Role of S-nitrosylation in regulating cardiac function and disease

National Heart, Lung, And Blood Institute

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Abstract

Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischemic preconditioning (IPC)-induced acute cardioprotection. We previously found C202 to be S-nitrosylated in cardioprotected hearts. We also showed in mouse embryonic fibroblasts showed that cysteine 202 of cyclophilin D (CyPD) is necessary for redox stress-induced activation of the mitochondrial permeability transition pore (PTP). C202 is highly conserved among species and can undergo redox-sensitive, post-translational modifications. We investigated whether C202 regulates the opening of PTP. We hypothesized that oxidation (e.g. perhaps the formation of a disulfide bond with the PTP) of C202 might target CypD to the PTP and that SNO of C202 blocks the oxidation that targets CypD to the PTP. This hypothesis would be consistent with data showing that oxidation targets CypD to the mitochondrial membrane. To test this hypothesis we developed a knock-in mouse model using CRISPR-Cas9 in which CypD-C202 was mutated to a serine (C202S). Infarct size is reduced in CypD- C202S Langendorff perfused hearts compared to WT. Cardiac mitochondria from CypD-C202S mice also have higher mitochondrial calcium retention capacity (CRC) compared to WT. Furthermore, isolated cardiac mitochondria subjected to oxidative stress exhibit less binding of CypD-C202S to F-ATPase, a proposed PTP component. Cysteine can also undergo S-acylation, a reversible post-translational lipid modification involving a thioester bond, and C202 matches a S-acylation motif. S-acylation of CypD-C202 was assessed using resin-assisted capture. We found that WT hearts are abundantly S-acylated on CypD C202 under baseline conditions indicating that S-acylation on C202 per se does not lead to PTP opening. CypD C202S knock-in hearts are protected from I/R injury suggesting further that lack of CypD S-acylation at C202 is not detrimental and does not induce PTP opening. The data are consistent with the hypothesis that either acylation, SNO or mutation of C202 can block the ability of CypD to activate PTP. All of these modifications would block oxidation of this cysteine. Interestingly, we find that ischemia leads to de-acylation of C202 and that calcium overload in isolated mitochondria promotes de-acylation of CypD. Furthermore, a high bolus of calcium in WT cardiac mitochondria displaces CypD from its physiological binding partners and possibly renders it available for interaction with the PTP. Taken together the data suggest that with ischemia CypD is de-acylated at C202 allowing the free cysteine residue to undergo oxidation during the first minutes of reperfusion which in turn targets it to the PTP.