Correspondence to: Daniel P. Fitzsimons, Department of Physiology, University of Wisconsin School of Medicine, 1300 University Avenue, Madison, WI 53706. Fax:(608) 265-5512 E-mail:fitzsimons{at}physiology.wisc.edu.
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Abstract |
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Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca2+ binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca2+ dependence of the rate constant of force redevelopment (ktr) in skinned single fibers in which cross-bridge and Ca2+ binding were also perturbed. Ca2+ sensitivity of tension, the steepness of the force-pCa relationship, and Ca2+ dependence of ktr were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, nonforce-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca2+, 3 µM NEM-S1 increased ktr to maximal values, and higher concentrations of NEM-S1 (6 or 10 µM) increased ktr to greater than maximal values. NEM-S1 also accelerated ktr at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 µM NEM-S1 increased ktr to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of ktr. These results show that ktr is not maximal in control fibers, even at saturating [Ca2+], and suggest that activation dependence of ktr is due to the combined activating effects of Ca2+ binding to TnC and cross-bridge binding to actin.
Key Words: cooperativity, regulation of contraction, skeletal muscle, myosin
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INTRODUCTION |
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In skeletal muscle, Ca2+ binding to low affinity sites on troponin C (TnC)1 initiates a series of molecular events that ultimately allow strong binding of cross-bridges to actin and subsequent force development (for review see
It is well established that the regulation of Ca2+-activated force in striated muscles involves cooperative interactions within the thin filament. This is evident, for example, in the biphasic form of the force-pCa relationship, which is steeper at low levels of Ca2+ than at high because of greater intermolecular cooperation at forces less than 0.50 Po (for review see
A model of activation proposed by
The present study was done to further investigate the mechanisms underlying the activation dependence of the rate of force development in skinned skeletal muscle fibers, which was done by varying the degree of molecular cooperation during activation. Fibers were treated with NEM-S1 to promote strong binding of endogenous cross-bridges (
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MATERIALS AND METHODS |
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Experimental Solutions
The composition of relaxing solution was as follows (in mM): 100 KCl, 20 imidazole, 4 MgATP, 2 EGTA, and 1 free Mg2+, pH 7.0 at 22°C. Activating solution contained (in mM): 79.2 KCl, 20 imidazole, 14.5 creatine phosphate, 7 EGTA, 5.42 MgCl2, and 4.68 ATP, with [Ca2+]free ranging from 1 nM (i.e., 9.0 pCa) to 32 µM (i.e., 4.5 pCa), pH 7.0 at 15°C and an ionic strength of 180 mM. A computer program (
Skinned Fiber Preparations
Fast-twitch skeletal muscle fibers were obtained from the psoas muscles of adult New Zealand rabbits. Bundles of 30 fibers were dissected from psoas muscles while in relaxing solution. Each bundle was tied with 4/0 suture to glass capillary tubes, placed in relaxing solution containing 1% Triton X-100 for 45 h at 4°C, and then stored in relaxing solution containing 50% glycerol at -20°C for up to 3 wk. Chemically skinned fibers were ready for experimental use 12 d after dissection.
Experimental Apparatus and General Protocols
Before each experiment, an individual skinned psoas fiber was carefully pulled from one end of a fiber bundle and mounted between a force transducer (model 400A; Aurora Scientific) and a DC torque motor (model 308B; Aurora Scientific) in an experimental apparatus similar to one described previously (2.35 µm in relaxing solution. During activation and relaxation, sarcomere length and fiber dimensions were recorded on videotape using a video camera (model WV-BL730; Panasonic) and VHS recorder (model SVO-1420; Sony).
Mechanical measurements were first performed on all fibers under control conditions (N = 33). Next, fibers were either subjected to (1) partial extraction of TnC (N = 15), (2) treatment with NEM-S1 (N = 18), or (3) both (N = 15); and mechanical measurements were repeated.
Experimental Treatments
Partial Extraction of Troponin C.
