Mitochondria and the Heart · Pathology and Pathophysiology of Cardiovascular Disease. 2001 ISBN 0-7923-7542-4 241. Liong Bing Liem, Eugene Downar (eds): Progress in Catheter Ablation

Mitochondria and the Heart

Developments in Cardiovascular Medicine

232. A. Bayes de Luna, F. Furlanello, BJ . Maron and D.P. Zipes (eds.):Arrhythmias and Sudden Death in Athletes. 2000 ISBN: 0-7923-6337-X

233. J-C. Tardif and M.G. Bourassa (eds): Antioxidants and Cardiovascular Disease.2000. ISBN: 0-7923-7829-6

234. J. Candell-Riera, J. Castell-Conesa, S. Aguade Bruiz (eds): Myocardium atRisk and Viable Myocardium Evaluation by SPET. 2000.ISBN: 0-7923-6724-3

235. M.H. Ellestad and E. Amsterdam (eds): Exercise Testing: New Concepts for theNew Century. 2001. ISBN: 0-7923-7378-2

236. Douglas L. Mann (ed.): The Role of Inflammatory Mediators in the FailingHeart. 2001 ISBN: 0-7923-7381-2

237. Donald M. Bers (ed.): Excitation-Contraction Coupling and CardiacContractile Force, Second Edition. 2001 ISBN: 0-7923-7157-7

238. Brian D. Hoit, Richard A. Walsh (eds.): Cardiovascular Physiology in theGenetically Engineered Mouse, Second Edition. 2001 ISBN 0-7923-7536-X

239. Pieter A. Doevendans, A.A.M. Wilde (eds.): Cardiovascular Genetics for Clinicians2001 ISBN 1-4020-0097-9

240. Stephen M. Factor, Maria A.Lamberti-Abadi, Jacobo Abadi (eds.): Handbook ofPathology and Pathophysiology of Cardiovascular Disease. 2001

ISBN 0-7923-7542-4241. Liong Bing Liem, Eugene Downar (eds): Progress in Catheter Ablation. 2001

ISBN 1-4020-0147-9242. Pieter A. Doevendans, Stefan Kaab (eds): Cardiovascular Genomics: New

Pathophysiological Concepts. 2002 ISBN 1-4020-7022-5243. Daan Kromhout, Alessandro Menotti, Henry Blackburn (eds.): Prevention

of Coronary Heart Disease: Diet, Lifestyle and Risk Factors in the SevenCountries Study. 2002 ISBN 1 -4020-7123-X

244. Antonio Pacifico (ed.), Philip D. Henry, Gust H. Bardy, Martin Borggrefe,Francis E. Marchlinski, Andrea Natale, Bruce L. Wilkoff (assoc. eds):Implantable Defibrillator Therapy: A Clinical Guide. 2002 ISBN 1-4020-7143-4

245. Hein J.J. Wellens, Anton P.M. Gorgels, Pieter A. Doevendans (eds.):The ECG in Acute Myocardial Infarction and Unstable Angina: Diagnosis and RiskStratification. 2002 ISBN 1-4020-7214-7

246. Jack Rychik, Gil Wernovsky (eds.): Hypoplastic Left Heart Syndrome. 2003ISBN 1-4020-7319-4

247. Thomas H. Marwick: Stress Echocardiography. Its Role in the Diagnosis and Evaluationof Coronary Artery Disease 2nd Edition. ISBN 1 -4020-7369-0

248. Akira Matsumori: Cardiomyopathies and Heart Failure: Biomolecular, Infectiousand Immune Mechanisms. 2003 ISBN 1-4020-7438-7

249. Ralph Shabetai: The Pericardium. 2003 ISBN 1-4020-7639-8250. Irene D. Turpie; George A. Heckman (eds.): Aging Issues in Cardiology. 2004

ISBN 1-40207674-6251. C.H. Peels; L.H.B. Baur (eds.): Valve Surgery at the Turn of the Millennium. 2004

ISBN 1-4020-7834-X252. Jason X.-J. Yuan (ed.): Hypoxic Pulmonary Vasoconstriction: Cellular and Molecular

Mechanisms. 2004 ISBN 1-4020-7857-9253. Francisco J. Villarreal (ed.): Interstitial Fibrosis In Heart Failure 2004

ISBN 0-387-22824-1254. Xander H.T. Wehrens; Andrew R. Marks (eds.): Ryanodine Receptors: Structure, function

and dysfunction in clinical disease. 2005 ISBN 0-387-23187-0255. Guillem Pons-Llado; Francesc Carreras (eds.): Atlas of Practical Applications of

Cardiovascular Magnetic Resonance. 2005 ISBN 0-387-23632-5256. Jose Marin-Garcia : Mitochondria and the Heart. 2005 ISBN 0-387-25574-5Previous volumes are still available


Edited by

Jose Marin-GarciaThe Molecular Cardiology and Neuromuscular Institute,Highland Park, New JerseyDepartment of Physiology & Biophysics,UMDNJ-Robert Wood Johnson Medical SchoolPiscataway, New Jersey

With the collaboration ofMichael J GoldenthalThe Molecular Cardiology Institute,Highland Park, New Jersey

4jj Springer

JosÈ MarÌn-GarcÌaThe Molecular Cardiology and Neuromuscular InstituteHighland Park, New JerseyDepartment of Physiology & BiophysicsUMDNJ-Robert Wood Johnson Medical SchoolPiscataway, New Jersey

Library of Congress Cataloging-in-Publication Data

A C.I.P. Catalogue record for this book is availablefrom the Library of Congress.

ISBN-10 0-387-25574-5 e-ISBN 0-387-25575-3ISBN-13 978-0387-25574-3 e-ISBN 978-0387-25575-0Printed on acid-free paper.

