Exchange of - for -Tropomyosin in Hearts of Transgenic Mice Induces Changes in Thin Filament Response to Ca, Strong Cross-bridge Binding, and Protein Phosphorylation

Kimberly A. Palmiter, Yoshimi Kitada, Mariappan Muthuchamy, David F. Wieczorek, R. John Solaro
1996 Journal of Biological Chemistry  
Despite its potential as a key determinant of the functional state of striated muscle, the impact of tropomyosin (Tm) isoform switching on mammalian myofilament activation and regulation in the intact lattice remains unclear. Using a transgenic approach to specifically exchange ␤-Tm for the native ␣-Tm in mouse hearts, we have been able to uncover novel functions of Tm isoform switching in the heart. The myofilaments containing ␤-Tm demonstrated an increase in the activation of the thin
more » ... by strongly bound cross-bridges, an increase in Ca 2؉ sensitivity of steady state force, and a decrease in the rightward shift of the Ca 2؉ -force relation induced by cAMP-dependent phosphorylation. Our results are the first to demonstrate the specific effects of Tm isoform switching on mammalian thin filament activation in the intact lattice and suggest an important role for Tm in modulation of myofilament activity by phosphorylation of troponin. The ability of myosin heads to react with actin in heart muscle occurs with a transition of the thin filament from an "off" to an "on" state that depends on complex alterations involving the tropomyosin (Tm) 1 molecule (for reviews see Refs. 1 and 2). These alterations include possible steric effects associated with changes in the position of Tm on the thin filament, as well as allosteric and cooperative effects associated with Tminduced changes in actin structure and reactivity with myosin (3-5). The steric and allosteric/cooperative alterations involving Tm are triggered by Ca 2ϩ binding to TnC, but also depend on myosin head binding (6). Ca 2ϩ binding to TnC promotes interactions with other Tn components, TnI (2) , an inhibitory protein that binds to actin, as well as TnT, a TnI-and Tmbinding protein. The steric model of activation (3) hypothesizes that Ca 2ϩ -TnC-induced movement of Tm or possibly TnI (2) reverses the off state by releasing actin sites for reaction with myosin. The allosteric model (4, 5) proposes that Ca 2ϩ -TnC itself cannot activate the thin filament, but acts as a co-factor shifting the equilibrium between off and on states of Tm such that strongly bound cross-bridges more easily activate the thin filament. Although recent considerations indicate that activation may involve both processes (2), the relative role of the steric and allosteric/cooperative mechanisms in turning on the activity of striated muscle remains unclear. Our perception of the role of Tm in the regulation of striated muscle, as well as it's structure/function relations, has come from a variety of approaches. These include x-ray diffraction of muscle preparations (7) and crystals (8, 9), reconstructions from electron micrographs (9, 10), and reconstitution studies of soluble systems with Tm (11-13), Tm peptides (14) , and mutants of Tm (15). In some cases, inferences regarding structure/ function relations have been made from comparisons of muscle fibers containing isoforms of Tm (16). However, interpretation of these studies is difficult in that there are multiple changes in myofilament proteins that occur along with the natural variations in Tm. A clearer understanding of the structure/function relations of Tm has been hampered by an apparent lack of methods for reversibly extracting Tm from the myofilament lattice in a force-generating system, as has proved so successful in the case of Tn components such as TnC and TnI (17). Thus, issues such as the role of Tm domains, covalent modifications, and the functional significance of isoform switching of Tm in the intact force-generating lattice of vertebrate-striated muscle have remained poorly understood. Delineating the functional differences between Tm isoforms has taken on new significance with the identification of missense mutations in the Tm gene causally linked to familial hypertrophic cardiomyopathy (18 -20). In the present experiments, we used a transgenic approach to overcome the difficulties of exchanging Tm isoforms in the intact myofilament lattice. Transgenic mice, which overexpress ␤-Tm in the heart, were generated as described previously (21) . This has permitted us to test explicitly the effects of alterations in Tm isoforms on myofilament activation. Our results provide the first unambiguous evidence that myofilament activation by Ca 2ϩ and strong cross-bridges is affected by the population of Tm isoforms present in the heart. Our results also indicate a role for Tm in the modulation of myofilament activation by phosphorylation of Tn. Some of our results have been published in abstract form (22). EXPERIMENTAL PROCEDURES Materials-ATP (from equine muscle), creatine phosphate, creatine phosphokinase, cAMP, EGTA, MOPS, saponin, and the protease inhibitors pepstatin A, leupeptin, and phenylmethylsulfonyl fluoride were
doi:10.1074/jbc.271.20.11611 pmid:8662805 fatcat:yzztk2wykvh7hfiivlm5htwdwa