Previous studies show that many interventions including endurance running exercise, pressure overload, and diabetes are capable of up regulating, to varying degrees, the expression of the beta myosin heavy chain (MHC) gene in the rodent heat. While previous studies, based primarily on in vitro cell culture models, suggest that cardiac beta MHC gene expression is chiefly regulated via transcriptional processes involving the interaction of specific transacting factors with cis regulatory elements in the beta MHC promoter, relatively little information is available concerning both the expression of and interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart, particularly under experimental conditions in which the beta MHC gene is being regulated by various patho-physiological stimuli. The primary aim of this proposal is to examine the involvement of key transcriptiona/nuclear factors affecting beta MHC gene expression via interaction with cis-elements of the beta MHC promoter in the models of chronic endurance running exercise, pressure overload, and diabetes, including the role that elevated carbohydrate utilization plays in the absence of insulin. We will test the general hypothesis that each of these interventions mediates an increase in transcription activity of the beta MHC promoter which involves divergent regulatory factors and cis-elements. To test this working hypothesis, we will integrate the following approaches. 1) Study in vivo Beta MHC gene transcriptional rate using both direct measurements via nuclear run on assay, and indirect measurement via reporter gene assays following direct myocardial injection of chimeric plasmid DNA containing specific beta MHC promoter fragments linked to a reporter gene CAT (chloramphenicaol acetyl transferase). Included in these analyses will be mutated constructs at specific sites thought to be essential for transcription in a given experimental model. 2) Determine the expression of known transacting factors (such as TRS, RXR, TEF-1, CNBP), at the protein and mRNA levels and correlate their expression with beta MHC gene transcription. 3) use protein-DNA interactions (gel mobility shift assays) between cardiac nuclear extract and critical beta MHC promoter segments (I.e., betae1, betae2, betae3, C-rich, beta MHC TRE etc.) In order to detect alteration in binding under specific experimental conditions, and further characterize these interactions via competition studies. These approaches will collectively delineate the molecular mechanisms responsible for the regulation of beta MHC expression in the in vivo setting.
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