Mechanisms of Maladaptation in Heart Failure

描述（由申请人提供）：b-肾上腺素能受体（bar）是G蛋白偶联受体，在心功能调节中起关键作用。已知b1ARs和b2ARs都能与Gs蛋白偶联激活腺苷酸环化酶并增加cAMP水平，然而b2ARs也能与Gi偶联，可以促进细胞存活信号。相反，b1AR的激活似乎主要对心肌梗死（MI）后心脏重构产生不利影响。我的实验室最近证明，b1ARs增强心肌梗死后的不良重塑，导致心功能下降，这与钙/钙调素依赖性蛋白激酶II （CaMKII）的激活有关。我们进一步发现，b1AR激活CaMKII需要受体的羧基末端尾部（C-tail）和一种称为b-阻滞蛋白的多功能调节分子。然而，许多问题仍然存在：如果b1AR和b2AR都结合b-阻滞蛋白，为什么只有b1AR激活CaMKII？这种b1AR的选择性激活是心肌梗死或压力过载后诱发不良心脏重构和心力衰竭的机制吗？我们提出了一个假设，即b1AR的羧基末端尾部（C-tail）的一个独特区域负责CaMKII的激活和心肌梗死和压力过载后的不良心脏重构，并提出了以下具体目标：目的1：确定b1AR c -尾的最小区域对CaMKII激活是必要的。我们将通过在b1AR和b2AR的同源c尾区域之间进行氨基酸“交换”实验，确定b1AR c尾中激活CaMKII所必需的最小氨基酸残基。目的2：证明在兔心肌细胞中，b1AR c尾部的最小结构域在过度表达时导致CaMKII激活。我们将创建过表达bAR交换突变体的腺相关病毒（AAV6）构建体，将这些构建体感染到兔心肌细胞中，并测量CaMKII活性、下游途径激活、细胞收缩力和Ca2+瞬态对bAR刺激的响应（与Don Bers博士合作）。目的3：为了证明b1AR的最小c尾结构域足以导致心肌梗死后不良的心脏重塑和随后的心功能下降。将含有b1AR/b2CTmin- dom和b2AR/b1CTmin-dom的AAV6注射到双b1AR/b2AR KO小鼠的心脏中，然后进行全面的生化和生理表型分析（与Wally Koch博士合作）。目的4：在体内测试表达无c尾CaMKII激活域的b1AR突变体的小鼠是否发生心肌梗死后不良心脏重构。我们将产生基因靶向小鼠，敲入突变的b1AR受体，没有c尾CaMKII激活域（b1AR/b2CTmin-dom），以确定体内心脏表型对病理刺激的反应，如慢性儿茶酚胺管理，压力过载和心肌梗死（与Oliver Smithies博士合作）。我们预计，拟议的研究将确定新的分子机制，通过bAR途径导致不良的心脏重塑。此外，所提出的研究将为治疗人类心脏肥厚和心力衰竭提供新的靶点。

DESCRIPTION (provided by applicant): b-Adrenergic receptors (bARs) are G protein-coupled receptors that play a critical role in the regulation of cardiac function. Both b1ARs and b2ARs are known to couple to Gs proteins to activate adenylyl cyclase and increase cAMP levels, however b2ARs that are also able to couple to Gi, can promote cell survival signals. In contrast, activation of b1AR appears to exert a primarily adverse influence on post myocardial infarction (MI) cardiac remodeling. My laboratory recently demonstrated that b1ARs potentiate adverse post-MI remodeling resulting in a decline in cardiac function, which is associated with activation of calcium/calmodulin-dependent protein kinase II (CaMKII). We further showed that activation of CaMKII by the b1AR requires the carboxyl- terminal tail (C-tail) of the receptor, and a multifunctional regulatory molecule known as b-arrestin. However, a number of questions remain: if both b1AR and b2AR bind b-arrestin why is it that only the b1AR activates CaMKII?; and is this selective activation of the b1AR the mechanism for induction of adverse cardiac remodeling and heart failure post MI or pressure overload? We propose the hypothesis that a unique region of the carboxyl-terminal tail (C-tail) of the b1AR is responsible for CaMKII activation and adverse cardiac remodeling post-MI and pressure overload and propose the following specific aims: Aim 1: To identify the minimal region of the C-tail of the b1AR necessary for CaMKII activation. We will identify the minimal amino acid residues within the C-tail of b1AR that are necessary for activation of CaMKII by performing amino acid "swap" experiments between homologous C-tail regions of the b1AR and b2AR. Aim 2: To demonstrate that the identified minimal domain of the b1AR C-tail leads to CaMKII activation when overexpressed in rabbit cardiac myocytes. We will create adeno-associated virus (AAV6) constructs that overexpress the bAR swap mutants, infect these constructs into rabbit cardiac myocytes and measure CaMKII activity, downstream pathway activation, cellular contractility and Ca2+ transients in response to bAR stimulation (in collaboration with Dr. Don Bers). Aim 3: To demonstrate that the minimal C-tail domain of b1AR is sufficient to lead to adverse cardiac remodeling and subsequent decline in cardiac function post MI. AAV6 containing the b1AR/b2CTmin- dom and the b2AR/b1CTmin-dom will be injected in hearts of double b1AR/b2AR KO mice followed by comprehensive biochemical and physiological phenotyping (in collaboration with Dr. Wally Koch). Aim 4: To test in-vivo whether post-MI adverse cardiac remodeling occurs in mice expressing the b1AR mutant without the C-tail CaMKII activation domain. We will generate gene-targeted mice with a knock-in of the mutated b1AR receptor without the C-tail CaMKII activation domain (b1AR/b2CTmin-dom) to determine the in-vivo cardiac phenotype in response to pathologic stimuli such as chronic catecholamine administration, pressure overload and myocardial infarction (in collaboration with Dr. Oliver Smithies). We anticipate that the proposed investigation will identify new molecular mechanisms by which bAR pathways lead to adverse cardiac remodeling. Furthermore, the proposed studies will lead to new targets to treat cardiac hypertrophy and heart failure in humans.