Mechanisms of Maladaptation in Heart Failure

心力衰竭适应不良的机制

• 批准号: 8185680
• 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/8185680
• 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. PUBLIC HEALTH RELEVANCE: b-Adrenergic receptors (bARs) play a critical role in the regulation of cardiac function. While acute stimulation of b1ARs augments cardiac contractility, chronic stimulation appears to exert an adverse effect on long-term cardiac remodeling. The adverse remodeling induced by b1ARs is associated with activation of calcium/calmodulin-dependent protein kinase II (CaMKII), which appears to play a key pathogenic role in the progression of heart failure. Our investigation into the molecular mechanisms of b1AR-mediated CaMKII activation will potentially lead to the discovery of novel ligands that selectively activate a subset of cellular pathways that promote beneficial heart remodeling and recovery from the syndrome of heart failure.

性状（由申请方提供）：b-肾上腺素能受体（bAR）是G蛋白偶联受体，在心脏功能调节中发挥关键作用。已知b1 AR和b2 AR两者都与Gs蛋白偶联以激活腺苷酸环化酶并增加cAMP水平，然而也能够与Gi偶联的b2 AR可以促进细胞存活信号。相反，b1 AR的激活似乎对心肌梗死（MI）后心脏重塑产生主要不利影响。我的实验室最近证明，b1 AR增强了MI后的不良重塑，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，通过b1 AR激活CaMKII需要受体的羧基末端尾（C-尾）和称为b-抑制蛋白的多功能调节分子。然而，仍然存在一些问题：如果b1 AR和b2 AR都结合b-抑制蛋白，为什么只有b1 AR激活CaMKII？b1 AR的选择性激活是否是心肌梗死或压力超负荷后诱导不良心脏重塑和心力衰竭的机制？ 我们提出的假设，一个独特的区域的羧基末端尾（C-尾）的b1 AR是负责CaMKII激活和不利的心肌梗死后心脏重构和压力超负荷，并提出以下具体目标：目的1：确定最小区域的C-尾的b1 AR必要的CaMKII激活。我们将通过在b1 AR和b2 AR的同源C-尾区域之间进行氨基酸“交换”实验来鉴定b1 AR的C-尾内激活CaMKII所必需的最小氨基酸残基。目的2：证明b1 AR C-尾的最小结构域在兔心肌细胞中过表达时导致CaMK II激活。我们将创建过表达bAR交换突变体的腺相关病毒（AAV 6）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、细胞收缩性和响应于bAR刺激的Ca 2+瞬变（与Don Bers博士合作）。目标3：证明b1 AR的最小C-尾结构域足以导致MI后不良心脏重塑和随后的心脏功能下降。将含有b1 AR/b2CTmin-dom和b2 AR/b1 CTmin-dom的AAV 6注射到双b1 AR/b2 AR KO小鼠的心脏中，随后进行全面的生物化学和生理学表型分析（与Wally Koch博士合作）。目标4：在体内检测表达b1 AR突变体而无C-尾CaMKII激活结构域的小鼠是否发生MI后不良心脏重塑。我们将产生基因靶向小鼠，敲入突变的b1 AR受体，而不敲入C-尾CaMKII激活结构域（b1 AR/b2 CTmin-dom），以确定体内心脏表型对病理刺激的反应，如慢性儿茶酚胺给药、压力超负荷和心肌梗死（与奥利弗史密斯博士合作）。 我们预计，拟议的调查将确定新的分子机制，bAR途径导致不利的心脏重塑。此外，拟议的研究将导致治疗人类心脏肥大和心力衰竭的新靶点。 公共卫生相关性：b-肾上腺素能受体（bAR）在心脏功能调节中发挥关键作用。虽然急性刺激b1 AR增强心肌收缩力，但慢性刺激似乎对长期心脏重塑产生不利影响。由b1 AR诱导的不良重构与钙/钙调素依赖性蛋白激酶II（CaMKII）的激活相关，CaMKII似乎在心力衰竭的进展中发挥关键的致病作用。我们对b1 AR介导的CaMKII激活的分子机制的研究将可能导致发现新的配体，这些配体选择性地激活促进有益的心脏重塑和从心力衰竭综合征中恢复的细胞通路的子集。