A five-year research program is proposed to examine the intracellular mechanisms regulating in vivo collagen metabolism in the normal and hypertrophying rat left ventricular myocardium, as well as in primary cultures of neonatal rat cardiac fibroblasts. The proposed experiments focus upon one particular aspect of collagen metabolism; namely, the intracellular degeneration of newly synthesized procollagens by rat cardiac fibroblasts both in vivo and in cell culture. The intracellular mechanisms regulating this important process are not fully understood, and the potential for pharmacological modulation of the """"""""targeting"""""""" of newly synthesized procollagen polypeptides for intracellular destruction would afford another means of reducing collagen accumulation in a variety of cardiovascular diseases associated with interstitial fibrosis. Specific experiments are outlined to estimate in vivo rates of collagen biosynthesis, accumulation, and degradation during normal physiological growth of rat left ventricular myocardium; and during the development of thyroxine-induced LV hypertrophy, and LV hypertrophy following abdominal aortic coarctation. Specific monoclonal antibody probes will also be developed for use in studies of procollagen polypeptide metabolism in cultured neonatal rat cardiac fibroblasts. These sequence- specific monoclonal antibodies (directed against the proaminopeptide domains of rat Type I and Type III procollagen polypeptides) will be used in specific radioimmunoassays for the quantitative analysis of intracellular and secreted procollagens, as well as in the production of immunoadsorbents for the quantitative isolation of newly synthesized and secreted procollagens from cultured cells and media. In addition, primary cultures of neonatal rat cardiac fibroblasts will be characterized with respect to procollagen biosynthesis, secretion, and intracellular degradation during """"""""normal"""""""" proliferation in culture, and during a variety of pharmacological interventions known to affect collagen metabolism in the cell culture systems. Finally, the intracellular mechanisms regulating cyclic AMP-dependent """"""""targeting"""""""" of newly synthesized procollagens for transport and degradation within fibroblastic lysosomes will be examined. If successful, these studies should provide new and important fundamental information regarding collagen turnover in the cardiac interstitium, as well as provide useful experimental tools for other investigators in this research program interested in cardiac interstitial collagen biochemistry, cellular and molecular biology.
