Thin filament Ca²⁺ binding properties and regulatory unit interactions alter kinetics of tension development and relaxation in rabbit skeletal muscle

Abstract

The influence of Ca²⁺ binding properties of individual troponin versus cooperative regulatory unit interactions along thin filaments on the rate tension develops and declines was examined in demembranated rabbit psoas fibres and isolated myofibrils. Native skeletal troponin C (sTnC) was replaced with sTnC mutants having altered Ca²⁺ dissociation rates (k_off) or with mixtures of sTnC and D28A, D64A sTnC, that does not bind Ca²⁺ at sites I and II (xxsTnC), to reduce near-neighbour regulatory unit (RU) interactions. At saturating Ca²⁺, the rate of tension redevelopment (k_TR) was not altered for fibres containing sTnC mutants with decreased k_off or mixtures of sTnC:xxsTnC. We examined the influence of k_off on maximal activation and relaxation in myofibrils because they allow rapid and large changes in [Ca²⁺]. In myofibrils with M80Q sTnC^F27W (decreased k_off), maximal tension, activation rate (k_ACT), k_TR and rates of relaxation were not altered. With I60Q sTnC^F27W (increased k_off), maximal tension, k_ACT and k_TR decreased, with no change in relaxation rates. Surprisingly, the duration of the slow phase of relaxation increased or decreased with decreased or increased k_off, respectively. For all sTnC reconstitution conditions, Ca²⁺ dependence of k_TR in fibres showed Ca²⁺ sensitivity of k_TR (pCa₅₀) shifted parallel to tension and low-Ca²⁺k_TR was elevated. Together the data suggest the Ca²⁺-dependent rate of tension development and the duration (but not rate) of relaxation can be greatly influenced by k_off of sTnC. This influence of sTnC binding kinetics occurs primarily within individual RUs, with only minor contributions of RU interactions at low Ca²⁺.

Contraction in striated muscle is a dynamic process that is dependent on Ca²⁺ binding kinetics, thin filament activation, and acto-myosin crossbridge cycling. Contractile activation involves a myofilament signalling cascade initiated by Ca²⁺ binding to troponin C (TnC), that alters interactions between troponin subunits and tropomyosin to reveal myosin binding sites on actin. This allows for strong crossbridge formation and cycling (reviewed in Gordon et al. 2000). Mechanical tension relaxation of myofilaments occurs with Ca²⁺ dissociation from TnC and crossbridge detachment (reviewed in Poggesi et al. 2005). The interactive kinetics of Ca²⁺-dependent thin filament activation and crossbridge binding are important for determining the speed of contraction and relaxation. However, an in-depth study of how Ca²⁺ binding kinetics of TnC influence tension development and relaxation in skeletal muscle has not been reported.

Both steady-state tension and the rate of tension redevelopment (k_TR) with a rapid release–restretch length transient vary greatly with [Ca²⁺] in demembranated skeletal muscle as previously demonstrated by us (Regnier et al. 1996, 1998, 1999; Moreno-Gonzalez et al. 2007) and others (Brenner, 1988; Metzger & Moss, 1990, 1991; Sweeney & Stull, 1990). k_TR has been proposed to measure the rates of transition of crossbridges between detached or weakly attached non-tension states and strong, tension generating states (Brenner & Eisenberg, 1986; Brenner, 1988). At saturating Ca²⁺, k_TR varies with myosin isoform (Metzger & Moss, 1990) or alterations of crossbridge cycling rate (Regnier & Homsher, 1998; Regnier et al. 1998), presumably when thin filament activation is not rate limiting. Additional evidence has shown that apparent rates of crossbridge transitions are limited at submaximal Ca²⁺ (Chalovich et al. 1981; Brenner, 1988). Altering the Ca²⁺-binding properties of skeletal TnC (sTnC) or thin filament activation have been shown to influence the Ca²⁺ dependence of k_TR (Regnier et al. 1996, 1999; Razumova et al. 2000; Moreno-Gonzalez et al. 2007). Together, these data suggest that Ca²⁺ binding dynamics of TnC have a strong influence on tension development kinetics.

While studying k_TR gives an indication of crossbridge kinetics during near-equilibrium of the thin filament, the process of Ca²⁺-mediated activation must be studied in a system that allows for rapid changes in [Ca²⁺]. Isolated skeletal myofibrils are preparations that permit the measurement of activation rate (k_ACT) with a rapid increase in Ca²⁺. k_ACT has been shown to be Ca²⁺ dependent (Tesi et al. 2002), similar to k_TR, but is different to k_TR because k_ACT includes the process of thin filament activation as well as crossbridge binding and tension generation. Further, mechanical measurements of tension using myofibril preparations also allow for the study of relaxation in the same myofibrils as activation by using a rapid decrease in Ca²⁺. Therefore, with this technique, both activation and biphasic relaxation can be studied under conditions of altered Ca²⁺ binding properties of sTnC. However, few studies (Luo et al. 2002; Piroddi et al. 2003) have been devoted to understanding the influence of TnC properties on both activation and/or relaxation kinetics.

An additional consideration in understanding activation of skeletal muscle is the influence of Ca²⁺-binding properties on cooperative activation of contraction. In skeletal muscle, near-neighbour regulatory unit (RU; 1 troponin + 1 tropomyosin + the number of actin monomers exposed with Ca²⁺ binding) interactions along the thin filament contribute greatly to the apparent cooperativity in the tension–pCa relationship (Brandt et al. 1984; Metzger & Moss, 1991; Regnier et al. 2002), but appear to have little influence on maximal k_TR or the Ca²⁺-dependent increase in k_TR (Moreno-Gonzalez et al. 2007). These data suggest that control of k_TR is local, i.e. modulated within individual RUs in the thin filaments of skeletal muscle. However, it is not known whether sTnC Ca²⁺ binding kinetics can influence the thin filament activation dependence of k_TR within individual RUs. It is also unknown how increased Ca²⁺-binding affinity of sTnC alters the Ca²⁺ dependence of k_TR in the presence or absence of near-neighbour RU interactions.

In this study we used both single demembranated rabbit skeletal fibres and myofibrils to examine the role of altered Ca²⁺ dissociation rate (k_off) of skeletal TnC (sTnC) on the kinetics of tension development (measured as k_ACT and k_TR) with or without near-neighbour RU interactions and on the kinetics of relaxation. We found that with decreased k_off, k_TR at low tension was increased in fibres without changing maximal k_TR both in the absence and presence of near-neighbour RU interactions. In skeletal myofibrils containing sTnC mutants, k_ACT and k_TR at maximal Ca²⁺ were unchanged with decreased k_off, but were decreased for a sTnC mutant with increased k_off. Interestingly, while the rates of relaxation were unaltered, the duration of the slow phase of relaxation was increased or decreased with sTnC mutants having decreased or increased k_off, respectively. To our knowledge, this is the first report of an influence of troponin Ca²⁺ binding kinetics on the isometric phase of relaxation. Preliminary reports of this work have been previously published (Kreutziger et al. 2004, 2006).

Methods

Fibre preparation

All animal procedures performed in the US were conducted in accordance with the US National Institutes of Health Policy on Humane Care and Use of Laboratory Animals and were approved by the University of Washington (UW) Animal Care Committee. Rabbits were housed in the Department of Comparative Medicine at UW and cared for in accordance with UW IACUC procedures. Male New Zealand white rabbits were anaesthetized with an intravenous injection of pentobarbital (40 mg kg⁻¹) in the marginal ear vein and when the animals had no reflexive response, were exsanguinated. The psoas muscle was exposed and small bundles of psoas fibres were excised at native length, demembranated and stored at −20°C for up to 6 weeks as previously described (Regnier et al. 2002). Segments of single fibres dissected from the fibre bundles were prepared for mechanical measurements by chemically fixing the ends with 1% gluteraldehyde in water (Chase & Kushmerick, 1988) and wrapped in aluminium foil T-clips for attachment to the mechanical apparatus.

Myofibril preparation

All animal procedures performed in Italy were conducted in accordance with the official regulations of the European Community Council on Use of Laboratory Animals (Directive 86/609/EEC) and protocols were approved by the Ethical Committee for Animal Experiments of the University of Florence. Male New Zealand white rabbits were killed by intravenous administration of pentobarbitone (120 mg kg⁻¹) through the marginal ear vein. The psoas muscle was exposed and small bundles of psoas fibres were excised at native length and tied to wooden sticks in ice-cold Ringer-EGTA solution, as previously described (Colomo et al. 1997). Bundles were transferred the next day to Rigor solution with 50% glycerol for 12 h, and then stored at −20°C. Myofibrils were prepared by homogenization of small pieces of glycerinated fibres in Rigor solution on ice, and used for up to 5 days as previously described (Colomo et al. 1997).

