Recent Advances in Mitocondrial Medicine and Coenzyme Q10 9781536131499, 1536131490

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MEDICINE AND BIOLOGY RESEARCH DEVELOPMENTS

RECENT ADVANCES IN MITOCHONDRIAL MEDICINE AND COENZYME Q10

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MEDICINE AND BIOLOGY RESEARCH DEVELOPMENTS

RECENT ADVANCES IN MITOCHONDRIAL MEDICINE AND COENZYME Q10

ANNA GVOZDJÁKOVÁ GERMAINE CORNÉLISSEN AND

RAM B. SINGH EDITORS

Copyright © 2018 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected]. NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data ISBN: H%RRN

Published by Nova Science Publishers, Inc. † New York

DEDICATION This book is dedicated to the memories of four world famous medical doctors and scientists, who contributed greatly to the development of clinical and experimental Mitochondrial Medicine, to knowledge of Coenzyme Q10 and to its applications in clinical and experimental conditions. Professor Ján Gvozdják, MD, DSc. FECS, (Slovakia), WHO expert in Cardiomyopathy, founder of Mitochondrial Medicine in Slovakia, died in 2000.

vi

Dedication

Professor Frederick Loring Crane, PhD (USA), discoverer of Coenzyme Q10, passed away in 2016.

Professor Franz Halberg, PhD (USA), known worldwide as the “Father of Chronobiology”, passed away in year 2013.

Dedication

vii

Professor Miroslav Mikulecký, MD, DSc. (Slovakia), medical doctor specialist in chronobiology, who wrote a textbook on statistics for his students, died in 2015.

Anna Gvozdjáková Germaine Cornélissen Ram B. Singh

CONTENTS

List of Tables

xiii

List of Figures

xv

Preface

xxi

Acknowledgments

xxv

List of Contributors

xxvii

Chapter 1

Mitochondrial Medicine Anna Gvozdjáková, Rafael de Cabo and Plácido Navas

1

Chapter 2

Mitochondrial Physiology Anna Gvozdjáková

13

Chapter 3

Mitochondrial Free Radicals and Antioxidants Anna Gvozdjaková, Zdeňka Ďuračková and Ingrid Žitňanová

31

Chapter 4

Chronobiology of Mitochondria Germaine Cornélissen, Anna Gvozdjáková, Cathy Lee Gierke, Lyazzat Gumarova and Linda Sackett Lundeen

37

Chapter 5

Mitochondrial Modulation of the Epigenome and the Emergence of Cardiometabolic Diseases Ram B. Singh and Anna Gvozdjáková

57

Chapter 6

Mitochondrial Diseases Anna Gvozdjáková and Ram B. Singh

71

Chapter 7

Mitochondrial DNA Diana Drobná and Peter Celec

81

Contents

x Chapter 8

Mitochondrial Neurology Anna Gvozdjáková, Jarmila Kucharská and Boris Mravec

101

Chapter 9

Autism Spectrum Disorder and Mitochondrial Dysfunction Katarína Babinská, Anna Gvozdjáková, Jarmila Kucharská, Lenka Vokálová, Gabriela Repiská, Dalibor Nakládal and Daniela Ostatníková

115

Chapter 10

Mitochondrial Cardiology Anna Gvozdjáková, Jarmila Kucharská, Naranjan S. Dhalla and Fedor Šimko

131

Chapter 11

Mitochondrial Diabetology Jarmila Kucharská, Anna Gvozdjáková, Ram B. Singh, Viliam Mojto and Germaine Cornélissen

145

Chapter 12

Mitochondrial Nephrology Katarína Gazdíková and Viliam Mojto

155

Chapter 13

Mitochondrial Immunology Katarína Gazdíková

187

Chapter 14

Mitochondrial Oncology Patrik Palacka

211

Chapter 15

Mitochondrial Reproductive Medicine Anna Gvozdjáková, Jozef Dúbravický and Ram B. Singh

229

Chapter 16

Methods for Diagnosing Mitochondrial Disturbances Anna Gvozdjáková, Jarmila Kucharská, Zuzana Rausová and Anna Hlavatá

241

Chapter 17

Respirometric Analysis of Mitochondrial Function in Human Blood Cells Zuzana Sumbalová, Luiz F. Garcia-Souza, Beáta Veliká, Chiara Volani and Erich Gnaiger

255

Chapter 18

Coenzyme Q10 Targeting Therapy of Mitochondrial Disturbances Anna Gvozdjáková, Jarmila Kucharská, Rafael de Cabo, Luca Tiano and Plácido Navas

269

Chapter 19

Targeting Mitochondria with Natural Antioxidants Zdeňka Ďuračková, Branislav Trebatický, Jana Trebatická and Jana Muchová

293

Chapter 20

Mitochondrial Transplantation Anna Gvozdjáková and Ram B. Singh

325

Contents

xi

Chapter 21

Melatonin as a Natural Protector of Mitochondria Fedor Šimko

335

Chapter 22

The Effects of a High-Protein Diet on Mitochondria Anna Gvozdjáková, Jarmila Kucharská, Zuzana Paduchová, Mária Kubalová and Jana Muchová

339

Chapter 23

Omega-3-PUFA, Omega-6-PUFA and Mitochondrial Dysfunction in Relation to Remodelling Ram B. Singh, Anna Gvozdjáková, Jaipaul Singh, Sergey Shastun, Naranjan S. Dhalla, Daniel Pella, Ján Fedačko and Germaine Cornélissen

Chapter 24

Chapter 25

353

Balneotherapy and the Effect of Natural Mineral Water on Mitochondrial Function Anna Gvozdjáková

369

The Effect of Polarized Light and Coenzyme Q10 on Mitochondrial Function Anna Gvozdjáková and Alfonz Smola

379

About the Editors

389

Index

393

LIST OF TABLES Table 7.1. Protein coding genes in the mitochondrial genome

84

Table 7.2. Name, product and location of mitochondrial genes encoding transfer RNA

85

Table 7.3. Name, position and products of genes coding ribosomal RNA in the mitochondrial genome

85

Table 7.4. Categorization of organisms according the mitochondrial genome organization

87

Table 11.1. Maximal activity of OXPHOS parameters in the heart of control and diabetic rats

151

Table 12.1. Clinical signs of mitochondrial diseases

169

Table 16.1. Differential diagnosis of primary lactic acidemia

244

Table 18.1. Effect of ubiquinol on plasma CoQ10-TOTAL concentration in children with autism

274

Table 22.1. Weight classification by BMI (kg/m2)

340

Table 22.2. High-protein diets and their macronutrient composition

341

Table 22.3. Dietary intake

346

Table 23.1. Effect of ω-3-PUFA and CoQ10 supplementation on mitochondrial function

359

Table 23.2. Effect of ω-3-PUFA and CoQ10 supplementation on diabetic mitochondrial function

361

Table 24.1. The effect of drinking medicinal water in rats on coenzyme Q in myocardium

373

xiv

List of Tables

Table 24.2. The effect of drinking medicinal water in rats on myocardium OXPHOS

373

Table 25.1. Effect of liposomal coenzyme Q10 (Li-Q-Sorb) and polarized light on mitochondrial function in rats

384

LIST OF FIGURES Figure 1.1. Proposed new coenzyme Q binding site in the VDAC outer membrane of the Mitochondria.

4

Figure 1.2. Frederick Loring Crane.

5

Figure 1.3. Signature of Frederick L Crane (January 17, 2013).

6

Figure 1.4. From Frederick L Crane, Metamora, IL 61548, USA (January 28, 2013).

7

Figure 1.5. From Frederick L Crane, Metamora, IL 61548, USA (July, 2013).

7

Figure 1.6. From Frederick L Crane, Germantown Hls, IL 61548-8600 (December 12, 2013).

8

Figure 1.7. From Frederick L Crane, Germantown Hills, IL 61548 (December 20, 2014).

8

Figure 1.8. From Frederick L Crane and Marilyn, Germantown Hills, IL 61548 (December 20, 2014).

9

Figure 2.1. Scheme of mitochondria.

15

Figure 2.2. Krebs cycle.

18

Figure 2.3. Carnitine shuttle.

19

Figure 2.4. Glycerophosphate shuttle.

20

Figure 2.5. Malate-aspartate shuttle.

21

Figure 2.6. Gluconeogenesis.

22

Figure 2.7. Respiratory chain.

23

Figure 2.8. “Q10-CLOCK” of control myocardium mitochondria.

25

Figure 2.9. “Q10-CLOCK” of control brain mitochondria.

25

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List of Figures

Figure 2.10. The spatial pattern of the respiratory chain (with permission).

27

Figure 2.11. Supercomplexes of the respiratory chain (with permission).

28

Figure 3.1. ROS formation in the mitochondrial respiratory chain

34

Figure 4.1a. Example of staggered lighting regimens

40

Figure 4.1b. Shift in phase of circadian rhythm in serum corticosterone

40

Figure 4.2. Definition of rhythm characteristics.

42

Figure 4.3. The same determination can be too high, acceptable, or too low depending on when it is taken. © Halberg Chronobiology Center.

50

Figure 5.1. The mitochondrial genome (adapted from www.mitomap.org).

61

Figure 5.2. Effects of interaction of epigenome and environment on epigenetic markers, resulting in chronic diseases.

63

Figure 5.3. Role of oxidative stress in epigenetic damage leading to chronic diseases.

65

Figure 6.1. Mitochondrial diseases.

72

Figure 7.1. Map of mitochondrial human genome.

84

Figure 7.2. The structure of mitochondrial transcription factor A.

88

Figure 7.3. The activation of immune system by mitochondrial DNA.

92

Figure 8.1. Reduced coenzyme Q10 production with age.

106

Figure 8.2. Mitochondrial Alzheimer’s disease.

107

Figure 8.3. Mitochondrial Huntington’s disease.

108

Figure 8.4. Brain structures.

110

Figure 8.5. Basal concentrations of CoQ10-OX and CoQ9-OX in brain structures.

111

Figure 9.1. Famous people in history who may have been on the autism spectrum.

117

Figure 9.2. The effects of ubiquinol on hyperactivity and aggressive behavior in children with ASD.

124

Figure 9.3. The effect of ubiquinol on self-injury and destructive behavior in children with ASD.

124

Figure 9.4. The effects of ubiquinol on playing with friends and verbal communication in children with ASD.

125

Figure 9.5. The effect of ubiquinol on food rejection and sleep disorder in children with ASD.

125

List of Figures

xvii

Figure 9.6. The proposed new binding site of coenzyme Q in VDAC in the external membrane of mitochondria.

126

Figure 10.1. Electron microscopy of cardiomyocyte.

132

Figure 10.2. Basal respiration of mitochondria (V1) in EMB of patients after heart transplantation in correlation with rejection degrees

138

Figure 10.3. Stimulated respiration of mitochondria (VADP) in EMB of patients after heart transplantation in correlation with rejection degrees.

138

Figure 10.4. Correlation between CoQ10 concentration and rejection degrees in EMB of patients after heart transplantation.

139

Figure 10.5. Effect of HTx rejection on oxidative phosphorylation of mitochondria in EMB.

139

Figure 11.1. Relationship between brain and myocardium mitochondrial coenzyme Q9 and coenzyme Q10 concentrations in control and diabetic rats.

148

Figure 11.2. The relationship between OPR in brain and myocardial mitochondria in control and diabetic rats.

149

Figure 11.3. Relationship between brain and myocardium for mitochondrial S3 function in control and diabetic rats.

149

Figure 11.4. Biological clock “Q10-CLOCK” in myocardial mitochondria of control and diabetic animals.

152

Figure 12.1. Mechanisms of mtDNA and nDNA mutations resulting in mitochondrial disease.

161

Figure 12.2. Diagram of mitochondrial dysfunction in pathogenesis of kidney disease.

162

Figure 12.3. Acute renal damage during acute tubular necrosis.

168

Figure 12.4. Participation of mitochondria in the pathogenesis of renal carcinoma.

171

Figure 12.5. Proposed mechanism of doxorubicin-induced nephropathy [68]

173

Figure 13.1. Mechanism of specific cellular and humoral immunity.

190

Figure 13.2. Participation of Toll-like receptors in the defense against infectious pathogens and induction of the innate immune anti-inflammatory response.

193

Figure 13.3. The role of mitochondria in activating the immune response.

194

Figure 13.4. Status of mitochondria in triggering antiviral signaling.

195

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List of Figures

Figure 13.5. Mitochondrial dysfunction caused by HCV infection.

200

Figure 15.1. Sperm structure.

230

Figure 15.2. Potential mechanism of infertility.

231

Figure 15.3. Sperm movement depends on the concentrations of total coenzyme Q10 and vitamin E.

232

Figure 15.4. The effect of CARNI-Q-NOL® on the amount of sperm in seminal fluid.

236

Figure 15.5. The effect of CARNI-Q-NOL® on % pathological sperm in seminal fluid.

237

Figure 15.6. The effect of CARNI-Q-NOL® on CoQ10-TOTAL plasma concentration in men with fertility disorder.

237

Figure 15.7. Proposed mechanism of action of CoQ10 and carnitine on mitochondrial function.

238

Figure 17.1. Oroboros O2k-FluoRespirometer. Copyright by Oroboros Instruments. Reproduced with permission;www.oroboros.at

259

Figure 17.2. Respiration of intact PBMC.

261

Figure 17.3. Schematic illustration of convergent electron transfer at the NADHjunction in the N-pathway, and at the Q-junction in the combined NSpathway

262

Figure 17.4. SUIT protocol with permeabilized PBMC.

264

Figure 18.1. Professor Fred L Crane.

270

Figure 18.2. Q10-CYCLE, part of mitochondrial respiratory chain.

271

Figure 18.3. Professor Karl Folkers.

271

Figure 18.4. A dynamic balance between ubiquinone and ubiquinol.

272

Figure 18.5. Comparison of bioavailability of ubiquinol and ubiquinone.

275

Figure 18.6. The absorption of ubiquinol versus oxidized CoQ10.

275

Figure 18.7. Ubiquinol supplementation significantly increases mitochondrial coenzyme Q10 concentrations in the cerebrum of mice.

276

Figure 18.8. Professor Franz Halberg.

277

Figure 18.9. Professor Miroslav Mikulecký.

277

Figure 18.10. Biological clock of CoQ9 and CoQ10.

277

List of Figures

xix

Figure 18.11. CoQ10 concentration in the blood of healthy subjects and in patients with various diseases.

278

Figure 18.12. Mechanism of statins effect on cholesterol, dolichol and coenzyme Q10 production.

281

Figure 18.13. Statins-induced mitochondrial myopathy.

283

Figure 18.14. Transfer of CoQ10 through the pores of the outer mitochondrial membrane.

284

Figure 19.1. Relation between mitochondrial production of reactive oxygen species and mitochondrial function/dysfunction.

296

Figure 19.2. Basic flavonoid structures.

298

Figure 19.3. Biological properties of flavonoids and polyphenolic compounds in the organism.

300

Figure 19.4. Mutual relations between ischemia/reperfusion and cell damage. Potential sites of polyphenols intervention.

308

Figure 23.1. Chemical structure of oleic acid.

356

Figure 23.2. Chemical structure of LNA.

356

Figure 23.3. Chemical structure of LA.

356

Figure 23.4. Metabolism of -3-PUFA and of -6-PUFA.

357

Figure 23.5. Mechanism of myofibrillar (MF) and mitochondria (MT) remodelling associated with defects in energy utilization and energy production followed by cardiac dysfunction and heart failure in diabetes.

362

Figure 24.1. The influence of drinking mineral water from Lúčky baths on heart OXPHOS in rats.

374

Figure 25.1. Variation in tissue penetration as a function of wavelength (Biotherapy, Czech Republic).

381

Figure 25.2. Biological effects of polarized light (Biotherapy, Czech Republic).

382

Figure 25.3. Effect of coenzyme Q10 and polarized light on the mitochondrial respiratory chain.

385

Figure 25.4. Effect of PL therapy on diabetic leg (Pálinkáš J).

386

PREFACE This book “Recent Advances in Mitochondrial Medicine and Coenzyme Q10” is an updated version of an earlier monograph entitled “Mitochondrial Medicine”, edited by A. Gvozdjáková and published in 2008 by Springer, The Netherlands and of another more recent book written in Slovak “Mitochondriálna Medicína a Koenzým Q10” (“Mitochondrial Medicine and Coenzyme Q10”), edited by A. Gvozdjáková and published in 2017 by HERBA s.r.o., Slovakia. We thank all authors who contributed to this monograph, most of them world experts in mitochondrial medicine. The monograph provides current knowledge of mitochondrial physiology, mitochondrial diseases, new non-invasive methods of diagnosis of mitochondrial disturbances using isolated thrombocytes, and a new perspective of targeted therapy of mitochondrial diseases, such as mitochondrial transplantation and mitochondrial replacement therapy. Individual chapters provide not only current knowledge in basic research but they also review their practical applications in clinical mitochondrial medicine. The monograph is divided into 25 chapters, focusing on the physiology, chronobiology and epigenetic of mitochondria, mitochondrial diseases, as well as noninvasive diagnostic posibilities in isolated thrombocytes and targeted therapy of mitochondrial disturbances with natural antioxidants, melatonin, coenzyme Q10, carnitine, or with new avenues such as mitochondrial replacement therapy and mitochondrial transplantation. Polarized light and natural mineral water are shown to improve mitochondrial energy production. Chapter 1 presents the history of mitochondrial medicine and is written in memory of the discoverer of coenzyme Q10, Professor Frederick Loring Crane. It is presented by Anna Gvozdjáková, Rafael de Cabo, and Placído Navas, President of the International Coenzyme Q10 Association. Mitochondrial physiology is supplemented by mitochondrial chronobiology, circadian cascades of oxidative phosphorylation, the “Q10-CLOCK”, and dynamics of mitochondria, mitochondrial supercomplexes and sirtuins, presented by Anna Gvozdjáková in Chapter 2.

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Anna Gvozdjáková, Germaine Cornélissen and Ram B. Singh

Chapter 3, by Anna Gvozdjáková, Zdeňka Ďuračková and Ingrid Žitňanská, reviews mitochondrial free radicals and antioxidants. In Chapter 4, Germaine Cornélissen, Anna Gvozdjáková, Cathy Lee Gierke, Lyazzat Gumarova and Linda Sackett Lundeen introduce chronobiology, the study of biological rhythms, and reviews their role in life, notably for mitochondrial function. The chapter covers broad time structures beyond circadian rhythms, and outline how a chronobiological approach serves both diagnosis and treatment. Mitochondrial epigenetics, modification of the genome and interactions of genes and the environment are presented by Ram B. Singh and Anna Gvozdjáková in Chapter 5. Chapter 6, presented by Anna Gvozdjáková and Ram B. Singh, provides basic information about mitochondrial disturbances and diseases, their genetic and biochemical classification. An overview of mitochondrial DNA is provided in Chapter 7 by Diana Drobná and Peter Celec. These authors focus on the mitochondrial genome, mtDNA mutations, and the immune system from the perspective of mitochondrial replacement therapy. Chapter 8 is provided by Anna Gvozdjáková, Jarmila Kucharská and Boris Mravec. Mitochondrial neurology includes mitochondrial dysfunction in Down´s syndrome, in relation to aging, in Alzheimer´s and Parkinson´s diseases. The effect of ubiquinol on brain structures is also examined under experimental conditions. Chapter 9 focuses on relationships between autism spectrum disorders, pathophysiology and mitochondrial dysfunction. Katarína Babinská, Anna Gvozdjáková, Jarmila Kucharská, Lenka Vokálová, Gabriela Repiská, Dalibor Nakládal and Daniela Ostatníková review the benefitial effect of ubiquinol in children with autism. Chapter 10, presented by Anna Gvozdjáková, Jarmila Kucharská, Naranjan S Dhalla and Fedor Šimko, provides a comprehensive view of mitochondrial disturbances in cardiology. Mitochondrial cardiomyopathy, decreased mitochondrial energy production, and coenzyme Q10 in endomyocardial biopsies of the myocardium in patients after heart transplantation are topics addressed in this chapter. Chapter 11, by Jarmila Kucharská, Anna Gvozdjáková, Ram B. Singh, Viliam Mojto and Germaine Cornélissen, examines mitochondrial metabolism in diabetes. Results from the experimental laboratory are discussed, based on correlations between diabetic mitochondrial function of the myocardium and brain as compared to relations found in healthy controls. Chapter 12, written by Katarína Gazdíková and Viliam Mojto, deals with mitochondrial nephrology. The survey presents both kidney physiological metabolism and mitochondrial nephropathy, its diagnosis and targeting therapy. Updated knowledge on mitochondrial immunology is presented in Chapter 13 by Katarína Gazdíková. Chapter 14, written by Patrik Palacka, presents current knowledge of mitochondrial oncology and discusses potential targeted therapy with coenzyme Q10.

Preface

xxiii

Chapter 15, presented by Anna Gvozdjáková, Jozef Dúbravický and Ram B. Singh is concerned with mitochondrial spermatopathy. It reviews beneficial effects of supportive treatment with coenzyme Q10 and carnitine in infertile men. Current diagnostic methods of mitochondrial disturbances are presented in Chapter 16 by Anna Gvozdjáková, Jarmila Kucharská, Zuzana Rausová and Anna Hlavatá. A new, non-invasive method, respirometric analysis of mitochondrial function in human blood cells, is presented in Chapter 17 by Zuzana Sumbalová, Louiz F. GarciaSouza, Beáta Veliká, Chiara Volani and Erich Gneiger. Chapter 18, by Anna Gvozdjáková, Jarmila Kucharská, Rafael de Cabo, Luca Tiano and Plácido Navas, discusses coenzyme Q10 targeting therapy of mitochondrial disturbances. Chapter 19, by Zdeňka Ďuračková, Branislav Trebatický, Jana Trebatická and Jana Muchová, deals with targeting mitochondria with natural antioxidants. Chapter 20, provided by Anna Gvozdjáková and Ram B. Singh provides a new prospective possibility of mitochondrial transplantation and mitochondrial replacement therapy in mitochondrial diseases. Fedor Šimko introduces a new natural protection of mitochondria by melatonin in Chapter 21. Chapter 22, provided by Anna Gvozdjáková, Jarmila Kucharská, Zuzana Paduchová, Mária Kubalová and Jana Muchová, shows how targeting ketogenic and high-protein diets improves mitochondrial respiration and energy production. Chapter 23, by Ram B. Singh Anna Gvozdjáková, Jaipaul Singh, Sergej Shastun, Naranjan S Dhalla, Daniel Pella, Ján Fedačko and Germaine Cornélissen, provides current information on the omega-3-PUFA, omega-6-PUFA and mitochondrial dysfunction in relation to remodeling. Chapter 24 by Anna Gvozdjáková documents the beneficial effect of natural mineral water on mitochondrial function, stimulation of antioxidants production, and reduction of oxidative stress. In Chapter 25, Anna Gvozdjáková and Alfonz Smola document a new prospective therapeutic method in the experimental laboratory; it involves the effect of polarized light on mitochondrial function. The healing effect of polarized light on the wound of a diabetic patient’s foot is shown in photographs. This monograph is intended for medical practitioners, pharmacists and biochemists. It also serves students within pre- and post-graduate education. Editors Anna Gvozdjáková Germaine Cornélissen Ram B. Singh

ACKNOWLEDGMENTS My sincere thanks go to all contributors of this book, specialists in mitochondrial medicine and coenzyme Q10, who accepted my request to write individual chapters. Several experimental and clinical projects were possible with several VEGA Grants from the Ministry of Education and APPV Grants of Slovakia. I extend my greatest thank to Dr. Raj K. Chopra, President of Tishcon Corp., USA, for support and donations of different forms of coenzyme Q10 (Q-GEL FORTE®), ACTIVE Q® – ubiquinol, CARNI-Q-NOL®, LI-QH®) for our experimental and clinical studies.

Dr. Raj K. Chopra

My thanks go out to the Dr. Hosoe and Dr. Funahashi from Japan for supplementary figures and information on ubiquinol.

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Anna Gvozdjáková

Help from Mary Sampson and Linda Sackett-Lundeen, from the Halberg Chronobiology Center, University of Minnesota, USA, is also greatly acknowledged. My deep thanks go to my son Peter, Ing. Architect – atelier 2 for preparing the Cover of book and several color figures in the book. Anna Gvozdjáková

LIST OF CONTRIBUTORS 1. Katarína Babinská, MD, PhD, Comenius University in Bratislava, Faculty of Medicine in Bratislava, Institute of Physiology , email: [email protected] 2. Rafael de Cabo, Senior Investigator, PhD, National Institute of Aging, Baltimore, MD, US, email: [email protected] 3. Peter Celec, Associate Professor, MD, Dipl Ing., Dr. Rer. Nat., DSc., MPH., Institute of Molecular Biomedicine, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 4. Germaine Cornélissen, Professor, PhD, Halberg Chronobiology Center University of Minnesota, Minneapolis, MN, USA; email: [email protected]; Website: http://halbergchronobiologycenter.umn.edu/ 5. Naranjan S Dhalla, PhD, MD, (Hon), DSc (Hon), Distinguished Professor, Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre, Winnipeg, Canada, email: [email protected] 6. Diana Drobná, Institute of Molecular Biomedicine, Medical Faculty, Comenius University in Bratislava, Slovakia 7. Jozef Dúbravický, MD, Department of Urology, University Hospital in Bratislava, Slovakia email: [email protected]

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List of Contributors

8. Zdeňka Ďuračková, Professor, Dipl. Ing, PhD, Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Medical Faculty, Comenius University in Bratislava, Slovakia email: [email protected], [email protected] 9. Ján Fedačko, Associate Professor, MD, PhD, FICC, FICN 1st Department of Medicine UPJS Medical Faculty and L. Pasteur University Hospital, Centre of Excellency for Atherosclerosis Research, Košice, Slovakia email: [email protected] 10. Luiz Felipe Garcia e Souza, Institute of Sport Science, University Innsbruck, Innsbruck, Austria email: [email protected] 11. Katarína Gazdíková, Professor, MD, PhD, MPH, Department of General Medicine, Medical Faculty and Institute of Nutrition, Faculty of Nursing and Professional Health Studies Slovak Medical University, Bratislava, Slovakia, email: [email protected] 12. Erich Gnaiger, Professor, PhD, Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery Medical University of Innsbruck, Innsbruck, Austria Oroboros Instruments Corp., Innsbruck, Austria, email: [email protected] 13. Lyazzat Gumarova, Associate Professor, PhD, Al‐Farabi Kazakh National University, Almaty, Kazakhstan email: [email protected] 14. Anna Gvozdjáková, Professor, Dr, PhD, DSc, Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia email: [email protected]; [email protected] 15. Anna Hlavatá, MD, PhD, MPH, University Hospital Bratislava, Department of Paediatrics, Slovakia, email: [email protected] 16. Mária Kubalová, Bc, Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 17. Jarmila Kucharská, PharmDr, PhD, Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia email: [email protected]

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18. Cathy Lee Gierke, Researcher, MS, Halberg Chronobiology Center, University of Minnesota, Minneapolis, MN, USA. email: [email protected] 19. Viliam Mojto, Associate Professor, MD, PhD, MHA, 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 20. Boris Mravec, Professor, MD, PhD, Institute of Physiology, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 21. Jana Muchová, Associate Professor, Dr, PhD, Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 22. Dalibor Nakládal, PharmDr, Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 23. Plácido Navas, Professor, PhD, Universidad Pablo de Olavide, Sevilla, Spain, email: [email protected] 24. Daniela Ostatníková, Professor, MD, PhD, Institute of Physiology, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 25. Zuzana Paduchová, Dr, PhD, Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 26. Patrik Palacka, MD, Bc, PhD, MPH, MBA, LL.M, 2nd Department of Oncology and National Cancer Institute, Medical Faculty,Comenius University in Bratislava, Slovakia, email: [email protected] 27. Daniel Pella, Professor, MD, PhD, 1st Department of Medicine UPJS Medical Faculty and L. Pasteur University Hospital, Centre of Excellency for Atherosclerosis Research, Košice, Slovakia, email: [email protected] 28. Zuzana Rausová, Dr, PhD, Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia email: [email protected]

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List of Contributors 29. Gabriela Repiská, Mgr, PhD, Institute of Physiology, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 30. Linda Sackett Lundeen, Researcher, MT(ASCP), Halberg Chronobiology Center, University of Minnesota, Minneapolis, MN, USA, email: [email protected] 31. Sergej Shastun, Professor, People’s Friendship University of Russia, Moscow, Russia, email: [email protected]; 32. Jaipaul Singh, Professor, School of Forensic and Applied Sciences, University of Central Lancashire, Preston, UK; email: [email protected]; 33. Ram Bahadur Singh, Professor, MD, Dr, FICN, Halberg Hospital and Research Institute, Civil Lines, Moradabad, (UP)244001, India, email: [email protected], cell: 91 9997794102 34. Alfonz Smola, Professor, Ing, PhD, FEI, Slovak Technical University, Bratislava, Slovakia, email: [email protected] 35. Zuzana Sumbalová, Dr, PhD, Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria Pharmacobiochemical Laboratory 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia email: [email protected] 36. Fedor Šimko, Professor, MD, PhD, Institute of Pathophysiology, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 37. Luca Tiano, Associate Professor, MD, PhD, Institute of Biochemistry Polytechic University of MARCHE, Ancona, Italy, email: [email protected] 38. Jana Trebatická, MD, PhD, Department of Paediatric Psychiatry, and Child University Hospital, Medical Faculty, Comenius University in Bratislava, Bratislava, Slovakia 39. Branislav Trebatický, MD, PhD, Department of Urology and University Hospital, Bratislava, Slovakia, Medical Faculty, Comenius University in Bratislava, email: [email protected]

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40. Beáta Veliká, PhD, Pavol Jozef Šafárik University in Košice, Faculty of Medicine, Department of Medical and Clinical Biochemistry, Košice, Slovakia, email: [email protected] 41. Lenka Vokálová, Mgr, Institute of Physiology, Medical Faculty, Comenius University in Bratislava, Slovakia, email: [email protected] 42. Chiara Volani, Department of Internal Medicine II, Infectious Diseases, Immunology, Rheumatology and Pneumology, Medical University of Innsbruck, Innsbruck, Austria; email: [email protected] 43. Ingrid Žitňanová, MD, PhD, Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Medical Faculty, Comenius University in Bratislava, Slovakia

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 1

MITOCHONDRIAL MEDICINE Anna Gvozdjáková1,, Rafael de Cabo2 and Plácido Navas3 1

Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 2 National Institute of Aging, Baltimore, MD, US 3 Universidad Pablo de Olavide, Sevilla, Spain

ABSTRACT Mitochondrial medicine is one of the fastest growing interdisciplinary fields in medicine. This chapter includes some history of mitochondrial medicine and coenzyme Q10. The chapter is dedicated to the memory of Frederick Loring Crane, who discovered coenzyme Q10 in 1957 with colleagues at the University of Wisconsin-Madison Enzyme Institute.

