Modulation of troponin C affinity for the thin filament by different cross-bridge states in skinned skeletal muscle fibers

Abstract

In vertebrate skeletal muscle, the C-domain of troponin C (TnC) serves as an anchor; the N-domain regulates the position of troponin-tropomyosin on the thin filament after changes in intracellular Ca2+. Another type of thin-filament regulation is provided by cross-bridges. In this study, we use skinned fibers reconstituted with chicken recombinant TnC (rTnC) to examine TnC-thin filament affinity when cross-bridges containing different ligands are formed. Dissociation and equilibrium binding of apo-TnC (i.e., lacking divalent cations) under different conditions were monitored by a standard test for maximum tension (P (o)). After 10 min in low-Mg2+ relaxing solution, rTnC dissociation (i.e., tension loss) was 80% vs only 45% in rigor. In rigor, adding myosin subfragment 1 (S1) reduced dissociation approximately twofold, whereas stretching to reduce filament overlap increased dissociation to nearly the value for relaxed fibers. Dissociation of rTnC after addition of Pi or MgADP to form A.M.Pi or A.M.ADP cross-bridges was significantly greater than with rigor (A.M) bridges. The increase in P (o) during equilibration with different concentrations of rTnC showed that the affinity for rTnC binding to the thin filament increased progressively with stronger cross-bridges: rTnC concentrations for half-maximal reconstitution (K (0.5)) were 8.1, 3.7, 2.9, and 1.1 microM for A + M.ADP.Pi, A.M.Pi, A.M, and A.M + S1. Cross-bridges containing MgADP(-) (A.M.ADP) were also less effective than rigor bridges in promoting rTnC binding. We conclude that cross-bridge state and number both modulate TnC affinity for the thin filament and that the TnC C-domain is a central element in this pathway.

A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy

“…ATPase activation, in the presence of Ca 2ϩ , demonstrated that D145E, located in the C-terminal domain, displayed a significant increase in the ability to activate actomyosin ATPase. Recently it was shown that different types of cross-bridges can modulate TnC affinity for the thin filament demonstrating the influence of myosin on the C-terminal domain of TnC (43). Here the C-terminal domain HCM mutation D145E strengthens this concept as it shows the influence of this domain on actomyosin function.…”

Effect of pulmonary TNF-α overexpression on mouse isolated skeletal muscle function

“…2, A and C), it is likely that Ca 2ϩ uptake rate could not be reduced (39). Thus, the reduced tension of Tg ϩ EDL was associated with a decreased cross bridge dissociation rate, which could be affected by either NO or ROS as shown in the literature (14,24,38,39). These results suggested that chronic production of TNF-␣ likely affected the cross bridge dissociation rate of Tg ϩ EDL under both PO 2 conditions, consistent with the hypothesis by Reid et al (31) that TNF-␣ negatively affected either myosin ATPase activity or the thin-filament Ca 2ϩ kinetics.…”

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

“…In comparison, the previously described HCM cTnC mutants A8V and D145E increased thin filament Ca 2ϩ affinity with (⌬pCa 50 s of ϩ0.14 and ϩ0.08), respectively (33). It is well known that the number of cross-bridges can modulate both Ca 2ϩ sensitivity and TnC affinity for the thin filament (41)(42)(43)(44)(45)(46)(47). Therefore, the addition of myosin S1 was used to assess whether strong cross-bridge formation indirectly (ϪATP) modulated the Ca 2ϩ affinity of the mutant containing thin and thick filament differently than the WT (41).…”