© 2005 Springer Science+Business Media, Inc.All rights reserved. This work may not be translated or copied in whole or in part withoutthe written permission of the publisher (Springer Science+Business Media, Inc., 233 SpringStreet, New York, NY 10013, USA), except for brief excerpts in connection with reviews orscholarly analysis. Use in connection with any form of information storage and retrieval,electronic adaptation, computer software, or by similar or dissimilar methodology nowknow or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks and similar terms,even if the are not identified as such, is not to be taken as an expression of opinion as towhether or not they are subject to proprietary rights.While the advice and information in this book are believed to be true and accurate at thedate of going to press, neither the authors nor the editors nor the publisher can accept anylegal responsibility for any errors or omissions that may be made. The publisher makes no

warranty, express or implied, with respect to the material contained herein.

Printed in the United States of America.

9 8 7 6 5 4 3 2 1 SPIN 11055679

springeronline.com

Dedication

This book is dedicated to mywife, Daniele, and daughter,Melanie, with love

Contents

1. An Introduction to Mitochondriaand the Heart

Overview 1What are mitochondria? 1How to study mitochondria: New and old 3Mitochondrial bioenergetics 6Mitochondrial biogenesis 7Cardiac mitochondrial changes during cardiac

growth and development 9Mitochondria: the primary site of ROS

generation and also a critical target of itsdamaging effects 10

Mitochondrial dysfunction in cardiovasculardisease 10

Defects in mtDNA 10Defects in nuclear-DNA encoded mitochondrial

proteins 12Myocardial ischemia and ETC 13Apoptosis and cell death 14Animal models of mitochondrial-associated

cardiovascular disease 15Diagnosis and treatment of mitochondrial-based

cardiac diseases 16The road ahead 16References 17

viii Mitochondria and the Heart

2. Mitochondrial Bioenergetics in the Heart 27

Overview 27Introduction 27Complex I (NADH-ubiquinone oxidoreductase) 28Enzyme structure 29Complex II (Succinate-ubiquinone oxidoreductase) 30Complex III (Ubiquinol-cytochrome c oxidoreductase) 32The Q cycle and bcl function 33Complex IV (Cytochrome c oxidase) 35COX enzyme structure 36COX subunit gene structure and expression 37COX activity regulation 39Complex V (F0-Fi ATPase; ATP synthase) 40Subunit composition, structure, and function 40ATP synthase defects and related pathologies 44Other enzymes bioenergetics 45Adenine nucleotide translocator (ANT) 45ANT structure 47ANT regulation in the heart 47ANT genes and their expression 47ANT dysfunction plays a key role in

cardiac pathology 48Creatine kinase 49Structure 50Creatine kinase and the heart 51PDH and the TCA (Krebs) cycle 52Overall regulation of mitochondrial bioenergetic

in the heart 53Supply and demand, substrates, and oxygen and

ATP-ADP levels 53References 56

CONTENTS ix

3, Heart Mitochondrial Biogenesis 63

Overview 63Introduction 63Structure of mtDNA 64MtDNA Function 66Replication 66Regulation of mtDNA replication 69Mitochondrial transcription and RNA processing 70Regulation of mitochondrial transcription and

processing 72Mitochondrial translation 73Mitochondrial ribosomes 73Initiation and elongation of translation 76Mitochondria DNA repair 76Nuclear participation in mitochondrial biogenesis 79Nuclear regulatory proteins and coordination

of transcriptional events 79Hormones affecting both mitochondrial and

nuclear transcription 82Mitochondria import and assembly of proteins 83Relevance of mitochondrial biogenesis 85References 85

4. ROS Generation, Antioxidants,and Cell Death 99

Overview 99Introduction 99The significance of ROS 100Generation of ROS 100Negative effect of ROS 102Antioxidant defense 104Role of ROS in cell signaling 106ROS and cardiac pathology 106ROS and apoptosis 108Apoptosis and cell death 109Myocardial apoptosis 112References 113


5. Myocardial Ischemia andCardioprotection 123

Overview 123Introduction 123Mitochondrial dysfunction in myocardial ischemia 124Oxidation-phosphorylation decline in ischemia 124Myocardial ischemia and oxidative stress 126Myocardial ischemia and mitochondrial

calcium flux 127Gene expression 128Reperfusion 129Cardioprotection 129Ischemic cardioprotection 129Cascade of mitochondrial events in IPC 131Signaling pathways 134Adenosine and other ligands 134Activation and translocation of PKC as a pivotal signaling event

in the genesis of IPC and CP 136Reactive oxygen species 137Cardioprotection: An emerging field 138Early and late IPC pathways 139Potential applications to clinical medicine 142References 143

6. Mitochondria Dysfunction inCardiomyopathy and Heart failure 157

Overview 157Introduction 157Mitochondria are the major source of bioenergy

in the cardiac cell 158What is the evidence for myocardial dependency on

mitochondrial function? 160Mitochondrial dysfunction and cardiomyopathy:

Support from human studies 160

CONTENTS xi

Clinical evidence of nuclear mutations in mitochondrialcomponents 165

Constribution of transgenic models to the study of mitochondria in heartdysfunction 166Evidence from animals models that mitochondrial

bioenergetic enzymes play a critical role in HF 168Mitochondrial dysfunction and other cellular pathways

in cardiomyopathy 169Mutations in contractile/sarcomere proteins and

mitochondrial function 170Mitochondrial function and cardiac hypertrophy 173Calcium signaling and mitochondrial function in HF 174Mitochondrial function and apoptosis in HF 174What events occurring in HF are truly tissue-specific? 176Future prospects 178References 179

1. Fatty Acid and Glucose Metabolism inCardiac Disease 197

Overview 197Introduction 197Role of fatty acids and their metabolism in the

normal cardiomyocyte: Structural and regulatoryroles in cardiac cell membranes 198

Fatty acid transporters and glucose carriers 198B ioenergetics of FAO 201Cellular location of FAO and glucose oxidation 204The effect of disorders of fatty acid and glucose metabolism on

cardiac structure/function 205Secondary effects on mitochondrial fatty acid (3-oxidation:

