REGULATION OF ANF AND BNP IN CARDIAC HYPERTROPHY

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Atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) have vasodepressor effects, while in the kidney they have potent natriuretic and diuretic activity. Although they have similar biological activity, each hormone has distinct features, most importantly differences in DNA regulatory sequences involved in gene expression. In adult hearts, the gene for ANF is preferentially expressed in the atria, and ventricular expression is less than 5% of the whole heart. In contrast, ventricular BNP mRNA represents greater than 70% of whole heart levels. ANF and BNP mRNA are augmented in the ventricle under various conditions of volume and pressure overload, including hypertension and heart failure. Given that the BNP gene is expressed primarily in the ventricles while the ratio of BNP to ANF is different in atria and ventricles there must be clear differences in the way ANF and BNP synthesis are regulated. The objective of this proposal is to define transcriptional and post-transcriptional mechanisms involved in differential regulation of the ANF and BNP genes during cardiac hypertrophy using 2 in vitro models of hypertrophy: adult feline cardiocytes stimulated to beat with isoproterenol and phenylephrine- or PMA-stimulated neonatal ventricular cardiocytes. Specifically, the hypotheses to be tested are that: 1) there are differences in the rates of transcription of the ANF and BNP genes, and cis-acting regulatory elements in the 5' flanking sequences (fS) of the BNP gene are distinct from those characterized for the ANF gene in neonatal cardiocytes; 2) during cardia hypertrophy, re-expression of the ANF and BNP genes involves cis-acting regulatory regions distinct from those involved in normal tissue-specific gene expression; 3) different trans-acting regulatory proteins affect ANF and BNP gene expression in normal vs. hypertrophied cardiac myocytes; and 4) BNP mRNA stability is regulated differently in normal and hypertrophied ventricles. These studies will employ cell culture models of hypertrophy, Northern blot of mRNA, transfection of chimeric promoter-reporter gene constructions, enzyme assays (luciferase and beta-galactosidase), polymerase chain reaction (PCR), DNA- and RNA-protein gel shift, and in vivo analysis of regulator elements (in vivo injection of DNA). Thus they will provide a detailed analysis of differences in ANF and BNP gene regulation and help us understand the relevance of each hormone in disease, such as hypertension and hypertrophy.