In Vitro 25. 607-616. 16. Vandenburgh, H.H. and S. Kaufman. 1979. Science 203. 265-268. 17. Vandenburgh, H.H. and S. Kaufman. 1980. In: Plasticity of Muscle. (D. Pette, ed) . Walter de Gruyter, Berlin. 493-506. 18. Vandenburgh, H.H. and S. Kaufman. 1981. J. Cell 109. 205-214.

Physiol.

19. Vandenburgh, H.H. 1983. J. Cell. Physiol. 116, 363-371. 20. Vandenburgh, H.H., P. Karlisch, and L. Farr. 1988. In Vitro 24, 166-174. 21. Hatfaludy, S., J. Shansky, and H.H. Vandenburgh. 1989. Am. J. Physiol. 256, C175-C181.

22. Yoshizato, K., T. Obinata, H. Huang, R. Matsuda, N. Shioya, and T. Miyata. 1981. Dev. Growth Diff. 23, 175-184. 23. Chiquet, M., E.C. Puri, and D.C. Turner. 1979. J. of Biol. Chem. 254, 5475-5482. 24. Vandenburgh, H.H. 1982. Develop.Biol 93, 438-443. 25. Vandenburgh, H.H. 1989., In Preparation. 26. Kennedy, J.M., S. Kamel, W.W. Tambone, G. Vrbova, and R. Zak. 1986. J. Cell Biology 103. 977-983. 27. Tomanek, R.J. 1975. Dev. Biol 42, 305-314. 28. Goldspink, D.F. 1977. J.Physiol. 264. 267-282. 29. Holly, R.G., J.G. Barnett, C.R. Ashmore, R.G. Taylor, and P.A. Mole. 1980. Am. J. Physiol. 238, C62-C71. 30. Sola, D.M. and A.W. Martin. 1953. Am. J. Physiol. 172. 325-332. 31. Vandenburgh, H.H., S. Hatfaludy, and J. Shansky. 1989. Am. J. Physiol. 256, C674-C682. 32. Vandenburgh, H.H., S. Hatfaludy, I. Sohar, and J. Shansky. 1989. Am. J. Physiol., In Revision. 33. King, S.W., B.E. Statland, and J. Savory. 1976. Clin. Chim. Acta 72, 211-218. 34. Hamosh, M., M. Lesch, J. Baron, and S. Kaufman. 1967. Science 157, 935-937. 35. Darr, K.C. and E. Schultz. J. Appl. Physiol. 63, 1816-1821. 36. Mackova, E.V. and P. Hnik. 1976. Physiol. Bohemoslovaca 25, 325-332.

ISOFORMS

Gurtej

OF

K.

TROPONIN

COMPONENTS

of

Basic

University

of

London,

troponin

troponin

and

These

characterise

and

muscle.

Troponin

classes

of

(2).

of

and

cardiac

studied

T by

difficult several

on is

of

to

the

gel

studies can

troponin

T but

variants

within

small each

antibodies

available

skeletal

to

exist

in

striated

are

usually

skeletal

three

cardiac by

in

have

with

in

far.

of

adult

less

size

and

it h a s

mobility.

the

In

classes

closely

the

be

as

different

major

always

can

been

certainty

of

muscle

extensive.

with

three

not

skeletal

similar

antibodies the

but

muscles

demonstrated

been

molecular

are

alternative

both

T composition have

These

differentially

T generated

studies

and

main

muscle.

and

described

variants

so

in

skeletal

skeletal

small

are

fast

adult

slow

differences class

I exists

skeletal,

electrophoresis

using

the

fast

muscle

distinguish

in

in

These

the

the

which

Troponin

proteins,

in

been

migrate

of

OTU.

C

troponin

all

proteins

NW1

different

troponin

have

College,

regulatory

I-filament

slow

slow

relatively

identify

other

Immunochemical

the

FIBRES

London,

isoforms

adult

developing

standard

specificities

of

of

in

and

(3-11).

in

the

the

shown

genes

three

Troponin

but

skeletal,

variants

of

of

normal

mechanisms

complexity

Troponin

MUSCLE

Veterinary

Street,

types.

(1).

been

in

muscles

studies

muscle

T has

fast

Multiple

in

exist

similar

different

in

splicing

but

located

types

is

isoforms

RNA

great

C,

Royal

College

composed

proteins

muscle

muscles

expressed

is

The

characteristic

cardiac

products

Royal

different

isoforms

cardiac

Sciences,

complex

I, T

myofibril.

three

DEVELOPING

Dhoot

Department

The

IN

related

apparent present

The Dynamic State of Muscle Fibers © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in Germany

of

with

most

study,

166 an i m m u n o c h e m i c a l distribution

of

investigation

troponin

was u n d e r t a k e n

T and t r o p o n i n

prepared whole

homogenates

immunoblotting

and i m m u n o c y t o c h e m i c a l

concluded

the m a j o r

chicken nor of

that

embryonic

the a d u l t

the

against

immunoblotting

to a d u l t

slow

predominate muscles

both Dr.

J.

42/25

all

and c a r d i a c breast

T of

fast

the

muscle

muscle

has

react with

Using

troponin

development isoform

of

and

a number

protein. I

similar to

these

during

T purified for

staining

of

from c h i c k e n

late

T (11).

cross

sections

of

T in

troponin

those

Unlike

antibody

either

of

the

reacted with

Tl/7,

to

troponin

both

to

the c h i c k e n

however,

specific

fast

T purified

the a d u l t

in

raised

provided

by

for (13).

raised

to

m u s c l e was f o u n d muscle with

type F24

adult

Tl/7 of

I

to

be

immunoperoxidase

antibody

isoforms for

Tl/7

muscles

F24,

antibody

by a n t i b o d y

two c a r d i a c

thus

CDC4 r a i s e d

stained

The

restricted

variants as

Antibody

skeletal

analysis,

muscle

troponin

T (kindly

breast

troponin

reaction

of

and c a r d i a c

fast

showed i t s

and was

(12).

designated

I n an i m m u n o b l o t t i n g of

isoforms

shown t o be s p e c i f i c

(8)

antibody,

specific

C)

early

h o w e v e r , was f o u n d

troponin

been

skeletal

monoclonal

troponin

F24

was

the

the c a r d i a c

this

by

antibodies

M. W i l k i n s o n )

troponin

type,

muscle-like

recognises

chicken

Another

not of

an i s o f o r m o f

embryonic

skeletal

of

skeletal

against

early

did

freshy

chicken

It

T in

neither

isoforms

muscle

in

of

procedures.

troponin

is

it

isoforms muscles

the

development.

Specificities Antibody

as

procedure,

during

and f a s t

embryonic

type

skeletal

of

muscle

adult

I

developing

isoform

skeletal

skeletal

antibodies

of

to study

II

fibres

stained fast

(Fig. F24

same

skeletal not

this

protein

skeletal

muscle.

from a d u l t

(Fig.l). the

3A & C ) .

did

and d e v e l o p m e n t a l

antibody

react (Fig.

with 3A &

Antibody

chicken isoforms

heart of

167

F i g u r e 1. S e r i a l s e c t i o n s o f r e d p o r t i o n o f g a s t r o c n e m i u s m u s c l e f r o m a d u l t c h i c k e n s t a i n e d w i t h a n t i b o d i e s CDC4 f o r s l o w t r o p o n i n T ( a ) , F24 f o r f a s t t r o p o n i n T ( b ) and LM5 f o r f a s t m y o s i n h e a v y c h a i n s ( c ) by t h e i m m u n o p e r o x i d a s e p r o c e d u r e . A n t i b o d y CDC4 s t a i n s t y p e I f i b r e s w h i l e a n t i b o d i e s F24 and LM5 s t a i n t h e t y p e I I f i b r e s . cardiac

troponin

troponin

T (14).

