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CELLULAR |
1 Department of Bioengineering, University of Washington, Seattle WA 98195 USA
2
Department of Biological Science and Program in Molecular Biophysics, and Department of Chemical and Biomedical Engineering, Florida State University, Tallahassee, FL 32306-4370, USA
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionAppendixReferences |
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(Received 1 November 2006;
accepted after revision 18 December 2006;
first published online 4 January 2007)
Corresponding author M. Regnier: University of Washington, Department of Bioengineering, Box 357962, Seattle, WA 98195-7962, USA. Email: mregnier{at}u.washington.edu
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Introduction |
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In rabbit psoas fibres, the relationship between Ca2+-activated steady-state force and ktr is highly curvilinear at 10–15°C, such that ktr is slow (1–2 s–1) and essentially constant at force levels <50% of maximal force (Fmax), while the magnitude increases 10–15-fold as force increases to Fmax. In maximally activated fibres, i.e. at saturating levels of Ca2+, the intrinsic rate of cross-bridge cycling is the primary determinant of ktr (Gordon et al. 2000). In contrast, we and others have shown that a Ca2+-dependent process is rate-limiting to force development during submaximal thin-filament activation in skeletal fibres (Brenner, 1986, 1988; Sweeney & Stull, 1990; Chase et al. 1994; Regnier et al. 1996, 1998b). We have previously shown that Ca2+ modulates ktr primarily through binding to TnC (Chase et al. 1994), leading to the hypothesis that the dynamics of thin-filament activation limits ktr at subsaturating levels of Ca2+ (Chase et al. 1994; Regnier et al. 1996, 1999b; Morris et al. 2001). In addition, the magnitude of the Ca2+-dependent increase in cardiac muscle ktr is less (only 2–4-fold) than in skeletal muscle, and we have provided evidence that even maximal ktr is limited by thin-filament activation kinetics (Regnier et al. 2004). This kinetic limitation (for skeletal and cardiac muscle) likely involves not only the Ca2+-binding properties of TnC (Regnier et al. 1996, 1999b), but also kinetic interactions of the other thin-filament regulatory subunits of Tn (Brenner & Chalovich, 1999) or Tm.
Additional influence of thin-filament dynamics on ktr could come from cooperative spread of activation between adjacent thin-filament regulatory units (RUs = 1 troponin, 1 tropomyosin, 7 actins) and/or allosteric modulation of strong cross-bridge formation during Ca2+ activation (Gordon et al. 2001; Regnier et al. 2002). The relationship between [Ca2+] and steady-state isometric force in skeletal muscle fibres is highly cooperative, and much of this cooperativity is due to interactions between neighbouring RUs (Regnier et al. 2002). The spread of activation and strong cross-bridge formation from activated RUs (those with Ca2+ bound to TnC) to neighbouring inactive RUs (those without Ca2+ bound to TnC) probably occurs via head-to-tail interactions of the neighbouring Tm molecules, or alternatively could be through actin itself in regulated thin filaments. There is no conclusive evidence that cooperative mechanisms between RUs are required to explain the Ca2+ activation dependence of ktr. However, there is some evidence to suggest that ktr may reflect events within individual RUs. Partial extraction of sTnC from skeletal fibres, that reduced maximal forces to as low as 0.13 Fmax resulted in little or no change in submaximal or maximal ktr as a function of [Ca2+] (Metzger & Moss, 1991). Chase et al. (1994) varied the degree of thin-filament activation in a Ca2+-independent manner by reconstituting fibres with mixtures of cTnC and a constitutively activated TnC (aTnC), and also found that ktr is not dependent on the level of force. Quantitative assessment of cooperative mechanisms in these earlier studies was limited because removing some of the native sTnCs or reconstituting the thin filaments with cardiac TnC may substantially alter the complex interactions that occur among TnC, TnI, TnT, Tm and actin (Piroddi et al. 2003; Clemmens et al. 2005). In addition, to determine the role of cooperative mechanisms in the Ca2+ activation of ktr, fibres should be activated by Ca2+ as in the normal activation process as opposed to the permanent activation with aTnC (Chase et al. 1994) or extraction of whole Tn (Metzger & Moss, 1991).
Mathematical models have also been used to investigate the possible role of cooperative mechanisms between RUs in the Ca2+ activation dependence of ktr. Campbell, (1997) and Razumova et al. (2000) have suggested that cooperative RU interactions can be used to explain both the Ca2+ dependence of force and ktr. However, Hancock et al. (1997), using a four-state model (Landesberg & Sideman, 1994) that coupled thin-filament states with a two-state cross-bridge cycle, showed that a non-linear ktr–force relationship could be simulated without including any cooperative mechanisms.
