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

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.