Keywords: mitochondrial medicine, coenzyme Q10, Frederick Loring Crane

1.1. INTRODUCTION Mitochondrial medicine is one of the fastest growing interdisciplinary fields of clinical and experimental medicine, contributing to elucidate the pathobiochemical mechanisms of various diseases linked to mitochondria. Over the last decade there has been a substantial increase of mitochondrial publications dealing with the function of mitochondria in health and disease. 

Corresponding Author Email: [email protected] .

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This book focuses on mitochondrial physiology, mitochondrial diseases, diagnostic methods and targeting therapy of mitochondrial disturbances.

1.2. HISTORY OF MITOCHONDRIAL MEDICINE The first reports of mitochondrial disorders were published between 1959 and 1962 [1]. Professor Luft first described the clinical symptoms of a 30-year old female patient whose daily caloric intake was extremely excessive, about 3,000 kcal, at a stable body weight of 38 kg and normal thyroid function. The dominant clinical symptoms included weakness of skeletal muscle. The main laboratory finding was an abnormally increased basal metabolic rate. In isolated mitochondria from striated muscle, an uncoupling effect of oxidative phosphorylation was found together with increased proteins, especially cytochrome a and cytochorme a3 and low endogenous coenzyme Q10 concentrations. High RNA content in muscle homogenate was evidence for increased mitochondrial synthesis. The architecture of mitochondria was changed; ragged red fibers in skeletal muscle were found. This disease was termed “pleoconial myopathy,” where mitochondria accumulated in the muscle. More recently and by taking advantage of emerging immunochemical, immunohistochemical, analytical techniques and nuclear magnetic resonance, many other mitochondrial disorders have been reported in humans [1, 2]. Mitochondrial disorders of the skeletal muscle in patients with neuromuscular disorders were termed Luft´s disease or Luft´s syndrome. The formal development of mitochondrial medicine was first published by Professor Rolf Luft in 1994 [2].

1.3. HISTORY OF COENZYME Q10 DISCOVERY The discovery of coenzyme Q has led to research by many investigators. In 1950, Dr. Green at the University of Wisconsin wanted to find out how the beta-oxidation enzymes of the fatty acids and the Krebs cycle were organized and how they contributed to the “coupling” of oxidative phosphorylation, and ultimately the formation of energy in the mitochondria. To do this, it was necessary to obtain a large amount of mitochondria. Dr. Green received 12 bovine hearts from a processing plant in Madison daily. From these hearts, 80-100 g of mitochondrial protein was extracted, from which two flavoproteins, succinate dehydrogenase and NADH dehydrogenase were isolated. Cytochrome c oxidase (cytochrome a and a3) were obtained by using deoxycholate [3]. In 1962, Hatefi et al., reconstructed the complete electron transport system of the mitochondria isolated from heart and proved the presence of several vitamins of the B-group and α-tocopherol [3].

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In 1957, Dr. Frederick Loring Crane isolated mitochondria from cauliflower, which was yellow, therefore assuming the presence of carotenoids in the cauliflower mitochondria [4]. Dr. Crane then published the first report on the isolation of quinones in mitochondria from bovine hearts [5]. In 1958, Dr. Karl Folkers, a biochemist, known as the “Father of Coenzyme Q10 in the World,” and his coworkers, determined the chemical structure of CoQ10, and suggested that quinones are essential for the transport of electrons in mitochondria [6]. The first successful clinical application of coenzyme Q10 in patients with congestive heart failure was reported in 1967 by Dr. Yamamura from Japan. In 1972, Littarru and Folkers showed there was a decrease of CoQ10 in patients with heart disease [6]. Professor Peter Mitchell of England received the Nobel Prize in Chemistry in 1978 for the detection of ATP transfer from mitochondria to the cell which also includes CoQ10 in energy transfer [7]. Professor Lars Ernster of Sweden pointed out the important antioxidant properties of CoQ10 [8]. Later, technology was developed in Japan to obtain a large amount of CoQ10 that could be used for clinical studies [9]. Since then, an HPLC method has been used to determine the CoQ10 concentration in blood, plasma and tissues of patients. During this period, there was a large boom in clinical and experimental studies using coenzyme Q10 as a supportive treatment of various diseases, such as: impaired immunity, encephalomyopathy or ataxia, Parkinson’s and Huntington’s disease, oncological diseases, diabetes, aging [10]. In 2006, KANEKA Corporation (Japan) introduced a stable form of reduced CoQ10 on the market, Ubiquinol, was used in many studies as a supportive treatment for many diseases. Ubiquinol is one of the strongest antioxidants able to regenerate other antioxidants such as Vitamin E and Vitamin C. Ubiquinol inhibits the initial oxidation degree of proteins and interferes with the promotion of lipid peroxidation [11].

1.4. DISCOVERER OF COENZYME Q10 – FREDERICK LORING CRANE (1925 -2016) With great humility, respect and appreciation, I recall and remember the precious man, Professor Frederick Loring Crane, the discoverer of the coenzyme Q10. He has worked with our team to learn about our findings of the effects of ubiquinol in children with autism. These results led to a hypothesis about an alternative function of coenzyme Q10 possibly in the mitochondrial outer membrane permeability regulation, which depends on the VDAC (Voltage Dependent Anion Channel), at the new CoQ binding site in the VDAC outer membrane of the mitochondria (Figure 1.1).

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Legend: CoQ - coenzyme Q; VDAC – Voltage Dependent Anion channel; ATP - adenosine triphosphate; ADP - adenosine diphosphate; Pi - inorganic phosphorus; I, II, III, IV, V - respiratory chain complexes; H+ - proton; e- -electron; Q-cycle - Coenzyme Q cycle; Cyt c - cytochrome c; NADH reduced nicotinamide adenine dinucleotide; NAD+ - nicotinamide adenine dinucleotide; FADH2 reduced flavin adenine dinucleotide; FAD+ - flavin adenine dinucleotide; O2.- - superoxide radical; H2O2 - hydrogen peroxide; OH. - hydroxyl radical; H2O - water; O2 - oxygen; Figure 1.1. Proposed new coenzyme Q binding site in the VDAC outer membrane of the Mitochondria [12].

Professor Frederick Loring Crane left us forever in 2016. In May 2014, his latest publication was published: Crane FL, Low H, Sun I, Navas P, Gvozdjáková A: Plasma membrane coenzyme Q: evidence for a role in autism. Biologics 2014; 8: 199-205. (http://iquaproject.org/inmemoriam/) International Coenzyme Q10 Association, 7.9.2016 – In Memoriam – Frederick Loring Crane).

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IN MEMORIAM – FREDERICK LORING CRANE Frederick Loring Crane, a wandering scientist and a wonderful person!

Figure 1.2. Frederick Loring Crane.

Fred L. Crane was born on December 3, 1925, in a hospital in Montague, Massachusetts. He obtained his PhD in Botany in 1953 from the University of Michigan, Ann Arbor. Fred’s seminal and transcendent discovery of coenzyme Q in the respiratory chain of mitochondria was done at the Institute of Enzyme Research, at the University of Wisconsin-Madison (FL Crane, Y Hatefi, RL Lester, C Widmer (1957) Isolation of a quinone from beef heart mitochondria. Biochim Biophys Acta 25: 220-221). His discovery catalyzed a tremendous advance in mitochondrial bioenergetics and opened the field to demonstrate the importance of coenzyme Q in many aspects of health and disease, particularly in mitochondrial diseases. From 1957 until his death, he never stopped collaborating, mentoring, visiting and influencing many scientists around the world. He was interested and pledged during his last years to demonstrate the importance of coenzyme Q in the plasma membrane and its role in autism. In fact, his last paper published in May 29, 2014 in Biologics completed an amazing, long and highly productive scientific career, which left behind a tremendous imprint with more than four hundred papers and thousands of friends (and fans), not only in the scientific community but with anyone that crossed his path. Fred Crane had a strong influence on scientists of all ages and during most

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of his life he enjoyed long-term visits in research institutes and universities around the world, from Australia to Stockholm. He also had a keen sense of humor. I clearly remember an anecdote from the time he was a visiting professor in Córdoba: Fred started to do his own grocery shopping and went to the market to buy fruits and vegetables by himself. The first day he called me and said: “Placido, I need to improve my Spanish substantially, because today, when I asked the shop clerk for bananas, he gave me artichokes.” Soon, they became friends. He received many awards during his career, among them: the American Chemical Society Eli Lilly Award in Biochemistry (1961), Fulbright Award in Australia (1971), NIH Career Award (1964-1994), Silver Medal in Biochemistry, from the University of Bologna (1989), Doctor Honoris causa of Medicine at the Karolinska Institute (1989), and Folkers Foundation Award for Research on Coenzyme Q (1996). Personally, I was thrilled when he accepted to be a visiting professor at the University of Córdoba (1994-1995), a visit that transformed our scientific life and more. He was among the pioneers that founded the “International Coenzyme Q10 Association,” where he was Vice-Chairman (1997-2002). Now, he has finished his last chapter in life, leaving behind his memories in his book “A Wandering Professor,” from which I copied the title of this memorial, but most importantly, he has left all of us a path to follow, a tremendous legacy of what it is to be a great scientist, mentor and a friend. He is leaving a void in our hearts but his legacy will live within our thoughts and actions forever! With our greatest memories Rafael de Cabo and Plácido Navas. A selection of our last communications with Professor Frederick Loring Crane (Figures 1.3. – 1.8.).

Figure 1.3. Signature of Frederick L Crane (January 17, 2013).

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Figure 1.4. From Frederick L Crane, Metamora, IL 61548, USA (January 28, 2013).

Figure 1.5. From Frederick L Crane, Metamora, IL 61548, USA (July, 2013).

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Figure 1.6. From Frederick L Crane, Germantown Hls, IL 61548-8600 (December 12, 2013).

Figure 1.7. From Frederick L Crane, Germantown Hills, IL 61548 (December 20, 2014).

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Figure 1.8. From Frederick L Crane and Marilyn, Germantown Hills, IL 61548 (December 20, 2014).

Our joint publications with Professor Fred L Crane: 1. Teske, B. F., Sun, I. L., Gvozdjáková, A., Low, H., Crane, F. L. Plasma membrane CoQ, Porin, and Redox Control of Autism. In: Price ER, Johnson SC (eds), Quinones, Nova Science Publishers, Inc. 2013; 157-172. ISBN: 978-1-62618-3230. 2. Gvozdjáková, A., Kucharská, J., Ostatníková, D., Babinská, K., Nakládal, D., Crane, F. L. Ubiquinol supportive therapy in children with autism. Conference of Nutritional Science and Therapy, Philadelphia, USA, July 15 – 17, 2013. 3. Gvozdjáková, A., Takahashi, T., Singh, R. B., De Meester, F., Wilson, D. G., Crane, F. L. New roles of coenzyme Q10 in cardiovascular diseases discovered by a single group. World Heart Journal 2013; 5/3: 159-171. 4. Crane, F. L., Löw, H., Sun, I., Navas, P., Gvozdjáková, A. Plasma membrane coenzyme Q: evidence for a role in autism. Biologics 2014; 8: 199-205. 5. Gvozdjáková, A., Kucharská, J., Ostatníková, D., Babinská, K., Nakládal, D., Crane, F. L. Ubiquinol improves symptoms in children with autism. HINDAWI, Oxidative Medicine and Cellular Longevity 2014; Article ID 798 957, 6 pages, http://dx.doi.org/10.1155/2014/798957.

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Anna Gvozdjáková, Rafael de Cabo and Plácido Navas 6. Gvozdjakova, A., Mikulecky, M., Crane, F. L., Kucharska, J., Cornelissen, G., Kumar, A., Palacka, P., Singh, R. B. Mitochondrial cardiomyopathy and coenzyme Q10. World Heart Journal 2014; 6(1): 29-46. 7. Gvozdjáková, A., Crane, F. L., Kucharská, J., Singh, R. B., Mojto, V., Cornélissen, G. Recent advances in mitochondrial medicine in relation to CVDS and coenzyme Q10. 25th National Conference of Indian Society of Hypertension BPCON, November 20-22, 2015; Faridabad, India, 2015; Abstract Book: 37-38. 8. Gvozdjáková, A. (ed). Mitochondrial Medicine, Springer, Netherlands, 2008, pp. 419. This monography was dedicated to Prof. Frederick Loring Crane: 50. Anniversary of discover of Coenzyme Q10 and to my husband Prof. Ján Gvozdják, MD, DSc, which left us forever in 2000.

REFERENCES [1]

Luft, R., Ikkos, D., Palmieri, G., Ernster, L., Afzelius, B. A case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of mitochondrial respiratory control: a correlated clinical, biochemical and morphological study. J. Clin. Invest. 1962; 41: 1776-1804. [2] Luft, R. The development of mitochondrial medicine. Proc. Nat. Acad. Sci. USA 1994; 91: 8731-8738. [3] Crane, F. Discovery of ubiquinone (coenzyme Q) and an overview of function. Mitochondrion 2007; 7S: S2-S7. [4] Crane, F. L. Electron transport and cytochromes of subcellular particles from cauliflower buds. Plant Physiol. 1957; 32: 619-625. [5] Crane, F. L., Hatefi, Y., Lester, R., Widmer, C. Isolation of a quinone from beef heart mitochondria. Biochim. Biophys. Acta 1957; 25: 220-221. [6] Littarru, G. P., Ho, L., Folkers, K. Deficiency of coenzyme Q10 in human heart disease. Part I. Int. J. Vitam. Nutr. Res. 1972; 42(2): 291-305. [7] Mitchell, P.: The vital protonmotive role of coenyzme Q: In: Folkers K, Littarru GP, Yamagi T (eds). Biomedical and Clinical Aspects of Coenzyme Q, vol. 6, Elsevier, Amsterdam, 1991; 3-10. [8] Ernster, L. Facts and ideas about the function of coenzyme Q10 in the mitochondria. In: Folkers K, Littarru GP, Yamagi T (eds). Biomedical and Clinical Aspects of Coenzyme Q, vol. 6, Elsevier, Amsterdam, 1991; 15-18. [9] Ramasarma, T. A touch history and a peep into the future of the lipid-quinone known as coenzyme Q and ubiquinone. Curr. Sci. 2012; 102/10: 1459-1471. [10] Gvozdjáková, A. (ed). Mitochondrial Medicine, Springer, Netherlands, 2008; pp. 419.

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[11] Littarru, G. P. Ubiquinol: New insights into the most active form of Coenzyme Q10. Medicatrix 2014; pp. 111. [12] Gvozdjáková, A., Kucharská, J., Ostatníková, D., Babinská, K., Nakládal, D., Crane, F. L. Ubiquinol improves symptoms in children with autism. HINDAWI, Oxidative Medicine and Cellular Longevity, Volume 2014; Article ID 798957; http://dx.doi.org/10.1155/978957.

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 2

MITOCHONDRIAL PHYSIOLOGY Anna Gvozdjáková Pharmacobiochemical Laboratory of the 3th Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia

ABSTRACT Mitochondria, as subcellular organelles, are central integrators of intermediary metabolism. Mitochondria play many important roles in the cell/body as: the main energy producers in the body from carbohydrates, fats and proteins and also one of the main sites of reactive oxygen radical production. Mitochondrial sirtuins are involved in the regulation of metabolism. Mitochondria are dynamic particles and their complexes of respiratory chains make up more stable supercomplexes. Mitochondria play a crucial role in apoptosis. A key component of the respiratory chain, coenzyme Q10, acts as a Q-cycle, with its own circadian “Q10-CLOCK”.

Keywords: metabolism, mitochondria, oxidative phosphorylation, coenzyme Q10, chronobiology, supercomplexes, sirtuins

2.1. INTRODUCTION Mitochondria are subcellular organelles found in the cytosol of all cells. The quantity of cell mitochondria is different in individual organs. It depends on the function of the cell and the energy demand of the given organ. Thrombocytes contain between two and six



Corresponding Author Email: [email protected].

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mitochondria; most organs contain between 500 and 2,000 mitochondria/cell. However, in cardiomyocytes, mitochondria make up about 38% of the cell volume; the ovum contains 2,000 – 20,000 mitochondria/cell and the oocyte possesses close to 100,000 mitochondria. Various shapes and sizes of mitochondria from ring to oval, from small to big, can be found in cells.

2.2. HISTORY OF MITOCHONDRIA Between 1850 and 1900, several light-microscopic studies reported the presence of small granules in cells, similar in shape and size to bacteria. From 1900 to 1930, the mitochondrion became well defined. At that time, some functions of mitochondria were suggested: mitochondria serve as centers of genetic information, protein synthesis, lipid synthesis and respiration. In the 1930s, the Krebs cycle (citric acid cycle) and the urea cycle were formulated. Electron microscopy allowed the identification of mitochondria in the 1950s. Fatty acid oxidation, respiratory chain, and oxidative phosphorylation were identified in mitochondria. In the 1960s, the complexes of the respiratory chain were characterized. Coenzyme Q and cytochrome c were identified as mobile compartments of the respiratory chain. In 1964, circular mitochondrial DNA (mtDNA) was discovered. In the 1970s, Mitchell`s chemiosmotic hypothesis and mitochondrial “Q-cycle” were accepted. In the 1980s, all genes of mammalian mtDNA and in vitro mitochondrial protein import were identified. The first molecular causes of mitochondrial diseases were identified and documented. The genetic basis of mitochondrial diseases was demonstrated in several conditions: diabetes mellitus, neurodegenerative disorders (Parkinson’s disease, Alzheimer’s disease) and aging. In the 1990s, the crucial role of mitochondria in apoptosis was established. The important mitochondrial membrane function in the transport of small molecules or ions was proved by discovering mitochondrial contact sites, called the mitochondrial permeability transition pore [1]. The first publication of “Mitochondrial Medicine” appeared in 1994 [2]. The beneficial effect of coenzyme Q10 in the early stages of Parkinson’s disease was reported [3]. The mitochondrial “Q10-CLOCK” and circadian cascade of oxidative phosphorylation were detected in rats in 2004 [4, 5].

2.3. MITOCHONDRIAL ULTRASTRUCTURE AND FUNCTION The mitochondrion consists of four compartments: two membranes, the intermembrane space, and the matrix within the inner membrane (Figure 2.1). The outer mitochondrial membrane (OMM) separates the cytosol from the intermembrane space. The

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OMM is responsible for interfacing with the cytosol and its interactions with cytoskeletal elements, which are important for the movement of mitochondria within a cell. This mobility is essential for the distribution of mitochondria during cell division and differentiation [6]. The inner mitochondrial membrane (IMM) separates the intermembrane space from the matrix. The folding of the IMM (cristae) serves to increase the surface area of this membrane. Mitochondria are moving along intermediate actin filaments, using kinesin and dynein. In muscles, mitochondria are localized between myofilaments – interfibrillar mitochondria (IFM). They are involved in muscle contraction and supply ATP for the actomyosin complex. The function of subsarcolemmal mitochondria (SLM) is to maintain the membrane electrical potential [7].

Figure 2.1. Scheme of mitochondria [8].

2.3.1. Composition of the Mitochondrial Membranes a) Lipid composition of mitochondrial membranes: phospholipids (phosphatidyl choline - 40%, phosphatidyl ethanolamine – 28.4%, cardiolipin – 22.5%, phosphatidyl inositol – 7%). b) Protein composition of the inner mitochondrial membrane: external proteins (loose association with the membrane, soluble in water): succinate dehydrogenase, NADH-dehydrogenase, F1-ATP-ase, cytochrome c, cytochrome c1, cytochrome oxidase – subunits 4,5,6,7. Internal proteins (insoluble in water): cytochrome b, cytochrome oxidase – subunits 1, 2, 3, iron protein of coenzyme QH2-cytochrome c reductase, subunits 5, 7, 9 of oligomycin-sensitive ATPase [9].

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2.3.2. Functions of the Mitochondrial Components Each of the four mitochondrial components has specific functions. The outer mitochondrial membrane (OMM) exerts several functions, importing protein from the cytosol to the interior of mitochondria. Smaller molecules can diffuse to the intermembrane space, larger molecules are actively transported into the intermembrane space and matrix [10]. Fusion of the inner and outer mitochondrial membranes establishes contact sites, which are involved in protein import through two complexes: TOM (complex of transport outer membrane) and TIM (complex of transport inner membrane). Proteins from the matrix are transported across the outer mitochondrial membrane and inner mitochondrial membrane via the TOM/TIM complex located at the contact sites [11, 12]. Contact sites for multiprotein complexes are called the mitochondrial permeability transition pore (MPTP). Activation of the MPTP leads to depolarization and release of cytochrome c into the cytosol, which activates caspase-dependent apoptosis. MPTP is an early regulator of apoptosis. Further functions of OMM are ion transfer, oxidation of neuroactive aromatic amines and cardiolipin synthesis. The inner mitochondrial membrane (IMM) folds into the matrix to form cristae. Integral membrane proteins represent five complexes of the electron transport respiratory chain (Complexes I-IV), ATP synthase (Complex V, F1F0-ATPase), and the adenine nucleotide translocase (ANT). Electron transport through the respiratory chain generates the electrochemical gradient ( H+) necessary for ATP production.  H+ is known as proton-motive force, which includes the membrane potential ( ) and proton gradient ( pH) [13]. The intermembrane space contains cytochrome c as a mobile electron carrier for the respiratory chain. Release of cytochrome c into the cytosol initiates caspase enzyme activation. The matrix contains the machinery necessary to transcribe and translate the 13 proteins of oxidative phosphorylation. Most of the proteins involved in oxidative phosphorylation are encoded by the nuclear genome, synthesized in the cytoplasm, and imported into the mitochondria. In the matrix, there are several metabolic pathways, such as pyruvate dehydrogenase complex (PDC), oxidation of pyruvate to acetyl CoA, further oxidation of ketone bodies, amino acids, initiation of the urea cycle, fatty acid oxidation, citric acid cycle, process of importing proteins, heme synthesis, and suppression of free radical damage through manganese-superoxide dismutase (Mn-SOD).

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2.4. MITOCHONDRIAL ENERGY METABOLISM Sources of mitochondrial energy production are carbohydrates, fats and proteins. Carbohydrate metabolism (glycolysis) generates pyruvate, which can cross the mitochondrial membrane. Pyruvate is oxidatively decarboxylated to acetyl-CoA, which enters the Krebs cycle. Another method for acetyl-CoA production is -oxidation of fatty acids. Acetyl-CoA is further oxidized to CO2 by the Krebs citric acid cycle [8, 14, 15].

2.4.1. Krebs Cycle Pyruvate and fatty acids are transported from the cytosol into the mitochondria and processed through the Krebs (citric acid) cycle. Pyruvate converts into acetyl-CoA, catalyzed by the pyruvate dehydrogenase complex (PDC). Acetyl-CoA condenses with oxaloacetate to form citrate and then isocitrate. After some structural changes, dehydrogenation, decarboxylation and CO2 loss, -ketoglutarate is formed. By further decarboxylation and CO2 loss, succinyl-CoA is formed. Hydrolysis of succinyl-CoA to succinate releases free energy for the synthesis of guanosine triphosphate (GTP) from guanosine diphosphate (GDP) and inorganic phosphate (Pi). GTP in turn phosphorylates adenosine diphosphate (ADP) to adenosine triphosphate (ATP). The dehydrogenation of succinate is followed by hydration to form malate and after further dehydrogenation oxaloacetate is formed and the cycle is completed. The resulting high-energy intermediates (nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and reduced flavin adenine dinucleotide (FADH2)) are utilized in the inner mitochondrial membrane to make ATP, Figure 2.2.

2.4.2. Fatty Acid Oxidation Fatty acid oxidation occurs primarily in the mitochondrial matrix, while synthesis of fatty acids occurs in the cytosol. Fatty acids are important components of biological membranes; their main function is energy production. The metabolism of free fatty acids is complex. Acyl-CoAs, formed in the cytoplasm from fatty acids, cannot cross the inner mitochondrial membrane to enter the mitochondrial matrix. Fatty acids are activated before they enter the mitochondrial matrix; activation requires ATP and acyl-CoA synthase. ATP is hydrolyzed to ADP and inorganic phosphate. End products of beta-oxidation are acetyl CoAs and reducing equivalents. The reducing equivalents are translocated to the mitochondrial respiratory chain and acetyl CoA enters the citric acid cycle. Fatty acids are oxidized to produce water in the respiratory chain and carbon dioxide in the citric acid cycle.

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Figure 2.2. Krebs cycle [8].

2.4.3. Shuttle Systems Electrons from cytoplasmic NADH enter mitochondria by shuttle systems. The inner mitochondrial membrane is impermeable to NAD+ and NADH. Several shuttle systems are located in the mitochondria.

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2.4.3.1. Carnitine Shuttle Oxidation of long-chain fatty acids is possible with carnitine, via the carnitine shuttle system. The produced acylcarnitine is translocated through both mitochondrial membranes into the matrix by carnitine-acyl transferase I, II and carnitine-acyl translocase. As one molecule of acylcarnitine is passed into the matrix, one molecule of carnitine is translocated back to the cytosol and acylcarnitine is converted back to acyl CoA. As a result of the carnitine shuttle system, NADH and FADH2 are formed. The acyl CoA can then enter the beta-oxidation pathway. NADH and FADH2 are able to provide protons required for ATP formation by the activity of the Krebs cycle, Figure 2.3.

Figure 2.3. Carnitine shuttle [8].

2.4.3.2. Glycerophosphate Shuttle Electrons from NADH formed by glycolysis in the cytoplasm are carried across the mitochondrial membrane by glycerol-3-phosphate. This cytoplasmic reaction is catalyzed by glycerol 3-phosphate dehydrogenase (GPDH). GPDH is reoxidized to dihydroxyacetone phosphate on the outer surface of the inner mitochondrial membrane. Electrons from glycerol 3-phosphate are transferred to FAD of the mitochondrial glycerol dehydrogenase. This enzyme uses FAD as an electron acceptor. Dihydroxyacetone phosphate is formed in the oxidation of glycerol 3-phosphate; it then diffuses back into the cytosol to complete the shuttle. Inside the mitochondria, FADH2 transfers its electrons to coenzyme Q, which enters the respiratory chain. Consequently, ATP is formed when cytoplasmic NADH (transported by the glycerol phosphate shuttle) is oxidized by the respiratory chain. This shuttle is irreversible, Figure 2.4.

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Figure 2.4. Glycerophosphate shuttle [8].

