Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that activates intracellular effectors; for example, it causes inositol phosphate (IP) and 1,2 diacylglycerol (DAG) accumulation, stimulating the release of intracellular calcium and the activation of the protein kinase Cs (PKCs). The activation of CaSR by ischemia/reperfusion (I/R) induces cardiomyocyte apoptosis through the mitochondrial apoptotic pathway; however, the underlying mechanisms remain unclear. In this study, rat hearts were subjected to 30 min of ischemia followed by 2 h of reperfusion in the presence of a CaSR activator, GdCl(3). Our results revealed that, under these conditions, the expression of CaSR was increased, the number of apoptotic cardiomyocytes was significantly increased (as shown by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay) and the cells with a typical apoptotic morphology were observed using transmission electron microscopy. Our data further showed that mitochondria isolated from hearts that had undergone I/R combined with GdCl(3) exhibited a significant increase in the translocation of phosphorylated PKCδ to the mitochondria, an increase in cytochrome c (cyt c) release from the mitochondria and a marked decrease in mitochondrial potential. The administration of rottlerin, an inhibitor of PKCδ, significantly reduced reperfusion-induced apoptosis, phospho-PKCδ translocation to the mitochondria and the release of cyt c from the mitochondria. Thus, the involvement of CaSR in cardiac apoptosis through the mitochondrial pathway during I/R with GdCl(3) is related to phospho-PKCδ translocation to the mitochondria.

Endoplasmic reticulum (ER) stress can be activated by ischemia/reperfusion (I/R) injury in cardiomyocytes. Persistent ER stress, with an increase in intracellular ca 2+ ([ca 2+ ]i) concentration, leads to apoptosis. Protein kinase c (PKc) has a key role in myocardial damage by elevation of [ca 2+ ]i. The calcium-sensing receptor (caSR), a G protein-coupled receptor, can increase the release of [ca 2+ ]i from the ER through the inositol triphosphate receptor (IP 3 R). Intracellular calcium overload has been demonstrated to cause cardiac myocyte apoptosis during I/R. However, the associations between PKc, caSR and ER stress are not clear. The present study examined the hypothesis that activation of PKcδ by caSR participates in ER stress-associated apoptotic pathways within myocardial I/R. Rat hearts were subjected to 30 min of ischemia in vivo, followed by reperfusion for 120 min. Gdcl 3 (a caSR activator) was used to elevate the intracellular ca 2+ concentration, but the ca 2+ concentration in the ER was significantly decreased during I/R. Following exposure to Gdcl 3 , expression levels of caSR, glucose-regulated protein 78 (GRP78), caspase-12, phosphorylated JNK and caspase-3 were increased, and the ratios of apoptotic myocardial cells were significantly increased. By contrast, following exposure to rottlerin, a PKcδ inhibitor, the expression levels of these proteins and the ratio of apoptotic myocardial cells were significantly reduced. The present study also demonstrated that PKcδ translocated into the ER to induce an ER stress response and participate in the ER stress-related apoptosis pathway. These results confirmed that caSR activated PKcδ to induce cardiomyocyte apoptosis through ER stress-associated apoptotic pathways during I/R in vivo.