Post-conditioning protects cardiomyocytes from apoptosis via PKCε-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum-mitochondria crosstalk

The intracellular Ca2+ concentration ([Ca2+](i)) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca2+](i), results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca2+](i) via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP3) to increase the release of intracellular Ca2+ from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca2+](i) through an interaction of PKC with CaR to alleviate [Ca2+](ER) depletion and [Ca2+](m) elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC epsilon translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca2+](i), the [Ca2+](ER) was not reduced and [Ca2+](m) was not increased in the PC and GdCl3-PC groups. Furthermore, there was no evident a dagger psi(m) collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl3-PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC epsilon interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the apoptotic pathway.