The emerging concept of mitochondrial cardiomyopathies☆☆☆

Oxidative phosphorylation and mtDNA 

OXPHOS subserves the fundamental purpose of transducing energy into adenosine triphosphate by the oxidation of fuel molecules5 through the activities of the respiratory electron transport chain. The large majority of the polypeptides of the respiratory chain complexes are encoded by nuclear DNA (nDNA) genes, synthesized in the cytoplasm, and imported into mitochondria. The mitochondrion, however, contains its own DNA, a double-stranded circle of 16,569 base pairs6 composed of 37 genes encoding the RNA components of the mitochondrial translation system (transfer RNA [tRNA] and ribosomal RNA [rRNA]) and 13 polypeptides belonging to four of the five complexes of the respiratory chain. Several genetic features differentiate mtDNA from nDNA. mtDNA is maternally inherited because during fertilization all mitochondria and mtDNAs are derived only by the oocyte. Affected mothers will pass mtDNAs to sons and daughters, but only daughters will transmit to their progeny. Moreover, mitochondria are polyploid, containing between 2 and 10 mtDNA molecules per organelle. At cell division mtDNAs are randomly distributed to daughter cells. In normal tissues all mtDNA molecules are identical (homoplasmy). Pathogenic mutations of mtDNA usually affect some but not all mitochondrial genomes within a cell, a tissue, or an individual (heteroplasmy). It follows that the degree of dysfunction of a cell or a tissue, and overall the pathogenic expression of a pathogenic mtDNA mutation, is largely determined by the relative abundance of normal and mutant genomes in different tissues. It also stands to reason that a minimum critical percentage of mutant mtDNA is required to be phenotypically expressed (threshold effect), but this threshold varies in different tissues depending on its specific dependence on oxidative metabolism. Hence the frequent involvement of tissues such as kidney, retina, brain, muscle, and heart is explained by their high OXPHOS demand. These characteristics have been amply verified by clinical observation of hundreds of patients. Clinical studies have also revealed that signs of cardiac involvement are frequent, either in association with neuromuscular symptoms or, less frequently, as the principal clinical feature, and take the form of cardiac rhythm abnormalities or myocardial dysfunction.7 We reviewed the current knowledge on the role of mtDNA alterations in heart diseases and used molecular genetics for an etiologic classification of these disorders.

Genetically defined mtDNA defects 

mtDNA mutations fall into three main categories: (1) single large-scale rearrangements of mtDNA, (2) point mutations resulting in missense mutations in protein encoding or RNA genes, and (3) mtDNA lesions resulting in abnormal nucleus-mitochondrion cross-talking, such as multiple mtDNA deletions. Single deletions of mtDNA are sporadic, point mutations are maternally transmitted, and multiple mtDNA deletions show mendelian inheritance.5

Point mutations of mtDNA 

Virtually all mtDNA point mutations (pm-mtDNAs) are maternally inherited. Most pm-mtDNAs involve tRNA genes and are associated with variable reduction of OXPHOS enzymes activities in skeletal muscle. Of the more than 50 pathogenic point mutations in mtDNA, about one fifth have been associated with cardiomyopathy, as shown in an updated morbidity map (Figure 1).

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Fig. 1. Morbidity circle of the mtDNA-related cardiomyopathies. The pathogenic mtDNA mutations associated with cardiomyopathy are shown. Numbers and surrounding letters refer to position and type of the mutated nucleotide. Mutations shown with an asterisk are still in the provisional status, indicating that only one group has reported the mutation and pathogenicity needs to be confirmed (see MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, Ga, www.gen.emory.edu/mitomap.html , 1999).

For still unknown reasons, the tRNALeuUUR and tRNAIle appear to be “hot spot” genes for mtDNA-associated cardiomyopathies. We will focus on the most frequent “cardiomyopathic” mutations.

Point mutations in the tRNALeuUURgene 

Mutation A3243G 

The A3243G mutation is commonly associated with the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).11 Normal early development, lactic acidosis, episodic vomiting, seizures, and recurrent cerebral episodes resembling strokes and causing hemiparesis or cortical blindness usually characterize the MELAS syndrome. The A3243G mutation has also been found in patients with CPEO, myopathy alone, or maternally inherited diabetes mellitus and deafness. Cardiac involvement occurs in 20% to 30% of patients with full-blown MELAS syndrome,12 usually in the form of hypertrophic cardiomyopathy but also as Wolf-Parkinson-White syndrome in the most compromised patients.9 Ventricular dysfunction in these patients has been observed by echocardiography, a simple investigation that should be performed in any candidate patient.13 Rarely, signs of cardiac involvement are predominant in patients harboring the A3243G mutation. For example, this was the case of a Portuguese boy who manifested a severe hypertrophic cardiomyopathy with secondary dilation as the sole clinical manifestation of the A3243G mutation.14 General examination revealed a cardiac systolic bruit and echocardiography showed a dilated left ventricle. Long-term electrocardiography showed some episodes of sinus arrhythmia at night. Skeletal muscle involvement was evidenced by the presence of mitochondrial proliferation by histochemistry.

