review: mitochondrial medicine – cardiomyopathy … · caused by defective oxidative...

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Address correspondence to Egil Fosslien, M.D., Departmentof Pathology (M/C 847), College of Medicine, University ofIllinois at Chicago, 1819 West Polk Street, Chicago IL 60612,USA; tel 312 996 7323; fax 312 996 7586; e-mail [email protected].

Review: Mitochondrial Medicine – CardiomyopathyCaused by Defective Oxidative Phosphorylation

Egil FosslienDepartment of Pathology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois

Abstract. During experimental hypertensive cardiac hypertrophy, the heart energy metabolism revertsfrom the normal adult type that obtains the majority of its requirement for adenosine triphosphate (ATP)from metabolism of fatty acids and oxidative phosphorylation (OXPHOS), to the fetal form, whichmetabolizes glucose and lactate. Mitochondrial synthesis and function require an estimated 1000polypeptides, 37 of which are encoded by mitochondrial (mt) DNA, the rest by nuclear (n) DNA. Inheritedor acquired aberrations of either mtDNA or nDNA mitochondrial genes cause mitochondrial dysfunction.Tissue expression of OXPHOS enzyme defects is often heterogeneous. As a result, cardiomyopathy andcardiac failure are frequent but unpredictable complications of mitochondrial encephalopathy, neuropathy,and myopathy. Several nuclear genes that encode mitochondrial proteins have been sequenced and specificdefects associated with nuclear genes that affect mitochondrial structure and function have been linked tohypertrophic and dilated cardiomyopathies and to cardiac conduction defects. Thyroid hormone andexercise stimulate expression of a nuclear respiratory factor (NRF) that induces the nuclear gene TFAM,which encodes the mitochondrial transcription factor A that controls mitochondrial replication andtranscription. TFAM-null mouse embryos lack mitochondria and fail to develop a heart. Mitochondrialdysfunction enhances the generation of radical oxygen species (ROS), which damage mtDNA, nDNA,proteins, and lipid membranes. Mice lacking the mitochondrial antioxidant enzyme manganese-superoxidedismutase (SOD) develop dilated cardiomyopathy. Palliative mitochondrial therapy with L-acetyl-carnitineand coenzyme Q10 improves cardiac function in patients with cardiomyopathy. Cure is only achievable bymitochondrial gene therapy. Experimental direct gene therapy uses vectors or targeting signal sequences toinsert genes into mtDNA; indirect gene therapy employs viral or non-viral vectors to introduce genes intonDNA. Clinical repair of damaged somatic and germline genes that encode mitochondrial proteins maysoon be within reach. (received 15 June 2003; accepted 23 June 2003)

Keywords: cardiomyopathy, mitochondria, mtDNA, nDNA, OXPHOS, statins, Q10, gene therapy.

I. Introduction

Cardiomyopathies are characterized by a decreasein cardiac function [1,2]. Hypertrophic cardio-myopathy is often caused by mutations in cardiacmyosin genes and dilated cardiomyopathy isassociated with aberrations of the dystrophin gene[3]. The fact that mitochondrial defects can causecardiomyopathy has been known for several decades

[4], but until recently, except for involvement ofmutations of the maternally inherited mitochondrialgenome, knowledge of the molecular pathology ofmitochondrial dysfunction as a cause of cardio-myopathy was incomplete [5].

The first patient with mitochondrial diseasecaused by a mutated nuclear-encoded mitochondrialgene was reported in 1995 [6]. The patient hadLeigh syndrome, which was caused by defectiveoxidative phosphorylation (OXPHOS) [6]. Sincethen, several nuclear genes that code for mito-chondrial proteins have been sequenced [7] andspecific defects associated with nuclear genes thataffect mitochondrial structure and function have

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been linked to hypertrophic and dilated cardio-myopathies and cardiac conduction defects [8-10].

Historically, when declining cardiac functionwas caused by a mutation of mtDNA, it was referredto as mitochondrial cardiomyopathy [11]. However,the nuclear genome encodes most proteins requiredfor mitochondrial synthesis and function, andmutation of a nuclear gene encoding a mitochondrialprotein can also cause cardiomyopathy. This hasbeen clearly shown in mouse gene knockout modelsand by studies of human tissues [12].

Mitochondrial dysfunction due to nuclear ormitochondrial gene mutations or toxic inhibitionof their products enhances oxidative stress, whichinterferes with mitochondrial and nuclear genomesand mitochondrial biosynthesis [13-15]. Theimportance of oxidative stress in the etiology ofcardiomyopathy is illustrated by a knockout modelof the gene that encodes an intra-mitochondrial free-radical scavenging enzyme, manganese-superoxidedismutase (Mn-SOD). The null mice developdilated cardiomyopathy and die within 10 days afterbirth [16].

The objective of this review is to elucidate therole of mitochondrial dysfunction in cardiomyo-pathy and cardiac failure. Evidence of dysregulationof mitochondrial function in cardiomyopathies hasbeen derived from embryology, studies on cardiactissues of patients with cardiomyopathy, clinical andautopsy findings, animal models, and in vitrofindings. Specifically, this paper focuses on the rolesof nuclear and mitochondrial genes in cardiomyo-pathy induced by dysfunction of mitochondrialoxidative phosphorylation (OXPHOS).

II. Cardiac energy metabolism

Disorders of mitochondrial OXPHOS are clinicallyand biochemically heterogeneous and lead to neuro-pathy, myopathy, or cardiomyopathy. The latter maybe the only clinical presentation; however, it is oftenpart of a wider spectrum of clinical manifestations.Cardiomyopathy should be especially suspected inpatients who present with myopathy [17].

The high energy demand of the myocardiumrequires an ample and secure supply of adenosinetriphosphate (ATP), which is mainly obtainable

from mitochondrial OXYPHOS [18]. As a result,cardiac mitochondrial dysfunction leading toreduced ATP generation causes cardiac contractiledysfunction and if severe, cardiac failure and death[8]. The fetal heart derives its energy mainly fromglucose and lactate. By comparison, the postnatalmyocardial contractile system depends primarilyupon energy provided by fatty acid metabolism [2].Over one third of the adult myocardial mass consistsof mitochondria, which supply most of the cardiacrequirement for ATP. Cardiac mitochondria of theadult heart preprocess fatty acids through β-oxidation and the Krebs cycle to provide high-energymetabolites as fuel for OXYPHOS.

A. Oxidative phosphorylationMitochondria developed from archebacteria.

The OXPHOS energy conversion system of mito-chondria therefore resembles that of bacteria. Duringevolution most of the bacterial genes were transferredto the nucleus of the host cell or were lost. As aresult, an intracellular dual genome symbiont wasformed, which developed into the present-daymitochondrion. Its double membrane structure,which consists of two bilipid membranes, is unique(as reviewed in [19]).

The mitochondrial OXPHOS system consistsof a fuel cell deriving energy from food metabolites,means for temporary storage of the harvested energyas a trans-membrane potential, and means for con-version of the stored energy into ATP or heat, or toenergize trans-membrane transport, which isrequired for importation of nuclear-encoded mito-chondrial proteins. Two fuel cell inputs are provided,one for electrons from NADH, and the other forelectrons from the Krebs cycle via flavin adeninedinucleotide (FAD2).

Energy is released from the fuel by electrons thatflow through the electron transport chain (ETC),consisting of 4 enzyme complexes and 2 electroncarriers to oxygen as the electron acceptor. The ETCpumps protons from the mitochondrial matrix tothe inter-membrane space, establishing a protongradient across the inner mitochondrial membrane.Electron flux is regulated to maintain a charge acrossthe membrane of about 150-160 mV (Fig. 1).

