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.

We have measured the apparent Ca2+ sensitivities of force development in skinned cardiac trabeculae at different sarcome lengths together with shifts in troponin (Tn) T subunits on specimens from the same hearts and drawn insights into the pathogenesis of myocardial dysfunction in the diabetic rat. The Ca(2+)-force relations were measured at a long (2.4-microns) and a short (1.9-microns) sarcomere length. In disease, compared with the control condition, the apparent Ca2+ sensitivity was greatly diminished at a sarcomere length of 1.9 microns but not affected at all at the long length (2.4 microns). We also examined the alterations in contractile regulatory proteins TnT and TnI by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots. The TnI band was largely unperturbed, but major changes were discerned in TnT. The normal rat heart indicated two major bands (TnT1 and TnT2) and a faint third band (TnT3); in the diabetic rat heart, there was a significant shift in intensity from TnT1 to TnT3. Since myosin isozyme shifts also accompany diabetes in the rat, we used a prototypical hypothyroid rat as well to evaluate the myosin influence in the length-induced effects on Ca2+ sensitivity. Myosin shifts during hypothyroidism were unaccompanied by significant changes in TnT, and there were also no length-dependent modifications in Ca2+ sensitivity. The findings raise the possibility that diabetic Ca(2+)-sensitivity changes in the myocardium are coupled with TnT alterations. A plausible explanation is offered whereby these TnT alterations modify the length dependence of Ca2+ sensitivity.

The force development by calcium-activated skinned frog skeletal muscle fibers and the motion on a slow time base after a quick decrease in load were studied at 0-1~ as a function of the ionic strength and the degree of activation. The ionic strength was varied between 50 and 190 mM by adding appropriate concentrations of KCI to the bathing solution. Under these conditions, the fibers could be maximally activated for several cycles at low ionic strength without developing residual tension. We found that the steady isometric force in fully activated fibers linearly decreased when the KCI concentration was increased from 0 to 140 mM. The steady isotonic motion at a given relative load in fully activated fibers was almost the same at KCI concentrations >--50 mM. In 0 and 20 mM KCI, the isotonic velocity decreased continuously for more than 300 ms. At a given relative load, the initial velocity of the motion in 0 and 20 mM KCI was about 0.6 and 0.9 times, respectively, that in 140 mM KC1. The initial velocity decreased further when residual tension developed; this observation provides additional evidence that residual tension may reflect the presence of an internal load. The effect of calcium on the motion was examined at 70 mM KCI. In this solution, the motion during the velocity transient at a given relative load appeared to be the same at different levels of activation. The speed of the subsequent motion was almost steady at high calcium levels but decreased continuously in low calcium levels. These results support the idea that at low ionic strength the response of the fiber to calcium is switch-like, but that other factors also affect the contraction mechanism under these conditions.

A B S TRA C T Calcium and ionic strength are both known to modify the force developed by skinned frog muscle fibers. To determine how these parameters affect the cross-bridge contraction mechanism, the isotonic velocity transients following step changes in load were studied in solutions in which calcium concentration and ionic strength were varied. Analysis of the motion showed that calcium has no effect on either the null time or the amplitude of the transients. In contrast, the transient amplitude was increased in high ionic strength and was suppressed in low ionic strength. These results are consistent with the idea that calcium affects force in skeletal muscle by modulating the number of force generators in a simple switchlike "on-off' manner and that the steady force at a given calcium level is proportional to cross-bridge number. On the other hand, the effect of ionic strength on force is associated with changes in the kinetic properties of the crossbridge mechanism.