Mutation A3260G 

A heteroplasmic A3260G mutation was first identified in a large pedigree characterized clinically by proximal muscle weakness, exercise intolerance, increased blood lactate production at rest and during exercise, and impaired cardiac ejection fraction.15 Severe hypertrophic cardiomyopathy was found in three individuals by Doppler echocardiography.

Mutation C3303T 

The C3303T mutation deserves special attention. Initially described in a large pedigree in which the proband and two siblings had died from severe infantile cardiomyopathy,16 the C3303T mutation was recently recognized in eight additional patients from four unrelated families. Presentation ranged from infantile-onset hypertrophic cardiomyopathy to moderate-severe cardiomyopathy and limb myopathy to isolated myopathy. In light of these new findings, the C3303T mutation should be considered in the differential diagnosis of infantile-onset cardiomyopathies.17

Point mutations in the tRNAIle gene 

Mutation A4269G 

This mutation was initially described in a patient who died in early adulthood from progressive heart failure. The clinical picture was multisystemic, but dilated cardiomyopathy appeared later in the course of the disease, precipitating the clinical course.18 The pathogenicity of the mutation was further confirmed by means of an “in vitro” cellular system in which the nucleus was obtained by a normal cell but the mitochondria (carrying the specific mtDNA mutation) were patient derived.19 This simple test is sufficient to demonstrate that a respiratory chain defect is borne by the mitochondrial genome. Interestingly, cells harboring 100% mutant mtDNAs showed significantly reduced OXPHOS activities and oxygen consumption compared with cells containing only normal mtDNAs.20

Mutation A4295G 

Mutation A4295G was detected in a 7-month-old girl who had had sudden onset of cyanotic spells and died as a result of complications of hypertrophic cardiomyopathy.21 Despite the hypertrophied left ventricle demonstrated by autopsy, the patient had had no symptoms of cardiac failure, exercise intolerance, or cyanosis until shortly before admission to the hospital. Ultrastructural studies of tissue at autopsy showed massive proliferation of mitochondria in heart and liver but not in skeletal muscle fibers. A brother was found to have concentric left ventricular hypertrophic cardiomyopathy, mild mitral regurgitation, and reduced ejection fraction at 2 years of age. An endocardial biopsy specimen showed mitochondrial hypertrophy. The brother subsequently underwent cardiac transplantation after a sudden deterioration of cardiac function and was “doing well” 8 months after transplant. Two additional children, aged 6 and 2 years, demonstrated no cardiac symptoms, but an elevated blood lactate level (an index of impaired OXPHOS) was found in one. Again extensive neurologic examinations were normal. The mother’s endomyocardial biopsy specimen showed no mitochondrial hypertrophy.

Mutation A4300G 

A case in point is the A4300G mutation described in a large Italian pedigree with maternally inherited cardiomyopathy (MICM) in which the proband had hypertrophic cardiomyopathy.22 Investigations in the extended pedigree showed symmetric hypertrophic cardiomyopathy in 10 family members by echocardiography. The illness had an unfavorable course. Progressive heart failure occurred in three subjects, who eventually died, whereas the proband underwent heart transplantation. Electrocardiographic or echocardiographic signs of cardiac hypertrophy in the absence of significant clinical complaints were observed in five subjects.23 Unlike other pm-mtDNAs, the heart was the only affected organ in this family.

Point mutations in the tRNALys gene 

Mutation A8344G 

Mutations in the tRNALysgene have consistently been associated with a syndrome characterized by myoclonic epilepsy, progressive cerebellar syndrome, ataxia, and myopathy (MERRF).24 A review of 62 patients with MERRF showed that 33% had clinical cardiopathy. Careful cardiologic evaluation of two patients showed cardiomegaly, electrocardiographic signs of ST depression, T-wave inversion, and ventricular premature beats and echocardiographic evidence of asymmetric septal hypertrophy with diffuse hypokinesis of the left ventricle.25

Mutation G8363A 

Two unrelated families with maternally inherited hypertrophic cardiomyopathy and hearing loss showed the G8363A mutation.26 The proband in the first family was a 16-year-old Hispanic man who developed normally until age 8 years, when he had heart failure and cognitive regression. The proband in the second family was a 44-year-old African American woman who showed progressive hearing loss, gait ataxia, shortness of breath after mild physical exercise, and a heart conduction defect. Cardiomyopathy was also found in a recently described Japanese family.27

Point mutations in the tRNAGly gene 

Mutation T9997C 

A single pedigree with maternally inherited nonobstructive cardiomyopathy had a heteroplasmic T9997C transition within the tRNAGly gene. Several members of the same pedigree showed also PEO and intestinal dismotility leading to pseudo-obstruction.28 This mutation is still awaiting pathogenic confirmation.

