Previously, we have defined cardioneuropathy (CN) as an entity characterized by cardiomyopathy, either dilated or hypertrophic, coexisting with neurologic disorders and variable mitochondrial phenotype. Some of these neurologic disorders may be primary and present with multisystemic involvement, including cardiac dysfunction arising from abnormalities in mitochondrial bioenergetic transduction. Several syndromes have been described, including MELAS and MERRF which appear to be caused by defined pathogenic mitochondrial DNA point mutations in specific mitochondrial transfer ribonucleic acids (tRNAs). These mutations affect mitochondrial DNA-encoded protein subunits of the respiratory chain complexes, which are pivotally involved in the generation of the cell’s bioenergy [1, 2]. Moreover, a group of multisystemic disorders with associated cardiomyopathy are attributable to mitochondrial defects originating in either mitochondrial DNA structural genes or nuclear DNA (e.g., Leigh disease, Friedreich’s ataxia [FRDA]). Other less well-characterized multisystemic disorders with cardiac involvement are more difficult to classify, albeit mitochondrial involvement appears to be frequently present.

To further understand the relationship between clinical and mitochondrial phenotypes in patients with this intriguing association, Marin-Garcia et al. [60] have reported a study of 28 young patients with both cardiomyopathy and neurologic disorders. These include seizures, dystonia, ophthalmoplegia, KSS, LD, and FRDA. All tissues (heart n  =  15 and skeletal muscle n  =  11) examined displayed marked defects in respiratory complex activities. Five patients had abundant large-scale mitochondrial DNA deletions, and one patient displayed a pathogenic point mutation previously reported with mitochondrial cytopathy. Patients with hypertrophic cardiomyopathy (HCM) (n  =  14) displayed a higher incidence of complex I defects, fewer DNA deletions, and mitochondrial structural abnormalities and were less often associated with developmental delay phenotype compared with patients with dilated cardiomyopathy (DCM) (n  =  14). Although structural abnormalities were present in a subset of patients, evaluation of respiratory enzyme activity was the most informative whether tissues examined were derived from heart or skeletal muscle (Table 12.2). One patient (16) had several signs indicative of KSS (i.e., ptosis, PEO, and atrioventricular conduction block), whereas a second case (13) had an atrioventricular conduction defect and hypotonia but no ptosis or PEO and died of cardiac failure at age 7 years. Two patients (1 and 2) shared both the neurologic signs and progressive cerebral deterioration symptomatic of Leigh ­disease. Two patients had a clinical presentation suggestive of FRDA (25 and 28). In the 25 patients for whom cardiac or skeletal muscle tissues were available, reduced levels of mitochondrial enzymatic activities were detected.

The spectrum of mitochondrial respiratory enzymes defects is shown in Table 12.2.

Reduced enzyme activity levels were found in complex I (18 cases), III (18 cases), IV (9 cases), and V (16 cases). Complex II and citrate synthase activity levels were in all cases similar to controls. Reduced levels of multiple respiratory complex activities were noted in 20 cases, and reductions in single respiratory complex activities were found in 5 cases, 4 of which were detected in cardiac tissue. Patients with MCM had increased incidence of complex I deficiency (12/13) compared with cases with DCM (6/13).

Pathogenic mitochondrial DNA point mutations [4, 6, 12, 41, 61–68], which have been considered responsible for encephalomyopathies including MELAS, MERRF, LD, and HCM, were found in one patient (a mutation at 8,993 in a case with LD). Five patients had significant levels of large-scale mitochondrial DNA deletions, including the common 5- and 7.4-kb deletions as well as several novel mitochondrial DNA deletions; two patients exhibited single deletions, and three patients had multiple deletions. Four of the five patients with mitochondrial DNA deletions had DCM; 1 had HCM. The location of these deletions in the human mitochondrial genome is shown in Fig. 12.3. The patient (16) with the classic KSS phenotype displayed multiple mitochondrial DNA deletions, including a novel 11-kb deletion that substantially deleted cytb and ND2 and entirely deleted the structural genes COI, COII, COIII, ATP6, ATP8, ND3, ND4, ND4 L, and ND5, as well as 13 transfer RNAs, including tryptophan, alanine, asparagine, cysteine, tyrosine, aspartic acid, lysine, glycine, arginine, histidine, serine, leucine, and glutamine, as well as the origin of replication for the light strand, as shown in Fig 12.3. The younger patient with KSS (13) did not show mitochondrial DNA deletions; however, severe defects in multiple mitochondrial enzymes, present in both skeletal and cardiac muscle, suggest the potential involvement of an unscreened pathogenic transfer RNA mutation. Two patients with clinical presentation consistent with FA harbored abnormalities in the frataxin gene; one (28) had homozygous mutant alleles [with a high level of GAA repeats (>1,500)]; the second patient (25) appears to be heterozygous with a mutant allele containing a high level of GAA repeats (>1,700) as well as a wild-type-sized allele; the latter also had marked deficiency in complex I and III activity levels in skeletal muscle. Structurally abnormal mitochondria were demonstrated by electron microscopy in 8 patients (of 16 examined). Normal organelles were mixed with strikingly abnormal mitochondria with stacks of paracrystalline bodies with characteristic geometric internal structure (Fig 12.4).