2.4.3.3. Malate-Aspartate Shuttle Electrons from cytoplasmic NADH (in heart and liver) are transferred to the mitochondria by the malate-aspartate shuttle. Electrons are transferred from cytoplasmic NADH to oxaloacetate, malate is produced; it enters the inner mitochondrial membrane and is reoxidized by NAD+ in the matrix to form NADH. Oxaloacetate is produced, which cannot cross the inner mitochondrial membrane, and is then reoxidized by NAD+ in the matrix to form NADH. Oxaloacetate, which cannot cross the inner mitochondrial membrane is formed. Aspartate, which can be transported to the cytosol, is formed by further transamination. This shuttle is reversible. In addition, NADH can be brought into mitochondria by the malate-aspartate shuttle only when the NADH/NAD+ ratio is higher in the cytosol than in the matrix of mitochondria, Figure 2.5.

2.4.4. Ketogenesis Another way for acetyl CoA to metabolize in the liver is its conversion to acetoacetate, which is degraded to acetone. 3-hydroxybutyrate is formed by the reduction of acetoacetate in the mitochondrial matrix, which reaction is dependent on the ratio NADH/NAD+ in mitochondria. The condensation of two molecules of acetyl CoA to acetoacetyl CoA occurs in the mitochondria. Acetoacetyl CoA then condenses with another acetyl CoA to form HMG CoA (hydroxy methylglutaryl CoA). HMG CoA is metabolized into acetoacetic acid and acetyl CoA. The acetoacetic acid is reduced into beta-hydroxybutyrate (3hydroxybutyrate). Ketones can be used in muscle and brain, when glucose is in short supply.

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Figure 2.5. Malate-aspartate shuttle [8].

2.4.5. Gluconeogenesis Gluconeogenesis occurs in the cytosol but depends on the mitochondrial compartments for its initial substrate, oxaloacetate. Gluconeogenesis occurs primarily in the liver and kidney. In the initial step, the malate-aspartate shuttle provides reducing equivalents to the mitochondrial compartment via metabolite exchange across the mitochondrial membrane. The key enzyme in gluconeogenesis is PEPCK (phosphoenolpyruvate carboxykinase). The control of gluconeogenesis rests in part with the mitochondrial malate-aspartate shuttle. This shuttle works to transport reducing equivalents into the mitochondria and is stimulated by the influx of ADP in exchange for ATP. Malate is transported into the mitochondria, whereupon it gives up two reducing equivalents and is transformed into oxaloacetate. Oxaloacetate cannot cross the mitochondrial membrane, so it is converted to alphaketoglutarate in a coupled reaction that also converts glutamate to aspartate. Aspartate travels out of the mitochondria in exchange for glutamate. In the cytosol, the reactions are reversed. Aspartate is reconverted to glutamate and alpha-ketoglutarate is reconverted to oxaloacetate. In turn, oxaloacetate can be reduced to malate or decarboxylated to form phosphoenolpyruvate, Figure 2.6.

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Figure 2.6. Gluconeogenesis [8].

2.4.6. Urea Cycle The urea cycle is initiated in the hepatic mitochondria and completed in the cytosol. Urea is liberated from arginine via arginase and released into the circulation, whereupon it is excreted from the kidneys in the urine. Ornithine, the other product of the arginase reaction, is recycled back to the mitochondrion, again to make citrulline.

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2.4.7. Respiratory Chain and Oxidative Phosphorylation Five protein complexes of the respiratory chain are located in the inner mitochondrial membrane. Complex I: NADH dehydrogenase-ubiquinone oxidase, Complex II: succinate dehydrogenase-ubiquinone oxidoreductase, Complex III: ubiquinone cytochrome c oxidoreductase, Complex IV: cytochrome c oxidase, Complex V: ATP synthase. Cytochrome c and coenzyme Q10 are mobile components of the respiratory chain. Coenzyme Q can occur in oxidized form (ubiquinone, Q), reduced form (ubiquinol, QH2) and radical form (Q.), called “Q-CYCLE”. The first four complexes receive electrons from the catabolism of carbohydrates, fats, and proteins, and generate a proton gradient across the inner mitochondrial membrane. Complex I and Complex II collect these electrons and transfer them to coenzyme Q10, Complex III and Complex IV. Complex I, III and IV utilize the energy in electron transfer to pump protons across the inner mitochondrial membrane, producing a proton gradient, which is used by Complex V for ATP production from ADP and inorganic phosphate. The produced ATP is translocated from the mitochondria into the cytoplasm by adenine nucleotide translocase (ANT), Figure 2.7.

Figure 2.7. Respiratory chain [8].

The inner and outer mitochondrial membranes have numerous contact sites. Both of them contain a large assortment of integral and peripheral proteins as well as numerous phospholipids. The electron transport chain (ETC; Complexes I – IV) is localized in the inner mitochondrial membrane, associated with electron transfer components, coenzyme

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Q and cytochrome c. In the matrix, pyruvate oxidation, -oxidation of fatty acids, and the TCA cycle pathways are associated. The mitochondrial permeability transition pore (MPT) includes key components, such as the adenine nucleotide translocator (ANT), porin, mitochondrial creatine kinase (CK), hexokinase (HEX), and the inner membrane phospholipid, cardiolipin (CL). The release of apoptogenic peptides, e.g., cyt c from mitochondria precedes caspase activation, leading to apoptotic cell death. The carnitine shuttle pathway for the mitochondrial import of fatty acids includes carnitine palmitoyl translocase I, II (CPT-I, CPT II) and carnitine-acylcarnitine translocase (CACT). The generation of reactive oxygen species (ROS) comes from the mitochondrial electron transport chain (ETC). Mitochondria contain antioxidants such as MnSOD and glutathione peroxidase (GPx). The mtDNA is shown with transcripts (mtRNA), which are translated on mitochondrial ribosomes (mtribosome), forming peptide subunits of Complexes I, III, IV, and V.

2.5. MITOCHONDRIAL “Q10 – CLOCK” Coenzyme Q10 (ubiquinone), as a crucial mobile component of the respiratory chain of the inner mitochondrial membrane, acts in three forms in the “Q-CYCLE:” CoQ (ubiquinone, oxidized form), CoQH2 (ubiquinol, reduced form), and CoQ. (ubisemiquinone, radical form). The dominant human form of coenzyme Q is CoQ10, the dominant rat form is CoQ9. The central role of CoQ10 is electron and proton transfer between Complex I and Complex III, and between Complex II and Complex III. The global functions of CoQ10 are in cellular bioenergetics (ATP production), “redox poise” (ratio of reduced to oxidized form), metabolic flux modulation, gene regulation, and oxygen radical production. According to the hypothesis of Linnane and Eastwood [16], “CoQ10 redox poise” changes determine the key metabolic control function in all subcellular membranes, resulting in the signaling process. Evidence on bioenergetics and antioxidant functions of coenzyme Q10 was presented in several papers [4, 5, 8]. CoQ10 concentrations in plasma, tissues, and mitochondria of various organs show biological circadian rhythms [4, 5, 8]. The comparison of the biological Q10-CLOCK in myocardium and brain are shown in Figures 2.8 and 2.9, respectively. Heart mitochondrial circasemidian (12 hours) and circadian (24 hours) components yield statistically significant maxima (PEAKS) only for CoQ10-ox at 15:27 hours and 3:30 hours, with minima (NADIRS) for CoQ10-ox at 10:00 hours and 20:51 hours. CoQ9-ox PEAKS are not statistically significant. Nocturnal activity of rats (22:00 - 10:00 hours) is reflected in higher peaks versus diurnal peaks (10:00 - 22:00 hours). Biological rhythms in humans are opposite to those in rats, Figure 2.8.

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Figure 2.8. “Q10-CLOCK” of control myocardium mitochondria.

Circadian rhythmicity in brain mitochondria of control rats had maximal CoQ10 (PEAK1) at 07:28 hours and (PEAK2) at 19:24 hours, with minima (NADIRS 1, 2) at 14:04 hours and 01:03 hours. Rats nocturnal activity was between 22:00 and 10:00 hours (inactivity between 10:00 and 22:00 hours), Figure 2.9.

Figure 2.9. “Q10-CLOCK” of control brain mitochondria.

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We assume that the cascade of OXPHOS of myocardial mitochondria and the biological clock “Q10-CLOCK” play an important role in the pathogenesis of altered cardiac function. The “Q10-CLOCK” in the mitochondria may be a key part for the regeneration of the mitochondrial membrane and for re-energization of cardiac mitochondria. The circadian rhythms of myocardial mitochondrial CoQ10 and of ATP can contribute to the understanding of the triggering of acute attacks of the heart muscle [8, 17]. Changes of biological clocks “Q10-CLOCK” of the myocardial and brain mitochondria may play an important role in human mitochondrial diseases.

2.6. DYNAMICS OF MITOCHONDRIA Mitochondria are very dynamic structures that are continuously subject to fusion and cleavage. The dynamics of mitochondria are controlled by processes that regulate the morphology of mitochondria: 1. Biogenesis of mitochondria: by dividing the mitochondria, new mitochondria are created, increasing their number and/or volume. 2. Old or damaged mitochondria are removed by mitochondrial cleavage (fission). 3. Removal of damaged mitochondria provides autophagy, which is called myopathy (mitophagy); there is a large number of small and circulating mitochondria. 4. A small number of large and prolonged mitochondria are formed by mitochondrial fusion. The fusion regulates two outer membrane proteins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) and one dynamin-guanosine triphosphate protein (OPA1 gene), which is located in the internal membrane. Mitochondrial fusions are important for mitochondrial functions [18, 19].

2.7. SUPERCOMPLEXES OF THE RESPIRATORY CHAIN Supercomplexes of the respiratory chain are also called respirasomes. These complexes are active and indispensable for the formation of stable respiratory complexes. Respirasomes are found in various species and tissues such as the brain, heart, liver, kidney, skeletal muscle, mushrooms, plants [20, 21, 22]. By using an electron microscope, complex 3D models of complex I were found. In all organisms, complex I is L-shaped, consisting of a membrane arm and a matrix arm. Complex III (ubiquinol): cytochrome c oxidoreductase or cytochrome bc1 is a dimer, located mainly in the matrix. Each monomer consists of 11 subunits. Complex IV in bacteria consists of 4 subunits, in mammals complex IV forms 13 subunits. Complex V (mitochondrial ATP synthase): 2 main structures: F0 = transport of protons in the inner membrane of mitochondria and catalytic

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F1 in the matrix of mitochondria. Mitochondrial ATP synthase forms another 9 subunits. The spatial pattern of the respiratory chain is shown in Figure 2.10.

Figure 2.10. The spatial pattern of the respiratory chain [20] (with permission).

Supercomplexes form a monomer of complex I (which is L-shaped, formed by a membrane and matrix arm), complex dimer III and complex IV, in different amounts of monomer copies. Complex II is not included in supercomplexes. Connections of the individual complexes to spatial, three-dimensional (3D) supercomplexes were identified: Complex III and IV are joined to the complex I membrane arm and are in contact one next to another. Complex III is a dimer, complex IV is a monomer. Quinone binds to Complex I in the matrix arm and is closely associated with the matrix / membrane arm. Rieske Fe-S protein and cytochrome b are present in ubiquinone complex III. These subunits of complex III are affected. Complex I surrounds and joins the matrix/membrane arm. Complex IV interacts with cytochrome c via the major subunit II, which is surrounded by the cytochrome c binding site of complex III. Using a three-dimensional representation, the mobile electron support (ubiquinone or cytochrome c) has been found to have a binding site on each complex and is surrounded by the corresponding binding site of the subsequent respiratory chain complex. In supercomplexes, the electrons have a short diffusion distance, so electron transfer is available via supercomplexes compared to the liquid model. Complex III and IV are essential for the stability of complex I. Electron microscopy has confirmed that ATP synthase dimers and respiratory chains form more stable supercomplexes (Figure 2.11.) [20]. The importance of respirasomes is not fully elucidated. It is believed that supercompaction reduces oxidative damage and increases the safety of metabolism. Interaction of the respiratory chain complexes within supercomplexes leads to an explanation for the causes of mitochondrial diseases due to lack of complexes (such as arteriosclerotic vascular disease; activity of complex I and III is reduced, but complex of I and IV is damaged in Parkinson’s disease) [20, 21, 22].

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Figure 2.11. Supercomplexes of the respiratory chain [20] (with permission).

Decreased functions of the respiratory chain as well as changes in coenzyme Q10 concentrations, “Q10-CLOCK,” may be important in the pathogenesis of altered brain and myocardial function. These changes can help clarify the mechanism that triggers an acute heart attack.

2.8. MITOCHONDRIAL SIRTUINS Sirtuins form a group of proteins that are dependent on NAD+- dependent deacylases. Intracellular NAD+ concentration and NAD+/NADH concentrations respond sensitively to energy metabolism in cells. Sirtuins are cell energy balance sensors, regulating the cell’s metabolic response to changes in the bioavailability of many tissues. Sirtuins are sensors that are involved in regulating metabolism for stress and aging, regulating ATP production, apoptosis, and cell signaling. NAD+ is the basic coenzyme for converting energy into a cell that exhibits circadian rhythms, oscillations in the oxidative metabolism of mitochondria. In mammals, 7 types of sirtuins (SIRT1-SIRT7) have been reported, which are localized in various subcellular parts. SIRT1, SIRT6 and SIRT7 are in the cell nucleus, SIRT2 in the cytoplasm and SIRT3, SIRT4 and SIRT5 in the mitochondria [23, 24]. Mitochondrial sirtuins are essential for the normal function of mitochondria through interaction and modification of the amount of mitochondrial proteins. Sirt3 is included in the normal function of various mitochondrial proteins, including oxidation of fatty acids, ketogenesis, oxidative phosphorylation, antioxidant and amino acid metabolism. In response to metabolic stress, acetylation of metabolic proteins such as tricarboxylic acid enzymes, fatty acid beta-oxidation enzymes, and mitochondrial respiratory chain complexes occur. SIRT3 interacts with complex I and II subunits, binds ATP synthase, and

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regulates mitochondrial translation. The involvement in electron transport of SIRT4 and SIRT5 are less understood than SIRT3. SIRT4 binds the adenine nucleotide translocator (ANT), which transports ATP from mitochondria to the cytoplasm and ADP into the mitochondria matrix, serving as a substrate for ATP synthase. SIRT5 interacts with cytochrome c. The biological significance of these interactions is unknown [25, 26].

REFERENCES [1]

Scheffler, I. E. A century of mitochondrial research: achievements and perspectives. Mitochondrion 2001; 1: 3-31. [2] Luft, R. The development of mitochondrial medicine. Proc. Nat. Acad. Sci. USA 1994; 91: 8731-8738. [3] Shults, C. W., Oakes, D., Kieburtz, K., Beal, M. F., Haas, R., Plumb, S., Juncos, J. L., Nutt, J., Shoulson, I., Carter, J., Kompoliti, K., Perlmutter, J. S., Reich, S., Stern, M., Watts, R. L., Kurlan, R., Molho, R., Harisson, M., Lew, M. Parkinson study group: Effects of coenzyme Q10 in early Parkinson diseases – evidence of slowing of the functional decline. Arch. Neurol. 2002; 59: 1541–1550. [4] Gvozdjáková, A., Kucharská, J., Cornélissen, G., Mikulecký, M., Singh, R. B., Halberg, F. Variation in cardiac mitochondrial coenzyme Q10 and oxidative phosphorylation. Int. J. Cardiol. 2004; 97(2): S15. Third International Congress on Cardiovascular Disease, Taipei, Taiwan, 26-28 November 2004. [5] Gvozdjáková, A., Kucharská, J., Cornélissen, G., Mikulecký, M., Singh, R. B., Halberg, F. Circadian and semicircadian variations of heart mitochondrial coenzyme Q in relationship to oxidative phosphorylation. Fourth Conference of the International Coenzyme Q10 Association, Los Angeles, USA, 2005; 14-17 April 2005, Abstract Book: 113-115. [6] Capetanaki, Y. Desmin cytoskeleton: a potential regulator of muscle mitochondrial behavior and function. Trends Cardiovasc. Med. 2002; 12: 339-348. [7] Williams, R. S., Rosenberg, P. Calcium-dependent gene regulation in myocyte hypertrophy and remodeling. Cold Spring Harb. Symp. Quant. Biol. 2002; 67:337344. [8] Gvozdjáková, A. (ed). Mitochondrial Medicine, Springer, Netherlands, 2008; pp. 409. [9] Stryer, L. Biochemistry. Third edition. Freeman WH and Company, New York, 1988; pp. 1089. [10] Yaffe, M. P. The machinery of mitochondrial inheritance and behavior. Science 1999; 283: 1493-1497.

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[11] Endo, T., Yamamoto, H., Esaki, M. Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles. J. Cell Sci. 2003; 116: 3259-3267. [12] Ziegelhoffer-Mihalovičová, B., Kolár, F., Jacob, W., Tribulová, N., Uhrík, B., Ziegelhoffer, A. Modulation of mitochondria contact sites formation in immature rat heart. Gen. Physiol. Biophys. 1998; 17: 385–390. [13] Fleury, C. H., Mignotte, B., Vayssiere, J. L. Mitochondrial reactive oxygen species and apoptosis. In: Ebadi M, Marwah J, Chopra R (eds). Mitochondrial ubiquinone (Coenzyme Q10), Prominent Press, 2001; pp 361-398. [14] Montgomery, R., Conway, T. W., Spector, A. A. Biochemistry. A case-oriented approach. Fifth edition. CV Mosby Company, St. Louis, Baltimore, Philadelphia, Toronto, 1990; pp. 905. [15] Tzagoloff, A. Mitochondria, Plenum Press, New York and London, 1982; pp. 334. [16] Linnanne, A. W., Eastwood, H. Cellular redox poise modulation; the role of coenzyme Q10, gene and metabolic regulation. Mitochondrion 2004; 4: 779–789. [17] Gvozdjáková, A., Mikulecký, M., Crane, F. L., Kucharská, J., Cornelissen, G., Kumar, A., Palacka, P., Singh, R. B. Mitochondrial cardiomyopathy and coenzyme Q10. World Heart Journal 2014; 6(1): 29-46. [18] Ni Hong-Min, Williams JA, Ding WX. Mitochondrial dynamics and mitochondrial quality control. Redox Biology 2015; 4: 6-13. [19] Otera, H., Ishihara, N., Mihara, K. New insight into the function and regulation of mitochondrial fission. Biochim. Biophys. Acta 2013; 1833: 1256-1268. [20] Vonck, J., Schäfer, E. Supramolecular organization of protein complexes in the mitochondrial inner membrane. Biochim. Biophys. Acta 2009; 1793: 117-124. [21] Schäfer, E., Dencher, N. A., Vonck, J., Parcej, D. N. Three-dimenstional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria. Biochemistry 2007; 46(44): 12579-12585. [22] Acín-Pérez, R., Fernánbdez-Silva, P., Peletao, M. L., Pérez-Martos, A., Enriquez, J. A. Respiratory active mitochondrial supercomplexes. Molecular Cell 2008; 32: 529539. [23] Osborne, B., Bentely, N. L., Montgomery, M. K., Turner, N. The role of mitochondrial sirtuins in health and disease. Free Rad. Biol. Med. 2016; http://dx.doi.org/10.1016/j.freeradbiolmed. 2016.04.197. [24] Li, X., Kazgan, N. Mammalian sirtuins and energy metabolism. Int. J. Biol. Sci. 2011; 7(5): 575-587. [25] Haigis, M. C., Sinclair, D. A. Mammalian sirtuins: biological insights and disease relevance. Annu. Rev. Pathol. 2010; 5: 253-295. [26] He, W., Newman, J. C., Wang, M. Z., Ho, L., Verdin, E. Mitochondrial sirtuins: regulators of protein acylation and metabolism. Trend in Endocrinology and Metabolism 2012; 23(9): 467-476.

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 3

MITOCHONDRIAL FREE RADICALS AND ANTIOXIDANTS Anna Gvozdjaková1,, Zdeňka Ďuračková2 and Ingrid Žitňanová2 1

Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 2 Institute of Medical Chemistry, Biochemistry and Clinical Biochemistry, Faculty of Medicine, Comenius University in Bratislava, Slovakia

ABSTRACT Mitochondrial free oxygen radical production and antioxidant systems are important for living cells. The production of reactive oxygen species (ROS) serves about 2-4% of the oxygen produced via the respiratory chain. Mitochondria are also a source of nitrogen radicals. Mitochondrial antioxidant enzymes, such as manganese superoxide dismutase (MnSOD) and glutathione peroxidase, neutralize free radicals. Cu/Zn-SOD is located in the cytoplasm, but also in the intermembrane space of the mitochondria. Cytosol antioxidant enzymes are catalase and glutathione. CoQ10 as an antioxidant interferes with the propagation of free radical-mediated chain reactions.

Keywords: mitochondria, free radicals, antioxidants



Corresponding Author Email: [email protected].

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3.1. FREE RADICALS OF MITOCHONDRIA Mitochondria, on the one hand, are the main sources of free radicals and their reactive metabolites (ROS), and on the other hand, they contain various antioxidants that can eliminate free radicals and ROS [1, 2]. There is a dynamic balance between free radical formation and antioxidant capacity of the organism. In complex IV of the mitochondrial respiratory chain, more than 95% of oxygen is converted into water. In complex III of the respiratory chain, however, 2-4% of oxygen gets out of the control of antioxidants, resulting in the formation of free radicals and ROS [3]. A small number of generated radicals and ROS play a positive role in the organism. They are engaged in cell signaling and regulate biological functions of cells and their further “fate” [4]. In case of damage to the architecture, genetics, and mitochondrial functions, various organ defects or mitochondrial disorders may arise, belonging to the area of mitochondrial medicine. Free radicals are atoms, molecules, or fragments containing one or more unpaired electrons. Free radicals are mostly highly reactive substances which can extract a free electron from other substances, resulting in their oxidation. Therefore, they are also called oxidants [3]. Mitochondria are one of the major sources of ROS; more than 90% of cellular ROS are formed in the respiratory chain of mitochondria during aerobic metabolism (Figure 3.1). These include superoxide radical, abbreviated as superoxide (O2.-), hydrogen peroxide (H2O2) and highly reactive hydroxyl radical (.OH). Other reactive oxygen species include singlet oxygen (1O2) and peroxynitrite (ONOO-). If the dynamic balance is disturbed by uncontrolled, excessive formation of free oxygen radicals, proteins, lipids, DNA, and to the function of respiratory chain enzymes may be damaged. Primarily, a small portion of oxygen is reduced to superoxide (O2.-), which can then be converted to H2O2 by the enzyme Mn-SOD (manganese superoxide dismutase, located in the mitochondrial matrix) or Cu/Zn-SOD (superoxide dismutase containing copper and zinc ions, located in the intermembrane space of mitochondria). From H2O2 a highly reactive hydroxyl radical (.OH) and hydroxyl anion (OH-) are produced during the catalytic oxidation of iron: SOD .2O2 → O2 + H2O2 2H+

Fe2+/Fe3+ H2O2 → HO. + OH-

Mitochondria are also a source of nitrogen radicals: the radical of nitric oxide (NO) and nitrogen dioxide (NOO.). Nitric oxide (NO.) (hereinafter referred to as NO) regulates several systems in the organism, e.g., the cardiovascular system, the central nervous system, and the immune system. Mitochondria are an important site of NO production that contributes to their biological functions. NO is produced by oxidation of L-arginine to L-

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citrulline in a reaction catalyzed by NADPH-dependent NO synthase (nitric oxide synthase, NOS). L-arginin + O2 + NADPH → L-citrulline + NADP+ + H2O + NO NOS consists of 3 major isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Each isoenzyme is involved in the oxidation of L-arginine, which produces a small amount of NO and L-citrulline. The fourth isoform of NOS is the mitochondrial Ca2+-sensitive NOS (mtNOS) catalyzing NO formation in mitochondria [58]. NO regulates the oxygen consumption in mitochondria and the transmembrane potential by reversible reaction with cytochrome c oxidase. NO can bind to the cytochrome oxidase and block the major pathway of oxygen and energy generation [9]. NO reduces oxygen consumption by mitochondria, electrochemical and proton gradient, thereby reducing ATP production and decreasing Ca2+ uptake by mitochondria [10]. Nitric oxide from mitochondria is consumed by three pathways: 1. With superoxide radical: Under aerobic conditions, about 85% of NO is consumed by the rapid reaction of NO. with O2.- when a peroxynitrite anion (ONOO-) is formed, abbreviated as peroxynitrite, which induces oxidative stress. Peroxynitrite is a relatively stable molecule; it can diffuse to more distant locations from the place of its formation, where it can decompose to form a hydroxyl radical. It is therefore a strong oxidant: NO + O2.- → ONOOONOO- + H+ → ONOOH → NO2 + .OH 2. With ubiquinol: The reaction of NO with ubiquinol (UQH-) generates ubisemiquinone (UQ.), which subsequently forms O2.- by autooxidation in the propagation reaction: NO + UQH- → UQ. + H+ + NOUQ. + O2 → UQ + O2.Formation of superoxide leads to rapid reaction with NO to toxic ONOO- generation (the reaction under point 1.) 3. With cytochrome oxidase: NO with cytochrome oxidase inhibits the main pathway of oxygen consumption and energy generation [10].

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Figure 3.1. ROS formation in the mitochondrial respiratory chain (modified from [2]).

3.2. ANTIOXIDANTS OF MITOCHONDRIA Mitochondria are protected against uncontrolled formation of reactive oxygen species by antioxidant system [4], including manganese-dependent superoxide dismutase (MnSOD), catalase, glutathione peroxidase, cytochrome c, and coenzyme Q10. a. Superoxide dismutase (SOD) is an enzyme that eliminates the superoxide radical. Three types of SOD are identified in mammals: Cu/Zn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3, extracellular SOD). The redox active centers of these SODs are Cu ions for Cu/Zn-SOD (Zn ion does not enter the redox reaction, and plays a stabilizing role) and EC-SOD and Mn for Mn-SOD. Cu/Zn-SOD is located in the cytoplasm, but also in the intermembrane space of the mitochondria. The mitochondrial matrix contains the Mn2+ ion in the SOD active site by which it can eliminate the superoxide radical formed in the matrix or at the inner side of the mitochondrial membrane by a dismutation reaction to oxygen and hydrogen peroxide.

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Mn-SOD in mitochondria can reduce the concentration of superoxide and thus prevent its reaction with NO, leading to prevention of the peroxynitrite (ONOO-) formation. Generated peroxynitrite leads to oxidative stress associated with diseases, neuronal degeneration, and aging, or it might be a signal for the cell to start apoptosis. This is the basis of superoxide function in mitochondria, "Mitochondrial theory of superoxide" [11]. A significant biological oxidant H2O2 is formed from the superoxide by the catalytic effect of SODs. In the biological system, the toxicity of hydrogen peroxide is eliminated by catalase and glutathione peroxidase enzymes. In the intermembrane space, the superoxide concentration is regulated by 3 different mechanisms: 1. Cu/Zn-SOD enzyme found mostly in the cytoplasm of eukaryotic cells. 2. Cytochrome c, which can be reduced by the superoxide, resulting in oxygen formation. 3. Reduced cytochrome c can transfer electrons on to the cytochrome oxidase. Some electrons that have escaped the O2.- producing respiratory chain can rereduce cytochrome c and contribute to the generation of energy required for the transport of H+ via complex IV of the respiratory chain. The spontaneous dismutation of O2.- in the intermembrane space is facilitated by the low pH caused by the release of H+ associated with respiration. Under certain conditions, hydrogen peroxide formed by dismutation may be the major precursor to the formation of .OH radical. b. Glutathione peroxidase, associated with the mitochondrial membrane, known as the phospholipid-hydroxyperoxide glutathione peroxidase, specifically reduces the lipid peroxides associated with the membrane. c. Catalase, the major enzyme that detoxifies H2O2, is found in peroxisomes. It is also present in the myocardial mitochondria. d. Cytochrome c, transferring electrons, has a detoxifying role against ROS. e. CoQ (ubiquinol), in the presence of succinate, is a reducing agent in the elimination of various peroxides. CoQ is a source of O2.- when it is partially reduced to a radical form of semi-quinone. At the same time, it is an antioxidant that interferes with the propagation of free radical chain reactions [12].

REFERENCES [1]

Gvozdjáková A. Mitochondrial Medicine, Springer, Netherlands, ed. A. Gvozdjáková, 2008, p. 409.

