Abstract
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.
Highlights
The regulation of striated muscle contraction involves Ca2ϩ activation of interactions between contractile proteins of the thick and thin filaments
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؉
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
Summary
The regulation of striated muscle contraction involves Ca2ϩ activation of interactions between contractile proteins of the thick and thin filaments. The thin filament protein troponin plays an integral role in activating contraction by binding Ca2ϩ that is released into the myoplasm during a twitch. During excitation-contraction coupling, Ca2ϩ binding to the low affinity sites on TnC initiates a series of events within the thin filaments that allow interaction of myosin with actin [2]. A fluorescent Ca2ϩ indicator (Fluo-3) and caged Ca2ϩ (DMnitrophen or NP-EGTA) were used to examine Ca2ϩ binding as a function of SL in bundles of fast skeletal muscle fibers and to directly assess the role of altered Ca2ϩ binding in conferring length dependence of Ca2ϩ sensitivity of tension
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