Here we investigated the contribution of TnC Ca2+ binding properties and the subsequent spread of activation along thin filaments in determining force redevelopment kinetics of rabbit psoas fibres. TnC Ca2+-binding properties were altered by replacing native TnC with cardiac TnC or a mutant sTnC (D28A) that does not bind Ca2+ at site I (xsTnC) (Moreno-Gonzalez et al. 2005). Cooperative spread of activation along thin filaments into near-neighbour RUs was reduced by replacing native TnC with different mixtures of purified native sTnC and a mutant sTnC (D28A, D64A) that does not bind Ca2+ at either N-terminal site (xxsTnC) (Regnier et al. 2002). Our results clearly demonstrate that reducing near-neighbour RU interactions greatly reduced Fmax, and had no effect on maximal ktr and little influence on the magnitude of the Ca2+-dependent increase of ktr. In contrast, cTnC or xsTnC in fibres reduced or eliminated the Ca2+ dependence of ktr and this reduction was correlated with increased Ca2+ dissociation kinetics of Tn (koff). Further experiments and modelling simulations suggest the Ca2+-binding properties of Tn influence the kinetics of both cross-bridge attachment and detachment. Thus our results strongly suggest that ktr is regulated at the level of individual RUs and that cooperative interactions between near-neighbour RUs have little influence on the magnitude of the Ca2+-dependent increase in ktr. Instead this increase appears to depend on the properties of TnC and its interactions with TnI or other proteins in the activation pathway. Preliminary reports of this work have been published previously (Regnier et al. 1999a; Moreno-Gonzalez et al. 2003).
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Methods |
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Chemically skinned segments of single fibres from rabbit psoas muscle were prepared as previously described (Regnier et al. 2002). Male New Zealand rabbits were housed in the Department of Comparative Medicine at the University of Washington (UW) and were cared for in accordance with the US National Institutes of Health Policy on Humane Care and Use of Laboratory Animals. The animals were killed with pentobarbital (120 mg kg–1) administered through the marginal ear vein. All protocols involving animals were approved by the UW Animal Care Committee.
For mechanical experiments, fibre segments were isolated from bundles immediately prior to use, and were further treated with 1% Triton X-100 (v/v) in relaxing solution to remove membranous residue. The fibre segment ends were chemically fixed by focal application of 1% glutaraldehyde in H2O to form artificial tendons that minimize end compliance (Chase & Kushmerick, 1988).
Experimental solutions
Compositions of relaxing and activating solutions for fibre experiments were calculated and solutions were made as previously described (Martyn et al. 1994; Regnier et al. 2002). Solutions were maintained at 0.17 M ionic strength and pH 7.0 at 15°C (experimental temperature), and contained (mM): 5 MgATP or 5 dATP, 15 phosphocreatine (PCr), 15 EGTA, at least 40 MOPS, 1 free Mg2+, 135 Na+ + K+, 1 dithiothreitol (DTT), at least 250 u ml–1 creatine kinase (CK), 4% w/v Dextran T-500. Ca2+ levels (given as pCa = –log[Ca2+]) were established by varying the amount of Ca(propionate)2.
Proteins
Native rabbit skeletal TnC, TnI, TnT were purified from rabbit skeletal back and leg muscles and native rat cTnC from rat hearts according to the method of Potter (1982) and Dong et al. (1996), respectively. The rabbit sTnC gene was cloned, and wild-type (WT) and mutants were expressed in E. coli as previously described (Regnier et al. 2002; Liang et al. 2003). Mutations were introduced at the x position of the low-affinity, N-terminal Ca2+-binding sites I and II as previously described (Regnier et al. 2002; Moreno-Gonzalez et al. 2005). Site I alone is inactive (D28A) in xsTnC, whereas both site I (D28A) and II (D64A) are inactive in xxsTnC. The purity of all native and recombinant proteins was assessed by SDS-PAGE and protein concentration was determined by UV-absorbance spectroscopy. Purified TnT and TnI were complexed with different TnCs, according to Potter (1982) for stopped-flow measurements.
Mechanical data acquisition and analysis
Mechanical measurements were performed in a previously described apparatus (Regnier et al. 2002; Moreno-Gonzalez et al. 2005). In all experiments, sarcomere length (SL) was set initially to 2.5 µm using HeNe laser diffraction and adjusted to between 2.4 and 2.6 µm during activations. To maintain structural and functional integrity, fibres were periodically (every 5 s) unloaded by rapid (10 LF s–1) 15% release of total fibre segment length (LF) for 
40 ms, followed by rapid restretch to the initial LF (Brenner, 1983). Force, LF and SL signals were digitized and analysed as previously described using custom data acquisition software (Chase et al. 1994; Regnier et al. 2002).
Steady-state force.
Isometric force was measured during the steady-state period just prior to the release/restretch of a digitized ktr record (see protocol below) (Sweeney et al. 1987; Chase et al. 1994; Martyn et al. 1994). Passive force was determined at pCa 9.0 and was subtracted from the total force at the various pCa levels to obtain the Ca2+-activated force that is reported. Passive force following reconstitution did not differ from pre-extracted values under any of the experimental conditions (data not shown). The Ca2+-activated force was normalized to fibre cross-sectional area, calculated from the diameter (58.5 ± 1.3 µm) assuming cylindrical geometry. Fmax (n = 57) was 286.0 ± 11.5 mN mm–2 prior to extraction of endogenous sTnC. A small number of fibres included in this study exhibited 15% rundown of Fmax, although the great majority exhibited <10%; all yielded similar results.
Kinetics of isometric tension (force) redevelopment (ktr).