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Ďuračková Z. Oxidants, antioxidants and oxidative stress. In: A. Gvozdjáková (ed.): Mitochondrial Medicine, Springer, Netherlands 2008; 19-49. [3] Ďuračková Z. Free radicals and antioxidants for non-experts. In: Systems Biology of Free Radicals and Antioxidants, Vol. 1. - Berlin: Springer, 2014. S. 3-38, ISBN 9783-642-30017-2. [4] Ďuračková Z. Some current insights into oxidative stress. Physiological Research 2010; 59: 459-469. [5] Tzagoloff A. Mitochondria, Plenum Press, New York and London, 1982; p. 334. [6] Endo T., Yamamoto H., Esaki M. Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles. J Cell Sci 2003; 116:3259-3267. [7] Fleury C. H., Mignone B., Vayssiere J. L. Mitochondrial reactive oxygen species and apoptosis. In: Ebadi M., Marwah J., Chopra R. (eds). Mitochondrial ubiquinone (Coenzyme Q10), Prominent Press, 2001:361-398. [8] Osborne B., Bentely N. L., Montgomery M. K., Turner N. The role of mitochondrial sirtuins in health and disease. Free Rad Biol Med 2016, http://dx.doi. org/10.1016/j.freeradbiolmed. 2016.04.197. [9] Schäfer E., Dencher N. A., Vonck J., Parcej D. N. Three-dimensional structure of the respiratory chain supercomplexes I1 III2 IV1 from bovine heart mitochondria. Biochemistry 2007; 46(44): 12579-12585. [10] Vonck J., Schäfer E. Supramolecular organization of protein complexes in the mitochondrial inner membrane. Biochim Biophys Acta 2009; 1793:117-124. [11] Otera H., Ishihara N., Mihara K. New insight into the function and regulation of mitochondrial fission. Biochim Biophys Acta 2013; 1833: 1256-1268. [12] Ni Hong-Min, Williams J. A., Ding W. X. Mitochondrial Dynamics and mitochondrial quality control. Redox Biology 2015; 4: 6-13. [2]

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 4

CHRONOBIOLOGY OF MITOCHONDRIA Germaine Cornélissen1,, Anna Gvozdjáková2, Cathy Lee Gierke1, Lyazzat Gumarova3 and Linda Sackett Lundeen1 1 2

Halberg Chronobiology Center, University of Minnesota, Minneapolis, MN, US Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 3 Department of Biophysics and Biomedicine, Al-Farabi Kazakh National University, Almaty, Kazakhstan

ABSTRACT Biological rhythms are the essence of life. They cover a wide range of frequencies, among which circadian rhythms have gained great interest in view of their ubiquity, partly endogenous nature now firmly documented at the molecular level, and wide-ranged involvement in health and disease. Circadian rhythms in organisms are synchronized by the lighting regimen and the feeding schedule. They are primarily orchestrated by a small brain area of roughly 20,000 neurons situated in the suprachiasmatic nuclei, while clock genes reside in almost every cell. Estimating quantitatively circadian (and other) rhythm characteristics is important in relation to both diagnosis and treatment since changes occurring during the development of a disease condition may affect the amplitude and/or phase as well as the average value and all rhythm parameters may be affected by treatment. After briefly examining circadian rhythms related to mitochondria, notably mitochondria of the heart and brain, we review early work on circadian, ultradian and infradian variation (components with a frequency higher or lower than one cycle per day, respectively). Consideration is given to the role of mitochondria in cellular metabolism, which is tightly coupled to circadian clocks.

Keywords: cellular metabolism, chronobiology, circadian, clock genes, feeding schedule, free-running, infradian, lighting regimen, mitochondria synchronizer, ultradian 

Corresponding Author Email: [email protected]; Website: http://halbergchronobiologycenter.umn.edu/.

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4.1. INTRODUCTION TO CHRONOBIOLOGY In addition to spatial organization, the temporal organization of all living organisms in time is very important. Biological rhythms are periodically recurring changes in the intensity and nature of biological processes and phenomena [1]. Biological rhythms are inherent to most if not all living organisms in one form or another and are noted at all levels of biological organization, from intracellular processes, tissues, organs and organ systems to the individual, populations, and the biosphere [1, 2]. Biological rhythms also span a broad frequency range, periods varying from milliseconds (activity of single neurons), seconds (heart beat and respiration), hours (about 90-minute basic rest-activity cycle), and days (circadian, circaseptan, and circatrigintan) to years and even decades [2]. Among them, the circadian rhythm plays a special role ever since its partly endogenous nature was placed on a solid molecular basis and core clock genes were shown to be implicated in major disease conditions [3]. For instance, circadian rhythms have been documented for the number of sex hormone receptors in the cytoplasm and the nuclei of hepatocytes in rats [4], the cytoplasmic glycogen volume and the axial ratio in mitochondria of rat hepatocytes [5], the volume of cytoplasm of acinar cells in rats [6], the mitotic index in the epithelium of the esophagus [7], and bioluminescence in Gonyaulax polyedra, known to be controlled by proton transfer from an acidic vacuole system to the scintillons [8].

4.1.1. Definition of Chronobiology Etymologically, chronobiology comes from the Greek “chronos” (time), “bios” (life), and “logos” (science). Chronobiology is a computer-aided objective quantification, mapping and investigation of mechanisms underlying biological rhythms, the fundamental mechanisms of life [9]. Its primary raison d’être is to replace a homeostatic view of physiology by an approach that accounts for rhythmic variation. In so doing, reference values can be refined by entering inside the conventional physiological range where earlier abnormalities of dynamic changes, such as the amplitude and phase of rhythmic variations, serve as additional endpoints to signal a heightened disease risk. When rhythms are mapped using inferential statistical methods, blunders can also be avoided by recognizing that physiological variables do not assume the same value at all times, and that a given intervention may lead not only to a difference in mean value but also to a difference in amplitude, phase, and/or period [9, 10].

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4.1.2. Circadian Rhythms Circadian rhythms were often referred to as 24-hour periodicities, found as if by accident, when investigators noticed consistent changes throughout the day while testing biological variables in blood or serum and physiological variables such as temperature or blood pressure. Early investigations by Franz Halberg studied the number of blood eosinophils at different times of the day in mice [11] and men [12]. It was not until around 1959 that the word circadian started to replace 24-hour periodicity. Franz Halberg wrote in 1959: “in discussions with Professor William McDonald (and others) of the Department of Classics at the University of Minnesota, the term ‘circadian’ was derived from ‘circa’ (about) and ‘dies’ (day); it may serve to imply that certain physiologic periods are close to 24 hours, if not of exactly that length.” [13]. The partly endogenous nature of circadian rhythms was first documented, statistically validated and quantified by objective numerical measures of the uncertainty of its characteristics, by Halberg in blinded mouse models [14, 15]. In blinded C mice and in mice born anophthalmic, the circadian period of rectal temperature deviated slightly but statistically significantly from 24 hours and from the period of control mice. Halberg’s experiments on mice established the phenomenon of free-running. They also documented the critical role of the eyes as transducers for the primary environmental synchronizer, the alternation between light and darkness. In other experiments, Halberg showed that the timing of circadian rhythms could be manipulated by changing the lighting regimen (i.e., the times when lights went ON and OFF in the experimental laboratory) [14]. For this reason, the time of light onset is often used as reference time. Known to many as “Zeitgeber” time, referring to it as “Hours After Light Onset (HALO)” time is preferred since the lighting regimen acts as a mere synchronizer of circadian rhythms rather than as a “Zeitgeber” or “time giver.” When animals have free access to food and water ad libitum, circadian rhythms are mostly synchronized by the lighting regimen. If food access is restricted during a portion of the day, this information needs to be specified as the feeding schedule is also a strong synchronizer of circadian rhythms [14-18]. The role of the lighting regimen as a synchronizer of circadian rhythms was very useful in many chronobiological studies which relied on staggered lighting regimens of 12 hours of light alternating with 12 hours of darkness (LD12:12), Figures 4.1a-b [14, 16]. Typically, prior to the start of study, a marker variable such as temperature is automatically monitored around the clock from animals in each room to make sure their circadian system has adjusted to their respective LD12:12 regimen. Circadian variation can then be obtained based on measurements taken during regular office hours from animals in each room. Caution needs to be taken, however, not to turn lights on when handling animals during the dark span, as this will disturb the circadian system (introducing phase shifts and reducing melatonin). Study designs based on two, three, or even six lighting regimens

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staggered by 12, 8 or 4 hours, respectively, were particularly useful to determine rhythmic changes in the susceptibility-resistance of animals to different anti-cancer drugs and to assess the circadian stage-dependent effect of other interventions [19]. Indeed, apart from the spontaneous rhythms, the response to a given stimulus applied under standardized controlled conditions of the laboratory also changes predictably depending on the circadian stage at which it is administered [15].

Figure 4.1a. Example of staggered lighting regimens consisting of 8 hours of light alternating with 16 hours of darkness. Six groups of animals are treated between 07:00 and 17:00 at one of 6 different circadian stages (at 2, 6, 10, 14, 18, and 22 Hours After Light Onset, HALO). From [16] (with permission).

Figure 4.1b. Shift in phase of circadian rhythm in serum corticosterone in female 15-month old D8 mice after inversion of lighting regimen. From [14] (with permission).

4.1.3. Core Clock Genes Underlying Circadian Rhythms Circadian rhythms are primarily orchestrated by a small brain area of roughly 20,000 neurons situated in the hypothalamus, directly above the optic chiasm, known as the suprachiasmatic nuclei (SCN). When both suprachiasmatic nuclei are destroyed, the circadian rhythm in telemetered temperature from freely moving animals exhibits a great

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amplitude reduction and a circadian acrophase advance [20, 21]. The SCN receive input from specialized photosensitive ganglion cells in the retina via the retinohypothalamic tract. Neurons in the ventrolateral SCN have the ability for light-induced gene expression. Melanopsin-containing ganglion cells in the retina have a direct connection to the ventrolateral SCN via the retinohypothalamic tract, where the signal allows synchronization of an organism’s circadian rhythms to the environmental light-dark cycle [22]. The circadian rhythm in the SCN is generated by a gene expression cycle in individual SCN neurons. This cycle has been well conserved through evolution and in essence is similar in cells from many widely different organisms that show circadian rhythms [22]. Core circadian “clock” genes are defined as genes whose protein products are necessary components for the generation and coordination of circadian rhythms. Evidence for a genetic basis of circadian rhythms at the molecular level in higher eukaryotes started with the discovery of the period (per) locus in Drosophila melanogaster in 1971 [23]. Analysis of per circadian mutants and additional mutations on Drosophila clock genes led to a model consisting of positive and negative autoregulatory feedback loops of transcription and translation. Similar models were later described in mammals and other organisms [24-26]. In mammals, including humans, the majority of identified clock components are transcriptional activators or repressors that modulate protein stability and nuclear translocation, and create two interlocking feedback loops [27]. In the primary feedback loop, CLOCK and BMAL1 heterodimerize in the cytoplasm to form a complex that, following translocation to the nucleus, initiates transcription of target genes such as the core clock genes “period” genes (PER1, PER2, and PER3) and two cryptochrome genes (CRY1 and CRY2). Negative feedback is achieved by PER:CRY heterodimers that translocate back to the nucleus to repress their own transcription by inhibiting the activity of the CLOCK:BMAL1 complexes [28]. Modern experimental approaches using systems biology have identified many novel components in biological clocks that suggest an integrative view on how organisms maintain circadian oscillation [29], but this is a topic beyond our scope herein. Studies in cyanobacteria suggested the presence of an alternative mechanism underlying circadian rhythms, since these single-cell organisms can maintain accurate 24hour timing in the absence of transcription. In other words, there is no requirement for a transcription-translation autoregulatory feedback loop for circadian rhythms to be manifested [30]. Circadian rhythms in cells lacking a nucleus, such as human red blood cells, also exist in the absence of transcription or genetic circuits, and therefore in the absence of a feedback loop [31]. Importantly, redox oscillations as demonstrated by peroxiredoxin rhythms have been described in different organisms covering the evolutionary tree, from eukaryotes to bacteria and archaea, suggesting that redox “clocks” may have preceded genetic feedback circuits, and that the latter may represent the major output mechanisms to coordinate cell and tissue physiology and behavior [32, 33].

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As noted above, a key feature of circadian rhythms is their ability to synchronize to external stimuli. The presence of cell autonomous oscillators in almost every cell in the body raises the question of how these oscillators are temporally coordinated. Major synchronizers of peripheral clocks in mammals include feeding, temperature, and oxygen. Feeding schedules and temperature cycles can synchronize peripheral clocks and even uncouple them from the SCN [34].

4.1.4. Estimating Circadian Rhythm Characteristics The simplest model representing changes recurring every day relies on the trigonometric sine and cosine functions, Figure 4.2. Such a model can be fitted to data by least squares, the principle underlying Halberg’s cosinor method [35]. Designed for the analysis of short and sparse time series focused on circadian rhythms, the cosine curve was selected so that light onset (0 HALO) or 00:00 could be equated to 0°. The model is characterized by four parameters: the MESOR (M, Midline Estimating Statistic Of Rhythm, a rhythm-adjusted mean), the amplitude (A, a measure of half the extent of predictable change within a cycle), the acrophase (, a measure of the timing of overall high values recurring in each cycle), and the period (, the duration of one cycle).

Figure 4.2. Definition of rhythm characteristics. The MESOR is a rhythm-adjusted mean; the double amplitude (2A) is a measure of the extent of predictable change within a cycle; the acrophase is a measure of the timing of overall high values recurring in each cycle, expressed in (negative) degrees in relation to a reference time set to 0°, with 360° equated to the period length; and the period is the duration of one cycle. © Halberg Chronobiology Center.

Often, in the case of circadian studies, the period can be anticipated to be 24 hours (or very close to 24 hours). In this case, regular least squares can be used to test the statistical

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significance of the rhythm and to estimate M, A and , with a measure of their uncertainties. In order for the acrophase to move clockwards, it needs to be expressed in negative degrees (with 360° ≡ 24 hours). As the cosinor method is applied to denser data and longer records, more complex signals can be modeled by the addition of harmonic terms, and additional time series analyses can be performed [36, 37].

4.2. CHRONOBIOLOGY OF MITOCHONDRIA Mitochondria are double membraned organelles found in the cells of all eukaryotic organisms. Eukaryotic cells (in single-cell and multiple-cell organisms) contain a defined nucleus (with a nuclear membrane surrounding the nucleus) and chromosomes, along with other organelles, each one with specific tasks to be performed in the cell. Mitochondria can be considered the power generators of the cell, converting oxygen and nutrients into adenosine triphosphate (ATP). ATP is a small molecule used in cells as a coenzyme and a nucleotide used in the formation of DNA and RNA. It is often referred to as the “molecular unit of currency” of intracellular energy transfer that powers the cell's metabolic activities [38]. Much was known about the mitochondrion as an organelle before its time structure was determined. Both circadian and circaseptan (about-weekly) variations in ATP have been documented [39]. In a series of experiments using staggered lighting regimens of 12 hours of light alternating with 12 hours of darkness, Gvozdjáková examined the circadian behavior of mitochondria in different organs [40-42]. Her team focused on the assessment of circadian rhythms of mitochondrial oxidative phosphorylation (OXPHOS) and coenzyme Q10 (CoQ10) in isolated mitochondria of the heart, brain and liver of experimental animals. Both 24-hour and 12-hour components were quantified based on samples collected at 6 different circadian stages, 4 hours apart. Two LD12:12 regimens staggered by 12 hours in two separate rooms were used in these experiments. In one room, lights were on from 10:00 to 22:00 and off from 22:00 to 10:00; an inverse lighting regimen was used in the other room.

4.2.1. Chronobiology of Myocardial Mitochondria Cardiac function relies on a time varying supply of oxidative energy produced by mitochondrial respiration. Mitochondrial dysfunctions in the heart muscle are associated with both structural and functional abnormalities [43]. Ablation of the circadian clock gene Bmal1 specific to the heart has been shown to result in cardiac mitochondrial defects, such as reduced enzymatic activities within the respiratory complex. Mice lacking cardiac Bmal1 function show decreased expression of genes associated with the fatty acid oxidative pathway, the tricarboxylic acid cycle, and the mitochondrial respiratory chain in the heart,

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and they develop severe progressive heart failure with age [43]. It has also been shown that similar changes in gene expression related to mitochondrial oxidative metabolism occur in C57BL/6J mice subjected to chronic reversal of the light-dark cycle; therefore, they show disrupted circadian rhythmicity. These findings indicate that the circadian clock system plays an important role in coordinating mitochondrial metabolism [43].

4.2.2. Circadian Variation of OXPHOS and CoQ10 in Myocardial Mitochondria ATP production in the mitochondrial respiratory chain in heart muscle and CoQ10 concentrations vary along the 24-hour scale in isolated mitochondria of the heart muscle of rats, as shown by Gvozdjáková et al. [40]. Changes in OXPHOS and CoQ10 myocardial mitochondria may be involved in the pathophysiology of the heart muscle. A low CoQ10 concentration and an impaired OXPHOS in heart mitochondria are considered to be a molecular basis for heart failure and can trigger an acute myocardial infarction, cardiac arrest, or stroke, all of which can end in sudden death [44]. Chest pain associated with acute myocardial infarction is more prevalent in the second quarter of the day [45]. In translating results from the experimental laboratory to humans, it should be noted that most circadian rhythms in diurnally-active humans have an opposite phase as compared to nocturnallyactive experimental animals (rats). Mitochondrial respiration takes place in the inner mitochondrial membrane in heart cells through a series of metabolic reactions using oxygen and nutrients in the cell to produce adenosine triphosphate (ATP), the source of energy in the cells. About 90% of the ATP in the heart is produced from these oxidation-reduction reactions in the mitochondria, with the mitochondria consisting of 20 to 40% of the cardiomyocyte volume [40]. A fatsoluble substance, also in the mitochondria, that participates in the cellular respiration process, generating energy in the form of ATP is CoQ10 [46]. The heart, along with the liver and kidney, are three of the organs in the body with the highest energy requirements; therefore, these organs have the highest concentrations of CoQ10 [47]. The function of the mitochondrial respiratory process in the heart muscle shows circadian variation in ATP production and in CoQ10 concentration [44]. In Gvozdjáková’s experiments, focus was placed on the 5 complexes of the mitochondrial respiratory process in the inner mitochondrial membrane. In particular, the circadian time structure of complex I (NADH dehydrogenase-ubiquinone oxidase) and complex II (succinate dehydrogenase-ubiquinone oxidase) was examined. The 24-hour and 12-hour components of isolated mitochondria in cardiac muscle of rats were estimated. The model for complex I and II shows two maxima and two minima within a 24-hour cycle. The function of OXPHOS can be evaluated in terms of several parts for complex I and complex II. The parts are S3 (S3 state; ADP-stimulated ATP production); S4 (State S4; basal

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respiration of mitochondria); OPR (oxidetive phosphorylation rate; the rate of ATP production); ADP:O (the coefficient of oxidative phosphorylation, which reflects coupling of oxidation and energy production); and RCI (the respiratory control index, which reflects the integrity of the mitochondrial membrane). These parts for complex I and II, as well as CoQ9 and CoQ10 are circadian stage-dependent. The phases of maxima and minima of the fitted model (24-hour and 12-hour components) characterize the “circadian cascade of oxidative phosphorylation” and the “biological clock of CoQ10-CLOCK and Q10-CLOCK.” Gvozdjáková and her team [40-42] reported maximal values of the OXPHOS parts for complex I to occur during rats’ inactivity and lasting 5 hours and 20 minutes, whereas during rats’ activity, they only lasted for 2 hours and 32 minutes. The opposite was true for complex II: during rats’ inactivity, they lasted only 2 hours and 47 minutes, but during their active span, they lasted 6 hours and 12 minutes. These results suggest that the respiratory chain of mitochondria could form super-complexes (complex I and II) in which activity of either complex I or complex II of OXPHOS is almost always high. A statistically significant circadian variation was reported only for CoQ10 (but not for CoQ9) in myocardial mitochondria of control animals. Circadian characteristics contribute to the understanding of the pathogenesis of altered cardiac function, such as the triggering of acute myocardial infarction [40, 44]. The “Q10CLOCK” in the mitochondria may play a key role in the regeneration of the mitochondrial membrane and for re-energizing cardiac mitochondria. In other studies, a number of components in the electron transport chain/OXPHOS in murine heart mitochondria have been found to exhibit a circadian peak around the middaylight hours, during the rats’ resting span [43]. When cardiomyopathy was induced with phenylephrine, the circadian rhythms were reportedly diminished in amplitude [43].

4.2.3. Circadian Brain Mitochondrial Cascade of OXPHOS and CoQ10 Circadian variations in mitochondrial OXPHOS cascade parameters were also reported in brain [44]. A two-component model consisting of cosine curves with periods of 24 and 12 hours was used to estimate the timing of the two maxima and two minima occurring along the 24-hour scale for complexes I and II, as reported elsewhere [44]. Circadian variation of the respiratory chain and of the “biological Q10-CLOCK” may contribute to the understanding of the pathogenesis of altered brain function and of mechanisms underlying the trigger of acute cerebral infarction. Mitochondrial dysfunction has a fundamental role in neurology, as the majority of patients with mitochondrial disease have some degree of neuropathology and this is usually degenerative in nature [48]. Damage to brain mitochondria at the metabolic level may be associated with age. Pathogenesis of mitochondrial disease involves damage of ATP, which is essential for life, apoptosis, production of reactive oxygen species, and for calcium physiology. Damage of

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brain mitochondrial oxidative phosphorylation and genetic disorders have a major impact on the dynamics of mitochondria, such as mobility, fission and fusion, and distribution. In the nervous system, the dynamics of mitochondria are essential for long-distance energy distribution. Astrocytes in the SCN display circadian rhythms in clock gene expression and extracellular accumulation of ATP. ATP release from astrocytes is a calcium-dependent process, and intracellular Ca++ concentrations fluctuate in an antiphase relationship with rhythmic ATP accumulation in cultures of immortalized rat suprachiasmatic nucleus cells (SCN2.2). Mitochondrial Ca++ rhythms were reported to be in almost exact antiphase with the peak in cytosolic Ca++. Given the calcium-dependent nature of ATP release, mitochondrial Ca++ seems to be integral to SCN rhythms [49]. Circadian fluctuations in ATP accumulation in the SCN have also been reported [49], exhibiting an average period of 23.7 hours. ATP concentrations in the rat SCN in vivo were marked by rhythmic variation during LD12:12 exposure or constant darkness, with peak accumulation occurring during the latter half of the dark phase or subjective night [49].

4.3. CHRONOBIOLOGY: BROAD TIME STRUCTURE CHARACTERIZING MITOCHONDRIA The importance of circadian rhythms in relation to mitochondria is apparent from experiments by Gvozdjáková and her team, and from studies published by others. Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Chronobiological aspects of mitochondria were studied at least since the 1960s. Both circadians and ultradians (variations with a frequency higher than one cycle in 20 hours) were reported. The prominent circadian rhythms involved in metabolism are important to ensure that development, survival, and reproduction remain synchronized to environmental changes along the 24-hour scale.

4.3.1. Early Work on Circadian Rhythms Related to Mitochondria Circadian variations in mitochondria were investigated by Heinz von Mayersbach, Karl Philippens, and their team as they used succinate dehydrogenase (SDH) as a marker enzyme to visualize mitochondria histochemically [50]. Using liver preparations, they documented a strong circadian variation in SDH activity, topographical distribution, and reaction-product appearance [50]. Philippens also found that the circadian rhythm in SDH activity was strongest in the periportal region of the liver lobules [51-53]. Circadian rhythms in liver SDH activity, albeit with opposite phase, were found in male and female

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rats, males peaking during the activity span and females during their rest span [51-53]. In rats fed ad lib, Philippens showed that inverting the lighting regimen for 6 weeks shifted the circadian rhythm of -glycerophosphate dehydrogenase (mGPDH) in isolated mitochondria in line with the shifted lighting regimen, while the circadian rhythm in SDH activity did not phase shift [54].

4.3.2. Ultradian Variations Related to Mitochondria Ultradian variations were extensively studied by Lloyd and coworkers [55, 56]. Lloyd et al. [55] reported respiratory oscillations in continuous yeast cultures that could be accounted for by cyclic energization of mitochondria, dictated by the demands of a temperature-compensated ultradian clock with a period of about 50 minutes. Electron transport components (NADH and cytochromes c and c oxidase) reportedly showed redox state changes as the organisms cycled between their energized and de-energized phases [55]. Lloyd and Murray [56] conclude that cellular auto-dynamism may be a function of a large ensemble of excitable intracellular components and that self-organization in time and space may encompass mitochondrial, nuclear, transcriptional and metabolic dynamics, coupled by cellular redox state. Their work suggests that ultradians are a widely occurring necessity for the coordination and coherence of living processes, rather than a curiosity only found in yeast [56].

4.3.3. Circadian Clocks and Cellular Metabolism Circadian rhythms in relation to mitochondria affect all fields of medicine. Their importance is apparent from numerous studies published in the areas of aging [57], brain [48], cardiac function [43], diabetes [58-60], metabolism [61-69], and mood disorders [70], among others. Circadian clocks are tightly coupled to cellular metabolism [71] and respond to lighting and feeding cycles, as originally shown by Franz Halberg [72]. Meal timing affects both circadian rhythms and metabolism [72, 73]. Caloric restriction achieved by means of intermittent energy restriction or time-restricted feeding reportedly forestalled and even reversed disease processes such as various cancers, cardiovascular conditions, diabetes, and neurodegenerative disorders [74]. Mitochondria are believed to play an integral part in these processes [74]. Nicotinamide adenine dinucleotide (NAD), involved in energy metabolism, may be involved in the interaction between the transcriptional/post-transcriptional delayed feedback loop and a complementary non-transcriptional-transcriptional coordination mechanism underlying the circadian system [75]. Numerous examples of inhibitors that

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affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms [76]. Evidence has recently been provided for self-sustained circadian oscillations of the hyperoxidation of the mitochondrial Peroxiredoxin, PrxIII, and cytosolic release of mitochondrial H2O2, which may constitute one biochemical output coupling metabolic changes and transcriptionalbased core clocks [77].

4.3.4. Circadian Rhythms as Indispensable Control: Methodological Considerations Establishing circadian rhythm characteristics in control animals is useful since they can then serve as reference for comparison of animals with different disease conditions and for testing the efficacy of different treatments, such as the supplementation with CoQ10 [9]. For such comparisons to yield useful information, circadian rhythm characteristics need to be estimated with a measure of uncertainty. The ranges in the timing of maxima and minima of different parts of the electron transport chain listed in studies by Gvozdjáková and her team [40-42, 44], however, are only point estimates. Such timings are associated with a fair amount of uncertainty, which depends on several factors: the rhythm’s prominence, the sample size (number of timepoints and number of animals per timepoint), and the amount of “noise.” The latter is contributed by several sources, including the amount of rhythm disturbance related to environmental conditions. In experiments by Gvozdjáková and her team, animals were housed in two different rooms kept on opposite LD12:12 lighting regimens. Lights were on from 10:00 to 22:00 and off from 22:00 to 10:00 in one room, while in the other room, they were on from 22:00 to 10:00. Since animals do not adjust instantaneously to a new lighting regimen, it is recommended to rely on a marker rhythm such as rectal temperature to make sure that animals have fully adjusted to their new routine before starting an experiment. This takes a minmum of one week, but in some experiments, up to 3 weeks were allowed for the animals to adjust before starting the study. Caution also needs to be taken not to disturb the animals once the study has begun. Turning lights on during the dark span can shift the phase of circadian rhythms. Usual noise from staff can also disturb the animals, and can provide them with unwanted clues about the 24-hour routine of workers in the laboratory. Such disturbances may have contributed to a larger-than-expected 12-hour component in the experiments by Gvozdjáková et al. [40-42, 44].

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4.3.5. Infradian Variations Related to Mitochondria The presence of intact food anticipatory activity in suprachiasmatic nucleus-ablated rodents or those lacking functional circadian oscillator genes [74] points to yet unidentified genes and circuits in eating pattern determination. It has been proposed that circadian coordination may be achieved by means of a distributed, decentralized system of oscillators, with contribution in gain setting by the metabolic hormones ghrelin and leptin [78]. The SCN may also be involved in the coordination of other-than-circadian rhythms, as suggested by a circaseptan amplification in dentin accretion after ablation of the SCN in Wistar rats [79]. Much evidence already supports the partly endogenous nature of circaseptan rhythms, which are most prominent in relation to growth, development, and repair [80]. Pertinent to the topic of this chapter, as demonstrated for the irradiation of four different kinds of tumor cells in culture, the about 7-day changes in relative ATP concentration were much larger than the circadian variation, higher tumor kill being achieved by irradiation at time of maximal -ATP [39]. These results open a new frontier for the optimization of treatment by timing (chronotherapy) according to circaseptan as well as circadian rhythms. Both components have already been implicated in various disease conditions, such as cardiovascular diseases, diabetes, immunology, and oncology [10].

4.3.6. Broad Chronobiological Applications for Diagnosis and Treatment Most physiological variables, including those related to mitochondria, are neither constant nor varying randomly. Rather, they follow rhythms with known periods, which render their variation predictable to some extent, the more so the larger their amplitude and the lesser the superimposed random variation. The rhythmic nature of these variables has important implications concerning the definition of reference values for screening and diagnostic purposes. First, the same value may be too high, acceptable, or too low depending on the time of its determination, Figure 4.3. The physiological range can thus be refined by accounting for rhythms. Second, abnormality does not need to be restricted to the mean value, as the amplitude, phase, and/or period of a rhythm may also change in the presence of disease or pre-disease [2, 3]. Screening for changes in all rhythm parameters is useful to diagnose disease earlier, thereby providing the opportunity to institute countermeasures earlier.

