Molecular Control of Cardiomyocyte Mitophagy by the RhoGAP GRAF1

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Summary Because mitochondrial dysfunction affects ATP production and promotes oxidative damage, these organelles are turned over every 2-3 weeks in healthy cardiomyocytes by a process that involves autophagy. Defects in mitochondrial quality control also enhance the progression of cardiac disease making it critical to identify the mechanisms that regulate this process. Several major pathways have been implicated that involve binding of the cytosolic E3 ubiquitin ligase, Parkin, to the outer mitochondrial membrane followed by the subsequent recruitment of mitochondria into double-membraned autophagosomes. This process is facilitated by the recruitment of the autophagosomal protein, LC3, by LC3-binding receptors that accumulate on damaged mitochondria providing a critical link between the cargo to be degraded and the autophagosome. Although evidence suggests that certain autophagy receptors promote the clearance of specific organelles or organelle components, little is known about the precise LC3-receptors that mediate cardiomyocyte mitophagy. Having previously identified GRAF1 as a critical regulator of cardiac form and function, our current data indicate that GRAF1 plays an important role in regulating cardiomyocyte mitochondrial clearance and metabolism. GRAF1 is expressed at high levels in the heart from E17 onwards and is poised to co-regulate actin- and lipid- dynamics by virtue of its multi-domain structure that includes a lipid binding/bending BAR domain, a phospholipid binding PH domain, a Rho-GAP domain, and a protein-interaction SH3 domain. Importantly, GRAF1 depletion in primary cardiomyocytes led to impaired mitochondrial OXPHOX-mediated ATP generation, mitochondrial membrane depolarization, increased mitochondrial-associated ROS, and increased ischemia/reperfusion-dependent myocyte death. GRAF1 depletion in cultured cardiomyocytes reduced LC3 mediated autophagic flux and led to the accumulation of mitochondria, and we observed similar effects in hearts from genetically modified GRAF1-deficient mice. Mechanistically, we showed that GRAF1 facilitates Parkin-dependent mitophagy by serving as a novel LC3 receptor. Our aims for this award are three-fold: In aim1, we will undertake a step-wise approach using sophisticated pH sensitive fluorophores to identify the precise mechanisms by which GRAF1 regulates cardiomyocyte mitochondrial homeostasis. In aim 2, we will use our newly developed cardiac-restricted GRAF1 knock-out mice to assess GRAF1’s contributions to cardiac metabolic reprogramming and in aim 3 we will use these mice to evaluate a Role of GRAF1-mediated mitophagy in cardioprotection. Results from the experiments proposed herein will provide the scientific foundation for the rational design of strategies to control cardiomyocyte mitophagy and could lead to novel approaches to treat ischemic heart disease.