No evidence was found of myofibril disarray, nuclear or sarcolemmal abnormalities (e.g., typical of dystrophy), or inflammatory or necrotic lesions. Also, one patient displayed scattered ragged red fibers on light microscopy. One patient also displayed a marked increase in the number of cardiac mitochondria. Patients with HCM tended to have less evidence of mitochondrial structural abnormalities (3/9) compared with patients with dilated cardiomyopathy (5/7).

In summary, defects in mitochondrial DNA and bioenergetics are frequently present in children with cardiomyopathy presenting in association with neurologic abnormalities. The high incidence and severity of the mitochondrial alterations in both cardiac and skeletal muscles of very young patients with cardioneuropathy suggest that a mitochondrial cytopathy plays an important and potentially primary role in the pathogenesis of these disorders.

MCM has often been found in association with certain “soft” clinical signs in patients and maternal relatives including hearing loss, hypotonia, and migraine-like headaches. Cardiac involvement includes both DCM and HCM, ventricular dysrhythmias, and conduction defects [70, 71]. Lactic acidosis is commonly present, and many patients display generalized muscle weakness [71, 72].

In a retrospective analysis of 90 young patients with mitochondrial myopathy, the incidence of cardiomyopathy was found to be near 6% and the prevalence of dysrhythmias around 22. Four patients received permanent pacemaker because of Adams–Stokes attack or bradydysrhythmias (mitochondriopathy diagnosis was made 1–3 years post pacemaker implantation in three cases). History of syncope, respiratory failure, right bundle branch block (RBBB), atrial fibrillation, and episodic ventricular tachydysrhythmias was presented in one patient with mitochondriopathy; another patient developed atrial tachydysrhythmias. Dysrhythmias were present in 14 patients including RBBB, bifascicular block, intraventricular block, Wolff–Parkinson–White (WPW) syndrome, and short PR interval syndrome. Eight patients exhibited a mtDNA 3243A-G mutation [73].

Reported abnormalities in mitochondrial structure include distended cristae, electron-dense and paracrystalline inclusions, as well as swollen or distended mitochondrial membranes Fig. 12.4 and Fig. 12.5.

In skeletal muscle, ragged red fibers (RRFs) are often present (Fig. 12.6) and may be associated with disorders of adult/adolescent onset [74], mainly in disorders caused by defects in mitochondrial protein synthesis and rarely in disorders associated with mitochondrial structural gene defects [75].

In addition, electron microscopy may allow the assessment of mitochondria number and structural abnormalities (Fig. 12.7).

Enzyme immunostaining is a useful adjunct to evaluate the relative amounts of specific enzyme activity in muscle fibers and specific enzyme content, e.g., cytochrome c oxidase [76]. Moreover, immunohistochemical studies of skeletal muscle biopsies of children with COX deficiencies have been used to distinguish between mtDNA- and nuclear ­DNA-specific defects, based on the levels of mtDNA-encoded subunits COXI and COXII relative to changes in nuclear DNA-encoded subunits COXIV and COXVA [77].

Defects in mitochondrial tRNAs, which play a key role in protein synthesis, are involved in the genesis of multisystemic diseases with associated cardiomyopathy, including MELAS [92], MERRF [93], and LS [94].

Parenthetically, a double-point mutation in mtDNA has been found in the proband, a 23-year-old woman with MERRF harboring 8356 T  >  C and 3243A  >  G transitions in mitochondrial tRNA genes and three relatives patients whose phenotypes were MERRF, MERRF/MELAS overlap syndrome, and asymptomatic carrier. It was suggested that the course of the phenotype of this family begins with MERRF and followed by MELAS. This double mutation was heteroplasmic in blood in each case but with different rates, the mt.8356 T  >  C appeared homoplasmic and the mt.3243A  >  G heteroplasmic in muscle of the two cases studied. No other mutations were detected in the total mtDNA sequence in this family [95]. To the best of our knowledge, no cardiac pathology has been so far identified.

