Mitochondrial dysfunction is increasingly recognized as a central contributor to cardiovascular diseases, including heart failure, ischemic heart disease, hypertension, and cardiomyopathy. The mitochondrial membrane protein ATPase family AAA domain-containing 3A (ATAD3A) plays a key role in maintaining mitochondrial structure, mitochondrial quality control, and cellular bioenergetics. Mutations in ATAD3A are associated with severe mitochondrial disorders and are frequently accompanied by neurological and cardiac manifestations. However, the molecular mechanisms linking ATAD3A deficiency to cardiac pathology remain poorly understood.
In this study, we investigated the pathogenic consequences of a novel ATAD3A(R109P) mutation identified in an infant with fatal cardiomyopathy using patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Human iPSC-CMs were cultured under two metabolic conditions: standard B27 medium, which primarily supports glycolytic metabolism, or a fatty acid-rich maturation medium (MM) that promotes the postnatal metabolic shift from glycolytic to oxidative metabolism. We systematically compared patient-derived iPSC-CMsPat to healthy donor-derived iPSC-CMscntrl.
iPSC-CMsPat showed reduced cellular ATP levels and a markedly elevated AMP/ATP ratio, especially under MM culture conditions, indicating chronic energetic stress. Despite preserved respiratory capacity, mitochondrial membrane potential, and mitochondrial mass, iPSC-CMsPat displayed increased proton leak. In addition, iPSC-CMsPat exhibited increased cellular ROS levels, indicating elevated oxidative stress. Mechanistically, ATAD3A facilitates the recruitment and processing of the mitophagy protein PINK1. We found that iPSC-CMsPat exhibited elevated PINK1 levels and increased lysosome-mitochondria colocalization, indicating activation of mitophagy and suggesting impaired PINK1 processing associated with the ATAD3A(R109P) mutation. The energetic stress phenotype was accompanied by increased PDK4 expression and enhanced PDH phosphorylation at Ser293, suggesting reduced pyruvate oxidation in mitochondria. In agreement with these findings, lactate accumulation was significantly increased in iPSC-CMsPat. Strikingly, iPSC-CMsPat showed significantly increased cell death under physioxia (6% O2) conditions compared to iPSC-CMsCntrl, indicating manifestation of the disease phenotype under physiologically relevant oxygen conditions.
Collectively, our findings highlight the key role of ATAD3A in mitochondrial quality control and cellular energy homeostasis in human cardiomyocytes. Our data suggest that both metabolic substrate availability and oxygen availability influence the manifestation of the disease phenotype, providing new mechanistic insight into the pathogenesis of ATAD3A-associated cardiomyopathy. Future studies aim to investigate the causal relationship between the ATAD3A(R109P) mutation and the observed disease phenotype via CRISPR-Cas9-mediated correction of the mutation to further define patient-specific disease mechanisms and to test potential therapeutic approaches.