The rate of isometric tension redevelopment was determined from the half-time of force recovery after a rapid release–restretch transient as previously described (Chase et al. 1994; Regnier et al. 1996, 1998b). In brief, after the development of steady-state force, the fibre was shortened by 15% LF with a 4 or 10 LF s–1 ramp, which reduced force to zero, followed by a rapid (300 µs) under-damped restretch to the initial LF. Force was reduced to baseline during shortening, transiently spiked with rapid restretch to LF, then the subsequent force redevelopment kinetics were characterized by an apparent rate constant derived from the half-time of force recovery (Eqn 1) (Chase et al. 1994). ktr traces where the force redevelopment started above 50% of steady-state force were not included in this study, to avoid over-estimation of the rate.
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| (1) |
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TnC was selectively extracted from fibres as previously described using trifluoperazine (TFP) (Regnier et al. 1999b, 2002; Moreno-Gonzalez et al. 2005). Fibres were placed in extracting solution for 30 s, followed by 10–15 s in relaxing solution (pCa 9.0), and this procedure was repeated in sets of five for an average of 9 min total in the extracting solution. Additional extraction was performed if Ca2+-activated force at pCa 4.5 (Fmax) was 
2% of pre-extracted Fmax. Extracted fibres were washed extensively in relaxing solution to remove residual TFP.
Reconstitution of Tn complexes in TnC-extracted fibres with cTnC, xsTnC or mixtures of sTnC: xxsTnC was achieved by 1–3 min incubations in 1 mg ml–1 (total) TnC in pCa 9.2 without CK or Dextran, as previously described (Regnier et al. 2002; Moreno-Gonzalez et al. 2005). Reconstitution was considered complete when force at pCa 4.5 no longer increased (rFmax) with subsequent incubations. We previously reported that additional incubation with sTnC for 1–3 min following cTnC or xsTnC reconstitution does not increase Fmax. This incubation time would normally completely reconstitute Tn complexes (Moreno-Gonzalez et al. 2005), thus suggesting that all troponin complexes were reconsituted. We have also previously demonstrated relatively equal binding affinities for sTnC and xxsTnC in the absence of Ca2+, suggesting the procedure for reconstitution with sTnC: xxsTnC mixtures should yield a random distribution of these two TnCs along individual thin filaments throughout the entire fibre diameter. (Regnier et al. 2002). Reconstitution of extracted fibres with 100% sTnC results in restoration of Fmax to >90% (often >95%) of the pre-extracted level, and reconstitution with 100% xxsTnC provides no active force even at pCa 4.0 (Regnier et al. 2002; Moreno-Gonzalez et al. 2005).
Ca2+ dissociation rates from troponin
The rate of Ca2+ dissociation (koff) from TnC was measured at 15°C in whole Tn complexes using an Applied Photophysics Ltd. (Leatherhead, UK) model SX.18 MV stopped-flow instrument with a dead time of 1.4 ms as previously described for TnC by Tikunova et al. (2002), and modified slightly for whole Tn. Three Tn complexes were created: sTnC–sTnI–sTnT, xsTnC–sTnI–sTnT, and cTnC–sTnI–sTnT. The Ca2+ koff was determined in a buffer containing (mM): 20 MOPS, 250 KCl, 1 DTT, 5 MgCl at pH 7.0. Ca2+ dissociation from Tn was quantified from an increase in fluorescence of the Ca2+ chelator Quin-2 (150 µM). Quin-2 was excited at 330 nm and fluorescence was monitored through a 510 nm broad band-pass interference filter (Tikunova et al. 2002). For N-terminal Ca2+-dissociation rates, each koff represents the mean of at least 15 averaged traces and was well fitted by a single exponential (r2 = variance less than 1.0 x 10–4). Values are reported as mean ± S.E.M. For TnC C-terminal Ca2+-dissociation rates, representative traces were obtained at a slower timescale and were well fitted by a double exponential. All TnC C-terminal Ca2+ koff rates were = 0.2 s–1.
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Results |
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For each muscle fibre, steady-state force and ktr were measured at varying levels of activating Ca2+ prior to extraction of endogenous sTnC and following replacement with either cTnC, xsTnC or mixtures of sTnC: xxsTnC. In all cases passive force (pCa 9.0) following reconstitution did not differ from pre-extracted values, indicating that thin-filament regulation was complete. Maximal steady-state force (Fmax) and rate of force redevelopment (maximal ktr) were compared for pCa 4.5 activations just prior to the extraction protocol and then following reconstitution. Figure 1 shows example pCa 4.5 force traces for fibres reconstituted with 100% cTnC (Fig. 1A) or 100% xsTnC (Fig. 1B). In both cases, maximal ktr was 15 s–1 prior to extraction of native TnC (control) and was reduced to 6 s–1 following reconstitution with cTnC or xsTnC. However, the reconstituted Fmax (rFmax) differed greatly for the fibre containing 100% cTnC (0.79 Fmax) versus the fibre containing 100% xsTnC (0.23 Fmax). To determine how reduction of near-neighbour RU interactions influence maximal ktr, fibres were reconstituted with mixtures of sTnC: xxsTnC that produced similar rFmax as that obtained in the cTnC- or xsTnC-reconstituted fibres. Reconstituted fibres with sTnC: xxsTnC mixtures ratios produced rFmax that was greater than proportionality between force and sTnC content, as we have previously reported (Regnier et al. 2002). Figure 1C shows a fibre reconstituted with 60% sTnC: 40% xxsTnC (rFmax = 0.80 Fmax) and Fig. 1D shows a fibre reconstituted with 5% sTnC: 95% xxsTnC (rFmax = 0.25 Fmax). In these fibres, control maximal ktr was 16 s–1 but, in contrast to fibres with cTnC and xsTnC, maximal ktr was not decreased even though rFmax was reduced.