Proteins

Rabbit skeletal TnC mutations F27W, M80Q, I60Q and D28A, D64A sTnC (xxsTnC) that does not bind Ca²⁺ at sites I and II were introduced by site-directed mutagenesis as previously described (Regnier et al. 2002; Kreutziger et al. 2007). WT or mutant sTnC was extracted and purified from E. coli (Dong et al. 1996). All recombinant proteins were used for myofibril sTnC replacement. Native rabbit sTnC, sTnI and sTnT were purified from ether powder of rabbit skeletal back and leg muscles (Potter, 1982). Purified sTnC (called ‘sTnC’ in the text) was used in fibres as a control for the effects of extraction–reconstitution of sTnC and for comparison to recombinant mutant sTnC. Note that ‘native’ sTnC refers to protein in the muscle preparation prior to any treatment. Whole sTn complexes used for stopped-flow measurements were formed using recombinant sTnC (WT or mutant) and purified sTnI and sTnT as previously described (Szczesna et al. 2000; Kreutziger et al. 2007). sTnC concentrations were calculated from peak absorbance at 280 nm with appropriate extinction coefficients (Kreutziger et al. 2007). Purity of native and recombinant troponin subunits was assessed by SDS-PAGE.

Ca²⁺ solutions for mechanical measurements

Experimental solutions for fibre mechanics were calculated as previously described (Martyn et al. 1994) and contained (mm): phosphocreatine 15, EGTA 15, 3-(N-mopholino) propanesulphonic acid (Mops) 80, free Mg²⁺ 1, (Na⁺+ K⁺) 135, ATP 5, dithiothreitol (DTT) 1, 250 units ml⁻¹ creatine kinase (Sigma, St Louis, MO, USA) and 4% (w/v) Dextran T-500 (Pharmacia, Piscataway, NJ, USA) at pH 7.0, 15°C, and an ionic strength of 0.17 m. For activating solutions, the Ca²⁺ level (expressed as pCa =−log [Ca²⁺]) was varied between pCa 9.0 and 4.0 by adjusting Ca(propionate)₂ concentration. Experimental solutions for myofibril mechanics were calculated as previously described (Brandt et al. 1998) and contained (mm): phosphocreatine 10, EGTA 10 (ratio of CaEGTA/EGTA set to obtain pCa values 8.0–3.5), Mops 10, free Mg²⁺ 1, (Na⁺+ K⁺) 155, MgATP 5, 200 units ml⁻¹ creatine kinase, and propionate and sulphate to adjust the final solution ionic strength to 0.2 m. Contaminant phosphate (P_i) was reduced in all experiments to < 5 μm by a P_i-scavenging enzyme system (purine-nucleoside-phosphorylase with substrate 7-methyl-guanosine) as previously described (Tesi et al. 2000).

Fibre mechanical data acquisition and analysis

For all muscle fibre experiments, mechanical data were collected from fixed and clipped fibre segments attached to an Aurora Scientific (Ontario, Canada) force transducer model 400A and a General Scanning model G120DT (Watertown, MA, USA) servo-motor (adjusted for 300 μs step time) by minutien pin hooks and mounted on a Nikon (Japan) inverted microscope as previously described (Regnier et al. 2002). Sarcomere length (SL) was initially set to 2.5 μm (L_o) and continuously monitored with helium–neon laser diffraction. Average unfixed L_o of gluteraldehyde-treated fibres was 1.27 ± 0.04 mm and diameter was 55 ± 1 μm (mean ±s.e.m.; n = 50). At 5 s intervals fibre segments were shortened by 15%L_o at 10 L_o s⁻¹, then rapidly restretched to initial L_o to maintain fibre integrity (Brenner, 1983; Sweeney et al. 1987; Chase & Kushmerick, 1988). The fibre preparation was moved between pCa solutions that were held in individual temperature-controlled troughs as previously described (Regnier et al. 2002). Measurement of steady-state isometric tension was made prior to the release–restretch transient used for measurement of k_TR at each Ca²⁺ level. Passive tension was determined at pCa 9.0 with a 15%L_o release–restretch square-wave protocol and was subtracted from total tension measured in higher Ca²⁺ solutions to obtain the active tension values reported. Maximal fibre tension (F_max) measured at pCa 4.5 just prior to sTnC extraction was 354 ± 12 mN mm⁻² (mean ±s.e.m., n = 50; assuming circular cross-sectional area). Fibres with greater than 12% loss of F_max prior to extraction were discarded. High-frequency sinusoidal stiffness was measured at steady-state tension by small-amplitude (0.05%L_o) high-frequency (1000 Hz) sinusoidal oscillations to examine changes in strong crossbridge binding as previously described (Dantzig et al. 1992).

k_TR–pCa data were fitted with the Hill equation

(1)

using non-linear least-squares regression analysis (SigmaPlot), where k_TR,max is the maximal k_TR at pCa 4.5, n_H is the slope of the curve (associated with the apparent cooperativity in the system), and pCa₅₀ is the pCa value at which half-maximal k_TR is obtained. Reported pCa₅₀ values for k_TR–pCa represent fits to the average data and are reported ±s.e.m. of the fits (Table 2), because fits for individual experiments were highly variable or not possible. For this reason, n_H values are not reported. Analysis of variance (ANOVA) was used to compare between fibre groups after reconstitution and when differences were significant, Student's paired or unpaired t tests were used with statistical significance set at P < 0.05.

Table 2.

k_TR,max and Ca²⁺ sensitivity of k_TR for rabbit psoas fibres reconstituted with sTnC mutants alone and in mixtures with xxsTnC

sTnC condition (n)	kTR,max (s−1)	pCa50 of kTR
Pre-extracted native sTnC (50)	14.0 ± 0.4	5.75 ± 0.02
sTnC (7)	13.5 ± 1.1	5.72 ± 0.02
sTnCF27W (9)	11.8 ± 0.4	5.78 ± 0.01
M80Q sTnC (6)	12.0 ± 1.2	5.90 ± 0.02
M80Q sTnCF27W (9)	13.2 ± 0.9	5.92 ± 0.03
sTnC:xxsTnC (6)	10.8 ± 1.0	5.29 ± 0.05
M80Q sTnC: xxsTnC (6)	10.1 ± 1.7	5.66 ± 0.06
M80Q sTnCF27W: xxsTnC (7)	9.5 ± 1.4	5.59 ± 0.08

n, number of fibres. k_TR,max is not different between all experimental groups by ANOVA. See Methods for curve fitting of k_TR–pCa data.

Myofibril mechanical data acquisition and analysis

For myofibril experiments, mechanical data were collected from single or small bundles of skeletal myofibrils attached between two glass microtools and perfused with solution that was rapidly changed as previously described (Colomo et al. 1997, 1998; Tesi et al. 1999, 2000). Briefly, myofibrils adhered strongly to the two glass microtools, a post and a calibrated tension probe (compliance 2.9–4.9 nm nN⁻¹; frequency response 2–3 kHz in experimental solutions). Sarcomere length (SL) was initially set 10–20% above slack length (Tesi et al. 1999) and was 2.60 ± 0.01 μm (mean ±s.e.m., n = 56) by calibrated visualization with a camera and monitor. At this SL, average myofibril L_o was 57 ± 2 μm and diameter was 2.14 ± 0.03 μm (1–4 myofibrils). Photoelectric detection of tension probe deflection was used to calculate tension. Motorized solution switching between two streams from a double-barrelled pipette and solution flow by gravity were as previously described (Tesi et al. 1999, 2000). The apparatus was constructed on a Nikon inverted microscope (Diaphot, Japan) with the glass post microtool attached to the lever arm of an electromagnetic length control motor (Colomo et al. 1994) used for rapid length changes of the preparation for measurement of k_TR (see below). Activation rate (k_ACT) and k_TR were estimated from mono-exponential fits as previously described (Tesi et al. 2000). Relaxation rate for the slow phase (k_REL,slow) was calculated from the slope of the regression line fitted to the tension trace normalized to the entire amplitude of the tension relaxation transient. The relaxation rate for the fast phase (k_REL,fast) was measured from a single exponential decay fitted to the data. For fitting, transition from the slow to rapid phase was determined subjectively from individual traces. The duration of the slow phase was measured from tension traces from the onset of solution change at the myofibril to the intercept of the regression line with the fitted exponential. Analysis of variance (ANOVA) was used to compare between myofibril groups after TnC exchange and when differences were significant, Student's unpaired t tests were used with statistical significance set at P < 0.05. All values reported in Table 3 for myofibril tension and kinetics are means ±s.e.m.

Table 3.