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Enzyme complexes I, III, and IV, which containproton pumps subunits encoded by mtDNA, arecompletely or partially embedded in the innermembrane. In contrast, complex II lacks a protonpump. Electrons from NADH or from the Krebscycle via FADH2 enter through complex I (NADHdehydrogenase, ND) and II respectively; they flowthrough ubquinone (coenzyme Q10, Q10) tocomplex III, then via cytochrome c to complex IV,the terminal enzyme of the electron transport chain,and finally to oxygen as the electron acceptor.Complex I, III, and IV associate to form super-complexes, also called “respirasomes,” whichimprove enzyme stabilities and reduce electrondiffusion distance [20].

Most of the energy that is dynamically storedin the membrane potential is coupled to phosphoryl-ation of adenosine diphosphate (ADP) to triphos-phate (ATP) by F0F1-ATPase (Complex V). Itstransmembrane F0 moiety channels protons backto the matrix where they join molecular oxygen toform water. F0 is connected by a stalk to the F1moiety, which protrudes into the matrix space. The

membrane energy released by F0 is utilized by F1for phosphorylation of ADP to ATP, most of whichis exported to the cytoplasm of the host cell.

Uncoupling may be due to membrane leakageor to heat production by uncoupling proteins(UCP1, UCP2, and UCP3), which participate inthe regulation of the membrane potential andprevent hyperpolarization of the membrane bychanneling protons back into the matrix. Loweringthe membrane potential lowers ETC oxygen radicalformation. The membrane potential is continuouslyconsumed and must be constantly replenished toprevent energy expenditure beyond supply anddepletion of the proton gradient. Dynamic main-tenance of the membrane potential requires vigilantregulation of the electron transport chain flux toreplenish lost energy, so as to keep the potentialwithin its physiological operating range.

III. Mitochondrial genetics

A. Dual genome symbiontMitochondrial replication is controlled by the

nucleus, but is not synchronized with nucleardivision in dividing cells. Even mitochondria ofpostmitotic cells (eg, the cardiomyocyte) arecontinuously degraded and must be constantlyreplaced independent of nuclear division, so-called“relaxed replication.” Enhanced mitochondrialsynthesis can offset a certain amount ofmitochondrial dysfunction, for instance induced byethanol consumption [21-23].

Remarkably, mitochondrial replication andfunction requires the synthesis of an estimated 1000polypeptides, all but 37 of which are coded bynDNA (Fig. 2). The electron transport chaincomprises almost 100 polypeptides including 13mtDNA-encoded structural subunits, which providehydrophobic proton pumping subunits, and 2subunits of complex V. Normally, a few thousandsmall, identical, circular, maternally inheritedmitochondrial genomes are present in each cell, so-called “homoplasmy.” By contrast, heteroplasmyexists when mitochondrial genomes with differentsequences simultaneously inhabit a cell, for exampledue to inherited or acquired mutation. Slowgeneration of antioxidant enzymes in cardiac

Fig. 1. Mitochondrial energy flux. The electron-transport-chain (1) stores energy from food metabolites (Fuel) as amembrane potential ∆Ψm (2). The potential is used forgeneration of adenosine triphosphate (ATP) (3, coupling, openarrow) or lost (uncoupling, solid arrow) as heat due tomembrane leak, uncoupling proteins, or opening of membranetransition pores and apoptosis.

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myocytes exposes the heart to a high risk of freeradical injury and development of mutations ordeletions [24]. Reduced synthesis of antioxidantenzymes during aging or exposure to environmentaltoxins or therapeutic agents that inhibit mitochon-drial energy conversion may exacerbate the risk ofdamage [25].

B. Mitochondrial transcription factorThe nuclear-encoded mitochondrial transcrip-

tion factor A (Tfam, mtTFA) is imported into mito-chondria where it regulates the rate of transcriptionof the mitochondrial genome [26]. It co-localizeswith nuclear-encoded mtDNA polymerase-γ(POLG), and phage T-7 gene 4-like protein with

intramitochondrial nucleoside location (Twinkle),forming dynamic nucleoid assemblies that are ableto divide, suggesting that they are the units of mito-chondrial inheritance [27].

The gene for Tfam, TFAM, is located on chrom-osome 10q21 [28]. A locus CMDIC at chromosome10q21-q23 has been linked to autosomal dominantfamilial dilated cardiomyopathy [29], but no reportthat CMDIC is TFAM or of involvement of TFAMmutation in a patient with cardiomyopathy has beenforthcoming. On the other hand, animal modelsshow that faulty TFAM expression causes cardio-myopathy. To prevent the development of cardio-myopathy, TFAM must be upregulated duringblastocyst implantation.

Fig. 2. Genetics of the dual genome symbiont: Synthesis of the mitochondrial oxidative phosphorylation system (OXPHOS)requires transcription of both mitochondrial genes (mtDNA) and nuclear genes (nDNA). Expression of nDNA-encodedmitochondrial proteins is regulated by nuclear respiratory factor genes (NRF1 and NRF2) in response to stimulation by thyroidhormone (T3) and OXPHOS-generated hydrogen peroxide (H2O2). Expression of the TFAM gene on chromosome 10q21 isalso essential form mitochondrial synthesis. The TFAM protein product, Tfam controls mtDNA replication and transcription.Chaperons (cpns) guide cytosolic proteins having mitochondrial targeting sequences to mitochondrial membrane import complexes,which have translocases of outer membranes (TOM) and inner membranes (TIM), for import into the inner membrane andinsertion into membrane enzyme complexes or for import into the mitochondrial matrix. NRFs coordinate regulation ofmitochondrial energetics genes, such as lipoprotein receptor genes and genes for fatty oxidation (FAO). For details see text.


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Evidence for transcription control by Tfam wasobtained by transfecting a monkey kidney cell line[30] with an expression vector harboring the TFAMgene, inserted in the sense- or antisense orientation.Transfection with the antisense plasmid causedreduced expression of TFAM and inhibited trans-cription of mtDNA [31]. Additional evidence of aregulatory role by Tfam was obtained usingheterozygous and homozygous TFAM knockoutmice. The heterozygous knockout reduces mtDNAcopy number and lessens cardiac mitochondrialelectron transport capacity. Homozygous knockoutmice die prior to embryonic day 10.5 after normalimplantation and gastrulation [32,33], showing thatadequate TFAM expression is essential for mtDNAreplication and transcription to ensure normal post-implantation development.

Such experiments have demonstrated that thetiming of onset of TFAM expression duringdevelopment is critical. This insight was gainedwhen the time of knockout was manipulated bycoupling its initiation to promoters that are activatedat different times during development. For example,muscle creatinine kinase, which is activated fromembryonic day 13, can serve to activate conditionalknockout. Such transgenic mice have normal cardiacmitochondrial electron chain transport function atbirth, but they develop postnatal cardiomyopathyafter the knockout is activated [34].

Transgenic mice with TFAM knockoutregulated by the α-myosin heavy chain, which isexpressed from embryonic day 8, show mitochon-drial cardiomyopathy during embryogenesis.Nevertheless, about 25% of TFAM heterozygousknockout mice survive the neonatal period but diefrom dilated cardiomyopathy, within several months,suggesting the presence of modifying genes. Studiesof mice intercrossed between long-lived knockoutsconfirm that modifying genes affect their life-span[34]. TFAMloxP-mice with conditional knockout ofcardiac TFAM have symptoms that resemble Kearns-Sayre syndrome. They show cardiac-specificOXPHOS deficiency, conduction block, dilatedcardiomyopathy, and die at 2-4 weeks of age [35].

