The long term objective of the proposed research is to understand how G- protein coupled receptors regulate growth of cardiac myocytes. The specific problem is to determine how alpha1-adrenergic receptor (alpha1-AdrR) stimulation effects increases in cell size and myofilament organization and induces expression of embryonic (ANF) and contractile protein (MLC-2) genes in neonatal rat cardiomyocytes. The alpha1-AdrR couples to the heterotrimeric G protein G-q to regulate phospholipase C (PLC). While G- q/PLC generated signals are required or induction of ANF expression, other pathways appear to be necessary, particularly for morphological responses. These pathways will be elucidated using biochemical assays, transient expression of inhibitory proteins along with reporter genes or reporter enzymes, microinjection of cell-impermeant antibodies and inhibitors into the cell, and expression of heterologous receptors. The first specific aim is to determine the extent to which G-alpha-q and PLC-generated mediators contribute to the genetic and morphologic responses to PE. The requirement for PLC will be assessed using PLC antibodies, G-alpha-q mutants and G- alpha-q gene knockouts, and that for protein kinase C (PKC) using inhibitor peptides and antisense oligonucleotides.
In Aim #2, involvement of beta- gamma subunits and the G-12 proteins will be examined using inhibitors of beta-gamma function and G-12 antibodies.
The third aim i s to examine the involvement of the Rho family small GTP-binding proteins (particularly Rho) in alpha-1-AdrR signalling, using application or microinjection of inhibitors (RhoGDI, Botulinum C3 transferase) and expression of dominant negative and activated mutants. Whether the activity of specific phospholipases or kinases are regulated by Rho function will also be determined.
The final aim i s to map the regions of G-q-linked receptors hat couple to effectors involved in hypertrophic responses. Wild-type and chimeric mAChR, including mAChR: alpha-1-AdrR chimeras, will be expressed transiently to define receptor determinants associated with various aspects of the hypertrophic response, and cloned into or delivered with adenoviral receptor vectors for high level expression needed to measure biochemical responses. The information obtained in these studies should contribute to an understanding of the basic mechanisms of G protein receptor function, regulation of gene expression, and the control of cardiac cell growth.
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