ARC Inhibits Cytochrome c Release From Mitochondria and Protects Against Hypoxia-Induced Apoptosis in Heart-Derived H9c2 Cells

Abstract

—Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart–derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome c from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome c release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome c release by acting upstream of caspase activation, perhaps at the mitochondrial level. The full text of this article is available at http://www.circresaha.org.

Enhanced Glycogen Synthase Kinase-3β Activity Mediates Hypoxia-induced Apoptosis of Vascular Smooth Muscle Cells and Is Prevented by Glucose Transport and Metabolism

“…We and others (2)(3)(4) have shown that hypoxia as well as hypoxia followed by reoxygenation can trigger cytochrome c release and apoptosis. Although release of cytochrome c and other proapoptotic factors could be due to generalized disruption of mitochondrial function and mitochondrial depolarization during hypoxia, our studies suggest that cytochrome c release is regulated specifically and occurs before mitochondrial failure (4,5). However, the specific upstream signaling mechanisms by which hypoxia causes cytochrome c release remain unidentified.…”

“…Apoptotic cells were identified based on nuclear condensation and/or fragmentation as reported previously (3)(4)(5). Briefly, cells were grown in 60-mm culture dishes and subjected to hypoxia (6 h) in the presence of specified chemical agents.…”

Autophagic program is regulated by miR-325

“…It can interact with Fas, FADD and Bax, 33,38 inhibit cytochrome c release 39 and maintain mitochondrial membrane potential. 40,41 These lines of evidence suggest that ARC is a critical factor for maintaining the cellular function.…”

Effect and mechanism of miR-126 in myocardial ischemia reperfusion

“…After 48 h of transfection, cells were confirmed to be an ischemia-reperfusion model according to the method described by Ekhterae and colleagues (Ekhterae et al, 1999). The cells were maintained in glucose-free and serum-free Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) in a hypoxia incubator for 10 h, and then cultured with DMEM containing 10% fetal bovine serum at normal oxygen levels for 2 h after 2 h of oxygen training.…”