Cases of mitochondrial disease caused by TFAMmutations have not been reported. However,abnormal Tfam levels have been found in a few

patients with mitochondrial disease caused bymtDNA mutations [36,28]. One patient presentedwith exercise intolerance, muscle wasting, and ocularmyopathy. Muscle biopsies showed ragged-red fibersindicating accumulation of a large number ofabnormal mitochondria. Heteroplasmy with thepresence of an mtDNA deletion between nucleotidepositions 8,300 and 12,400 was detected in musclecells [36]. The increased Tfam level in this patientmay have been a compensatory response to mtDNAdeletion, causing reduced ATP generation.

C. Nuclear respiratory factorsNuclear respiratory factors (NRF) -1 and -2

regulate Tfam expression. NRF-1 expression isessential for implantation and growth of theimplanted blastocyst. Complete lack of NRF-1 islethal. The NRF-1(-/-) blastocyst shows very lowmtDNA levels and lacks the usual enhancement ofmtDNA replication required to support rapidgrowth at the time of implantation. NRF-1 nullblastocysts die within 3.5-6.5 days post-conception.In comparison, the NRF-1(+/-) blastocyst hasmitochondria that show reduced rhodaminestaining, which indicates reduced mitochondrialmembrane potential compared with mitochondriain the wild-type blastocyst. Nevertheless, when it isallowed to progress, the NRF-1(+/-) blastocystdevelops into a surviving mouse [37].

Muscle contractile activity or thyroid hormone(T3) enhances the rate of mitochondrial proteinimport. T3 stimulates expression of NRF-1 and thetranscriptional coactivator peroxisome proliferators-activated receptor-γ coactivator (PGC)-1α [38]. Theheart has high constitutive tissue levels of PGC-1αand exercise causes a transient increase in itstranscription [39,40]. T3 increases mitochondrialimport of the nuclear-encoded mitochondrial heatshock protein (mtHsp)-70, and the increased matrixlevel of this chaperon enhances import of othermitochondrial proteins from the cytosol [41].

PCG-1α and cytochrome c oxidase subunit VIIheart isoform promoter regions contain binding sitesfor myocyte enhancer factor-2 (MEF2). In themouse, MEF2 is activated by embryonic day 8.5[42]. In the rat model of cardiac myocyte responseto pressure overload, MEF2 is an important signaling


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molecule in gene activation leading to cardiac hyper-trophy [43]. Cardiac-specific conditional knockoutof MEF2 is usually lethal, demonstrating theimportance of MEF2 expression for myocytedifferentiation and the maintenance of cardiacmitochondrial function [44,45].

The promoter region of the cardiac α-myosinheavy-chain gene contains binding sites for MEF2and thyroid hormone receptor, which synergisticallyinduce transcription of the gene [46]. Moreover,the UCP3 gene promoter region contains bindingsites for MEF2 and thyroid receptor [47]. The genefor MEF2A, the isoform expressed predominantlyin cardiac tissue, is located on chromosome 15q26[48]. No aberration in this gene with linkage toeither dilated or hypertrophic cardiomyopathy hasbeen identified [42]. However, a disease gene atregion 15q24-q26 has been linked to 5 cases ofcongenital ataxia, optic atrophy, and mentalretardation in a consanguineous family [49].

Human genes that contain binding sites forNRF-1, NRF-2, or both, include TFAM atchromosome 10q21, and OXPHOS genes such asATPase β and γ subunits, and complex IV subunitsVb, Vic, and VIIa [44]. Furthermore, the gene forthe low-density lipoprotein related receptor protein(LRP), which is important for cellular uptake offatty-acid-rich lipoproteins, harbors NRF-1-bindingsites in its promoter region [50]. In the mouse, thegene for vascular endothelial cadherin, which isessential for angiogenesis, contains binding sites forNRF-1 [51]. Such findings suggest that NFR-1 andNRF-2 coordinate mitochondrial replication, energygeneration, embryonal vasculogenesis, and angio-genesis to supply oxygen and nutrients, and providefor the removal of cellular waste products. Thepresence of such binding sites for NRF-1 in the LRPpromoter region and in other genes involved inprocessing high energy lipids suggests that NRF-1also regulates the supply of fuels for mitochondrialenergy conversion.

D. Mitochondrial importNuclear-encoded mitochondrial proteins are

synthesized with mitochondrial targeting sequences.Cytosolic chaperon molecules guide such proteinsto translocases of the mitochondrial outer membrane

(Tom complexes), where they are unfolded [52,53].Translocases of the inner membrane (Timcomplexes) sort proteins to the various mitochon-drial sub-compartments. Translocase Tim22 insertsproteins that lack a matrix targeting signal peptideinto the inner membrane for assembly intomultimeric OXPHOS enzyme complexes or carrierproteins such as mitochondrial nucleotide trans-locator (ANT) -1 [54]. The membrane potential-driven Tim23 presents proteins that carry a positivelycharged N-terminal mitochondrial-matrix signalingpeptide to mitochondrial matrix mtHsp70, whichpulls proteins into the matrix. Next, a mitochondrialprocessing peptidase cleaves the targeting sequence,whereupon mtHsp60 and matrix chaperons refoldthe shortened sequences into functional proteins orprotein subunits [55,53].

IV. Molecular pathology

A. Defective OXPHOS enzyme activityOXPHOS enzyme complexes have been

analyzed using simple enzyme complex antibodiesor specific subunit antibodies for immunohisto-chemistry on explanted cardiac tissues or endocardialbiopsies, or immunoblot assay [56]. Enzymesubunits have been separated by two-dimensionalelectrophoresis (2-DE) or 2-DE followed by high-resolution analysis using mass spectrometry [57,58].The following selected findings exemplify thediversity of OXPHOS enzyme defects in patientswith cardiomyopathy and illustrate the great varietyof clinical symptoms and syndromes with whichcardiomyopathy may be associated.

Defective complex I activity causes about onethird of cases of OXPHOS disease in humans [59].Cases with isolated complex I disease with cardio-myopathy are illustrated in Fig. 3. The enzymedefects were diagnosed using cultured skin fibro-blasts. All patients died within 2 years of age. 4 ofthe patients were diagnosed at birth. The shortesttime between diagnosis and death occurred in apatient whose complex I deficiency was traced to amutation in nuclear mitochondrial gene NDUFS8,located at chromosome 11q13 [60].

Of 101 children diagnosed to have OXPHOSdisease, 17 had abnormal cardiac enzyme activities


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and suffered from cardiomyopathy. The enzymecomplex that was defective varied greatly, as did theclinical diagnoses, which included Alpers, Sengers,and Kearns-Sayre syndromes. Moreover, the age ofthe patients at the onset of cardiomyopathy and atthe time of death varied greatly (Fig. 3) [61,62].

A 14-year-old boy with MELAS (mitochondrialencephalomyopathy, lactic acidosis, and stroke-likesymptoms [63]) had cardiomyopathy and markedlydecreased complex I and IV enzyme activities, butthe activities of complex III and V were onlymoderately lower than normal [64]. No defect was

found in complex II activity in this patient, but rarecases of cardiomyopathy due to reduced activity ofcomplex II have been reported. Thus, complex IIactivity was significantly reduced in cardiac biopsiesfrom 2 brothers with hypertrophic cardiomyopathyand gait abnormalities [65]. Muscle biopsy from a25-year-old female with Kearns-Sayre syndrome(KSS) revealed complex II deficiency, cardiacconduction defects, ragged-red fibers, andmitochondrial inclusions. KSS patients typicallyhave progressive degeneration of the retina, shortstature, dementia, and heart block [66]. By

Fig. 3. Illustration shows patients with cardiomyopathy due to mitochondrial oxidative phosphorylation system (OXPHOS)enzyme deficiency. Bar graph shows the age at the time of diagnosis and the age at the time of death. Age is shown in years andin days at the top and bottom of the graph respectively. A, top shaded area: Cardiomyopathy due to isolated complex I deficiency.In one case the defects was caused by a mutation of the nuclear mitochondrial gene NDUFS8, which codes for a nuclear-encodedsubunit of complex I. [Data from Loeffen JL et al. Hum Mutat 2000;15:123-134]. B, lower section: Patients with cardiomyopathycaused by a variety of OXPHOS enzyme deficiencies and presenting with diverse clinical syndromes. Enzyme complexes indicatedby roman numerals. In this study the time of onset and the time of death varied greatly. KSS: Kearns-Sayre syndrome; MERF:myoclonus epilepsy with ragged red fibers, IMM: infantile mitochondrial myopathy with complex IV deficiency; MEM:mitochondrial encephalomyopathy; Alpers: Alpers syndrome; Sengers: Sengers syndrome. [Data from Holmgren D et al. EurHeart J 2003;24:280-288]. For further details see text.


