Preservation of mitochondrial integrity is critical for maintaining cellular homeostasis. Mitophagy is a mitochondria-specific type of autophagy which eliminates damaged mitochondria thereby contributing to mitochondrial quality control. Depolarization of the mitochondrial membrane potential is an established mechanism for inducing mitophagy, mediated through PINK1 stabilization and Parkin recruitment to mitochondria. Hexokinase-II (HK-II) which catalyzes the first step in glucose metabolism, also functions as a signaling molecule to regulate cell survival, and a significant fraction of cellular HK-II is associated with mitochondria (mitoHK-II). We demonstrate here that pharmacological interventions and adenoviral expression of a mitoHK-II dissociating peptide which reduce mitoHK-II levels lead to robust increases in mitochondrial Parkin and ubiquitination of mitochondrial proteins in cardiomyocytes and in a human glioblastoma cell line 1321N1, independent of mitochondrial membrane depolarization or PINK1 accumulation. MitoHK-II dissociation-induced mitophagy was demonstrated using Mito-Keima in cardiomyocytes and in 1321N1 cells. Subjecting cardiomyocytes or the in vivo heart to ischemia leads to modest dissociation of mitoHK-II. This response is potentiated by expression of the mitoHK-II dissociating peptide, which increases Parkin recruitment to mitochondria and, importantly, provides cardioprotection against ischemic stress. These results suggest that mitoHK-II dissociation is a physiologically relevant cellular event that is induced by ischemic stress, the enhancement of which protects against ischemic damage. The mechanism which underlies the effects of mitoHK-II dissociation can be attributed to the ability of Bcl2-associated athanogene 5 (BAG5), an inhibitor of Parkin, to localize to mitochondria and form a molecular complex with HK-II. Overexpression of BAG5 attenuates while knockdown of BAG5 sensitizes the effect of mitoHK-II dissociation on mitophagy. We suggest that HK-II, a glycolytic molecule, can function as a sensor for metabolic derangements at mitochondria to trigger mitophagy, and modulating the intracellular localization of HK-II could be a novel way of regulating mitophagy to prevent cell death induced by ischemic stress.
Mitophagy, a mitochondria-specific form of autophagy, removes dysfunctional mitochondria and is hence an essential process contributing to mitochondrial quality control. PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are critical molecules involved in stress-induced mitophagy, but the intracellular signaling mechanisms by which this pathway is regulated are unclear. We tested the hypothesis that signaling through RhoA, a small GTPase, induces mitophagy via modulation of the PINK1/Parkin pathway as a protective mechanism against ischemic stress. We demonstrate that expression of constitutively active RhoA as well as sphingosine-1-phosphate induced activation of endogenous RhoA in cardiomyocytes result in an accumulation of PINK1 at mitochondria. This is accompanied by translocation of Parkin to mitochondria and ubiquitination of mitochondrial proteins leading to recognition of mitochondria by autophagosomes and their lysosomal degradation. Expression of RhoA in cardiomyocytes confers protection against ischemia, and this cardioprotection is attenuated by siRNA-mediated PINK1 knockdown. In vivo myocardial infarction elicits increases in mitochondrial PINK1, Parkin, and ubiquitinated mitochondrial proteins. AAV9-mediated RhoA expression potentiates these responses and a concurrent decrease in infarct size is observed. Interestingly, induction of mitochondrial PINK1 accumulation in response to RhoA signaling is neither mediated through its transcriptional upregulation nor dependent on depolarization of the mitochondrial membrane, the canonical mechanism for PINK1 accumulation. Instead, our results reveal that RhoA signaling inhibits PINK1 cleavage, thereby stabilizing PINK1 protein at mitochondria. We further show that active RhoA localizes at mitochondria and interacts with PINK1, and that the mitochondrial localization of RhoA is regulated by its downstream effector protein kinase D. These findings demonstrate that RhoA activation engages a unique mechanism to regulate PINK1 accumulation, induce mitophagy and protect against ischemic stress, and implicates regulation of RhoA signaling as a potential strategy to enhance mitophagy and confer protection under stress conditions.
An established feature of cardiac disease involves an increasing presence of dysfunctional mitochondria in the heart. Mitophagy is a key player in maintaining mitochondrial quality and thereby regulates cellular homeostasis. For instance, inhibition of mitophagy has been shown to exacerbate cardiac injury induced by oxidative stress. RhoA is a small G‐protein that serves as a proximal downstream effector of numerous GPCRs and is also responsive to oxidative stress. Activation of RhoA has been previously shown to protect the heart against stress; however, its specific role in mitophagy has not been determined. Here, we explored the possibility that RhoA signaling regulates mitophagy and in turn, provides cardioprotection. Adenoviral overexpression of RhoA in neonatal rat ventricular myocytes increased both endogenous and exogenous PINK1 levels as well as mitochondrial Parkin levels, leading to mitophagy assessed by EM analysis. Although RhoA expression did not affect PINK1 mRNA levels, it did inhibit PINK1 protein degradation, thus stabilizing PINK1 protein. Depolarization of the mitochondrial membrane potential is well established to stabilize PINK1 at the mitochondria. Interestingly, however, RhoA expression did not change the mitochondrial membrane potential. Thus the basis for PINK1 accumulation at mitochondria in response to RhoA appears to be unique. siRNA‐mediated knockdown and pharmacological inhibition of PKD, an established downstream effector of RhoA, reversed RhoA‐induced stabilization of PINK1. However, overexpression of PKD alone was not sufficient to stabilize PINK1. Active RhoA accumulates at mitochondria and this response is completely inhibited by PKD inhibition, suggesting a potential role of PKD dependent mitochondrial RhoA distribution in regulating PINK1 stability. While extrapolating the role of RhoA signaling in cardiac disease, myocardial infarction induced by ligation of the left anterior descending artery was reduced in adult mice expressing constitutively active RhoA through cardiac‐specific MLC2v promoter driven adeno‐associated virus serotype 9 when compared to that of wild‐type (WT) mice. Furthermore, although both WT and cardiac specific RhoA knockout (KO) mice around 18 weeks old revealed no overt signs of cardiac dysfunction, at around 45 weeks old, KO mice not only showed hypertrophy and fibrosis, but also revealed contractile dysfunction. Currently, we are investigating the specific contribution of RhoA‐induced mitophagy in these cardiac disease models. Taken together, our results suggest that RhoA stabilizes PINK1 through its PKD‐mediated translocation to the mitochondria, subsequently inducing mitophagy in a manner uniquely independent of the conventional pathway and thus, could confer cardioprotection against stresses. Support or Funding Information This work was supported by American Heart Association grant 19TPA34910011 to S. Miyamoto.
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