T present

in

by t h e

isoforms type

I

fibres

electrophoretic

LM5 r e c o g n i s e s

all

isoforms

of

heavy

myosin

(Fig.

the a d u l t

Antibody

fast

could

procedure in

antibody

skeletal

and t h e r e f o r e

immunoblotting

stained

addition,

the slow

i s o f o r m had much f a s t e r cardiac

In

the

muscle. mobility

easily

chain.

be

4 & 5).

skeletal

adult

CDC4

and

The

recognised slow

than

the

distinguished Antibody

muscle

CDC4

(Fig.

developmental

la).

168

I

J •Slow "Fast

F i g u r e 2. I d e n t i f i c a t i o n of f a s t and slow i s o f o r m s of t r o p o n i n I i n d e v e l o p i n g c h i c k e n p e c t o r a l muscle u s i n g a n t i b o d y 42/25 by t h e i m m u n o b l o t t i n g p r o c e d u r e . Samples: a - f = p e c t o r a l muscle f r o m p o s t h a t c h s t a g e s 50, 30, 6 , 3 and 1 week, g - k = 20, 17, 14, 12 and 11 day i n o v o . Distribution

of

troponin

I in developing chicken

muscle

of

troponin

I i n t h e c h i c k e n embryonic

fibres. The i s o f o r m s

muscles i n t h e p r e s e n t technique

of

s t u d y were i n v e s t i g a t e d

immunoblotting.

Both f a s t

I were d e t e c t e d d u r i n g embryonic

skeletal

muscle.

Although

this

t h e samples of

the

and slow i s o f o r m s

troponin

quantitative,

by

skeletal

technique

development is

not

slow s k e l e t a l

s t a g e s b u t t h e predominance of

I during l a t e r

development

Distribution striated

(Fig.

of

muscle-like

muscles

during early troponin

s t a g e s of embryonic

isoform

and p o s t

fast hatch

2).

isoforms

of t r o p o n i n T i n t h e a d u l t

chicken

muscle

A l a r g e number of

fast

both the p e c t o r a l

and l e g muscles by t h e

procedure (L3-L5)

of

strictly

b o t h l e g and p e c t o r a l

showed predominance of embryonic

of

(Fig.

3).

isoforms

of

t r o p o n i n T were a p p a r e n t

Using a n t i b o d i e s

and two minor

immunoblotting

F24 and T l / 7 ,

( L I and L2) v a r i a n t s

in

of

this

three

major

protein

169 were

detected

antibodies

of

different

fibres T

and

mass

the

adult

fast

increased resulted

The

the

of

fast

leg

of

variants

antibody

CDC4

could

one

in

this

in

immunoblotting

(ALD),

a

isoform

slow

troponin

showed Tl/7

the

and

cardiac ref.

of

skeletal

embryonic antibody

the

and

the

the

T

in

fast

only

a

slow T

isoform

identified

the

adult

case

muscle

this two

in

the

for

the

(11).

The

region

adult

a

studies

tonic

(Fig.

containing one

detected

the

often

pectoral

4

& 5).

slow of

adult detected

The

fibres

analysis

also

troponin

single

chicken

of

muscle,

T.

Both

isoform

of

(Fig.

3 and

heart

5,

case

Tl/7

with

by

pectoral

a

troponin

antibody

muscle

of

samples

of

of

F24,

showed

immunoblotting

amounts

muscle

class

T

in

the

developing

muscle

skeletal

the

presence

of

be

muscle

variants

14).

chicken

twice

muscles

antibodies

troponin

13,

was

leg

presence

CDC4

Studies

As

of

adult

type

muscle.

dorsi

several

leg

the

leg

in

the

troponin variants,

in

this

T

of

two

I

molecular

also

of

troponin

Bl,

latissimus

single

major

major

of

varied

type

higher

particularly

anterior

of

variants

regions

type

had

these

T

of

The

composed

three

was

containing of

T

troponin

(8,11).

was

however, This

absence

T

the

of

of

the

proportion

variants

troponin

T,

the

neither

itself,

isoforms on

muscles

two

of

Although isoform

muscle

These

muscle.

the

fast

mixed

amount

amount

troponin

use

3).

slow

depending

the

fibre

in

the

various

troponin

slow

muscles.

pectoral

small

pectoral

deeper

type

in

the

all A

with

the

(Fig.

than

(L1-L5). of

in

B2

leg

muscles

present

species

B1

the

reacted

proportions in

in

small muscle

embryonic

from

post

number was

not

extracts

little

or

procedure muscle

hatch

of

the

fast

no

despite

(Fig.

isoforms

detected

until

in mid

early

reaction

extracts

stages

of

with

loading

compared 3).

The

both

the

embryonic

with

leg

170

B1 B2 •

J a b c

N1. N2-

d e

f g h i j

k t m n o p q r s

.mm* **

L

( » A

rs H - *

^

w i t tf .

B1& -

N2 N1-

mrnm

cardiacdevelopmental B2 & —

cardiac adult

c

nuif M

UN»

F i g u r e 3 . I d e n t i f i c a t i o n of f a s t and c a r d i a c i s o f o r m s of t r o p o n i n T i n t h e d e v e l o p i n g c h i c k e n s t r i a t e d m u s c l e s . Two Western b l o t s w i t h i d e n t i c a l samples of whole muscle e x t r a c t s s e p a r a t e d i n SDS t r i s g l y c i n e p o l y a c r y l a m i d e g e l s s t a i n e d w i t h a n t i b o d i e s F24 (A) and T l / 7 ( C ) by the i m m u n o p e r o x i d a s e p r o c e d u r e . B o t h i m m u n o b l o t s a f t e r p h o t o g r a p h i n g were r e s t a i n e d w i t h amido b l a c k to r e v e a l t h e p r e s e n c e o f u n s t a i n e d p r o t e i n s (B and D ) . W h i l e a n t i b o d y F24 o n l y r e a c t s w i t h t h e f a s t i s o f o r m s , a n t i b o d y T l / 7 r e c o g n i s e s b o t h t h e f a s t and c a r d i a c i s o f o r m s o f t r o p o n i n T. S a m p l e s : a = l e g m u s c l e from 1 day o l d c h i c k e n , b - f = l e g m u s c l e s f r o m 1 7 , 1 4 , 1 3 , 12 and 11 day i n ovo i n t h a t o r d e r , g = c a r d i a c m u s c l e f r o m 13 day i n o v o , TT~= c a r d i a c m u s c l e f r o m a d u l t , i - o = p e c t o r a l m u s c l e s f r o m S2 week, 34 week, 6 week, 3 week, 2 week and 1 day o l d c h i c k e n s , p - s = p e c t o r a l m u s c l e f r o m 1 7 , 1 4 , 12 and 11 day i n o v o i n t h a t o r d e r . N1 and N2 = n e o n a t a l t y p e t r o p o n i n T ; B1 and B2 = p e c t o r a l muscle type t r o p o n i n T; L = l e g muscle type t r o p o n i n T . The s t a i n i n g o f a l o w e r m o l e c u l a r mass p e p t i d e i n t h e c a r d i a c s a m p l e s w i t h a n t i b o d y T l / 7 i s due to some d e g r a d a t i o n of the c a r d i a c i s o f o r m s .