These example maximal ktr traces were representative of the measurements obtained for all fibres reconstituted with the various TnCs and sTnC: xxsTnC mixtures. The data for all experiments are summarized in Fig. 2, with sTnC: xxsTnC fibres grouped by force increments (see legend). Maximal ktr stayed nearly constant as rFmax progressively decreased to 0.10 Fmax (0.13 ± 0.03) when fibres were reconstituted with decreasing fractions of functional sTnC (
). In fact only fibres with rFmax < 0.20 Fmax had a significantly reduced maximal ktr compared with control (
) or fibres reconstituted with 100% sTnC (&). In contrast, maximal ktr was reduced by >50% for fibres reconstituted with 100% cTnC (
) or 100% xsTnC (
). Interestingly, as in the example fibres in Fig. 1, maximal ktr for fibres reconstituted with cTnC or xsTnC was the same even though rFmax in xsTnC-reconstituted fibres (0.25 ± 0.02, n = 13) was only 37% of that in cTnC-reconstituted fibres (0.68 ± 0.03, n = 9). Figure 2 illustrates two salient points. First, maximal Ca2+-activated ktr has little or no dependence on the fraction of functional RUs or the level of steady-state force, i.e. maximal ktr does not appear to depend on interactions between near-neighbouring RUs (Regnier et al. 2002). Second, while maximal ktr does not depend on the level of isometric force per se, it is greatly influenced by the Ca2+-binding properties of TnC, which also affect steady-state force (Moreno-Gonzalez et al. 2005).
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The dependence of ktr on [Ca2+] in the bathing solutions (ktr–pCa relationship) is summarized in Fig. 3. The steady-state force–pCa relationship is shown as fitted lines for comparison (data reported in (Regnier et al. 2002 and Moreno-Gonzalez et al. 2005)). Figure 3A shows that prior to extraction of native TnC (control) the Ca2+ dependence of ktr, defined as the magnitude increase in ktr from the first measurable value to maximal Ca2+ activation, was 10-fold as Ca2+ was varied. However, the Ca2+ dependence of ktr varied only 2-fold following reconstitution with 100% cTnC (Fig. 3A) and was eliminated in fibres reconstituted with 100% xsTnC (Fig. 3B). We report only the magnitude of Ca2+-dependent increase in ktr because it was so diminished in fibres containing cTnC or xsTnC that it was impossible to determine the Ca2+ sensitivity (pCa50) of this rate. These reductions in the Ca2+ dependence of ktr resulted from both a decreased maximal ktr and an elevated ktr at low levels of Ca2+, demonstrating that the properties of Tn complexes can play a prominent role in determining ktr.
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0.70 Fmax (Fig. 3C) and 0.20 Fmax (Fig. 3D). For these experiments, fibres reached Fmax at pCa 5.5 under control conditions, even though ktr was still rising. To minimize the time of Ca2+ activation and to maintain fibre integrity prior to extraction of native TnC, the preparation was then placed in a pCa 4.5 solution to obtain maximal ktr. Following reconstitution, measurements were also made over the pCa range 5.5–5.0 because steady-state force continued to increase over this range of [Ca2+]. Interestingly, the Ca2+-dependent range of ktr was minimally reduced for fibres with the different fractions of functional (sTnC-containing) versus non-functional (xxsTnC-containing) Tn. In fibres reconstituted with sTnC: xxsTnC mixtures that produced 0.70 Fmax (Fig. 3C; mixture ratios given in legend), the magnitude of the Ca2+-dependent increase in ktr was minimally reduced due to a small elevation at low levels of Ca2+. This contrasts with the much larger reduction in cTnC-containing fibres (Fig. 3A) that produced a similar rFmax. For fibres reconstituted with sTnC: xxsTnC mixtures that produced 0.20 Fmax (Fig. 3D; mixture ratios given in legend), the Ca2+-dependent range of ktr values was reduced by 40%, due to some elevation of ktr at low levels of Ca2+. This contrasts with the complete elimination of the Ca2+-dependent range of ktr values for fibres reconstituted with xsTnC (Fig. 3B), even though rFmax was similar. These results indicate that near-neighbour RU interactions are not the primary determinant of the Ca2+ dependence of ktr. Instead the major determinant of both maximal ktr and the Ca2+ dependence of ktr appear to be the local thin-filament activation by Ca2+ and subsequent strong cross-bridge formation.