Tension generation and relaxation parameters for rabbit psoas myofibrils exchanged with sTnC mutants at 15°C

Myofibril sTnC batch	Tension generation			Relaxation
	Fmax (mN mm−2)	kACT (s−1)	kTR (s−1)	Slow phase		Fast phase kREL (s−1)
				Duration (ms)	kREL (s−1)
Sham-treated	340 ± 22 (9)	8.48 ± 0.62 (9)	8.80 ± 0.61 (9)	75 ± 7 (9)	1.72 ± 0.25 (8)	38.1 ± 6.0 (9
WT sTnC	330 ± 43 (11)	8.17 ± 0.40 (11)	8.23 ± 0.49 (11)	76 ± 5 (11)	1.43 ± 0.14 (11)	44.2 ± 5.2 (11)
M80Q sTnCF27W	331 ± 36 (13)	8.06 ± 0.37 (13)	8.36 ± 0.45 (13)	102 ± 8 (13)#†	1.53 ± 0.22 (13)	29.1 ± 2.9 (12)†
I60Q sTnCF27W	176 ± 16 (13)*°	3.25 ± 0.32 (13)*°	3.32 ± 0.33 (13)*°	42 ± 3 (13)*°	1.53 ± 0.21 (13)	49.6 ± 2.9 (13)

Number in parentheses is number of myofibrils. F_max, maximum isometric tension; k_ACT, rate constant of tension rise following step-wise pCa decrease (8.0–3.5); k_TR, rate constant of tension redevelopment following release-restretch of myofibril; k_REL, rate constant of tension relaxation for slow and fast phases following step-wise pCa increase (3.5–8.0). *P < 0.01 versus sham-treated; °P < 0.01 versus WT sTnC; #P < 0.05 versus sham-treated; †P < 0.05 versus WT sTnC.

Rate of tension redevelopment (k_TR)

The rate of tension redevelopment (k_TR) was determined from tension traces following a rapid release–restretch transient as previously described for fibres (Chase et al. 1994; Regnier et al. 1996, 1998) and for myofibrils (Tesi et al. 2000). Briefly, once steady-state tension was achieved, the fibre was shortened by 0.15 L_o at a 10 L_o s⁻¹ ramp, held at this position for 20 ms, then followed by a rapid (300 μs) under-damped restretch to 1.05 L_o (held for 1 ms) and finally released back to L_o. Myofibrils were shortened by 0.30 L_o with a step length change, held at this position for 35 ms, then rapidly restretched to L_o. Tension in both fibres and myofibrils was reduced to zero during shortening, transiently spiked with rapid restretch, then redeveloped to steady-state levels characterized by an apparent rate constant. The apparent rate constant, k_TR, was estimated for myofibrils from mono-exponential fits (see above) and was calculated for fibres from the half-time of tension recovery (t_1/2) after restretch as

(2)

Corresponding tension values at a given pCa are normalized to the maximum for each condition (Figs 1A and B, and 4) or to the maximum just prior to extraction of sTnC (Figs 1C, 3 and 5).

Figure 1. Rate of tension redevelopment (k_TR) in single permeabilized rabbit psoas fibres reconstituted with purified sTnC, recombinant mutant M80Q sTnC^F27W, or mixtures of D28A, D64A sTnC (xxsTnC) with either sTnC or M80Q sTnC^F27W to ∼0.2 F_max at saturating Ca²⁺ (pCa 4.5; A and B) and maximal k_TRversus relative reconstituted F_max for all thin filament reconstitution conditions (C).

A and B, example tension traces comparing k_TR in different fibres after extraction of native sTnC and reconstitution with 100% purified sTnC, 100% M80Q sTnC^F27W, or mixtures of sTnC and xxsTnC or M80Q sTnC^F27W and xxsTnC to recover ∼0.2 F_max. Reconstituted F_max was 1.00 for 100% sTnC, 0.82 for 100% M80Q sTnC^F27W, 0.18 for sTnC: xxsTnC, and 0.18 for M80Q sTnC^F27W: xxsTnC. Traces for 100% sTnC and 100% M80Q sTnC^F27W were normalized relative to their own maximal tension at maximal Ca²⁺ (pCa 4.5). Tension traces of maximal k_TR with xxsTnC mixtures are shown at their reconstituted tension levels, ∼0.2 F_max. Values of k_TR are given in the figure. Dotted line indicates zero tension. Pre-extracted F_max was 435 mN mm⁻² for 100% sTnC, 257 mN mm⁻² for 100% M80Q sTnC^F27W, 242 mN mm⁻² for sTnC: xxsTnC, and 365 mN mm⁻² for M80Q sTnC^F27W: xxsTnC. C, relationship between maximal k_TR (pCa 4.5) and reconstituted F_max for fibres reconstituted with sTnC (○), M80Q sTnC (▿), sTnC^F27W (◊), or M80Q sTnC^F27W (□) and fibres reconstituted with xxsTnC in mixtures with sTnC (grey circles), M80Q sTnC (grey arrowheads) or M80Q sTnC^F27W (grey squares) to ∼0.2 F_max. Reconstituted F_max for each condition was normalized relative to pre-extracted F_max (•). Maximal k_TR values and number of fibres for each group are given in Table 2. Reconstituted F_max was 0.96 ± 0.01 for sTnC, 0.88 ± 0.02 for sTnC^F27W, 0.85 ± 0.03 for M80Q sTnC, 0.81 ± 0.01 for M80Q sTnC^F27W, 0.23 ± 0.03 for sTnC: xxsTnC, 0.20 ± 0.01 for M80Q sTnC, and 0.20 ± 0.01 for M80Q sTnC^F27W: xxsTnC as previously reported (Kreutziger et al. 2007). Maximal k_TR values are not different between all groups by ANOVA. Some error bars are smaller than symbols.

Figure 4. Example tension traces and Ca²⁺ sensitivity of k_TR for fibres with 100% sTnC or 100% M80Q sTnC^F27W (A–D) or reduced near-neighbour RU interactions (E and F).

A, example tension traces at pCa 6.0 for fibres reconstituted with 100% sTnC or 100% M80Q sTnC^F27W. Traces are normalized to their own F_max. B, example tension traces at different pCa values but similar tension levels at ∼0.88 F_max and ∼0.35 F_max. Reconstituted F_max was 0.97 for 100% sTnC and 0.82 for 100% M80Q sTnC^F27W. The trace for M80Q sTnC^F27W at pCa 6.0 is the same for panels A and B. In A and B, dotted line indicates zero tension and pre-extracted F_max was 308 mN mm⁻² for 100% sTnC and 257 mN mm⁻² for 100% M80Q sTnC^F27W. C–F, k_TR–pCa relationships are shown for fibres prior to sTnC extraction (•) and following reconstitution with sTnC (○; C), M80Q sTnC^F27W (□; D), sTnC: xxsTnC (grey circles; E), and M80Q sTnC^F27W: xxsTnC (grey squares; F). Reconstituted F_max values are given in the legend to Fig. 1C. Some error bars are smaller than symbols. *Maximal values just after reconstitution (in a back-to-back comparison with pre-exchange F_max and k_TR,max). ^#Maximal values at the end of the pCa curve (demonstrating extent of fibre run-down).

Figure 3. Tension–pCa relationship in skeletal fibres reconstituted with I60Q sTnC.

Extraction of native sTnC was followed by reconstitution with I60Q sTnC (▵), yielding 0.59 F_max at saturating Ca²⁺ (pCa 3.5; n = 4 fibres). Normalized tension–pCa shows curve fits to the Hill equation for purified sTnC and M80Q sTnC^F27W as previously published (dashed and dotted curves, respectively; Kreutziger et al. 2007). Fibres with I60Q sTnC had a reduced Ca²⁺ sensitivity (continuous curve; pCa₅₀= 4.55 ± 0.07) and slope (n_H= 1.7 ± 0.2) versus purified sTnC (pCa₅₀= 5.96 ± 0.03; n_H= 3.2 ± 0.4; Kreutziger et al. 2007).

Figure 5. Relationship between k_TR and relative steady-state tension as pCa was varied.

k_TR values at each pCa in Fig. 4 were re-plotted versus the steady-state isometric tension achieved at that pCa (i.e. data were binned by pCa). Tension was normalized relative to the pre-extracted F_max for each group. Reconstitution conditions for each panel and the symbols are the same as in Fig. 4. Some error bars are smaller than symbols. *Maximal values just after reconstitution. ^#Maximal values at the end of the pCa curve.

