Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.
Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.
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DOI：
10.1152/ajpheart.00129.2010
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发表时间：
2010-09
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影响因子：
--
通讯作者：
Eaton P
中科院分区：
文献类型：
--
作者：
Madhani M;Hall AR;Cuello F;Charles RL;Burgoyne JR;Fuller W;Hobbs AJ;Shattock MJ;Eaton P
磷酸二酯酶5型抑制剂西地那非对缺血再灌注损伤具有强大的心脏保护作用。PKG介导的信号转导与这种保护有关，尽管这种激酶的机制和下游靶点仍有待充分阐明。在这项研究中，我们评估了磷酸磷脂酶（PLM）的作用，激活Na+/K+-ATP酶，在再灌注期间给予西地那非提供的心脏保护。在再灌注的前10分钟，0.1 μM西地那非处理对离体灌注小鼠心脏的梗死（四唑染色指数）具有最佳保护作用。延长西地那非治疗（再灌注30，60或120分钟）并没有改变所提供的保护程度。这种保护是PKG依赖性的，因为它被KT-5823阻断。使用磷酸化特异性抗体对PLM进行的Western印迹分析表明，西地那非在再灌注时不调节PLM Ser 63或Ser 68磷酸化，但显著增加Ser 69磷酸化。用西地那非或8-溴-cGMP（PKG激动剂）处理分离的大鼠心室肌细胞增强PLM Ser 69磷酸化，这是双吲哚马来酰亚胺（PKC抑制剂）敏感的。膜片钳研究表明，西地那非治疗也激活Na+/K+-ATP酶，这是根据PLM Ser 69磷酸化预期的。再灌注期间Na+/K+-ATP酶的激活会减弱此时的Na+超负荷，这为西地那非如何在此时防止损伤提供了分子解释。事实上，使用火焰光度法和铷摄取到离体小鼠心脏，我们发现，西地那非增强Na+/K+-ATP酶活性在再灌注。在这项研究中，我们提供了一个分子解释如何西地那非后卫对心肌缺血后再灌注损伤。
The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na+/K+-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 μM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na+/K+-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na+/K+-ATPase activation during reperfusion would attenuate Na+ overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na+/K+-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.
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