To determine if Ca2+-dependent increases in ktr resulted from differences in the number of cross-bridges available to participate in thin-filament activation and force generation, the data in Fig. 3 were replotted as ktr versus steady-state force produced at each level of [Ca2+] (Fig. 4). This allows comparison of ktr at similar levels of steady-state force, under different experimental conditions, independent of the [Ca2+]. At force levels <0.20 Fmax, fibres reconstituted with cTnC (Fig. 4A) or xsTnC (Fig. 4B) had similar elevated ktr, which was constant within that range of forces. With cTnC-reconstituted fibres, ktr increased with increasing force but remained elevated above control conditions over the range of forces from 0.20 to 0.70 Fmax, as previously observed (Chase et al. 1994). In contrast, fibres with sTnC: xxsTnC mixtures that produced 0.70 Fmax (Fig. 4C) had similar or only slightly increased ktr at steady-state force levels below 0.20 Fmax compared with pre-extracted controls values, and ktr was elevated at forces >0.20 Fmax. In comparison, fibres with sTnC: xxsTnC mixtures that produced 0.20 Fmax (Fig. 4D) had slightly elevated ktr at the lowest force, but there was a steep increase towards maximal ktr as force approached 0.20 Fmax at maximal Ca2+ activation. However, ktr remained constant over the range of forces <0.20 Fmax under control conditions. This difference is probably due to the fact that obtaining similar forces required much more Ca2+ when fibres contained the sTnC: xxsTnC mixture. Combined, the data in Figs 3 and 4 suggest that ktr at submaximal levels of Ca2+ depends much more on the Ca2+-activation properties of Tn than on interactions between near-neighbouring RUs along the length of thin filaments.
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At submaximal levels of Ca2+ activation, ktr is correlated with Ca2+ koff measured from isolated TnCs (Johnson et al. 1994) for fibres reconstituted with TnC mutants (Regnier et al. 1999b) or under the effects of Ca2+-sensitizing agents (Regnier et al. 1996). However, koff of isolated TnCs (Johnson et al. 1994) is quite rapid and not indicative of the rates in whole Tn complexes (Table 1) or in skeletal muscle fibres. To more closely approximate fibre conditions, we complexed recombinant cTnC, xsTnC, xxsTnC or native sTnC with purified native sTnI and sTnT for measurements of N-terminal Ca2+-dissociation kinetics using stopped-flow spectrofluorimetry with Quin-2. Table 1 shows that Ca2+ koff increased in the order sTnC–Tn < cTnC–Tn < xsTnC–Tn. As expected, we were unable to detect a signal associated with Ca2+ binding to N-terminal site I and II in Tn containing xxsTnC, suggesting Ca2+ binding was minimal or non-existent at these sites.
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2-fold difference in Ca2+ koff between sTnC–Tn and xsTnC–Tn may be enough to eliminate the Ca2+-dependent increase of ktr. Therefore, these data clearly suggest an important role for Ca2+-dissociation kinetics of Tn in determining both steady-state force and the kinetics of force development. Influence of increasing cross-bridge cycle rate on ktr
In skeletal muscle fibres, ktr is limited by the dynamics of thin-filament activation during submaximal levels of Ca2+ (Chase et al. 1994; Regnier et al. 1996, 1999b), while maximal ktr is thought to be determined predominantly by the intrinsic rate of cross-bridge cycling (Brenner & Eisenberg, 1986). If reduction of maximal ktr and elevation of ktr at low levels of Ca2+ activation (force) result from the faster koff for cTnC–Tn and xsTnC–Tn (Figs 3A and B and 4A and B, Table 1), increasing the rate of cross-bridge cycling should have little or no effect on the ktr–force relationship except during maximal Ca2+ activation. To test this hypothesis we substituted 2-deoxy-ATP (dATP) for ATP. We have previously demonstrated that dATP increases (by 10–30%) the rate of actomyosin NTPase in solution, in vitro motility of actin filaments, and maximal ktr and unloaded shortening (but not Fmax) in skinned skeletal muscle fibres (Regnier & Homsher, 1998; Regnier et al. 1998a, 1997b; Clemmens & Regnier, 2004). Figure 5 summarizes the effect of dATP on maximal ktr. As previously reported (Regnier et al. 1998b), dATP increased maximal ktr by 15% in control conditions. A similar increase was seen for fibres with cTnC but not for fibres with xsTnC, suggesting that the faster koff of xsTnC–Tn (Table 1) may limit maximal ktr.
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; pre-extraction) conditions and following reconstitution with cTnC in 5 mM ATP (). The elevation of ktr at low forces and reduction of the maximal value can be seen with cTnC, as in Fig. 4A. These fibres were then exposed to the same Ca2+ activating solutions, but with 5 mM dATP replacing ATP (
). Under this condition, there was an increase in the maximal ktr as well as in the level of steady-state force attained at saturating [Ca2+] (compare left-most two *). In contrast, at submaximal Ca2+ activation, there was no change in ktr at equivalent levels of steady-state force obtained with ATP. This implies that submaximal ktr was not limited by cross-bridge kinetics because otherwise it should have been elevated by dATP. Combined, the results in Figs 5 and 6 support the hypothesis that elevation of ktr at submaximal steady-state force levels results not from altered actomyosin cycling rates per se, but from rate-limiting Ca2+-dependent interactions in thin-filament activation that may alter cross-bridge cycling.
