ROLE OF IGF-I IN PREVENTING CARDIOMYOCYTE APOPTOSIS

该指导临床科学家发展奖的主要目的是为申请人作为心脏研究独立研究者的职业生涯做好准备。为了建立在其之前的生物化学培训和研究炎症介质的经验基础上，申请人建议在当前拟议研究的背景下获得分子生物学和体细胞基因转移方面的额外技能。心肌细胞凋亡已被证明在许多临床上重要的条件，包括缺血性心脏病和心力衰竭。胰岛素样生长因子-I（IGF-I）对心肌细胞功能和存活具有有益作用。然而，在心肌缺血再灌注损伤中，控制心肌细胞凋亡的细胞内机制和IGF-I的抗凋亡作用仍在很大程度上未被探索。IGF-I与其受体（IGF-IR）的结合启动了两种细胞内信号传导途径：1）胰岛素受体底物（IRS）-I的磷酸化，同时激活磷脂酰肌醇（PI）3-激酶，随后激活Akt（一种丝氨酸-苏氨酸激酶），和2）Ras/MAP（促分裂原活化蛋白）激酶途径。在许多系统中，PI 3-激酶的激活对于IGF-I的抗凋亡作用是至关重要的。本研究基于以下四个假设：1）心肌细胞凋亡在缺血再灌注（IR）诱导的心肌损伤中起重要作用; 2）IR诱导的心肌细胞凋亡是通过特异的细胞内信号通路介导的，其中包括caspase; 3）通过体细胞基因转移介导的。IGF-I可在心肌局部高水平过表达，并作为“旁分泌”介质保护周围心肌细胞免受IR损伤，和4）IGF-I在IR诱导的损伤中的有益作用是通过PI-3激酶和Akt的活化以及随后的caspase依赖性细胞凋亡的抑制来介导的。为了验证这些假设，我们将使用腺病毒载体表达人IGF-I、CrmA和Akt。在具体目标1中，我们将开发和表征必要的载体。在特定目标2中，我们将通过检查单独和与其他构建体组合的人IGF-I的病毒表达对心肌细胞凋亡和功能的功能后果来探索IGF-I的抗凋亡作用的分子机制。在具体目标3中，我们将研究IGF-I的病毒表达在体内的保护作用，并探索IR诱导的大鼠损伤模型中心脏细胞凋亡的潜在机制。了解特定的细胞内分子如半胱天冬酶在心脏损伤的分子发病机制中的作用，并确定IGF-I通过体细胞基因转移的有益作用的潜在机制，可能为许多临床重要疾病的管理提供新的治疗方法。

The primary purpose of this Mentored Clinical Scientist Development Award is to prepare the applicant for a career as an independent investigator in cardiac research. To build on his previous training in biochemistry an experience in studying inflammatory mediators, the applicant proposes to acquire additional skills in molecular biology and somatic gene transfer in the context of the current proposed study. Cardiomyocyte apoptosis has been documented in many clinically important conditions including ischemic heart disease and heart failure. Insulin-like growth factor-I (IGF-I) has beneficial effects on both cardiomyocyte function and survival. However, the intracellular mechanisms controlling cardiomyocyte apoptosis and the antiapoptotic effect of IGF-I in cardiac ischemia-reperfusion injury remain largely unexplored. Binding of IGF-I to its receptor (IGF-IR) initiates two intracellular signaling pathways: 1) phosphorylation of insulin receptor substrate (IRS)-I with activation of phosphatidylinositol (PI) 3-kinase and subsequent activation of Akt, a serine-threonine kinase, and 2) the Ras/MAP (mitogen-activated protein) kinase pathway. In many systems, activation of PI 3-kinase is critical for the anti-apoptotic effect of IGF-I. This proposal is based on four hypotheses: 1) that cardiac apoptosis plays an important role in ischemia-reperfusion (IR)-induced cardiac injury; 2) that cardiac apoptosis induced by IR is mediated through specific intracellular signaling pathways involving caspases; and 3) that through somatic gene transfer. IGF-I can be overexpressed locally in myocardium at high level and function as a "paracrine" mediator protecting the surrounding cardiomyocytes against IR injury, and 4) that the beneficial effects of IGF-I in IR-induced injury are mediated through activation of PI-3 kinase and Akt with subsequent inhibition of caspase- dependent apoptosis. To test these hypotheses we will use adenoviral vectors to express human IGF-I, CrmA, and Akt. In Specific Aim 1, we will develop and characterize the necessary vectors. In Specific Aim 2, we will explore the molecular mechanisms of the anti-apoptotic effect of IGF-I by examining the functional consequences of viral expression of human IGF-I, alone and in combination with other constructs, on cardiomyocyte apoptosis and functions. In Specific Aim 3, we will study the protective effect of viral expression of IGF-I in vivo and explore the underlying mechanisms in cardiac apoptosis in the rat model of IR- induced injury. Understanding the role of specific intracellular molecules such as caspases in the molecular pathogenesis of cardiac injury and defining the underlying mechanisms involved in the beneficial effect of IGF-I through somatic gene transfer may provide novel therapeutic approaches for the management of many clinically important disorders.