Point mutations in the 12S rRNA gene 

Mutation A1555G 

A family with maternally inherited restrictive cardiomyopathy showed the A1555G mutation in the mtDNA 12S rRNA gene. The mutation was heteroplasmic in several tissues from the proposita, including heart muscle, and in leukocytes from her two daughters, one of whom was still asymptomatic at age 6 years.29 Skeletal muscle biopsy in the proposita showed “minicores” and reduced cytochrome c oxidase levels.

Point mutations in polypeptide-encoding mtDNA genes 

Changes involving mtDNA genes encoding protein subunits of the respiratory electron transport chain, such as those associated with the syndrome LHON (Leber’s hereditary optic neuroretinopathy), or MILS (maternally inherited Leigh’s syndrome) occasionally manifest with cardiac conduction abnormalities or heart pathologic features. In a few patients Wolf-Parkinson-White or Lown-Ganong-Levine syndromes,30 prolonged QT interval,31 and childhood-onset hypertrophic cardiomyopathy32 have been reported.

mtDNA lesions transmitted in a mendelian manner 

Nuclear gene alterations affecting mtDNA housekeeping functions, such as replication and biogenesis, have been hypothesized in mitochondrial disorders associated with mendelian inheritance. In these conditions the genetic defect lies in the nDNA and secondarily affects mtDNA in the form of multiple deletions. Multiple mtDNA deletions were first described in several pedigrees presenting with autosomal dominant PEO, a syndrome that also includes proximal muscle weakness and wasting and sensory motor peripheral neuropathy. Idiopathic dilated cardiomyopathy was reported in a mother and her son with multiple mtDNA deletions in both skeletal muscle and heart and in two consanguineous families from Saudi Arabia with PEO and severe, intractable hypertrophic cardiomyopathy.33 A recent clinical assessment of 12 unrelated AD-PEO families harboring multiple mtDNA deletions found cardiac abnormalities in 18 of 48 patients.34

Mitochondrial DNA depletion syndrome is an autosomal inherited disease associated with grossly reduced cellular levels of mtDNA in infancy. Most patients are normal at birth, but symptoms develop in the early neonatal period, in the form of myopathy and liver failure, but hypertrophic CM and severe loss of OXPHOS activities are found in some patients.35 It has been suggested that a quantitative defect of mtDNA resulting from an unknown nuclear gene(s) likely affects copy number.

Are mtDNA defects a “common” cause of primary cardiomyopathies? 

Although current findings link abnormalities in mtDNA structure and function with a broad group of cardiac pathologic features, the pathophysiologic events that give rise to specific forms of heart diseases associated with mtDNA mutations still remain obscure. This is partly due to the occurrence in rare families often with little clinical characterization and practically no follow-up. However, the frequent heritable nature of familial DCM deems it important to undertake a diligent search for all potentially affected genes, including mtDNA. Mutations in the mtDNA were found more frequently in 58 unrelated patients with dilated cardiomyopathy (DCM) than in control subjects, suggesting that multiple mutations may exert a cumulative effect on heart function.36 Of the 43 mutations identified, four were heteroplasmic and affected evolutionarily conserved regions. Conversely, in 52 adult patients with DCM and 10 with HCM, molecular analyses ruled out seven of the many “cardiomyopathic” mutations proposing that mtDNA defects are not common in primary CM, at least in adults.37 A more complete study analyzed endomyocardial biopsy specimens from 601 patients with DCM and showed ultrastructurally abnormal mitochondria in the form of increased number and size, abnormal cristae, and inclusion bodies in 85 cases (14%). Nineteen patients (3% of total) harbored likely pathogenic mtDNA mutations and significantly lower mean levels of the respiratory chain complexes I and IV.38 It was concluded that, by altering the function of respiratory enzyme subunits or tRNA genes, mtDNA point mutations could be relevant for the pathogenesis of dilated cardiomyopathy. Although it cannot be excluded a priori that patients carried mtDNA alterations as the only DNA defect, it is intriguing to consider that in a subgroup of patients with DCM the dose of mutant mtDNA in the myocardium may constitute the basis for, or contribute to, the development of slowly but inexorably progressive, chronic illnesses such as DCM and congestive heart failure. This might be by acting as cofactors39 or as a second “genetic hit”40 to pathogenic nuclear gene mutations. A similar hypothesis has recently been proposed in an experimental murine model where the expression of a gene regulating transcription and replication of mtDNA was manipulated.41