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Discussion |
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Maximal ktr and the Ca2+ dependence of ktr are properties of individual RUs
We found that maximal ktr was independent of Fmax as the number of interacting RUs was progressively reduced (Fig. 2), and that the magnitude of Ca2+-dependent increase in ktr was minimally altered (Figs 3C and D and 4C and D). These observations indicate that ktr is determined primarily by thin–thick-filament protein interactions within the confines of individual RUs, and that interactions between RUs have little influence on ktr in skinned skeletal muscle fibres. This contrasts the strong influence of near-neighbour RU interactions on setting the level of Ca2+-activated steady-state isometric force (Regnier et al. 2002). Comparable results have been obtained by others. Metzger & Moss (1991) found that partial extraction of sTnC from skinned rabbit psoas fibres had little effect on maximal ktr and ktr at low levels of Ca2+ activation. However, these studies were not definitive because regions of thin filaments with incomplete Tn complexes could have altered the interactions that occur between TnI, TnT, Tm and actin, potentially affecting the spread of activation along thin filaments. An alternative approach was used by Morris et al. (2001), who extracted native sTnC and reconstituted Tn complexes with mixtures of cTnC and a cTnC mutant that does not bind Ca2+ at N-terminal site II (CBMII). This approach is similar to our experiments in which fibres were reconstituted with mixtures of sTnC: xxsTnC (Figs 1C and D, 2, 3C and D and 4C and D), with an important difference being the type of functional TnC used, i.e. cardiac TnC versus skeletal TnC. In their study using cTnC: CBMII mixtures, maximal ktr was not affected by the fractional content of cTnC. However, maximal ktr and the Ca2+ dependence of ktr in fibres reconstituted with 100% cTnC were diminished from pre-extracted values, similar to what we observed (Figs 2, 3A and 4A).
The length of thin filament activated by Ca2+ binding to each Tn may also be diminished by replacement of native sTnC with cTnC and xsTnC. In a previous study (Regnier et al. 2002), we estimated this length to be 10–12 actin monomers for skeletal fibres with sTnC. If this length is less in fibres with cTnC or xsTnC, the interaction between neighbouring RUs may be reduced because fewer actins are available for myosin binding when Ca2+ binds to one Tn. This is suggested by a large decrease in the Hill coefficient (nH) and reduced Ca2+ sensitivity of the force–pCa relationship seen when these proteins replace native sTnC (dotted lines in Fig. 3A and B) (Moreno-Gonzalez et al. 2005). Additionally, we find that the length of thin filament activated by Ca2+ binding to Tn is smaller in cardiac muscle (containing cTnC) than in skeletal muscle (unpublished observations). Thus, our data and other studies support the hypothesis that maximal ktr and the Ca2+ dependence of ktr are primarily determined by the properties of the Tn complex at the level of individual functional RUs, with no observable dependence on Fmax and little or no influence of near-neighbour RU interactions along thin filaments.
Factors that regulate ktr within individual RUs
The rate that force develops is dependent on both myosin and thin-filament properties (Gordon et al. 2000). Maximal Ca2+-activated ktr in skinned skeletal fibres is primarily determined by the intrinsic rate of actomyosin cross-bridge cycling, when the contribution of thin-filament dynamics should be least limiting (Brenner, 1986; Metzger & Moss, 1990). Brenner & Eisenberg (1986) proposed that ktr reflects the sum of the forward (fapp) and reverse (gapp) rates of cross-bridge transitions from weak-binding to strong-binding, force-generating states. They also proposed that the steep relationship between ktr and Ca2+-activated force (as seen in Fig. 4) reflected a Ca2+ dependence of fapp. However, we and others have shown that this model is too simplistic to predict results from experiments when either cross-bridge cycling rate or thin-filament activation are altered independently of [Ca2+] (Chase et al. 1994; Regnier et al. 1998b, 1999b; Fitzsimons et al. 2001). These experiments led us to conclude that ktr is controlled by a complex kinetic interaction between cross-bridge cycling and Ca2+-dependent thin-filament dynamics. This idea is further supported by our present experiments showing that cTnC and xsTnC in skeletal fibres can reduce or eliminate the magnitude of Ca2+-dependent increase in ktr (Figs 3A and B and 4A and B).
