Calcium-sensing receptors regulate cardiomyocyte Ca(2+ )signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca(2+ )directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP(3)Rs) 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 Ca(2+ )signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP(3)Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP(3)Rs were located in the SR. [Ca(2+)]i, [Ca(2+)](m )and [Ca(2+)](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 [Ca(2+)](SR), increased [Ca(2+)](i )and [Ca(2+)](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 Ca(2+ )release from the SR into the mitochondria through IP(3)Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.