Allen M. Samarel - US grants

A five year research plan is presented that is targeted towards the general issue of the regulation of protein turnover during pre- and postnatal cardiac development. The overall aims of this study are to examine the relationship between measured rates of total and specific cardiac protein synthesis and degradation, relative mRNA levels, and net protein anabolism and catabolism during late fetal and early postnatal development of the rabbit heart. The techniques to be employed involve the use of continuous and flooding infusion methods of tracer amino acid administration to whole animals, isolation and amino acid analysis of specific cardiac proteins, methods to identify specific proteins and products of cell-free translation derived from cardiac mRNA, and immunochemical, biochemical, and morphological studies of cardiac lysosomal function during fetal and neonatal life. Of particular interest are the adaptive changes in myofibrillar protein synthesis and degradation during growth and remodeling of the left and right ventricles. The hypothesis to be tested is that rates of specific and total cardiac protein degradation as well as synthesis are vitally important to the regulation of cardiac protein mass during physiological adaptation to postnatal hemodynamic changes. Thus, these studies will provide a basis for comparing neonatal functional adaptation to the hypertrophic response of the adult heart to pathological conditions producing pressure or volume overload.

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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.

A five-year research plan is proposed with the long-term objective of elucidating the rate-limiting steps and routes of catabolism responsible for the selective degradation of specific contractile proteins in cardiac muscle cells. The proposed investigations at the cellular level are a logical extension of the whole animal research conducted during Years 01-05 of this project, which defined the relative importance of contractile protein synthesis and degradation during physiological growth of the left and right ventricles, and during experimental interventions leading to hypertrophy, regression from hypertrophy, and primary atrophy of the rabbit heart. In the proposed studies, the intracellular events responsible for contractile protein degradative processing will be examined in a well-characterized cell culture system that synthesizes and degrades pro- teins at rates comparable to those observed in vivo. Specific experiments are outlined to estimate rates of myosin heavy chain and light chain biosynthesis, accumulation and degradation in neonatal rat ventricular myocytes maintained under serum-free conditions. Particular emphasis will be placed on the effect of spontaneous contractile activity and contractile arrest on the rates of myosin subunit turnover. Pulse-chase biosynthetic labeling experiments will also be used to evaluate the kinetics of myosin subunit degradation, and to provide evidence for (or against) the existence of kinetic precursors of myofibrillar protein subunits. In vitro studies will also be performed to examine whether one form of spontaneous "damage" to the essential myosin light chain of ventricular muscle alters its susceptibility to proteolysis by purified proteases, and to degradation by the ubiquitin-mediated, ATP-dependent proteolytic system. Finally, a mass microinjection system will be used to assess the rate-limiting steps and routes of catabolism of native and damaged ventricular myosin light chain 1 incorporated into living cardiac muscle cells. If successful, these studies will provide new and fundamental information regarding the manner in which specific cardiac contractile proteins are targeted and processed for intracellular destruction. These studies will help to define the steps in a physiologically important, metabolic pathway that is intimately involved in the structural adaptation and remodeling of both the neonatal and adult heart.

A 5-year research program is outlined with the broad, long-term objective of elucidating the molecular mechanisms responsible for altered Ca2+ transporter gene expression in patients with cardiac hypertrophy and heart failure. There is now substantial evidence to indicate that expression levels of SERCA2 and NCX1, the major Ca2+ transporters in cardiac muscle, are profoundly altered in the failing human ventricular myocardium. These changes may result in reduced contractile function and increased susceptibility to ventricular arrhythmias. However, the underlying intracellular me4chanisms responsible for these changes, and the signal transduction pathways involved are only now being elucidated. Four specific aims are outlined to clarify these mechanisms in cultured cardiomyocytes, and related them to what may be occurring in hypertrophy and heart failure in experimental animals and man. First, previous work and preliminary data indicate a critical role of PKC activation in SERCA2 down-regulation during hypertrophy and heart failure. We will therefore use molecular biological techniques to over- express and down-regulate specific PKC isozymes to ascertain which PKC isozymes is responsible. Second, we will characterize the [Ca2+]i and Ras-dependent signaling pathways that regulate SERCA2 gene expression. Studies will focus on the non-receptor protein tyrosine kinase PYK2 that is activated by [Ca2+]i and PKC, and that may link G1- coupled receptor activation to the Ras-Raf-MEK-ERK protein kinase cascade. Third, preliminary data indicate that the 3' untranslated region of the SERCA2 mRNA regulates its stability in response to mechanical and neurohormonal stimuli that activate PKCs. Therefore, a series of experiments is outlined to define the cis-acting sequences and trans-acting factors that are involved. Fourth, we will test the hypothesis that activation of PKCs by either neurohormonal or mechanical stimuli (or their combination) up-regulates NXC1 mRNA and protein levels, and begin to analyze the signaling pathways responsible for these changes. The proposed experiments should substantially contribute to our understanding of the mechanisms responsible for altered Ca2+ transporter gene expression in heart failure Future therapeutic strategies targeted towards prevention or reversal of these changes require a thorough understanding of the responsible intracellular mechanisms.