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 methylb- 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. Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischemic preconditioning (IPC)-induced acute cardioprotection. The majority of proteins that show increased SNO following IPC are localized to the mitochondria, and our recent studies suggest that caveolae transduce acute NO/SNO cardioprotective signaling in IPC hearts. Due to the close association between subsarcolemmal mitochondria (SSM) and the sarcolemma/caveolae, we tested the hypothesis that SSM, rather than the interfibrillar mitochondria (IFM), are major targets for NO/SNO signaling derived from caveolae-associated eNOS. Following either control perfusion or IPC, SSM and IFM were isolated from Langendorff perfused mouse hearts, and SNO was analyzed using a modified biotin switch method with fluorescent maleimide fluors. In perfusion control hearts, the SNO content was higher in SSM compared to IFM (1.330.19, ratio of SNO content Perf-SSM vs Perf-IFM), and following IPC SNO content significantly increased preferentially in SSM, but not in IFM (1.720.17 and 1.070.04, ratio of SNO content IPC-SSM vs Perf-IFM and IPC-IFM vs Perf-IFM, respectively). Consistent with these findings, eNOS, caveolin-3 and connexin-43 were detected in SSM, but not in IFM, and IPC resulted in a further significant increase in eNOS/caveolin-3 levels in SSM. Interestingly, we did not observe an IPC-induced increase in SNO or eNOS/caveolin-3 in SSM isolated from caveolin-3-/- mouse hearts, which could not be protected with IPC. In conclusion, these results suggest that SSM are the major target for protein SNO in the IPC mouse heart, suggesting that the SSM may be the preferential target of sarcolemmal signaling-derived post-translational protein modification (caveolae-derived eNOS/NO/SNO), thus providing an important role in cardioprotection. We have also begun to explore the role of sulfhydration in cardioprotection. hydrogen sulfide (H2S) and nitric oxide (NO), play important roles in postconditioning (PostC)-induced cardioprotection. The emerging data suggest that both H2S and NO could regulate protein function through redox-based protein post-translational modification on cysteine residue(s), i.e., S-sulfhydration (SSH) and S-nitrosylation (SNO), respectively. In this study, we examined whether there is a synergistic protective effect in pharmacological PostC mouse hearts with H2S and NO donors using Langendorff perfused heart model. After 20 min of equilibrium perfusion and 20 min of no-flow global ischemia, the heart was subjected to pharmacological PostC at the beginning of reperfusion for 7 min with either 0.1 mmol/L NaHS (H2S donor), 10 micromol/L SNAP (NO/SNO donor), or both, followed by reperfusion with regular perfusion buffer for total 90 min. Compared to control, PostC with either NaHS or SNAP significantly reduced post-ischemic contractile dysfunction, the post-ischemic heart rate pressure product (RPP) recovery was 52.34.8% for PostC-NaHS, 51.73.9% for PostC-SNAP vs 36.42.5% for control (n=8 in each group). The post-ischemic myocardial infarction was decreased from 49.91.4% for control to 34.62.9% for PostC-NaHS and 35.22.9% for PostC-SNAP. Interestingly, PostC simultaneously with two donors together had a synergistic protective effect, post-ischemic RPP recovery was 72.24.2% and infarct size was 19.73.0%.
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