Ca++-sensitizing mutations in troponin, P(i), and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity

Abstract

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca(++)-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity (VRTF) at both maximal and submaximal Ca(++) levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca(++)-sensitizing mutation in the Ca(++)-binding subunit of Tn (TnC) increased VRTF at submaximal Ca(++) under acidic conditions but had no effect on VRTF at maximal Ca(++) levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca(++). Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate.

Keywords: 2-deoxy-ATP; fatigue; phosphate; regulation; troponin C; troponin I.

Fig. 1.

Simplified consensus model of the cross-bridge cycle. The key steps of the ATPase cycle of myosin linked to the mechanical events. The first step, inorganic phosphate (P_i) release, is linked to actin strong binding and the movement of the lever arm that results in a unitary displacement (d). During this step, tropomyosin moves over the surface of actin to allow actomyosin binding, and this is thought to be the step regulated by Ca⁺⁺, i.e., the weak-to-strong transition (28). There are likely two actomyosin state with ADP bound in the active site (AM-ADP) states (60), with acidosis thought to regulate the transition from the first (AM-ADP) to the second (AM-ADP) (17). We also believe that, when P_i is elevated, the powerstroke may not be reversed, but rather myosin dissociates from actin in a postpowerstroke state to account for P_i-induced increase in regulated thin-filament velocity (V_RTF) (17, 18). Thus there are likely many more steps than represented here, but for clarity we have reduced the key steps.

Fig. 2.

Location of mutations within troponin (Tn) structure. A: rabbit skeletal crystal structure of the NH₂-terminal region of Ca⁺⁺-binding subunit of Tn (TnC) shown with V43 labeled in blue, coordinates from Soman et al. (61). V43 is part of the hydrophobic patch in TnC thought to swing away from the center helix of TnC, allowing inhibitory subunit of Tn (TnI) to bind in the presence of Ca⁺⁺ (73). B: TnI (dark gray) binds to the NH₂-terminal of TnC (white) in the Ca⁺⁺-activated state. The R156 TnI used in the present study represents a truncation mutant that eliminates the last 26 residues. The COOH terminus of TnI is not defined in the crystal structure, so the figure is truncated at residue 143. Both structural images were rendered with Polyview 3D. C: sequence of the COOH terminus of TnI for wild-type (WT) and for the TnI R156 variant illustrating the truncated residues.

Fig. 3.

Effect of mutations in Tn on velocity-pCa relationship at different pH. V_RTF determined at normal (7.4) and low pH (6.8) using filaments reconstituted with WT Tn, Tn with a V43Q mutation in TnC, and Tn with TnI truncated beyond R156. V_RTF plotted as a function of the -log of the [Ca⁺⁺] (pCa). Data points represent means ± SE. Data were fit with Hill equation with fit parameters displayed in Table 1. *V_RTF with V43Q is significantly different from control at the given pCa value. #V_RTF with R156 is significantly different from control at the given pCa value.

Fig. 4.

A–C: effect of 15 mM P_i on the velocity-pCa relationship at different pH. V_RTF with WT Tn plotted as a function of the pCa and pH in the presence and absence of 15 mM P_i. Data points represent means ± SE. Data were fit with Hill equation with fit parameters displayed in Table 2. *V_RTF with 15 mM P_i is significantly different from control at the given pCa value.

Fig. 5.

A–C: effect of 2-deoxy ATP (dATP) of the velocity-pCa relationship at different pH. V_RTF with WT Tn plotted as a function of the pCa and pH in the presence of dATP. Data points represent means ± SE. Data were fit with Hill equation with fit parameters displayed in Table 3. *V_RTF with dATP is significantly different from control at the given pCa value.

Fig. 6.

Effect of pH, P_i, and dATP on WT V_RTF. V_RTF for filaments reconstituted with WT Tn at pCa 5.0 as a function of pH, in the presence and absence of 15 mM P_i, and dATP. Values represent means ± SE and were analyzed using a 3-way ANOVA. All pairwise comparisons were significant except the effect of P_i (open bar) vs. dATP (shaded bar) at each pH level. *Significantly different from control; #significantly different from Pi condition; ‡significantly different from dATP condition. The interactions for pH × ATP type and P_i by ATP type were both significant (P < 0.05), indicated by §, but the P_i × pH interaction only trended toward significance (P = 0.07). The 3-way interaction (pH × P_i × ATP type) was not significant (P = 0.564).