Determining the Effects of Different Cross-Bridge States on the Rate of Calcium Dissociation from Troponin C Mutants in Rabbit Ventricular Myofibrils

Abstract

The molecular events required for thin filament activation and deactivation in cardiac muscle are generally thought to be in rapid equilibrium. Stopped-flow measurements obtained with reconstituted thin filaments suggest that the rate of Ca2+ dissociation from cardiac TnC can be modulated by myosin S1 binding to actin and is not always rapid, but approaches the rate of mechanical relaxation at 15oC. To further probe the effects that myosin contributes to thin filament deactivation, we have utilized rabbit ventricular myofibrils as a more physiological biochemical system to examine the kinetics of Ca2+ dissociation from TnC. Incorporating a fluorescent TnC (C35S, C84S, T53C) labeled with IANBD into the myofibrils allows us to follow both the rates of Ca2+ dissociation from TnC, as well as what we think is cross-bridge (CB) detachment, through a change in IANBD fluorescence from TnC. However, CB detachment is only reported by the wild type TnC construct when there is no Ca2+ on the thin filament, while in the presence of ATP, Ca2+ dissociation is no longer observed. CB detachment can be observed in the presence of Ca2+ when Ca2+ binding to TnC is inactivated by D65A TnC. Additionally, the rate of Ca2+ dissociation from TnC can be observed when CBs are detached (presence of ATP) by the Ca2+ sensitizing mutations V44Q or L48Q TnC, but not when Ca2+ binding is desensitized by D73N TnC. Thus, weak and strong binding states of the thick filament differentially influence those of the thin filament within the cardiac myofibril, and are not necessarily in rapid equilibrium.