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Figure 4.3. The same determination can be too high, acceptable, or too low depending on when it is taken. © Halberg Chronobiology Center.

The effect of external stimuli on physiology is also circadian stage-dependent [15]. This means that treatment can be optimized by timing according to rhythms. The aims of chronotherapy are two-fold: maximizing treatment efficacy and minimizing negative side effects. Chronotherapy targets effects on all rhythm parameters in an attempt to restore healthy rhythmic patterns. For instance, it is common to find disease conditions associated with a dampened circadian variation, while a more robust circadian system reflects good health [3]. Optimal treatment timing is therefore best implemented individually, where chronotherapy is determined based on the chronodiagnosis in an approach known as chronotheranostics [10].

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[18] Cornélissen G. When you eat matters: 60 years of Franz Halberg's nutrition chronomics. The Open Nutraceuticals Journal 2012; 5(Suppl 1–M1): 16. [19] Halberg F, Klein P, Kabat H. Circadian rhythms and drug dosing. In: Biological Rhythms, Documenta Geigy, Basel, Switzerland, Ciba-Geigy Ltd., 1980; pp. 4–6. [20] Powell EW, Halberg F, Pasley JN, Lubanovic W, Ernsberger P, Scheving LE. Suprachiasmatic nucleus and circadian core temperature rhythm in the rat. Journal of Thermal Biology 1980; 5: 189–196. [21] Powell EW, Pasley JN, Scheving LE, Halberg F. Amplitude-reduction and acrophase-advance of circadian mitotic rhythm in corneal epithelium of mice with bilaterally lesioned suprachiasmatic nuclei. The Anatomical Record 1980; 197: 277-281. [22] https://en.wikipedia.org/wiki/Suprachiasmatic_nucleus. [23] Konopka RJ, Benzer S. Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci USA 1971; 68(9): 2112–2116. [24] Ralph MR, Menaker M. A mutation of the circadian system in golden hamsters. Science 1988; 241(4870): 1225–1227. [25] Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K, Lee CC, van der Horst GT, Hastings MH, Reppert SM. Interacting molecular loops in the mammalian circadian clock. Science 2000; 288(5468): 1013–1019. [26] Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ. An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 2001; 291: 1040–1043. [27] Ko CH, Takahashi JS. Molecular components of the mammalian circadian clock. Human Molecular Genetics 2006; 15(Spec No 2): R271–R277. [28] Lowrey PL, Takahashi JS. Mammalian circadian biology: elucidating genome-wide levels of temporal organization. Annual Review of Genomics and Human Genetics 2004; 5: 407–441. [29] Baggs JE, Price TS, DiTacchio L, Panda S, Fitzgerald GA, Hogenesch JB. Network features of the mammalian circadian clock. PLoS Biology 2009; 7(3): e52(13 pp.). [30] Tomita J, Nakajima M, Kondo T, Iwasaki H. No transcription-translation feedback in circadian rhythm of KaiC phosphorylation. Science 2005; 307(5707): 251–254. [31] O'Neill JS, Reddy AB. Circadian clocks in human red blood cells. Nature 2011; 469(7331): 498–503. [32] Edgar RS, Green EW, Zhao Y, van Ooijen G, Olmedo M, Qin X, Xu Y, Pan M, Valekunja UK, Feeney KA, Maywood ES, Hastings MH, Baliga NS, Merrow M, Millar AJ, Johnson CH, Kyriacou CP, O'Neill JS, Reddy AB. Peroxiredoxins are conserved markers of circadian rhythms. Nature 2012; 485(7399): 459–464. [33] Loudon AS. Circadian biology: a 2.5 billion year old clock. Current Biology 2012; 22(14): R570–R571.

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[47] Aberg F, Appelkvist EL, Dallner G, Ernster L. Distribution and redox state of ubiquinones in rat and human tissues. Archives of Biochemistry and Biophysics 1992; 295(2): 230–234. [48] Brown GK. Congenital brain malformations in mitochondrial disease. J Inherit Metab Dis 2005; 28(3): 393–401. [49] Burkeen JF, Womac AD, Earnest DJ, Zoran MJ. Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes. J Neurosci 2011; 31(23): 8432–8440. [50] von Mayersbach H. An overview of the chronobiology of cellular morphology. In: Reinberg A, Smolensky M, eds. Biological Rhythms and Medicine – Cellular, Metabolic, Physiopathologic, and Pharmacologic Aspects, New York, SpringerVerlag, 1983; pp. 47–78. [51] Philippens K. Twenty-four hour periodicity of succinodehydrogenase in rat liver. III. Int Congr Histochem Cytochem New York, 1968; pp. 206–207. [52] Philippens K. Tages rhythmische Schwankungen im Succino-dehydrogenase system. [Daily rhythmic fluctuations in the succino-dehydrogenase system.] In: Hettler LH, ed. Abhdlg Dtsch Akad Wiss Berlin. Berlin: Akademie Verlag, 1970; pp. 607–610. [53] Philippens K. Vergleichende Untersuchungen uber biochemische Aktivitatsbestimmungen an Mitochondrien und histochemischem Reaktionsausfall. [Comparative studies on biochemical activity determinations on mitochondria and histochemical reaction failure.] Acta Histochem Suppl 1971; 10: 323–332. [54] Philippens K. Circadian activity patterns of two rat liver mitochondrial enzymes. Succinatedehydrogenase (SDH) and -glycerophosphate dehydrogenase (mGPDH). Int J ChronobioI 1973; 1: 350. [55] Lloyd D, Eshantha L, Salgado J, Turner MP, Murray DB. Respiratory oscillations in yeast: clock-driven mitochondrial cycles or energization. FEBS Letters 2002; 519: 41–44. [56] Lloyd D, Murray DB. The temporal architecture of eukaryotic growth. FEBS Letters 2006; 580: 2830–2835. [57] Gong C, Li C, Qi X, Song Z, Wu J, Hughes ME, Li X. The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver. Chronobiol Int 2015; 32(9): 1254–1263. [58] Lee J, Liu R, de Jesus D, Kim BS, Ma K, Moulik M, Yechoor V. Circadian control of β-cell function and stress responses. Diabetes Obes Metab 2015; 17(Suppl 1): 123–133. [59] Vieira E, Merino B, Quesada I. Role of the clock gene Rev-erb in metabolism and in the endocrine pancreas. Diabetes Obes Metab 2015; 17(Suppl 1): 106–114. [60] van Moorsel D, Hansen J, Havekes B, Scheer FA, Jörgensen JA, Hoeks J, Schrauwen-Hinderling VB, Duez H, Lefebvre P, Schaper NC, Hesselink MK, Staels

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In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 5

MITOCHONDRIAL MODULATION OF THE EPIGENOME AND THE EMERGENCE OF CARDIOMETABOLIC DISEASES Ram B. Singh1, and Anna Gvozdjáková2 1

2

Halberg Hospital and Research Institute, Civil Lines, Moradabad, India Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia

ABSTRACT Mitochondria – the major energy sources of the cell, producing energy (ATP) through oxidative phosphorylation (OXPHOS) – are heterogeneous and essentially contribute to cellular functions. Mitochondrial dysfunction compromises overall cell functioning, and is associated with tissue damage and diseases. Mitochondrial OXPHOS is coordinated by both mitochondrial DNA and nuclear genomic DNA. In many cellular processes, including apoptosis, aging and oxidative metabolism, mitochondria perform a central role, which varies in different tissues. The brain, heart and muscle are considered tissues with high energy usage and contain a large number of mitochondria, allowing them to be more susceptible to a reduction in aerobic metabolism. In mitochondrial dysfunction, the mitochondrial electron transport chain (ETC) is injured or there are mutations of mitochondrial DNA. The human mitochondrial genome is a 16.6 kb circular DNA encoding for 13 proteins in the ETC and the displacement loop (D-Loop). Mitochondrial DNA consists of 37 genes, 22 encoding for transfer RNAs (tRNAs), two for ribosomal RNAs (rRNAs), and 13 encoding for proteins important in the electron transport chain. Each of these 13 proteins is directly involved in the coordination of cellular respiration, generating the majority of ATP required for the process. Epigenetic modifications of the genome occur due to interactions of genes and the environment, via the biological 

Corresponding Author Email: [email protected].

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Ram B. Singh and Anna Gvozdjáková regulatory system without altering the DNA sequence. Of all the epigenetic processes, DNA methylation is perhaps the best understood epigenetic adaptation and most common DNA modification. This mechanism plays an important role in regulating the gene expression of many biological processes and has wide-ranging effects on health. Epigenetic damage and its dysfunction have been described in many human diseases with a relevant role of epigenetic marks with obesity and its co-disease susceptibility. While nuclear DNA methylation is a well-established feature, very little attention has been devoted to mitochondrial epigenetics. Aging presents specific epigenetic markers, which may be part of physiopathological processes undergone during the onset of chronic diseases. The next challenge will be the manipulation of this mitochondrial modified epigenome by the use of molecules which may modulate oxidative stress that damages the epigenome, resulting in to DNA methylation, histone modification and mRNA alterations.

Keywords: epigenetics, mitochondria, oxidative stress, environmental risk factors, inflammation

5.1. INTRODUCTION The ultrastructures of cells and genomes were unknown to Darwin. Despite this lack of knowledge, Darwin wrote in The Origin of Species: “Natural selection can act only by taking advantage of slight successive variations; it can never take a leap, but must advance by the shortest and slowest steps.” Julian Huxley, a British scientist, published his landmark Monograph; ‘Evolution: The Modern Synthesis in 1942’. This monograph brought Darwin’s ideas into the 20th century and incorporated a knowledge of genes that was emerging in this century in the light of Gregor Mendel’s experiments on inheritance (Monograph: Experiments with Plant Hybrids) at Hynčice (Vražné) in the now Czech Republic. In the mid-century, Barbara McClintock discovered transposable elements, where parts of the genome can jump around and cause mutations or alter gene expression, skewing Mendelian ratios and inheritance patterns. Epigenetic changes may occur in the same generation due to environmental factors and manifest health or disease in the near future [1-4]. Epigenetic alterations during fertilization of the egg can cause obesity and metabolic syndrome in the offspring, which may be a metabolic manifestation of mitochondrial DNA alteration [3-6]. Genotype manifests due to interactions of the environment on genes and epigenes, which appear to play important roles in the development of phenotypes. In the last two decades, environmental factors in general and nutrition in particular, have been shown to be important in the pathobiology of genetic variations and epigenetic inheritance, with underlying mitochondrial alterations, leading to the emergence of chronic diseases of affluence [1-6]. In diverse eukaryotic lineages, the evolution of the mitochondrial genome has taken radically different pathways; the organelle itself is increasingly viewed as a genetic and functional mosaic, with the bulk of the mitochondrial proteome having an evolutionary

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origin [1]. During mitochondrial evolution, the question may be asked whether the mitochondrion originated after the eukaryotic cell arose, as assumed in the classical endosymbiont hypothesis, or whether this organelle had its beginning at the same time as the cell containing it [1, 2]. The genetic function of mitochondrial DNA was first fully revealed by complete sequencing of the ∼16-kb mitochondrial genome from several mammalian species [7, 8]. Previous studies reported that mitochondrial DNA encodes a small number (13 in mammals) of protein subunits of the mitochondrial electron transport chain and ATP synthase, as well as the ribosomal RNA (rRNA) and transfer RNA (tRNA) components of a mitochondrial translation system [7, 8], (see Chapter 7). Further investigations of mitochondrial DNA from non-animal species showed marked variation in size, physical form, coding capacity, organizational patterns, and modes of expression across the eukaryotic domain, indicating multiple functions of mitochondria [8]. Only 27 mitochondrial proteins are encoded by the gene-rich mitochondrial genome, and only three by the most gene-poor mitochondrial DNA. The vast majority of mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol, and imported back into the organelle [8, 9]. The differences in genome structure are reflected in the mito-transcriptome [10]. This review aims to examine the available evidence on the mitochondrial epigenetics in the pathogenesis of cardio-metabolic diseases (CMD).

5.2. MITOCHONDRIAL FUNCTION AND DYSFUNCTION Mitochondria are the major energetic sites of the cell, producing energy (ATP) through oxidative phosphorylation (OXPHOS). They are heterogeneous and essentially contribute to cellular functions. Mitochondrial dysfunction compromises overall cell functioning, tissue damage, and diseases. Mitochondrial OXPHOS is coordinated by both mitochondrial DNA and nuclear genomic DNA [11] (see Chapter 2). In many cellular processes, including apoptosis, aging, and oxidative metabolism, mitochondria perform a central role, which varies in different tissues [11, 12]. The brain, heart, and muscle are considered tissues with high energy usage and contain a large number of mitochondria, allowing them to be more susceptible to a reduction in aerobic metabolism. In mitochondrial dysfunction, the mitochondrial electron transport chain (ETC) is injured, or there are mutations of mitochondrial DNA [11]. These changes are important factors in the pathogenesis of mitochondrial diseases, aging, cancer and a range of other human disorders (see Chapter 6). Among various animals, the organization of mitochondrial genomes is similar, but several different rearrangement operations shape animal mitogenomes. Recent studies indicate that exposure to pollutants and non-alcoholic fatty liver disease may be associated with variations in mitochondrial DNA, which may play a role in mitochondrial gene expression regulation [11]. Hyperglycemia can also predispose to mitochondrial DNA methylation in retinal endothelial cells, and negatively

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regulate mitochondrial gene expression. Mitochondrial epigenetics may modulate nuclear DNA and nuclear DNA epigenetics may affect mitochondrial DNA, which may offer a therapeutic potential for the management of metabolic diseases [11, 12]. Most pathogenic mitochondrial DNA mutations induce defects in mitochondrial OXPHOS and thereby affect ATP synthesis. A large degree of phenotypic variation has been demonstrated in association with different mitochondrial DNA mutations, which are difficult to reconcile with OXPHOS defects as the sole pathogenic factor, implying that additional mechanisms must contribute to the phenotype, including epigenetic modifications [11, 12]. Due to advances in mitochondrion biology, there is a marked increase in our understanding of mitochondrial dynamics, bioenergetics, and redox homeostasis, and subsequently of their functions in tissue homeostasis and diseases, including cardiometabolic diseases (CMDs) [12]. Mitochondrial functions mainly rely on the enzymes in their matrix. Sirtuins are a family of NAD+-dependent deacylases and ADPribosyltransferases, three members of the sirtuin family (SIRT3, SIRT4, and SIRT5) located in the mitochondria [12] (see Chapter 2). These mitochondrial sirtuins coordinate energy and redox metabolism as well as mitochondrial dynamics in the mitochondrial matrix; they are involved in cardiovascular function and CMDs. Our understanding of mitochondrial sirtuins in this context, including cardiac remodeling, pulmonary artery hypertension, and vascular dysfunction, needs further studies. Work is also needed to examine potential therapeutic strategies targeting mitochondrial sirtuins to improve mitochondrial function in CMDs [12].

5.3. THE HUMAN MITOCHONDRIAL GENOME Mitochondria have their own circular genome, approximately 16.6 kb in size. The human mitochondrial genome is a 16.6 kb circular DNA encoding for 13 proteins in the ETC and the displacement loop (D-Loop) [2]. Mitochondrial DNA consists of 37 genes, 22 encoding for transfer RNAs (tRNAs), two for ribosomal RNAs (rRNAs), and 13 encoding for proteins important in the electron transport chain. Each of these 13 proteins is directly involved in the coordination of cellular respiration, generating the majority of ATP required for the process. Mitochondria also have an array of other important cellular roles, such as calcium homeostasis and neural stem cell differentiation [2]. As such, abnormal mitochondrial function, dynamics and trafficking have been associated with a number of brain disorders, including Alzheimer’s disease schizophrenia, bipolar disorder, and major depressive disorder. Two rRNAs and 22 tRNAs are necessary for the translation of mitochondrial genes [2] (Figure 5.1). Mitochondrial DNA represents less

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than 1% of total cellular DNA, and the number of mitochondria within a particular cell varies from tissue to tissue (see Chapter 7). Within mitochondrial DNA (np 16024–516), the D –loop is a noncoding region that contains cis-acting regulatory elements which are required for replication and transcription of DNA. Mitochondrial dysfunction usually arises after reaching a minimal threshold of heteroplasmy due to over a thousand copies of mitochondrial DNA [2]. That threshold is apparently different for different tissues and is dependent on energetics and other mitochondrial functions. The mitochondrial gene products are essential for normal cellular function; within a single cell, mitochondrial DNA generally has identical sequences, which are described as homoplasmic. In response to somatic mutations, heteroplasmy, or different mitochondrial DNA sequences within mitochondria of the same cell, can occur. The mitochondrial proteins, including those involved in the replication, transcription, and translation of mitochondrial DNA, are nuclear-encoded. Nuclear-encoded proteins are imported by specialized protein complexes on the inner and outer mitochondrial membrane. The mammalian mitochondrial DNA contains no introns and lacks histones.

Figure 5.1. The mitochondrial genome (adapted from www.mitomap.org).

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5.4. INTERACTIONS OF EPIGENOME AND ENVIRONMENT Epigenetic modifications of the genome occur due to interactions of gene and environment, via the biological regulatory system, without altering the DNA sequence [11]. Of all the epigenetic processes, DNA methylation is perhaps the best understood epigenetic adaption and most common DNA modification [11]. This mechanism plays an important role in coordinating the gene expression of many biological processes and has wide-ranging effects on health. Epigenetic damage and its dysfunction have been described in many human diseases with a relevant role of epigenetic marks with obesity and its codisease susceptibility. While nuclear DNA methylation is a well-established feature, very little attention has been devoted to mitochondrial epigenetics. Obesity is often associated with a state of mild chronic inflammation characterized by an abnormal production of proinflammatory and pro-oxidant mediators that have been linked to a mitochondrial dysfunction [11]. The energy restriction therapy devised to lose weight induces an increase in the intracellular ATP content and mitochondrial-related gene expression, concomitantly with a decrease in oxidative stress and inflammatory markers. Mitochondrial dysfunction and the increment of mitochondrial ROS production are important benchmarks of the aging and metabolic disturbances, indicating that mitochondrial dysfunction may be critically involved in the pathogenesis of obesity [11]. Mitochondrial epigenetics is a novel mechanism to understand the pathobiology of diseases with a mitochondrial dysfunction via epigenetic involvement in mitochondrial DNA. Evolutionary biology, biochemistry, genomics, developmental biology, systems biology and the impact of the environment on genes concerning the mechanism of evolution have grown significantly. However, it is not yet clear if the increase in chronic diseases is due to epigenetic inheritance or to the development of phenotypes, such as proinflammatory cytokines and transcription factors, due to interactions of thrifty genes (developed during scarcity) with environmental factors, such as excess fat or refined carbohydrates in the diet (Figure 5.2) [11-14]. Alteration in the methylation of genes may influence epigenetic inheritance. It has been proposed that dramatic adaptations can also occur, for instance in the form of major morphological changes, which appear to be the cause of changes in structure from apes to man and various human races. It is not clear whether such adaptations may be responsible for the development of diseases or for the development of humans. Some lineages may have greater ‘evolvability’ than others, independent of how much genetic variation is present at the outset. In this connection, heritable phenotype variations may depend on the biology and biochemistry of the genes, as well as on their status of methylation and remethylation. Some populations have more genetic variations and greater susceptibility to environmental factors and enormous evolvability for adaptations than others. Therefore, they are expected to generate phenotypic variation more rapidly within a few generations. The influence of photosynthesis, flight and multicellularity can enhance the evolvability,

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resulting in rapid inheritance. The ability to evolve at a different speed than other species needs some particular characteristics related to epigenetic inheritance. The environment can have adverse effects on human health and bring about disease in many harmful ways [14]. Many epidemiological studies have been conducted with the aim of elucidating the association between environmental exposure and human disease at the molecular and pathological levels. Such associations can often occur through induced epigenetic changes. One such mechanism is through environmental factors that increase oxidative stress in the cell; this stress can subsequently lead to alterations in DNA molecules. The two cellular organelles that contain DNA are the nucleus and mitochondria, and the latter are particularly sensitive to oxidative stress, with mitochondrial functions often disrupted by increased stress. Over the past decade, there has been a substantial increase in the number of epigenetic studies investigating the impact of environmental exposures upon genomic DNA, but to date there is insufficient attention paid to the impact on mitochondrial epigenetics when studying human disease in relation to environmental exposure.

Figure 5.2. Effects of interaction of epigenome and environment on epigenetic markers, resulting in chronic diseases.

Environments hazardous to human health can range from natural conditions to pollutants generated by human activities, such as those associated with air, water, temperature, and food. Humans are exposed to a multitude of environmental pollutants at differing intensities during their lifetimes. The list of diseases linked to different types of pollutants is continuously being updated. Individuals have different susceptibilities to these

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environmental exposures; part of the general population may be protected from their effects, or only develop mild symptoms, while a variable proportion may go on to present severe disease phenotypes. There is growing evidence supporting a link between mitochondrial dysfunction and environmental pollutants known to contribute to common human diseases. The symptoms of such diseases may become more severe with chronic exposure to these pollutants. Mitochondrial oxidative damage, DNA copy number, and DNA mutations have been widely studied in relation to environmental exposure and disease outcomes, but many mitochondrial diseases cannot be fully understood solely by genetic studies due to their non-Mendelian inheritance. Epigenetic elements can affect gene expression without changing the DNA sequence. Interestingly, epigenetics can be reversibly modified by internal and external cellular stimuli, including but not limited to pollutants, oxidative stress, temperature, nutrients, UV, and aging. More focus should thus be placed on mitochondrial epigenetics in order to better understand the development of non-Mendelian mitochondrial dysfunction in response to environmental exposures implicated in CMDs. Environmental pollutants may cause damage at the molecular level in the cell, which can disrupt cellular function. The most widely studied impact of environmental exposures is that of DNA damage [11-14]. There are two cellular organelles that contain DNA: the nucleus, which contains genomic DNA; and the mitochondria, each of which contains multiple copies of its own genome of approximately 16 kb in length. Mitochondria play an important role in the cellular response to environmental stressors. The normal mitochondrial DNA copies in cells, and the damaged mitochondrial DNA co-exist and may influence the mitochondrial DNA mutations that can range from normal, mild and severe according to the proportion of abnormal DNA copies. The proportion of these diseased mitochondria that are passed on from mother to child is random. If the child inherits fewer diseased than normal mitochondria from the mother, disease symptoms may not appear in the child but this non-Mendelian mitochondrial disease can be maternally inherited, or can also be acquired in later life [14-17]. Human mitochondria are maternally inherited, leading to the ‘bottleneck’ theory. As humans are exposed to the environment throughout their lifetime, the onset of diseases related to mitochondrial function can be at any stage, and with different phenotypes. Epigenetic modifications or mechanisms have been recognized as important factors regulating gene expression. Three broad categories of epigenetic modifications include genomic DNA methylation, post-translational modification of histone tails within higherorder chromatin, and regulation of gene expression by non-coding RNAs (ncRNAs). Perturbed epigenetic mechanisms have been associated with a variety of human pathologies, but their role in the pathogenesis of mitochondrial diseases is unclear. The importance of the epigenome in the pathogenesis of mitochondrial diseases is likely to be as significant as any other human disease where epigenetic mechanisms are affected.

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Recently, mitochondrial studies identifying defects in the mitochondria have led to the discovery that mitochondrial dysfunction induces or is associated with epigenetic alteration within the nuclear genome, which could contribute to the perplexing complexity associated with mitochondrial diseases; atherosclerosis, osteoporosis, carcinogenesis, and neuronal plasticity (Figure 5.3). Epigenetics and mitochondrial function in fetal tissues may be molecular signatures responsive to in utero tobacco smoke exposure [18]. Maternal smoking during pregnancy results in an increased risk of low birth weight through perturbations in the utero-placental exchange. The ENVIRONAGE birth cohort study, involving 255 nonsmokers and 65 former smokers and 62 smokers, examined the effect of self-reported tobacco smoke exposure during pregnancy on birth weight and the relation to placental tissue markers. The smokers delivered newborns with a birth weight on average 208 g lower (p = 0.0002) than mothers who did not smoke during pregnancy. In the smoker group, the relative mitochondrial DNA content was 21.6% lower (p = 0.01) than in the non-smoker group, while absolute mitochondrial DNA methylation levels of MT-RNR1 were higher (0.62%, p = 0.003). Lower CpG-specific methylation of CYP1A1 in placental tissue (−4.57%, p < 0.0001) were observed in smokers as compared to non-smokers.

Figure 5.3. Role of oxidative stress in epigenetic damage leading to chronic diseases [17].

Nevertheless, no mediation of CYP1A1 methylation nor any other investigated molecular signature was observed for the association between tobacco smoke exposure and birth weight. Mitochondrial DNA content, methylation of specific loci of DNA, and CYP1A1 methylation in placental tissue may serve as molecular signatures for the

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association between gestational tobacco smoke exposure and low birth weight. Mitochondrial DNA methylation is receiving ever-increasing interest, particularly in diseases characterized by mitochondrial dysfunction; however, most studies have been limited to the investigation of specific target regions [19]. DNA methylation is an important epigenetic mechanism involved in gene regulation, with alterations in DNA methylation in the nuclear genome being linked to numerous complex diseases. Mitochondrial genetic studies have been previously confounded by nuclear-mitochondrial pseudogenes. A recent study identified 74 nominally significant differentially methylated regions (p 0.19 μmol/L determined prior to systemic treatment in patients with metastatic urothelial carcinoma is a good prognostic factor. Overall survival of these patients is significantly longer. Cardiotoxicity and hepatotoxicity are characteristic for anthracycline antibiotics. Coenzyme Q10 moderated the toxicity of anthracyclines under experimental conditions. However, no study led to conclusive evidence for coenzyme Q10 to be used as support therapy in order to moderate side effects associated with doxorubicin. A group of small molecules inhibiting the activity of the respiratory chain complex III significantly reduces tumor growth under experimental conditions. Before they are introduced into clinical practice, they will naturally need to be tested in all three phases of clinical studies. Perspective: developing a novel therapeutic approach based on mitochondrial dynamics and in vivo cell reprogramming will be important for improving the quality of human life [9].

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Warburg O: On the origin of cancer cells. Science 1956; 123: 309-314. Warburg O: On respiratory impairment in cancer cells. Science 1956; 124: 269-270. Yeung SJ, Pan J, Lee MH: Roles of p53, MYC and HIF-1 in regulating glycolysis – the sevents hallmark of cancer. Cell Mol Life Sci 2008; 65: 3981-3999. Prieto J, Torres J: Mitochondrial Dynamics: In cell reprogramming as it is in cancer. Hindawi, Stem Cells International, Volume 2017, Article ID 8073721, 11 pages; https://doi.org/10.1155/2017/8073721. Massie R. Mitochondrial structure, function, and genetics. . Hardie RA, van Dam E, Cowley M, Han TL, Balaban S, Pojic M, Pinese M, Iconomou M, Shearer RF, McKenna J, Miller D, Wadell N, Pearson JV, Grimmond SM, Sazanov L, Biankin AV, Villas-Boas S, Hoy AJ, Turner N, Saunders DN. Mitochondrial mutations and metabolic adaptation in pancreatic cancer. Cancer and Metabolism 2017; 5: 2 DOI: 10.1186/s40170-017-0164-1 Santidrian AF, Matsuno-Yagi A, Ritland M a spol. Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression. J Clin Invest 2013; 123(3): 1068-1081. Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J Gen Physiol 1927; 8(6): 519-530. Yizhak K, Le Dévédec SE, Rogkoti VM a spol. A computational study of the Warburg effect identifies metabolic targets inhibiting cancer migration. Mol Syst Biol 2014; 10: 744. Vozza A, Parisi G, De Leonardis F a spol. UCP2 transports C4 metabolites out of mitochondria, regulating glucose and glutamine oxidation. Proc Natl Acad Sci USA 2014; 111(3): 960-965. Marin-Valencia I, Cho SK, Rakheja D a spol. Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors. NMR Biomed 2012; 25(10): 1177-1186. Pedersen PL, Mathupala S, Rempel A a spol. Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention. Biochim Biophys Acta 2002; 1555(1-3): 14-20. Chapiro J, Sur S, Savic LJ a spol. Systemic delivery of microencapsulated 3bromopyruvate for the therapy of pancreatic cancer. Clin Cancer Res 2014; 20(24): 6406-6406. Ye J, Mancuso A, Tong X et al. Pyruvate kinase M2 promotes de novo serine synthesis to sustain mTORC1 activity and cell proliferation. Proc Natl Acad Sci USA 2012; 109(18): 6904-6909. Maddocks OD, Berkers CR, Mason SM a spol. Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells. Nature 2013; 493(7433): 542546. Zirath H, Frenzel A, Oliynyk G a spol. MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells. Proc Natl Acad Sci 2013; 110(25): 10258-10263. Wise DR, DeBerardinis RJ, Mancuso A a spol. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction. Proc Natl Acad Sci USA 2008; 105(48): 18782-18787.