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comparison, a mycotoxin, neurotoxin, and foodcontaminant, 3-nitropropionic acid (3-NPA), asuicide inhibitor of complex II [67], reduces ATPgeneration and induces bradycardia in the isolatedbeating heart [68]. In the mouse model, 3-NPAreduces cardiac complex II activity to 20-30% ofnormal [69].

Often, mitochondrial enzyme defects showremarkable tissue differences in their expression. Asexamples, examination of autopsy tissues from a caseof fatal infantile cardiomyopathy revealed severe

reduction in complex I and IV activities in the heartas well as the skeletal muscle, but activities ofcomplex II and III were significantly lower in theheart compared to skeletal muscle [70]. Markeddecrease of either complex I, IV, or both, was foundin endocardial biopsies in 15 of 32 infants diagnosedwith idiopathic hypertrophic cardiomyopathy [71].

In 8 children with cardiomyopathy, examinationof OXPHOS enzyme activities revealed defects ofcomplexes I, III, IV, V in 2, 5, 3, and 4 casesrespectively, but no defect was detected in complex

Fig. 4. Mitochondrial (mt) DNA point mutations and deletions detected in mitochondrial cardiomyopathies. Top area showslocations of structural mtDNA oxidative phosphorylation system (OXPHOS) genes, which encode enzyme subunits. Genelocations are illustrated relative to a linearized scale of the circular human mitochondrial genome. Genes for subunits of complexesI, III, and IV are shown in separate lanes for clarity. mtDNA gene location data from www.mitomap.org, 2003. Complex II iscompletely encoded by nDNA. Lower area shows locations of some mtDNA mutations and deletions that have been reported tocause cardiomyopathy. [Sources of data: A: Turner et al. Eur Heart J 1998;19:1725-1729; B: Goldstein JD et al. Pediatr DevPathol 1999;2:78-85; C: Casali C et al. Biochem Biophys Res Commun 1995;213;588-593; D: Terasaki F et al. Jpn Circ J2001;65:691-694; E: Bobba A et al. Clin Chem Acta 1995;243:181-189.] The 4977 nucleotide (nt) deletion removes complexI genes ND3-ND5 plus ND4L; the COIII gene of complex IV, and the ATPase8 subunit gene of complex V. The 7,436 deletionin addition removes the following subunit genes: ND6, complex I; cytochrome b (cytb), complex III; COII, complex IV; andATPase6, complex V. For details see text.


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II activity [72]. In contrast, a study involving 157cases of mitochondrial disorders in childhooddetermined that enzyme defects were most commonin complex I and complexes I + IV, and were oftenassociated with parental consanguinity [73].

Low activities of cardiac complex III activitieswere found in studies of patients with idiopathicdilated cardiomyopathy. For example, in 10 hearttransplant patients, who had been diagnosed withdilated cardiomyopathy, myocardial activities ofcomplex III and IV, both associated with cytochromec, were reduced, but activities of complex I and IIwere unaffected [74]. These results probably excludethe presence of an mtDNA deletion reaching fromthe cytochrome b gene (complex III) to the COIIIgene (complex IV), because such a deletion wouldremove several ND-subunits of complex I as well(Fig. 4). The findings are more consistent with aloss of cytochrome c, because it interacts with bothcomplex III and IV. Loss of cytochrome c suggeststhat apoptosis had a significant role in thepathogenesis of cardiomyopathy in these patients.

Another study of patients with dilatedcardiomyopathy reported that 37 of 55 patients hadreduced enzyme activity of at least one OXPHOSenzyme. Complex III was most frequently affected,followed in declining frequency by complex V, IV,and I; again, complex II was unaffected. Patientsless than 18 years old were more likely to have singledefects. Patients 19-64 years old had more multipleenzyme activity defects [75].

A third study involved 3 patients with lowcardiac complex III activity compared to donorhearts. Comparison of cardiac mtDNA cytochromeb gene sequences in these patients with theCambridge mtDNA sequence [76] revealed onlyneutral polymorphism. The investigators concludedthat decreased complex III activity was most likelydue to secondary causes such as oxidative stress [77].

Defective complex IV activity in the left ventric-ular wall was detected in explanted hearts from 6patients with ischemic and 8 patients with non-ischemic dilated cardiomyopathy. The activitydecrease correlated with diminished ejectionfraction. It was suggested that defective complexIV activity resulted in reduced cardiac ATPgeneration, which contributed to the cardiomyo-

pathy [78]. In a group of 16 patients with end-stage cardiomyopathy, 10 children and 2 neonatesshowed decreased cardiac mitochondrial activity ofenzyme complex I, III, IV, and V. Sequence analysisrevealed heteroplasmy in 4 patients with mutationsin the ND5 gene, in cytochrome b, and mitochon-drial transfer RNA (tRNAArg). Because mutationof the latter affects only mitochondrial transcription,lack of deficiency in complex II, which only containsnDNA-encoded subunits, is not surprising [79].

By comparison, whereas activity of complex Iwas significantly decreased in terminally failingmyocardium in a series of 43 explanted failing heartscompared with 10 donor hearts, no general mito-chondrial genetic abnormality could be detected.The amounts of mtDNA, mtRNA and Tfam wereunaltered compared with donor hearts [80]. Thesefindings suggest the presence of either a mutationof a nuclear mitochondrial gene or inhibition ofcomplex I by a toxin or other agent. For example,many insecticides and neurotoxins inhibit complexI either at the PSST or ND1 subunit of complex I,which are involved in electron transfer from complexI to Q10 [81-84].

B. Abnormal expression of mitochondrial genome

1. Inherited mtDNA point mutation. Manydifferent types of abnormalities in mtDNA thatcause defective OXPHOS enzymes have beendetected in patients with hypertrophic and dilatedcardiomyopathy [85]. Mutations of the mitochon-drial genome frequently affect several organs.Accordingly, patients suffer from various disorderssuch as neurological disease, diabetes, andcardiomyopathy [86] or combinations of theseafflictions. Cardio-myopathy is a relatively frequentcompanion of mitochondrial neuromusculardisorders. Thus, in 8 patients diagnosed with spinalmuscular dystrophy, abnormal electrocardiogramsand thickening of the myocardium were commonfindings [87].

Occasional cases of cardiomyopathy as a resultof homoplasmic inherited mutation with thepresence of only mutated mtDNA in all cells havebeen reported [88, 89], but most patients withmitochondrial disease caused by inherited


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mitochondrial DNA mutations exhibit hetero-plasmy. Typically, mitochondria in affected cellscontain a mixture of wild-type mtDNA and over20% mutated mtDNA, but the level of mutatedmtDNA is occasionally much higher. For example,an infant son of a mother who carried a T8993Gmutation of the MTATP6 gene in her leukocytespresented with Leigh syndrome and hypertrophiccardiomyopathy. MTATP6 encodes the ATPsynth-ase6 subunit of complex V [90]. The heteroplasmyin the infant reached 90% in his fibroblasts andskeletal muscle. This case illustrates the variabilityof tissue expression of mtDNA gene defects. Themutation was present in the patient’s heart, but notin his liver [91]. A brother had died at age 2 fromsudden infant death syndrome [92].