Relationship to mitochondrial respiration and OXPHOS 208Fatty acid metabolism defects and their association with

cardiomyopathy and arrhythmias 208Cardiomyopathy 208Arrhythmias and conduction defects 209

xii Mitochondria and the Heart

Fatty acids, glucose, and cardiac apoptosis 210Abnormalities in mtDNA and their association with both

diabetes and cardiomyopathy 211Molecular players in fatty acid-related cardiac diseases;

modulation of gene expression 211MCAD 211VLCAD 212CPT-II 212MTP 212PPAR 213Peroxisome proliferator-activated receptor y

coactivator (PGC- la) 215Animal models of defective fatty acid metabolism and

cardiac failure 216Advances in diagnostics and treatment of fatty acid/cardiac

disease 217References 218

8. Mitochondria in Pediatric Cardiology 229

Overview 229

MlTOCHONDRIAL CARDIOMYOPATHY 229

Introduction 229Diagnosis 232Clinical signs 233Histological and electron microscopic (EM) analysis 233Biochemical analysis 235MtDNA analysis 236Mitochondrial tRNA mutations 236Mitochondrial structural gene mutations 241ATP synthase (ATPase6) 241Cytochrome b (cytb) 242Mutations in COX and ND subunits 243MtDNA depletion 244

MITOCHONDRIA AND CONGENITAL HEART DEFECTS (CHD) 245

Structural and functional cardiac defects 245Cardiac arrhythmias 246

CONTENTS xiii

Other congenital cardiomyopathies with mitochondrialdefects 246

Congenital heart defects and mitochondrial respiration 247Conclusions 248References 249

9. Mitochondria and the Aging Heart 263

Overview 263Introduction 264Bioenergetics and gene expression in the aging heart 265Mitochondrial function and gene expression in the

aging heart 266ROS in the aging heart 268Mitochondrial DNA damage in the aging heart 270Rat model of cardiac aging: Defects in ETC 273Gene expression 275DNA copy number and damage analysis 275Comments 276On mitochondrial dysfunction 276On mitochondrial DNA damage 277On gene expression of mitochondrial stress proteins and cellular

programming 277On mitochondrial PT pore 278Conclusions 278Potential approaches to reverse mitochondrial dysfunction

in the aging heart 279References 281

10. Heart Mitochondria SignalingPathways 289

Overview 289Introduction 289Mitochondrial abnormalities, signaling defects, and

myocardial disease 290

xiv Mitochondria and the Heart

Mitochondrial signaling in myocardial ischemiaand cardioprotection 291

Mitochondrial signaling and myocardialhypertrophy 294

Signaling the mitochondria: Key players 296Nuclear gene activation 296Protein kinases 298Calcium signaling 300Mitochondrial receptors 301Signals of survival and stress impact heart

mitochondria 302Survival signals and apoptosis 304Stress signals 306Metabolic signals 307Future prospects: Therapeutic targets and directions 307The essential role played by mitochondriain in cytoprotective

signaling and CP 307Mitochondrial metabolic intermediates 308Summary 309References 310

11. Treatment of Mitochondrial-BasedCardiac Diseases: Targeting theOrganelle 323

Overview 323Introduction 323Treatment of respiratory and metabolic defects 324Use of antioxidants 325Treatment of FAO disorders, arrhythmias, and CHF 327Cardioprotective agents 329New approaches in treatment 333Gene therapy in cardiovascular diseases 333Mitochondria and gene therapy 336Targeting mitochondria using nucleic acids 336Targeting mitochondria using bioactive compounds 339References 340

CONTENTS xv

12. Future Frontiers in MitochondrialCardiac Biology 351

Overview 351Introduction 351Disease gene identification: Diagnostic application 353Modifying factors, Epigenetics, and SNPs 354Animal models of mitochondrial-based heart

disease 360Cellular engineering 363Applications of stem cells in mitochondrial defects and

toxicology 365References 367

Glossary 377

Index 388

Preface

Mitochondria have been pivotal in the development of some of themost important ideas in modern biology. Since the discovery that theorganelle has its own DNA and specific mutations were found inassociation with neuromuscular and cardiovascular diseases and withaging, an extraordi-nary number of publications have followed, and theterm mitochondrial medicine was coined. Furthermore, ourunderstanding of the multiple roles that mitochondria play in cardiaccell homeostasis opened the door for intensive experimentation tounderstand the pathogenesis and to find new treatments forcardiovascular diseases.

Besides its role in adenosine triphosphate generation, mitochondriaregu-late a complex network of cellular interactions, involving (1)generation and detoxification of reactive oxygen species, includingsuperoxide anion, hy-drogen peroxide, and hydroxyl radical; (2)maintenance of the antioxidant glutathione in a reduced state andadequate level of mitochondrial matrix superoxide dismutase; (3)cytoplasmic calcium homeostasis, particularly under conditions ofcellular calcium loading; (4) transport of metabolites betweencytoplasm and matrix; (5) both programmed (apoptosis) and necroticcell death; and (6) cell growth and development. It is therefore notsurprising that this organelle has come to be the center stage in manycurrent investigations of cardiovascular diseases, aging, and aging-related disease. Concomitant with these advances, an impressive effortis under- way for the development of new tools and methodologies tostudy mitochondrial structure and function, including powerful ways tovisualize, monitor, and alter the organelle function to assess the geneticconsequences of these perturbations.

Because the heart is highly dependent for its function on oxidativeenergy that is generated in mitochondria—primarily by fatty acid p-oxidation, respiratory electron chain, and oxidative phosphorylation(OXPHOS)—it is understandable that defects in mitochondrialstructure and function can be found in association with cardiovasculardiseases.