171

period with the

leg

either

type

simultaneously hatching.

hatch

pectoral

type

of

latter as

The

a large

expression

mobility

during

that

only

age.

The p r o p o r t i o n

a trace

pectoral

the

of

slow

No s i g n i f i c a n t

skeletal chicken

and s l o w

skeletal

position

of

late

the

isoforms

same a p p a r e n t muscle.

had

detected

leg

type

level

gradually

after

of

decreased

three

troponin

the mass

additional

B1 and B2 and t h e i r period

in

slower

months

so

of

T variants

in

early

post

period

isoforms

of

during

hatch

the

antibody

CDC4 t h a t

recognises

isoforms

slow

skeletal

embryonic slow

or

isoform

in

a large

number

the a d u l t

muscles or

did

of

post

amount o f b o t h embryos

A faint period

the e a r l y

similar

to t h a t

ALD o r l e g m u s c l e s .

samples

f r o m the e a r l y

n o t show any number cardiac

of

staining

pectoral

muscle

T in

(Fig. of

the

were

cardiac the

5).

The muscle

corresponding

The W e s t e r n embryonic the

muscle

detected

developing

blots of

a

T

(Fig.

trace

10-12

developed

or

either

troponin in

of

pectoral

region

samples,

was d e t e c t e d

immunoblots

both

not

i s o f o r m of c a r d i a c

isoforms

when t h e

in

skeletal

band i n

i s o f o r m was

hatch

T in

developmental

a small

(Fig.4). muscle

troponin

troponin

muscle embryonic

to be g e n e r a l l y

the a d u l t

The two

variants

leg

molecular

the e a r l y

appeared

chicken

Two o f

3).

of

of

In

of

and c a r d i a c

mobility

4).

of

region

skeletal

reaction

was o b s e r v e d w i t h

leg

region

the

the

adult.

developing

until

the

show

the

m u s c l e was much h i g h e r

in

Studies the

of

not only

post

in

hatch

amount was

until

the e a r l y

in

the

than

of

isoforms

pectoral

the p o s t

All

variants

T (Fig.

the a d u l t

3).

appear

not o b s e r v e d

hand d i d

N1 and N2 named n e o n a t a l

electrophoretic

than

four

appeared to have

B1 and B2 i n

variants

number o f

(Fig.

not

studies

on the o t h e r

troponin

used

did

complement was

T but a l s o

type

class

T variants

immunoblotting

muscle

troponin

pectoral

t h e two a n t i b o d i e s

troponin

and a f u l l

after

presence

of

fast

day

for

old

longer

172

^ a b c d e f g h j j k t m

n

o

p

q

r

s

cardiac . developmental cardiac adult ' slow skeletal

F i g u r e 4. A n a l y s i s of s l o w a n d c a r d i a c i s o f o r m s of t r o p o n i n T in t h e d e v e l o p i n g c h i c k e n s t r i a t e d m u s c l e . W e s t e r n b l o t o f w h o l e m u s c l e e x t r a c t s s e p a r a t e d in S D S t r i s g l y c i n e 1 0 % p o l y a c r y l a m i d e g e l s s t a i n e d w i t h a n t i b o d y C D C 4 by t h e i m m u n o p e r o x i d a s e p r o c e d u r e ( A ) . B is a s a m e b l o t as A restained with amido black. Samples: a - d = pectoral muscles f r o m d a y 1 1 , 1 2 , 14 a n d 17 in o v o , e - k = p e c t o r a l muscles f r o m 1 d a y , 1 w e e k , 2 w e e k , 3 w e e k , 6 w e e k , 34 w e e k a n d 52 w e e k o l d c h i c k e n s in t h a t o r d e r . 1 = c a r d i a c m u s c l e f r o m a n a d u l t c h i c k e n , m = c a r d i a c m u s c l e f r o m 13 d a y c h i c k e m b r y o , n = A L D m u s c l e f r o m a 12 w e e k o l d c h i c k e n , o - s = l e g m u s c l e s f r o m 1 1 , 1 2 , 1 3 , 14 a n d 17 d a y in o v o . time

(Fig.

embryonic presence was

5).

of

this

further

pectoral

isoforms

the

of

isoform

analysis

muscles, troponin

Immunocytochemical

To the

confirm

presence

muscle

and

of

of

the

cardiac

the

T

at

extracts

of

to

troponin

T

isoforms

the

the level

The

the

as

in

protein

detect

antibody

embryonic

leg

cardiac

well

(Fig.3).

T

immunoblotting

troponin

the

Tl/7.

failed

mRNA

(4,5),

antibody

this

of

type

documented

troponin

however,

studies

of

well

using

T with

extend

distribution

is of

investigated

immunoblotting and

As

skeletal

observations was

further,

investigated

in

173

b c d

q

f

g

h i j

k

I

m

n

cardiac developmental cardiac adult slow skeletal

f M

> ft j

M

I ftÜ

F i g u r e 5. A n a l y s i s of s l o w a n d c a r d i a c i s o f o r m s of t r o p o n i n T in s o m e s a m p l e s of the c h i c k e n d e v e l o p i n g s t r i a t e d m u s c l e . W e s t e r n b l o t of w h o l e m u s c l e e x t r a c t s s e p a r a t e d in S D S t r i s g l y c i n e 10% p o l y a c r y l a m i d e g e l s s t a i n e d w i t h a n t i b o d y C D C 4 by the i m m u n o p e r o x i d a s e p r o c e d u r e (A). Samples: a & i = ALD m u s c l e f r o m 12 w e e k o l d c h i c k e n , b & h = c a r d i a c m u s c l e s f r o m 13 day c h i c k e n e m b r y o , c - g = c h i c k e n p e c t o r a l m u s c l e f r o m 9, 1 1 , 12, 14 a n d 17 day in oyo in t h a t o r d e r , j - o = c h i c k e n leg m u s c l e s f r o m 11, 13, 14, 17 day in o v o a n d 1 day p o s t - h a t c h . B = I m m u n o b l o t A r e s t a i n e d w i t h ami do b l a c k . tissue F24

sections

shown

muscles

to s t a i n

(Fig.

embryonic procedure.

using

immunoperoxidase

type

II f i b r e s

1 a n d 6) s h o w e d

skeletal Antibody

muscle LM5

to

in a d u l t

little

(Fig. fast

procedure

6b,

or no

chicken by

of m y o s i n

Antibody

skeletal

reaction

7b) e v e n

class

(1).

with

early

this heavy

chains

174

F i g u r e 6. F r o z e n s e c t i o n s from a c o m p o s i t e b l o c k of a d u l t c h i c k e n p e c t o r a l ( P ) , a d u l t g a s t r o c n e m i u s (G) and e m b r y o n i c p e c t o r a l m u s c l e f r o m 13 day i n o v o ( e ) s t a i n e d w i t h a n t i b o d y LM5 ( a ) and F24 ( b ) by the i m m u n o p e r o x i d a s e p r o c e d u r e . A n t i b o d y LM5 r e a c t s w i t h m y o s i n h e a v y c h a i n s p r e s e n t i n b o t h e m b r y o n i c and a d u l t f a s t s k e l e t a l m u s c l e s . A n t i b o d y F24 i n contrast reacts with troponin T present in adult skeletal m u s c l e b u t shows o n l y a weak r e a c t i o n w i t h 13 day e m b r y o n i c pectoral muscle. in

contrast

stained

all

pectoral

muscle

muscles,

however,

was

level

staining

with

of

gradually 7d) fast

even

increased though

troponin

Antibody

its

to

failure

to

that to

present

in

13 day

Weak s t a i n i n g

of

some

observed

as e a r l y

antibody

F24

in

subsequent

complement

T was n o t o b s e r v e d

of

muscles

detect

cardiac

after

F24

isoforms

isoforms

of

in

ovo.

growth isoforms

(Fig. of

hatching. of

intensity (Fig. in

The

sections

investigations

antibody

cardiac

embryonic

skeletal

10 day

embryonic

showed s t a i n i n g

observed with recognise

as

tissue

the a d u l t

until

immunohistochemical

skeletal

ability

myotubes

6a).

during

a full

T1 /7 i n

embryonic similar

(Fig.

