MOLECULAR/CELLULAR BIOLOGY OF CARDIOMYOCTE DEVELOPMENT

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The goal of our research program is to understand the morphogenesis of embryonic cardiac muscle in vivo, particularly the assembly of myofibrils in that tissue. The central focus of the project is the role of two myosin-binding proteins(MyBPs) termed MyBP-C and MyBP-H (C and H protein, respectively), which we hypothesize to be necessary for the lateral alignment of thick myofilaments in nascent A bands. In work accomplished over the past 5 years, we have: a) cloned, sequenced and identified the myosin binding domains of both proteins and pinpointed the region of MyBP-C both necessary and sufficient for targeting of this protein to the A band; b) shown that a truncation mutant lacking the myosin binding domain acts as a dominant negative for myofibril assembly; c) demonstrated that MyBP-C is expressed significantly after sarcomeric myosin in the embryonic heart and in cultured skeletal myotubes, and shown that the appearance of MyBP-C correlates with the emergence of cross-striations in those cells; d) prepared replication-defective, retroviral vectors which have been used to trace the lineage of cardiomyocytes, precursors of the coronary vessels, and the cardiac conduction system; e) proposed plausible models to explain the morphogenesis of the myocardium, coronary vessels and the peripheral conduction in vivo; f) constructed modified versions of these retroviral vectors for gene delivery to the embryonic myocardium or to coronary precursors; g) used those vectors to prove that FGF signaling is required for early myocyte proliferation but not for myocyte differentiation; h) created cadherin constructs which disrupt adherent junctions; i) isolated a full-length mouse genomic clone encoding MyBP-H and prepared the vector needed for homologous recombination in embryonic stem (ES) cells and subsequent mouse gene knockout. Based on these accomplishments we now propose two sets of in vivo studies, one in embryonic chickens and the other in transgenic mice, to test the functions of MyBP-C and -H in the embryonic heart. 1. Using replication-deficient, retroviral vectors, we will target recombinant forms - both wild-type and mutant - to the precardiac mesoderm or to the forming myocardium at selected stages of embryonic chick development. Our goal will be to test whether: a) precocious expression of wild-type MyBP-C; b) anti-sense suppression of wild-type MyBP-C; c) or the expression of mutant forms of MyBP-C alter myofibril assembly and cardiac morphogenesis in ovo. Subsequent experiments will focus on comparable analyses of MyBP-H. 2. We will prepare transgenic mouse knockouts for skeletal MyBP-H, cardiac MyBP-H and cardiac MyBP-C. Our goal will be to analyze the development and function of cardiac and skeletal muscle in mice either heterozygous or null for these genes.