REGULATION OF RRNA SYNTHESIS DURING CARDIAC HYPERTROPHY

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Cardiac hypertrophy occurs normally in neonatal hearts and, in adult hearts, is induced by various physiological and pathological conditions such as ischemia, hypertension, and thyrotoxicosis. During this process the cardiac muscle cells increase is size but not in number. The increase in size is associated with an increased rate of protein synthesis. To increase the cellular capacity for protein synthesis, the cells increase the number of ribosomes by synthesizing more of them. In this process the rate of transcription of the ribosomal RNA genes (rDNA) is increased. We will measure both the amounts and activities of the components of the rDNA transcription system in order to determine how these factors are regulated during the process of hYPertrophy. Molecular and immunological probes will be developed for the analysis of the regulation of expression of the factors at both the mRNA and protein levels during cardiac hypertrophy. Constructs of cloned rat DNA carrying various mutations, such as deletion and substitution mutations, in both the known and putative cis-acting regulatory elements of the rDNA promoter, such as the UBF binding site, the SL-l binding sites, CRE, and AP-1 sites, will be used to map the cis-acting regulatory elements involved in the regulation of rDNA transcription during cellular hypertrophy. These experiments will involve the cotransfection of mutant and pseudo, wild-type promoters into cultured neonatal rat cardiac myocytes. In the initial series of experiments to determine the role of these cis-acting elements, the transfected cells will be exposed to hypertrophic stimuli, such as phorbol ester and forskolin. Once a specific element is identified, we will examine its role in the hypertrophic response exhibited by spontaneously contracting cells in comparison to KC1 arrested, non-contracting cells. The behavior of the mutant and pseudo, wild-type promoters will be assayed by oligonucleotide directed primer extension using RNA isolated from induced and noninduced transfected cells. These experiments should (1) demonstrate how the rRNA genes are regulated during cardiac hypertrophy and (2) lead to experiments that will allow the elucidation of the signal transduction pathway(s) that lead from the initiating stimulus to the process of cardiac hypertrophy.