A new cardiotonic agent, (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), has been developed and screened for its ability to bind to cardiac troponin C. In perfused hearts, low concentrations of 0.03 or 0.1 mumol/L Levosimendan increased +dP/dt, but did not affect the speed of relaxation and produced only a slight increase in spontaneous heart rate in the hearts perfused with 0.1 mumol/L of the drug. In these same hearts, perfusion with 0.03 mumol/L Levosimendan did not alter the 32P incorporation into troponin I or C protein, whereas a slight but significant increase was noted for phospholamban, with no detectable change in tissue cAMP levels. Administration of 0.1 or 0.3 mumol/L Levosimendan significantly increased myocardial cAMP levels as well as the phosphorylation of phospholamban, troponin I, and C protein. Levosimendan (0.03 to 10 mumol/L) reversibly increased force generated by detergent-extracted fiber bundles over a range of submaximally activating free Ca2+ concentrations with no significant effect on maximum force or on Ca2+ binding to myofilament troponin C. There was no direct effect of Levosimendan on Ca2+ uptake by vesicles of sarcoplasmic reticulum (SR). In contrast, under conditions optimal for cAMP-dependent phosphorylation, Levosimendan slightly but significantly lowered the concentration of Ca2+, yielding half-maximal uptake rates by the SR vesicles. Our results indicate that at low concentrations Levosimendan acts preferably as a Ca2+ sensitizer, whereas at higher concentrations its action as a phosphodiesterase inhibitor contributes to the positive inotropic effect.

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 ...