Ca2+ binding to troponin C in skinned skeletal muscle fibers assessed with caged Ca2+ and a Ca2+ fluorophore. Invariance of Ca2+ binding as a function of sarcomere length.

Ca2+ sensitivity of tension varies with sarcomere length in both skeletal and cardiac muscles. One possible explanation for this effect is that the Ca2+ affinity of the regulatory protein troponin C decreases when sarcomere length is reduced. To examine length dependence of Ca2+ binding to troponin C in skeletal muscle, we developed a protocol to simultaneously monitor changes in sarcomere length, tension, and Ca2+ concentration following flash photolysis of caged Ca2+. In this protocol, [Ca2+] was rapidly increased by flash photolysis of caged Ca2+, and changes in [Ca2+] due to photolysis and the subsequent binding to troponin C were assessed using a Ca2+ fluorophore. Small bundles of fibers from rabbit skinned psoas muscles were loaded with Ca2+ fluorophore (Fluo-3) and caged Ca2+ (dimethoxynitrophenamine or o-nitrophenyl-EGTA). The bundles were then transferred to silicone oil, where [Ca2+]free, tension, and sarcomere length were monitored before and after photolysis of caged Ca2+. Upon photolysis of caged Ca2+, fluorescence increased and then decayed to a new steady-state level within approximately 1 s, while tension increased to a new steady-state level within approximately 1.5 s. After extracting troponin C, fibers did not generate tension following the flash, but steady-state post-flash fluorescence was significantly greater than when troponin C was present. The difference in [Ca2+]free represents the amount of Ca2+ bound to troponin C. In fibers that were troponin C-replete, Ca2+ binding to troponin C did not differ at short (approximately 1.97 microm) and long (approximately 2.51 microm) sarcomere length, yet tension was approximately 50% greater at the long sarcomere length. These results show that the affinity of troponin C for Ca2+ is not altered by changes in sarcomere length, indicating that length-dependent changes in Ca2+ sensitivity of tension in skeletal muscle are not related to length-dependent changes in Ca2+ binding affinity of troponin C.