Approximately 50% of endogenous TnC was specifically extracted from thin filaments of psoas fibers using a modification of a method reported previously (2.35 µm. Maximal Ca2+-activated force was significantly reduced after partial extraction of TnC, ranging from 0.15 to 0.65 of the Po measured in the preextracted fiber. Measurements of ktr and force were subsequently obtained as functions of pCa in the partially TnC-extracted fibers. Control experiments demonstrated that incubating the partially TnC-extracted fibers for
30 s in relaxing solution containing 0.6 mg skeletal TnC/ml resulted in complete recovery of steady-state force and restored Hill coefficients to control values (data not shown).
Preparation and Use of NEM-S1.
Myosin subfragment 1 (S1) was purified from rabbit fast-twitch skeletal muscle and modified with N-ethylmaleimide (NEM) as described previously (
Specific Experimental Protocols
Rate of Tension Redevelopment.
The rate constant of force redevelopment (ktr) in skinned psoas fibers was assessed using a modification of an experimental protocol described previously (
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Force-pCa Relationship.
During measurements of ktr, each skinned psoas fiber was exposed to solutions of varying pCa and allowed to develop steady-state force. The difference between steady-state force and the force baseline after the slack step was measured as the total force at that pCa. Active force was obtained by subtracting Ca2+-independent force, measured in solution of pCa 9.0, from the total force. Force-pCa relationships were determined by expressing submaximal force (P) at each pCa as a fraction of maximal force (Po) determined at pCa 4.5, i.e., P/Po. The apparent cooperativity in the activation of force development was inferred from the steepness of the force-pCa relationship at forces <0.50 Po, and was quantified using a Hill plot transformation of the force-pCa data (
Quantification of Partial TnC Extraction by SDS-PAGE
The extent of TnC extraction was assessed using SDS-PAGE and ultrasensitive silver staining (0.75 mm of fiber segment was placed in a microfuge tube containing SDS sample buffer (10 µl/mm segment length) and stored at -80°C until analyzed for TnC content. The proportion of TnC present in the fiber was determined by densitometric analysis of silver-stained gels using a GS-670 imaging densitometer and Molecular Analyst software (BioRad Laboratories). To quantify the amount of TnC extracted, the ratio of TnC/(MLC1 + MLC3) was determined for each fiber (
Statistics
All data are expressed as mean ± SEM. Where appropriate, either a two-tailed t test for independent samples or a paired t test was used as a post hoc test of significance, with significance set at P < 0.05.
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RESULTS |
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The Effects of NEM-S1 and Partial Extraction of TnC on Steady-state Mechanical Properties
Our studies of cooperative mechanisms in skinned skeletal muscle fibers involved two perturbations that previously have been shown to alter cooperation in the activation of contraction. NEM-S1 was used to mimic the effects of strong-binding cross-bridges to further activate Ca2+-dependent tension and the rate of tension development (
Treatment with NEM-S1.
NEM-S1 had pronounced effects on maximal Ca2+-activated tension (Po), Ca2+-independent tension, Ca2+ sensitivity of tension (pCa50), and Hill coefficient (n2) in single skinned psoas fibers, as reported earlier (
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NEM-S1 also potentiated submaximal Ca2+-activated force in a concentration-dependent manner (Fig 3). Mean pCa50 was significantly increased after treatment with either 6 µM NEM-S1 (pCa50 = 0.07 ± 0.01, P < 0.05) or 10 µM NEM-S1 (
pCa50 = 0.12 ± 0.03, P < 0.05), so that Ca2+ sensitivity of force was increased. Such increases are consistent with the idea that, at low levels of Ca2+, NEM-S1 promotes the formation of strongly bound, force-generating cross-bridges (
Treatment with NEM-S1 and Partial Extraction of TnC.
To further examine the mechanisms of cooperative activation of thin filaments, single fibers were first incubated with 200 µM TFP to partially extract TnC and were subsequently treated with NEM-S1. Such an approach allows dissection of the relative roles of cross-bridge binding and near-neighbor interactions in the thin filament in activation of contraction. In these experiments, segments of each fiber were analyzed by SDS-PAGE (Fig 4) before TnC extraction (lane 1), after partial TnC extraction (lane 2), after TnC extraction plus NEM-S1 (lane 3), and after readdition of skeletal TnC to a previously extracted fiber (lane 4). Densitometric scans of the gels indicated that 50% of the endogenous TnC was extracted from psoas fibers under the conditions used. Furthermore, the extraction procedure was specific for TnC since the relative amounts of other myofibrillar proteins (e.g., myosin light chains, TnI, and TnT) were unchanged.
