According to Starling's law of the heart, the force of contraction during the ejection of blood is a function of the end-diastolic volume. To seek the molecular explanation of this effect, a study was made of the effects of length on Ca2+ sensitivity during tension development by isolated demembranated cardiac muscle in which the cardiac form of troponin C was substituted with skeletal troponin C. The results of troponin C exchange were compared at sarcomere lengths of 1.9 and 2.4 micrometers. Enhancement of the myocardial performance at the stretched length was greatly suppressed with the skeletal troponin C compared with the cardiac troponin C. Thus the troponin C subunit of the troponin complex that regulates the activation of actin filaments has intrinsic molecular properties that influence the length-induced autoregulation of myocardial performance and may be a basis for Starling's law of the heart.

SUMMARY1. Skinned fibre preparations of right ventricular trabeculae, psoas and soleus muscles from hamster and rabbit were activated by Ca2' and the length dependencies of their pCa (-log [Ca2+])-force relationships were compared.2. Ca2+ sensitivity of the myocardium was higher at 22-24,tm 7. Using mutant CBM2A, in which site 2 was inactivated, the activation of cardiac muscle by both Ca2+ and Sr2+ ions was completely blocked. This is the expected N1S 8789 J. GULATI, E. SONNENBLICK AND A. BABU result, since both regulatory sites were now inactive, regulatory site 1 being normally inactive in cardiac muscle. Also, when this mutant was loaded into a moderately extracted fibre, the length dependence remained at the reduced level observed after partial TnC extraction. This shows that the modified state of the thin filament following such partial extraction occurs in response to the loss of active TnC rather than the vacancy per se in the thin filament.8. The results of this study firmly indicate a direct role of TnC in the modified length dependence of cbardiac function when compared with that in skeletal muscle, and further, provide direct evidence that site 1 of the N-terminus of TnC is a key component of the length sensing instrument in the myocardium. This novel function of cardiac TnC in the length-sensing mechanism is additional to its classical role as the Ca21 switch.

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Contraction of isolated, intact frog muscle fibers under increasing tonicity of the external solution was studied by adding (i) effectively impermeant sodium chloride and sucrose and (ii) permeant potassium chloride. Force of isometric contraction decreased as a function of tonicity, independent of the permeability of the solute. In contrast, cell volume changed with tonicity in impermeant solutes and was constant with potassium chloride. The results are evidence that ionic strength in the sarcoplasm directly influences the contraction mechanism. Also, the findings show that force development is unaffected by changes in fiber volume, suggesting that the force per cross-bridge is constant at different distances between the thin and myofilaments. Finally, in light of the length-force relation, the results support the idea that cross-bridges are independent force generators.

Effects of intracellular ionic strength on the isotonic contraction properties of both intact fibers and skinned fibers give insights into the cross-bridge mechanism, but presently there is fundamental disagreement in the results on the two fiber preparations. This paper, which studies the effects on contraction of varying the osmotic pressure of the bathing medium with impermeant and permeant solutes, explains the above controversy and establishes the physiological significance of the previous results on skinned fibers. Fast-twitch fibers, isolated singly from tibialis and semitendinosus muscles of frogs, were activated by a temperature-jump technique in hyperosmotic solutions with either 100 or 150 mM sucrose (impermeant), or 50 or 75 mM KCl (permeant). Intracellular ionic strength was expected to rise in these solutions from the standard value of approximately 190 to 265 mM. Cell volume and the speed of unloaded shortening both decreased with sucrose and were constant with KCl. On the other hand, isometric force decreased equally with equiosmolar addition of either solute; this is additional evidence that contractile force decreases with ionic strength and is independent of fiber volume. Therefore, for the main cross-bridges, force per bridge is constant with changes in the lateral separation between the myofilaments. The next finding, that at a fixed cell volume the contraction speed is constant with KCl, provides clear evidence in intact fibers that the intrinsic speed of shortening is insensitive to increased ionic strength. The data with KCl are in agreement with the results on skinned fibers. The results suggest that in the cross-bridge kinetics in vivo the rate-limiting step is different for force than that for shortening. On the other hand, the decrease in speed with sucrose is associated with the shrinkage in cell volume, and is explained by the possibility of an increased internal load. A major fraction of the internal load may arise from unusual interactions between the sliding filaments; these interactions are enhanced in the fibers compressed with sucrose, but this does not affect the intrinsic kinetics of the main cross-bridges.