Different mutations may be expressed as thesame clinical syndrome. For instance, Leber’shereditary optic neuropathy (LHON) may be causedby single point mutation of ND4 or ND6, or bymutations in other parts of the mitochondrialgenome [93,94]. Because complex I is the largestOXPHOS enzyme complex with the most subunits,7 encoded by mtDNA and 36 encoded by nDNA,patients who present with Leigh syndrome or exhibitother clinical symptoms of OXPHOS disease shouldbe screened for complex I gene defects, even ifenzyme activities are normal in cultured skinfibroblast. This recommendation is based upon thefinding of mutated NDUFV1 in 6 patients withcomplex I disease [95].

Investigation of a series of 28 patients withidiopathic dilated cardiomyopathy and markedreduction of OXPHOS enzymes in cardiac tissuesrevealed point mutations involving the ND5 subunitgene, the COII and COIII genes for subunits ofcomplex IV, and the mtDNA gene for cytochromeb [96]. In a sequencing study of the mtDNA D-loop control region, which includes nucleotides 110-570, critical mutations were found in 8 of 47 patientswith cardiomyopathy. These mutations wereunrelated to aging, based on a comparison with 40age-matched control subjects who had no historyof heart disease [97].

The importance of defective mitochondrialgenome translation is illustrated by the A8344Gmutation, which results in abnormal tRNALys and

faulty translation, and causes myoclonus epilepsywith ragged red fibers (MERRF). Comparativeproteomic studies showed that this mutation lowersthe synthesis of all mitochondrial proteins thatnormally harbor lysine in their structure [58]. It istherefore not surprising that the A8344G mutationmay cause a variety of additional symptoms such asataxia, mental and motor retardation, ophthalmo-plegia, and cardiomyopathy [98]. Another suchmutation, A8296G, which affected the tRNALys

gene, was detected in an 8-day-old patient who wasdiagnosed with hypertrophic cardiomyopathy byechocardiography. The infant died of cardiac failure.The mutation was present in all tissues examinedafter autopsy [99].

A variable phenotype is also noted in maternally-inherited myopathy with cardiac involvement(MIMyCa syndrome), which can be caused byheteroplasmic A3260GLeu mutation, which causesa significant decrease in transcription of mtDNAencoded leucine-containing subunits for complex Iand IV [71]

2. Somatic point mutations. Humans accum-ulate somatic mutations during aging, but they areusually present in a much lower proportion (0.01%-1%) than in the inherited mitochondrial diseases[86,63]. It has been proposed that by relaxedreplication, a low level of mutated mtDNA ormtDNA with deletion may gradually reach athreshold concentration beyond which the cellularenergy metabolism is insufficient for maintenanceof normal cellular function and disease or deathensues [100,86].

Another suggestion is that mitochondria formcellular genetic networks and exchange genesequences. When this assumption was included ina computer model of single post-mitotic cells itshowed that, even in cases of an initially rare somaticmutation, relaxed replication could lead to randomdrift with increased proportion of the accumulatedmutant mtDNA within the cell [101]. However, invitro findings in cell lines harboring full-lengthmitochondrial genomes with point mutations,indicate that mutated mtDNA does not repopulatemitochondria at a faster rate than the wild typegenome [102].


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3. Deletions. The number of cardiac mtDNAdeletions increases with normal aging and deletionsemerge at earlier ages in patients with cardio-myopathy. Significant increase of a 7.4kilobase (kb)deletion (deleting nucleotides 8637-16084) but notof a 5kb deletion (deleting nucleotides 8469-13447)was found in myocardium of patients 41 years andolder with idiopathic dilated cardiomyopathycompared with controls. In this study the 5kbdeletion was also detected in 3 of 7 controls [103].By comparison, the 7.4 kb deletion was present inall 15 and the 5kb deletion in 7 endocardial biopsiesfrom 15 patients with idiopathic dilatedcardiomyopathy. But similar deletions were foundin control subjects, suggesting that the deletions werenot the cause of the cardiomyopathy [104]. On theother hand, presence of inherited deletions has beenreported in cases of dilated cardiomyopathy [105].Whether or not such deletions are generally primaryand pathogenic or occur as secondary events remainsa controversial question [106].

In any case, oxidative stress is the main etiologiccandidate as the cause of the deletions. Normalmitochondrial respiration produces a small amount(1-3%) of oxygen radicals that are normallyneutralized by anti-oxidant enzymes. During mito-chondrial OXPHOS dysfunction due to enzymedefects or enzyme inhibition, oxidative stress issignificantly increased. Besides, during humanaging, the levels of antioxidant enzymes decline.Oxygen radicals that are not neutralized byantioxidant enzymes damage nucleic acids andproteins, and cause lipid peroxidation [107]. Thusoxidative stress may induce mtDNA mutations,fragmentation, and deletion of part of themitochondrial genomic sequence, resulting insmaller genomes (deleted mtDNA, δmtDNA). Invitro, dmtDNA replicates faster and repopulatesmitochondria more efficiently. It has been proposedthat the increased rate of replication is due to theirsmaller size compared with wild-type mtDNA [102].

Experimental evidence that oxidative stressinduces mtDNA damage in vivo was obtained usingmice lacking the heart and skeletal muscle-specificisoform of adenine nucleotide translocator-1(ANT1). Lack of ANT1 uncouples OXPHOS byabolishing exchange of ADP and ATP across mito-

chondrial membranes. In the ANT1-null mouse,the electron transport chain responded with astriking increase in hydrogen peroxide generation.This was compensated by a marked increase in theantioxidant enzyme manganese-superoxidedismutase (Mn-SOD) in skeletal muscle but muchless so in the heart, which resulted in a major upsurgeof cardiac mtDNA rearrangements [108].

Because the level of mutated mtDNA in agingcardiac tissue is relatively low, alternative etiologieshave been proposed to explain aging-related cardio-myopathy due to declining mitochondrial function.It has been reported that thiol proteases, which arepivotal in mitochondrial degradation, are partic-ularly sensitive to damage by radical oxygen speciesgenerated by the electron transport chain [109].Besides, perinuclear lipofuscin or “aging pigment,”which indicates abnormal mitochondrialdegradation, is frequently observed in aging cardiacmyocytes [110]. These findings corroborate thenotion that faulty mitochondrial degradation mayplay a role in the development of cardiomyopathyin older patients.

C. Abnormal expression of nuclear OXPHOS genesThe nuclear genes that code for mitochondrial

proteins are distributed over several chromosomes(Table 1). At first glance, the distribution mayappear random. However, in a few cases, a clusteringof these genes with other disease-related genes isevident at chromosome locations 1q21, 7q32,10q24, and 15q25, but most conspicuously atchromosome region 11q13. Interestingly, severalcancer suppressor genes or oncogenes have beenmapped to the same chromosome regions as thegenes for nuclear-encoded mitochondrial proteins,suggesting an association between abnormalmitochondrial synthesis and function and neoplasia.Also, some clusters are located at or close tochromosomal viral integration points or fragile sites.

Mutations of two nuclear genes, NDUFV2 andNDUFS2, encoding complex I subunits werestrongly linked with early onset hypertrophiccardiomyopathy in 3 siblings in a consanguineousfamily. The mutation caused a marked decrease inthe amount of nuclear encoded subunit and complexI activity [111]. Mutation of NDUFV2 has also


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Table 1. Selected nuclear mitochondrial genes involved in the synthesis and function of the mitochondrial oxidative phosphorylationsystem (OXPHOS). The genes, which are located on many different chromosomes, have either been shown to cause cardiomyopathy,or are located at or close to other genes critical for mitochondrial synthesis and maintenance. Sources of further gene data areavailable from OMIM™ (On-line Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=OMIM).For further detail see text.


