CARDIOPROTECTIVE EFFECTS OF PDE-5 INHIBITORS

描述（由申请人提供）：磷酸二酯酶-5（PDE-5）抑制剂西地那非（万艾可）已知可增强男性勃起功能，对心脏缺血再灌注损伤（I/R）具有强大的心脏保护作用。我们建议对西地那非和其他临床使用的PDE-5抑制剂触发导致心脏保护的信号通路的机制进行进一步深入研究。假设1：新型PDE-5抑制剂触发心脏和肌细胞对I/R的保护。我们将研究短效（西地那非，维地那非）或长效（他达拉非）PDE-5抑制剂对I/R和细胞凋亡的保护作用。假设二：释放预处理介质，包括腺苷，缓激肽和一氧化氮介导PDE-5抑制剂诱导的心脏保护作用。采用腺苷A1、缓激肽B2受体、eNOS、nNOS和iNOS的药理学抑制剂和去甲肾上腺素基因敲除小鼠。我们将确定这些介质在PDE-5抑制剂的心脏保护中的直接作用。假设三：PDE-5抑制剂可选择性激活PKC亚型，从而诱导心肌保护作用，PKC亚型在eNOS/iNOS/nNOS基因转录中起重要作用。我们将研究PDE-5抑制剂治疗后PKC同工酶的易位。利用PKC同工酶的选择性肽阻断剂，我们将确定它们在eNOS、iNOS或nNOS的转录中的作用。假设4：PDE-5抑制剂刺激鸟苷酸环化酶并升高cGMP，导致蛋白激酶G（PKG）活化，作为mitoKATP通道开放的前奏。利用腺病毒基因转移PKG剪接变体或其无效突变体，我们将确定它们在心肌细胞和完整心脏中对I/R的保护作用。假设5：PDE-5抑制剂通过转录因子HSF-1的活化，通过HSP的过表达来保护I/R。利用siRNA，我们将确定HSF-1在PDE-5抑制剂的小鼠心脏保护中的因果关系。这些研究首次将为PDE-5抑制剂对I/R损伤的保护作用提供大量新信息。预计这些研究将对将长期研究的缺血和药物预处理现象带到临床前沿产生巨大影响。

DESCRIPTION (provided by applicant): Phosphodiesterase-5 (PDE-5) inhibitor, sildenafil (Viagra), which is known to enhance erectile function in men, induces powerful cardioprotective effect against ischemia-reperfusion injury (I/R) in heart. We propose to perform further in depth studies on the mechanisms by which sildenafil and other clinically used PDE-5 inhibitors trigger signaling pathways that lead to cardioprotection. Hypothesis 1: Novel class of PDE-5 inhibitors trigger protection against I/R in heart and myocytes. We will study the effect of short acting (sildenafil, verdenafil) or long-acting (tadalafil) PDE-5 inhibitors on protective effect against I/R and apoptosis. Hypothesis 2: Release of preconditioning mediators, including adenosine, bradykinin and nitric oxide mediate cardioprotection induced by PDE-5 inhibitors. Using pharmacological inhibitors and gone knockout mice of adenosine A1, bradykinin B2 receptors, eNOS, nNOS and iNOS. we will determine the direct role of these mediators in cardioprotection by PDE-5 inhibitors. Hypothesis 3: PDE-5 inhibitors selectively activate PKC isoforms that play an essential role in the gene transcription of eNOS/iNOS/nNOS to induce cardioprotective effect. We will study translocation of PKC isozymes after treatment with PDE-5 inhibitors. Using selective peptide blockers of PKC isozymes, we will determine their role in gone transcription of eNOS, iNOS or nNOS. Hypothesis 4:PDE-5 inhibitors stimulate guanylate cyclase and elevate cGMP causing activation of protein kinase G (PKG) as a prelude to the opening of mitoKATP channels. Using adenoviral gene transfer of splice variants of PKG or their null mutants, we will determine their role in protection against I/R in cardiomyocytes and intact heart. Hypothesis 5:PDE-5 inhibitors protect against I/R by overexpression of HSPs through activation of transcription factor, HSF-1. Using siRNAs, we will determine the cause and effect relationship of HSF-1 in cardioprotection by PDE-5 inhibitors in mice. These studies, first of its kind, will provide tremendous amount of new information on protective effect of PDE-5 inhibitors against I/R injury. It is expected that the studies will have enormous impact on bringing the long-studied phenomenon of ischemic and pharmacologic preconditioning to the clinical forefront.