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.
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.
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