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been linked to Parkinson’s disease [112], anassociation that may illustrate the large variation ofclinical expression of mitochondrial disorders.Others have noted mis-sense mutations ofNDUFS2, which is located on chromosome 1q23,a breakpoint found in Ebstein-Barr virus (EBV)-related neoplasia [113,114]. Transgenic miceexpressing EBV nuclear-antigen-leader peptideconsistently develop dilated cardiomyopathy [115].The product of the NDUFS4 gene is necessary forcomplex I assembly as evidenced by non-sensemutations of the gene, which prevents assembly ofcomplex I in the inner mitochondrial membrane[116]. Clinically, NDUFS4 mutations are associatedwith Leigh-like syndrome or fatal neurologicalsyndrome [117,118].

An interesting complex I gene, NDUFA2 islocated at 5q31 [119]. A study of 7 pesticide sprayers(average age 36 years) with long exposure topesticides revealed an aphidicolin-sensitive fragile siteat chromosome region 5q31. This region is also abreakpoint found in cancer [120]. NDUFA6,NDUFA7, and NDUFB8, are located at chromo-some regions 22q13.1, 19p13.2, and 10q23.2-23.33, respectively. NDUFV1 and NDUFS8 arelocated at chromosome 11q13. NDFUV1 encodesa complex I subunit that provides a binding site foriron cluster N-2 [121,122].

Chromosome locations of these nuclearmitochondrial genes are not only of interest toelucidate the pathogenesis of cardiomyopathy, butalso because of findings of mutations andbreakpoints occurring at these locations in benignand malignant tumors. For example, the tumorsuppressor PTEN is located within the regions towhich NDUFB8 has been mapped. It has beenassociated with prostate, endometrial, and thyroidcancers, and many other tumors. The cluster atchromosome 11q13 includes UCP2, UCP3, andMEN1 (menin-1); the latter is associated withendocrine neoplasia. Why are several genes involvedin energy metabolism located at or near tumorsuppressor genes?

A partial answer may be provided by obser-vations of a direct link between OXPHOS diseaseand neoplasia in complex II defects. Complex II(succinate dehydrogenase, EC 1.3.99.1), consists of

4 protein subunits, all encoded by nuclear DNA.The two subunits encoded by genes SDHC andSDHD span the inner membrane. They areconnected on the matrix side to flavoprotein andiron-sulfur protein subunits encoded by genesSDHB and SDHA respectively. SDHB, SDHC,and SDHD encoded subunits are involved in theassembly of the holoenzyme, and may partake inoxygen sensing and signaling. Mutations of thesegenes are associated with formation of hereditaryparaganglioma and pheochromocytoma. The flavo-protein is the catalytic subunit that participates inthe Krebs cycle converting succinate to fumarate.It carries electrons from FADH to Q10. Mutationof SDHA reduces Krebs cycle activity and OXPHOSenergy conversion [123,124]. The first cases of mito-chondrial disease caused by a mutated nuclear-encoded gene involved the mutation of SDHA in 2first cousins who presented clinically with Leighsyndrome [6].

Complex III (ubiquinol cytochrome c reductase,E.C. 1.10.2.2) consist of cytochrome b, a mtDNAencoded subunit, and nDNA-encoded subunitscytochrome c1, 2 core proteins, an iron-sulfurprotein, a hinge protein, 3 low molecular weightproteins, and QP-C, the ubiquinone cytochrome creductase binding subunit encoded by the nucleargene UQCRB, which must be imported from thecytoplasm.

Isolated complex III defects are uncommon, buta small number of cases have been reported. In aconsanguineous patient, a deletion was found inUQCRB. The parents were heterozygous for thedeletion, but none of 55 control subjects carried thedeletion [125]. Patients with cardiomyopathyassociated with isolated complex III deficiencyillustrate the importance of normal cytochrome bexpression for the activity of the complex. Five suchpatients had less than half the normal enzymeactivity owing to a C15452G transition of thecytochrome b gene [126]. A different patient whodeveloped exercise intolerance carried a G15242Amutation of the cytochrome b gene, but mutationsof any of the 10 nDNA encoded subunits wereabsent [127].

Deficiency of complex IV (cytochrome coxidase, EC 1.9.3.1) is often caused by a defect in


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its synthesis [128]. The holoenzyme consists of 3mtDNA-encoded and 10 nDNA-encoded subunitproteins. It carries electrons from cytochrome c tomolecular oxygen, which is reduced to water. Energyfrom the electron flux through the 3 prokaryoticcatalytic subunits powers the built-in proton pump,which helps to maintain the inner membranepotential as it pumps protons from the matrix tothe intermembrane space [129,130]. Two copper-containing prosthetic groups, cytochrome a, andcytochrome a-3, are essential for the enzymaticfunction of the holoenzyme. Lack of copper hindersheme insertion into the complex and results incomplex IV deficiency [131]. Copper deprivationleads to complex IV defect and increased oxidativestress in cultured cells [132].

The eukaryotic proteins SCO1 and SCO2 arerequired for assembly of the complex. Experimentalmutations of either protein further demonstrate therequirement for copper in proper complex IVfunction. For example, mutation of yeast SCO1(Sco1) hampers inclusion of a mitochondrialcytochrome c oxidase subunit and foils integrationof copper into the enzyme complex. Significantly,copper supplementation rescues complex IV activityin yeast and bacteria that harbor mutated Sco1[128]. Also, patients with mutation of the copper-binding assembly protein SCO2 suffer from loss ofintegration of mtDNA-encoded subunits and lackof inclusion of copper into complex IV. They havesevere complex IV deficiency, affecting especiallycardiac and skeletal muscle, and die of fatal infantilecardiomyopathy [133]. Importantly, it has recentlybeen demonstrated that copper supplementation ofmyoblasts and fibroblasts from patients with SCO2mutations restores nearly normal complex IVactivity. This suggests that copper supplementationcould conceivably be used to treat patients with suchmutated mitochondrial assembly protein genes[134].

The variable clinical presentation caused byabnormal complex IV assembly is illustrated by howdifferent SCO2 mutations affect the clinicalpresentation and pathology of affected patients. Acase of a patient with a SCO2 mutation that resultedin a truncated protein showed the overlap betweenclinical symptoms often observed in mitochondrial

diseases. The patient had lactic acidosis, hypotonia,generalized weakness, and hypertrophic cardio-myopathy. The clinical presentation resembledspinal muscular atrophy, known as Werdnig-Hoffmann disease, which, next to cystic fibrosis, isthe most lethal autosomal recessive disease inCaucasians. At autopsy, loss of motor neurons andastrocytosis were noted in the ventral horns of thespinal cord. Spinal muscular atrophy is caused by amutation of 1 of 3 SMA genes located at or near5q12-q13. However, no mutation of the SMA genecould be detected in this patient [135].

Loss-of-function mutations of SURF1, anothercomplex IV nuclear-encoded assembly protein,presents clinically as Leigh syndrome. In the mousemodel, nearly all Surf1 knockout animals die postimplantation. Mice that survive show complex IVdeficiency in liver and heart, lack of motor strength,and early-onset post-natal mortality [136]. Ahomozygous missense mutation of COX10 leadingto complex IV deficiency was detected in aconsanguineous family. The gene, which maps tochromosome 17p12-p11.2, close to the SCO1 gene,encodes an enzyme required for the synthesis of theheme A prosthetic group of complex IV [137].