The Ca2+ dependence of ktr within RUs could occur via regulation of thin-filament activation kinetics and/or thin-filament effects on cross-bridge-binding and -cycling kinetics. The Ca2+-binding kinetics of TnC are probably not rate limiting per se, at least in skeletal muscle (Brenner & Chalovich, 1999). Ca2+ binding to TnC is rapid and close to diffusion limited in the Tn complex or in regulated thin filaments. However, the transmission of the Ca2+-binding signal through the interaction of TnC with TnI, and the subsequent movement of Tm could limit thin-filament activation kinetics. This is supported by the much slower apparent kinetics of TnC Ca2+ binding when complexed in Tn, compared to isolated TnC (Dong et al. 1996, 1997a, 1997b). In the current study, when native skeletal TnC was replaced with either cTnC or xsTnC, the interaction between TnC and TnI may well have been affected. Both of these TnCs have a single N-terminal Ca2+-binding site. Ca2+ binding to cTnC results in much less hydrophobic patch exposure for interaction with TnI than found for the skeletal isoform (Gagne et al. 1995; Li et al. 1999). In the presence of Ca2+, weaker interaction between TnC and TnI could shift cTnI or sTnI binding towards increased interaction with actin, thereby decreasing thin-filament activation. It could also increase the probability of de-activation if cross-bridge binding is not sufficient to maintain the activated state. The faster Ca2+ koff from cTnC–Tn and xsTnC–Tn in solution (Table 1) and the correlated decrease in rFmax and ktr (Fig. 2) support this idea. The inverse correlation between rFmax and koff suggests that the ability of Tn to activate thin filaments is in the order of fibres containing sTnC > cTnC > xsTnC. This provides an explanation for the lower maximal ktr seen with cTnC- or xsTnC-reconstituted fibres (Fig. 2). It does not, however, explain why maximal Ca2+-activated ktr is similar for cTnC- versus xsTnC-reconstituted fibres, while rFmax with xsTnC-reconstituted fibres is only one-third of that with cTnC (Fig. 2). This cannot be explained by a difference in the extent of Tn complex reconstitution for xsTnC versus cTnC, as our control measurements demonstrated that most or all of the Tn complexes are complete (see Methods). Additionally, it is not readily apparent how increased koff can explain the elevation of ktr (compared with control) at low levels of [Ca2+] (Fig. 3) and at similar levels of Ca2+-activated force (Fig. 4). The potential coupling between Ca2+ koff and gapp as a possible explanation for these issues is discussed below and in the Appendix.
In addition to determining the dynamics of Ca2+-induced changes in thin-filament protein interactions, thin-filament regulatory proteins may more directly affect cross-bridge cycling. We (Gordon et al. 1997; Kohler et al. 2003; Clemmens & Regnier, 2004) and others (Homsher et al. 1996; 2000,) have demonstrated that regulatory proteins increase actomyosin kinetics in the in vitro motility assay, and this is best explained by thin-filament regulatory proteins increasing cross-bridge detachment rate (gapp). Microneedle force measurements in the in vitro motility assay suggest that regulatory proteins may also affect fapp (Clemmens & Regnier, 2004). In addition, there is growing evidence that alterations in the molecular structure of regulatory proteins influence cross-bridge cycling and force production in muscle fibres (Regnier et al. 1999b; Homsher et al. 2000; Morris et al. 2001; Piroddi et al. 2003; Chandra et al. 2005; Hernandez et al. 2005; Kruger et al. 2005; Moreno-Gonzalez et al. 2005). As such, our data lead us to hypothesize that a faster Ca2+ koff from Tn may somehow result in an increased gapp in skeletal muscle fibres, especially at low levels of Ca2+ activation.
We (Regnier et al. 1996) previously reported that the Ca2+-sensitizing compound calmidazolium (CDZ), which reduces Ca2+ koff from isolated TnC (Johnson et al. 1994), increases ktr at submaximum levels of Ca2+ activation. In contrast, here we show even greater elevation of submaximal ktr in fibres reconstituted with either cTnC or xsTnC (Fig. 4A and B), both of which increased (rather than reduced) Ca2+ koff when measured in whole Tn complexes containing sTnI and sTnT (Table 1). In the model by Hancock et al. (1997), slower koff elevated ktr at submaximal [Ca2+], as with CDZ. In the Hancock study, however, koff was based on solution measurements using isolated TnC (Johnson et al. 1994). In contrast, here we measured koff for Ca2+ bound to whole Tn. We propose that skeletal Tn containing cTnC or xsTnC has a lower apparent affinity for the switch region of TnI in the presence of Ca2+, thereby favouring TnI binding to actin and reducing thin-filament activation. Since TnI and cross-bridges compete for binding sites on thin filaments, this competition may increase the apparent rate of cross-bridge detachment (gapp), thereby increasing ktr (fapp + gapp) (Brenner, 1986; Brenner & Eisenberg, 1986). This is a reasonable explanation because in the absence of Ca2+, the TnI–actin interaction is strong enough to prevent cross-bridge transition from weak to strong, force-generating states. In contrast, slowed koff of sTnC by CDZ should shift TnI binding towards TnC within an RU, elevating thin-filament activation at submaximal [Ca2+] by prolonging exposure of cross-bridge-binding sites on actin and increasing submaximal ktr. In this case, faster ktr would result from increased fapp.
In conclusion, our experimental data demonstrate that loss of near-neighbour RU interactions does not greatly affect maximal ktr or the magnitude of Ca2+-dependent increase in ktr (Figs 2, 3 and 4). Instead, ktr is strongly influenced by the Ca2+-binding properties of individual thin-filament RUs, without the involvement of cooperative mechanisms between RUs. In contrast, steady-state isometric force depends strongly on near-neighbour RU cooperative interactions (Regnier et al. 2002; Moreno-Gonzalez et al. 2005). Taken together our results indicate that in skinned skeletal fibres, ktr is determined by a balance of actin-binding site availability, the kinetics of cycling cross-bridges, and the influence of regulatory proteins on the apparent rate constants in the cross-bridge cycle.
