**6.3 Mitochondrial metabolic myopathy resulting from respiratory chain complex deficiency**

Mitochondrial cardiomyopathy, a distinct myocardial condition is denoted by improper cardiac muscle structure and function accompanied by genetic defects in the mitochondrial respiratory chain. Various genetic anomalies are detected in genes regulating mitochondrial activities or in genes coding for ETC complexes or in genes that code for proteins necessary in assembling and transporting.

Peroxisomal adenine nucleotide transporter-1 and 2 (ANT-1 and ANT-2) in humans are ADP/ATP carriers with two main functions; to catalyze mitochondrial-cytosol ATP/ ADP exchange and regulation of the mitochondrial inner membrane mitochondrial permeability transition pore (mtPTP). The ATN-1 isoform influencing the mtPTP towards a more open state while ATN-2 is involved in the guiding the closure of the mtPTP. Within the heart, muscles, and brain ANT-1 is the predominant isoform while the second isoform ANT-2 is expressed systemically. However, ablation of ANT-1 isoform does not impair fetal development; but does result in hypertrophic cardiomyopathy in the postnatal period. In contrast, a study published in 2016 by Kokoszka et al. [125] identified the embryonic lethality of ANT-2 null mutations to be associated with a distinct cardiac developmental defect consistent with LVNC/LVHT; characterized by swollen mitochondria in cardiomyocytes and hyperproliferating cardiomyocytes. In these studies, mice with ANT-2 mutations, cardiac maturation progresses during embryonic days E9.5 and E13.5. However, by day E14.5 mice exhibit embryonic lethality. Histologic analysis of the heart revealed hyper-proliferating cardiomyocytes, decreased apoptosis, and swollen mitochondria with few cristae, and mtPTP open for transport. These findings suggest that loss of ANT-2 regulation of mtPTP closure results in cardiomyocytes maintenance in an immature state with a reduction in total number of contractile fibers and loss of organization thus paving the way for the continued immature cardiomyocyte proliferation resulting in LVNC and embryonic lethality [37].

#### **6.4 Mitochondrial genome mutations in LVNC**

In most of the reported cases, LVNC is related to the alterations in nuclear DNA encoded mitochondrial genes. Defects in the mediator of a mitochondrial fusion protein, Mitofusins 1 & 2 (MFN 1 & 2) affect the mitochondrial morphology during embryogenesis. Mediated by MFN1 and 2, mitochondrial fusion facilitates the exchange between the contents of mitochondrial membrane and matrix to help in maintaining the mitochondrial function. Mutations in any of the MFN's results in mitochondrial fragmentation that affects the symmetry of fusion and fission events [126]. As cell apoptosis is significantly influenced by the mitochondrial fusion and fission, cells lacking apoptosis related genes such as caspases 3, 7, 8, FADD, and c-FLIP are also susceptible to develop cardiac noncompaction. Caspases 3 and 7 are potential regulators of mitochondrial apoptosis. A3243G transition in tRNALeu(UUR) gene is another common mitochondrial mutation [127, 128].

The tRNALeu(UUR) gene encodes for mitochondrial transfer ribonucleic acid (tRNA) for leucine (tRNALeu) and this nucleotide alteration was studied in both HCM and LVNC [129]. Other studies show that A3243G mutation related disorders

cause alterations in nearly 56 different genes, thus having a broad spectrum of impact. The A to G transition at the 3243rd position in tRNALeu (UUR) gene results in diminished glucose oxidation rate, NADH response, and mitochondrial membrane potential that ultimately affects ATP production [127, 130]. Lastly, a A8381G transition in ATPase subunit 8 genes (ATPase 8) was observed in a patient having LVNC complications [43, 102, 115]. These arising missense mutations impact within the ETC complex V that could alter the ATPase complex stability, directing to reduced ATP production [43, 131].

#### **6.5 Mitochondrial DNA transitions in LVNC**

Single mutations arising from mtDNA transitions are responsible for an abnormal diseased phenotype. mtDNA transitions mutations occur when there is a replacement of the complementary nucleotide (A replaced with G, T replaced with C and vice versa). One example is found in the mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 1 (mt-ND1) encoding gene for the NADH dehydrogenase 1 enzyme that is part of complex ETC complex I, which is active in mitochondria. Here, a single nucleotide transition (T → C) at a highly conserved nucleotide position (T3398C) is within the region of the *ND1* gene of the mt genome. This particular mtDNA transition results in the conversion of methionine to threonine at the 31st amino acid position was identified in patients suffering from cardiomyopathy [39]. A case report presented by Finsterer et al. [40] investigated the combination of A → G transition at the nucleotide position of 15,662 in novel mitochondrial cytochrome B and three of the known mutations (T3398C, T4216C, G15812A) in *ND1* gene. These above-mentioned mutations were found to be in association with left ventricular hyper trabeculation and some clinical conditions. Later studies determined that a homoplasmic A → G transition at the nucleotide position of 8381 in the ATPase8 gene, investigated by Finsterer et al. [129] resulted in an amino acid substitution of alanine to threonine. Their findings revealed the correlation of LVNC with A8381G mtDNA transition that develop later in adulthood.

#### **6.6 Mutations in overlapping regions in LVNC**

The ETC complex V, known as ATP synthase, is made up of 13 protein subunits. Two of the gene mutations in mitochondrial genome or the nuclear genes result in complex V deficiency. The mitochondrial-encoded protein subunits of complex V, ATPase 6, and ATPase 8 overlap in the mitochondrial genome and undergo polycistronic fashion of transcription. The nucleotide position 8528 is within the overlapping region that codes for ATPase 6 and ATPase 8 subunits. The phenotypic representation of point mutation affects ATPase 6 and ATPase 8 genes of complex V. This is commonly seen in identical mitochondrial mtDNA mutations in children [42]. Here, the nucleotide alteration of m. T8528C, causes a M1T in the start codon in ATPase 6 subunit (One might expect to have alterations in the protein translation initiation. However, it has not been reported so far, thus making the function of altered ATPase 6 unclear) and W55R missense mutation in ATPase 8 subunit. Additionally, 2 other overlapping mutations in G8529A and C8558T affecting both the subunits have been reported in patients exhibiting cardiomyopathies [101, 115, 132]. This kind of overlapping pathogenic mutations could cause reduced ATP synthesis by affecting mitochondrial energy production and complex I, II and III for which the mechanism is still unknown [42].
