Mechanisms of Maladaptation in Heart Failure

心力衰竭适应不良的机制

• 批准号: 8677941
• 负责人: Howard A Rockman
• 金额: $ 38.47万
• 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/8677941
• 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-肾上腺素能受体（BAR）是G蛋白偶联受体，在心脏功能的调节中起着至关重要的作用。众所周知，B1AR和B2AR都可以将其与GS蛋白相对，以激活腺苷酸环化酶并增加cAMP水平，但是也可以将其与GI相对的B2AR可以促进细胞存活信号。相反，B1AR的激活似乎主要对心肌后梗塞（MI）心脏重塑产生不利影响。我的实验室最近证明，B1ARS增强MI后重塑后，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的CAMKII激活需要受体的羧基末端尾巴（C-Tail），而多功能调节分子被称为B- arrestin。但是，仍然存在许多问题：如果B1AR和B2AR都绑定B-arrestin，为什么只有B1AR激活Camkii？这种选择性激活B1AR是诱导心脏重塑和心力衰竭后MI或压力超负荷的机制吗？ 我们提出了这样一个假设，即B1AR的羧基末端尾部（C-Tail）的独特区域负责CAMKII激活和不良心脏重塑后MI和压力超负荷，并提出了以下特定目的：目标1：确定B1AR C-tail所需的CAMKKII CAMKII必需的最低区域。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，从而确定B1AR的C尾中最小氨基酸残基。目标2：证明在兔心肌细胞中过表达时，确定的B1AR C-tail的最小结构域会导致CAMKII激活。我们将创建与腺相关的病毒（AAV6）构造，以过表达条形交换突变体，将这些构建体感染到兔子心肌细胞中，并测量CAMKII活性，下游途径激活，细胞收缩力和Ca2+瞬变，以响应与酒吧刺激（与Don Bers协作）。 AIM 3：证明B1AR的最小C尾域足以导致心脏重塑不良，并随后MI后心脏功能下降。含有B1AR/B2CTMIN-DOM和B2AR/B1CTMIN-DOM的AAV6将注入双B1AR/B2AR KO小鼠的心脏中，然后是全面的生化和生理表型（与Wally Koch博士合作）。 AIM 4：测试体体是否发生在没有C-尾CAMKII激活域的B1AR突变体的小鼠中，是否发生了MI后心脏重塑。 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). 我们预计拟议的研究将确定条条通路导致心脏不良重塑的新分子机制。此外，拟议的研究将导致新的靶标治疗人类心脏肥大和心力衰竭。