Troponin C replacement

Replacement of native sTnC in fibres was accomplished by extracting native sTnC, followed by reconstituting troponin complexes with recombinant or purified sTnC. sTnC extraction solution contained (mm): Mops 10, EDTA 5 and trifluoperazine (TFP) 0.5 at pH 6.6 at 15°C. Selective sTnC extraction was completed to 1.3 ± 0.1%F_max (n = 50) with repeated incubations in extraction solution (30 s) and pCa 9.0 solution (10 s) as previously described (Regnier et al. 1999; Moreno-Gonzalez et al. 2005). Reconstitution with 1 mg ml⁻¹ of total sTnC protein in pCa 9.0 solution was completed with two incubations of 2 min and 1 min. If F_max did not increase after the second incubation, reconstitution was considered complete. Reported reconstituted F_max values (Fig. 1C) are from back-to-back measurements in pCa 4.5 comparing just prior to extraction and immediately following reconstitution. To ensure that Tn complexes were complete after reconstitution with I60Q sTnC, an additional 1 min incubation in purified sTnC was performed and tension measured. No increase in F_max at pCa 3.5 (to ensure saturation of tension; Fig. 3) indicated that all Tn complexes were complete and the observed reduction in F_max was in fact due to the sTnC properties and not incomplete reconstitution. ANOVA and Student's t tests were used to compare reconstituted F_max between fibre groups. Replacement of native sTnC in myofibrils was accomplished by passive sTnC exchange. Exchange of sTnC in myofibrils was carried out at 4°C overnight by incubating homogenized myofibrils in relaxing solution added with 0.05 mg ml⁻¹ recombinant sTnC. Batches of myofibrils prepared each time included untreated myofibrils (as a control for quality of preparation), sham-treated myofibrils (as a control for the exchange protocol; ‘Sham-treated’ in Table 3), and sTnC-treated myofibrils (test groups) including WT sTnC (as a control for sTnC mutant-exchanged myofibrils), M80Q sTnC^F27W, I60Q sTnC^F27W and xxsTnC (which does not bind Ca²⁺ at either site I or site II, as a control for extent of exchange in myofibrils). Following this exchange protocol no myofibrils developed any significant tension in relaxing solution at 2.6 μm SL and no significant difference was found in F_max and kinetic parameters between untreated, sham-treated and WT sTnC-exchanged myofibrils. This indicates that the sTnC-exchange protocol per se is not affecting myofibril mechanics, kinetics and regulation. Myofibrils exchanged with xxsTnC resulted in ∼5%F_max values compared with sham-treated or WT sTnC-exchanged myofibrils, suggesting that > 90% of sTnC was exchanged. Qualitatively similar results were obtained by an extraction–reconstitution protocol previously described (Piroddi et al. 2003). In the current study we chose to use the exchange protocol because tension levels were better maintained compared with controls.

Ca²⁺ dissociation rate from sTn

Ca²⁺ dissociation rate (k_off) from whole sTn (containing I60Q sTnC or I60Q sTnC^F27W and native purified sTnI and sTnT) was measured at 15.0°C using an Applied Photophysics Ltd (Leatherhead, UK) model SX-18MV stopped-flow instrument as previously described (Tikunova et al. 2002; Gomes et al. 2004; Kreutziger et al. 2007). Briefly, Ca²⁺ was removed from sTn using the fluorescing Ca²⁺ chelator Quin-2 (Calbiochem) with excitation at 330 nm. Increases in Quin-2 fluorescence were monitored through a 510 nm broad bandpass interference filter (Oriel) and two to four raw data traces averaged before being fitted with a single exponential curve. Reported k_off values represent the mean ±s.e.m. of the rate reported from six to eight average traces for each sTn with variance less than 3.3 × 10⁻³ (Table 1). Whole sTn (6 μm) with no additional Ca²⁺ was rapidly mixed with 150 μm Quin-2. The buffer used was (mm): Mops 10, KCl 150, MgCl₂ 1, DTT 1, pH 7.0 at 15°C as previously described (Kreutziger et al. 2007). Newly reported k_off for I60Q sTnC-sTn and I60Q sTnC^F27W-sTn was measured at the same time and under the same conditions as measurements for M80Q-containing mutants, first reported in Kreutziger et al. (2007), allowing for direct comparisons between k_off values between all of these whole sTn complexes. Values for k_off from whole sTn complexes are repeated in Table 1 taken from Kreutziger et al. (2007) for ease of comparison for WT sTnC-sTn, M80Q sTnC-sTn, sTnC^F27W-sTn and M80Q sTnC^F27W-sTn.

Table 1.

Ca²⁺ dissociation rate (k_off) measured in whole sTn with sTnC mutants at 15°C

sTnC in sTn (n)	koff (s−1)
WT sTnC (6)	5.57 ± 0.04*
sTnCF27W (7)	4.57 ± 0.02*
M80Q sTnC (7)	3.25 ± 0.01*
M80Q sTnCF27W (7)	2.16 ± 0.02*
I60Q sTnC (8)	79.69 ± 2.33
I60Q sTnCF27W (8)	62.01 ± 1.06

^*Previously reported in Kreutziger et al. (2007) and repeated here for comparison. n, number of traces (see Methods).

Results

Maximal rate of tension redevelopment in skeletal fibres

To determine how increased Ca²⁺-binding affinity of sTnC influences contractile kinetics, we measured the rate of tension redevelopment (k_TR) following a release–restretch length transient in single skinned rabbit psoas fibres. Example tension traces at saturating Ca²⁺ (pCa 4.5) are shown in Fig. 1 and are used to determine the maximal k_TR (k_TR,max). The top trace in Fig. 1A is from a fibre reconstituted with 100% sTnC from which k_TR was calculated to be 12.7 s⁻¹. To study how an increased affinity of sTnC altered k_TR, fibres were reconstituted with 100% M80Q sTnC^F27W This sTnC mutant has a 61% slower Ca²⁺ dissociation rate (k_off) and an increased steady-state Ca²⁺ affinity (K_d) as we previously reported (Table 1 and Kreutziger et al. 2007). k_TR,max following reconstitution was 13.0 s⁻¹ (Fig. 1B, top trace), suggesting no difference compared with fibres containing 100% sTnC.

To reduce near-neighbour regulatory unit (RU) interactions in the thin filament, which are a major source of cooperative activation in skeletal muscle (Regnier et al. 2002; Moreno-Gonzalez et al. 2005; Kreutziger et al. 2007), fibres were reconstituted with mixtures of sTnC and D28A, D64A sTnC (xxsTnC; which does not bind Ca²⁺ in N-terminal sites I and II). A mixture ratio of ∼20% sTnC and ∼80% xxsTnC (denoted 20: 80 sTnC: xxsTnC) was used to achieve ∼0.2 of the pre-extracted maximal Ca²⁺-activated tension (F_max). An example tension trace from a fibre reconstituted to 0.18 F_max shown in Fig. 1A (bottom trace) resulted in a k_TR,max of 11.0 s⁻¹. This agrees with our previous report suggesting that near-neighbour RU interactions have little or no effect on k_TR,max (Moreno-Gonzalez et al. 2007). Reconstitution of fibres with M80Q sTnC^F27W: xxsTnC required using a ∼40: 60 ratio mixture to achieve 0.2 F_max (see Kreutziger et al. 2007) for explanation of mixture content versus reconstituted F_max). An example tension trace for a fibre reconstituted to 0.18 F_max resulted in a k_TR,max of 11.2 s⁻¹ (Fig. 1B, bottom trace). This suggests that increased Ca²⁺-binding affinity of sTnC does not influence k_TR,max even with severely reduced near-neighbour RU interactions.

k_TR,max data for all fibre groups are shown in Fig. 1C as a function of reconstituted F_max to examine how kinetics are related to total tension generating capacity in skeletal fibres. In addition to 100% sTnC and 100% M80Q sTnC^F27W, some fibres were reconstituted with two other mutants, sTnC^F27W and M80Q sTnC. k_off values for sTnC mutants in whole sTn complex decreased in the order of WT sTnC > sTnC^F27W > M80Q sTnC > M80Q sTnC^F27W, as previously reported (Table 1 and Kreutziger et al. 2007). In spite of the differences in k_off, k_TR,max did not differ between fibre groups (Table 2). k_TR,max was also unaffected for fibres reconstituted with mixtures of xxsTnC and native or mutant sTnC to obtain ∼0.2 F_max (Fig. 1C). All fibre groups showed slight reductions in k_TR,max by paired t test versus pre-extracted k_TR,max, possibly due to fibre run-down over the course of the experiment. Statistical analysis of the reconstituted k_TR,max values with an ANOVA test showed no difference between any of the reconstituted values, including those with reduced near-neighbour RU interactions. The combined data suggest that increasing Ca²⁺ affinity of sTnC does not increase k_TR,max in skeletal fibres and that k_TR,max in skeletal muscle is not dependent on F_max or thin filament near-neighbour RU interactions, as previously suggested (Moreno-Gonzalez et al. 2007).