7)

generally despite

addition.

troponin

T in

The

175

F i g u r e 7 . F r o z e n s e c t i o n s f r o m 10 day (a - c ) and 15 day (d and e) c h i c k e n e m b r y o n i c p e c t o r a l m u s c l e s t a i n e d w i t h d i f f e r e n t a n t i b o d i e s a g a i n s t t r o p o n i n T (a - d) and f a s t m y o s i n h e a v y c h a i n s ( e ) by t h e i m m u n o p e r o x i d a s e t e c h n i q u e , a = s t a i n e d w i t h a n t i b o d y CDC4, b and d w i t h a n t i b o d y F 2 4 , c = with Tl/7. significant confirmed

amounts

in

embryonic

by l i t t l e

or

no r e a c t i o n

the e a r l i e r of

early

indicated slow

stages

embryonic

of

troponin

development

skeletal

the absence

skeletal

or

muscle

only

with

m u s c l e was

antibody

(Fig.

7).

with

The

non

antibody

low l e v e l

also

CDC4

reaction

CDC4

expression

during

of

also adult

T.

Discussion Both

fast

and s l o w

isoforms

embryonic

skeletal

muscle.

during

the

proportion

early of

embryonic

large

primary

of

troponin

I were o b s e r v e d

The a b u n d a n c e period

of

slow

may r e f l e c t

generation

fibres

the at

in

isoform high these

stages

176

but

it

does

present

not p r e c l u d e

in

small

predominance muscle (15).

fast to

in which It

whether

is

detected

the

this

in

the

late

isoform

in

or

is

slow

study of

in

skeletal

the c h i c k e n

the

variants

leg muscles,

also

detected

shown i n

the

troponin

T was

The l a c k

of

(that

of

reaction

I)

striated

muscle in

troponin

the e a r l y

This

during

troponin

embryonic none o f

reacted with

T variant

in

troponin

the it

were

some

troponin T gene

in

T was in

generates

also

been

i s o f o r m of

skeletal

slow

to

antibodies

a

unique studies

reported

both

leg

used

in

observed

to

and

isoform

the predominance muscle

of

from o t h e r

been of

and

adult

the e x i s t e n c e differs

muscle

myosin

antibodies

T has

the embryonic

the

antibodies

has

the embryonic

but

of

variants

study.

development of

as

contains

T indicates

The n a t u r e as

it

T

particularly

embryonic

observation

type

type

a single

the monoclonal

is

studies

Not o n l y

chicken

present

It

embryonic

troponin

muscle,

only

T as

in

I.

muscle-like

earlier

some l e g fast

I

distinct

two d i f f e r e n t

the

the

troponin

leg

muscles.

investigation

in

troponin

which

known a t p r e s e n t

of

all

isoform.

predominate pectoral

with

the in

myotubes

(12).

(8,11).

of

(9,16,17),

observed

must c o n t a i n

(18,19)

While

are

fast

pectoral

isoforms

rabbit

troponin embryonic

adult

region.

number

muscles

of

period

investigation

detected

and e x t e n d s number

The

ovo

troponin

skeletal

being

skeletal

all

troponin

muscle

detected with

the

of

muscle

in

rat

in

to a d u l t

slow

the p r e s e n c e

in

red s t r i p

a large

a large

as w e l l .

of

isoforms

cardiac

confirms

identification additional

stages

isoform to

isoform

the l a t e r

detected

skeletal

similar

this

from p r e s e n t

slow

identical

the embryonic

The p r e s e n t

the

or

the embryonic

muscle

foetal

of

fibres

during

however,

fast

variants

I

i s o f o r m was

n o t known w h e t h e r

skeletal

generation

troponin

not c l e a r ,

either

embryonic also

secondary

of

may be s i m i l a r

the p o s s i b i l i t y

is

not

this of by

leg

type

other

177

investigators

(18,19)

electrophoretic

mobilities.

immunochemically

troponin

phenotype

during

in

the

troponin

T is

isoforms

in

antibodies

development

unique

both

to

was a l i t t l e

of

at only

in

is

monoclonal

antibodies

study

developmental

amounts.

b u t was

not

used

and a d u l t of

i s o f o r m of

The p r e s e n c e studies

using

either

type

been polyclonal

of

the

two

investigation.

The

be d e t e c t e d

troponin

T at

described

skeletal this

the c h i c k e n time

factors

period

of

seems

of

It

is

not

known, of

muscle.

variants

a particularly

the c o m p l e t i o n

in

distinct

isoforms

skeletal

several

both

samples,

must be

embryonic

embryonic

affecting

muscle

stage

isoforms.

or m u l t i p l e

and d i s a p p e a r a n c e

a long

study

also

could

in

to

into

isoforms

T exist T over

either

of c a r d i a c

has

is

cardiac

a single

troponin

discrepancy

not t r a n s l a t e d

the p r e s e n t

(5)

amounts

the

property

reports

the p r o t e i n

this

however,

with

trace

whether

appearance

the e a r l i e r

mRNA i s

is

muscle

although

from a l l

gradual

this

the embryonic

previously

developmental

of

the case w i t h in

isoforms,

that

however, troponin

for

or

level,

demonstrated

proportion

the major

that

low l e v e l

quantitative

skeletal

view o f

reason

is

unlike

period.

species

by some i m m u n o h i s t o c h e m i c a l (20)

a small

The

the p r o t e i n

antibodies present

in

however,

cellular

about

distinct

embryonic

this

the

their

that

muscle

and n e o n a t a l

possible

very

isoform,

modify

striated

isoforms

in

therefore

by b r i n g i n g

the e a r l y

significant

T at

suggested

it

that

expressing

surprising.

but

troponin

in

o f mRNA f o r

not c l e a r

appears

adult

embryonic

expressed protein

of

cardiac

the p r e s e n c e

It

components

levels

The non r e a c t i o n of

The e m b r y o n i c

distinct.

the o t h e r changes

may be due t o s i m i l a r i t i e s

of

The

of useful

muscle

specialisation.

Acknowledgements: Medical B r i t a i n.

Research

This Council

work was

supported

and M u s c u l a r

by g r a n t s

Dystrophy

from

Group o f

the Great

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Res.

179 17. H a r t n e r K . , B. K i r s c h b a u m , B i o c h e m . 17j), 3 1 - 3 8 .

D. P e t t e .

18. M a t s u d a il-19.

R., T. O b i n a t a ,

Y. S h i m a d a .

19. S h i m i z u

N.,

1985.

20. T o y o t a

Y. S h i m a d a .

N . , Y. S h i m a d a .

Dev.

1989. 1981.

Biol.

1 9 8 1 . J . Cell

Eur. Dev.

n_l,

Biol.

J. Biol.

82,

324-334.

91 , 4 9 7 - 5 0 4 .

