Zhou Yue, Wang Daying, Gao Xiufang, Lew Karsheng, Richards Arthur Mark, Wang Peipei
Cardiovascular Research Institute, National University of Singapore, Singapore, Singapore.
Department of Cardiology, Putuo Hospital, Shanghai, China.
PLoS One. 2014 Jun 20;9(6):e99665. doi: 10.1371/journal.pone.0099665. eCollection 2014.
Hydrogen sulfide (H2S) is known to have cardiac protective effects through Akt activation. Akt acts as a 'central sensor' for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i) normoxia, (ii) global ischemia and (iii) ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH) release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×10(3) vs. 10±3×10(3) mmHg/min, p<0.05) and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05) compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05). Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism. These data suggest mTORC2 phosphorylation of Akt is a key mediator of H2S-induced cardioprotection in I/R.
已知硫化氢（H₂S）通过激活Akt发挥心脏保护作用。Akt作为心肌细胞存活或死亡的“中央传感器”；其活性受多种激酶（包括PI3K、mTORC2、PDK1）和多种磷酸酶（包括PTEN、PP2A和PHLPPL）调节。基于先前的研究发现，PI3K抑制剂LY294002可消除H₂S诱导的Akt磷酸化和心脏保护作用，人们认为PI3K是H₂S诱导的Akt磷酸化的介导因子。然而，LY294002同时抑制PI3K和mTOR，而PI3K仅将Akt募集到膜上，在膜上Akt被Akt激酶磷酸化。我们进行了一系列实验，以进一步评估mTORC2、PDK1、PTEN、PP2A和PHLPPL在H₂S诱导的Akt磷酸化和心脏保护中的作用，我们认为此前尚未对此进行过研究。分离成年Sprague-Dawley大鼠的心脏，并在有或没有50 μM H₂S供体硫氢化钠（NaHS）的情况下使其经历（i）常氧、（ii）整体缺血和（iii）缺血/再灌注。评估心脏机械功能和乳酸脱氢酶（LDH）释放。在灌注结束时，所有心脏还进行了Western分析，检测Akt以及一组合适的Akt调节因子和靶点。与未处理的心脏相比，用50 μM NaHS预处理的心脏在再灌注结束时功能得到改善（速率压力乘积；19±4×10³ vs. 10±3×10³ mmHg/min，p<0.05），细胞损伤减轻（LDH释放19±10 vs. 170±87 mU/ml，p<0.05）。NaHS显著增加了再灌注心脏中的磷酸化Akt、磷酸化mTOR、磷酸化Bim和Bcl-2（P<0.05）。此外，我们使用H9c2细胞证明，NaHS预处理可减少缺氧/复氧后的细胞凋亡。重要的是，特异性mTOR抑制剂PP242消除了离体心脏中的心脏保护作用和蛋白磷酸化，并降低了H9c2细胞中的凋亡效应。仅在再灌注期间用NaHS处理心脏通过类似机制产生的心脏保护作用较小。这些数据表明，Akt的mTORC2磷酸化是H₂S在缺血/再灌注中诱导心脏保护作用的关键介导因子。
