Abstract
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.
Highlights
Reperfusion therapy with either intravenous thrombolysis or coronary revascularization is effective strategy to reduce infarct size and improve clinical outcome after the onset of acute myocardial infarction [1]
To verify the in vivo model of myocardial I/R injury in mice, infarct size was evaluated by TTC/Evans Blue staining and myocardial apoptosis was assessed by transferase-mediated dUTP nick-end labeling (TUNEL) assay 24 h after the end of reperfusion
The present study found that myocardial I/R injury resulted in the up-regulation of Stromal interaction molecule 1 (STIM1)/Orai1/Transient receptor potential channel 1 (TRPC1) in the in vivo mouse model
Summary
Reperfusion therapy with either intravenous thrombolysis or coronary revascularization is effective strategy to reduce infarct size and improve clinical outcome after the onset of acute myocardial infarction [1]. The restoration of blood to the ischemic myocardium may cause ischemia/reperfusion (I/R) injury, leading to reduced benefit from reperfusion [2]. The underlying pathophysiological mechanisms of myocardial I/R injury have not yet been fully elucidated. As an important second messenger, intracellular calcium (Ca2+) has demonstrated to be implicated in the signaling networks that regulate pathological conditions, in addition to the process of myocardial contraction.
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