AIMS: Both mitochondria and nitric oxide (NO*) contribute to cardioprotection by ischaemic preconditioning (IPC). IPC causes mild uncoupling of mitochondria via uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT), and mild uncoupling per se is cardioprotective. Although electrophilic lipids are known to activate mitochondrial uncoupling, the role of such species in IPC-induced uncoupling and cardioprotection is unclear. We hypothesized that endogenous formation of NO*-derived electrophilic lipids (nitroalkenes such as nitro-linoleate, LNO2) during IPC may stimulate mitochondrial uncoupling via post-translational modification of UCPs and ANT, thus affording cardioprotection. METHODS: Hearts from male Sprague-Dawley rats were Langendorff-perfused and subjected to IPC. Nitroalkene formation was measured by HPLC-ESI-MS/MS. The effects of exogenous LNO2 and biotin-tagged LNO2 on isolated heart mitochondria and cardiomyocytes were also investigated. RESULTS: Nitroalkenes including LNO2 were endogenously generated in mitochondria of IPC hearts. Synthetic LNO2 (<1 microM) activated mild uncoupling, an effect blocked by UCP and ANT inhibitors. LNO2 (<1 microM) also protected cardiomyocytes against simulated ischaemia-reperfusion injury. Biotinylated LNO2 covalently modified ANT thiols and possibly UCP-2. No effects of LNO2 were attributable to NO* release, cGMP signalling, mitochondrial KATP channels, or protective kinase signalling. CONCLUSION: Components of a novel signalling pathway are inferred, wherein nitroalkenes formed by IPC-stimulated nitration reactions may induce mild mitochondrial uncoupling via post-translational modification of ANT and UCP-2, subsequently conferring resistance to ischaemia-reperfusion injury.
AIMS: Both mitochondria and nitric oxide (NO*) contribute to cardioprotection by ischaemic preconditioning (IPC). IPC causes mild uncoupling of mitochondria via uncoupling proteins (UCPs) and the adenine nucleotide translocase (ANT), and mild uncoupling per se is cardioprotective. Although electrophilic lipids are known to activate mitochondrial uncoupling, the role of such species in IPC-induced uncoupling and cardioprotection is unclear. We hypothesized that endogenous formation of NO*-derived electrophilic lipids (nitroalkenes such as nitro-linoleate, LNO2) during IPC may stimulate mitochondrial uncoupling via post-translational modification of UCPs and ANT, thus affording cardioprotection. METHODS: Hearts from male Sprague-Dawley rats were Langendorff-perfused and subjected to IPC. Nitroalkene formation was measured by HPLC-ESI-MS/MS. The effects of exogenous LNO2 and biotin-tagged LNO2 on isolated heart mitochondria and cardiomyocytes were also investigated. RESULTS:Nitroalkenes including LNO2 were endogenously generated in mitochondria of IPC hearts. Synthetic LNO2 (<1 microM) activated mild uncoupling, an effect blocked by UCP and ANT inhibitors. LNO2 (<1 microM) also protected cardiomyocytes against simulated ischaemia-reperfusion injury. Biotinylated LNO2 covalently modified ANT thiols and possibly UCP-2. No effects of LNO2 were attributable to NO* release, cGMP signalling, mitochondrial KATP channels, or protective kinase signalling. CONCLUSION: Components of a novel signalling pathway are inferred, wherein nitroalkenes formed by IPC-stimulated nitration reactions may induce mild mitochondrial uncoupling via post-translational modification of ANT and UCP-2, subsequently conferring resistance to ischaemia-reperfusion injury.
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