Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C

Abstract

Understanding the effects of thin and thick filament proteins on the kinetics of Ca(2+) exchange with cardiac troponin C is essential to elucidating the Ca(2+)-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac troponin C at a novel engineered Cys-53 with 2-(4'-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of troponin C upon incorporation into the troponin complex. Neither tropomyosin nor actin alone affected the Ca(2+) binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca(2+) sensitivity ( approximately 7.4-fold) and accelerated the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca(2+) sensitivity ( approximately 6.2-fold) and decreased the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac troponin I with slow skeletal troponin I led to higher Ca(2+) sensitivities and slower Ca(2+) dissociation rates from troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac troponin C to sense and respond to Ca(2+). These results imply that both cross-bridge kinetics and Ca(2+) dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.

FIGURE 1

behaves biochemically and physiologically similar to wild-type and endogenous cTnC. Panel A shows a ribbon representation of the regulatory domain of cTnC in the apo state (1SPY (53)) and in Ca²⁺ saturated cTn (1J1E; TnI, and TnT have been omitted for clarity (31)) utilizing the software Rasmol (54). The regulatory domain of cTnC contains five helices denoted as N, A, B, C, and D. As shown in panel A, helices B and C (BC subdomain) move away from the N, A, and D helices (NAD subdomain) upon Ca²⁺ and TnI binding. The D-helix is pointing out of the page, with the NAD subdomain colored light gray, the BC subdomain colored dark gray, and the Ca²⁺ binding loop indicated by an asterisk (*). Thr-53 is depicted in a stick representation. Panel B shows the IAANS emission spectra of the apo state (solid line) and Ca²⁺ saturated state (dashed line, pCa 3.0) for in the cTn complex (B1), in reconstituted thin filaments (B2), plus myosin S1 (B3), and plus ATP (B4). The emission fluorescence of the spectra was calculated relative to the peak fluorescence of each respective apo state, which was considered 100%. Panel C shows the time course of Ca²⁺ dissociation from wild-type cTn directly followed by an increase in quin-2 fluorescence. Panel C also shows the EGTA-induced time courses of Ca²⁺ dissociation from and reported by an increase in IAANS fluorescence. Overlaid with the kinetic traces are the fitted exponential curves to the data (smooth curves, which may be difficult to discern). Panel D shows the Ca²⁺-dependent increase in force development in skinned rat trabeculae containing endogenous cTnC (▴), wild-type cTnC (▵), and (▪).

FIGURE 2

Effects of ssTnI on the IAANS emission spectra of in systems of increasing complexity. Panels A–D show the IAANS emission spectra of the apo state (solid line) and Ca²⁺-saturated state (dashed line, pCa 3.0) for in the ssTn complex (A), in reconstituted thin filaments (B), plus myosin S1 (C), and plus ATP (D). The emission fluorescence of the spectra was calculated relative to the peak fluorescence of each respective apo state, which was considered 100%.

FIGURE 3

Ca²⁺ sensitivities and dissociation rates from and Panel A shows the Ca²⁺-dependent decrease in IAANS fluorescence of (▪) and (•). Panel B shows the time courses of Ca²⁺ dissociation from unlabeled cTn^T53C and ssTn^T53C directly followed by an increase in quin-2 fluorescence. Panel B also shows the EGTA-induced time courses of Ca²⁺ dissociation from and reported by an increase in IAANS fluorescence.

FIGURE 4

Ca²⁺ binding properties and Ca²⁺ dissociation rates from the Tn complexes in the presence of cTm, or actin ± myosin S1 without cTm. Panel A shows the Ca²⁺-dependent decrease in IAANS fluorescence of (▪) and (•) in the presence of cTm. Panel B shows the EGTA-induced time courses of Ca²⁺ dissociation from (0.3 μM) in the presence of cTm (0.9 μM) or actin (4 μM) ± myosin S1 (0.57 μM) reported by an increase in IAANS fluorescence. Panel C shows the EGTA-induced time courses of Ca²⁺ dissociation from (0.3 μM) in the presence of cTm (0.9 μM) or actin (4 μM) ± myosin S1 (0.57 μM) reported by an increase in IAANS fluorescence. Increasing the concentration of or to 0.5 μM did not alter the results (data not shown).

FIGURE 5

Ca²⁺ sensitivities and dissociation rates from and reconstituted thin filaments. Panel A shows the Ca²⁺-dependent increase in IAANS fluorescence of (□) and (○) reconstituted thin filaments. Panel B shows the EGTA-induced time courses of Ca²⁺ dissociation from and reconstituted thin filaments reported by a decrease in IAANS fluorescence.

FIGURE 6

Effects of myosin S1 on the Ca²⁺ sensitivities and dissociation rates from and reconstituted thin filaments. Panel A shows the Ca²⁺-dependent decrease in IAANS fluorescence of (▪) and (•) reconstituted thin filaments in the presence of myosin S1. Panel A also shows the Ca²⁺-dependent increase in IAANS fluorescence of (□) and (○) reconstituted thin filaments in the presence of myosin S1 and ATP. Panel B shows the EGTA-induced time courses of Ca²⁺ dissociation from and reconstituted thin filaments in the presence of myosin S1 reported by changes in IAANS fluorescence. The EGTA-induced decrease in fluorescence as opposed to the increase in fluorescence is due to the different emission filters used to capture the signals. Panel C shows the EGTA-induced time courses of Ca²⁺ dissociation from and reconstituted thin filaments in the presence of myosin S1 and ATP reported by an increase in IAANS fluorescence.