This study describes the effects of exchanging native cardiac troponin C (CTnC) from the right ventricular muscle of Syrian hamster for purified skeletal (S) TnC from fast twitch muscles in triggering cardiac contraction.Ca2+ sensitivity of the myocardium became decreased with STnC to 62% of the original value with CTnC. Furthermore, the slope of the pCa-force curve of cardiac muscle was found to be increased with STnC. The results show that the TnC moiety, as part of the switching mechanism during activation, also regulates thin-filament cooperativity in muscle. Modifications in both the Ca2+ sensitivity and cooperativity are associated with alterations in the primary structure of TnC.

Force development by skinned frog semitendinosus fibers was studied at various levels of lateral compression to compare the results with intact fibers and to evaluate the limits on cross-bridge movements during isometric contraction. The skinned fibers were compressed osmotically using a high molecular weight polymer, dextran T500. Ca-activated force remained constant down to 58% of the fiber width (w0) after skinning, corresponding to a nearly twofold change in separation between the thin and thick filaments in the myofilament lattice. This agrees with the earlier result on intact fibers, and gives additional evidence that the cross-bridge mechanism for force generation is relatively insensitive to large changes in interfilament separation. Further compression, below 0.58 w0, produced a sharp drop in force, and the force was practically zero at a fiber width of 50%. The effect at high compression was the same at all pCa's, which indicates that the Ca sensitivity of the myofilaments is unaffected by radial compression. The stiffness of the fiber remained high in rigor in the presence of dextran, which indicates that the rigor cross-bridge attachment is not inhibited, and actually may be improved, with decreases in the interfilament space. Also, the drop in active force with the highest compression was similar when the compressed fibers were put in rigor before contraction, which suggests that the force drop also was not due to a hindrance to cross-bridge attachment. The results appear to exclude large motions such as tilting and rocking of the bridge as a rigid molecule, but suggest that at least some molecular movement is essential for force development; they also raise the possibility that there is a critical interfilament separation in the fiber, below which the cross-bridge cannot function.

This study addresses a long-standing controversy on the effects of the degree of activation on cross-bridge kinetics in vivo, by utilizing isolated intact and skinned fiber preparations . Steady force levels ranging from 0.1 to 0 .76 Po were achieved at 0°C with temperature-step stimulation of intact fibers by varying the amount of caffeine in the bathing medium. The speed of unloaded shortening (by slack test) was found to be practically constant, which suggests that intracellular Ca" in the intact preparation has relatively little effect on isotonic shortening . Along with the results on tetanically stimulated fibers (force, Po), we observed a minor but significant trend for the speed to decline with lowered force levels . This trend is explained by the presence of a constant internal load equaling -1% Po. The effect of Ca" on the shortening behavior of skinned fibers was examined at 0 and 10'C. At 0'C, there was practically no effect of Ca" on the shortening response in slack tests. At 10°C, there was also no Ca" effect during the first activation cycle, but in subsequent cycles the speed of shortening was reduced during partial activation, which indicates that there were permanent changes in the fiber properties under these experimental conditions. The latter result could be explained if the internal load had increased to^-5% Po in the modified skinned fiber (compared with 1 % Po in intact fiber) . These findings show that isotonic contraction of frog fibers is intrinsically unaffected by the variations in intracellular Ca" that modulated the force over a nearly complete range. The results provide support for the idea that Ca" influences the force development in vivo by on-off switching mechanisms .