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depicts an individual thin-filament RU and the associated cross-bridges. Its features have been described in detail elsewhere (Hancock et al. 1997; Regnier et al. 1998b; 1999b,) and therefore, will only be described briefly here. In this scheme the apparent rate constants (fapp and gapp) that control cross-bridge entry into and out of force-generating states (States 2 and 3) are coupled with a thin-filament activation component that is controlled by Ca2+ binding to (kon) and dissociation from (koff) Tn. State 1
2 is a rapid equilibrium between detached and weak cross-bridge attachment. Transitions between states 3 4 and 4 1 are necessary because Ca2+ can dissociate from Tn while cross-bridges are in attached, force-bearing conformations. While this model greatly simplifies cross-bridge chemo-mechanical transitions, it is sufficient to explain the ktr versus force relationship for conditions where the kinetics of Ca2+ binding to TnC (Chase et al. 1994; Regnier et al. 1996; 1999b,) or cross-bridge cycling (Regnier et al. 1998b) are varied independently.
To simulate control steady-state force and ktr at 15°C, the apparent rate constants regulating cross-bridge cycling (fapp, f'app, gapp and g'app) were set to generate a maximal ktr of 13 s–1 (controlled by fapp + gapp) and a minimal ktr of 1.5 s–1 (controlled by gapp) (as in Fig. 4). To simulate varying [Ca2+] we initially assumed that Ca2+ affects only thin-filament activation kinetics, and values for kon and koff were selected in the following manner. A wide range of koff values could be used to simulate the control ktr versus force relationship, as kon was varied to simulate changing [Ca2+]. Even though the koff value obtained for sTn in solution (Table 1) was within this range, we selected a somewhat larger value (50–100 s–1). This allowed us to simulate the ktr versus force relationship of fibres reconstituted with cTnC or xsTnC by increasing koff in a proportional amount as indicated from our stopped-flow measurements (Table 1). An apparent second-order binding constant of Tn for Ca2+ was calculated (107 M–1s–1) that is within the range reported by Rosenfeld & Taylor (1985) for thin filaments in solution, and this allowed us to simulate a ktr versus force relationship. Simulation values for ktr at low and high force are shown in Fig. 4B (
control, 
xsTnC).
To simulate results from skeletal muscle fibres containing cTnC and xsTnC, gapp was increased from 1.5 s–1 to 3–4 s–1 and Ca2+ koff was increased 2-fold. Using these values, simulated Fmax was reduced by 30% and maximal ktr was reduced by 50%, similar to levels seen for cTnC-reconstituted fibres (Fig. 2). In addition, simulations of ktr for low [Ca2+] were increased 2-fold, as we also observed in cTnC-reconstituted fibres (Fig. 4A). Further increasing Ca2+ koff (4-fold) and increasing gapp to 6 s–1 reduced Fmax down to 25% of control Fmax. Interestingly, these simulations had little further effect on maximal ktr (10% reduction) but almost completely eliminated the Ca2+ dependence of ktr, reproducing our results of xsTnC-reconstituted fibres (Fig. 4B). No combination of altering kon and koff (without increasing gapp) was able to simulate elevated ktr at low force (Ca2+) levels. Additionally, no combination of altering fapp and gapp (without changing Ca2+ kon and/or koff) was able to simulate a similar maximal Ca2+-activated ktr with a greatly different Fmax, as observed in fibres reconstituted with cTnC versus xsTnC (Fig. 2). Thus, we conclude that the simplest explanation of our data is that faster Ca2+ koff with cTnC and xsTnC, compared with sTnC (Table 1), is coupled to an increased rate of cross-bridge detachment (gapp). This apparent coupling reduces or eliminates the Ca2+ dependence of ktr, as observed in Figs 3 and 4. Using this model we also tested whether, when Ca2+ koff is increased (to simulate cTnC- or xsTnC-reconstituted fibres), increases in fapp + gapp alter ktr at low levels of Ca2+ activation. They did not, in agreement with our results when cross-bridge cycling rate was increased with dATP (Fig. 6).
As mentioned above, Scheme 1 describes a simple coupled relationship between activation of the thin filament and cross-bridge cycling. As such, we have not incorporated any cooperative mechanisms of Ca2+ or cross-bridge binding on the thin-filament activation steps. Thus we cannot model the cooperativity observed in the relationship between steady-state force and pCa. Campbell et al. (1997) and Razumova et al. (2000) have suggested from modelling studies that cooperative interactions between near-neighbour RUs can be used to explain both the Ca2+ dependence of force and ktr. However, using the simple model depicted in Scheme 1, we were able to predict the ktr versus force relationship as [Ca2+] is varied (Fig. 4).
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, 56 fibres). Fibres reconstituted with various mixtures of sTnC and xxsTnC to give different rFmax levels were binned in 0.2 or 0.3 rFmax increments even when the proportions of sTnC (10–100%) and xxsTnC (0–90%) varied within some groups. Note that maximal ktr 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 ktr under control conditions (
). & Fibres reconstituted with 100% sTnC. Relative rFmax between any group (except for &) and control Fmax (
) is statistically significant (P < 0.01). Relative maximal ktr values among sTnC: xxsTnC groups are not statistically significant. Relative maximal ktr between cTnC and xsTnC is not statistically significant.
) (C, 8 fibres and D, 13 fibres). Fibres reconstituted with mixtures of sTnC: xxsTnC were grouped according to rFmax (
) shows that dATP extends the curve beyond maximal values of force and ktr 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.