Cross-Talk between Neighboring Troponins in Ventricular Myofibrils

Troponin C (TnC) is responsible for Ca2+-dependent activation and inactivation of the thin filament during contraction and relaxation of the heart. To study the Ca2+ binding properties of TnC, we designed a human cardiac TnC (C35S T53C C84S) ± the L48Q mutation. L48Q TnC has an increased Ca2+ sensitivity and decreased rate of Ca2+ dissocation as previously shown in multiple biochemical systems. We exchanged IANBD labeled control or L48Q Tn into rabbit ventricular myofibrils and used a stopped-flow apparatus to measure the fluorescence change related to Ca2+ dissociation. Unlike the mono-phasic Ca2+ dissociation curve of control myofibrils (25.3/s ± 0.7), Ca2+ dissociation from L48Q TnC myofibrils was biphasic, with rates of 17.3 ± 0.5/s and 4.73 ± 0.07/s, at 15oC. The apparent Ca2+ affinities of the fast and slow phases for L48Q TnC corresponded to the steady-state Ca2+ binding affinities of control and L48Q myofibrils, respectively. We hypothesized that the biphasic rate of L48Q Tn exchanged myofibrils results from the influence of the un-exchanged endogenous Tn. Consistent with this hypothesis, reconstituted thin filaments containing 100% L48Q Tn had a mono-phasic Ca2+ dissociation curve. Whereas, Ca2+ dissociation from reconstituted thin filaments containing IANBD labeled L48Q Tn and unlabeled control Tn lead to a biphasic Ca2+ dissociation rate, confirming the crosstalk between Tn subunits. Interestingly, the biphasic characteristic of L48Q TnC was absent when incorporated into diseased human ventricular myofibrils, possibly due to an altered thin filament state. Fluorescently labeled L48Q TnC provides insight into the interactions occurring between neighboring Tn subunits. Thus, L48Q TnC may be a useful tool to probe myofilament protein interactions along the thin filament.