Abstract 1459: Connexin 43 Hemi Channels are Permeable to Exogenously Added NAD: Implications in Protection of Cardiomyocytes against PARP-mediated Cell Death
Robust activation of poly (ADP ribose) polymerase-1 (PARP) following oxidative stress and DNA damage is a major cause of caspase independent cardiomyocyte cell death. PARP-mediated cell-death is associated with cellular NAD depletion. Connexin 43 (Cx43) is a major gap junction protein which forms hemi channels in cardiomyocytes. This study was designed to investigate whether exogenously added NAD can enter into cardiomyocytes via Cx43 hemi channels, and protect cells from PARP-mediated cell death. Primary cultures of neonatal rat heart myocytes were treated at various time points with a free-radical generating mixture of H2O2 and FeSO4 (0.1mM each). To examine permeability of cells to NAD, myocytes were treated with FITC-tagged-NAD (NAD-FITC) and intracellular localization of NAD was determined by confocal microscopy. We found that following oxidative stress myocyte cell death was proportionally related to intensity of PARP activation and NAD depletion. In these cells with PARP activation, exogenously added NAD-FITC (green) readily entered into the cell interior. This NAD transport was abolished when myocytes were treated with gap junction blockers such as carbenoxolone or 18β-glycyrrhetinic acid. To further confirm a role of Cx43 in NAD transport, we utilized HeLa cells in which Cx43 was either epigenetically silenced or constitutively over expressed. We found that HeLa cells lacking Cx43 had no NAD transport activity, while cells over expressing Cx43 showed abundant entry of NAD into the cells, thus validating a role of Cx43 in cellular NAD transport. Addition of NAD into culture media prior to PARP activation also rescued cells from PARP-mediated cell death. Cellular NAD levels are known to control the activity of the longevity factor SIRT1 deacetylase. To test if SIRT1 could be involved in mediating the beneficial effects of NAD, we did an identical cell death experiment with myocytes in which SIRT1 levels were reduced 50% by siRNA silencing. In these cells we found no beneficial effect of NAD following oxidative stress, thus indicating that SIRT1 is taking part in cell survival effects of NAD. These results demonstrate that exogenously added NAD can enter into cardiomyocytes via Cx43 hemi channels and protect cells against PARP-mediated cell death.