Atrial natriuretic factor (ANF) is a potent endogenous anti-hypertensive hormone derived from cardiac myocytes. During pressure-overload, ANF expression is strongly induced and the cardiac myocytes increase in size, suggesting a close mechanistic link between ANF gene expression and cardiac growth processes. Since uncontrolled cardiac growth can lead to the pathological condition of cardiac hypertrophy, a further understanding of ANF gene expression may yield a broader understanding of this process potentially leading to new therapeutic strategies. The novel observation that forms the basis of this proposal is thai in creased ANF production occurs during long-term (24-72 hour) electrical pacing of contractions in cultured neonatal rat ventricular and atrial myocytes. Cultured myocytes represent the most appropriate model system for further study of this since factors such as contraction rate, hormonal and nutrient environment, and mechanical stress (via the use of deformable culture substrates) can be precisely controlled. We hypothesize that the calcium transients that drive the contractions also activate ANF gene expression via the activation of calcium-sensitive protein kinases that ultimately regulate ANF gene transcription. Furthermore, since contracting myocytes exert tension on themselves and neighboring cells, pace-induced ANF expression may be closely related to stretch-induced ANF expression which has also been demonstrated to occur in cultured myocytes.
The Specific Aims are: 1) To identify the cis-elements and trans-acting factors that confer pace- inducibility to ANF expression. 2) To define the second messenger system linking pacing to ANF expression. 3) To characterize the relationship between pace- and stretch-induced ANF expression. Transient transfection techniques utilizing ANF enhancer:reporter gene constructs will be used to examine pacing-induced ANF transcription, in vitro kinase assays will be performed on cell extracts to test for pacing activation of protein kinases, and a device with which the myocytes can be simultaneously paced and stretched will be utilized in comparisons of these stimuli. We will also consider the potential role of autocrine and paracrine factors in pace-induced ANF expression. The proposed experiments will potentially elucidate mechanisms by which contractile activity may modulate cardiac gene expression.