In our and other institutions, children with cardiomyopathy are screened for evidence of tRNA mutation using a variety of techniques such as single-strand conformation polymorphism (SSCP) analysis [96], restriction enzyme digestion [97], and automated DNA sequencing [98]. Point mutations in tRNA genes (e.g., tRNA^Leu, tRNA^Ile, tRNA^Val, tRNA^Lys, tRNA^Gly, and tRNA^cys) have been associated with CM and HF [64, 66, 68, 99–103] (a number of which are shown in Table 12.3). Molecular data concerning the location of these mutations, biochemical findings concerning the enzymes affected, the inheritance pattern, the relative proportion of mutant relative to wild-type alleles (heteroplasmy), and pertinent clinical findings are also presented in Table 12.3. For certain mutations, e.g., 3243 in tRNA^Leu and 8344 in tRNA^Lys, clinical manifestations are often delayed occurring primarily in adolescents or adults. On the other hand, atypical, early presentation of cardiomyopathy has been reported in cases with mutation at nt 3243 [104] and in a case of fatal infantile histiocytoid cardiomyopathy with a sporadic nt 8344 mutation in heart and liver [85]. For other less frequent tRNA mutations, particularly those associated with fatal infantile cardiomyopathy (e.g., 4269 tRNA^Ile, 4317 tRNA^Ile, 4320 tRNA^Ile, and 3303 tRNA^Leu), given the limited number of cases reported thus far, it is presently unknown whether or not the clinical outcome will invariably be the same. The majority of the aforementioned studies detected tRNA mutations using mtDNA derived from biopsied skeletal muscle; in some instances, the amount of the mutant allele is high enough to be detected in blood as well [105]. However, more often, analysis of the patient’s blood has provided limited information since hematopoietic cells may lose mtDNA mutations [1, 7]. Moreover, different quantities of both normal and mutant alleles may be found in the patient (i.e., heteroplasmy), an intracellular mixture of wild-type and mutant genomes which may be different dependent on the tissue examined. This is in contrast with situations where only one allele or the other is found (i.e., homoplasmy). Only when the percentage of mutant tRNA genes increases above a certain tissue-specific threshold does protein synthesis become inhibited, resulting in decreased activity levels of specific respiration enzymes and abnormal cellular phenotypes [1, 2]. Young individuals (<20 years of age) appear to have higher thresholds requiring as much as 95% of mutant mtDNAs in order to express the disease phenotype.

Moderately and severely deleterious mtDNA mutations are likely to be heteroplasmic, while mildly deleterious mtDNA mutations are homoplasmic [106]. For example, the 3303 tRNA^Leu mutation was described as homoplasmic in the proband and a close relative both who died of fatal infantile cardiomyopathy; however, maternal relatives with milder symptoms were heteroplasmic for this allele [4]. A homoplasmic mutation at 12192 in tRNA^His has been reported in several patients with DCM [107]. A pathogenic mutation in tRNA^Ile at nt 4300 was homoplasmic in all samples from several unrelated children affected with severe HCM as well as their maternal relatives [108]. Such findings suggest that caution is warranted in assessing pathogenicity as a function of heteroplasmy.

Detection of specific tRNA mutations has been made relatively simple by the use of restriction fragment digestion of PCR-amplified products containing the gene sequence of interest. Defective mtDNA genes can be characterized by either a loss or a gain of a restriction enzyme site. For the analysis of sequence changes which do not alter natural restriction sites, a restriction site can be created using “mismatched” primers which will distinguish between the presence of the wild-type and mutant alleles [109]. All of the point mutations in tRNA noted in Table 12.3 have been analyzed by restriction digestion. This approach has made possible the detection of each of the defective alleles described above using minimal tissue, limited technology, and resources. Similar diagnostic tests can also be performed with genes, e.g., beta-myosin heavy chain (β-MHC) and acyl-CoA dehydrogenase in which a number of specific mutations have been identified that are associated with a poor clinical outcome in patients with HCM [110]. These molecular genetic tests in combination with an analysis of pedigree/inheritance (e.g., an autosomal dominant pattern for β-myosin defects) can be used in excluding these defects, which are particularly common in cases of familial HCM [110, 111], in the differential diagnosis of MCM.

A number of mitochondrial tRNA mutations have been reported whose association with cardiomyopathy remains to be confirmed. For instance, a heteroplasmic mutation in the tRNA^Pro at 15990 converting the tRNA^Pro anticodon to that of tRNA^Ser has been reported in a child with a “pure myopathy” characterized by progressive weakness of the extremities [112]. While mitochondrial respiratory complex activity levels in the patient’s biopsied skeletal muscle were severely decreased, there was no evidence of cardiac involvement.

