Mechanisms of Maladaptation in Heart Failure

心力衰竭适应不良的机制

• 批准号: 8469543
• 负责人: Howard A Rockman
• 金额: $ 37.37万
• 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/8469543
• 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(申请人提供)：B-肾上腺素能受体(BARS)是G蛋白偶联受体，在心脏功能调节中起关键作用。已知b1ARs和b2ARs都能与Gs蛋白偶联以激活腺苷环化酶并增加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)后的心脏重构产生不利影响。我的实验室最近证实，b1ARs增强了心肌梗死后不利的重塑，导致心功能下降，这与钙/钙调蛋白依赖的蛋白激酶II(CaMKII)的激活有关。我们进一步证明，由b1AR激活CaMKII需要受体的羧基末端尾巴(C-Tail)和一个名为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都与b-arresin结合，为什么只有b1AR激活CaMKII？这种选择性激活b1AR是诱导心肌梗死或压力超负荷后不利心脏重构和心力衰竭的机制吗？我们提出的假设是，b1AR的羧基末端尾部的一个独特区域负责CaMKII的激活和心肌梗死后的不利心脏重构和压力超负荷，并提出以下具体目标：目的1：确定CaMKII激活所需的b1AR的C-尾部的最小区域。我们将通过在b1AR和B2AR的同源C-尾区之间进行氨基酸“交换”实验，确定b1AR C-尾中激活CaMKII所需的最小氨基酸残基。目的：证明b1AR C-Tail的最小结构域在兔心肌细胞中过表达时可导致CaMKII的激活。我们将创建腺相关病毒(AAV6)结构，过表达bar交换突变体，将这些结构感染到兔心肌细胞，并测量CaMKII活性、下游通路激活、细胞收缩能力和钙离子瞬变对bar刺激的响应(与Don Bers博士合作)。目的3：证明b1AR的最小C-尾区足以导致心肌梗死后心脏的不良重构和随后的心功能下降。将含有b1AR/b2CTmin和B2AR/b1CTmin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鉴定(与Wally Koch博士合作)。目的：在体内测试表达b1AR突变体而不表达C-ail CaMKII激活域的小鼠是否发生心肌梗死后不利的心脏重构。我们将产生敲入突变的b1AR受体而没有C-ail CaMKII激活结构域(b1AR/b2CTmin-dom)的基因靶向小鼠，以确定在慢性儿茶酚胺给药、压力超负荷和心肌梗死等病理刺激下的体内心脏表型(与Oliver Smithies博士合作)。我们预计，拟议的研究将确定BAR通路导致不利心脏重构的新的分子机制。此外，拟议的研究将导致治疗人类心肌肥大和心力衰竭的新靶点。