Maximal activation and relaxation kinetics in skeletal myofibrils

Single or small bundles of rabbit psoas myofibrils were used to measure tension activation with a rapid increase in Ca²⁺ and then relaxation with a rapid decrease in Ca²⁺ for the same preparation. k_TR can also be measured during this activation–relaxation cycle, which enables comparison of activation rate (k_ACT) and k_TR. Measurements were made at maximal Ca²⁺ (pCa 3.5) to determine if maximal k_ACT could be increased by a sTnC mutant with greater Ca²⁺ affinity even though k_TR,max is unchanged. Example traces of maximal activation–relaxation cycles are shown in Fig. 2A for myofibrils exchanged with wild-type (WT) sTnC, M80Q sTnC^F27W and I60Q sTnC^F27W. A rapid solution change from pCa 8 to pCa 3.5 (first arrow) activated the myofibrils, causing a rise in tension that is characterized by k_ACT (shown normalized to F_max for each trace in Fig. 2B). Once steady-state tension was reached, a length transient was used to measure k_TR (length trace is shown below tension trace in Fig. 2A). Bi-phasic relaxation occurred with rapid solution change from pCa 3.5 to pCa 8 (second arrow in Fig. 2A) and is shown with greater temporal resolution and normalized to F_max for each trace in Fig. 2C. Relaxation is characterized by a slow phase rate (k_REL,slow) and duration and by a fast phase rate (k_REL,fast; see Methods for parameter estimation). Sham-treated myofibrils produced an F_max of 340 ± 22 mN mm⁻² and were not different from untreated myofibrils (data not shown). F_max was not different for WT sTnC-exchanged myofibrils (Table 3). To determine the extent of sTnC exchange, a subgroup of myofibrils was exchanged with 100% xxsTnC for each experimental batch. These myofibrils produced 17 ± 9 mN mm⁻² tension (n = 10) or ∼5% of sham-treated or WT F_max, suggesting that greater than 90% of native sTnC was exchanged for exogenous sTnC. Sham-treated and WT sTnC-exchanged myofibrils did not differ for any kinetic parameters (Table 3). Parameters describing bi-phasic relaxation (k_REL,slow, duration and k_REL,fast; reviewed in Poggesi et al. 2005) for sham-treated and WT sTnC-exchanged myofibrils were similar to previously reported values (Tesi et al. 2002; de Tombe et al. 2007). These control experiments suggest the exchange procedure did not alter myofibril mechanics. As the M80Q sTnC^F27W mutant had the most dramatic reduction of k_off, we focused on this sTnC mutant to examine how decreased Ca²⁺ dissociation from sTnC affected tension kinetics. For M80Q sTnC^F27W-exchanged myofibrils, there was no change in k_ACT or k_TRversus WT sTnC (or sham-treated myofibrils) at maximal Ca²⁺, similar to comparisons for F_max (Table 3; Fig. 2B). These data suggest that in the presence of saturating Ca²⁺, a higher affinity of sTnC for Ca²⁺ (with M80Q sTnC^F27W) does not alter the relationship between k_ACT and k_TR. During relaxation with M80Q sTnC^F27W, there was no change in k_REL,slow and a small decrease in k_REL,fast (P < 0.05 versus WT sTnC; although t test versus sham-treated indicates no significant difference). Surprisingly, the duration of the slow phase of relaxation was increased by 34% for M80Q sTnC^F27W (P < 0.05; Fig. 2C; Table 3). These data suggest that increasing Ca²⁺ affinity of sTnC (via decreased k_off) prolongs thin filament activation upon rapid removal of Ca²⁺, thus delaying the onset of the fast phase of relaxation, without altering the rate at which isometric relaxation occurs. To further examine how Ca²⁺ affinity of sTnC affects tension development and relaxation kinetics in myofibrils, we used a mutant sTnC with an increased Ca²⁺-k_off. I60Q sTnC and I60Q sTnC^F27W had k_off values that were 14-fold and 11-fold faster, respectively, in whole sTn complex at 15°C versus WT sTn (Table 1). Characterization of I60Q sTnC in single skinned psoas fibres (Fig. 3) showed reduced F_max (by ∼40%), pCa₅₀ (by 1.41 pCa units) and k_TR,max (by ∼70%; Tanner, 2007). Due to the severe loss of Ca²⁺ sensitivity with I60Q sTnC, pCa 3.5 was used to saturate the thin filament and Ca²⁺-activated tension, which is saturated and well-fitted by the sigmoidal Hill equation (Fig. 3). These changes in tension–pCa were similar to previous experiments using other TnCs with increased k_off (Moreno-Gonzalez et al. 2007), and should be similar for I60Q sTnC^F27W (not characterized in fibres) because of the comparable increase in k_off. In skeletal myofibrils, our goal was to determine if faster k_off differentially affected activation (k_ACT) and relaxation kinetics. Exchanging I60Q sTnC^F27W into myofibrils resulted in 0.53 F_maxversus WT sTnC myofibrils (Table 3). With an increased k_off, I61Q sTnC^F27W slowed both k_ACT and k_TR by ∼60% at maximal Ca²⁺, in contrast to when k_off was reduced with M80Q sTnC^F27W (Table 3). Examining relaxation showed that the duration of the slow phase of relaxation was reduced by 45% with I60Q sTnC^F27W. However, increasing k_off with I60Q sTnC^F27W had no effect on k_REL,slow and k_REL,fast, similar to results when k_off was reduced with M80Q sTnC^F27W. Combined, the data indicate that k_off has little influence on relaxation rates but does influence the duration of the isometric slow phase. In addition, decreases in k_off do not influence tension development rates, but large increases in k_off can dramatically decrease both steady-state tension and the rate of tension development.

Figure 2. Activation, k_TR and relaxation in single rabbit psoas myofibrils upon rapid change in pCa for a WT sTnC-exchanged myofibril, a M80Q sTnC^F27W-exchanged myofibril, and a I60Q sTnC^F27W-exchanged myofibril.

A, example tension traces of activation–relaxation cycles in single or small bundles of myofibrils show tension through time. Activation (characterized by k_ACT) upon rapid increase in Ca²⁺ from pCa 8 to pCa 3.5 (first arrow), k_TR with a release–restretch length transient, and relaxation with rapid decrease in Ca²⁺ (second arrow) are shown. Changes in myofibril length are seen in bottom trace for k_TR measurement. B, expanded time scale with tension normalized to maximum for each condition during activation (pCa 8.0 to 3.5; arrow) shows a reduced k_ACT for I60Q sTnC^F27W. C, expanded time scale with normalized tension traces during relaxation (pCa 3.5 to pCa 8.0; arrow) shows a prolongation of slow phase duration with M80Q sTnC^F27W and a reduction of slow phase duration with I60Q sTnC^F27W. Myofibril diameter, L_o and SL were 1.91, 42 and 2.57 μm, respectively (WT sTnC); 2.00, 49.4 and 2.63 μm (M80Q sTnC^F27W); 1.72, 29.8 and 2.55 μm (I60Q sTnC^F27W). Tension and kinetic parameters are reported in Table 3.

Ca²⁺ sensitivity of the rate of tension redevelopment in skeletal fibres

At submaximal levels of Ca²⁺ activation, altering the Ca²⁺ affinity of sTnC had striking effects on steady-state tension and k_TR. As with myofibril studies, we focused on the M80Q sTnC^F27W mutant because it had the most dramatic reduction of k_off. Reconstitution of fibres with 100% M80Q sTnC^F27W resulted in higher tension and faster k_TR at most submaximal levels of Ca²⁺. For the example tension traces in Fig. 4A, steady-state tension was greater by 2/3 and k_TR was more than doubled for the M80Q sTnC^F27W reconstituted fibre compared with 100% sTnC at pCa 6.0 (P < 0.01 for unpaired t test). The elevated k_TR with M80Q sTnC^F27W was not completely explained by the higher tension level, as demonstrated by tension-matched traces in Fig. 4B. At a lower tension level (0.35 F_max) M80Q sTnC^F27W had an elevated k_TR at 3.8 s⁻¹versus 2.2 s⁻¹ with sTnC. However, this effect was eliminated at higher tensions, as shown by the upper two traces at 0.88 F_max in Fig. 4B. These data suggest that the decreased Ca²⁺-k_off of M80Q sTnC^F27W may increase k_TR at lower, but not higher, levels of submaximal thin filament activation.

The k_TR data as Ca²⁺ was varied for all fibres reconstituted with 100% sTnC or 100% M80Q sTnC^F27W are summarized in Fig. 4C and D, respectively. Plots of k_TR–pCa are shown in each panel for pre-extracted (filled circles) and reconstituted fibres. Data of average values were well-fitted by the Hill equation (eqn (1); fits not shown) and pCa₅₀ values are reported in Table 2. In control experiments, the full range of Ca²⁺ dependence of k_TR was maintained following reconstitution with 100% sTnC and values were similar at all pCa levels (Fig. 4C). The rise in steady-state tension with increasing Ca²⁺ preceded that of k_TR (Kreutziger et al. 2007). Fibres reconstituted with 100% sTnC showed no change in Ca²⁺ sensitivity (pCa₅₀) of k_TRversus pre-extracted value (Table 2), demonstrating the sTnC extraction–reconstitution protocol did not alter the k_TR–pCa relationship. k_TR was fairly constant at low Ca²⁺ (≥ pCa 6.0) and the rise in k_TR lagged behind that of tension and did not increase above ∼2 s⁻¹ until tension was ≥ 0.5 F_max. Overall, for all thin filament reconstitution conditions (1) the k_TR–pCa data maintain a large range of k_TR values from low Ca²⁺ to maximum Ca²⁺ (i.e. strong Ca²⁺ dependence of k_TR; Fig. 4C–F), and (2) the change in Ca²⁺ sensitivity of k_TR (pCa₅₀ of k_TR) is similar in direction and magnitude to the change in pCa₅₀ of tension (Kreutziger et al. 2007). For fibres reconstituted with 100% M80Q sTnC^F27W, Ca²⁺ dependence of k_TR (defined as the range of k_TR values from low to high Ca²⁺) was reduced due to a small but significant elevation of low-Ca²⁺k_TR values (P < 0.05 by paired t test at each pCa > 6.1; Fig. 4D). pCa₅₀ of k_TR increased with M80Q sTnC^F27W by ∼0.2, shifting k_TR data to the left (Fig. 4D; Table 2). As noted above, k_TR,max was reduced for all reconstitution conditions versus pre-extracted k_TR,max (by paired t test), but there was no difference in k_TR,max between reconstitution groups by ANOVA. These data demonstrate that M80Q sTnC^F27W has a greater effect on k_TR at lower Ca²⁺ levels.

