Sulphenylation of CypD at Cysteine 104: A Novel Mechanism by Which SO2 Inhibits Cardiomyocyte Apoptosis

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.

Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259

“…SO 2 increased mitochondrial membrane potential and decreased the opening of the mitochondrial permeability transition pore. After that the release of cytochrome c and the activities of caspase-3 and -9 were reduced, thereby preventing cardiomyocyte apoptosis [ 12 , 50 ]. These studies indicated that SO 2 played a cardioprotective effect.…”

“…Notably, these modifications occur at cysteine residues, indicating that cysteine residues within the 1-322 region may play critical roles in the following aspects of STAT3 activation. Our previous studies showed that SO 2 exerted cardiovascular protective effects through cysteine sulphenylation of target proteins, such as AAT, Smad3, nuclear factor-κB p65, and cyclophilin-D [ 12 , 22 , 26 , 64 ]. In this research, we observed that endogenous SO 2 sulphenylated the STAT3 protein at Cys259 in cardiomyocytes.…”

“…SO 2 content in H9c2 cells was determined using the SS-1 probe as described in previous studies [ 12 , 22 ]. SS-1 is a chemo-selective fluorescent probe for in situ visualization of SO 2 , kindly provided by professor Kun Li (Sichuan University, China).…”

“…STAT3 sulphenylation was determined in cell and purified human STAT3 protein (ab43618, Abcam, USA) using biotin switch assay as described in previous studies [ 12 , 22 ]. H9c2 were lysed in non-denaturing lysis buffer (Pulley, China) for 20 min.…”

“…Supplementation with SO 2 ameliorated AngII-induced cardiac hypertrophy by inhibiting autophagy, and alleviated isoproterenol-induced myocardial injury by inhibiting oxidative stress. These findings suggested the significant cardioprotective role of endogenous SO 2 [ [11] , [12] , [13] ]. Additionally, it was reported that AAT2 knockdown in pancreatic ductal adenocarcinoma cells led to increased levels of ROS and caused cell cycle arrest [ 14 ].…”