The beta-adrenergic receptors (beta-ARs) are important modulators in the sympathetic control of various metabolic processes in the central and peripheral nervous system, including the heart. Several cardiovascular diseases, including cardiac hypertrophy and myocardial ischemia, are modulated by the desensitization of the beta-adrenergic receptor-adenylyl cyclase system in the heart. While much is known about the physiological responses of beta-adrenergic receptors to catecholamines, there is a limited understanding of the molecular mechanisms that regulate beta-AR subtype activation, expression, and desensitization in the normal and failing heart. The beta1-adrenergic receptor (beta1-AR) subtype is localized predominantly in the heart and is believed to be the principal modulator of the rate and force of cardiac contraction. My laboratory has cloned, sequenced, and expressed the rat and rhesus macaque beta1- adrenergic receptor genes. We have defined the beta1-AR transcriptional start sites, promoter elements, and polyadenylation sites used for expression in the C6 glioma cell line, and have identified potential enhancer and repressor regions that influence beta1-AR expression. In addition, we have defined the primary beta1-AR promoter in cardiac myocytic cells, and have determined that beta1-AR transcription in the heart occurs predominantly from downstream start sites. The overall focus of the research proposed in this application will be to precisely identify functional regulatory elements and factors involved in the regulation of the beta1-adrenergic receptor gene in myocytic cells and within the heart, and to analyze the transcriptional activation of the beta1-adrenergic receptor subtype during receptor desensitization and hormonal stimulation. We will employ site-directed mutagenesis, luciferase reporter systems, transfection in myocardial cells and established cell lines, DNA injection and delivery of promoter-luciferase recombinants into the rat heart, gel shift and DNase l footprinting analyses, and molecular cloning approaches to achieve the following Specific Aims: 1. Precise identification of beta1-adrenergic receptor promoter and enhancer elements activated during basal expression in C6 and primary' myocytic cells. 2. Identification of beta1-adrenergic receptor transactivator elements expressed during agonist-mediated receptor activation and desensitization and upon hormone stimulation. 3. Determination of exact beta1-adrenergic receptor transcriptional start sites utilized within the rat peripheral system, including the heart. 4. Determination of beta1-adrenergic receptor subtype promoter activity and enhancer responsiveness in vivo within the rat heart. 5. Identification and molecular cloning of the beta1-adrenergic receptor transactivator expressed in C6 and primary myocytic cells. This research will utilize unique molecular tools to provide insight in the molecular mechanisms of beta-AR regulation, and may provide the basic framework for understanding the role of abnormal beta-AR function in cardiovascular disease.
