Mechanisms of Maladaptation in Heart Failure

心力衰竭适应不良的机制

• 批准号: 8321456
• 负责人: Howard A Rockman
• 金额: $ 39.25万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-24 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321456
• 关键词: Acute; Adenylate Cyclase; Adrenergic Receptor; Adverse effects; Amino Acids; Arrestins; Binding; Biochemical; Calcium/calmodulin-dependent protein kinase; Cardiac; Cardiac Myocytes; Catecholamines; Cell Survival; Chronic; Collaborations; Cyclic AMP; Dependovirus; Development; Functional disorder; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Targeting; Goals; Heart; Heart Hypertrophy; Heart failure; Human; In Vitro; Infarction; Investigation; Knock-in Mouse; Laboratories; Lead; Ligands; Measures; Mediating; Methods; Molecular; Molecular Conformation; Mus; Mutate; Myocardial; Myocardial Infarction; Oryctolagus cuniculus; Pathologic; Pathway interactions; Phenotype; Physiological; Play; Proteins; Recovery; Regulation; Role; Signal Pathway; Signal Transduction; Stimulus; Syndrome; Tail; Testing; base; calmodulin-dependent protein kinase II; in vivo; mutant; novel; overexpression; pressure; prevent; protein activation; receptor; research study; response

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.

描述（由申请人提供）：b-肾上腺素受体（bAR）是G蛋白偶联受体，在心脏功能的调节中发挥关键作用。已知 b1AR 和 b2AR 均可与 Gs 蛋白偶联以激活腺苷酸环化酶并增加 cAMP 水平，但 b2AR 也能够与 Gi 偶联，从而促进细胞存活信号。相比之下，b1AR 的激活似乎主要对心肌梗死 (MI) 后心脏重塑产生不利影响。我的实验室最近证明，b1AR 会增强不良的心肌梗死后重塑，导致心脏功能下降，这与钙/钙调蛋白依赖性蛋白激酶 II (CaMKII) 的激活有关。我们进一步表明，b1AR 激活 CaMKII 需要受体的羧基末端尾（C 尾）和称为 b-arrestin 的多功能调节分子。然而，仍然存在一些问题：如果 b1AR 和 b2AR 都结合 b-arrestin，为什么只有 b1AR 激活 CaMKII？ b1AR 的这种选择性激活是否是 MI 或压力超负荷后诱导不良心脏重塑和心力衰竭的机制？ 我们提出假设，b1AR 的羧基末端尾部（C 尾）的独特区域负责 CaMKII 激活以及 MI 和压力超负荷后的不良心脏重塑，并提出以下具体目标： 目标 1：确定 CaMKII 激活所需的 b1AR C 尾的最小区域。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”实验来鉴定 b1AR C 尾内激活 CaMKII 所必需的最小氨基酸残基。目标 2：证明已鉴定的 b1AR C 尾最小结构域在兔心肌细胞中过表达时会导致 CaMKII 激活。我们将创建腺相关病毒 (AAV6) 构建体，该构建体过度表达 bAR 交换突变体，将这些构建体感染到兔心肌细胞中，并测量 CaMKII 活性、下游通路激活、细胞收缩性和 Ca2+ 瞬态响应 bAR 刺激（与 Don Bers 博士合作）。目标 3：证明 b1AR 的最小 C 尾结构域足以导致不良的心脏重塑以及随后 MI 后心功能的下降。含有 b1AR/b2CTmin-dom 和 b2AR/b1CTmin-dom 的 AAV6 将被注射到双 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）。目标 4：体内测试表达不含 C 尾 CaMKII 激活结构域的 b1AR 突变体的小鼠是否会发生 MI 后不良心脏重塑。我们将生成基因靶向小鼠，敲入突变的 b1AR 受体，但不带 C 尾 CaMKII 激活结构域 (b1AR/b2CTmin-dom)，以确定体内心脏表型，以响应病理刺激，如慢性儿茶酚胺给药、压力超负荷和心肌梗死（与 Oliver Smithies 博士合作）。 我们预计拟议的研究将确定 bAR 途径导致不良心脏重塑的新分子机制。此外，拟议的研究将产生治疗人类心脏肥大和心力衰竭的新目标。