ROS, Inflammation, and Cardioprotection in Type 2 Diabetes

描述（由申请人提供）：2型糖尿病（T2D）正迅速成为一种全球性流行病，预计到2025年，全球将有超过3亿人患有该疾病。这种疾病是心脏病发作和心肌细胞死亡的主要独立危险因素。目前的药物治疗不足以控制这种毁灭性疾病及其严重的心血管并发症。基于我们丰富的初步数据，本项目旨在验证以下中心假设：慢性抑制磷酸二酯酶-5 （PDE-5）通过AMPK/Sirt1/PGC-1¿信号转导网络抑制心肌氧化应激和炎症体形成，从而保护T2D心脏，从而诱导线粒体抗氧化酶-谷胱甘肽s -转移酶1 （GSTK1）和改善线粒体功能。我们提出以下三点具体目标。目的1：他达拉非抑制PDE-5激活AMPK/Sirt1/PGC-1信号通路，减轻t2dm心肌缺血/再灌注（I/R）后细胞死亡（坏死/凋亡）和线粒体呼吸链功能障碍。使用db/db和高脂饮食（HFD）喂养的T2D小鼠，我们将证明他达拉非治疗对a) AMPK磷酸化、eNOS和一氧化氮生成的影响；b) I/R后AMPK¿2显性阴性转基因小鼠的心脏保护功能可能丧失；c) AMPK在激活Sirt1和PGC-1¿中的作用；d) cgmp依赖性蛋白激酶（PKG）和AMPK/Sirt1/PGC-1¿信号转导网络在I/R后心肌细胞凋亡/坏死和线粒体呼吸链功能障碍的衰减中的潜在相互作用。目的2:PDE-5抑制可恢复T2D心脏中线粒体GSTK1的抑制水平，并通过其抗氧化和脂质调节特性减轻I/R损伤。使用GSTK1基因敲除小鼠的hfd诱导的T2D，我们将证明GSTK1在PDE-5抑制剂诱导的T2D心脏保护和脂质调节特性中的致病作用。我们还将通过直接在心内注射腺病毒GSTK1诱导GSTK1靶向过表达，以证明GSTK1在减轻T2D心脏I/R损伤中的直接因果关系。目的3:PDE-5抑制剂通过抑制糖尿病心脏炎症小体和抑制caspase-1来减轻炎症。我们将研究：a) PDE-5抑制剂在心脏和心肌细胞I/R后减轻炎性体形成并随之抑制caspase-1活性中的作用；b) AMPK和Sirt-1在抑制炎性小体形成中的作用；c) AMPK、GSTK1和PKG1¿参与I/R后炎性体形成的预防。总的来说，拟议的研究将为一种潜在的新型药物治疗方法铺平道路，以对抗由T2D疾病引起的炎症和心脏损伤。

DESCRIPTION (provided by applicant): Type 2 diabetes (T2D) is rapidly becoming a global pandemic and is projected to afflict more than 300 million individuals worldwide by the year 2025. This disease constitutes a major independent risk factor for heart attack and myocardial cell death. The current lines of drug therapy are insufficient to control this devastating disease and its serious cardiovascular complications. Based on our ample preliminary data, the current project is designed to test the following central HYPOTHESIS: Chronic inhibition of phosphodiesterase-5 (PDE-5) protects T2D hearts through suppression of myocardial oxidative stress and inflammasome formation via the AMPK/Sirt1/PGC-1¿ signal transduction network that leads to induction of mitochondrial antioxidant enzyme - glutathione S-transferase kappa 1 (GSTK1) and improvement of mitochondrial function. We propose the following 3 specific aims. Aim 1: PDE-5 inhibition with tadalafil activates AMPK/Sirt1/PGC-1¿ signaling pathway and attenuates cell death (necrosis/apoptosis) and mitochondrial respiratory chain dysfunction following myocardial ischemia/reperfusion (I/R) in T2D. Using db/db and high fat diet (HFD)-fed T2D mice, we will demonstrate the effect of tadalafil treatment on a) AMPK phosphorylation, eNOS and nitric oxide generation; b) the possible loss of cardioprotection in the AMPK¿2 dominant negative transgenic mice following I/R; c) the role of AMPK in activating Sirt1 and PGC-1¿; and d) the potential interplay of cGMP-dependent protein kinase (PKG) and AMPK/Sirt1/PGC-1¿ signal transduction network in attenuation of cardiomyocyte apoptosis/necrosis and mitochondrial respiratory chain dysfunction following I/R. Aim 2: PDE-5 inhibition restores depressed level of mitochondrial GSTK1 in the T2D hearts and attenuates I/R injury via its antioxidant as well as lipid-regulating properties. Using GSTK1 knockout mice with HFD-induced T2D, we will demonstrate the causative role of GSTK1 in PDE-5 inhibitor-induced cardioprotection and lipid regulating properties in T2D. We will also induce targeted overexpression of GSTK1 by direct intramyocardial injection of adenoviral GSTK1 to demonstrate the direct cause and effect relationship of GSTK1 in reducing I/R injury in T2D hearts. Aim 3: PDE-5 inhibitors attenuate inflammation through suppression of inflammasome and inhibition of caspase-1 in diabetic heart. We will examine: a) the role of PDE-5 inhibitors in attenuating inflammosome formation with consequent inhibition of caspase-1 activity following I/R in the heart and cardiomyocytes; b) the role of AMPK and Sirt-1 in attenuation of inflammasome formation; c) the involvement of AMPK, GSTK1, and PKG1¿ in prevention of inflammasome formation following I/R. Overall, the proposed studies will pave the way to a potentially novel pharmacotherapy against inflammation and cardiac injuries resulting from T2D conditions.