This work is focused on the clinical problem of myocardial hypertrophy, specifically the molecular mechanisms which regulate this process. A new model system was developed to study hypertrophy, employing neonatal heart myocytes in serum-free culture. The novel observation was made that stimulation of the alpha 1-adrenergic receptor on these cells by the catecholamine norepinephrine induces cell enlargement or hypertrophy, without DNA synthesis. The alpha 1-adrenergic receptor is the first receptor shown to regulate cardiac myocyte hypertrophy. Recent work has shown that the alpha 1 receptor regulates mRNA expression during hypertrophy. In particular, alpha 1 stimulation induces two contractile protein iso-mRNAs characteristic of early cardiac development and pressure-load hypertrophy in vivo, skeletal alpha-actin iso-mRNA and B-myosin heavy chain iso-mRNA. This alpha 1 receptor effect is selective and is mediated at the level of iso-gene transcription. Thus, the critical question is the mechanism for induction of transcription by the alpha 1 receptor. The present experiments will test the hypothesis that the skeletal alpha- actin iso-gene and the B-myosin heavy chain iso-gene contain one or more alpha 1 response elements, i.e., DNA sequences required specifically for transcription of these genes in response to alpha 1-adrenergic stimulation. Two complementary experimental approaches for functional identification alpha 1 response elements are proposed: (1) transfection of hybrid genes in a transient assay system; and (2) DNase I hypersensitivity mapping. The necessary cloned genes are available to begin these experiments and preliminary studies provide evidence that the work is feasible. The long-range goal is to define the intracellular pathway from the alpha 1 receptor at the cell surface to gene transcription. The overall hypothesis is that receptor stimulation activates a pre-existing protein or proteins that binds to the alpha 1 response element in a transcriptional complex with RNA polymerase II. Identification of an alpha 1 response element or elements in the present work will provide the foundation for subsequent identification of the protein or proteins.