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[18] Hu S1, Balakrishnan A, Bok RA a spol. 13C-pyruvate imaging reveals alterations in glycolysis that precede c-Myc-induced tumor formation and regression. Cell Metab 2011;14(1): 131-142. [19] Weinberg F, Hamanak R, Wheaton WW a spol. Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci USA 2010; 107(19): 8788-8793. [20] Son J, Lyssiotis CA, Ying H a spol. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature 2013; 499(7459): 504. [21] Porporato PE, Payen VL, Pérez-Escuredo J a spol. A mitochondrial switch promotes tumor metastasis. Cell Rep 2014; 8(3): 754-766. [22] Jolliet P, Simon N, Barré J a spol. Plasma coenzyme Q10 concentrations in breast cancer: prognosis and therapeutic consequences. Int J Clin Pharmacol Ther 1998; 36(9): 506-509. [23] Iwase S, Kawaguchi T, Yotsumoto D a spol. Efficacy and safety of an amino acid jelly containing coenzyme Q10 and L-carnitine in controlling fatigue in breast cancer patients receiving chemotherapy: a multi-institutional, randomized, exploratory trial (JORTC-CAM01). Support Care Cancer 2016; 24(2): 637-646. [24] Palan PR, Mikhail MS, Shaban DW a spol. Plasma concentrations of coenzyme Q10 and tocopherols in cervical intraepithelial neoplasia and cervical cancer. Eur J Cancer Prev 2003; 12(4): 321-326. [25] Rusciani L, Proietti I, Rusciani A a spol. Low plasma coenzyme Q10 levels as an independent prognostic factor for melanoma progression. K Am Acad Dermatol 2006; 54(2): 234-241. [26] Palacka P, Kucharská J, Mego M, Luha J, Sumbalová Z, Světlovská D, Rejleková K, Mardiak J, Gvozdjáková A: Novel prognostic factors in metastatic urothelial carcinoma (MUC). J Clin Oncol 2015; 33(15): e15525. (ASCO Annual Meeting 2015; Chicago, 29.05. – 02.06. 2015). [27] Brea-Calvo G, Rodríguez-Hernández A, Fernández-Ayala DJ a spol. Chemotherapy induces an increase in coenzyme Q10 levels in cancer cell lines. Free Radic Biol Med 2006; 40(8): 1293-1302. [28] Chen PY, Hou CW, Shibu MA a kol. Protective effect of Co-enzyme Q10 on doxorubicin-induced cardiomyopathy of rat hearts. Environ Toxicol 2016; doi: 10.1002/tox.22270. [29] Roffe L, Schmidt K, Ernst E. Efficacy of coenzyme Q10 for improved tolerability of cancer treatments: a systematic review. J Clin Oncol 2004; 22(21): 4418-4424. [30] Fiorillo M, Lamb R, Tanowitz HB a spol. Repurposing atovaquone: Targeting mitochondrial complex III and OXPHOS to eradicate cancer stem cells. Oncotarget 2016; doi: 10.18632/oncotarget.9122.

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[31] Zhou J, Duan L, Chen H a spol. Atovaquone derivatives as potent cytotoxic and apoptosis inducing agents. Bioorganic and Medicinal Chemistry Letters 2009; 19: 5091-5094. [32] Blein S, Barjhoux L, Damiola F a spol. Targeted Sequencing of the Mitochondrial Genome of Women at High Risk of Breast Cancer without Detectable Mutations in BRCA1/2. PloS One 2015; 10: e0136192. [33] Chihara N, Amo T, Tokunaga A a spol. Mitochondrial DNA alterations in colorectal cancer cell lines. Journal of Nippon Medical School 2011; 78: 13-21. [34] Polyak K, Li Y, Zhu H a spol. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nature Genetics 1998; 20: 291-293. [35] Dasgupta S, Hoque MO, Upadhyay S a spol. Mitochondrial cytochrome B gene mutation promotes tumor growth in bladder cancer. Cancer Research 2008; 68: 700706. [36] Dasgupta S, Hoque MO, Upadhyay S a spol. Forced cytochrome B gene mutation expression induces mitochondrial proliferation and prevents apoptosis in human uroepithelial SV-HUC-1 cells. International Journal of Cancer 2009; 125: 28292835. [37] Li G, Fu D, Liang W a spol. CYC1 silencing sensitizes osteosarcoma cells to TRAILinduced apoptosis. Cellular Physiology and Biochemistry 2014; 34: 2070-2080. [38] Jung HJ, Kim KH, Kim ND a spol. Identification of a novel small molecule targeting UQCRB of mitochondrial complex III and its anti-angiogenic activity. Bioorganic and Medicinal Chemistry Letters 2011; 21: 1052-1056. [39] Jung HJ, Cho M, Kim Y at al.: Development of a novel class of mitochondrial ubiquinolcytochrome c reductase binding protein (UQCRB) modulators as promising antiangiogenic leads. J Med Chem 2014; 57: 7990-7998. [40] Obre E, Rossignol R: Emerging concepts in bioenergetics and cancer research: Metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy. Int J Biochem Cell Biol 2015; 59: 167-181. [41] Vyas S, Zaganjor E, Haigis MC: Mitochondria and cancer. Cell 2016; 166/3: 555566. [42] Allen BG, Bhatia SK, Anderson CM, Eichenberger-Gilmore JM, Sibenaller ZA, Mapuskar KA et al.: Ketonic diets as an adjuvant cancer therapy: history and potential mechanism. Redox Biol 2014; 2C: 963-970. [43] Branco AF, Ferreira A, Simoes RF, Magalhaes-Novais S, zehowski C, Cope E, Silva AM, Pereira D, Sardao VA, Cunha-Oliveira T: Ketogenic diets: from cancer to mitochondrial diseases and beyond. Eur J Clin Invest 2016; 46/3: 285-298. [44] Hursting SD, Lavigne JA, Berrigan D, Perkins SN, Barret JC: Calorie restriction, ageing, and cancer prevention: mechanisms of action and applicability to humans. Annu Rev Med 2003; 54: 131-152.

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 15

MITOCHONDRIAL REPRODUCTIVE MEDICINE Anna Gvozdjáková1,, Jozef Dúbravický2 and Ram B. Singh3 1

Pharmacobiochemical Laboratory of the 3th Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 2 University Hospital in Bratislava, Department of Urology, Bratislava, Slovakia 3 Halberg Hospital and Research Institute, Moradabad, India

ABSTRACT reproductive system to achieve pregnancy after 12 months of unprotected sexual activity Fertility disorder (infertility) is a disease defined by the failure of the. Male infertility is one of the major stressors affecting about 40% of couples. Supplementary therapy with coenzyme Q10 and carnitine improved sperm function.

Keywords: infertility, sperm, mitochondria, coenzyme Q10, carnitine

15.1. SPERM MITOCHONDRIAL FUNCTION Sperm contains a number of mitochondria, which are spirally arranged around the middle of the axomenum. The amount of mitochondrial in the cell is individual, depending on the energy requirements of the cells (figure 15.1). For energy generation (ATP) in sperm mitochondria, two endogenous substances with antioxidant properties are key: coenzyme Q10 and carnitine. Cells with high ATP requirements have higher amounts of mitochondria. 

Corresponding Author Email: [email protected].

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Figure 15.1. Sperm structure.

The oocyte contains approximately 100,000 mitochondria, while the mature sperm has 22-28 mitochondria. The key role of mitochondria in sperm is the generation of energy required for sperm mobility. ATP regulates cell survival, cell dynamics during division, and sperm motility [1, 2]. Several pathobiochemical mechanisms are involved in male fertility disorders, such as uncontrolled free oxygen radical production (ROS) and reduced antioxidant protection of the organism (coenzyme Q10, carnitine, superoxide dismutase, glutathione peroxidase, catalase, vitamin E). Oxidative stress in the testis or sperm can be caused by infection, inflammation, smoking, chemotherapy, drugs, varicocele, and leukocytes [2-6]. Factors leading to women's fertility disorders include stress, depression, reduced antioxidant protection, chronic fatigue, flawed bowel status, and muscle imbalance. The removal of the pelvic floor slows metabolic processes, resulting in various gynecological problems. Rehabilitation exercises, according to Mojzes, can favorably influence many women's problems, such as external genital pain, irregular and painful menstruation, poor eggshell quality, repeated abortions, and attempts to conceive [3, 7-9] (Figure 15.2).

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Figure 15.2. Potential mechanism of infertility.

15.2. MITOCHONDRIAL SPERMATOPATHY The pathobiochemical mechanisms of male fertility disorders include reduced mobility and sperm quality, damage to the balance between free radicals of oxygen and nitrogen, reduced total body antioxidant capacity, mtDNA fragmentation, and disorders of energy production in sperm mitochondria. Supportive antioxidant and energetic treatments with vitamin E, vitamin C, coenzyme Q10 and carnitine may be beneficial in men with fertility disorders [10, 11]. Physiological sperm function requires only minimal amounts of ROS, but uncontrolled ROS production causes decreased sperm motility and sperm amount, increases the percentage of pathological sperm, reduces energy production in sperm mitochondria, and leads to fragmentation of mitochondrial DNA (deoxyribonucleic acid). These changes are included in the term “mitochondrial spermatopathy" [7]. Sperm movement is used as an indicator of sperm quality and male infertility. Reduction of sperm motility, lack of ATP in sperm mitochondria, and mtDNA fragmentation are the causes of fertility disorders in men [12].

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Figure 15.3. Sperm movement depends on the concentrations of total coenzyme Q10 and vitamin E.

According to the WHO (World Health Organization), sperm motility is determined by the following steps: Grade a + b = rapid forward movement; Stage b + c = fast and slow forward movement; Stage c = moving sperm on site; Stage d = immobile sperm [13]. Figure 15.3 shows the dependence of sperm movement on their concentration of CoQ10 and α-tocopherol [8].

15.3. OXIDATIVE STRESS AND SPERM FUNCTIONS Oxidative stress is a serious cause of fertility disorders in both men and women. However, for the preservation of sperm functions, the formation of ROS is necessary to a small extent. The formation of a high concentration of nitrogen oxygen species (NOS) can be toxic to sperm function. Effects of the NO.radical depends on its concentration and interaction with H2O2, which is toxic to human sperm. The primary mechanism of nitric oxide is in the induction of sperm damage at the level of mitochondria and DNA synthesis. High ROS production due to large amounts of leukocytes in sperm leads to peroxidation of lipids and damage to sperm functions, including mobility, survival and fertilization. High concentrations of ROS were detected in sperm in 25% of infertile men, but not in sperm of fertile men. ROS induces a chain of changes that lead to immobilization of sperm in the absence of ATP [14]: oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA fragmentation, in both the core and the mitochondrial genome. Sperm is protected from oxidative stress by the presence of antioxidant enzymes in seminal plasma [10, 15].

15.4. ANTIOXIDANTS AND SPERM FUNCTIONS Sperm contains several antioxidants: glutathione peroxidase [16], superoxide dismutase [1] and catalase [17]. The lack of superoxide dismutase is associated with immobility of the sperm [18], and a lack of catalase with astenozoospermia [17]. Prooxidants and antioxidants are present in both seminal plasma and sperm.

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The beneficial effect of substances with antioxidant and energetic properties was shown in male infertility. Supplementation with vitamin C and vitamin E and intramuscular administration of glutathione improves sperm quality [3, 4, 11, 19]. Glutathione is important not only for sperm vitality as an antioxidant but is essential for the formation of glutathione peroxidase, an important enzyme that belongs to the structural proteins in the middle part of adult sperm. Glutathione deficiency can lead to instability of the middle part of the sperm, resulting in a mobility disorder. Reduced concentrations of glutathione during sperm formation cause damage of sperm membrane integrity as a result of increased oxidative stress. Glutathione peroxidase and glutathione reductase, as antioxidant enzymes, can be directly involved in the inhibition of sperm lipoperoxidation. Supplementation with folic acid and zinc increases sperm count [11].

15.5. EFFECT OF COENZYME Q10, CARNITINE AND VITAMIN E ON FERTILITY DISORDERS Supportive antioxidant and energy treatments with vitamin E, vitamin C, coenzyme Q10 and carnitine can have a beneficial effect in men with fertility disorders.

15.5.1. Coenzyme Q10 Coenzyme Q10 is an essential part of every cell of the human organism. CoQ10 is present in the body in 3 forms in Q10-CYCLE: in the oxidized form (ubiquinone forms, approximately 10%); semiquinone (the radical); and in reduced form (ubiquinol, which accounts for about 90% of the total CoQ10 content in the body) [18]. The beneficial effect of coenzyme Q10 and carnitine was shown in a pilot study [3, 7, 8, 20-22]. Ubiquinol was found to have a beneficial effect on reproductive hormones in serum.

15.5.2. Carnitine Carnitine is an endogenous component of the human organism. The highest carnitine concentration (95%) occurs in the heart and skeletal muscle, 4% in other organs, and 1% in the extracellular space. Carnitine is a naturally occuring compound in the body; it belongs to a group like vitamins. The concentration of carnitine in skeletal muscle is about 70% higher than in plasma. From the subcellular particles, the highest carnitine concentration was found in the mitochondria, dominantly in liver and kidney mitochondria [22].

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Carnitine sources for the human body are external, i.e., from foods (as meats: sheep, mutton, lamb, beef, pork; cow‘s milk and breast milk) and internal, i.e., carnitine is produced in the kidneys and liver. Carnitine biosynthesis in the body occurs in a series of metabolic reactions, which require two amino acids (L-methionine and L-lysine) and vitamin C, vitamin B6, niacin, and iron. Carnitine is a 3-hydroxy-4-N-trimethyl amino butyric acid. Various types of carnitine are known: L-carnitine, L-carnitine fumarate, Lcarnitine tartarate, acetyl-L-carnitine, propionyl-L-carnitine and aminocarnitines. A third source consists of nutritional supplements (CARNI-Q-GEL®, CARNI-Q-Nol®).

15.5.2.1. The Physiological Role of Carnitine Carnitine is peresent in biological materials as free carnitine (in relatively high concentrations) and as acylcarnitines (metabolic products of reactions utilizing acyl CoA catalyzed by carnitine acyltransferases). The acyl groups in the acylcarnitines range from short-chain (acetyl) to long-chain (such as palmitoyl). In 1955, acetylcarnitine was shown to be formed from acetyl CoA and carnitine in the liver, and that carnitine stimulated fatty acid oxidation in the liver. A great amount of mitochondrial research activity from 1960 on was performed in many laboratories once acylcarnitines were shown to be substrates for long-chain fatty acid oxidation by mitochondria. Carnitine is involved in several physiological functions: L-carnitine acts as an antioxidant (as free radical scavenger) L-carnitine is essential for the transfer of long-chain fatty acid from the cytoplasm to the mitochondria L-carnitine acts as scavenger system for acyl groups L-carnitine removes lactic acid from blood and tissues L-carnitine is involved in the formation and utilization of ketone bodies L-carnitine is involved in branched-chain amino acid metabolism L-carnitine increases ammonia conversion into urea, which is excreted in the urine L-carnitine is involved in membrane stabilization. The heart and skeletal muscles obtain most of their energy demands by fatty acids oxidation, which occurs primarily in the mitochondrial matrix. Carnitine is an essential factor for the transport of long-chain fatty acids (acyl CoA) from the cytosol to the mitochondrion. Without carnitine, the oxidation of long-chain fatty acids cannot proceed. Acyl CoA can cross the mitochondrial membrane only after transfer of the acyl group to carnitine; then acylcarnitine and CoA are produced. The basic, reversible reaction is: Acyl CoA + Carnitine  Acylcarnitine + CoA

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The transport of acylcarnitine into the mitochondrial matrix requires enzyme activity from carnitine palmitoyltransferase I and II (CPT I and CPT II, exclusive mitochondrial enzymes), and from carnitine translocase. Under physiological conditions, long-chain acyl carnitines are formed mostly in the intermembrane space and imported into the matrix, whereas short-chain acylcarnitines are formed within the matrix and exported into the cytosol [23, 24].

15.5.2.2. Deficit of Carnitine Deficiencies of carnitine in humans were first noted in 1973. Reasons for the carnitine deficit in the human body vary. The primary deficiency is a result of a damaged transport mechanism of carnitine in the plasma membrane. Carnitine is eliminated in the urine. Damaged transport of long-chain acylcarnitines into the matrix inhibits -oxidation of fatty acids. Reduced carnitine content was found in patients with kidney disease, particularly those undergoing dialysis, who may lose up to 80% of their serum carnitine per day. Insufficiency of carnitine can develop and progress to cardiomyopathy, myopathy (low carnitine in skeletal muscles) or encephalomyopathy. Other reasons of carnitine deficiency are associated with genetic metabolic disturbances (defects of -oxidation fatty acid, defects in respiratory chain function), or aquired deficiency (hemodialysis, or valproic acid effect in epilepsy therapy). 15.5.2.3. Deficit of Carnitine and Vegetarians Although in healthy people carnitine deficiency is rare, in vegans (pure vegetarians), lysine and methionine are lacking because their diet lacks carnitine. Both child and adult vegetarians have lower carnitine concentrations in their plasma. Individuals on strict macrobiotic diets have carnitine deficiency; they lose weight, develop weakness, and suffer from several nutritional deficiencies, leading to the initiation of disease development [25]. 15.5.2.4. Supplementation of Carnitine Carnitine supplementation is beneficial in physiology conditioning (e.g., in trained athletes) and in pathological conditions. In patients with neuromuscular diseases, carnitine supplementation can improve ankle and knee function and handgrip strength. In hypertensive patients with anemia, carnitine supplementation (in daily doses of 1g for 3 months) improved myocardial fatty acid utilization. Supplementation with carnitine improves quality of life of chronically hemodialysed patients. Patients with human immunodeficiency virus (HIV) experienced a beneficial effect from carnitine supplementation (6g daily for 6 months) [26]. A synergetic effect of L-carnitine fumarate (880mg/day) with 60 mg hydrosoluble CoQ10 decreased male infertility after 3-12 months [22].

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In our studies, we recruited 60 infertile couples over a 6-month period. Patients were screened prior to entering the study and were followed-up after 3 and 6 months. The men received 2-3 CARNI-Q-Nol® capsules daily (2-3x 440 mg L-carnitine, 2-3x 30 mg ubiquinol, 2-3x 50.34 mg vitamin E) or CARNI-Q-GEL® (2-3x 440 mg L-carnitine, 2-3x 30 mg ubiquinone, 2-3x 50.34 mg vitamin E). The best results were for men and women who used antioxidants as a supportive energy treatment. Supportive treatment consisted of 1-2 capsules of daily ACTIVE Q® (50-100 mg ubiquinol) or Q-GEL-ULTRA® (2x60 mg of ubiquinone). The couples were divided into 3 groups: men using CARNI-Q-Nol® and partners (N = 20); couples (N = 20) using 1-2 CARNI-Q-NOL® capsules daily; and couples (N = 20) receiving combined CARNI-Q-NOL® supportive treatment and Mojžiš method [7]. Significant results include sperm improvement in men, reduction in oxidative stress, and increase in sperm counts by 48.9% after 3 months, and by 80.7% after 6 months of supportive treatment (Figure 15.4). Sperm motility increased and the percentage of pathological sperm decreased after 3 months from 47.78% to 35.56%, which represents a decrease in pathological sperm by 25.58% (Figure 15.5). The CoQ10-TOTAL concentration significantly increased in the plasma of men with fertility disorder after a 3- and 6-month supportive treatment with CARNI-Q-NOL® (Figure 15.6).

Figure 15.4. The effect of CARNI-Q-NOL® on the amount of sperm in seminal fluid.

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Figure 15.5. The effect of CARNI-Q-NOL® on % pathological sperm in seminal fluid.

Figure 15.6. The effect of CARNI-Q-NOL® on CoQ10-TOTAL plasma concentration in men with fertility disorder.

Pregnancy was confirmed in 45% of women who were on antioxidant and energetic support, combined with Mojzes method of exercise. For pregnancy of several women, supportive treatment was stopped after 4 to 6 months, and for some other women after 1 to 2 months for pregnancy [7].

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Figure 15.7. Proposed mechanism of action of CoQ10 and carnitine on mitochondrial function [7].

Legends: Acyl-CoA: acyl-coenzyme A; CPT I: carnitine phosphate transpeptidase; CPT II: carnitine phosphate transpeptidase II; CoQ: coenzyme Q; VDAC: Voltage Dependent Anion Channel; ATP: adenosine triphosphate; ADP: adenosine diphosphate; Pi: inorganic phosphorus; I, II, III, IV, V: respiratory chain complexes; H+: proton; e-: electron; Q-cycle: Coenzyme Q cycle; Cyt c: cytochrome c; NADH-reduced nicotinamide adenine dinucleotide; NAD+: nicotinamide adenine dinucleotide; FADH2: reduced flavin adenine dinucleotide; FAD: flavin adenine dinucleotide; O2.-: superoxide radical; H2O2: hydrogen peroxide; OH.: hydroxyl radical; H2O: water; O2: oxygen.

15. 6. MECHANISMS OF SUPPORTIVE TREATMENT WITH COENZYME Q10 AND CARNITINE CoQ10 and carnitine act synergistically on energy production in sperm mitochondria, reducing oxidative stress. Professor Frederick L. Crane predicts that CoQ10 passes through the VDAC (Voltage Dependent Anion Channel) of the outer mitochondrial membrane (OMM) into the respiratory chain of the inner mitochondrial membrane (IMM) [5] (Figure 15.7).

CONCLUSION New options for supportive energy and antioxidant treatment of couples with fertility disorder include water-soluble nutritional supplements with the highest bioavailability

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such as Carni-Q-Nol®, Carni-Q-GEL®, Active Q®, Q-GEL- ULTRA® (manufactured by Tishcon Corp., USA).

REFERENCES [1]

Jansen, R. P. S., Burton, G. J. Mitochondrial dysfunction in reproduction. Mitochondrion, 2004; 4: 577-560. [2] Thorburn, D. R. Mitochondrial reproductive medicine. In: DiMauro, S., Hirano, M., Schon, E. A. (eds), Mitochondrial medicine. Informa Healthcare 2006; 214-259. [3] Gvozdjáková, A., Kucharská, J., Lepieš, P., Braunová, Z., Malatinský, E. Decreased level of sperm coenzyme Q10, mitochondrial respiration and energy production in infertile patients. Therapeutic effect of coenzyme Q10 (A pilot study). 1st Conference of the International Coenzyme Q10 Association, Boston, Mass, USA, May 1998, abtract book: 137-138. [4] Sanocka, D., Miesel, R., Jedrzeczak, P., Chemlonska-Soyta, A., Kurpisz, M. Efrect of reactive oxygen species and the activity of antixoidant systems of human semen; association with male infertility. Int. J. Androl., 1997; 20: 255-264. [5] Aitken, R. J. Free radicals, lipid peroxidation and sperm function. Reprod. Fertil. Dev., 1995; 7: 659-668. [6] Aitken, R. J., Baker, M. A. Oxidative stress, sperm survival and fertility control. Mol. Cell Endocrinol., 2006; 250: 66-69. [7] Gvozdjáková, A., Kucharská, J., Dubravický, J., Mojto, V., Singh, R. B. Conzyme Q10, α-tocopherol, and oxidative stress could be important metabolic biomarkers of male infertility. Hindawi, Disease Markers, Volume 2015, article ID 827941, 6 pages. [8] Gvozdjáková, A., Kucharská, J., Lipková, J., Bartolčičová, B., Dúbravický, J., Voržáková, M., Černáková, I., Singh, R. B. Importance of the assessment of coenzyme Q10, alpha-tocopherol and oxidative stress for the diagnosis and therapy of infertility in men. Brat. Med. J., 2013; 114(11): 607-609. [9] Thakur, A. S., Littaru, G. P., Funahashi, I., Painkara, U. S., Dange, N. S., Chauhan, P. Effect of ubiquinol on serum reproductive hormones of Amenorrhic patients. Int. J. Clin. Biochem., 2016; 31(3): 342-348. DOI 10.1007/s12291-015-0542-9. [10] Littaru, G. P., Tiano, L. Clinical aspects of coenzyme Q10 in relationship with its bioenergetic and antioxidant properties. In: Mitochondrial Medicine, ed. A. Gvozdjáková, Springer, Netherlands, 2008; 301-323. [11] Sheweita, S. A., Tilmisany, A. M., Al-Sawaf, H. Mechanisms of male infertility: role of antioxidants. Curr. Drug Metab., 2005; 6: 495-501. [12] Agarwal, A., Said, T. M.: Oxidative stress, DNA damage and apoptosis in male infertility: a clinical approach. BJU Int., 2005; 95: 503-507.

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[13] World Health Organization, WHO Laboratory Manual for the examination and processing of Human Semen, WHO Press, Geneva, Svitzerland, 5th edition, 2010. [14] Aitken, J., Fisher, H. Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. BioEssays, 1994; 16(4): 259-267. [15] Kobayashi, T., Miyazaki, T., Natori, M., Nozawa, S. Proetective role of superoxide dismutase in human sperm motility: superoxide dismutase actitivy and lipid peroxide in human seminal plasma and spermatozoa. Hum. Reprod., 1991; 6: 987-991. [16] Alvarez, J. G., Sorey, B. T. Role of glutathione peroxidase in protecting mammalian spermatozoa fromloss of motility caused by spontaneous lipid peroxidation. Gamete Res., 1989; 23: 77-90. [17] Jeulin, C., Soufir, J. C., Weber, P., Laval-Martin, D., Calvayrac, R. Catalase activtiy in human spermatozoa and seminal palsma. Gamete Res., 1989; 24: 185-196. [18] Kobayashi, T., Miyazaki, T., Natori, M., Nozawa, S. Proeftive role of superoxide dismutase in human sperm motility: superoxide dismutase actitivy and lipid peroxide in human seminal plasma and spermatozoa. Hum. Reprod., 1991; 6: 987-991. [19] Angelitti, A. G., Colacicco, L., Calla, C., Arizzi, M., Lippa, S. Coenzyme Q: potentially useful index of bioenergetic and oxidative status of spermatozoa. Clin. Chem., 1995; 41(2): 217-219. [20] Gvozdjáková, A., Kucharská, J., Bartolčičová, B., Dubravický, J., Voržáková, M., Lipková, J. Effect of Carni-Q-Nol and Mojzis exercise on sperm function and pregnancy. 7st Conference of the International Coenzyme Q10 Association, Seville, Spain, 2012, abstract book: 90-91. [21] Gvozdjáková, A., Kucharská, J., Bartolčičová, B., Lipkova, J., Dubravicky, J., Singh, R. B. Metabolic biomarkers of CARNI-Q-Nol supportive therapy in human fertility disturbances, BIT’s a 2nd World Congress of MolMed and BIT’s 3rd Annual World Congress of Biomarkers, Guangzhou, China, December 2012, abstract book: 067. [22] Gvozdjáková, A. Carnitine. In Mitochondrial Medicine, ed. A. Gvozdjáková, Springer Netherlands, 2008, p.357-362. [23] Bartelli, A., Ronca, G. Carnitine and coenzyme Q10: biochemical properties and functions, synergism and complementary action. Int. J. Tiss. React., 1990; XII/3: 183-186. [24] De Jong, J. W., Ferrari, R. (eds). The carnitine system. A new therapeutical approach to cardiovascular diseases. Kluwer Academic, Dordrecht, Boston, London 1995; pp. 393. [25] Ferrari, R., DiMauro, S., Sherwood, G. L-carnitine and its role in medicine. From function to therapy. Academic Press, Harcourt Brace Jovanovich, London, San Diego, New York, Boston, Sydney, Tokyo, Toronto 1992; pp. 433. [26] De Simone, M. C., Tzantzoglou, S., Jirillo, E. Carnitine deficiency in AIDS. AIDS, 1992; 6: 203-205.

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 16

METHODS FOR DIAGNOSING MITOCHONDRIAL DISTURBANCES Anna Gvozdjáková1,, Jarmila Kucharská1, Zuzana Rausová1 and Anna Hlavatá2 1

Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 2 Department of Paediatrics, University Hospital Bratislava, Slovakia

ABSTRACT Diagnostic methods for mitochondrial diseases – the biochemical analysis of blood, muscle biopsy (skinned fibers), isolated mitochondria and thrombocytes for respiratory chain and oxidative phosphorylation analysis, histochemistry, electron microscopy, molecular, MRS analysis and family history – are all presented within this chapter.