Abnormalities in the organelle structure and function are beingincreas-ingly reported in association with conditions such as dilated andhypertro-phic cardiomyopathy, cardiac conduction defects and sudden

xviii Mitochondria and the Heart

death, ischemic and alcoholic cardiomyopathy, myocarditis, andneuromuscular diseases associated with cardiac disease and aging.Some of the mitochondrial abnormalities may have a genetic basis(e.g., mitochondrial DNA changes might lead to abnormal OXPHOS,and fatty acid oxidation defects might be due to specific nuclear DNAmutations), while other abnormalities may be due to a more sporadic orenvironmental cardiotoxic insult or may not yet be characterized.

To understand the role that mitochondria play in cardiovasculardisease, we discuss the biogenesis and function of cardiac mitochondriaduring normal growth, development, and aging. Within this context, wethen examine the interaction and characterization of mitochondria andmitochondrial abnormalities in cardiac diseases, their diagnosis,therapeutic options currently available, future directions for research,and new frontiers in treatment. While aberrations in the bioenergeticfunction of the mitochondria are frequently related to cardiacdysfunction, the specific defect causing the bioenergetic abnormalitiesoften resides in a nonbioenergetic pathway (e.g., signaling between themitochondria and nucleus) or in the overall mitochondrial biogenesis ordegradation pathways. Understanding these pathways and the effectsthat mitochondrial defects have in cardiac pathology is extremelyimportant in establishing the diagnosis and treatment of mitochondrial-based cardiac diseases.

As mitochondria's role in the field of cardiology is strengthenedand as research on the multiple functions of this organelle continues itsexpansion, the time seems appropriate for a book that may integrateknown facts, what is developing and what will be known in the nearfuture. In addition to providing a recount of past discoveries, the bookdeals with areas that are of emerging interest to researchers andclinicians, eyeing potential alternatives that may improve currentlyavailable therapies and interventions in the management ofcardiovascular diseases in general and the cardiovascular pathology ofaging in particular.

It is hoped that this work will further advance the field ofmitochondrial medicine.

"New discoveries, fragments of the past, parts of the future."

Jose Marin-GarciaHighland Park, New Jersey

Chapter 1

An Introduction to Mitochondria and theHeart

Overview

This introductory chapter to mitochondria and the heart describesmitochondria and their components, their multiple functions (includ-ing their pathology in cardiovascular diseases), and briefly their rolein myocardial ischemia/cardioprotection, apoptosis/cell death, andcurrently available animal models of mitochondrial-based cardiac de-fects. Most of these subjects merit a more comprehensive discussionand have chapters dedicated to them.

What are mitochondria?

Mitochondria, the powerhouse of the cell, are double-membranedorganelles located in the cytoplasm, and their primary cellular role isthe generation of bioenergy.

The components of the mitochondria (Figure 1.1) include a compactmitochondrial genome and their own class of ribosomes. Structural,regulatory, and functional proteins are involved in a variety of tasksthat range from enzymatic constituents of bioenergetic pathways—e.g., oxidative phosphorylation (OXPHOS) and respiration, thetricarboxylic acid (TCA) cycle, fatty acid oxidation (FAO); substrate,ion, and nucleotide transport membrane channels; biosynthesis ofmitochondrial components e.g., mitochondrial RNA and DNA(mtDNA); the import and assembly of the various protein complexes;and critical elements that are involved in intercellular communicationand cell-death pathways (e.g., redox signaling and apoptoticprogression).

While most biological membranes have approximately a 50:50 ratioof protein to lipid, the inner mitochondrial membrane is somewhatexceptional because it exhibits a ratio of 75:25, which is indicative ofmore densely packed proteins. In cardiac tissue, the anionic phospho-

MITOCHONDRIA AND THE HEART

ATP

PTPORE

carnitineshuttle

nuclear DNAfragmentation

PyruvateCytosol

Fatty acids APOPTOTICCELL DEATH

Figure 1.1. The mitochondrial organelle. The inner and outer mitochondrialmembranes have numerous points of contact (termed contact sites) and contain alarge assortment of integral and peripheral proteins as well as numerousphospholipids. The electron transport chain (ETC) is composed of inner-membranelocalized respiratory complexes I-V with associated electron-transfer components,coenzyme Q (CoQ) and cytochrome c (cytc) as shown. Also shown are the matrixassociated pyruvate oxidation, fatty acid (5-oxidation (FAO), and the TCA cyclepathways. The apoptosis-associated mitochondrial permeability transition (PT)pore is shown including key components such as the adenine nucleotidetranslocator (ANT), porin, mitochondrial creatine kinase (CK), hexokinase (HEX)and the inner membrane phospholipid, cardiolipin (CL). The release of apoptogenicpeptides (e.g., cyt c) from mitochondria precedes caspase activation, leading toapoptotic cell death. Also depicted is the carnitine shuttle pathway for the mitchon-drial import of fatty acids including carnitine palmitoyltransferases (CPT-I andCPT-II) and carnitine translocase (CAT), as is the pyruvate transporter (PyT). Thegeneration ofROSfrom mitochondrial ETC is depicted as are the mitochondrial an-tioxidants superoxide dismutase (MnSOD) and glutathione peroxidase (GPx). ThemtDNA is shown with transcripts( mtRNA) which are translated on mitochondrialribosomes( mtribosomes) forming peptide subunits of complex I,III, IV, and V.

lipid, cardiolipin, is particularly prevalent in the mitochondrial innermembrane and at the contact sites. Pores in the outer membrane

An Introduction to Mitochondria and the Heart 3

facilitate the transport of most small molecules (< 5000 kDa) in andout of the mitochondria and are composed primarily of the abundantprotein, porin.The inner membrane contains the components of themitochondrial respiratory electron transport chain (ETC), OXPHOS,and adenine nucleotide transport (ANT). Because the proton andelectron flux are located in the inner mitochondrial membrane, theorganelle is primarily responsible for the electrochemical potentialgradient associated with ATP synthesis and its coupling with electrontransport. The inner membrane forms highly folded lamellarstructures that are termed cristae and that extend the surface area ofthe inner membrane to within the matrix; the number of cristaesharply increases in highly respiratory-active cardiomyocytes (i.e.,threefold more than found in hepatocytes).

