The Ca2+-Induced Structural Changes in Troponin In-Situ and In-Vitro: A FRET Study in Permeabilized Cardiac Muscle Fibers and Reconstituted Thin Filaments

Abstract

Ca2+ binding to N-domain of cardiac troponin C (N-cTnC) initiates a cascade of structural changes within the thin filament. Among these changes, Ca2+-induced structural changes in troponin play a critical role in the allosteric regulation of the force-generating acto-myosin interactions. Although previous in-vitro studies have provided insight into these structural changes, how they are modulated by geometric and mechanical constraints conferred by the sarcomeric lattice structure in muscle tissue remains unknown. Recently we have developed a technique to quantitatively measure these structural changes in chemically-skinned muscle fibers using time-resolved FRET. In this study, we use this technique to monitor the Ca2+-induced N-cTnC opening in skinned muscle fibers and compare the obtained results to in-vitro studies. FRET donor (AEDANS) and acceptor (DDPM) modified double-cysteine mutant cTnC(13C/51C)AEDANS-DDPM was reconstituted into skinned muscle fibers to examine the N-cTnC opening. To study the effects of crossbridge and sarcomere-length based activation on this structural change, simultaneous measurements of fluorescence intensity decays and isometric tension were performed. Our results show that at steady-state, the Ca2+-induced N-cTnC opening is proportional to muscle force development, but the level of opening varies depending on the presence or absence of strong crossbridges (XB). For instance, in the presence of sodium vanadate (inhibitor of XB) no Ca2+-induced force response was observed, but Ca2+-induced opening of N-cTnC in the fibers was evident but with a smaller magnitude. Furthermore, when compared to in-vitro measurements, results from muscle fibers suggest that N-cTnC may adopt a more compact structure in muscle fibers due to the constraints imposed by the myofilament lattice structure. The effects of the myofilament lattice structure on structural dynamics of N-cTnC are also discussed in terms of FRET distance distributions.