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

The intracellular Ca 2+ concentration ([Ca 2+ ] 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 [Ca 2+ ] i , results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca 2+ ] i via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP 3 ) to increase the release of intracellular Ca 2+ 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 [Ca 2+ ] i through an interaction of PKC with CaR to alleviate [Ca 2+ ] ER depletion and [Ca 2+ ] 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 ε translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca 2+ ] i , the [Ca 2+ ] ER was not reduced and [Ca 2+ ] m was not increased in the PC and GdCl 3 –PC groups. Furthermore, there was no evident ∆ψ 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 GdCl 3 –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 ε 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.