Reduction of near-neighbour RU interactions in the thin filament using sTnC: xxsTnC mixtures allowed for examination of how these cooperative interactions alter k_TR–pCa. Further, mixtures of xxsTnC and the mutant M80Q sTnC^F27W (with a decreased k_off) were used to examine how Ca²⁺ binding properties of sTnC are coupled to near-neighbour RU interactions for setting k_TR. Reconstitution to 0.2 F_max with sTnC: xxsTnC slightly reduced the Ca²⁺ dependence of k_TR via decreased k_TR,max (P < 0.05 by paired t test; not different from reconstitution groups by ANOVA) and via slightly elevated k_TR at low Ca²⁺ (not significant for pCa 6.2–5.8), similar to previous reports (Fitzsimons et al. 2001; Moreno-Gonzalez et al. 2007). pCa₅₀ of k_TR was reduced by ∼0.4 with sTnC: xxsTnC, which is probably due to both a rightward shift and loss of slope of the k_TR–pCa relationship (Fig. 4E; Table 2). Reconstitution to 0.2 F_max with M80Q sTnC^F27W: xxsTnC (to reduce near-neighbour RU interactions and increase Ca²⁺ affinity of sTnC) also reduced Ca²⁺ dependence of k_TR via decreased k_TR,max (P < 0.05 by paired t test; not different from reconstitution groups by ANOVA) and via elevated k_TR at low Ca²⁺ (P < 0.01 for pCa 6.4–6.0; P < 0.02 for pCa 5.9). pCa₅₀ of k_TR was reduced by ∼0.1 in fibres reconstituted with M80Q sTnC^F27W: xxsTnC mixtures (Fig. 4F; Table 2), which was less than the loss of Ca²⁺ sensitivity of k_TR in fibres with sTnC: xxsTnC. Interestingly, the change in pCa₅₀ of k_TR (ΔpCa₅₀) between M80Q sTnC^F27W and sTnC was 0.2 and between mixtures M80Q sTnC^F27W: xxsTnC and sTnC: xxsTnC was 0.3. These ΔpCa₅₀ values suggest that increased Ca²⁺ affinity of sTnC can increase the Ca²⁺ sensitivity of k_TR in both the presence and absence of near-neighbour RU interactions.

To examine how Ca²⁺-dependent increases in k_TR depended on the number of strong-binding crossbridges available for tension generation and thin filament activation, the data of Fig. 4C–F were replotted as k_TRversus steady-state tension where data were binned by pCa value (Fig. 5). This relationship exhibits changes in k_TR at similar levels of steady-state tension under different sTnC-reconstitution conditions and independent of Ca²⁺ level. Pre-extracted data (filled circles) followed a concave curve because steady-state tension increased prior to the increase in k_TR with increasing Ca²⁺. Fibres reconstituted with sTnC exhibited the same curvilinear relationship for k_TR–tension (Fig. 5A), verifying that the protocol for incorporating sTnC did not alter the relationship between tension and k_TR in fibres. Reconstitution with M80Q sTnC^F27W increased k_TR at all values of relative tension (open squares, Fig. 5B). As reconstituted F_max was reduced with M80Q sTnC^F27W, it is not clear whether or not the apparent elevation of k_TR at higher, submaximal relative tension levels is due to the k_off of M80Q sTnC^F27W or some other mechanism (like run-down) that reduced F_max. At tension levels < 50%, k_TR was elevated with M80Q sTnC^F27W, suggesting that decreased k_off allowed for faster k_TR at low levels of crossbridge binding and of thin filament activation. For fibres reconstituted with sTnC: xxsTnC, while reduced near-neighbour RU interactions resulted in much lower F_max, k_TR was somewhat elevated at all relative tension levels (Fig. 5C). These data suggest that spread of activation between near-neighbour RUs may limit k_TR at low Ca²⁺ and low tension levels but probably have little effect on k_TR at high Ca²⁺. M80Q sTnC^F27W: xxsTnC reconstituted fibres showed a slightly greater increase in low-tension k_TR values (not significant; Fig. 5D) than either 100% M80Q sTnC^F27W (Fig. 5B) or sTnC: xxsTnC (Fig. 5C). Therefore, Ca²⁺ binding kinetics of sTnC may influence k_TR at low levels of thin filament activation independent of near-neighbour RU interactions, and certainly both near-neighbour RU interactions and k_off of sTnC contribute to k_TR at low Ca²⁺ and low tension.

To better understand the mechanism by which k_TR was increased at low tension levels (< 50%F_max) for these variations to the thin filament activation properties, we examined fibre stiffness, which includes components of both crossbridge and filament stiffness. High-frequency sinusoidal stiffness was not altered in fibres reconstituted with 100% sTnC (Fig. 6A, white circles) versus pre-extracted control measurements (black circles), suggesting that the extraction–reconstitution protocol did not alter the stiffness–tension relationship. In fibres containing M80Q sTnC^F27W, stiffness increased at Ca²⁺ activated tension levels < 0.2 F_max (P < 0.05) for tension-matched k_TR at < 0.2 F_max (Fig. 6A, white squares) versus pre-extracted native sTnC (black squares). These data suggest that altered Ca²⁺-binding kinetics of sTnC altered crossbridge and/or filament stiffness. Reduction of near-neighbour RU interactions with xxsTnC mixtures also elevated the low-tension versus stiffness relationship (P < 0.05) at < 0.3 F_max for sTnC: xxsTnC (grey cirles) or M80Q sTnC^F27W: xxsTnC (grey squares) for tension-matched comparisons with pre-extracted native values (black symbols; Fig. 6B). Interestingly, this elevation of stiffness at low tension due to reduced near-neighbour RU interactions was independent of whether mixtures contained sTnC or M80Q sTnC^F27W. These observed effects of k_off and near-neighbour RU interactions on fibre stiffness could contribute to the elevation of k_TR at low levels of Ca²⁺ activation. All together, the data suggest that sTnC's Ca²⁺-binding properties in individual RUs and interactions between neighbouring RUs can both influence k_TR at low Ca²⁺ or low tension levels.

Figure 6. Relationship between normalized stiffness and tension as pCa was varied in the presence (A) and absence (B) of near-neighbour RU interactions.

High-frequency sinusoidal stiffness was measured in a subset of experiments prior to extraction for each group (black symbols) and after reconstitution with 100% sTnC (white circles; n = 6 fibres; A), 100% M80Q sTnC^F27W (white squares; n = 4 fibres; A), sTnC: xxsTnC (grey circles; n = 3 fibres; B), and M80Q sTnC^F27W: xxsTnC (grey squares; n = 4 fibres; B) and normalized to maximal stiffness for each condition.

Discussion

The main goal of this study was to examine how altering the Ca²⁺ dissociation rate (k_off) of sTnC influenced the kinetics of tension activation and relaxation as Ca²⁺ was varied and as near-neighbour regulatory unit (RU) interactions were reduced in skeletal muscle. Both single fibre and myofibril preparations were used because of their individual advantages. Single fibres offer the ability for multiple Ca²⁺ activations, but their size limits the ability to investigate activation and relaxation kinetics due to diffusion limitations. Myofibrils do not have these diffusion limitations because of their size, but the quality of mechanical measurements decays after a few activations. Our main findings are that decreasing k_off of sTn does not change the maximal rates of tension development (measured as k_ACT, k_TR) or relaxation (k_REL,slow and k_REL,fast), but increases k_TR at low tension levels in the absence or presence of near-neighbour RU interactions. However, the duration of slow phase, isometric relaxation is prolonged. In contrast, increasing the k_off of sTn reduces maximal tension development kinetics and shortens the duration of slow phase relaxation. Additionally, reduced near-neighbour RU interactions had no effect on maximal tension development kinetics but increased k_TR and stiffness at low tension levels, and there was little or no effect on the k_TR–tension relationship when k_off was decreased (Fig. 5C and D). These results suggest that native sTn may be optimized to provide maximal activation kinetics and to modulate the rate of force development with small changes in intracellular Ca²⁺. This appears to be primarily a function of individual sTn complexes, with some modulation of rate by near-neighbour RU interactions at low Ca²⁺. Additionally, we have shown for the first time that increasing k_off of sTn affects both activation and relaxation, while decreasing k_off only affects relaxation. Below we discuss results with altered k_off of sTnC in thin filaments with the full complement of functional regulatory units, followed by a discussion of reduced near-neighbour RU interactions.