BIOCHEMICAL PROPERTIES OF THE DIAPHRAGM DURING DEVELOPMENT OF RESPIRATORY FUNCTION IN THE RAT A.M.KELLY1, B.W.C.ROSSER3. N.A.RUBINSTEIN2 AND P.M.NEMETH . DEPARTMENTS OF PATHOBIOLOGY, SCHOOL OF VETERINARY MEDICINE1 AND ANATOMY, SCHOOL OF MEDICINE2, UNIVERSITY OF PENNSYLVANIA, PHILADELPHIA, PA. 19104, AND THE DEPARTMENT OF NEUROLOGY3, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MO. 63110

Introduction

The diaphragm is the most important muscle of respiration and although it is a voluntary muscle it is unlike other voluntary muscles in a number of respects.

For example, it

is reported to be a composite structure chiefly formed from the septum transversum and the pleuroperitoneal folds and not to be somitic in origin (1).

In addition, the primordium of

the diaphragm is distinctive as membrane junctions including adhering junctions and gap junctions commonly

interconnect

primary myotubes whereas these membrane specializations are comparatively uncommon in the primordia of limb muscles (Ling, Kelly and Franzini-Armstrong, in preparation).

We believe

these

with

membrane

specializations

are correlated

early

diaphragmatic function in at least two ways, (a) to coordinate initial "practice" respiratory movements before the tissue is fully

innervated

and

(b) to

ensure

that

the

primordium

functions as a robust structural unit partitioning the pleural from

the

peritoneal

cavities

prior

to

the deposition

of

connective tissue. In

the

present

study

we

describe

the

metabolic

and

contractile properties of the fetal, neonatal and adolescent diaphragm and relate these properties to the development of respiratory function.

In particular we are interested to

The Dynamic State of Muscle Fibers © 1990 Walter de Gruyter& Co., Berlin • New York • Printed in Germany

182

learn how the fetal diaphragm is prepared so that it can abruptly support aerobic respiration at birth and sustain the extraordinarily high metabolic and ventilatory demands of the neonate without fatigue. We also outline the changes in the contractile and metabolic programming of the diaphragm as metabolic rates decline and respiratory rates slow with maturity. In this study we have investigated the expression of MHCs by three different methods; (a) by Northern blots of MHC mRNAs using oligonucleotide probes complementary to unique 3 1 untranslated regions of embryonic, neonatal and adult fast lib MHC genes (2) , (b) by pyrophosphate gel electrophoresis of actomyosins (3) and (c) by immunochemistry using monoclonal antibody N0Q7 5 4D, specific to slow MHC (4) and SC 711 specific to adult fast Ila MHC (5) . Since myosin isozymes and metabolic enzymes are likely to move coordinately throughout development we have also analyzed enzymes representing the major energy generating pathways in muscle; B-hydroxyacyl CoA dehydrogenase (BOAC for fatty acid oxidation), malate dehydrogenase (MDH) for the citric acid cycle and lactic dehydrogenase (LDH) for glycolysis. The Fetal Diaphragm

MHC compared

expression to

in the

limb muscles.

fetal For

diaphragm example,

is

precocious

transcripts

for

neonatal myosin are plentifully expressed by the diaphragm at 18 days gestation (Figure 1), the earliest stage we can confidently isolate this muscle in the fetus, but they must have been present at an even earlier stage since the translation product, neonatal MHC isozyme, is also accumulated in the diaphragm at 18 days

(Figure 2 ) .

By contrast, only

traces of neonatal MHC mRNA are present in the 18 day fetal gastrocnemius

(Figure 1) and this protein product

is not

detected in the gastrocnemius until 21 days gestation (6).

183

Figure 1. Developmental expression of neonatal MHC mRNA in the diaphragm (a) compared to the gastrocnemius (b) . Northern blot uses an oligonucleotide probe complementary to unique 3' UTR sequences of rat neonatal MHC mRNA. Transcripts for neonatal MHC occur in the diaphragm at 18 days gestation and at 21 days in the gastrocnemius.

18f

21 (

0

5

10

15

30

Figure 2. Pyrophosphate gel showing myosin isozyme composition of the euthyroid diaphragm from 18 days gestation (18f) to maturity. Isozymes fl to f4 refer to embryonic and neonatal myosin, FM1 to FM3 refer to adult fast myosin and SM refers to slow myosin. Neonatal myosin, fl to f3, is present from 18 days gestation and the adult fast isozyme FM3 is present from 21 days gestation.

184

In addition to expression of neonatal MHC, the adult fast myosin isozyme, FM3, can be detected in non-denaturing gels of the diaphragm at least by 21 days gestation (Figure 2) whereas adult

fast

isozymes

are

not

found

in

the

developing

gastrocnemius until 10 to 14 days post partum (3, 6 & 7) . We can show by immunochemistry that early accumulation of FM3 in the diaphragm is correlated with expression of adult fast Ila MHC (Figure 5). One

way

of

accounting

for

these

differences

is by

recognizing that the primordium of the diaphragm is modelled before that of the hind limb muscles.

However, precocity of

the diaphragm is more complex than this since the pattern of isozyme switching in the sternomastoideus, a neck muscle lying rostral to the diaphragm more closely simulates that of limb muscles than the diaphragm (8). In addition switching

to

its precocity,

the developing

diaphragm

the program

of

is remarkable

myosin for the

gradual way that one isozyme replaces another (Figure 2 and 7) despite the abrupt shift from intra- to extra-uterine life. This is unlike the hastier pattern of transition seen in limb muscles

(Figure 1 and 6) and suggests that

diaphragmatic

development is designed to ensure that necessary adjustments to MHC expression are minimized as the neonate copes with its new environment.

Moreover, this cautious pattern of exchange

may permit a broad interval of safety in case of premature delivery. These advanced patterns of MHC expression appear to be linked with maturational shifts in the capacity of various metabolic

pathways

in

the

diaphragm

(Figure

3)

and

we

hypothesize that the two systems are essentially coordinated so that vital,"practice," respiratory movements made by the developing

diaphragm

(9) are not jeopardized

by

fatigue.

Enzymes representing oxidative, glycolytic and high energy phosphate metabolism are all higher in the fetal diaphragm compared

to

interesting

fetal

limb

difference

muscles

(8) .

However,

is in the capacity

the

most

for fatty acid

185 oxidation.

A t 19 d a y s g e s t a t i o n ,

a c t i v i t y of

B-hydroxyacyl

C o A (BOAC) is t h r e e t i m e s h i g h e r a n d at b i r t h six t i m e s h i g h e r in t h e

diaphragm

than

in limb

muscles

(Figure

3) .

We

are

impressed by these findings since they show that mitochondrial f u n c t i o n i n c r e a s e s in t h e d i a p h r a g m late in g e s t a t i o n d e s p i t e t h e c o n s t r a i n t s o n o x y g e n s u p p l y at t h i s stage. increasing

beta

oxidation

the

fetal

diaphragm

a n t i c i p a t e s t h e n e o n a t a l p e r i o d of p h y s i o l o g i c a l that accompanies suckling.

M o r e o v e r , by clearly

hyperlipemia

In v i e w of t h e h i g h y i e l d of A T P

a c h i e v e d b y b e t a o x i d a t i o n , c h o i c e of t h i s m e t a b o l i c p a t h w a y s e e m l i k e l y to c o n t r i b u t e to the f a t i g u e r e s i s t a n t p r o p e r t i e s of t h e d i a p h r a g m in t h e

neonate.

