CA2-C IN SKINNED SKELETAL-MUSCLE FIBERS ASSESSED WITH CASED CA2+ AND A CA2+ FLUOROPHORE - INVARIANCE OF CA2+ BINDING AS A FUNCTION OF SARCOMERE-LENGTH( BINDING TO TROPONIN)

Ca2+ sensitivity of tension varies with sarcomere length in both skele tal and cardiac muscles, One possible explanation for this effect is t hat the Ca2+ affinity of the regulatory protein troponin C decreases w hen sarcomere length is reduced. To examine length dependence of Ca2binding to troponin C: in skeletal muscle, we developed a protocol to simultaneously monitor changes in sarcomere length, tension, and Ca2concentration following sash photolysis of caged Ca2+. In this protoco l, [Ca2+] was rapidly increased by flash photolysis of caged Ca2+, and changes in [Ca2+] due to photolysis and the subsequent binding to tro ponin C were assessed using a Ca2+ fluorophore. Small bundles of fiber s 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+](fre e), tension, and sarcomere length were monitored before and after phot olysis of caged Ca2+. Upon photolysis of caged Ca2+, fluorescence incr eased and then decayed to a new steady-state level within similar to 1 s, while tension increased to a new steady-state level within similar to 1.5 s, After extracting troponin C, fibers did not generate tensio n following the flash, but steady-state post-flash fluorescence was si gnificantly greater than when troponin C was present, The difference i n [Ca2+](free) represents the amount of Ca2+ bound to troponin C. In f ibers that were troponin C-replete, Ca2+ binding to troponin C did not differ at short (similar to 1.97 mu m) and long (similar to 2.51 mu m ) sarcomere length, yet tension was similar to 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 tha t length-dependent changes in Ca2+ sensitivity of tension in skeletal muscle are not related to length-dependent changes in Ca2+ binding aff inity of troponin C.