Suppression of Stim1 reduced intracellular calcium concentration and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cells

OBJECTIVE Previous studies have demonstrated Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) contributes to intracellular Ca2+ accumulation. The present study aimed to investigate the expression of STIM1 and its downstream molecules Orai1/TRPC1 in the context of myocardial ischemia/reperfusion injury (MIRI) and the effect of STIM1 inhibition on Ca2+ accumulation and apoptosis in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R). METHODS Expression of STIM1/Orai1/TRPC1 was determined by RT-PCR and Western blot in mice subjected to MIRI and H9C2 cardiomyocytes subjected to H/R. To knock-down STIM1, H9C2 cardiomyocytes was transfected with Stealth SiRNA. Apoptosis was analyzed by both flow cytometry and TUNEL assay. Cell viability was measured by MTT assay. Intracellular Ca2+ concentration was detected by laser scanning confocal microscopy using Fluo-3/AM probe. Furthermore, the opening of mitochondrial permeability transition pore (mPTP) was assessed by coloading with calcein AM and CoCl2, while ROS generation was evaluated using the dye DCFH-DA in H9C2 cardiomyocytes. RESULTS Expression of STIM1/Orai1/TRPC1 significantly increased in transcript and translation level after MIRI in vivo and H/R in vitro In H9C2 cardiomyocytes subjected to H/R, intracellular Ca2+ accumulation significantly increased compared with control group, along with enhanced mPTP opening and elevated ROS generation. However, suppression of STIM1 by SiRNA significantly decreased apoptosis and intracellular Ca2+ accumulation induced by H/R in H9C2 cardiomyocytes, accompanied by attenuated mPTP opening and decreased ROS generation. In addition, suppression of STIM1 increased the Bcl-2/Bax ratio, decreased Orai1/TRPC1, and cleaved caspase-3 expression. CONCLUSION Suppression of STIM1 reduced intracellular calcium level and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cardiomyocytes. Our findings provide a new perspective in understanding STIM1-mediated calcium overload in the setting of MIRI.