Influence of k_off on the maximal rate of tension development

We found that maximal k_TR (k_TR,max) was not altered by sTnC mutants having a decreased Ca²⁺ dissociation rate (k_off) in fibres and myofibrils (Fig. 1C; Tables 2 and 3). This agrees with previous reports using calmidazolium or bepridil to decrease Ca²⁺–k_off (Regnier et al. 1996; de Tombe et al. 2007) and suggests that at saturating Ca²⁺, k_TR,max cannot be increased above that measured with native sTnC by altering the Ca²⁺-binding properties of sTnC. Since k_TR,max is not limited by a lack of available Ca²⁺ or Ca²⁺-binding properties of sTnC, other processes following Ca²⁺ binding that lead to tropomyosin movement or the kinetics of crossbridge transitions must determine k_TR,max (the latter explanation has been suggested previously (Brenner & Eisenberg, 1986; Brenner, 1988; Metzger & Moss, 1990)). However, k_TR,max can be decreased with a sTnC mutant having an increased k_off (I60Q sTnC^F27W) in myofibrils (Table 3). This agrees with our previous reports using cTnC or a sTnC mutant with a single N-terminal Ca²⁺ binding site where k_off is increased relative to native sTnC (Morris et al. 2001; Piroddi et al. 2003; Moreno-Gonzalez et al. 2007). The k_TR data in myofibrils with I60Q sTnC^F27W at saturating Ca²⁺ (Table 3) can be simulated using a four-state model described by Regnier et al. (1999), as was done by Moreno-Gonzalez et al. (2007). Although increases in k_off alone were not sufficient to simulate reduced k_TR,max and reduced F_max, increases in both k_off and crossbridge detachment rate (g_app) did simulate these results well. This suggests that thin filament regulatory proteins may alter crossbridge attachment/detachment kinetics.

How the activation process regulates the rate of tension development in skeletal muscle is not well understood. There is some question of whether k_TR is a measure that accurately describes the temporal sequence of myosin–thin filament interactions that occur during the contractile activation process. Another measure of tension development kinetics, the rate of activation (k_ACT) in myofibrils, occurs with a rapid increase from low to high Ca²⁺ (Fig. 2) and takes into account the Ca²⁺ activation pathway of thin filament proteins. Our data in skeletal myofibrils showed that k_ACT did not differ from k_TR at saturating Ca²⁺ for sTnC, M80Q sTnC^F27W or I60Q sTnC^F27W (Table 3). Thus, our data demonstrating that k_ACT is equal to k_TR suggest that thin filament activation kinetics are not limiting to the rate of tension development in skeletal muscle at high levels of Ca²⁺ and low P_i for either native sTnC or M80Q sTnC^F27W On the contrary, when k_off of sTnC was increased (with I60Q sTnC^F27W), both k_ACT and k_TR decreased to a similar extent, suggesting that the Ca²⁺ binding dynamics of I60Q sTnC^F27W had become limiting for both types of measurements. Perhaps the reduced activation rates were due to the severe compromise in Ca²⁺ binding (increased k_off) that did not allow for maximal activation of the thin filament. Indeed, changes in k_off affect Ca²⁺ affinity and the steady-state level of thin filament activation level. Experimental evidence suggests that Ca²⁺ binding and tropomyosin mobility during activation is in rapid equilibrium preceding tension generation (Robertson et al. 1981; Kress et al. 1986). Our data suggest this rapid activation process is maintained even when k_off of sTnC is altered, so that k_ACT and k_TR at high Ca²⁺ are determined by the Ca²⁺-binding equilibrium and resulting activation level of thin filaments.

Influence of k_off on relaxation

To our knowledge, this is the first report suggesting that thin filament Ca²⁺ dissociation alters isometric relaxation from maximal Ca²⁺ activation. We found no change in the kinetics of relaxation (k_REL,slow and k_REL,fast; Table 3), but discovered that the duration of the slow, isometric phase of relaxation was modulated by altered Ca²⁺–k_off of sTnC in skeletal myofibrils. This result was very intriguing, as little is known about the role of thin filament regulatory protein dynamics in tension relaxation. The finding that the rate of slow phase relaxation (k_REL,slow; Table 3) was unchanged by sTnC mutants supports the existing hypothesis that k_REL,slow is primarily determined by the crossbridge detachment rate at maximal Ca²⁺ (Poggesi et al. 2005). In addition, the finding that the rate of fast phase relaxation (k_REL,fast) was not different between WT and mutant sTnCs suggests that the phenomenon of sarcomeric ‘give’ itself is not dependent on Ca²⁺ dissociation kinetics of sTnC and how they influence thin filament de-activation (Stehle et al. 2002; Tesi et al. 2002; Poggesi et al. 2005). Surprisingly, M80Q sTnC^F27W (decreased k_off) increased the duration of slow phase by 34%, while I60Q sTnC^F27W (increased k_off) decreased the duration of the slow phase by 45% (Table 3). These data suggest that while the k_REL,slow is independent of the Ca²⁺ binding kinetics of troponin, the duration of the slow phase and thus the onset of sarcomeric ‘give’ (fast phase) can be influenced by thin filament de-activation via changes in Ca²⁺–k_off. Indeed, the isometric phase of relaxation involves not only Ca²⁺ dissociating from TnC but a regressing population of cycling crossbridges (Gordon et al. 2000), providing a mechanism whereby altered k_off of sTnC could affect isometric relaxation duration without altering the rate of tension decline.

Few studies have probed how changes to Ca²⁺ kinetics of sTnC affect relaxation. Lou et al. (2002) found that the slow phase rate of relaxation from partial Ca²⁺ activation at low (5 μm) Pi was decreased when native sTnC was replaced with chicken M82Q sTnC in skinned skeletal fibres. Our measurements showing no change in slow phase rate, but a prolonged duration were from fully Ca²⁺ activated myofibrils to complete relaxation. Recently published results in skeletal myofibrils using bepridil showed no change in slow phase duration (de Tombe et al. 2007), contrary to our current results where duration of the slow phase was increased with M80Q sTnC^F27W. Bepridil is a Ca²⁺-sensitizing agent that has been characterized in cardiac TnC to bind in the N-terminal domain in the presence of Ca²⁺ and decreases k_off of isolated cardiac TnC (Solaro et al. 1986; MacLachlan et al. 1990; Li et al. 2000; Wang et al. 2002). However, the effect of bepridil on k_off has not been measured either in skeletal TnC or in whole troponin complex, where k_off is greatly slowed from values obtained with isolated TnC. Since M80Q sTnC^F27W also has a decreased Ca²⁺–k_off, we may have expected similar results to the bepridil experiments. The difference in how bepridil and M80Q sTnC^F27W affected slow phase duration may have arisen from differences in the specificity of each of these Ca²⁺ sensitizers. It has been reported that bepridil binds to cardiac TnC in the presence of Ca²⁺ but not in its absence (MacLachlan et al. 1990; Li et al. 2004), thus its action during a step decrease in Ca²⁺ may be complex. The rapid reduction of Ca²⁺ to initiate relaxation in skeletal myofibrils could also initiate bepridil dissociation. Further study in this area is needed to examine how Ca²⁺ dissociation kinetics are related to the kinetics of thin filament de-activation and how these influence relaxation, particularly at submaximal Ca²⁺ levels. In summary, our current data suggest that k_off of native sTnC may be optimal for allowing rapid activation and relatively rapid relaxation (i.e. a fairly short relaxation duration dominated by the fast phase).

Influence of k_off on the rate of tension development (k_TR) at low Ca²⁺

We found that incorporating I60Q sTnC^F27W into myofibrils reduced F_max by ∼50% and reduced k_TR,max by ∼60% to 3.3 s⁻¹, suggesting that compromising thin filament activation level can limit tension development kinetics. Indeed, k_TR in fibres reconstituted with 100% sTnC at Ca²⁺ levels that produced ∼0.5 F_max was 3–4 s⁻¹ (Fig. 5A), which is much less than maximal k_TR but similar to k_TR with I60Q sTnC^F27W in myofibrils. Other studies using the cardiac isoform of TnC (with increased k_off) reconstituted into psoas fibres (Moreno-Gonzalez et al. 2007) or myofibrils (Piroddi et al. 2003) have reported reduced tension and k_TR, similar to our data with I60Q sTnC^F27W in myofibrils and I60Q sTnC in fibres. We recently reported that skeletal troponin containing cardiac TnC has an approximately 50% faster k_off than troponin with a full complement of fast skeletal subunits (Moreno-Gonzalez et al. 2007). Together these data support the idea that the Ca²⁺ binding kinetics of sTnC can become limiting to k_TR if k_off is increased, even at saturating Ca²⁺.

