Abstract Body: Mitochondria are essential for cardiomyocyte contraction by generating ATP and orchestrating metabolic pathways. Understanding how mitophagy—selective removal of damaged mitochondria in lysosome—and metabolic flexibility are coordinated is crucial for cardiac function under physiological and pathological conditions. Our study explores the molecular mechanisms behind this coordination in cardiomyocytes.
We identify GRAF1(Arhgap26) as a pivotal mediator in PINK1-Parkin dependent mitophagy, facilitating damaged mitochondria clearance and regulating mitochondrial substrate flexibility. Depletion of GRAF1 in neonatal rat ventricular cardiomyocytes (NRVCMs) compromises mitochondrial function, evidenced by reduced membrane potential, impaired oxidative phosphorylation, and elevated cleaved Caspase 3 levels. Moreover, GRAF1 depletion attenuates LC3II activation and increases mitochondrial mass following toxin-induced stress. Using tamoxifen-inducible cardiomyocyte-restricted GRAF1 knockout mice (GRAF1^CKO), we observe normal cardiac function under basal conditions. However, upon isoproterenol (ISO) treatment, GRAF1^CKO mice display cardiac dysfunction and reduced mitophagy. Metabolomics analysis reveal distinct metabolic signatures between ISO-treated control and GRAF1^CKO mice, with impaired fuel flexibility observed in GRAF1^CKO mice, as highlighted by impaired glucose utilization relative to control mice. Mechanistically, phosphorylation of specific sites (S668, T670, and S671) within the proline-rich region of GRAF1 by PINK1 or PINK1-dependent kinases regulates its dual functions. This phosphorylation event releases the autoinhibitory state of the SH3 domain, enabling it to interact with ABI2 and WAVE2 complex for actin remodeling necessary for efficient mitochondria clearance. Additionally, phosphorylation enhances GRAF1’s affinity for Malonyl-CoA Decarboxylase (MCD), promoting MCD degradation and subsequent elevation of cellular malonyl-CoA levels. This rise in malonyl-CoA inhibits carnitine pamitoyl transferase-I (CPT-I), thereby suppressing fatty acid oxidation while promoting glucose oxidation in mitochondria, contributing to the mitochondrial metabolic flexibility.
In summary, our findings elucidate GRAF1's role in orchestrating mitophagy and metabolic flexibility in stressed cardiomyocytes. GRAF1 phosphorylation serves as a molecular switch, ensuring efficient mitochondrial clearance and substrate utilization to maintain cardiac energetics and function.