Troponin C occurs as two isoforms, one (sTnC) expressed in fast skeletal muscle and the other (cTnC) expressed in cardiac and slow skeletal muscle. On the basis of subunit exchange experiments it has been suggested that cTnC may play a specific role as a length-sensing molecule. In this study we have compared skinned fibers from bovine ventricle and slow rabbit soleus muscle with respect to the effects of force and sarcomere length on Ca2+ binding to troponin C. A double-isotope technique was used to measure Ca2+ binding concurrent with force generation. The phosphate analogue vanadate was used to regulate force independent of free Ca2+ concentration. To determine the effect of sarcomere length, muscle fibers were released from longer sarcomere length to shorter sarcomere length, and bound Ca2+ was determined either before or after the release. Reduction in force or length was associated with reduced binding of Ca2+ to cTnC in cardiac muscle, but no effect of these interventions was seen in soleus muscle. Thus the nature of the mechanical feedback on the regulatory Ca(2+)-binding sites appears to be a property of the myofilament system rather than the troponin C isoform.

The steep relationship between systolic force and end diastolic volume in cardiac muscle (Frank-Starling relation) is, to a large extent, based on length-dependent changes in myofilament Ca(2+) sensitivity. How sarcomere length modulates Ca(2+) sensitivity is still a topic of active investigation. Two general themes have emerged in recent years. On the one hand, there is a large body of evidence indicating that length-dependent changes in lattice spacing determine changes in Ca(2+) sensitivity for a given set of conditions. A model has been put forward in which the number of strong-binding cross-bridges that are formed is directly related to the proximity of the myosin heads to binding sites on actin. On the other hand, there is also a body of evidence suggesting that lattice spacing and Ca(2+) sensitivity are not tightly linked and that there is a length-sensing element in the sarcomere, which can modulate actin-myosin interactions independent of changes in lattice spacing. In this review, we examine the evidence that has been cited in support of these viewpoints. Much recent progress has been based on the combination of mechanical measurements with X-ray diffraction analysis of lattice spacing and cross-bridge interaction with actin. Compelling evidence indicates that the relationship between sarcomere length and lattice spacing is influenced by the elastic properties of titin and that changes in lattice spacing directly modulate cross-bridge interactions with thin filaments. However, there is also evidence that the precise relationship between Ca(2+) sensitivity and lattice spacing can be altered by changes in protein isoform expression, protein phosphorylation, modifiers of cross-bridge kinetics, and changes in titin compliance. Hence although there is no unique relationship between Ca(2+) sensitivity and lattice spacing the evidence strongly suggests that under any given set of physiological circumstances variation in lattice spacing is the major determinant of length-dependent changes in Ca(2+) sensitivity.

The sensitivity of skinned cardiac muscle bundles to Ca2+ is a function of sarcomere length. Ca2+ sensitivity is increased as fiber length is extended along the ascending limb of the force-length curve and it has been suggested that this phenomenon makes a major contribution to the steep force-length relationship that exists in living cardiac muscle. To gain greater insight into the mechanism behind the length dependence of Ca2+ sensitivity isotopic measurements of Ca2+ binding to detergent-extracted bovine, ventricular muscle bundles were made under conditions in which troponin C was the only major Ca2+ binding species. Experiments were designed to determine whether 1) Ca2+-troponin C affinity varies in the sarcomere length range corresponding to the ascending limb of the force-length curve, and 2) Ca2+ binding correlates with length per se or with changes in the number of length-dependent cross-bridge attachments. Measurements were made of Ca2+ binding in the rigor and relaxed states. The latter state was produced by suppressing actin-myosin interaction with the phosphate analogue, sodium vanadate. After vanadate treatment it is possible to obtain a complete Ca2+ saturation curve in the presence of physiological MgATP concentrations and at constant sarcomere length. The results show that the binding of Ca2+ to the regulatory site of cardiac troponin C is length dependent but this length dependence is actually a dependence on the number of attached cross bridges.

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The duration of activation in cardiac muscle is a function of the load. On the basis of studies of Ca2+ transients in muscles subjected to quick release, it has been suggested that force or shortening-mediated changes in Ca2+-troponin C affinity may provide a mechanism for a contraction-activation feedback. This study was designed to test the hypothesis that the formation of force-generating complexes between actin and myosin enhances the affinity of cardiac troponin C for Ca2+. This was done by first establishing the normal relationship between Ca2+ binding and force development in chemically skinned bovine ventricular muscle bundles and then comparing the Ca2+-saturation curves obtained with relaxed and contracting muscle bundles. A double isotope technique was used to measure Ca2+ binding during ATP-induced force generation and during relaxation maintained by the phosphate analogue vanadate. The results showed that the generation of force was associated with an enhanced binding of Ca2+ to the Ca2+-specific regulatory site of cardiac troponin C. These data provide direct evidence that feedback between force and activation in the heart may be mediated by the Ca2+-regulatory site of troponin C.

The steep relationship between active force and length in cardiac muscle is based on a length dependence of myofilament Ca(2+) sensitivity. However, it is not muscle length but the lateral spacing between actin and myosin filaments that sets the level of Ca(2+) sensitivity, mainly through modulation of myosin-mediated activation of the thin filament.

On the basis of isotopic methods it has been found that force generation promotes increased Ca2+ binding to troponin C in cardiac muscle [P. Hofmann and F. Fuchs. Am. J. Physiol. 253 (Cell Physiol. 22): C541-C546, 1987] but not in skeletal muscle (J. Muscle Res. Cell Motil. 6: 477, 1985). However, studies with skinned rabbit psoas muscle fibers containing substituted fluorescent troponin C analogues indicate that force-generating cross bridges do promote increased Ca2+ binding in skeletal muscle (K. Güth and J. D. Potter. J. Biol. Chem. 262: 13627-13635, 1987). We have reexamined this question using a modified contraction-relaxation protocol in which Ca2+ binding to detergent-treated rabbit psoas fibers was measured either during steady-state force development or after relaxation was induced by one of two myosin ATPase inhibitors, vanadate or 2,3-butanedione monoxime. A standard double-isotope technique was used to measure Ca2+ binding. Another set of experiments was done in which force was reduced by releasing muscle fibers from sarcomere lengths of 2.4-2.6 microns to 1.5-1.7 microns, and bound Ca2+ was determined either before or after the release. No statistically significant effect of force generation or sarcomere length on Ca(2+)-troponin C affinity was observed. Thus the discrepancy remains between results obtained with isotopic and fluorescence methods. It is possible that in skinned fibers emission from fluorescence probes is more closely related to protein-protein interactions than to the amount of Ca2+ bound to troponin C.

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