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We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

We compared sarcomere length (SL) dependence of the Ca2+‐force relation of detergent‐extracted bundles of fibres dissected from the left ventricle of wild‐type (WT) and transgenic mouse hearts expressing slow skeletal troponin I (ssTnI‐TG). Fibre bundles from the hearts of the ssTnI‐TG demonstrated a complete replacement of the cardiac troponin I (cTnI) by ssTnI. Compared to WT controls, ssTnI‐TG fibre bundles were more sensitive to Ca2+ at both short SL (1.9 ± 0.1 μm) and long SL (2.3 ± 0.1 μm). However, compared to WT controls, the increase in Ca2+ sensitivity (change in half‐maximally activating free Ca2+; ΔEC50) associated with the increase in SL was significantly blunted in the ssTnI‐TG myofilaments. Agents that sensitize the myofilaments to Ca2+ by promoting the actin‐myosin reaction (EMD 57033 and CGP‐48506) significantly reduced the length‐dependent ΔEC50 for Ca2+ activation, when SL in WT myofilaments was increased from 1.9 to 2.3 μm. Exposure of myofilaments to calmidazolium (CDZ), which binds to cTnC and increases its affinity for Ca2+, sensitized force developed by WT myofilaments to Ca2+ at SL 1.9 μm and desensitized the WT myofilaments at SL 2.3 μm. There were no significant effects of CDZ on ssTnI‐TG myofilaments at either SL. Our results indicate that length‐dependent Ca2+ activation is modified by specific changes in thin filament proteins and by agents that promote the actin‐myosin interaction. Thus, these in vitro results provide a basis for using these models to test the relative significance of the length dependence of activation in situ.

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 filament 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 -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 ...