Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup

Abstract

Mutations of both nuclear and mitochondrial DNA (mtDNA)-encoded mitochondrial proteins can cause cardiomyopathy associated with mitochondrial dysfunction. Hence, the cardiac phenotype of nuclear DNA mitochondrial mutations might be modulated by mtDNA variation. We studied a 13-generation Mennonite pedigree with autosomal recessive myopathy and cardiomyopathy due to an SLC25A4 frameshift null mutation (c.523delC, p.Q175RfsX38), which codes for the heart-muscle isoform of the adenine nucleotide translocator-1. Ten homozygous null (adenine nucleotide translocator-1(-/-)) patients monitored over a median of 6 years had a phenotype of progressive myocardial thickening, hyperalaninemia, lactic acidosis, exercise intolerance, and persistent adrenergic activation. Electrocardiography and echocardiography with velocity vector imaging revealed abnormal contractile mechanics, myocardial repolarization abnormalities, and impaired left ventricular relaxation. End-stage heart disease was characterized by massive, symmetric, concentric cardiac hypertrophy; widespread cardiomyocyte degeneration; overabundant and structurally abnormal mitochondria; extensive subendocardial interstitial fibrosis; and marked hypertrophy of arteriolar smooth muscle. Substantial variability in the progression and severity of heart disease segregated with maternal lineage, and sequencing of mtDNA from five maternal lineages revealed two major European haplogroups, U and H. Patients with the haplogroup U mtDNAs had more rapid and severe cardiomyopathy than those with haplogroup H.

Fig. 1.

Extended Mennonite cardiomyopathy pedigree, homozygosity mapping, ANT1 (SLC25A4) frameshift mutant identification, and mtDNA haplogroup determination. (A) Genealogical analysis permitted connection of all 10 ANT1^−/− patients across 13 generations. Among seven affected sibships, there were two haplogroups (H and U) encompassing four different mtDNA subhaplotypes: U2 (red), H5 (violet), H6 (light blue), and H1 (dark blue). (B) To map the chromosomal mutant locus, five affected individuals were screened for regions of shared homozygosity using the Affymetrix 10,000-marker SNP genotyping array. Chromosome blocks are separated by downward ticks along the horizontal axis, with chromosome 4 indicated. The vertical axis indicates the number of serial homozygous SNPs shared by all five patients (yellow) or the location score (violet), a calculated value that incorporates population-specific allele frequencies to determine the likelihood that shared blocks are autozygous. A single shared 4.7 Mb region on chromosome 4q35, flanked by SNPs rs1113122 and rs1388935, had the highest location score and contained 48 RefSeq genes, including SLC25A4. (C) Regional sequence of the mutant ANT1 (SLC25A4) gene showing the c.523delC single base deletion that results in a frame shift (p.Q175RfsX38) that destroys the enzyme (Fig. S1A).

Fig. 2.

Cardiac dysfunction of ANT1 c.523delC mutant patients and the association between mtDNA haplogroup and cardiomyopathy severity and progression. (A) VVI echocardiography showing vector analysis of ANT1^+/+ versus ANT1^−/−, mtDNA haplogroup U2 enzyme null heart. VVI tracks the vectorial movement of endocardial borders of the LV in apical, four-chamber, and short axis views for obtaining longitudinal (Top), circumferential (Middle), and radial (Bottom) deformations. LV wall shows concentric hypertrophy with attenuation of peak strains in all three directions reminiscent of comparable data generated for Ant1^−/− knockout mice (14). (B and C) Representative electrocardiogram tracings from a 33-y-old woman with H haplogroup mtDNA (B) and a 25-y-old man with U haplogroup mtDNA (C) show tall R and S wave voltages in precordial leads. Seven of nine ANT1 patients had repolarization abnormalities with inverted T waves in V1 and V6, which could be seen at all ages and in both haplotype groups (arrowheads). Shortened PR intervals were observed (asterisks). Only the two oldest U haplogroup patients had prolonged QRS duration (arrow), in this case showing a right bundle-branch block pattern, whereas QRS duration was normal in younger patients from both mitochondrial haplogroups. (D) Cardiac mass index increases progressively in control subjects (gray shaded area, mean ± SD, slope 0.81 ± 0.12, r² = 0.92) and homozygous ANT1-deficient patients. Cardiac enlargement is more rapid in patients who have mitochondrial U2 haplogroup (purple circles, slope 3.5 ± 1.4, r² = 0.56) versus those with H haplogroups (orange squares, slope 2.1 ± 0.88, r² = 0.88); the slopes differ significantly (F = 19.5, P < 0.0001). (E) There was a strong correlation (r_s = 0.94, P = 0.017) between LV mass index and MPI in affected U haplogroup subjects, suggesting that cardiac energetics declined in parallel with tissue hypertrophy.

Fig. 3.

Myocardial histopathology of explanted heart from a 15-y-old ANT1^−/−, haplogroup U2, patient showing severe cardiomyocyte and mitochondrial abnormalities. The explanted ANT1^−/−, haplogroup U heart weighed 868 g (510 g/m²) with left posterior and interventricular septal walls measuring 20 and 28 mm, respectively. (A–D) Ventricle sections stained with H&E and examined by light microscopy and (E and F) ventricle sections examined by electron microscopy. (A) In areas of relative myocardial sparing, fiber orientation was distorted. (B and C) In most LV regions, myocytes were sparse, hypertrophic, and disoriented (sometimes with markedly enlarged nuclei) and showed evidence of balloon degeneration or frank necrosis (arrowheads). (D) There was extensive interstitial fibrosis, especially in subendocardial regions, where myocytes were almost completely replaced by connective tissue. Striking intimal and medial hypertrophy of arteriolar smooth muscle (arrow) resulted in narrow coronary vessels. (E and F) High mitochondrial density together with a paucity of myofibrils that are irregularly shaped and disoriented. (G) Cristae disfiguration and regions of empty mitochondria devoid of cristae (asterisks). (H) Dark granules of unclear composition occasionally seen within mitochondria.