Previous studies have shown a role for nitric oxide and S-nitrosylation in postconditinoing (PostC), however no S-nitrosylated proteins have been identified. The goal of this study was to identify proteins that are S-nitrosylated withPostC. We examined SNO signaling in PostC in a Langendorff perfused mouse heart model. After 20 min of equilibrium perfusion and 25 min of global ischemia, PostC was applied at the beginning of reperfusion with six cycles of 10 seconds of perfusion and 10 seconds of ischemia. The total period of reperfusion was 90 min. Compared to control, PostC significantly reduced post-ischemic contractile dysfunction and infarct size. PostC-induced protection was blocked by treatment with either L-NAME (10 M, a constitutive NO synthase inhibitor) or ascorbate (1 mM, a reducing agent to decompose SNO), but not by ODQ (10 M, a highly selective soluble guanylyl cyclase inhibitor). We used a modified biotin switch metho with CyDye maleimide mono-reactive sulfhydryl-reactive fluorescence dyes to identify SNO proteins. PostC led to a 25% or greater increase in SNO of 13,which was blocked by treatment with L-NAME. These results suggest that NO-mediated SNO signaling is critical in PostC-induced cardioprotection and the data provide the first set of candidate SNO proteins. In previous studies we have found that mitochondria are key regulators of preconditioning and most proteins showing an increase in SNO with IPC are mitochondrial. However, it is not clear how IPC transduces NO/SNO signaling to mitochondria. Using Langendorff perfused mouse hearts, we found that IPC-induced cardioprotection was blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor), ascorbic acid (a reducing agent to decompose SNO), or methyl-b-cyclodextrin (MbCD, a cholesterol sequestering agent to disrupt caveolae). IPC not only activated AKT/eNOS signaling but also led to translocation of eNOS to mitochondria. MbetaCD treatment disrupted caveolae structure, leading to dissociation of eNOS from caveolin-3 and blockade of IPC-induced activation of the AKT/eNOS signaling pathway. A significant increase in mitochondrial SNO was found in IPC hearts compared to perfusion control, and the disruption of caveolae by MbetaCD treatment not only abolished IPC-induced cardioprotection, but also blocked IPC-induced increase in SNO. In conclusion, these results suggest that caveolae transduce IPC-induced eNOS/NO/SNO acute cardioprotective signaling in the heart. Oxidative stress and membrane damage following myocardial ischemia/reperfusion injury are important contributors to cardiomyocyte death and the loss of myocardial function. Our previous study identified cysteine 144 (C144) of tripartite motif-containing protein 72 (TRIM72) as a potential site for S-nitrosylation (SNO). TRIM72 is a cardioprotective membrane repair protein that can be both activated and targeted for degradation by different oxidative modifications. Consistent with the potential regulation of TRIM72 by various oxidative modifications, we found that SNO levels increased at C144 of TRIM72 with ischemic preconditioning. Therefore, to investigate the role of C144 in the regulation of TRIM72 function, we mutated C144 of TRIM72 to a serine residue (TRIM72(C144S)), and expressed either TRIM72(WT) or TRIM72(C144S) in HEK-293 cells, which lack endogenous TRIM72, in order to examine the effect of this mutation on the functional stability of TRIM72 and on cell survival. We hypothesized that SNO of TRIM72 stabilizes the protein, thus allowing for membrane repair and enhanced cell survival. Upon treatment with hydrogen peroxide (H2O2), we found that TRIM72(WT) levels were decreased, but not TRIM72(C144S) and this correlated with increased H2O2-induced cell death in TRIM72(WT) cells. Additionally, we found that treatment with the cardioprotective S-nitrosylating agent S-nitrosoglutathione (GSNO), was able to preserve TRIM72(WT) protein levels and enhance TRIM72(WT)-mediated cell survival, but had no effect on TRIM72(C144S) levels. Consistent with our hypothesis, GSNO was also found to increase SNO levels and inhibit H2O2-induced irreversible oxidation for TRIM72(WT) without affecting TRIM72(C144S). In further support of our hypothesis, GSNO blocked the ischemia/reperfusion-induced decrease in TRIM72 levels and reduced infarct size in a Langendorff-perfused heart model. The results of these studies have important implications for cardioprotection and suggest that SNO of TRIM72 at C144 prevents the oxidation-induced degradation of TRIM72 following oxidative insult, therefore enhancing cardiomyocyte survival.
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