ROLE OF CARDIAC TROPONIN C AND TROPONIN I IN THE MYOFILAMENT LENGTH DEPENDENT ACTIVATIO
Myocardial contractility stems from the sliding movement between thin and thick myofilaments produced by the interactions between myosin and actin involved in the crossbridge cycle, which in turn is controlled by thin filament regulatory proteins. As intracellular [Ca2+] rises, the N-domain of cTnC opens and initiates protein conformational transitions in cTnI and other thin filament proteins through direct and allosteric effects that activate crossbridge cycling in a process known as “regulatory switching.” Despite many insights having come from in vitro studies, a molecular level understanding of how regulatory switching works inside the physiological environment of the sarcomere has remained elusive. Furthermore, numerous in situ studies point to the ability of force generating crossbridges to modulate regulatory switching, and evidence is growing that the regulatory switching process can also be affected by sarcomere length. A series of fluorophore-modified cTnC and cTnI mutants were engineered to monitor structural transitions and conformational dynamics of cTn in reconstituted thin filaments and permeabilized muscle fibers. In attempt to connect molecular events to cardiac muscle function, we propose a new experimental approach that integrates FRET and TCSPC with muscle mechanics approaches. Chemical perturbations of crossbridge cycling and laser diffraction will be used to explore the effects of in situ sarcomeric constraints, modulatory feedback from crossbridge cycling, and length-dependent activation on Ca2+-dependent regulatory switching. The overall objective of this study was to investigate the structural mechanisms involved in the Ca2+ induced, force-generating (strong) crossbridge modulated and length dependence mediated pathways to thin filament activation chemically skinned cardiac muscle preparations. Specific Aim 1 was to identify the effect of mechanical/geometric constraints and highly organized proteins environment found in muscle cells on structural transitions of N-domain of cTnC and C-domain of TnI. Specific Aim 2 was to determine effect of length-dependence and crossbridge cycling on structural transitions of N-cTnC and C-TnI.