F i g u r e 3. E n z y m e a c t i v i t i e s in w h o l e m u s c l e h o m o g e n a t e s of rat d i a p h r a g m a n d E D L m u s c l e s d u r i n g t h e p e r i n a t a l p e r i o d . Values a r e m e a n +/_ S D a c t i v i t i e s , expressed as mol/kg p r o t e i n / h r a t 21°C. A l l t i m e p o i n t s a r e t h e m e a n s of at l e a s t 5 animals. T i m e p o i n t s are d a y s in u t e r o .

186 25

35

/J-hydroxyacyl-CoA Dehydrogenase

Malate Dehydrogenase

30

20

l

25 15

10

5

é

0 —E -2

0

J

15

i

10

1

1

1

1

I

4

6

8

10

200

o—
2S>~

s IC Ca Cb A+l A B A C C IC

It It -HCI

30d 60d C 'lt It P -1F

-«2F -3F

Fig. 1. Heavy (upper panel) and light (lower panel) chain analyses from single fibers in rabbit soleus (sol), 30d and 60d stimulated tibialis anterior, and contralateral tibialis anterior (TA) muscles. Slow (HCI) and fast (HCIIa) heavy chains were electrophoretically separated using 5% SDS-polyacrylamide gels according to Rushbrook and Stracher (2 9). Microelectrophoresis for myosin light chain analysis was performed according to Laemmli (30). p = myosin extract from psoap m. s= myosin extract from soleus m. IF, 2F, and 3F = fast light chains. ISa, lSb, and 2S = slow light chains. Key for fiber types: I, type I; IC, type IC; C, type IIC; A, type IIA; B, type IIB; It, transformed type I; A+I, co-electrophoresis of type IIA + type I. Ca and Cb are analyses from two different parts of the same fiber (histochemically classified as type IIC) dissected from the contralateral TA of a 30d stimulated animal.

319

Slow-twitch fibers contain the slow myosin heavy chain HCI (31,32) which corresponds to the cardiac heavy chain BHCcard (33). The other slow isoform, HCIton, is associated with slow, tonic fibers in extraocular muscles (34) , tensor tympani muscle (25), and intrafusal fibers (36,37). Two of the fast heavy chains, HCIIa and HCIIb, are expressed in type IIA and IIB fibers, respectively (23,38). An additional fast myosin heavy chain, HCIId or HC2x, has been described in small mammals (39-41, see also Schiaffino et al. and Termin et al., this volume) . Two other specific fast heavy chains have been detected in super-fast contracting fibers of extraocular muscles, HCIIeom (42,43), and muscles derived from the first branchial arch (jaw closing muscles and the tensor tympani muscle), HCIIm (15,35). Adding to the list of fast heavy chain isoforms, Rushbrook et al. (44) have recently identified four heavy chain species in adult fast avian muscle. Two unique heavy chain isoforms, HCemb and HCneo, are expressed at specific stages of development (29,45). These "developmental" myosin heavy chains are also expressed in normal adult muscle, e.g. extraocular muscles (42,43), murine masseter muscle (46), and intrafusal fibers (36,37,47). Additional embryonic heavy chain isoforms have been found in developing chicken pectoralis muscle (48, 49) .

Table 2. Myosin Heavy Chains in Adult Mammalian Skeletal Muscle. slow

fast

developmental

HCI (BHCcard) HCIton

HCIIa HCIIb HCIId HCIIm HCeom

HCemb HCneo

320

Coexpression of heavy chain isoforms in single fibers. Various combinations of more than one heavy chain isoform have been found in single fibers of normal, adult mammalian muscle (Table 3).

These "hybrid" fibers commonly express two heavy

chains, e.g. HCIIa/HCIIb

(24,50,51), HCIIb/HCIId

(40,52), or

HCIIa/HCI (27), and appear to correspond to the histochemical fiber types: type IIAB, IIBD, or C (IC and IIC) fibers, respectively.

In addition, protein and immunochemical analyses

have revealed the coexpression specific heavy chain isoforms, e.g. HCneo/HCfast, HCneo/HCemb, and HCeom/HCIIa, in single fibers of extraocular muscles (42, 43) .

Likewise, the majority

of fibers in the masseter muscle express more than one heavy chain and may coexpress up to three heavy chains (HCfast/HCslow/HCneo)(20).

Intrafusal fibers also represent a

special fiber population which coexpress different combinations of HCneo/HCf ast/HCslow/HC I ton (36,37) and HCe\ib (47) in the adult.

Recently, under conditions of induced transformation in

rat EDL muscle, up to four myosin heavy chain isoforms have been found in a single muscle fiber (HCI/HCIIa/HCIId/HCIIb)

Table 3.

(53).

Single Fibers Coexprsssing MHC's in the Adult.

IC (IB, IM) , IIC AB BD EOM masseter m intrafusal stim. EDL (rat)

HCI/HCIIa HCIIa/HCIIb HCIId/HCIIb HCneo/HCfast, HCneo/HCemb, HCeom/HCIIa HCneo/HCfast/HCslow HCneo/HCf ast/HCslow/HCIton/HCemb HCI/HCIIa/HCIId/HCIIb

Isomyosins The formation of isomyosins is the result of the hexameric structure of myosin.

Three fast isomyosins have been

electrophoretically separated under nondenaturing conditions: FM3, FM2, FMI (54,55).

These correspond, respectively, to a

321

pair of identical fast heavy chains (either HCIIa or HCIIb) plus three different light chain combinations: 1) LC1F homodimer/LC2F homodimer, 2) LC1F/LC3F heterodimer/LC2F homodimer, and 3) LC3F homodimer/LC2F homodimer (Table 4). Likewise, three slow isomyosins have been detected (SM2, SMI, and SMI') (56,57). SM2 is a "true" slow isomyosin consisting of only slow HCI and the slow light chains (LC1S, LC2S), whereas SMI contains, in addition to these slow components, LC1F (25). The composition of this isomyosin is similar to many slow, type I fibers in human muscle (23) and to the transformed slow fibers, termed type It, found in chronically stimulated rabbit tibialis anterior muscle (58). The third slow isomyosin (SMI1) has been described in rat hindlimb muscles (56,57) and appears to exist in small quantities. Another fast isomyosin (termed IM because of an electrophoretic mobility between SMI' and FM3) has been demonstrated in rat and mouse soleus muscle (25, 57) . This fast isomyosin is associated with histochemically-classified type IIA fibers in certain muscles (59) and consists of the fast myosin heavy chain HCIIa (25) and the fast and slow light chains LC1F, LC2F, LC1S, and LC2S (25,57). Single fiber analyses have revealed that the type IIA fibers in rat (26) and rabbit (13) (Fig. 1) soleus muscle express this same complement of heavy and light chains. In addition to these fast and slow isomyosins, "developmental" isomyosins are expressed in adult muscle (Table 4). Adult masseter and extraocular muscles contain neonatal myosin heavy chain, which is apparently resolved into three neonatal bands under nondenaturing conditions (20,42). Because neonatal myosins contain a different heavy chain but the same light chains as those found in fast muscle (54), these three neonatal myosin bands may be the result of the three light chain combinations: LC1F homodimer, LC1F/LC3F heterodimer, and LC3F homodimer.

322 "Special" muscle-specific isomyosins have also been detected. Adult extraocular muscle contains a fast migrating isomyosin band which does not correspond to any of the known adult fast, slow, neonatal, or embryonic bands (42).

This extraocular

isomyosin may contain HCeom. Similarly, special fast and slow isomyosins have been reported during postnatal development in the cat temporalis muscle (60, 67) . Although not yet electrophorectically demonstrated, other unique heavy (e.g. HCIIm, HCIton) and light (LClemb, LC2emb LC2s', LClSa, LClSb) chain isoforms should increase the number of possible isomyosins.

