Abstract 5874: Roles of Ischemia-inducible G-protein Activator in Hypoxia-Induced Apoptosis of Cardiomyocytes and Its Regulation of Connexin 43
Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein signaling pathway. Activator of G-protein signaling 8 (AGS8) is a G-protein activator induced in the myocardium subjected to repetitive ischemia. The induction of AGS8 in ischemic myocardium may suggest a role of AGS8 in the adaptation process of cardiomyocytes to ischemia/hypoxia. As an initial approach to address this issue, we investigated involvement of AGS8 in the survival of rat neonatal cardiomyocytes (NCM) exposed to hypoxia or hypoxia/reoxygenation. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas over-expression of AGS8 increased apoptosis. Immunoprecipitation and immunofluorescence studies indicated that AGS8 formed complexes with the channel protein connexin 43 (CX43), which regulates cell permeability to small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a Gβγ-dependent manner by providing a scaffold composed of Gβγ and CX43. AGS8siRNA blocked internalization of CX43 in the plasma membrane following exposure of NCM to repetitive hypoxia. The internalization of CX43 was associated with decreased dye flux through CX43 (34.1±10.6%, p<0.05 vs. normoxia, mean±SEM) and this was prevented by AGS8siRNA. Subsequent experiments indicated that the effects of AGS8siRNA were mimicked by a Gβγ signal inhibitor Gallein. In contrast, AGS8siRNA or Gallein did not influence epidermal growth factor-mediated internalization of CX43. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-Gβγ influences the sensitivity of cells to hypoxia via regulating the permeability of CX43 in the membrane. In the face of hypoxic stress, this previously undefined regulatory mechanism may play critical roles in the survival of cardiomyocytes.