Mutations have also been found in the anticodon stem and loop of tRNA^Thr at 15923 and 15924 in 2 unrelated cases of fatal infantile respiratory failure [113] and in tRNA^Thr at 15928 in several cases of cardiomyopathy [114]. Although the pathogenicity of these mutations has been called into question, since the 15924 tRNA^Thr and 15928 tRNA^Thr mutations have been observed in control tissues [115], the mutant allele at nt 15924 has been confirmed in patients with cardiomyopathy rather than in normal subjects [116].

Leigh disease (LD) is a rapidly progressive neurodegenerative childhood disease with variable clinical manifestations including seizures, ataxia, basal ganglia lesions, and muscle weakness that can be associated with cardiomyopathy [12, 80]. A mutation in ATPase6 has been reported in several independent cases of Leigh syndrome with associated cardiomyopathy. The ATPase6 mutation is a T  →  G transversion at np 8993 converting a highly conserved hydrophobic leucine to arginine [117]. In biochemical studies with cultured lymphocytes derived from patients with LD, a pronounced defect was noted in the proton channel of the H⁺-translocating ATP synthase, thus inhibiting ATP generation from OXPHOS [118]. Abnormal activity of complex V was also found in the heart and skeletal muscles of an unrelated individual with Leigh cardiomyopathy containing the same mutation [80]. This mutation has been shown to present itself heteroplasmically in maternal relatives, while the patient had >95% mutant mtDNA in several tissues, e.g., heart, brain, and skeletal muscles. The relative concentration of the mutant allele correlates with the clinical severity of the disease.

A severe phenotype (“true” Leigh syndrome) occurs with a higher percentage of mutant genomes; lesser amounts of the mutant allele are associated with milder symptoms often termed NARP syndrome (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) [119]. A second heteroplasmic, maternally inherited mutation located at the same position 8993 (T  →  C) resulting in a leucine  →  proline change has also been reported [107]. Although the T8993C mutation is generally considered to be clinically milder than the T8993G mutation when the level of heteroplasmy exceeds 90%, progressive neurodegeneration has been reported [120]. Mutations at a variety of other genetic loci can also cause Leigh syndrome, e.g., point mutations at several sites including the mitochondrial tRNA^Lys gene [94], nuclear genes encoding subunits of pyruvate dehydrogenase [82], the flavoprotein subunit of complex II [121], and COX assembly factor SCO2 [52].

Several mutations in the cytochrome b (cytb) gene have been identified in children with cardiomyopathy. The first, an A  →  G mutation at nt 14927 in cytb, converting a highly conserved threonine to an alanine residue, was found in the heart mtDNA of a 1-year-old male with marked cardiomegaly and HF [11]. Also, a mutation at nt 15236 converting a highly conserved isoleucine to a valine at residue 164 has been reported in 2 unrelated patients [49]: a 7-year-old female with familial DCM and a 19-year-old male presenting with HCM. However, data concerning the extent of heteroplasmy, inheritance pattern, and respiratory enzyme activity analysis were not reported in these cases. Another cytb mutation, a C  →  A at nt 15452 in cytb, converted a leucine to an isoleucine at residue 236 [122]. This mutation was heteroplasmic, present in 7 of 33 cases of DCM screened and not found in controls (18 individuals), with four of the 7 mutant alleles detected in infants under 2 years of age. In these patients, there was a striking reduction in cardiac complex III activity with levels ranging from 0 to 40% of the control value. Since this mutation does not result in changing a highly conserved amino acid residue, its presence in several cases of Leber’s hereditary optic neuropathy (LHON) was previously considered nonpathogenic [123]. Nevertheless, to rigorously prove the pathogenicity of this mutation, its relationship to both the defective complex III activity and to the cellular phenotype will require the use of cybrids in which defective mitochondria containing the mutant gene are introduced into cultured cells by cell fusion [124].

In a child with COX deficiency and displaying severe histiocytoid cardiomyopathy, a mutant allele in cytb at nt 15498 was detected [125]. This mutation results in the replacement of a neutral amino acid (glycine) with an acidic one (aspartic acid) at a highly conserved residue (251), altering the interactions of cytochrome b with other subunits thereby affecting complex III assembly and proton transfer function [125, 126]. Another study identified a cytb mutation at nt 15243 in a patient with severe HCM and complex III deficiency. This mutation changes a highly conserved glycine to a glutamic acid residue situated within the cd2 helical region of cytb in close proximity to the hinge region of the Rieske iron–sulfur protein and was found to profoundly alter the subunit interactions, conformation of the bc1 complex, and complex III stability and activity [127, 128]. A cytb mutation at nt 15508 in a highly conserved aspartic with a glutamic acid residue was found in a female infant with DCM in association with marked complex III deficiency [114].

