Thin-filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres

Abstract

Thin-filament regulation of isometric force redevelopment (k(tr)) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca(2+)-activated force (rF(max)) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal k(tr) was unaffected until rF(max) was <0.2 of pre-extracted F(max). In contrast, reconstitution with cTnC or xsTnC reduced maximal k(tr) to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF(max) of 0.68 +/- 0.03 and 0.25 +/- 0.02 F(max). The k(tr)-pCa relation of fibres containing sTnC: xxsTnC mixtures (rF(max) > 0.2 F(max)) was little effected, though k(tr) was slightly elevated at low Ca(2+) activation. The magnitude of the Ca(2+)-dependent increase in k(tr) was greatly reduced following cTnC or xsTnC reconstitution because k(tr) at low levels of Ca(2+) was elevated and maximal k(tr) was reduced. Solution Ca(2+) dissociation rates (k(off)) from whole Tn complexes containing sTnC (26 +/- 0.1 s(-1)), cTnC (38 +/- 0.9 s(-1)) and xsTnC (50 +/- 1.2 s(-1)) correlated with k(tr) at low Ca(2+) levels and were inversely related to rF(max). At low Ca(2+) activation, k(tr) was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k(tr) at any [Ca(2+)] and suggest Ca(2+) activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.

Figure 1. Kinetics of force redevelopment (k_tr) in single permeabilized rabbit psoas muscle fibres at saturating [Ca²⁺] (pCa 4.5) before native sTnC extraction and after reconstitution with either cTnC (A) or mutant sTnCs (B–D)

Force records comparing k_tr in four different example fibres prior to extraction of endogenous TnC (control) and after reconstitution with 100% cTnC (A), 100% sTnC,D28A (xsTnC) (B), or mixtures of sTnC and sTnC,D28A,D64A (xxsTnC) (C and D). Force traces were normalized relative to maximal force under control conditions (F_max) for each fibre. Reconstituted F_max (rF_max) and k_tr for each trace are given next to the respective force record. Example fibres with similar reconstituted steady-state force levels were paired (A–C and B–D) to demonstrate that k_tr is very similar between control conditions and when fibres are reconstituted with varied mixtures of sTnC: xxsTnC, but not when they are reconstituted with either cTnC or xsTnC. Control force for each trace is as follows: 366.5 mN mm⁻² (A), 429.6 mN mm⁻² (B), 317.3 mN mm⁻² (C), and 322.4 mN mm⁻² (D).

Figure 2. Relationship between maximal k_tr

and reconstituted maximal isometric force (rF_max) (pCa 4.5) for fibres reconstituted with cTnC (□, 9 fibres), xsTnC (▿, 13 fibres) or sTnC: xxsTnC mixtures (•, 34 fibres) Maximal k_tr and rF_max of TnC-reconstituted fibres were normalized to maximal k_tr and F_max, respectively, obtained in the same fibre prior to TnC extraction (control , 56 fibres). Fibres reconstituted with various mixtures of sTnC and xxsTnC to give different rF_max levels were binned in 0.2 or 0.3 rF_max increments even when the proportions of sTnC (10–100%) and xxsTnC (0–90%) varied within some groups. Note that maximal k_tr does not depend on the level of reconstituted force but on the properties of TnC. Values are means ±s.e.m.; some error bars are smaller than the symbols. Data for sTnC: xxsTnC mixtures were fitted with a linear regression (solid line);. *P < 0.01 versus maximal k_tr under control conditions (). & Fibres reconstituted with 100% sTnC. Relative rF_max between any group (except for &) and control F_max () is statistically significant (P < 0.01). Relative maximal k_tr values among sTnC: xxsTnC groups are not statistically significant. Relative maximal k_tr between cTnC and xsTnC is not statistically significant.

Figure 3. Ca²⁺ dependence of k_tr

Summary of k_tr–pCa data for fibres prior to native TnC extraction (•) and after reconstitution with cTnC (□) (A, 6 fibres), xsTnC (▿) (B, 10 fibres), or sTnC: xxsTnC mixtures (○) (C, 8 fibres and D, 13 fibres). Fibres reconstituted with mixtures of sTnC: xxsTnC were grouped according to rF_max (∼0.70 F_max in panel C using 20: 80 or 60: 40 sTnC: xxsTnC mixtures, or ∼0.20 F_max in D using 10–15% sTnC and 80–90% xxsTnC mixtures) to compare with fibres reconstituted with 100% cTnC or 100% xsTnC, respectively. Notice the substantial reduction in (A) or elimination of (B) the Ca²⁺ dependence of k_tr with cTnC or xsTnC. Values are means ±s.e.m.; some error bars are smaller than the symbols. Force–pCa curves previously reported (Regnier et al. 2002; Moreno-Gonzalez et al. 2005) are included for control (dashed lines) and experimental conditions (dotted lines) for visualization of the effect on pCa₅₀ and Hill coefficient for steady-state isometric force.

Figure 4. Relationship between k_tr and steady-state isometric force as pCa is varied

k_tr data from Fig. 3 were replotted against steady-state force, normalized relative to pre-extracted F_max (control •), for fibres reconstituted with 100% cTnC (□) (A, rF_max= 0.73 ± 0.03), 100% xsTnC (▿) (B, rF_max= 0.23 ± 0.02), or sTnC: xxsTnC mixtures (○) (C, rF_max= 0.69 ± 0.05 and D, rF_max= 0.21 ± 0.02). Data were binned by pCa. Values are means ±s.e.m.; some error bars are smaller than the symbols. In B, k_tr simulation values (see Appendix) for control (⋆) and xsTnC (⋆) conditions at low and high force are included for comparison with experimental data.

Figure 5. Effect of dATP on maximal k_tr for fibres reconstituted with cTnC (6 fibres) and xsTnC (3 fibres)

Maximal k_tr with 5 mm ATP (black bars) or 5 mm dATP (grey bars) as the contractile substrate was normalized to maximal k_tr obtained in the same fibre prior to TnC extraction (control – sTnC) with ATP. Values are means ±s.e.m.*P < 0.01 versus maximal k_tr with ATP. #Values from Regnier et al. (1998b). Note that dATP increases maximal k_tr under control conditions and in fibres reconstituted with 100% cTnC, but not with 100% xsTnC.

Figure 6. Effect of dATP on the relationship between k_tr and steady-state isometric force for fibres reconstituted with 100% cTnC (□, 6 fibres)

k_tr data from Fig. 4A were replotted for ATP conditions (control • and cTnC □). In addition, the relationship between k_tr and force with dATP for those cTnC-reconstituted fibres () shows that dATP extends the curve beyond maximal values of force and k_tr with ATP at high levels of Ca²⁺ activation. *Maximal values under each condition. Data were binned by pCa. Values are means ±s.e.m.; some error bars are smaller than the symbols.