description): The goal of this proposal is to define the signaling pathways involved in beta-adrenergic stimulation of hypertrophy and apoptosis. Akt phosphorylation and inactivation of GST 3 beta (and upregulation of GATA4) is proposed to initiate hypertrophic signaling via the beta AR. In cardiac apoptosis, Gs alpha is proposed to mediate beta AR induced apoptosis. In the first Specific Aim, the role of Akt-GSK3 beta in hypertrophy and apoptosis will be determined in adenoviral infected and transiently transfected cultures of neonatal rat cardiac myocytes. In the second Specific Aim, the role of the Akt-GSK3 beta pathway in GATA 4 regulation will be elucidated to understand regulation of this important transcription factor in cardiac hypertrophy. 1) Significance: Recent data demonstrating that beta-adrenergic receptor blockade improves ventricular systolic function and energetics suggest that prolonged stimulation of the sympathetic nervous system leads to pathological cardiac remodeling, hypertrophy, and myocardial dysfunction. The finding that chronic stimulation of in vitro cultures of neonatal cardiac myocytes (NVM) also leads to cardiac hypertrophy, as well as cell death, provides a well-controlled system to study the molecular mechanism by which hypertrophy and its converse, cardiac apoptosis, is initiated. The data also suggest that developmental differences in adrenergic response are present between the neonatal and adult rat. 2) Approach: As background for Specific Aim 1.1 and 1.2, the PI provides preliminary data demonstrating that isoproterenol stimulates hypertrophy and ANF transcriptional activation in NVM. These effects were due to activation of beta-1 receptors. Co-expression of ANF-luciferase and Gs alpha modestly activates ANF transcription; expression of G beta gamma gives rise to a larger and dose-dependent activation of ANF. An absence of suppressive effects of dominant negative Ras or DN-MEKK1 on transcriptional activation of ANF by isoproterenol suggested a lack of involvement of the Ras-MAPK pathways (or PKA see below). There do appear to be modest effects on PKA-mediated ANF Luc activity; it is possible that the low activity may depend on differences in the temporal patterns of expression of ANF-Luc in ISO-exposed vs. transfection of a PKA expression vector (Fig. 6A and see below). Activation of Akt by beta AR appears to occur via Ca signaling and Ca/calmodulin kinase. Constitutively active Akt stimulated ANF expression where dominant negative Akt inhibited isoproterenol-induced ANF transcription. In the proposed studies, adenoviral-mediated gene delivery of a variety of constructs is planned to determine the role of the Akt-GSK pathway in hypertrophy and apoptosis. Cardiac hypertrophy will be characterized at the protein and mRNA level of """"""""fetal genes"""""""" following virus infection and application of isoproterenol. Hypertrophic phenotypes will be examined to determine which phenotype is regulated by the Akt-GSK 3 beta pathway and if this phenotype corresponds to the cellular consequences of beta-adrenergic stimulation.
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