Table 4. Light and Heavy Chain Combinations in Specific Isomyosins (25,56,59). slow muscle isomyosins and number of theoretical combinations: SM2 SMI SMI'

(HCI/LC1S/LC2S) (HCI/LC1S/LC1F/LC2S) ? (HCI/LC1S/LC1F/LC3F/LC2S/LC2F)

1 3 12

fast muscle isomyosins and number of theoretical combinations: IM FM3 FM2 FMI EOM IIM

(HCIIa/LClS/LClF/LC2F) 3 (HCIIa/HCIIb/LClF/LC2F) 3 (HCIIa/HCIIb/LClF/LC3F/LC2F) 9 (HCIIa/HCIIb/LC3F/LC2F) 3 ?(HCeom/LClS/LClF/LC3F/LC2S/LC2F) .... 12 ?(HCIIm/LClS/LClF/LC3F/LC2S/LC2F) .... 12

developing muscle isomyosins and number of combinations: fM5-eM4. fM4-eM3. fM3-eM2. fM2-eMl. fMl

(HCemb/LCemb/LC2F) (HCemb/LClF/LC2F) (HCneo/LClF/LC2F) (HCneo/LClF/LC3F/LC2F) (HCneo/LC3F/LC2F)

1 1 1 3 1

Theoretical isomyosins: Considering only the heavy chains HCI and HCIIa and the slow (LC1S and LC2S) and fast (LC1F, LC2F, and LC3F) light chains, a

323 total of 54 isomyosins is theoretically possible (13,24,62,63). Such a large number is not only the result of certain fibers coexpressing specific light and heavy chains, but also is the result of theoretical heavy chain heterodimers. The formation of heavy chain heterodimers has been observed in rat cardiac ventricular myosin (64). If heterodimers do not exist in skeletal muscle, the two heavy chains and the slow and fast light chains mentioned above would yield a maximum of 36 isomyosins based only upon the different light chain combinations (13,24). The number of possible isomyosins in adult mammalian skeletal muscle is far greater if the additional myosin light and heavy chain variants so far detected are considered. Also, the relative concentrations of the individual components (e.g. variable ratios of HCI/HCIIa expression within the C fiber population, Fig.l) and their location within the fiber increase the complexity of isomyosin composition and contribute to the dynamic nature of muscle. The distributional pattern of various isomyosins within the same fiber is not known. Several locations are possible: 1) a random distribution with A-bands composed of different thick filaments each containing only one isomyosin or by thick filaments containing more than one isomyosin, 2) a sarcomeric distribution with homogeneous thick filaments within a sarcomere, but heterogeneity along the length of the myofibril, and 3) a myofibrillar distribution with isomyosin homogeneity throughout the length of each myofibril. The possibility that thick filaments may be composed of different myosins has been reported for nematode muscle (65) and in developing chicken pectoralis muscle (66). However, segmental differences in contractile properties (67) and myofibrillar ATPase activity (68) along the length of single frog muscle fibers have been described. Similarly, nonuniform myosin expression along the length of single fibers in chronically stimulated and contralateral rabbit

324 tibialis muscle (69)(Fig. 1), in multiply innervated muscle fibers (70,71), in muscle spindles (36,37,47), and in denervated (72) and regenerating rat muscle (73,74) has been described. Regional variations.in myosin expression may also occur along the length of normal human muscle fibers (unpublished observations). Heterogeneous myosin expression along a muscle fiber could point to either a loss of nuclear coordination or the existence of regional control by specific nuclear domains (75, 76) .

Summary The existence of a large number of light and heavy chain isoforms indicates the possibility of numerous isomyosins. In addition, single fibers coexpressing isoforms in varying ratios and the possibility of regional distribution along the length of a muscle fiber increase the complexity of isomyosin composition. To date, only a limited number of isomyosins has been identified. It is not known if this is the result of preferential affinities between specific myosin light and heavy chains or if this is the result of insufficient resolution of the applied methods. Improved methodology, as well as widening the scope of muscles and species investigated, will no doubt increase the list of defined isomyosins. Taken together, the multiplicity of myosin light and heavy chain combinations illustrates the dynamic state of skeletal muscle fibers.

Acknowledgments This study was supported by the Deutsche Forshungsgemeinschaft, Sonderforschungsbereich 156, and by the Alexander von HumboldtStiftung.

The authors would like to thank Linda Watkins and

Roxanne Dicken for techincal assistance with the manuscript.

325

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MUSCLE FIBER TYPES EXPRESSING DIFFERENT MYOSIN HEAVY CHAIN ISOFORMS. THEIR FUNCTIONAL PROPERTIES AND ADAPTIVE CAPACITY

S. Schiaffino, L. Gorza, S. Ausoni CNR Unit for Muscle Biology and Physiopathology, Institute of General Pathology, 35100 Padova, Italy R. Bottinelli, C. Reggiani Institute of Human Physiology, University of Pavia, 27100 Pavia, Italy L. Larson, L. Edstrom Departments of Clinical Neurophysiology and Neurology, Kaiolinska Hospital, Karolinska Institute, Stockholm, Sweden K. Gundersen, T. L0mo Institute of Neurophysiology, University of Oslo, 0162 Oslo 1, Norway

In this review we present recent results of a collaborative study aimed at 1) identifying the myosin heavy chain (MHC) isoforms expressed by various fiber types in rat and mouse skeletal muscle, 2) correlating the MHC profile with functional properties, 3) identifying factors responsible for modulating the fiber type phenotype and 4) defining the adaptive capacity of different fiber types with respect to MHC isoform expression. This report is mainly focused on a new major subset of type 2 muscle fibers defined by the presence of a distinct MHC isoform, that we have recently identified in rat and mouse skeletal muscle. 1. Type 2 Muscle Fibers: Three Major Populations Containing Different MHC Isoforms Some years ago we reported the existence of three distinct populations of type 2 muscle fibers in rat skeletal muscle (1, 2). The three fiber types were identified using several monoclonal antibodies (mAbs) specific for myosin heavy chains (MHCs) and were called type 2A, type 2B and type 2X. Type 2X fibers are widely distributed in rat skeletal muscles; they represent a major population in the normal diaphragm and are the predominant fiber type in the soleus muscle after high frequency electrical stimulation (3, 4). The existence of a distinct MHC isoform in type 2X fibers was demonstrated by immunoblotting experiments after electrophoretic separation of MHCs in porous gels (4). Immunoperoxidase staining of rat skeletal muscle with some of these antibodies is illustrated in Fig. 1. With the exception of SC-75 which is specific for the SI fragment of the MHC, all these antibodies are specific for the rod portion of the MHC, as determined by immunoblotting and enzyme immunoassay with proteolytic myosin fragments (4). Anti-myosin antibodies that distinguish three populations of type 2 skeletal muscle fibers were recently described also by other groups (5-7). A particularly important result has been the

The Dynamic State of Muscle Fibers © 1990 Walter de Gruyter & Co., Berlin • New York • Printed in Germany

330

1 2 A 2X 2 6

331

electrophoretic separation of three type 2-MHCs (8) and their distribution in isolated single muscle fibers (9). It appears that the type IID fibers described by Termin et al. (9) correspond to the type 2X fibers identified with our monoclonal antibodies (see also Termin et al., this volume).