The matrix compartment enclosed within the inner membrane con-tains the mtDNA, ribosomes, transfer, and ribosomal RNAs as well asa multitude of enzymes required for the oxidation of pyruvate, fattyacids, and TCA cycle metabolites.The number and morphology of mitochondria within the cardiomyo-

cyte can change as a function of diverse physiological stimuli (e.g.,exercise, hormones/cytokines, electrical stimulation, etc.), the stage incardiac development and pathophysiological insult (e.g., cardiachypertrophy). The population of mitochondria in the cardiomyocytesis controlled by autophagy, a process by which lysosomes regulatethe different cellular components.

Under some conditions, more than 1,000 mitochondria can be pres-ent in a cardiomyocyte. Typically distributed in a uniform fashionalong the entire length of the myofibrillar apparatus (Figure 1.2), theyhave variable shapes that are likely related to the extent ofmitochondrial fusion and division. Mitochondria are involved incardiac contraction to provide a constant supply of ATP to thesarcomeres. Information concerning the movement of mitochondriawithin the cardiac cell and its regulation is currently limited; however,it is possible that cardiac mitochondrial movement is mediated byboth actin myofilaments and microtubules as has been demonstratedwith neural cells [1].

How to study mitochondria: New and old

The in vitro growth, culturing, and passaging of cardiomyocytesderived from either neonates or adults have furnished highly informa-

MITOCHONDRIA AND THE HEART

Figure 1.2. Mitochondria of different shapes (arrows) are distributed alongmyofibers.

tive models to study cardiomyocyte phenotype, molecular andbiochemical events in response to physiological stresses, the additionand deletion of specific genes, and pharmacological treatments.Moreover, cardiomyocyte culture has proven to be a convenientsystem for examining rapid signaling changes in membrane channels,mitochondria, transducing kinases, and the receptors associated withmyocardial hypertrophy, hypoxia, and apoptosis. In addition, recent-ly developed cardiac myocyte cell lines have provided researchers theopportunity to evaluate the expression of several transcription factorsassociated with early cardiac development.

Over the last decade, great advances have been made in cytochem-ical techniques for evaluating mitochondria, both at the ultrastruc-tural and functional levels, including the use of fluorescent dyes. Thedevelopment of fluorescent imaging technology is improving ourability to measure precisely levels of specific ions and metabolites inthe mitochondrial organelle of living cells as well as its sub-compartments, such as the mitochondrial matrix. For example, by


specifically targeting the Ca++ probe aequorin (a Ca++ binding photo-protein) to the mitochondrial matrix, free mitochondrial Ca++ levelscan be determined [2]. A variety of fluorescent probes andpotentiometric dyes (listed in Table 1.1) have been used increasinglyto evaluate quantitatively overall cardiomyocyte mitochondrialnumber, membrane potential, oxidative stress and Ca++ levels [3-8].

Table 1.1. Fluorescent dyes

Fluorescent Dye To evaluate ReferenceMitotracker green Mitochondrial number [8]JC-1 Membrane potential [5]CMX-Ros Membrane potential [4]Fura-2 Ca++ levels [7]Dihydrorhodamine 123 Oxidative stress [6]

Other techniques that are available include the fractionation and iso-lation of mitochondria and their membrane-bound subcomparmentsby differential centrifugation to recover specific membrane fractions;the identification of specific markers of mitochondria and potentiallycontaminating subcellular organelles (e.g., ER, lysosomes); the devel-opment and availability of a large armamentarium of specific anti-bodies to mitochondrial proteins for use in both immunocytochemicalanalysis and western immunoblot analysis; easily accessed andupdated databases with molecular information to furnish specificprobes for molecular genetic and gene expression analysis; a varietyof amplification and mutation detection techniques to screen formaternally inherited mtDNA point mutations and Mendelian inheritednuclear DNA mutations; quantitatively accessing large-scale mtDNAdeletions; gauging mitochondrial copy number; and improvedtechniques for the analysis of mtDNA damage. Several excellentbooks containing updated methods are commercially available.

A recent and critical development in mitochondrial methodologyinvolves the use of cell hybrids (cybrids) to study the effects ofspecific mutations on mitochondrial function. The development ofcultured mammalian cells that lack mtDNA due to growth in lowconcentrations of ethidium bromide was pivotal in the developmentof the cybrid technique [9-10] These cells are comparable to yeastpetite cells that lack mtDNA and are similarly termed rho° cells.These cells lack mtDNA, exhibit defective respiration, and adopt ananaerobic phenotype. Cytoplasts containing mitochondria can be

° MITOCHONDRIA AND THE HEART

prepared from a wide variety of enucleated cells (e.g., platelets andfibroblasts) and fused with rho° cells lacking mtDNA to form cybrids,essentially changing the combination of nucleus and mitochondria.Cybrids containing normal mitochondria regain functional respira-tion, manifest an aerobic phenotype, and can be readily distinguishedfrom cybrids with defective mitochondria. Cybrids can be maintainedin culture using the appropriate media supplementation and have beensuccessfully employed to study nuclear-cytoplasmic interactions, aswell as the effects of specific mitochondrial mutations in differentnuclear backgrounds.