Conclusions 

The mechanisms leading to cardiac dysfunction in mitochondrial disorders are still unclear. This is mostly because OXPHOS alterations and mtDNA mutations are associated with multisystem disorders, and much rarer is the case of isolated cardiomyopathy resulting from mtDNA defects. It is worthwhile to reiterate that the frequency of pathogenic mtDNA mutations in patients with DCM is low and that a role of mtDNA is unlikely in the majority of these patients, in spite of the findings reported by Arbustini et al.38 Nonetheless, the search of mtDNA alterations in patients with hypertrophic forms of cardiopathy not harboring sarcomeric protein genes mutations is important, especially if maternal inheritance is present. Our original description of the A4300G mutation was the successful result of this strategy. Knowing the basis of a disorder will have immediate application to genetic counseling. For example, finding a pathogenic mtDNA mutation in an affected man allows him to ensure that his progeny will not inherit the disease. On the contrary, women are at risk of transmitting the disorder to all their offspring, regardless of the sex. More rational therapies could also be adopted.

The question of the selective, often predominant, heart involvement in specific mtDNA-related diseases is still unanswered and reflects a more general ignorance about pathogenesis. As for all diseases characterized by heteroplasmic mutations, selective cardiac dysfunction might be explained by the higher level of mutant mtDNAs in heart (above the threshold level). The late occurrence of cardiomyopathy in KSS may reflect the low abundance of rearranged mtDNAs in the myocardium compared with skeletal muscle. The reason for the exquisite involvement of the conduction system in KSS might be explained by the findings that the sinus node, the AV node, or the bundle branches harbor a percentage of deleted species higher than the contractile myocardium.42 However, a role for tissue-specific modulating factors cannot be completely ruled out. As far as mtDNA point mutations are concerned, the frequency of alterations in “hot-spot” tRNAs is rather impressive and may have some still unexplained pathogenic significance. Also, the position of the mutation within the tRNA structure may be more important than the type of tRNA in determining organ specificity. Notably, four of the “cardiopathic” tRNA mutations map to the acceptor stem of the molecule (Figure 2) and likely result in similar pathogenic consequences adversely affecting the general cloverleaf structure.

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Fig. 2. The site of the mtDNA mutations associated with cardiomyopathy is illustrated in the generic cloverleaf of the tRNA gene for isoleucine (circled bases). The position in the cloverleaf secondary structure of other cardiomyopathy mutations in tRNAs is also shown (boxed base changes).

Novel experimental approaches are now possible to understand the pathogenic mechanisms of mitochondrial cardiomyopathies. As an example, Graham et al44 have produced in mice a phenotype characterized by intensive proliferation of mitochondria in skeletal and cardiac muscle, with concomitant defective OXPHOS and increased free radicals production, by knocking out the mouse gene for ANT-1. This gene encodes the tissue-specific cardiac isoform of the enzyme adenine-nucleotide translocator and is a straightforward candidate for oxidative-related cardiac alterations. This work can provide considerable information for the process leading to cardiac hypertrophy. In addition, it may help to clarify the role of excessive free radical formation. Free radical accumulation, in the form of reactive oxygen species, is a major causative factor in many disease states, including neurodegeneration and cardiovascular diseases, likely through mtDNA damage and further OXPHOS impairment.

Although mouse modeling will probably prove useful to understand the pathophysiologic mechanisms in general terms, patient material is still a better source in individual cases, especially if one considers mtDNA-related cardiomyopathies. An obstacle is the difficulty in obtaining permanent cell lines carrying a given mtDNA mutation. Cell lines derived from patients usually display very poor growth, making it very difficult to generate sufficient material for analysis. Furthermore, unknown nuclear factors could be involved in the expression of the disease, adding to the complexity of the analyses. In this scenario, the use of an endomyocardial biopsy specimen has been proposed as the method of choice for early detection of OXPHOS defects.38, 45 This is, however, a procedure not always easy to propose, often yielding tiny fragments not suitable for complete molecular and biochemical analyses, if not performed in specialized research laboratories. As a result, new experimental systems are necessary. The mentioned transmitochondrial “in vitro” system has already been applied with success in other mtDNA-associated disorders.19 It lets a given mitochondrial genome be immortalized, analyzing mitochondrial function in a neutral nuclear background, examining in detail the mechanisms by which the mutation results in phenotypic changes. Extending its use to the mtDNA-related cardiomyopathies, the eventual use of a cardiac specific cell line will make it possible to address the exact metabolic requirements for cells with impaired respiratory chain function and to test different growth conditions or treatments in both mutated and wild-type mtDNAs. It will also allow correlation of these changes with either pharmacologic or physiologic tests specific for that tissue and possible strategies to preferentially damage or inhibit one type of mtDNA in its replication.