Abstract
Objectives: The study was designed to explore the role of endogenous gaseous signaling molecule sulfur dioxide (SO2) in the control of cardiomyocyte apoptosis and its molecular mechanisms. Methods: Neonatal mouse cardiac myocytes (NMCMs) and H9c2 cells were used in the cell experiments. The endogenous SO2 pathway including SO2 level and the expression of SO2-generating enzyme aspartate aminotransferase 1/2 (AAT1/2) were detected in NMCMs. The apoptosis of cardiomyocytes was examined by a TUNEL assay. The cleavage and the activity of apoptotic proteins caspase9 and caspase3 were measured. The content of ATP, the opening of mitochondrial permeability transition pore (mPTP), and the cytochrome c (cytc) leakage were detected by immunofluorescence. The sulphenylation of cyclophilin-D (CypD) was detected by biotin switch analysis. The four CypD mutant plasmids in which cysteine sites were mutated to serine were constructed to identify the SO2-affected site in vitro. Results: ISO down-regulated the endogenous SO2/AAT pathway of cardiomyocytes in association with a significant increase in cardiomyocyte apoptosis, demonstrated by the increases in apoptosis, cleaved-caspase3/caspase3 ratio, and caspase3 activity. Furthermore, ISO significantly reduced ATP production in H9c2 cells, but the supplement of SO2 significantly restored the content of ATP. ISO stimulated mPTP opening, resulting in an increase in the release of cytc, which further increased the ratio of cleaved caspase9/caspase9 and enhanced the protein activity of caspase9. While, the supplementation of SO2 reversed the above effects. Mechanistically, SO2 did not affect CypD protein expression, but sulphenylated CypD and inhibited mPTP opening, resulting in an inhibition of cardiomyocyte apoptosis. The C104S mutation in CypD abolished SO2-induced sulphenylation of CypD, and thereby blocked the inhibitory effect of SO2 on the mPTP opening and cardiomyocyte apoptosis. Conclusion: Endogenous SO2 sulphenylated CypD at Cys104 to inhibit mPTP opening, and thus protected against cardiomyocyte apoptosis.
Highlights
Myocardial injury is an important pathophysiological process in a variety of cardiovascular diseases (Jin et al, 2013), including hypertension, heart failure, and coronary heart disease (Leong et al, 2017)
Our previous animal study showed that the down-regulated endogenous SO2/AAT pathway might be involved in the mechanisms underlying isoproterenol (ISO)induced myocardial damage, and the protective role of SO2 might be related to the enhancement of myocardial antioxidant capacity in rats (Liang et al, 2011)
ISO-stimulated Neonatal mouse cardiac myocytes (NMCMs) showed a marked decrease in SO2 content as compared with the control group as evidenced by a significant decrease in SO2-specific green fluorescence, and an obvious decrease in AAT1 and AAT2 protein expression, and AAT activity in the ISO-stimulated NMCMs (Figures 1A–C), accompanied by a significant increase in cell apoptosis, as evidenced by an increase in apoptotic cells, the cleaved-caspase3/caspase3 ratio, and caspase3 activity demonstrated by colorimetric assay (Figures 1D–F)
Summary
Myocardial injury is an important pathophysiological process in a variety of cardiovascular diseases (Jin et al, 2013), including hypertension, heart failure, and coronary heart disease (Leong et al, 2017). Cardiomyocyte apoptosis plays a crucial part in cardiovascular diseases (Singh et al, 2011). Clarifying the mechanisms underlying cardiomyocyte apoptosis in cardiovascular diseases has always been the focus of the research (Abbate and Narula, 2012). Our previous animal study showed that the down-regulated endogenous SO2/AAT pathway might be involved in the mechanisms underlying isoproterenol (ISO)induced myocardial damage, and the protective role of SO2 might be related to the enhancement of myocardial antioxidant capacity in rats (Liang et al, 2011). Jin et al (2013) found that myocardial injury is related to the inhibition of the bcl2/cytc/caspase9/caspase pathway mediated by SO2. The molecular mechanisms by which endogenous SO2 protected cardiomyocyte injury and apoptosis are completely not clear
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