D. Electron carriers, ANT, and frataxinThe electron carrier coenzyme Q10 is an

antioxidant. Q10 deficiency has been associatedwith congestive heart failure and cardiomyopathy[138]. But no depletion of Q10 and no increasedrate of cell death were observed in cultured fibroblastfrom patients harboring a mutation of the fibroblastATPase6 gene or in the nuclear gene SDHA that, asnoted above, encodes the catalytic Krebs-cycleflavoprotein of complex II [139]. On the otherhand, idebenone treatment restored OXPHOSfunction and dramatically improved the clinicalpicture of patients with deficiencies of complex I -III, all of which interact with the Q10 pool [140].By comparison, the electron carrier cytochrome creceives electrons from the heme group of cyto-chrome b of complex III to its own heme group andthen delivers the electrons to cytochrome c oxidase,the terminal enzyme of the electron transport chain(for graphics, refer to www.rcsb.org/pdb/molecules/pdb36_1.html).


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Sengers syndrome is characterized by hyper-trophic cardiomyopathy, myopathy, cataracts, andlactic acidosis [141]. It is associated with markedreduction in the protein content of ANT1. However,no genetic defect has been identified [142]. Of 12patients with Sengers syndrome, 3 died as neonatesand 6 died in early adulthood [143]. The FRDAgene on chromosome region 9q13 encodes themitochondrial protein frataxin. A gene located inthis region is associated with familial dilatedcardiomyopathy suggesting that this gene may beFRDA.

Mutation of FRDA leads to a deficit in frataxin,which causes mitochondrial iron accumulation andFriedreich’s ataxia, an autosomal recessivedegenerative disorder with failure of cardiacmitochondrial energy metabolism. Treatment usingQ10 or idebenone plus vitamin E considerablyameliorates the symptoms of the disease [144].

E. Abnormal uncouplingIn rodent postnatal myocardium, UCP-2 is

induced by fatty acids and UCP-3 by peroxisomeproliferator-activated receptor-α (PPARα) [145].The latter serves as a “lipostat” that regulatesexpression of several genes, which are required forcardiac mitochondrial fatty oxidation as evidencedby myocyte fat accumulation in the PPARα(-/-)mouse [146]. The non-steroidal anti-inflammatorydrug (NSAID) sulindac sulfide, which has anti-cancer properties, induces PPARα [147-149].

Uncoupling can occur during opening of themitochondrial permeability transition (MPT) pores.For example, reperfusion after cardiac ischemiaopens the pores, depolarizes the membrane, andinduces apoptosis [150]. Several NSAIDs or theirmetabolites induce MPT pore opening. Salicylate,a metabolite of aspirin, opens MPT pores in vitro.The induced membrane depolarization maycontribute to the pathogenesis of in vivo aspirin-induced myocardial injury in Reye’s syndrome [151].

F. Defective protein importDefects of the mitochondrial protein import

system can lead to defective oxidative phos-phorylation. The genes for the human pre-proteintranslocase subunit of the outer membrane complex

(TOM22) and inner membrane subunit (TIM10)have been mapped to 22q12-q13 and 11q12.1-q12.3, respectively. In yeast, the Tim22 complexharbors Tim9, Tim10, and Tim12; these proteinsstructurally resemble the deafness/dystoniasyndrome peptide (DDP) whose gene TIMM8A hasbeen mapped to chromosome Xp22. Mutations ofTIMM8A are associated with Jensen syndrome andMohr-Tranebjærg syndrome (MTS), two rare neuro-degenerative diseases. MTS affects post-mitotictissues leading to cortical blindness, dystonia, andmental deficiency. Its clinical presentation resemblessyndromes caused by defects in mitochondrialoxidative phosphorylation [152,153] and illustratesthat abnormal import of nuclear-encoded mito-chondrial proteins affect OXPHOS function.

Metaxin, another karyotic protein that isassociated with mitochondrial protein import, isessential for embryogenesis. MTX, the gene formetaxin, is located at chromosome 1q21, a non-random human cytomegalovirus integration locus[154-156]. This chromosome region harbors theglucocerebrosidase gene involved in Gaucher’sdisease, a storage disease which can affect the heart[157]. However, no case of cardiomyopathy causedby MTX mutation has been reported.

G. Dual genome diseaseMutation of Twinkle [158], ANT1 [159], or

the nuclear gene for the mitochondrial DNApolymerase-γ subunit (POLG) [160], result in anunusual form of inheritance, exhibiting Mendelianinheritance combined with clinical expression ofmaternally inherited mitochondriopathies. Proteinsencoded by these genes support mtDNA stability;mutation of any of the genes causes multiplemtDNA mutations and loss of mtDNA with form-ation of multiple deletions that lead to significantpathology [161]. For example, mutation of theTwinkle gene causes progressive externalophthalmoplegia (PEO) of the autosomal dominanttype [158]. Twinkle has been mapped tochromosome 10q24, and clusters with the COX15gene, mutation of which is associated withhypertrophic cardiomyopathy [162]. Moreover,10q24 is an integration site for human papilloma


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virus type 6 (HPV6AI1) [163], which has beenlinked to the development of benign neoplasms.

Childhood onset of PEO, autosomal recessivetype, is associated with multiple mtDNA deletionsand with severe cardiomyopathy that requires hearttransplantation [164]. Besides Twinkle, PEO can becaused by mutations of POLG and ANT1 [159].Mutation of POLG is implicated in male infertility[165], illustrating the unpredictable clinicalconsequences of mutations that affect mitochondrialfunction. Noteworthy, but of unknown significance,is that fact that POLG clusters with the COX5Agene at chromosome 15q25 [166].

V. Therapy

Patients with cardiomyopathy caused bymitochondrial defects, especially those with cardiac-specific defects, can benefit from cardiac trans-plantation [167]. By comparison, gene therapy formitochondrial disorders was, until a few years ago,considered highly speculative and theoretical [168].However, several novel approaches have beenreported to restore normal mitochondrial functionin vitro. Direct mitochondrial gene therapy uses avector or targeting signal sequences to insertmitochondrial genes into mitochondria. Indirectmitochondrial gene therapy uses viral or non-viralvectors to introduce genes into the nucleus fornuclear coding of mitochondrial proteins [169].

Synthetic biotinylated polynucleobase molec-ules are taken up by cells in vitro and entermitochondria when attached to the mitochondrialtarget sequence of the nuclear-encoded COX7subunit [170]. Repair of defective ATP generationin LHON mitochondria harboring a G11778Atransition was achieved in vitro by importing asynthetic ND4-subunit into mitochondria inLHON cybrids. The synthetic subunit, whichincluded a mitochondrial targeting sequence,entered the defective mitochondria, successfullyrestored complex I function, and normalized ATPgeneration [171]. In vitro, direct microinjection ofrestorative mtDNA sequences into the mouse oocytecan correct mitochondrial defects [172]. Repair ofmitochondria in the human germline or eveninsertion of mitochondrial sequences that have been

found to be associated with longevity now appearscientifically feasible. However, religious and ethicalconsiderations may influence the decision whetheror not to implement these approaches to alter thehuman germline [173].

Cardiac oxidative damage increases with age inrodent models of aging. However, dietary restrictionreduces oxidative damage in both the rat and mousemodels [174]. The pineal sleep hormone andantioxidant, melatonin, may have anti-agingproperties and other beneficial effects [175]. It dose-dependently increases the activities of complexes Iand IV of mitochondria from rat liver and brainand enhances ATP synthesis [176].

