Background: ATP is produced across the inner mitochondrial membrane via oxidative phosphorylation (OXPHOS) and is the main energy source of cardiomyocytes. Hence, mitochondrial ATP synthase activity is critical for meeting cardiomyocyte energy requirements and ensuring proper contractile function. Dysfunctional mitochondria and disturbance of OXPHOS with loss of ATP are detrimental and often cause cardiovascular disease. Improving cardiac respiration and available ATP levels during cardiac stress is crucial, but remains challenging. This is largely due to the lack of knowledge regarding mechanistic insights to ATP synthase regulation. An important mediator of mitochondrial homeostasis is BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3). BNIP3 is suggested to regulate the metabolic efficiency of mitochondria. Here, we aim to elucidate the impact of BNIP3 on cardiac bioenergetics in respect to ATP synthase activity.

Methods & Results: Native gel-electrophoresis revealed BNIP3 in higher oligomeric complexes at similar molecular weight as ATP synthase in C57BL/6J mice. The proximity of BNIP3 and ATP synthase within the inner mitochondrial membrane was further determined by a proximity ligation assay and electron microscopy. Co-immunoprecipitation verified the direct interaction of BNIP3 and ATP synthase for the first time. The BNIP3 binding site of ATP synthase was then identified using a microarray approach and fluorescence anisotropy being ATP synthase subunit f within the membrane bound region of ATP synthase. Along this novel interaction, BNIP3 ablation altered the mitochondrial morphology by increasing ATP synthase dimer formation and cristae density. Further, the basal membrane potential was altered in BNIP3 KO mice compared to C57BL/6J mice. Extracellular flux technology revealed an increased mitochondrial respiratory capacity in BNIP3 depleted mice. This negative regulatory impact of BNIP3 on ATP synthase activity was in line with increased tissue ATP levels in mice lacking BNIP3 activity. Finally, the hydrolytic activity of ATP synthase was decreased by BNIP3 inhibition augmenting the hypothesized regulatory role of BNIP3 on ATP synthase.

Conclusion: Inhibition of BNIP3 activity serves as a promising target for the treatment of cardiovascular disease. Here, genetic ablation of BNIP3 improves the respiratory phenotype of cardiomyocytes and leads to elevated ATP levels in cardiac tissue. Based on the novel finding of the interaction of BNIP3 and ATP synthase in oligomeric complexes, our findings attribute a negative regulatory impact of BNIP3 on mitochondrial bioenergetics.