Alterations in the Ca2+ sensitivity of tension development by single skeletal muscle fibers at stretched lengths

The apparent length dependence in the calcium sensitivity of tension development in skeletal muscle has been investigated in the present study. At sarcomere lengths of 2.46–2.62 micron, the Hill plot of tension-pCa data is well fit by not one but two straight lines, suggesting the possible involvement of more than a single class of Ca2+-binding site in tension development. On the other hand, increasing the sarcomere length to 3.00–3.25 micron yielded Hill plots that were described by a single straight line, which indicates that at long lengths tension might be regulated by the binding of Ca2+ to a single class of Ca2+-binding sites, presumably the low affinity sites of TnC. This length-dependent transformation of the tension pCa relation occurred at free Mg2+ concentrations of both 0.05 and 3.2 mM. Although the mechanism of this effect is uncertain, plausible explanations for the biphasic Hill plot at the shorter lengths include the possible involvement of Ca2+ activation of the thick filaments and/or myosin LC2 phosphorylation in the process of tension development

Calcium alone does not fully activate the thin filament for S1 binding to rigor myofibrils

Skeletal muscle contraction is regulated by calcium via troponin and tropomyosin and appears to involve cooperative activation of cross-bridge binding to actin. We studied the regulation of fluorescent myosin subfragment 1 (fS1) binding to rigor myofibrils over a wide range of fS1 and calcium levels using highly sensitive imaging techniques. At low calcium and low fS1, the fluorescence was restricted to the actin-myosin overlap region. At high calcium and very low fS1, the fluorescence was still predominantly in the overlap region. The ratio of nonoverlap to overlap fluorescence intensity showed that increases in the fS1 level resulted in a shift in maximum fluorescence from the overlap to the nonoverlap region at both low and high calcium; this transition occurred at lower fS1 levels in myofibrils with high calcium. At a fixed fS1 level, increases in calcium also resulted in a shift in maximum fluorescence from the overlap region to the nonoverlap region. These results suggest that calcium alone does not fully activate the thin filament for rigor S1 binding and that, even at high calcium, the thin filament is not activated along its entire length

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+