It is noteworthy that a relatively large number of pathogenic mutations in cytb have been found in cardiomyopathic tissues. While a subset of these mutations represent either neutral amino acid substitutions or likely polymorphisms, it remains to be seen whether there is some additive or synergistic effect of these mutations on mitochondrial function. These findings also imply that cytb represents a hitherto uncharacterized “hotspot” for mtDNA defects some of which could play an important role in the pathogenesis of cardiomyopathy adding it to a list that includes several mitochondrial tRNA genes (e.g., tRNA^Leu, tRNA^Ile, and tRNA^Lys) and ATPase6 [1, 2, 7].

Obayashi et al. [11] found a mutation at COII at nt 7673 converting a highly conserved isoleucine to valine at residue 30 in a 1-year-old patient with cardiomegaly. Ozawa et al. [49] reported a mutation in COI at nt 6521 causing a change from highly conserved isoleucine to methionine at residue 206 in a 7-year-old female with DCM. However, lack of information concerning allele heteroplasmy and enzymatic data ­precluded a complete evaluation of these mutations’ pathogenicity.

Several potentially pathogenic mutations have been reported in mtDNA genes encoding complex I subunits. One in ND5 at nt 13258 converted a highly conserved serine to a cysteine residue in a child with HCM [11] and a second mutation in ND1 at nt 3394 converting a highly conserved threonine to a histidine residue in a child with DCM [49]. Another mutation in ND1 at nt 3310 has been described in a case of HCM in conjunction with type 2 diabetes [129]. As with the COX mutations described above, no biochemical data were available. A severe reduction of complex I activity was found in association with a mutation in ND5 at nt 14069, resulting in a serine to leucine transition in a female of 14 years old with DCM [75] [114].

Mitochondrial DNA depletion appears to be a potential cause of mitochondrial cardiomyopathy, and depletion of wild-type mtDNA may be the primary defect in patients with mitochondrial myopathy and respiratory enzyme defects. The basis for the respiratory enzyme abnormalities found in some cases with MCM can be explained as a result of either pathogenic mtDNA mutation or mtDNA depletion. These alterations may be tissue specific and autosomally transmitted [130–133]. Several nuclear loci have been identified as likely responsible for mtDNA depletion, although some drugs like azidothymidine (AZT), which inhibits both the viral DNA polymerase and mitochondrial DNA POLG, abrogating mtDNA replication, are known to elicit mtDNA depletion and associated myopathies [57]. Also, autosomal-recessive mutations in enzymes that play a role in mitochondrial nucleotide metabolism (e.g., thymidine kinase, thymidine phosphorylase, and deoxyguanosine kinase) have been identified in a subset of patients with mtDNA depletion and represent an example of “dual genome” disorders. To confirm the pathogenic role that mtDNA depletion play in cardiomyopathy a systematic and accurate estimation of total undeleted mitochondrial genomes and their relationship to cardiac respiratory enzyme defects is needed. Dual-genome oligonucleotide array-based comparative genomic hybridization has been applied to the molecular diagnosis of mitochondrial DNA deletion and depletion syndromes by Chinault et al. [134]. This custom array can reliably detect mitochondrial DNA deletion with identification of the deletion break points and the percentage of heteroplasmy. Simultaneous detection of copy number changes in both nuclear and mitochondrial genomes potentially makes this dual genome of significant value in the diagnoses of mitochondrial DNA depletion syndromes.

Ultrastructural changes and abnormal mitochondrial number have been found in a variety of CHD, including ventricular septal defect, PDA, and right ventricular dysplasia [135, 136]. In addition, DiGeorge/velocardiofacial syndrome, which can present with tetralogy of Fallot, results from a large chromosomal deletion ranging from 1.5 to 3 Mb in chromosome 22 excising over 30 genes, some of which appear to be involved in mitochondrial function [137]. The loss of the gene for the mitochondrial citrate transporter was initially proposed as contributing to the distinct phenotype of this syndrome [137]. However, recent evidence implicated the deletion of the gene encoding the T-box transcription factor TBX1 in the cardiovascular abnormalities present in velocardiofacial/DiGeorge syndrome [138].