Nadtochiy Sergiy M, Baker Paul R S, Freeman Bruce A, Brookes Paul S
Department of Anesthesiology, Box 604, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Cardiovasc Res. 2009 May 1;82(2):333-40. doi: 10.1093/cvr/cvn323. Epub 2008 Dec 2.
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.
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.
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.
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.
线粒体和一氧化氮（NO*）均参与缺血预处理（IPC）介导的心脏保护作用。IPC通过解偶联蛋白（UCPs）和腺嘌呤核苷酸转位酶（ANT）导致线粒体轻度解偶联，而轻度解偶联本身具有心脏保护作用。虽然已知亲电子脂质可激活线粒体解偶联，但此类物质在IPC诱导的解偶联及心脏保护中的作用尚不清楚。我们推测，IPC过程中内源性生成的源自NO*的亲电子脂质（如硝基油酸酯等硝基烯烃，LNO2）可能通过对UCPs和ANT进行翻译后修饰来刺激线粒体解偶联，从而提供心脏保护。
采用Langendorff灌注法对雄性Sprague-Dawley大鼠心脏进行灌注，并施加IPC。通过高效液相色谱-电喷雾串联质谱（HPLC-ESI-MS/MS）测定硝基烯烃的生成。同时研究了外源性LNO2和生物素标记的LNO2对离体心脏线粒体和心肌细胞的影响。
包括LNO2在内的硝基烯烃在IPC处理的心脏线粒体中内源性生成。合成的LNO2（<1 microM）激活轻度解偶联，UCP和ANT抑制剂可阻断该效应。LNO2（<1 microM）还可保护心肌细胞免受模拟缺血-再灌注损伤。生物素化的LNO2共价修饰了ANT的巯基，可能还有UCP-2。LNO2的作用不归因于NO*释放、cGMP信号传导、线粒体ATP敏感性钾通道或保护性激酶信号传导。
推断出一条新的信号通路，其中IPC刺激的硝化反应形成的硝基烯烃可能通过对ANT和UCP-2进行翻译后修饰诱导线粒体轻度解偶联，进而赋予对缺血-再灌注损伤的抗性。