Keywords: diagnosis, methods, mitochondrial diseases, mitochondria, biopsy, respiratory chain, oxidative phosphorylation

16.1. INTRODUCTION The diagnosis of inherited mitochondrial diseases is difficult. Mitochondrial diseases should be considered in a differential diagnoses when there are unexplained features, especially when these occur in combination with encephalomyopathy: seizures, 

Corresponding Author Email: [email protected].

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developmental delay or regression (including early and late-onset dementia), myoclonus, movement disorders (dystonia, dyskinesias, chorea, etc.), complicated migraine, stroke, neuropathy, cardiac conduction defects or cardiomyopathy, hearing deficits, short stature, disorders of extraocular muscles (including ptosis, acquired strabismus and ophthalmoplegia), diabetes, renal tubular disease, visual loss (retinitis), and lactic acidosis (which can be mild). More than one organ may be involved in mitochondrial diseases, particularly when the functions of tissues with high energy requirement (brain, heart, skeletal muscles, liver, kidneys, etc.) are impaired. Affected subjects often show various combinations of signs and symptoms; thus in cases of multi-organ involvement, a mitochondrial disorder should be considered. Clinical manifestations of mitochondrial disease may occur even before birth and into late adult life. In neonates (up to one month), nonspecific findings are seen, such as lethargy, irritability, hyperactivity, failure to feed well, hypothermia or fever, cyanosis, seizures, vomiting, jaundice, diarrhea, and abdominal distension. Further manifestations are ketoacidotic coma with recurrent apnea, severe hypotonia, liver enlargement, proximal tubulopathy, severe neonatal sideroblastic anemia, concentric hypertrophic cardiomyopathy, and hepatic failure. In infancy (one month to two years), the manifestations include failure to thrive, recurrent episodes of acute myoglobinuria, proximal tubulopathy (de Toni Debre Fanconi syndrome), severe trunk and limb dwarfism, early-onset insulin-dependent diabetes mellitus (Wolfram syndrome), rapidly progressive encephalomyopathy, and subacute necrotizing encephalomyopathy (Leigh’s disease). In childhood (over two years) and adulthood, the following neuromuscular clinical features may be present: muscle weakness with myalgia and exercise intolerance, progressive sclerosing poliodystrophy (Alper’s disease), encephalomyopathy with myoclonus, ataxia, hearing loss, muscle weakness and generalized seizures (MERRF), progressive external ophthalmoplegia (PEO), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Leber’s hereditary optic neuroretinopathy (LHON), neurogenic muscle weakness, ataxia, retinitis pigmentosa and variable sensory neuropathy with seizures, mental retardation or dementia (NARP), mitochondrial myopathy and peripheral neuropathy, encephalopathy and gastrointestinal encephalopathy (MNGIE) [1]. Most cases have deficient activity of the oxidative phosphorylation complex or a disorder of the pyruvate dehydrogenase complex. In some patients, an isolated disorder of a single complex is found, while in other cases a combined disorder with deficient activity of several complexes is present.

16. 2. METABOLIC ANALYSIS Standard screening tests include the determination of:

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plasma lactate; lactate/pyruvate molar ratio = redox status in the cytoplasm; ketonemia (“paradoxical” elevation in fed individuals); -hydroxy butyrate/acetoacetate molar ratio = redox status in mitochondria; blood glucose and free fatty acids; urinary organic acid = lactate, ketone bodies, citric acid cycle intermediates.

When standard tests are inconclusive, the following determinations should be made: 1. blood determinations in fasted individuals and one hour after food intake: glucose, lactate, pyruvate, ketone bodies and their molar ratios at 15 min, 30 min, 45 min, 60 min, and 90 min; 2. lactate/pyruvate molar ratios in the CFS (only when no elevation of plasma lactate is observed); 3. redox status in plasma following exercise.

16.2.1. Differential Diagnosis of Lactic Acidemia Elevated plasmatic lactate value is the diagnostic marker of disturbed mitochondrial metabolism and of respiratory chain disorders. Normal lactate values depend on age and laboratory method. Normal plasmatic lactate value is 0.7 - 2.1 mmol/l. Normal plasmatic pyruvate value is 0.034 - 0.102 mmol/l. Normal plasmatic 3-hydroxy-butyrate value is 0.030 - 0.300 mmol/l. Determination of the lactate:pyruvate ratio in body fluids is another important parameter. Elevation of this ratio over 25 can be a sign of oxidative phosphorylation disorder. Low values of this ratio are a sign of pyruvate dehydrogenase complex disturbance. Elevation of alanine in plasma and CSF is a sign of mitochondrial inherited metabolic disorders. Determination of acetoacetate and 3-hydroxy-butyrate is useful for quantifying ketosis in the diagnosis of pyruvate carboxylase deficiency. Blood sampling technique for lactate determination. The blood sample is taken after fasting and after a meal from an uncuffed vein or artery. False elevation (up to 4-6 mmol/l) is often due to increased muscular activity in a struggling child, in patients on assisted ventilation, or it may be due to seizures.

16.2.2. Lactic Acidemia Primary lactic acidemia is a common sign in different inherited metabolic disorders. It can be caused by the following group of disorders:

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Anna Gvozdjáková, Jarmila Kucharská, Zuzana Rausová et al. 1. Mitochondrial disorders: respiratory chain disorders, PDH (pyruvate dehydrogenase) deficiency, HHH syndrome (hyperornitinemia, hyperammonemia, homocitrullinuria); 2. Gluconeogenesis defects: PCD (pyruvate carboxylase deficiency), PEPCK (phosphenolpyruvate carboxykinase) deficiency, fructose-1,6-biphosphatase deficiency; 3. Glycogen storage disease: GSD Ia, Ib, III; 4. Organic acidemias: methylmalonic acidemia, propionic academia; 5. Fatty acid oxidation defects: CPT I, II, VLCAD, LCHAD, MCAD, SCAD, glutaric aciduria type II; 6. Other: biotinidase deficiency, Krebs cycle defects. Table 16.1. Differential diagnosis of primary lactic acidemia

Lactate fasting

Lactate after meal

Lactate: 3-OHpyruvate butyrate: fasting acetoacetate

Ketones

Blood sugar fasting

Typical for

(N)- 

(Rise)

(N)-  (N)- 

()-

N

(N)- 

Rise

N

N

N

N

Respiratory chain disorders PDH

(N)- 

Fall

(N)- ↓



(↓)

PCD

(N)-  N

Fall

(N)-  N

N

N-()

↓↓

GSD I

(Rise)

N

N

↓↓

GSD III

N- 

(Fall)

N

N

- ↓↓

↓↓

(N)- 

(Rise)

N- 

N- 

- 

↓-

Fatty acid oxidation defects OA

Secondary lactic acidemia: causes of elevated lactate are due to deficiencies in blood taking techniques; elevated lactate can be a sign of other disorders as well. Secondary causes of elevated lactate may be due to the following groups of disorders:         

vascular: shock (hypovolemic, post-hemorrhagic); infectious: septicemia, malaria; respiratory: hypoxemia, hyperventilation, status asthmaticus; neurological: seizures, spasticity, perinatal asphyxia; cardiac: cardiomyopathy, heart failure; diabetes mellitus: ketoacidosis, biguanidines; gastrointestinal: chronic diarrhea, malabsorption, short bowel syndrome; renal: chronic renal insufficiency, renal tubular acidosis, urinary tract infection; hepatic: acute liver failure, liver cirrhosis, chronic hepatopathy;

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malignancies: leukemia, lymphoma, (Hodgkin’s disease, NHL); drugs: biguanidines, salicylates, probiotics, statins, stavudine; nutritional: thiamine deficiency; intoxications: ethanol, methanol, CO (table 16.1.).

Of the imaging methods, nuclear magnetic resonance of the brain or NMR spectroscopy is used. CT of the brain is of lesser diagnostic value in this group of disorders [2, 3, 4].

16.3. MUSCLE BIOPSY Muscle biopsy can be used for histochemical, electron microscopic, biochemical or genetic analysis. Histopathology and electron microscopy. Histochemical features often correlate with specific metabolic and genetic changes. Ragged red fibers, presented in biopsy of skeletal muscle, indicate a defect of respiratory Complexes I – IV. Increased lipid within myofibers is a regular feature of Kearns-Sayre syndrome and progressive external ophthalmoplegia (PEO), but not in myoclonic epilepsy with ragged red fibers (MERRF) and mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Total deficiency of succinate dehydrogenase indicates a severe defect in Complex II. Total absence of cytochrome-c-oxidase activity in all myofibers correlates with a severe deficiency of Complex IV, or deficiencies in cytochrome c oxidase activity, or deficiency of coenzyme Q10 [5]. Electron microscopy may confirm abnormal appearance of mitochondria. Immuno-histochemistry. Defects in the presence or absence of specific proteins can rule out other diseases or confirm loss of respiratory chain proteins. Biochemistry. In most cases, deficient activity of the oxidative phosphorylation complex or disorder of the pyruvate dehydrogenase complex is found. In some patients, an isolated disorder of a single complex is observed, while in other cases a combined disorder with deficient activity of several complexes is present. For evaluation of mitochondrial metabolism in the organism, the determination of other parameters is necessary, such as plasmatic alanine, lactate, pyruvate and 3-hydroxy-butyrate, and that repeatedly during the day (before and after meals), alanine in CSF, profile of urinary acylcarnitines and elevated urinary excretion of Krebs cycle products (citrate, aconitase, isocitrate, oxoglutarate, succinate, fumarate, malate) and dicarboxylic aciduria [6, 7].

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16. 4. MITOCHONDRIA AND OXIDATIVE PHOSPHORYLATION In isolated mitochondria or skinned fibers from tissue biopsy, activities of mitochondrial enzymes and respiratory chain function, connected with oxidative phosphorylation, can be measured. A new noninvasive method for measurement of mitochondrial oxidative phosphorylation was developed (see Chapter 17).

16.4.1. Isolation of Mitochondria Mitochondria from heart muscle, skeletal muscle, liver and brain can be isolated by means of differential centrifugation [8, 9]. An isolation solution for heart and skeletal muscle mitochondria contains 180 mmol.l-1 KCl, 4 mmol.l-1 EDTA and 0.1% of albumin with addition of Nagarase 2.5 mg.g-1 of the tissue. Sedimented mitochondria are washed twice in isolation solution without albumin. Liver mitochondria are isolated in a solution containing in mmol.l-1: mannitol 225, saccharose 75 and EDTA 0.2. For isolation of brain mitochondria, a solution containing in mmol.l-1: saccharose 320, K+EDTA 1 and Tris-HCl 10 is used [10].

Respiratory Chain Analysis and Oxidative Phosphorylation Mitochondrial oxidative phosphorylation is measured at 30°C by means of an Oxygraph (Gilson), using a Clark oxygen electrode [11] in a solution containing in mmol.l1 : HEPES 10, KH2PO4 5, KCl 120, EDTA 0.5 and dextran 2%. The function of Complex I is measured with the substrate glutamate (5 mmol.l-1) or glutamate + malate (2.5 + 2.5 mmol.l-1). The function of Complex II is measured with succinate (10 mmol.l-1) and rotenone is used to inhibit Complex I. Respiration is measured before and after addition of 600 nmol ADP, and expressed as state 3 and 4, respectively. Mitochondrial Function Parameters For purity and functional characterization of intact mitochondria, the following parameters are used: RCI (S3/S4): respiratory control index, an indicator of mitochondrial membrane functional integrity; State 3, with ADP stimulated mitochondrial oxygen consumption (in the presence of substrate and ADP added); State 4, basal mitochondrial respiration (after consumption of the added ADP); ADP:O, coefficient of oxidative phosphorylation (indicator of coupling oxidation and phosphorylation); OPR, oxidative phosphorylating rate (indicator of ATP production rate) is calculated [12].

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16. 5. SKINNED FIBERS PREPARATION AND OXIDATIVE PHOSPHORYLATION Endomyocardial biopsy, skeletal muscle biopsy 3 – 5 mg (or spermatozoa) are used for the preparation of skinned fibres and measurements of mitochondrial respiratory chain function.

16.5.1. Isolation of Saponin-Skinned Fibers Wet tissue is cut into small slices 5-6 mm long. Bundles of fibers are placed into 1 ml of an ice-cold incubation medium containing in mmol/l: 20.0 HEPES, 8.0 MgCl2, 0.5 dithiotreitol, 220.0 saccharose, 5.3 ATP, 15.00 creatine phosphate, 5.00 phosphate, 1.0 g albumin – in 1.00 ml, pH 7.4. After 20-minute exposure to saponin (50 microgram), skinned fibers are received; all procedures are carried out at 0-4o C. For saponin removal 10.0 mg/1 ml of isolation solution is used [13, 14, 15].

Respiration Measurements Mitochondrial oxidative phosphorylation in skinned fibers is measured by using Clark oxygen electrode (polarographic method) in a medium consisting of 5.0 mM, KH2PO4, 120.0 mM KCL, 0.5 mM EDTA, 2% dextran, pH 7.2. As substrate, glutamate, pyruvate, succinate can be used in a concentration of 10.0 mM. Mitochondrial ATP production is stimulated with 500 nmol of ADP [11]. Parameters of Oxidative Phosphorylation in Skinned Fibers Basal respiration: Vo (as state 4 in isolated mitochondria of differential centrifugation); Stimulated respiration with ADP: VADP (as state 3, ATP production in isolated mitochondria); Ratio of stimulated and basal respiration: VADP/Vo (as RCI in isolated mitochondria) [16, 17].

16.6. MEASUREMENT OF MITOCHONDRIAL ENZYME ACTIVITY 16.6.1. Citrate Synthase Citrate synthase is a Krebs cycle enzyme located in the mitochondrial matrix. It may be used as an internal control of functional mitochondria [5]. Citrate synthase activity assay is done according to the method of Coore et al., [18].

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16.6.2. Analysis of Enzymatic Activity of Respiratory Chain Complexes Complex I assay (NADH-Ubiquinone Oxidoreductase): Complex I activity is measured by determining the decrease in NADH absorbance at 340 nm, resulting in reduction of ubiquinone to ubiquinol [19]. Complex II assay (Succinate-Ubiquinone Oxidoreductase): Complex II activity is measured by the secondary reduction of a dye (6,6-dichlorophenolindophenol) by the ubiquinol formed, monitored at 600 nm [20]. Complex III assay (Ubiquinol Cytochrome C Reductase): Complex III: enzyme donates electrons from ubiquinol to cytochrome c, leading to reduction of cytochrome c, monitored at 550 nm [19]. Complex IV assay (Cytochrome C Oxidase) is measured by evaluating the oxidation of cytochorme c as a decrease in absorbance at 550 nm [21]. Complex V assay (ATP-synthase): The enzymatic activity of mitochondrial ATPsynthase is monitored at 660 nm, according to the method of Taussky and Shorr [22].

16.7. MOLECULAR TESTS The mitochondrion has its own DNA (mtDNA) with a single, circular structure. The mtDNA communicates with nuclear DNA (nDNA). Five complexes of respiratory chain are encoded by nDNA and mtDNA: Complex I: 41 subunits (mtDNA encoded: 7, nDNA encoded: 34 subunits); Complex II: 4 subunits (all 4 are encoded by nDNA); Complex III: 10 subunits (mtDNA encoded: 1, nDNA encoded: 9 subunits); Complex IV: 13 subunits (mtDNA encoded: 3, nDNA encoded: 10 subunits); Complex V: 12 subunits (mtDNA encoded: 2, nDNA encoded: 10 subunits). Disorders due to mutations in mtDNA and disorders due to mutations in nDNA are included in mitochondrial diseases (encephalomyopathies) [6]. Pathological alterations of mtDNA are involved in three major classes: point mutations, rearrangements, and copy number mutations (depletions). Point mutations include amino acid substitutions and protein synthesis mutation (mRNA, tRNA). Most of these are maternally inherited and heteroplasmic, but they are associated with a striking variety of clinical phenotypes (LHON, MERRF, MELAS, NARP, Leigh‘s syndrome, diabetes and deafness). The second class of mtDNA diseases are deletions with duplications of the mt genome. They are usually sporadic and heteroplasmic, occurring during early development (KSS, Pearson‘s syndrome, CPEO, diabetes, deafness). The third class of mtDNA diseases are mtDNA depletions due to copy number mutations.

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Finally, while detection of mtDNA rearrangement or base substitution confirms the genetic origin of the disease, negativity of these investigations does not rule out an mtDNA mutation nor does it represent a clue that a nuclear mutation is involved. Recently, it was demonstrated that damage to mtDNA and loss of the mitochondrial membrane potential participate in apoptotic cell death [23].

16.8. MAGNETIC RESONANCE SPECTROSCOPY Phosphorus magnetic resonance spectroscopy (MRS) of the muscles and brain allows scientists to study muscle and brain energy metabolism in vivo. Inorganic phosphate (Pi), creatine phosphate (PCr), adenosine mono-di- or tri-phosphate (AMP, ADP, ATP), and intracellular pH may be measured. The Pi/PCr ratio is a most useful parameter that may be monitored at rest and during exercise and recovery. An increased ratio is found in most patients and MRS is becoming a useful tool in the diagnosis of mitochondrial diseases as well as in monitoring therapeutical trials.

16.9. FAMILY HISTORY Histories of family members can sometimes indicate inheritance patterns by noting “soft signs” in unaffected relatives. These include deafness, short stature, migraine, headaches and PEO.

16.10. COENZYME Q ANALYSIS 16.10.1. Determination of Coenzyme Q Homologues Concentrations of coenzyme Q9 and Q10 are determined in isolated mitochondria by the HPLC method [24] with some modifications [25]. Mitochondrial suspension is extracted twice by mixture of hexane/ethanol (5/2 v/v), the organic phases are collected and evaporated under nitrogen, and the residue dissolved in ethanol is injected on to the column SGX C18 7 m (Tessek). The mobile phase consists of methanol/ acetonitrile/ethanol (6/2/2 v/v/v, Merck). Concentrations of the oxidized form of coenzymes Q9 and Q10 are detected spectrophotometrically at 275 nm using external standards (Sigma) and calculated in nmol.mg of proteins-1. Mitochondrial proteins are determined [26].

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For mitochondrial content of cytochromes b, c1, c, aa3, a spectrophotometric method is used at 605 – 630 nm [27]. Cytochrome c release from mitochondria is used as apoptotic signal [28].

16.10.2. Determination of Total CoQ10 in Whole Blood and Plasma Concentration of CoQ10-TOTAL (oxidized + reduced CoQ10) in human blood and plasma can be determined by a modified HPLC method with spectrophotometric detection [24, 25]. For the oxidation of ubiquinol to ubiquinone, 100 µl of 1,4-benzoquinone (2 mg/1 ml double distilled water - daily fresh) is added to 500 µl of blood or plasma and centrifuged for 10 seconds [29]. After 10-minute incubation at room temperature, 2 ml of the mixture hexane/ethanol (5/2 v/v) is added, shaken for 5 minutes, and centrifuged at 1.000 x g for 5 minutes. The hexane layer is separated and the extraction procedure is repeated with 1 ml of the extraction mixture. Collected organic layers are evaporated under nitrogen at 50°C. The residues are taken up in 99.9% ethanol and injected into a reverse phase HPLC column (SGX C18, 7 µm, Tessek Ltd). Elution is performed with methanol/acetonitrile/ethanol (6/2/2, v/v/v) at a flow rate 0.9 ml/min. The concentrations of CoQ10-TOTAL are measured by a UV detector at 275 nm, with an external standard. Data are collected and processed using a CSW32 chromatographic station (DataApex Ltd). Concentrations of CoQ10-TOTAL in blood and plasma are calculated in mol/l-1.

16.10.3. Determination of Total CoQ10 in Isolated Human Platelets Isolated human platelets (100 - 200 µl) were disintegrated with 500 µl of cold methanol [30]. Oxidation of ubiquinol to ubiquinone was performed with 1,4-benzoquinone as described for plasma extraction. The cell suspension was extracted with 2 ml hexane by shaking for 5 min. After centrifugation (1000 x g, 5 min) organic layer was separated and evaporated under nitrogen. The residue was taken up in ethanol and injected into HPLC column. The concentrations of CoQ10-TOTAL were measured, detected and data processed as described above. Concentrations of CoQ10-TOTAL in platelets were calculated in pmol/cells x 109.

16.10.4. Determination of Total CoQ10 in Human Skeletal Muscle Biopsy Biopsy samples from human skeletal muscles (5 - 15 mg) were homogenized using an Ultra-Turrax in 1 ml of double distilled water for 10 seconds. Extraction mixture hexane/ethanol (5/2 vv, 2 ml) and 1,4-benzoquinone (50 µl) were added, and samples were

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mixed and incubated at room temperature for 10 min. After shaking for 5 min and centrifuging, an organic layer was separated, evaporated and measured by HPLC under conditions as described above. Concentrations of CoQ10-TOTAL were calculated in pmol/mg wet weight tissue.

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Munnich, A. Defects of the respiratory chain. Mitochondrial Medicine 2001, Program Book,1-24, San Diego, USA, March 2-4, 2001. Blau, N., Duran, M., Gibson, K. M., Blaskovics, M. E. eds.: Physician´s guide to the laboratory diagnosis of metabolic diseases, 2nd ed. Heidelberg: Springer, 2002. Fernandes, J., Saudubray, J. M., Van den Berghe, G., eds: Inborn Metabolic Diseases, 3rd ed. Heidelberg: Springer, 2000. Hoffmann, G. F., Nyhan, W. L., Zschocke, J., Kahler, S. G., Mayatepek, E. eds.: Inherited Metabolic Diseases, ed. USA: Lippincott Williams  Wilkins, 2002. Sarnat, H. B., Marin-Garcia, J. Pathology of mitochondrial encephalomyopathies. Can. J. Neurol. Sci. 2005; 32: 152-166. Barshop, B. A. Metabolomic approach to mitochondrial diseases: correlation of urine organic acid. Mitochondrion 2004; 4: 521-527. Esterhuizen, K., van der Westhuizen, F. H, Louw, R. Metabolomics of mitochondrial disease. Mitochondrion 2017; 35: 97-110. Palmer, J. W., Tandler, B., Hoppel, C. L. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J. Biol. Chem. 1977; 252: 8731-8739. Sarma, J. S., Ikeda, S., Fischer, R., Maruyama, Y., Weishaar, R., Bing, R. J. Biochemical and contractile properties of heart muscle after prolonged alcohol administration. J. Mol. Cell Cardiol. 1976; 8: 951-972. Bosetti, F., Brizzi, F., Barogi, S., Mancuso, M., Siciliano, G., Tendi, A. A., Murri, L., Rapoport, S. I., Solaini, G. Cytochrome c oxidase and mitochondrial F1F0-ATPase (ATP synthase) activities in platelets and brain from patients with Alzheimer´s disease. Neurobiology of Ageing 2002; 23: 371-376. Rouslin, W., Millard, R. W. Canine myocardial ischemia: defect in mitochondrial electron transfer complex I. J. Mol. Cell Cardiol. 1980; 12: 639-645. Darley-Usmar, V. M., Rickwood, D., Wilson, M. T. Mitochondria, a practical approach, 1987, IRL Press Limited, England, pp. 321. Kunz, W. S., Kuznetsov, A. V., Schulze, W., Eichhorn, K., Schild, L., Striggow, F., Bohnensack, R., Neuhof, S., Grasshoff, H., Neumann, H. W., Gellerich, F. N. Functional characterization of mitochondrial oxidative phosphorylation in saponinskinned human muscle fibers. Biochim. Biophys. Acta 1993; 1144: 46-53.

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[14] Kuznetsov, A. V., Winkler, K., Kirches, E., Lins, H., Feinster, H., Kunz, W. S. Application of inhibitor titrations for the detection of oxidative phosphorylation defects in saponin-skinned muscle fibers of patients with mitochondrial diseases. Biochim. Biophys. Acta 1997; 1360: 142-150. [15] Veksler, V. I., Kuznetsov, A. V., Sharov, V. G., Kapelko, V. I., Saks, V. A. Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim. Biophys. Acta 1987; 892: 191-196. [16] Gvozdjakova, A., Kucharska, J., Kuznetsov, A. V., Gvozdjak, J. Use of myocardial skinned fibres for the study of mitochondrial oxidative phosphorylation during ischaemia. Cor. Vasa 1990; 32(4): 343-350. [17] Gvozdjakova, A., Kucharska, J. Implication of coenzyme Q depletion in heart transplantation. In: Coenzyme Q: Molecular mechanisms in health and disease, Kagan VE, Quinn PJ (eds), CRC Press, USA, 2001; 293-230. [18] Coore, H. G, Denton, M. R., Martin, B. R., Randle, P. J. Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones. Biochem. J. 1971; 125: 115127. [19] Ragan, C. I., Wilson, M. T., Darley-Usmar, V. M., Lowe, P. N. Subfractionation of mitochondria, and isolation of the proteins of oxidative phosphorylation, in Mitochondria, a Practical Approach. London: IRL Press, 1987: 79-112. [20] Hatefi, Y., Stiggal, D. L. Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Methods Enzymol. 1978; 53: 21-27. [21] Wharton, D. C., Tzagoloff, A. Cytochrome oxidase from beef heart mitochondria. Methods Enzymol. 1967; 10: 245-250. [22] Taussky, H. H., Shorr, E. A micro colometric method for the determination of inorganic phosphorus. J. Biol. Chem. 1953; 248: 558-595. [23] Santos, J. H., Hunakova, L/, Chen, Y., Bortner, C., Van Houten, B. (Cell- sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death. J. Biol. Chem. 2003; 278: 1728-1734. [24] Lang, J. K., Gohil, K., Packer, L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Anal. Biochem. 1986; 157: 106-116. [25] Kucharska, J., Gvozdjakova, A., Mizera, S., Braunova, Z., Schreinerova, Z., Schramekova, E., Pechan, I., Fabian, J. Participation of coenzyme Q10 in the rejection development of the transplanted heart. Physiol. Res. 1998; 47: 399-404. [26] Lowry DH, Rosenbrough NY, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193: 265-276. [27] Rickwood, D., Wilson, M. T., Darley-Usmar, V. M. Isolation and characteristics of intact mitochondria. In: Darley-Usmar VM, Rickwood D, Wilson TM (eds): Mitochondria, a Practical Approach. London: IRL Press, 1987: 1-17.

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[28] Zhang, D., Mott, J. L., Farrar, P., Reyrse, J. S., Chang, S. W., Stevens, M., Denniger, G., Zassenhaus, H. P. Mitochondrial DNA mutations activate the mitochondrial apoptotic pathway and cause dilated cardiomyopathy. Cardiovasc. Res. 2003; 57(1): 147-157. [29] Mosca, F., Fattorini, D., Bompadre, S., Littarru, G. P. Assay of coenzyme Q10 in plasma by a single dilution step. Anal. Biochem. 2002; 305: 49-54. [30] Niklowitz, P., Menke, T., Andler, W., Okun, J. G. Simultaneous analysis of coenzyme Q10 in plasma, erythrocytes and platelets: comparison of the antioxidant level in blood cells and their enviroment in healthy children and after oral supplementation in adults. Clin. Chim. Acta 2004; 342: 219-226.

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 17

RESPIROMETRIC ANALYSIS OF MITOCHONDRIAL FUNCTION IN HUMAN BLOOD CELLS Zuzana Sumbalová1,2,*, Luiz F. Garcia-Souza3, Beáta Veliká4, Chiara Volani5 and Erich Gnaiger1 1

Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria 2 Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 3 Institute of Sport Science, University Innsbruck, Innsbruck, Austria 4 Department of Medical and Clinical Biochemistry, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, Slovakia 5 Department of Internal Medicine II, Infectious Diseases, Immunology, Rheumatology and Pneumology, Medical University of Innsbruck, Innsbruck, Austria

ABSTRACT Respirometric analysis of mitochondrial function in human immune cells and platelets has become increasingly attractive, since such tests can be accomplished within a few hours and may thereby support early diagnosis of diseases with mitochondrial dysfunctions. Here we discuss methodological aspects of blood cell preparation, show examples of measurements of mitochondrial respiration in intact and permeabilized platelets and peripheral blood mononuclear cells, and give a short overview of the current literature on blood cell bioenergetics in health and disease.

*

Corresponding Author Email: [email protected].

