Recently, we identified a novel receptor-independent G-protein activator, Activator of G-protein signaling 8 (AGS8), from a cDNA library of rat hearts subjected to repetitive transient ischemia. AGS8 mRNA was induced in the ischemic myocardium and it regulated Gβγ signaling. In the face of myocardial ischemia and/or ischemia/reperfusion, AGS8 may be involved in signaling events associated with adaptation of the cardiomyocyte to ischemic events. As an initial step to address this issue, we examined the influence of AGS8 on survival of cultured cardiomyocyte following hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia for 6h, 24 h or hypoxia (6h)/reoxygenation (18h) following transfection of AGS8 siRNA. Apoptosis was determined by DNA end-labeling or detection of changes of mitochondrial membrane potential. Interestingly, silencing of AGS8 inhibited apoptosis of cardiomyocytes induced by hypoxia alone (57.8±3.4%, p<0.05 vs. control-siRNA, at 24 h, mean±SEM) as well as hypoxia/reoxygenation (36.4±4.0%, p<0.05 vs. control-siRNA, mean±SEM). Transfection of pcDNA::AGS8 by virus envelope increased apoptosis of NCM (151%±9.6%, p<0.05 vs. vector group, mean±SEM) following hypoxia/reoxygenation, but not NCM cultured in normoxia. Affinity purified AGS8 antibody recognized immunoreactive signals enriched at the cell-cell interface of ventricular cardiomyocytes and NCM, regions also enriched in the gap junction protein connexin 43 (CX43). Indeed, AGS8 and CX43 co-immunoprecipitated from left ventricle lysates. The level of CX43 protein was decreased by overexpression of AGS8 in NCM, whereas suppression of AGS8 expression increased the level of CX43. Subsequent studies indicated that AGS8 stimulated phosphorylation of CX43 in a Gβγ dependent manner following transient expression in COS7 cells. As the level and/or phosphorylation of CX43 may influence apoptosis of cardiomyocytes, these studies suggest a potential mechanism by which AGS8 induction in the face of ischemia may provide a component of the signaling events involved in adaptation and suggest that targeted disruption of the AGS8 cascade may protect the myocardium from ischemic injury.