ROLE OF IGF-I IN PREVENTING CARDIOMYOCYTE APOPTOSIS

这个有指导的临床科学家发展奖的主要目的是为申请者作为心脏研究的独立研究员的职业生涯做好准备。为了在他以前的生物化学培训的基础上积累研究炎症介质的经验，申请人建议在目前拟议的研究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。心肌细胞凋亡在包括缺血性心脏病和心力衰竭在内的许多临床重要疾病中都有报道。胰岛素样生长因子-I(IGF-I)对心肌细胞功能和存活都有有益的影响。然而，控制心肌细胞凋亡的细胞内机制以及IGF-I在心肌缺血再灌注损伤中的抗凋亡作用仍未得到很大程度的了解。胰岛素样生长因子-I(IGF-I)与其受体(IGF-IR)的结合启动了两条细胞内信号通路：1)胰岛素受体底物(IRS)-I的磷酸化，激活磷脂酰肌醇(PI)3-激酶，随后激活丝氨酸-苏氨酸激酶Akt；2)Ras/MAP(丝裂原激活蛋白)通路。在许多系统中，PI3-激酶的激活对IGF-I的抗凋亡作用至关重要。这一假设基于四个假设：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涉及caspase的特定细胞内信号通路介导的；3)通过体细胞基因转移。IGF-I可在心肌局部高水平高表达，发挥旁分泌介质的作用，保护周围心肌细胞免受IR损伤；4)IGF-I在IR损伤中的有利作用是通过激活PI-3激酶和Akt，从而抑制caspase依赖的细胞凋亡。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，我们将开发和描述必要的载体。在特定的目标2中，我们将通过检测病毒表达的人IGF-I单独和与其他结构物联合对心肌细胞的凋亡和功能的影响，来探索IGF-I抗凋亡作用的分子机制。在具体目标3中，我们将研究病毒表达的IGF-I在体内的保护作用，并探讨IGF-I在IR损伤大鼠心肌细胞凋亡中的潜在机制。了解caspase等特定细胞内分子在心脏损伤的分子发病机制中的作用，并明确IGF-I通过体细胞基因转移发挥有益作用的潜在机制，可能为许多临床重要疾病的治疗提供新的治疗方法。

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.