Exercise can significantly increase SOD activityin young rats; however, in old rats, vitamin Esupplementation is required to achieve similarbenefits. Exercise training in young rats enhancesthe activity of tissue SOD of both the left and rightventricle, compared with sedentary controls. Bycontrast, old rats fail to show this response toexercise. Administration of oral vitamin E to oldrats is required to remedy their lack of SOD activityenhancement and to reduce lipid peroxidation.Levels of cardiac catalase are also affected by exerciseand vitamin E supplementation. In the left ventricleof young rats’ hearts, exercise enhances catalaseactivity compared with non-supplemented sedentaryrats.

A remarkable decrease in cardiac catalase activitywas observed in 22-month-old sedentary ratscompared to 4-month-old sedentary rats. In theyoung rats, vitamin E supplement-ation aloneincreased left ventricular catalase activity by about80% compared to non-supplemented controls, butonly by about 4% in old rats. In the latter, exerciseenhanced catalase activity by 17% and whencombined with oral vitamin E by 23%, comparedto sedentary age-matched controls [24].

Some drugs induce adverse mitochondrial sideeffects. For example, drugs such as acidic NSAIDsinhibit mitochondrial function [177]. Azido-thymidine (AZT) reduces ATP and glutathionelevels in cultured cells [178]. Patients receiving AZTtherapy may develop mitochondrial myopathy; thereis one reported case of LHON associated with AZTtherapy [179].


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The rate of substrate production by the meval-onate pathway can enhance or decrease the velocityof Q10 synthesis [180] (Fig. 5). Iatrogenic inhib-ition may occur with therapeutic use of adriamycin,gemfibrozil, or statins. The latter inhibit β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA)reductase, [181]. Importantly, inhibition of Q10synthesis may be tissue-specific, affecting high-energy consuming organs more than other organs.In a rat model of the effects of inhibition by a statindrug, synthesis of ATP in the heart was decreased,but ATP production in liver was unaffected [182].

One possible reason for the different effects ofstatins on different organs may be variations in theuptake of Q10 by different tissues. After iv Q10administration in the guinea pigs, Q10 rapidlyaccumulates in the liver from which it is redistributedby blood lipoproteins to kidney, heart, brain, andadrenals, with the organ uptake peaking at 8, 24,

and 168 hr respectively. The adrenal exhibits thehighest level of Q10 uptake, compared to the otherorgans [183]. Q10 treatment can improve cardiacfunction in patients with cardiomyopathy. Whenstatins that inhibit Q10 synthesis are administeredto patients, Q10 supplementation is recommended.Furthermore, oral Q10 therapy should be supple-mented with agents that are essential for endogenoussynthesis of the Q10 aromatic ring structure fromtyrosine (Fig. 5).

As noted above, thyroid hormone plays animportant role in the regulation of mitochondrialsynthesis. However, a surplus of thyroid hormonereduces cardiac ATP levels and induces cardiacdysfunctions. A study of 20 hyperthyroid patientsdemonstrated an inverse correlation between serumlevels of thyroid hormones and Q10. In hyper-thyroid patients with cardiac dysfunction, oral Q10supplementation (120 mg/day) reversed the cardiac

Fig. 5. The illustration outlines the central role of the electron carrier coenzyme Q10 (Q10) for the operation of the oxidativephosphorylation system (OXPHOS). Synthesis of Q10, also called ubiquinone, requires two sources, tyrosine for the aromaticring of Q10, and farnesyl for synthesis of its prenylated side chain. Farnesyl is derived from the cholesterol-synthesis pathway andQ10 side-chain synthesis is therefore regulated by β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase, which isinhibited by statins, agents frequently used to lower serum cholesterol levels. Estrogen stimulates expression of the reductase gene.Adenosine triphosphate (ATP) inhibits HMG-CoA reductase activity. Several vitamins are essential for the synthesis of thearomatic ring structure of Q10. Rare inherited deficiencies of enzymes required for this conversion lead to greatly reduced musclestrength, but Q10 administration restores function. For further detail see text.


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impairment [184]. Further evidence of beneficialeffects of Q10 in hyperthyroidism was obtained ina rabbit model. Sustained administration of L-thyroxine (T4) to 29 animals induced cardiacdysfunction. Examination of the rabbit heartsshowed reduced levels of ATP. By contrast, co-treatment of a comparison-group of animals withQ10 protected ATP levels and prevented T4-inducedcardiac dysfunction [185].

Dietary lipids affect the lipid composition ofthe mitochondrial membrane and the function ofoxidative phosphorylation enzymes. Dietary lipidshave a relatively short half-life in the membranes.In the rat model they are rapidly altered by changesin the composition of dietary fatty-acids. In an 11-day crossover study, rats were feed either onlysoybean oil or rapeseed oil. The group of rats thatwere changed from soybeen oil to rapeseed oil rapidlydeveloped a membrane lipid composition resemb-ling that found in rats fed only rapeseed oil [186].Rapeseed oil contains erucic acid, which lowers therespiratory capacity of heart mitochondria,presumably by an effect on the mitochondrialmembrane [187].

Erucic acid is normally not detectable in humantissues. However, when present in vegetable oil infood or animal feed it accumulates in human oranimal tissues, such as the heart, where it can inducelipid accumulation and cardiac fibrosis [188,189].Another vegetable oil acid, linoleic acid, an 18:2 acidand a constituent of margosa oil, has been implicatedin the etiology of Reye’s syndrome. Peroxidized lino-leic acid reduces rat liver mitochondrial ADP-stimulated (state 3) respiration, and uncouplesrespiration [190].

Of relevance to therapeutic options is the recentdemonstration of mitochondrial DNA sequences inplasma or serum of cancer patients [191]. Thisobservation suggests that sensitive analyticalmethods might be devised for early detection ofmitochondrial dysfunction due to nDNA-encodedmitochondrial gene abnormalities or mtDNAmutations and deletions. Such an approach mightprovide opportunities for early intervention andprevention of cardiomyopathy.

VI. Conclusions

The tissue expression of OXPHOS enzymes inpatients with cardiomyopathy caused by mito-chondrial defects is often heterogeneous. Initialdiagnosis is based on finding abnormalities inmetabolites in body fluids or biopsies, followed byanalysis of OXPHOS enzyme activities or molecularanalysis of inherited or acquired gene defects [192].Many findings of altered mitochondrial function incardiomyopathies have been based upon studies ofmitochondrial enzyme activities only, and the exactnature of genetic causes of defective enzyme functionmay have been only partially investigated. Centralto the pathogenesis of cardiomyopathies is adisturbance of the mitochondrial membranepotential, which is normally strictly regulated toprovide close tracking between depletion of theproton gradient and its restoration.

Whereas many nuclear genes for mitochondrialproteins have now been sequenced, in many casestheir structure, promoter regions, and regulationhave not yet been completely clarified. Dividingcells loose daughter cells with inefficient, deletedmtDNA, but post-mitotic cardiac myocytes lack thisoption.

Enhanced oxidative stress in the heart is a highrisk factor for development of increasing hetero-plasmy, particularly during the later decades ofhuman life. When heteroplasmy reaches the cardiactissue threshold level, mitochondrial energygeneration becomes insufficient for normalphysiological myocardial functions, and cardiomyo-pathy develops.

Several palliative therapeutic approaches arecurrently available for patients with cardiomyopathy,including heart transplantation, use of regimens ordrugs that prevent mitochondrial damage (especiallydamage caused by oxidative stress), supplementatsthat protect or restore the mitochondrial oxidativephosphorylation enzymes, and the avoidance of toxicfoods and environmental agents (such as certainpesticides) that inhibit mitochondrial function.

The cure of cardiomyopathies is only feasiblethrough gene therapy. Methods for the repair ofdamaged mitochondrial genes or nuclear genes thatencode mitochondrial proteins are almost within


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reach. Whereas modification of the human germlineto repair mitochondrial defects now appears to bescientifically possible, clinical applications of suchgene therapy may be limited to some extent byreligious and ethical considerations.

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