The long-term objective is to understand the molecular mechanisms that initiate and maintain the hypertrophic response of the myocardium. Of particular interest are the regulatory events that trigger the altered expression of gene networks associated with the hypertrophic response. To do this, an understanding of the events that govern cardiac gene expression is required. The main focus of this grant remains to understand the molecular mechanisms that regulate gene expression in the heart. A second focus is to determine if helix-loop-helix (HLH) proteins participate in the establishment of pathologically induced cardiac hypertrophy.
The specific aims are to: 1) Characterize the proximal alpha-MHC enhancer/promoter and identify cardiac-specific regulatory elements therein; 2) Isolate and characterize of a clone encoding alpha- MHC binding factor-2 (BF-2); and 3) Establish a link between mRNA levels of particular HLH proteins and cardiac-hypertrophy. Promoter fusion and site-directed mutagenesis experiments are proposed to identify elements regulating cardiac-specific expression. Activity will be assessed in primary neonatal cardiomyocytes, in the adult animal, in a cardiac cell line and in skeletal muscle cells. In vivo and in vitro techniques will be used to identify critical regulatory factors. DNA binding activity of these factors, and mRNA levels where possible, will be examined during development and establishment of hypertrophy. The DNA binding site for BF-2 will be used to isolate a clone encoding BF-2 from a neonatal rat heart cDNA expression library. The BF-2 clone will be characterized and used in functional studies to determine the effect of over and under expression of BF-2 on gene expression in the heart. Cardiac HLH clones will be used to determine the levels of several HLH mRNAs in the normal and hypertrophied adult heart. This information will be correlated with the presence of binding sites in genes which are regulated during cardiac-hypertrophy.
