Four New Muscle Myosin <scp>II</scp> Binding Properties of Titin: Implications for Myofibrillogenesis

Before muscle specific proteins are expressed in precursor cells of cardiac muscle, only the nonmuscle myosin II isoform of myosin II is present in the cells. It provides contractile force for the cell divisions that occur, and it is present together with actin in fibers in the cytoplasm. When expression of muscle proteins occurs, both muscle and nonmuscle isoforms of myosin II co-exist in the same cells. Nonmuscle myosin II isoforms are organized in minisarcomeric bands in premyofibrils with alternating bands of muscle-specific alpha-actinin. In the same cell, muscle myosin II is present in mature myofibrils that are unassociated with nonmuscle myosin II isoforms. There is an intermediate group of fibrils, i.e., nascent myofibrils, in which both isoforms of myosin II are present in the cardiomyocytes. Since muscle and nonmuscle myosin II form copolymers in solution, we asked whether any of the myofibril proteins that interact with muscle myosin could prevent nonmuscle and muscle myosin from copolymerizing, thus explaining the absence of nonmuscle myosin II in mature myofibrils. We examined the effects of two myosin-binding proteins involved in cardiomyopathies: C-Protein (myosin binding protein or MyBP-C) and titin, on the filament forming properties of the two different types of myosin II. In filament forming conditions, neither C-protein nor titin bind nonmuscle myosin II. Both C-protein and titin, as expected, bind muscle myosin II. C-Protein does not inhibit the copolymerization of the two different types of myosin IIs. Co-polymerization of nonmuscle and muscle myosin IIs is prevented in the presence of either full length titin isolated from cardiac muscles or a bacterially expressed titin peptide containing just one myosin-binding region. In the presence of titin, paracrystals of Light Meromyosin (LMM) change their normal 14 nm periodicities in pure LMM paracrystals to 42 nm in copolymerization of LMM and full-length titin. Our experiments suggest four novel roles of titin in myofibrillogenesis: one: prevention of copolymerization of nonmuscle myosin II and muscle myosin II in mature myofibrils, and two: formation of thick filaments with linear cross-bridges separated by 42 nm repeats in the absence of C-Protein. Two additional properties of titin in myofibrillogenesis would be titin's capture and transport of copolymers of myosin filaments to premyofibrils to form nascent myofibrils, and the start of release of nonmuscle myosin II from copolymeric myosin II filaments resulting in mature myofibrils lacking nonmuscle myosins.