Mitochondrial bioenergetics

Mitochondria are abundant in energy-demanding cardiac tissueconstituting 20% to 40% of cellular volume. Mitochondrial energyproduction depends on both nuclear and mtDNA-encoded geneticfactors that modulate normal mitochondrial function (includingenzyme activity and cofactor availability) and on environmentalfactors (including substrate availability—such as sugars, fats, andproteins— and oxygen). Several interacting bioenergetic pathwayscontribute to mitochondrial energy metabolism (shown in Figure 1.1),including pyruvate oxidation, the TCA cycle, the (3-oxidation of fattyacids, and the common final pathway, OXPHOS, which generatesapproximately 80% of cellular ATP. OXPHOS is performed bycomplexes of proteins located at the mitochondrial inner membrane,including the respiratory ETC complexes I-IV, ATP synthase(complex V), and the adenine nucleotide translocator (ANT). Fattyacids are the primary energy substrate for ATP production byOXPHOS in postnatal and adult cardiac muscle. To be fully utilizedfor bioenergetic production via mitochondrial fatty acid (3-oxidation,fatty acids need to be effectively transported into the cardiomyocyteand subsequently into the mitochondria, a process requiring severaltransport proteins including the carnitine shuttle (carnitine acyltrans-ferase and two carnitine palmitoyltransferases as well as carnitine).Fatty acid (3-oxidation and the oxidation of carbohydrates via theTCA cycle generate the majority of intramitochondrial NADH andFADH2, the direct source of electrons for ETC/OXPHOS. The supplyof ATP from other sources (e.g., glycolytic metabolism) is limited innormal cardiac tissue. In addition to these bioenergetic pathways and


metabolic intermediates, the heart also maintains stored high-energyphosphates (e.g., phosphocreatine) produced by mitochondrial cre-atine kinase using ATP from closely associated ANT and mitochon-drial ATP synthase.

Mitochondrial biogenesis

Human mitochondria have their own double-stranded circular DNAencoding 13 protein components of 4 of the enzyme complexes (i.e.,I, III, IV, and V) involved in electron transport and OXPHOS. Theseprotein-encoding mtDNA genes are transcribed into specific mRNAsthat are translated on mitochondrial specific ribosomes. The mtDNAalso encodes part of the mitochondrial protein synthesis machinery,including 2 ribosomal RNAs (rRNA) and 22 transfer RNAs (tRNA),as shown in Figure 1.3 [11].

D-loop12s cytb

16sND6

ND5

ND4

COIcom

COII ATPase6ATPase8

Figure 1.3. Human mitochondrial double-stranded circular DNA. This circularDNA encodes 13 protein components of 4 of the 5 enzyme complexes involved inelectron transport and OXPHOS, two rRNAs (12S and 16S), and 22 tRNAs, asshown. The noncoding D-loop region is also shown.

In general, the cardiomyocyte contains multiple mitochondria (50to 100 per cell) and each mitochondrion contains multiple copies ofmtDNA (1 to 10 copies per mitochondrion).

Pathogenic point mutations and large-scale deletions in mtDNA aswell as generalized depletion of mtDNA levels have severe conse-

8 MITOCHONDRIA AND THE HEART

quences for organs such as the heart, since ATP derived fromOXPHOS is needed to maintain myocardial contractility. The nucleargenome encodes the entire complement of proteins involved inmtDNA replication and transcription, protein components ofmitochondrial ribosomes, multiple structural and transport proteins ofthe mitochondrial membranes, and remaining peptide subunits of therespiratory complexes. Synthesized on cytosolic ribosomes, thesenuclear-encoded proteins are targeted to mitochondria and importedby a complex but well characterized process involving signal peptiderecognition, membrane receptors, proteases, and an array of molecularchaperones. Cardiac-specific regulation of a number of the nucleargenes encoding OXPHOS proteins can be mediated by variable geneexpression sensitive to a number of physiological and developmentalstimuli and by the presence of tissue-specific isoforms for specificpeptides. Therefore, mutations in nuclear genes involved inmitochondrial biogenesis might be expected to contribute to theobserved mitochondrial cardiac enzyme and mtDNA defects,including an increased incidence of large-scale mtDNA deletions andmtDNA depletion associated with cardiac disorders. However, thusfar only a limited number of mutations have been identified in nucleargenes affecting mitochondrial biogenesis and leading to cardiacdisorders. While the nuclear genome controls mitochondrialbiosynthesis, mtDNA genes have a much higher mutation ratebecause of a lack of histones, limited DNA repair, and exposure toreactive oxygen species (ROS) generated by the ETC. Nevertheless,the search for mutations in both nuclear and mitochondrial genomesas well as mutations that affect the cross-talk between the genomes ispresently expanding.

Cardiac mitochondrial biogenesis is increased during myocardial hy-pertrophy, treatment with a variety of agents (e.g., thyroxin and xe-nobiotics), electrical stimulation, and exercise [12], but the mole-cular mechanisms that regulate cardiac-specific mtDNA levels andoverall mitochondrial number are not known [13].


Cardiac mitochondrial changes during cardiac growthand development

Variations in mitochondrial substrate utilization, enzyme andmembrane activities, growth program, and gene expression occurduring the course of cardiac development and in aging, and theyreflect changes in cardiac function [14]. Moreover, the role (anddemand) for ATP production is clearly modulated during cardiac de-velopment as well as during physiological transitions and perturba-tions.

Among the multiple metabolic changes that occur in cardiac musclewith advancing age are modifications in membrane fatty acid andlipid composition, including increased levels of saturated fatty acidsand decreased levels of polyunsaturated fatty acids and cardiolipin[15-16]. Cardiolipin, the most unsaturated cellular phospholipid, is amajor component of the mitochondrial inner membrane; plays anintrinsic role in cardiac mitochondrial membrane transport function,fluidity, and stability; and is a facilitator of key mitochondrial innermembrane bioenergetic enzymes. In addition, marked reductions incarnitine and acetylcarnitine levels have also been reported in theaging heart.

A by-product of mitochondrial bioenergetic activity is generationof ROS, including superoxide, hydroxyl radicals, and hydrogenperoxide (H2O2) [17]. Side reactions of the mitochondrial ETC withmolecular oxygen directly generate the superoxide anion radical.Superoxide radicals can be converted to H2O2 (in the presence of theenzyme superoxide dismutase), which can further react to form thehydroxyl radical. Normally, these toxic by-products, which arepowerful cell-damaging oxidants, are neutralized by antioxidant en-zymes, some of which are found in the mitochondria (e.g., manganesesuperoxide dismutase [MnSOD] and glutathione peroxidase) andothers of which are found in the cytosol (e.g., CuSOD or catalase) orare scavenged by glutathione. Increased generation of ROS occursduring aging and can also result from myocardial ischemia/reperfu-sion, inflammation, and impaired antioxidant defenses. This maycause profound effects on cardiac cells, including increasedperoxidative damage effecting primarily the membrane phospholipidsand proteins. Oxidative damage also targets nucleic acids (particular-ly mtDNA), causing induction of strand breaks, base modificationsand subsequently, point mutations, and deletions [18].