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Keywords: immune cells, lymphocytes, platelets, PBMC, oxidative phosphorylation, respirometry

17.1. INTRODUCTION Assessment of mitochondrial function in human blood cells represents a rapidly expanding area of translational research. Although various pathologies are clinically manifested primarily in a specific organ, bioenergetics may be affected in several organs simultaneously. Blood cells represent an attractive source of mitochondria which can be obtained much less invasively compared to tissue biopsies. Therefore, substantial efforts have been made during the past 20 years to investigate their usefulness as a potential substitute for tissue biopsies for functional diagnostics and monitoring the progress of treatment in metabolic diseases. Blood contains a wide array of circulating cell types: erythrocytes, platelets, and a heterogeneous group of leukocytes which includes mononuclear cells (monocytes, lymphocytes) and polymorphonuclear cells (neutrophils, eosinophils, basophils, mast cells). The physiology and bioenergetics differ considerably between different cell types, thus making some more suitable for translational research in various pathologies than others [1, 2]. Since human mature erythrocytes do not contain mitochondria and bioenergetics of neutrophils also depends mainly on glycolytic ATP formation [3, 4], platelets, monocytes and lymphocytes are the main target of investigation of mitochondrial function in translational research. Thrombocytes (platelets, PLT) are cytoplasmic fragments of discoid shape with 2-3 µm diameter, released from megakaryocytes in bone marrow. They circulate in the blood for 7 to 10 days and are essential for blood coagulation. Platelets are metabolically very active in the resting state and upon their functional activation, glycolytic and oxidative metabolism increase even further [5]. When activated, platelets release pro- and antiinflammatory factors and micro-particles into the circulation, recruiting leukocytes to injured or inflamed sites, and promoting clot formation concurrently. These direct or chemokine-mediated platelet-platelet and platelet-leukocyte interactions play a central role in inflammation and immune response [6]. Lymphocytes are a heterogeneous population comprising the smallest leukocytes (Tand B-cells, involved in the adaptive immune response) with 6-9 µm diameter, and natural killer (NK) cells. Originating from the bone marrow, B-cells subsequently migrate to the spleen, while T-cells mature in the thymus. In healthy humans, T-cells represent 65-85% of peripheral blood lymphocytes. There are 3 main subpopulations of T-cells: helper Tcells, suppressor T-cells, and cytotoxic T-cells (killer cells). The individual types of B- and T-cells can be distinguished by immunostaining of different cell surface markers (CD antigens) expressed by these cells. Lymphocytes stay in the circulation for rather long

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periods of time [7]. Importantly, activation of lymphocytes is essential for their immunological functions and is accompanied by an increase of glycolytic activity and mitochondrial respiration [8]. Monocytes represent the largest circulating leukocytes with a diameter up to 20 µm, involved in the immediate defense against infection. Derived from monoblasts, they develop in the bone marrow and circulate in the blood upon release for about 1 to 3 days. After this time, they usually migrate to the tissues, where they differentiate to macrophages or dendritic cells. Lymphocytes and monocytes are frequently studied, mainly due to the availability of isolation methods, as a mixed population of immune cells. They are collectively referred to as peripheral blood mononuclear cells (PBMC), with lymphocytes representing about 80% of the PBMC population.

17.2. ISOLATION OF BLOOD CELLS Isolation of blood cells from whole blood collected by venipuncture is, in principle, straightforward. However, the quality and purity of isolated cells are affected by numerous factors. It is therefore strictly advisable to follow established practical guidelines for handling blood samples and isolating blood cells [9]. Platelets can be isolated from platelet-rich plasma obtained after low g centrifugation of the whole blood [4, 10], or from the mixed PBMC-platelet layer obtained after centrifugation of diluted blood on density gradient medium. Different cell populations of PBMC (monocytes, T- and B-lymphocytes) can be further separated by staining with antibodies and separation using magnetic beads labeled with anti-antibody and Magnetic Activated Cell Sorting (MACS) separator [4]. Alternatively, surface markers can be stained with fluorescence dyes and cells can be separated using Fluorescent Activated Cell Sorting (FACS). Currently, a consensus is lacking among diagnostic and research laboratories on standardized methods for the isolation of blood cells used in respirometric measurements (for PBMC and PLT, see [11]). Specific differences include the choice of anticoagulants, duration of the storage of collected blood, temperature until and during the isolation procedure, media for cell isolation and gradient centrifugation, centrifugation speeds, brakes and the number of washing steps. These factors affect significantly the quality, purity and viability of isolated cells and contribute to considerable biases or irreproducibility of reported results on cell respiration. Up to date, there is no firm evidence for the impact of different isolation protocols on mitochondrial physiology of blood cells. The purity of the target cell fraction is crucial for respiratory measurements, as contaminating cells may significantly contribute to respiration, which is typically expressed per cell count of the target cells. PLT and neutrophils are the main contaminants in the PBMC fraction. To assess the purity of a preparation, a device counting all cell types

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has to be used. For this purpose, a hematology analyzer routinely used for clinical analyses (e.g., Sysmex XN-350, Sysmex Corporation) is most suitable.

17.3. MEASUREMENT OF CELLULAR BIOENERGETICS IN BLOOD CELLS Assessment of human mitochondrial function is usually limited by the availability of only small amounts of sample. During the past 25 years, development of highly sensitive instruments made the measurements of respiration in blood cells possible. Sufficient numbers of PLT and PBMC for respirometric measurements can be isolated from 9 ml of whole blood. Such functional tests are generally performed with fresh samples and can be completed within hours. Alternatively, respirometric competence has been reported for cryopreserved PBMC, with obvious advantages for diagnostic applications [12]. Two devices are most commonly used in the literature for such studies: (1) the O2kFluoRespirometer (Oroboros Instruments, Austria), and (2) the Seahorse XFe Extracellular Flux Analyser (Agilent, USA). Critical evaluation of instrumental specifications is necessary for planning a research project. (1) The O2k device is designed for High-Resolution FluoRespirometry. Oxygen consumption is measured polarographically in a suitable medium in two 2-ml Durane glass chambers under continuous stirring and tight temperature control. Mitochondrial function can be assessed in suspended cells [13-16], permeabilized muscle fibers, tissue homogenates, and isolated mitochondria [15, 16]. Extended substrate-uncoupler-inhibitor titration (SUIT) protocols can be applied with multiple consecutive titrations [15-18]. (2) The Seahorse XFe Extracellular Flux Analyser provides measurement of oxygen consumption and extracellular acidification in microplates (24 or 96 wells) using fluorescent probes. Up to four titrations per well can be programmed automatically in correspondingly simple experimental protocols. This multi-well system is designed for high-throughput screening of cellular metabolism in attached cells [19]. Suspension cells or isolated mitochondria must be adhered to the bottom of the micro-wells before starting the measurements. The impact of artificial adherence of naturally suspended blood cells is not clear and should be taken into account. Notably, the volume-specific oxygen flux in experiments with blood cells is rather low, which requires rigorously tested corrections for oxygen background flux, two-point oxygen signal calibration, and adequate number of data points, all of which is available only with the first instrument.

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17.4. ASSESSMENT OF MITOCHONDRIAL FUNCTION BY HIGH-RESOLUTION RESPIROMETRY The O2k-FluoRespirometer (Oroboros Instruments, Austria; Figure 17.1) is a sensitive device to measure mitochondrial function in small samples. Mitochondria are not only essential for cellular metabolism and energy production, they are also involved in many physiological and pathological processes, including programmed cell death, reactive oxygen species metabolism, and signaling. High-Resolution FluoRespirometry (HRFR) has the potential to resolve questions for current scientific challenges in mitochondrial pathophysiology by assessing respiratory function in structurally intact organelles. Mitochondrial function can be assessed in intact cells or in cells after selective permeabilization of plasma membranes.

Figure 17.1. Oroboros O2k-FluoRespirometer. Copyright by Oroboros Instruments. Reproduced with permission; www.oroboros.at.

17.4.1. Measurement of Respiration in Intact Cells Isolated intact cells offer an advantage of studying respiration under near-physiological conditions, but limit the application of substrates to those that are permeable through the plasma membrane [15, 16]. In intact cells, ROUTINE respiration, oligomycin-induced LEAK respiration, uncoupler-stimulated electron transfer (ET) capacity, and ET inhibitorinduced residual oxygen consumption (Rox) can be determined [15-17]. Respiratory coupling states can be evaluated in the presence of external substrates or in their absence

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with respiration fueled by internal substrate stores (endogenous respiration). External substrates are taken up by living cells to various extents and intracellular metabolism of external and internal substrates supports mitochondrial respiration. Frequently, respiration of intact cells is measured in cell culture media containing non-physiological high concentrations of glucose, L-glutamine and other accessible substrates, which can fuel or inhibit (Crabtree effect) respiration. A representative measurement performed simultaneously in 2 chambers of an O2k-FluoRespirometer is presented in Figure 17.2. ROUTINE respiration is controlled by cellular energy demand, energy turnover and the degree of coupling of oxidation to phosphorylation. LEAK respiration or LEAK oxygen flux, compensating for proton leak, proton slip, cation cycling, and electron leak, is a dissipative component of respiration, which is not available for performing biochemical work. LEAK respiration in intact cells is assessed upon inhibition of ATP synthase by oligomycin, which can cross the cell membrane. ET-capacity is the oxidative capacity in the noncoupled state at optimum uncoupler concentration. This optimum concentration is obtained by stepwise titration of an established protonophore (such as CCCP, FCCP or DNP) to induce maximum oxygen flux as the determinant of ET-state. As shown in Figure 2, the presence of oligomycin may exert an inhibitory effect on ET-capacity, therefore, for proper evaluation of ET-capacity, measurements without addition of oligomycin should be performed in parallel. Residual oxygen consumption, Rox, is respiration due to oxidative mitochondrial and non-mitochondrial side reactions remaining after inhibition of the ET system. For evaluation of ET-linked mitochondrial respiration, O2 flux in all respiratory coupling states should be corrected for Rox. From these measured parameters, flux control ratios, excess ET-ROUTINE capacity, and coupling efficiency are calculated to characterize the bioenergetics of the intact cell [16, 17]. Small changes in cellular respiration and minor alterations in respiratory control may indicate significant mitochondrial defects, severe injuries of mitochondrial proteins or mtDNA, or alterations in the state of mitochondrial signaling cascades [15, 16].

17.4.2. Measurement of Mitochondrial Respiration in Permeabilized Cells For detailed respiratory OXPHOS analysis, cells must be permeabilized or mitochondria should be isolated to make mitochondria accessible to external fuel substrates, inorganic phosphate and ADP. Cell membranes can be selectively permeabilized by detergents such as saponin or digitonin. These compounds have a high

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a

b Figure 17.2. Respiration of intact PBMC. A) Traces from the measurement in cell culture medium RPMI 1640 without glutamine at 37 °C in 2 mL chambers of the Oroboros O2k-FluoRespirometer. The blue line represents oxygen concentration in chamber A, green and red traces represent oxygen consumption as flow per 106 cells (chambers A and B, respectively). A coupling control protocol was applied in parallel with 4·106 PBMC per chamber (2·106 per mL). After stabilization of ROUTINE respiration, oligomycin (ATP-synthase inhibitor; 2.5 µM) was added in chamber B (red trace) to induce LEAK respiration (in chamber A the solvent for oligomycin, ethanol, was added in this step). Uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was titrated stepwise to evaluate maximum ET-capacity. Complex I inhibitor rotenone inhibited respiration of intact cells. Subsequently, Complex II substrate succinate (10 mM) was added for evaluation of integrity of the cell membrane. The intact plasma membrane is impermeable to succinate, which therefore can stimulate respiration only in cells with a damaged cell membrane but functional mitochondria, which are counted as dead cells. Finally, Complex III inhibitor antimycin A was added to fully inhibit mitochondrial respiration for evaluation of residual oxygen consumption (Rox) in the ROX state. Since the intact plasma membrane is permeable to all applied inhibitors and the uncoupler, there is no need for cell membrane permeabilization [15]. Experiment 2016-05-19 P7-03.DLD. B) Coupling/pathway control diagram illustrating respiratory states obtained step-by-step in the applied coupling control protocol.

affinity to cholesterol and the plasma membrane can be selectively permeabilized at an optimum concentration, leaving mitochondrial membranes intact. After permeabilization of cells, substrates linked to different ET-pathways can be titrated sequentially to measure respiratory capacities in different coupling and pathway control states (Figure 17.3). Such

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SUIT protocols are designed to obtain extended information about the mitochondrial respiratory system from a single experimental run. An expanding library of SUIT protocols exploring different aspects/pathways of the OXPHOS system is available [20]. Respirometric OXPHOS analysis with SUIT protocols allows the discrimination of specific respiratory defects, which may provide the diagnostic basis for applications of targeted therapies.

Figure 17.3. Schematic illustration of convergent electron transfer at the NADH-junction in the Npathway, and at the Q-junction in the combined NS-pathway (modified from [17]). The NADHjunction is a junction for convergent electron flow from NADH-linked substrates through the mitochondrial NADH pool to Complex I (CI), and further transfer through the Q-junction to Complex III (CIII). Representative type N substrates are pyruvate (P), glutamate (G) and malate (M). The corresponding dehydrogenases (PDH, GDH, MDH) and some additional TCA cycle dehydrogenases (isocitrate dehydrogenase, oxoglutarate dehydrogenase) generate NADH, the substrate of Complex I (CI). Succinate (S) is the direct substrate of Complex II (succinate dehydrogenase). S supports electron transfer through Complex II to CII-bound flavin adenine dinucleotide (FADH2) and to Q. NS electron input or the NS-pathway control state is electron input from a combination of substrates for the N- and S-pathway through Complexes CI and CII simultaneously into the Q-junction. NS electron input reconstitutes TCA cycle function with convergent electron flow through the ET-pathways.

In typical measurements, 4·106 PBMC or 200·106 platelets are added to the 2 mL O2k chambers. The optimal concentration of digitonin is tested for each cell type in a separate respirometric protocol [16]. Figure 17.4 shows a SUIT protocol for evaluation of respiratory capacities with separate electron flow through the N- or S-pathway and with convergent electron flow through the NS-pathway [17, 18].

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17.5. MITOCHONDRIAL FUNCTION OF BLOOD CELLS IN HEALTH AND DISEASE A growing body of literature suggests that mitochondrial function of blood cells is a sensitive marker of various systemic disorders. In patients with amyotrophic lateral sclerosis, Complex IV (CIV) is impaired in both PLT and PBMC [22], reflecting the systemic nature of this disease. In patients with Alzheimer´s disease CIV activity is decreased in brain mitochondria and PLT [23], and decreased N-OXPHOS-capacity is found in permeabilized lymphocytes [24]. Interestingly, mitochondrial respiration of intact PBMC [12] and PLT [25] is decreased in patients with major depression. Bioenergetics of PBMC can reflect the health of an organism, since these cells are activated by the release of stress factors into the circulation. Accordingly, respiration of PBMC and PLT normalized to cell count is increased in patients with sepsis, an effect accompanied by increased density of mitochondria per cell in PBMC, but not in PLT [26, 27]. Different responses of both cell types in systemic inflammation might be attributed to their different physiology and function. PBMC are primarily involved in immune defense, contain a nucleus and can increase mitochondrial mass according to acute metabolic requirements. In contrast PLT, essential for blood clotting lack a nucleus, and are consequently more limited in their potential for metabolic reprogramming. In diabetic patients, PLT often exhibit hypersensitivity and enhanced reactivity in response to agonists [28]. In an animal model of type 1 diabetes mellitus, increased mitochondrial membrane potential and elevated mitochondrial respiration was found in PLT, corresponding to their activated state [29]. Adverse effects of high doses of statins on cellular energy metabolism was reflected by diminished respiration of both intact and permeabilized PLT in rats, and even therapeutic doses of statins in humans caused a decrease in respiration of permeabilized PLT [30]. Recent studies indicate a positive correlation between respiration of PBMC and physical function and strength in overweight elderly adults [31, 32]. An increase in PBMC respiration corresponding to activation of PBMC was reported in healthy adults with low vitamin D content and high concentrations of inflammatory cytokines. These data suggest that PBMC respiratory function is a sensitive marker for proinflammatory conditions [33]. In addition, respiration of monocytes is sensitive to oxidative stress and may be potentially used as a functional biomarker of systemic oxidative stress in patients with metabolic disorders [34].

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a

b Figure 17.4. SUIT protocol with permeabilized PBMC. A) Trace from the measurement performed at 37°C. PBMC (4·106) were added to the 2-mL chamber of the Oroboros O2k-FluoRespirometer containing respiration medium MiR05 [21]. After stabilization of ROUTINE respiration, NADH-linked substrates pyruvate (5 mM) and malate (2 mM) were added to fuel the N-pathway upon cell membrane permeabilization with 32 µg digitonin (8 µg/106 cells). Next, a kinetically saturating concentration of ADP (1 mM) was injected and stimulated respiration to OXPHOS-capacity. Subsequently, NADHlinked substrate glutamate (10 mM) was added to further stimulate the N-pathway. CII substrate succinate (10 mM) stimulated convergent electron flow to the Q-junction, now from both the Npathway and succinate (NS). Titration of uncoupler CCCP allowed evaluation of maximal NS- ETcapacity. Subsequent inhibition of CI by rotenone allowed evaluation of S-ET-capacity. Addition of CIII inhibitor antimycin A enabled evaluation of residual oxygen consumption (Rox) subtracted from all fluxes for correction. Experiment 2015-08-11 PS-02.DLD. B) Coupling/pathway control diagram illustrating respiratory states obtained step-by-step in the applied SUIT protocol. Metabolic control steps are shown by arrows linking respiratory states, with vertical arrows indicating coupling control, and horizontal arrows indicating pathway control [17, 18].

CONCLUSION Various populations of blood cells, primarily lymphocytes, monocytes and platelets are well suited and accessible for analysis of mitochondrial respiration in humans.

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Metabolic and bioenergetic characteristics of various local and systemic pathologies can be identified by assessment of cellular respiratory function of PBMC and PLT. Detailed, high-resolution measurements are required to provide quantitatively reliable data. Importantly, there is a need for harmonization of protocols applied for isolation of blood cells, cell counting and respirometric analyses. In addition, normalization of respiration per cell count and per mitochondrial markers should be standardized to make datasets comparable within and across different research groups. Further studies are required to define the cell types which are most suitable as biomarkers in a spectrum of human diseases. These will contribute to the establishment of standardized functional mitochondrial assays, as required in preventive medicine and diagnosis of mitochondrial diseases.

AUTHOR CONTRIBUTIONS ZS and EG designed the experiments and wrote the manuscript. LFGS co-wrote the manuscript, BV and CV revised the manuscript, ZS, BV and LFGS carried out the experiments and data analysis. CV performed the experiment for Figure 17.3. EG was responsible for the resources.

ACKNOWLEDGMENTS We thank Verena Menz and Martin Burtscher for taking the blood samples and for the ethics application, Verena Laner for project administration, Stephanie Droescher for technical assistance, Gerhard Krumschnabel and András Mészáros for critical comments to the manuscript. Contribution to K-Regio project MitoFit funded in part by the Government of Tyrol within the program K-Regio of Standortagentur Tirol. Supported by Action of Austria-Slovakia (Beáta Veliká). Contribution to COST Action CA15203 MitoEAGLE.

DECLARATION OF CONFLICTING INTERESTS Competing financial interests: Erich Gnaiger is founder and CEO of Oroboros Instruments, Innsbruck, Austria.

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[24] Leuner K, Schulz K, Schütt T, Pantel J, Prvulovic D, Rhein V, Savaskan E, Czech C, Eckert A, Müller WE. Peripheral mitochondrial dysfunction in Alzheimer's disease: focus on lymphocytes. Mol Neurobiol 2012; 46: 194-204. [25] Hroudová J, Fišar Z, Kitzlerová E, Zvěřová M, Raboch J. Mitochondrial respiration in blood platelets of depressive patients. Mitochondrion 2013; 13: 795-800. [26] Sjövall F, Morota S, Persson J, Hansson MJ, Elmér E. Patients with sepsis exhibit increased mitochondrial respiratory capacity in peripheral blood immune cells. Crit Care 2013; 17: R152. [27] Sjövall F, Morota S, Asander Frostner E, Hansson MJ, Elmér E. Cytokine and nitric oxide levels in patients with sepsis-temporal evolvement and relation to platelet mitochondrial respiratory function. PLOS ONE 2014; 9: e97673. Corrected and republished in: PLOS ONE 2014; 9: e103756. [28] Watala C. Blood platelet reactivity and its pharmacological modulation in (people with) diabetes mellitus. Curr Pharm Des 2005; 11: 2331-2365. [29] Siewiera K, Kassassir H, Talar M, Wieteska L, Watala C. Higher mitochondrial potential and elevated mitochondrial respiration are associated with excessive activation of blood platelets in diabetic rats. Life Sci 2016; 148: 293-304. [30] Vevera J, Fišar Z, Nekovářová T, Vrablík M, Zlatohlávek L, Hroudová J, Singh N, Raboch J, Valeš K. Statin-induced changes in mitochondrial respiration in blood platelets in rats and human with dyslipidemia. Physiol Res 2016; 65: 777-788. [31] Tyrrell DJ, Bharadwaj MS, Jorgensen MJ, Register TC, Molina AJA. Blood cell respirometry is associated with skeletal and cardiac muscle bioenergetics: Implications for a minimally invasive biomarker of mitochondrial health. Redox Biology 2016; 10: 65-77. [32] Tyrrell DJ, Bharadwaj MS, Van Horn CG, Kritchevsky SB, Nicklas BJ, Molina AJA. Respirometric Profiling of Muscle Mitochondria and Blood Cells Are Associated with Differences in Gait Speed Among Community-Dwelling Older Adults. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 2015; 70: 1394-1399. [33] Calton EK, Keane KN, Soares MJ, Rowlands J, Newsholme P. Prevailing vitamin D status influences mitochondrial and glycolytic bioenergetics in peripheral blood mononuclear cells obtained from adults. Redox Biol 2016; 10: 243-250. [34] Chacko BK, Zhi D, Darley-Usmar VM, Mitchell T. The Bioenergetic Health Index is a sensitive measure of oxidative stress in human monocytes. Redox Biol 2016; 8: 43-50.

In: Recent Advances in Mitochondrial Medicine ... ISBN: 978-1-53613-149-9 Editors: A. Gvozdjáková, G. Cornélissen et al. © 2018 Nova Science Publishers, Inc.

Chapter 18

COENZYME Q10 TARGETING THERAPY OF MITOCHONDRIAL DISTURBANCES Anna Gvozdjáková1,*, Jarmila Kucharská1, Rafael de Cabo2, Luca Tiano3 and Plácido Navas4 1

Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, Medical Faculty, Comenius University in Bratislava, Slovakia 2 National Institute of Aging, Baltimore, MD, US 3 Institute of Biochemistry Polytechic University of MARCHE, Ancona, Italy 4 Universidad Pablo de Olavide, Sevilla, Spain

ABSTRACT Coenzyme Q10 (CoQ10) is an essential mobile component of the mitochondrial respiratory chain, which is present in all tissues and organs, including blood. CoQ10 transports electrons and protons in the respiratory chain, contributing to aerobic energy production; it has antioxidant properties, and it is an obligatory substrate for pyrimidine nucleotide synthesis and β-oxidation of fatty acids. Two major forms of CoQ10 are present in the body: ubiquinone, the oxidized lipophilic form which represents about 10%, and ubiquinol, the reduced form, which represents about 90% of the total. Ubiquinol is one of the strongest antioxidants; it reduces oxidative stress, free radicals production, and its bioavailability can be higher than ubiquinone in specific formulations. CoQ10 concentration decreases with age, after statin treatment, and in various mitochondrial diseases grouped as CoQ10 deficiency syndrome. Supplementary and targeting CoQ10 mitochondrial therapy is being recommended for improving health and quality of human life.

*

Corresponding Author Email: [email protected].

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Keywords: coenzyme Q10, ubiquinone, ubiquinol, targeting therapy, mitochondria

18.1. COENZYME Q10 AS UBIQUINONE AND UBIQUINOL CoQ10 was found in all tissues of the body; higher concentrations were found in tissues with very active oxidative metabolism, such as brain, heart, skeletal muscle, and kidney. CoQ10 was first isolated by Professor Frederick Loring Crane in 1957 (Figure 18.1) as a golden crystalline substance from the bovine heart mitochondria at the Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin USA [1.

Figure 18.1. Professor Fred L Crane. (5th Conference of the International CoQ10 Association, Kobe, Japan, 2007).

The yellow substance was found to be reversibly reduced losing its color; the major finding was to demonstrate the essential function of CoQ10 in the respiratory chain of beef heart to produce ATP (Figure 18.2). CoQ10 resembles a vitamin and is sometimes called “vitamin Q.” Professor Karl Folkers (Figure 18.3) determined the chemical structure of CoQ10 in year 1958 [3. The structure of CoQ10 is similar to vitamin K (chemical name: 2,3-dimethoxy-5-methyl-6decaprenyl-1,4-benzochinone). The benzoquinone ring is linked to the long side isoprenyl chain that in humans consists of 10 isoprenyl units. CoQ10 is a lipophilic antioxidant capable of recycling and regenerating other antioxidants, such as vitamin E and ascorbic acid, with special effect on plasma membrane [4; its supplementation causes changes in gene expression [5. CoQ10 as an antioxidant occurs in all cellular membranes as well as in high- and low-density lipoproteins [6.

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Abbreviations: Cell cytoplasm; outer mitochondrial membrane; inner mitochondrial membrane; intermembrane space; complexes of respiratory chain: I, II, III, IV, V; Q-cycle – coenzyme Q10 cycle; cyt c – cytochrome c; e- electrone; NADH – reduced nikotinamide adenine dinucleotide; NAD+nikotinamide adenine dinucleotide; FADH2 – reduced flavine adenine dinucleotide; FAD+ – flavine adenine dinucleotide; O2- - superoxide radical; H2O2 – hydrogen peroxide; .OH- hydoxyl radical; proteins; lipids; DNA – deoxyribonucleic acid; O2 – oxygen; H2O – water; ADP – adenosine dinucleotide phosphate; ATP – adenosine trinucleotide phosphate; Pi – inorganic phosphate. Figure 18.2. Q10-CYCLE, part of mitochondrial respiratory chain [2.

Figure 18.3. Professor Karl Folkers.

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Two major forms of CoQ10 are present in the body: ubiquinone, the oxidized form, and ubiquinol, the reduced form. The redox cycle between these two forms is one of the strongest antioxidants that prevents oxidative stress, reduces free radical formation, and contributes to prevent damage in lipids, proteins and DNA, CoQ10 occurs in Q10-CYCLE [7, Figure 18.2. The Q-cycle is considered the movement of electrons among the Q molecules inside complex III. The reduction of ubiquinone by the transplasma membrane redox system enzymes cytochrome b5 reductase and NQO1 contributes to the antioxidant cell protection [8, which can involve CoQ10 semiquinone [9. The reduction of ubiquinone in the respiratory chain is required for ATP biosynthesis by oxidative phosphorylation; a specific CoQ10 cycle is present in complex III to improve high-energy electron transit [10. In an experimental study, decreased specific activity of NQO1 (DT-diaphorase) was found with age in the mouse and in the rat liver [11, 12. Other non-mitochondrial enzymes, e.g., thioredoxin reductase, which was significantly decreased in kidneys of aged rats, were able to reduce ubiquinone [13. Ubiquinol supplementation overcomes the impaired reducing capability [14, 15. Kaneka confirmed that ubiquinol is stable in acidic condition, in low pH as demonstrated by Kaneka´s patent [16. In the organism, there is a dynamic balance between ubiquinone and ubiquinol, Figure 18.4.

P. Lambrechts, Kaneka, Brussels, 2011. Figure 18.4. A dynamic balance between ubiquinone and ubiquinol.

The content of CoQ10 in tissues and organs is different; it is highest in the heart and its concentration and biosynthesis rate depends on age and nutrition [17. From the total CoQ10 concentration in the cell, mitochondria contain 40-50% (25-30% plasma membrane, 1520% endoplasmic reticulum and 5-10%, cytoplasm).

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The central role of CoQ10 is in the “redox poise,” which represents the ratio of ubiquinol to ubiquinone, and determines its key role in metabolic control, energy production, and gene regulation, as the “orchestra leader” of metabolic pathways in the body [18. The antioxidant benefit of CoQ10 is in the reduction of free oxygen radical production and inhibition of inflammatory and oxidative damage to endothelial cells [19, 20. CoQ10 modulates endothelial dysfunction and inhibits atherosclerosis through microRNAs (miRNAs) that are involved in key biological processes in protein translation [21. The human organism can acquire CoQ10 from exogenous sources, such as meals (pork and beef, fish, broccoli, melon, red grapefruit, grapes, red wine, sunflower, and beans). Daily CoQ10 intake from a meal ranges from 3-5 mg. However, most of the CoQ10 comes from the endogenous biosynthesis in all cells. CoQ10 is not soluble in water, so it is slowly absorbed from the small intestine, passes into the lymph, then into the blood and tissues. Absorption of exogenous CoQ10 and its bioavailability depend on the type of CoQ10 preparation. Improvement of the absorption of CoQ10 has been reported when it is consumed with grapefruit juice [22. Ubiquinol is the most active form of CoQ10; when given orally, it has been shown to have higher bioavailability than ubiquinone [23. The maximal absorption time is from 6 to 10 hours [24, 25. All supplemental CoQ10 products are better absorbed when taken with meals. In our study, we found a significantly increase (p