Another line of evidence suggests that Ca²⁺ binding kinetics of sTnC can modulate k_TR. k_TR remained at a constant low value with increasing Ca²⁺ until tension was > 0.5 F_max with native sTnC (Fig. 5A). However, when k_off was decreased (with M80Q sTnC^F27W), k_TR was elevated at tension levels < 0.5 F_max (Fig. 5B). In these experiments, we also measured high-frequency (1000 Hz) sinusoidal stiffness and steady-state force. Control measurements (Fig. 6A, circles) show that the extraction–reconstitution protocol did not affect stiffness–force. However, reconstitution with M80Q sTnC^F27W showed that at tension < 0.2 F_max, stiffness increased relative to force (Fig. 6A, white versus black squares). These data suggest that M80Q sTnC^F27W altered filament and/or crossbridge contributions to stiffness, which agrees with Regnier et al. (1995) where we suggested an increase in stiffness relative to force was indicative of an increase in the population of bound crossbridges in pre-force states (i.e. weakly bound or strongly bound, low-force states). Further, at low levels of Ca²⁺ activation, a greater portion of the length change for measuring stiffness is transferred to the crossbridges versus at high Ca²⁺ activation (Martyn et al. 2002), suggesting that changes in crossbridge distributions could influence the stiffness–force relationship. In particular, the sinusoidal stiffness measurements made with M80Q sTnC^F27W may be detecting increased weak and/or strong pre-force crossbridge binding, thus increasing stiffness relative to force at low Ca²⁺ (Fig. 6A). Therefore, the mechanism of increased k_TR at low tension could involve increased crossbridges in pre-tension states (weakly or strongly bound), as k_TR is an apparent rate of tension generation. This suggests that in native skeletal muscle, the fast k_off of sTnC may limit weak and/or strong pre-force crossbridge binding, contributing to the slow k_TR at tension < 0.5 F_max.

Interestingly, either decreasing or increasing k_off of sTnC increases k_TR at low levels of Ca²⁺ activation, but this probably occurs by different mechanisms. How this might occur was discussed in a recent paper (Moreno-Gonzalez et al. 2007), but it bears some repetition here in the context of the current work. The time that any individual RU remains activated may be increased with decreased k_off and decreased with increased k_off. When k_off is decreased, as for M80Q sTnC^F27W, the effect at low Ca²⁺ is probably greater affinity (compared with WT sTnC) for TnI, and consequently a lower affinity of TnI for actin. This should result in maintaining tropomyosin in a position permissive to crossbridge binding. The possible means of an increased k_off to increase k_TR at low Ca²⁺ are less obvious. We previously reported a positive correlation between whole sTn k_off and elevation of k_TR at low Ca²⁺ when comparing native sTnC with cTnC or D28A sTnC, which both have increased k_off (Fig. 5B in Moreno-Gonzalez et al. 2007). However, for these proteins, both maximal tension and k_TR (pCa 4.0) were greatly reduced, in contrast to our results with M80Q sTnC^F27W (Fig. 1C). This suggests a lower ability to activate the thin filament, even at high Ca²⁺. This could result from lower affinity of cTnC and D28A sTnC for TnI in rabbit psoas fibres at any given [Ca²⁺] and, consequently, greater affinity of TnI for actin. If so it could lead to an active role of Tn to re-stabilize tropomyosin in an inhibitory position and, thus, increase the crossbridge dissociation rate (Clemmens et al. 2005). While testing these ideas is beyond the scope of this study, our past and current data demonstrate the ability of altered k_off to modulate crossbridge cycling kinetics at low levels of Ca²⁺ activation. In addition, the data from current and past studies strongly suggest kinetic coupling between thin filament and crossbridge cycling, especially at low levels of Ca²⁺ activation.

Influence of near-neighbour RU interactions on the rate of tension development (k_TR)

A unique and important aspect of our study was to examine how the Ca²⁺-binding properties of sTnC act locally, within a regulatory unit (RU) of the thin filament, compared with the ensemble effects of troponin complexes along the length of thin filaments. This allowed us to separate the effects of decreased k_off of sTnC from near-neighbour RU cooperative mechanisms on k_TR. We and others have previously shown that near-neighbour RU interactions along the length of the thin filament are the dominant form of cooperative activation in skeletal fibres (as indicated by the slope, n_H, of the tension–pCa relationship; Metzger & Moss, 1991; Regnier et al. 2002). As we previously found (Gillis et al. 2007; Moreno-Gonzalez et al. 2007), loss of near-neighbour RU interactions does not change k_TR,max (Fig. 1), indicating that when the thin filament is saturated with Ca²⁺, near-neighbour RU interactions have little role in determining k_TR. At the lowest levels of Ca²⁺, k_TR is slightly elevated for sTnC: xxsTnC above pre-extracted values (Fig. 4E; not significant by paired t test), similar to results found by Fitzsimons et al. (2001) with partial extraction of sTnC. These data suggest that disrupting near-neighbour RU interactions may have released a limitation on the rate tension can redevelop. In addition, stiffness increased at tension < 0.3 F_max for both sTnC: xxsTnC and M80Q sTnC^F27W: xxsTnC (Fig. 6B), where near-neighbour RU interactions were diminished. This suggests that filament stiffness may have increased with a combination of xxsTnC mixtures in the thin filament and low levels of activating Ca²⁺, as thin filament flexural rigidity increases (and presumably axial stiffness increases) at low [Ca²⁺] with regulated thin filaments (Isambert et al. 1995). Perhaps, under normal conditions, near-neighbour RU interactions increase filament compliance, which could limit k_TR at low Ca²⁺ levels (with little effect at saturating Ca²⁺, as suggested by Razumova et al. 2000). However, our results show that the contribution of near-neighbour RU interactions to determining k_TR is small and the Ca²⁺ dependence of k_TR remains large in the absence of near-neighbour RU interactions (Fig. 4E). This remaining Ca²⁺ dependence may be a direct result of the level of activating Ca²⁺, since more Ca²⁺ is required to achieve a given relative tension level when near-neighbour RU interactions are reduced. This increased Ca²⁺ level may contribute to the elevation of k_TR at the lower end of the k_TR–tension relationship with diminished near-neighbour RU interactions (Fig. 5C), as previously suggested (Moreno-Gonzalez et al. 2007).

The finding that loss of near-neighbour RU interactions does not affect maximal k_TR and only moderately elevates low-tension k_TR demonstrates that much of the Ca²⁺ dependence of k_TR resides within individual RUs. pCa₅₀ of k_TR was reduced by 0.13 (shifted to the right) for fibres reconstituted with M80Q sTnC^F27W: xxsTnC and by 0.43 for fibres reconstituted with sTnC: xxsTnC versus reconstitution with 100% sTnC. These data suggest that increasing the Ca²⁺ affinity of sTnC increases k_TR at submaximal Ca²⁺ levels within RUs in the thin filament. At low Ca²⁺ levels for fibres reconstituted with M80Q sTnC^F27W: xxsTnC, k_TR may be elevated to a greater extent than in sTnC: xxsTnC fibres (Fig. 4E and F), suggesting that the Ca²⁺ binding properties of sTnC and near-neighbour RU interactions act independently of one another to influence k_TR at low Ca²⁺. This results in an extended Ca²⁺ dependence (i.e. range) of k_TR in native skeletal muscle. In other words, the Ca²⁺ binding kinetics of sTnC and near-neighbour RU interactions combine to slow k_TR for tension levels less than 0.5 F_max, and as Ca²⁺ increases and activates more RUs in the thin filament, these two processes become less limiting to the rate of tension development. At the individual RU level, crossbridge binding probability is probably related to time Ca²⁺ is bound to sTnC, but could also include mechanisms of crossbridge-facilitated additional crossbridge binding. However, detailed study at this level is beyond the scope of the current experiments.

In conclusion, we have demonstrated that k_TR in skeletal muscle is primarily determined within RUs of the thin filament, with minor contributions of near-neighbour RU interactions to reducing k_TR at low Ca²⁺. Further, the Ca²⁺ binding kinetics of native sTnC act within RUs to allow for a large Ca²⁺-dependent range of tension development rates. Decreasing (current work) or increasing (Moreno-Gonzalez et al. 2007) k_off increases low-Ca²⁺k_TR and diminishes its Ca²⁺ dependence. Finally, we have demonstrated for the first time that increasing or decreasing k_off can influence the duration of the slow, isometric phase of relaxation in skeletal myofibrils, and that increasing k_off affects activation kinetics. Comparisons between fibre and myofibril data suggest that Ca²⁺ binding to sTnC may not limit the rate of tension rise during activation, but may influence the duration of relaxation. These data suggest that the Ca²⁺ dissociation kinetics of native sTnC are optimized both for maximizing the range of tension development kinetics over all activating levels of Ca²⁺ and for minimizing the duration of relaxation in skeletal muscle.