Mitochondria: The primary site of ROS generation, andalso a critical target of its damaging effects

The mitochondrial respiratory chain, located in the inner membrane,is often damaged, resulting in further increase in ROS generation andleading to a vicious cycle of diminished mitochondrial function. Inaddition to the well characterized cell-damaging effects of ROS,mitochondrial ROS generation and oxidative stress are recognized ashaving important regulatory functions. Oxidative species (e.g., H2O2)can function as a potent signal sent from mitochondria to othercellular sites rapidly and reversibly, eliciting an array of intracellularcascades that lead to different physiological end-points for thecardiomyocyte (e.g., apoptosis, necrosis, cardioprotection, and cellproliferation).

Mitochondrial dysfunction in cardiovascular disease

Defects in mtDNA

Discrete mitochondrial OXPHOS defects or respiratory chain enzymeactivity deficiency have been documented in both dilated (DCM) andhypertrophic (HCM) cardiomyopathies [19-20]. In addition,cardiomyopathy can present with specific pathogenic mtDNA pointmutations. Pathogenic mtDNA mutations are generally located innucleotides that are highly conserved in evolution, usuallyaccompanied by reduced levels of myocardial specific respiratoryenzyme activity, and frequently present in heteroplasmic fashion (amixed population of both mutant and wildtype mtDNA genomes)[21-23], albeit recent data suggest that certain pathogenic mtDNAmutations can also be homoplasmic [24].

Mutations in a number of mitochondrial tRNA genes have been de-tected in association with cardiomyopathy. Specific tRNA genesincluding Leu, He, and Lys appear to be hot spots for mutations incardiomyopathic patients. In patients with either HCM or DCM,mutations at other mtDNA loci have also been identified (e.g., othertRNAs, rRNA, and in a variety of mtDNA protein-encoding genes).In addition, multisystemic mitochondrial diseases with a widespectrum of clinical manifestations, which include cardiomyopathy,


have been reported with pathogenic mtDNA mutations. Thesedisorders, including Leigh, MELAS (mitochondrial encephalomyopa-thy with lactic acidosis and strokelike episodes) and MERRF(myoclonic epilepsy with ragged red fibers) syndromes are maternallyinherited and often present with a variable cardiac phenotype and arange of neurological symptoms [21]. Specific mtDNA mutationsfound in association with primary cardiomyopathy can also be presentin patients with different combinations of clinical phenotypes. Thevariability between mitochondrial genotype and phenotype may berelated to the involvement of unidentified genetic or environmentalcofactor(s) capable of modulating the effect of mtDNA mutations.Similarly, specific phenotype(s) may be caused by a variety ofdifferent nuclear and/or mtDNA mutations. For example, Leighsyndrome can be caused by mutations in ATPase-6 subunit [25], pointmutation in mitochondrial tRNALys gene [26], nuclear DNA mutationsin pyruvate dehydrogenase [27], nuclear-encoded complex II subunits[28], or mtDNA depletion [29].

Sporadic large-scale rearrangements of mtDNA can also be associ-ated with cardiac disorders. In Kearns-Sayre syndrome (KSS), cardiacconduction abnormalities typically coexist with somatic large-scalemtDNA deletions [30-31]. The majority of mtDNA deletions in KSSare of a single type; they are not inherited and are mainly detected inskeletal muscle and rarely in blood. In contrast, a multiple mtDNAdeletion phenotype associated with DCM may result from geneticdefects in unidentified autosomal nuclear loci that can be eitherdominantly or recessively inherited [32-33]. The mtDNA deletionsdetected in KSS and in the autosomal disorders tend to be abundantcompared to the less abundant, large-scale mtDNA deletions revealedby polymerase chain reaction (PCR) amplification in cardiac tissuesof many primary cardiomyopathies [34]. These less abundantdeletions reflect specific mtDNA damage (probably as a consequenceof ROS) and often occur in an age-dependent manner; however, theirrole in cardiac pathogenesis is unclear.

Depletion in cardiac mtDNA levels has also been reported in pa-tients with isolated cardiomyopathy, either DCM or HCM [35-36],and also can be induced by drugs such as zidovudine (AZT) andadriamycin (doxorubicin) [37-38].

1 2 MITOCHONDRIA AND THE HEART

Defects in nuclear DNA-encoded mitochondrialproteins

Mutations in a wide array of nuclear genes encoding mitochondrialproteins can also cause cardiomyopathy. For example, cardiomyopa-thy may occur as a consequence of mutations in mitochondrialtransport proteins that facilitate the passage of critical metabolitesacross the inner mitochondrial membrane. Mutations in a mitochon-drial transport protein, frataxin, which is involved in mitochondrialiron accumulation, cause Friedreich ataxia (FRDA) with HCM [39].Moreover, mutations in nuclear genes encoding factors required forthe assembly and functioning of the multiple-subunit respiratorycomplexes have been implicated in some mitochondrial-based dis-eases such as Leigh syndrome. Mutations in the SCO2 gene encodinga copper chaperone that takes part in complex IV assembly can resultin cardiomyopathy [40].