2. Correlation of MHC Isoform Distribution with ATPase and Oxidative Enzyme Activities We have correlated antimyosin reactivity with ATPase and succinate dehydrogenase (SDH) histochemical staining in rat, mouse and guinea pig skeletal muscles (2, 4, 10). In rat skeletal muscle type 2X fibers stain like type 2B fibers in the ATPase reaction after preincubation at pH 4.6, whereas they stain like type 2A fibers in the ATPase reaction after formaldehyde-alkali (Fig. 1). They display moderate to strong SDH staining whereas true 2B fibers, defined by antimyosin staining, react very weakly for oxidative enzymes. In most enzyme histochemical studies fiber typing has been based on the 4.6 ATPase reaction: type 2B and 2X fibers were thus lumped together in the same "2B" group. As a consequence, when myosin ATPase activity was correlated with oxidative enzyme activity, type 2B fibers were described as a heterogeneous population comprising fibers with both high and low oxidative capacity (11,12). Our interpretation is that type 2B fibers identified by the 4.6 ATPase reaction in rat skeletal muscle comprise two distinct fiber populations: true 2B fibers which have uniformly low oxidative activity and 2X fibers which display moderate to strong reaction for oxidative enzymes. In mouse skeletal muscle, on the other hand, type 2X fibers stain darker than 2B fibers in the 4.6 ATPase reaction. In previous histochemical studies the darker type 2 fibers were classified as type 2B fibers and, since these fibers stain strongly for SDH activity, this has led to the conclusion that in the mouse, in contrast to other species, 2B fibers have higher oxidative activity than 2A fibers (13). However, a correlated enzyme hystochemical and immunohistochemical analysis (10) shows that this conclusion is erroneous and that fibers classified as 2A represent mostly 2B fibers, while fibers classified as 2B correspond to 2X fibers, 2A fibers being relatively rare in mouse fast-twitch skeletal muscle (Fig. 2 and 3). Type 2X fibers were identified in mouse skeletal muscle by the same antibodies used for rat muscle (4, see Fig. 1), and by a new anti-MHC mAb, BF-34 (unpublished clone), that in adult mouse skeletal muscle stains selectively 2A and 2X fibers.

Figure 1. Identification of type 2X fibers by immunocytochemistry and enzyme histochemistry. Serial transverse sections of rat tibialis anterior muscle were stained with mAbs against MHCs or processed for the histochemical demonstration of succinate dehydrogenase and myosin ATPase activity, a , scheme showing the pattern of reactivity of seven anti-MHC mAbs with different fibre types, as determined by immunoperoxidase staining. The filled and open boxes indicate positive and negative reaction, respectively, b -h , immunoperoxidase staining with mAbs SC-75 (6 ), BA-D5 (c ), BF-32 {d), BF-F3 (e ), BF-35 (f), RT-D9 (g ) and SC-71 (h ). Type 1 (1), type 2A (A), type 2B (B) and type 2X (X) fibres are indicated, i, histochemical staining for succinate dehydrogenase activity, k-m, myosin ATPase staining after alkali-formaldehyde pretreatment (£), and after, acid pretreatment at pH 4.6 (/) or 4.3 (m). Asterisk in m marks a hybrid type 2C fibre that contains both type 2 (see b and h ) and type 1 (see c ) myosin. From Schiaffino et al. (4).

332

Figure 2. Serial transverse sections of the superficial portion of the mouse tibialis anterior muscle. Histochemical staining for myosin ATPase activity after acid pretreatment at pH 4.6 (p ), myosin ATPase after alkaliformaldehyde pretreatment(6), succinate dehydrogenase (c ); immunoperoxidase staining with mAbs BF-32 (ft), BF-3S (e ), BF-34 I f ) . Note that there are no type 1 and 2A fibers in this part of the muscle, as shown by lack of reactivity with mAb BF-32, which is specific for type 1- and 2A-MHCs. Under these conditions, mAb BF-3S, that reacts with all MHCs except 2X-MHC, is specific for 2B-MHC, and mAb BF-34, that in adult mouse muscle reacts with 2A- and 2X-MHC, is specific for 2X-MHC. The larger 2B fibers stain weakly for SDH, whereas the smaller 2X fibers stain strongly for SDH. Fibers co-expressing 2B- and 2X-MHC are also present

333 3. Intermediate Fiber Types Containing Multiple MHC Isoforms Fibers containing two or even three different MHCs are regularly seen in rat, mouse and guinea pig skeletal muscles. Combinations of MHC isoforms are not random but appear to follow precise rules. In the rat and mouse slow-twitch soleus muscle three combinations of MHCs coexisting in the same fiber can be detected: type 1- and 2A-MHC, type 2A- and 2X-MHC, or type 1 - , 2A- and 2X-MHCs. Fibers containing type 1- and 2X-MHC are usually not seen. Fibers containing 2A- and 2X-MHC are regularly found in rat and mouse fast-twitch skeletal muscle. Fibers containing 2B- and 2X-MHC are frequent in mouse skeletal muscle (Fig. 3) and are probably present also in rat muscle but are difficult to detect with our antibodies. In

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Figure 3. Serial transverse sections of the deep portion of the mouse tibialis anterior muscle. Immunoperoxidase staining with mAbs BF-F3 (a), BF-32 {b ), BF-35 (c), BF-34 {d). This portion of the muscle consists of a few type 2A fibers (b), and a major proportion of type 2B (a,c ) and type 2Xfibers(d ). Several fibers appear to co-express 2B- and 2X-MHC (arrows).

334

contrast, fibers containing 2A- and 2B-MHC are never observed in rat and mouse muscles. If we assume that the combinations of MHCs detected with anti-MHC antibodies reflect preferential or obligatory pathways of MHC transitions in skeletal muscle fibers, the following sequence could account for the observed combinations: 1 4w

C

Figure 1. Time to peak twitch contraction for TA (hatched columns) and EDL (clear columns) for 4 weeks' stimulation (4w-S), 6 weeks' stimulation (6w-S) and 6 weeks' stimulation with >4weeks' recovery (R>4w), with their respective control groups (4w-C, 6w-C, C). Error bars are S.E.M. (For statistical analysis see Table 1.)

4w-S 4w-C 6w-S 6w-C R>4w

C

Figure 3. Twitch:tetanus ratios for the TA muscles. Same groups as Figure 1. Error bars are S.E.M. (For statistical analysis see Table 1.)

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Figure 2. Time from peak to half-relaxation for TA (hatched columns) and EDL (clear columns). Same groups as Figure 1. Error bars are S.E.M. (For statistical analysis see Table 1.)

4w-S 4w-C 6w-S 6w-C R>4w Figure 4. Maximum rate of rise of tetanic tension (%Po/ms) for the TA muscles. Same groups as Figure 1. Error bars are S.E.M. (For statistical analysis see Table 1.)

406 muscles exerted maximum forces that frequently exceeded the linear range of our transducers. Figure 3 depicts measurements of the twitch:tetanus ratio in TA muscles of the same three groups; Figure 4 shows the corresponding rates of rise of tetanic tension measured at the stimulation frequency for which each was maximal. Table 1 gives the results of two-tailed Student's t-tests between the unpaired experimental and control muscle data; n=5 in each group except the EDL recovery group, in which n=9. Experimental and control muscles differed very significantly in the second (6-week) group, but in the third (recovery) group the difference was either insignificant or, at best, barely significant. The exception was maximum rate of rise of tetanic tension, which was still significant at p4w

TA: Time to peak twitch Time to half-relaxation Twitch:tetanus ratio Max. rate of rise

0.0007 0.016 0.0001 0.099

0.0001 0.0001 0.0001 0.0001

0.035 0.61 0.49 0.005

EDL: Time to peak twitch Time to half-relaxation

0.0015 0.043

0.0001 0.0001

0.16 0.84

1.50