Identifying gene variant-specific cardiomyocyte features in MT-ATP6-related mitochondrial cardiomyopathy (DGK-Forschungsstipendium)

Complex V-related cardiomyopathy, a rare but serious condition, is caused by irregular mitochondrial-driven ATP production. Mutations in MT-ATP6 gene, a subunit of Complex V encoded in the mitochondrial genome (mtDNA), have been identified as a causative factor in cardiac disease. While ATP6 mutations can cause mitochondrial cardiomyopathy, it is not a universal feature, meanly depending on the type of variant and the amount of mtDNA mutant load present in cardiac tissue. Both m.8993T>G and m.9176T>C ATP6 variants have been reported as triggers of cardiac disease. However, the link between ATP6 deficiency and intracellular modulators of pathological signaling remodeling remains elusive. Aiming to explore efficient treatments for this cardiomyopathy, the goal of the present work is to investigate the main mediators that play a role in cardiac remodeling triggered by different ATP6 variants. First, we successfully differentiated induced pluripotent stem cells (iPSCs) with homoplasmic mutations of m.8993T>G and m.9176T>C ATP6 variants, into iPSC-derived cardiomyocytes that preserved the initial mutant mtDNA load (100%). The analysis of these cells revealed a substantial increase in the cell size of iPSC-CM expressing the ATP6 variants (n=4; p < 0.0001, for m.8993T>G and m.9176T>C) versus control group or WT, along with a significant rise in hypertrophic cardiomyopathy-related proteome (n=5). Mitochondrial respiration and membrane potential did not show a notable difference from the control group (n=5). However, a reduction in total complex V protein levels and irregular mitochondrial shape, evidenced by a swollen architecture, were identified through stimulated emission depletion microscopy (STED) in m.8993T>G and m.9176T>C ATP6 variants. We did not observe any difference in the level of markers indicating alterations in cardiogenesis processes. However, contractile properties as detected in a 3D modelling by generation of engineered heart myocardium (EHM) with either ATP6-deficient or WT IPSC-CM, and assembled with human fibroblasts from healthy donors, showed a notable decrease in the contraction force of m.9176T>C mutation but not in m.8993T>G or WT. Our findings indicate that the homoplasmic content of the m.8993T>G and m.9176T>C MT-ATP6 variants contributes to a hypertrophic phenotype, evidenced by morphological and molecular changes. Nevertheless, the m.9176T>C variant demonstrably weakens contractile function, implying a potentially more severe pathological phenotype linked to this mutation. Taken as a whole, the available evidence forms a strong basis for the exploration of tailored therapeutic strategies specific to this condition.