HEART FIBROBLAST AND COLLAGEN TYPE III REGULATION

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The development of pathological hypertrophy in the myocardium has been frequently associated with abnormal accumulation of extracellular matrix proteins (ECM) (mostly collagens) in the interstitium. The abnormal accumulation of fibrillar collagens results in an altered ventricular stiffness and can ultimately contribute to the development of myocardial failure. Cardiac collagens are comprised of types I and III and to a lesser extent of types IV and V. It has been demonstrated that during the early phases of cardiac pressure overload hypertrophy the ratio of type III to type I collagen increases. Results obtained from in vitro experiments performed with cardiac fibroblasts also suggest that III collagen gene expression is more responsive (vs. that of type I) to challenges derived from treating cells with either growth factors or mechanical stimulation. However, the molecular mechanisms by which collagen type III is regulated in the cardiac fibroblast are currently unknown. The overall aim of this proposal is to examine the regulation of the collagen type III gene by factors that are postulated to play an important role in directing extracellular matrix protein production in the myocardium. We propose to address the following questions: 1) Does transforming growth factor beta-1, angiotensin II (ANG II) and mechanical deformation result in an increase in type III collagen production and deposition in a culture of mouse cardiac fibroblasts? If so through what mechanisms? For this purpose, we would determine if stimulation of mouse cardiac fibroblast with these factors results in an increased expression of collagen type III mRNA and protein production using Northern blot and ELISA techniques. In order to determine the nature of the molecular mechanisms responsible for the increase in ECM protein production nuclear runoffs, mRNA stability and transcriptional (CAT-transfection) assays will be performed; 2) Does increased expression of collagen type III depend on the interaction between the angiotensin II receptor, ANG II, TGFbeta-1 and/or stretch? (a) Using binding assays we would explore if stimulation of mouse cardiac fibroblasts using the above described manipulations regulate the expression (number) of angiotensin II receptors. (b) Biochemical techniques would be used to examine if ANG II and/or stretch increases the levels of TGFbeta-1 gene expression and release. (c) Using the techniques described in Aim #1, we will examine if the simultaneous mechanical and growth factor stimulation of cardiac fibroblasts results in enhanced levels of collagen type III, and; 3) Does regulation of collagen type III gene expression by these factors depend on specific cis-acting elements contained within the promoter region of the gene? Utilizing transfection assays based on a CAT-collagen type III mouse promoter construct (and selective deletions) we would like to determine what are the specific cis-acting elements contained within the gene that might mediate the transcriptional activation by each of these different factors (TGFbeta-1, ANG II and stretch).