Cardiomyopathy may also be a primary manifestation of several in-herited disorders of mitochondrial FAO [41]. Deficiencies in verylong-chain acyl-CoA dehydrogenase (VLCAD) [42], long-chain 3-hydroxylacyl-CoA dehydrogenase (LCHAD) [43], short-chain acyl-CoA dehydrogenase (SCAD) [44], and the mitochondrial trifunctionalprotein (MTP) [45] have been reported to cause cardiomyopathy inyoung children. Also, defects in carnitine transport into cells and inthe carnitine-acylcarnitine shuttle, which is responsible for fatty acidtransport into mitochondria, can be associated with cardiomyopathy[46]. The cardiac pathogenesis of these inherited disorders of fattyacid (3-oxidation and carnitine metabolism likely includes both adeficient bioenergetic supply to the heart and the accumulation oftoxic levels of fatty free acids with subsequent cardiac dysfunction.These disorders primarily occur in early childhood and are usuallyprecipitated by infectious illness or fasting when the heart hasincreased dependence on FAO for energy. Many of the inherited FAOdisorders described above can result in sudden neonatal death [41].

Cardiac conduction defects also occur in patients with specific de-fects in FAO [47]. Both ventricular and atrial arrhythmias areassociated with deficiencies in CPT-II, carnitine translocase, andMTP activities.

Long-chain acylcarnitines possess detergent-like properties andaccumulation can extensively modify membrane proteins and lipidswith toxic effects on cardiac membrane electrophysiologicalfunctions, including ion transport and gap-junction channel activity


[48-49]. Moreover, the accumulation of long-chain fatty acidmetabolites plays a pivotal role in the production of ventriculararrhythmias occurring during myocardial ischemia.

Arrhythmias, cardiac failure, and severe DCM with abnormal mito-chondria are also common features of Barth syndrome, an X-linkeddisorder also characterized by cyclic neutropenia and neonatal onset[50-51]. The protein tafazzin responsible for Barth syndrome isencoded by the G4.5 gene and likely belongs to a family ofacyltransferases involved in phospholipid synthesis. Its defect resultsin cardiolipin depletion [52].

Myocardial ischemia and ETC

When the supply of O2 becomes limited (as occurs with myocardialischemia, OXPHOS and mitochondrial ETC flux decline), creatinephosphate is rapidly depleted, fatty acid and pyruvate oxidationdecrease, and ATP production is impaired. The increased hydrolysisof ATP and accumulation of lactate lead to lower intracellular pH andintracellular acidosis, with a direct inhibitory effect on cardiac con-tractile function. In addition, myocardial ischemia results in reducedactivity levels of respiratory complex IV and V and increased levelsof mtDNA deletions [53-56]. Sustained ischemia will lead to ATPdepletion and finally to necrotic cell death.

Paradoxically, functional mitochondria can exacerbate ischemic da-mage, especially at the onset of reperfusion. Increased OXPHOScauses increased ROS accumulation with increased lipid peroxidation;this results in lower cardiolipin levels in the inner membrane withconsequent effect on complex IV activity [57]. Reperfusion injurymediates an increased opening of the permeability transition (PT)pore, a dynamic megachannel located at contact sites between themitochondrial inner and outer membranes, which can result insubsequent triggering of apoptotic cell death [58].

As discussed more comprehensively in Chapter 5, a cardioprotec-tive mechanism can be elicited by ischemic preconditioning (IPC)with short bouts of ischemia applied prior to a more prolongedischemic insult [59-61]. This cardioprotective effect may be mediatedby improved ATP production, lowered Ca++ overloading in themitchondrial matrix, and increased ROS generation leading to proteinkinase C activation and has been proposed to involve the opening ofmitochondrial ATP-sensitive KATP (mitoKATp) and PT pore channels


[62-66]. Several drugs that specifically activate the mitoKATP channelopening (e.g., diazoxide, nicorandil) mimic ischemic preconditioning[67-68]. Evidence for mitoKATP channels as effectors of myocardialpreconditioning has also been demonstrated in human subjects [69].

Apoptosis and cell death

Apoptosis (programmed cell death) leading to cardiac cell loss and toextensive left ventricular remodeling has been shown to occur incardiac failure both in patients with DCM and in animal models.Mitochondria play a pivotal role in the early events of apoptosis. Acentral outcome in the mitochondrial apoptotic pathway is the releasefrom the intermembrane space into the cytosol of a group of proteins(e.g., cytochrome c, Smac, AIF, and endonuclease G) that subsequent-ly triggers a cascade of cytoplasmic changes depicted in Figure 1.1[70-72]. These apoptogenic mitochondrial proteins form an apopto-some complex on release in the cytosol and are involved in thesubsequent activation of downstream cysteine-aspartate proteases(caspases) initiating cell self-digestion and nuclear DNA fragmenta-tion by endonucleases, and leading to apoptotic cell death [73]. Therelease of the mitochondrial intermembrane peptides occurs primarilyas a result of outer membrane permeabilization regulated by thecomplex interactions of different members of the Bcl-2 family, in-cluding Bax, Bid, Bcl-2, and Bcl-X (L) [74]. Proapoptotic membrane-binding proteins (e.g., Bax, Bid, and Bad), translocated from thecytosol to mitochondria, potentiate cytochrome c release, whereasantiapoptotic proteins antagonize this event. The release of theseproteins is also associated and potentially regulated by the opening ofthe PT pore, a critical early step of apoptosis preceding the caspasecascade [75]. PT pore opening is promoted by elevated Ca++ influxinto mitochondria, excessive mitochondrial ROS production,prooxidants, fatty acids, and nitric oxide [73]. PT pore opening isalso accompanied by the dissipation of the mitochondrial membranepotential and depolarization. Changes in membrane potential can beeither a cause or the result of PT pore opening, since extensive protoninflux occurs at this site. There is a continual dynamic in the balanceof the proapoptotic proteins (e.g., Bax) and antiapoptotic Bcl-2 factorsin modulating the progression of the apoptotic events within themitochondria.

Documents

Mitochondria and the Heart · Pathology and Pathophysiology of Cardiovascular Disease. 2001 ISBN 0-7923-7542-4 241. Liong Bing Liem, Eugene Downar (eds): Progress in Catheter Ablation