Abstract 9901: Cardiac Protection From Hypoxic Injury by a Novel Synthetic Peptide Targeting the Activator of G-Protein Signaling 8-Mediated Gβγ Signal

Abstract

Ischemia-reperfusion injury of the heart is associated with activation of multiple signaling pathways including those of heterotrimeric G-protein. We identified an ischemia-inducible G-protein activator, activator of G-protein signaling 8 (AGS8), which interacts directly with Gβγ and regulates Gβγ signaling in cells. The knockdown of AGS8 by siRNA dramatically blocked hypoxia-induced apoptosis of cardiomyocytes, suggesting AGS8 triggered a critical cascade leading to cell death, which could be a novel therapeutic target. To investigate this possibility further, we attempted to protect cardiomyocytes from hypoxia-induced apoptosis by utilizing a synthetic peptide which disrupts AGS8-mediated Gβγ signaling. We first developed a peptide (AGS8-peptide) that blocked the association of Gβγ with AGS8 based on amino acid sequences of the Gβγ binding domain of AGS8. We then investigated the effects of the AGS8-peptide on cardiomyocytes exposed to hypoxic stress. Cultured rat neonatal cardiomyocytes were exposed to hypoxia (6 h)/reoxygenation (18 h) following transfection with AGS8-peptide. Interestingly, AGS8-peptide effectively blocked apoptosis in cardiomyocytes, as determined by DNA end-labeling (9.7±1.5% of control, p<0.05, mean±SEM) and an increase in cleaved caspase-3 (36.7±6.5% of control, p<0.05, mean±SEM). Because AGS8-mediated Gβγ signaling influenced apoptosis of cardiomyocytes via channel protein connexin 43 (CX43), the effects of AGS8-peptide on permeability/localization of CX43 were examined. The permeability of CX43 following the exposure of cells to hypoxia, as determined by a flux of fluorescence dye (Lucifer yellow), was decreased (34.1±4.3%, p<0.05 vs. normoxia control, mean±SEM). However, AGS8-peptide blocked the decrease of membrane permeability (93.0±12.3%, p<0.05 vs. control group, mean±SEM). AGS8-peptide also blocked hypoxia-induced internalization of CX43 in cardiomyocytes (8.3±1.3% of control, p<0.05, mean±SEM). These data indicate the importance of AGS8-mediated Gβγ signaling in cardiomyocytes and suggest that the targeted disruption of the AGS8 signal will provide a novel approach for protecting the myocardium against ischemic injury.