Partial extraction of TnC had reversible effects on maximal Ca2+-activated tension (Po), Ca2+-independent tension, Ca2+ sensitivity of tension, and the Hill coefficient (n2), which are summarized in Table 1. As reported previously (
Partial extraction of TnC also affected the Ca2+ sensitivity of tension and reduced the responsiveness of fibers to NEM-S1 (Fig 5). Mean pCa50 in control fibers was 5.97 ± 0.01, which decreased to 5.75 ± 0.02 after extraction of TnC (pCa50 = 0.22 ± 0.03, P < 0.05). Subsequent treatment with 3 µM NEM-S1 did not alter the Ca2+ sensitivity of force (
pCa50 = 0.01 ± 0.01); however, 6 µM NEM-S1 increased the Ca2+ sensitivity of force, i.e., pCa50 increased to 5.89 ± 0.02 (
pCa50 = 0.15 ± 0.01; P < 0.05), a mean value near that observed in TnC-replete fibers when treated with NEM-S1 (Table 1). Partial extraction of TnC and successive treatment with increasing concentrations of NEM-S1 resulted in progressive reductions in the steepness of the force-pCa relationship (Table 1), suggesting that the effects of these two treatments on cooperativity are additive and most likely involve different molecular mechanisms.
Effects of NEM-S1 and Partial Extraction of TnC on the Rate of Force Redevelopment
According to
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Consistent with earlier results, ktr was found to vary 10-fold as Ca2+ activation was varied from near threshold to maximal levels. As shown in Fig 6 A for a single submaximal pCa (pCa 6.1), the rate of force redevelopment at submaximal concentrations of Ca2+ was accelerated by NEM-S1, with the degree of acceleration increasing with the concentration of NEM-S1 (Fig 6 A, traces bd), compared with control (trace a). Under identical experimental conditions, another fiber was subjected to partial TnC extraction and subsequent treatment with 6 µM NEM-S1 (Fig 6 B). As summarized previously, ktr under control conditions varied with the level of Ca2+ activation, increasing when Ca2+ concentration was increased from pCa 5.9 (Fig 6 B, trace b) to pCa 4.5 (Fig 6 B, trace a). After partial extraction of TnC, the steady force decreased at each pCa, but the rate of force redevelopment (Fig 6 B, trace c) at pCa 5.9 was nearly equivalent to that of the preextracted fiber at the same pCa. Subsequent treatment with 6 µM NEM-S1 significantly increased steady force at pCa 5.9 and markedly accelerated the rate of force redevelopment (Fig 6 B, trace d).
The relationship between ktr and steady-state isometric force (as a measure of the level of activation due to Ca2+ binding and cooperative mechanisms) was variably affected by interventions to increase numbers of strongly bound cross-bridges (i.e., NEM-S1) and to disrupt near-neighbor cooperativity in the thin filament (partial extraction of TnC). As shown in Fig 7 A, ktr during maximal activation was unaffected by NEM-S1, but ktr values at low levels of activation were increased to levels either identical to (at 3 µM NEM-S1) or greater than (at 6 or 10 µM NEM-S1) the values obtained in maximally activated fibers. At intermediate levels of activation, NEM-S1 increased ktr to greater than the control values, but ktr was still less than maximal. Similar effects of NEM-S1 were observed in fibers that were subjected to sarcomere length control during measurements of ktr (Fig 8). Progressive increases in the concentration of NEM-S1 further increased the value of ktr at intermediate levels of activation, but even the highest concentration used (10 µM) did not accelerate ktr to maximal values. In the absence of Ca2+ (pCa 9.0), application of NEM-S1 induced active tension development (Fig 9 and Table 3); under these conditions, ktr was supramaximal when compared with the maximal values measured at pCa 4.5 under control conditions.
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Partial extraction of TnC also increased ktr at intermediate levels of activation as compared with the control (Fig 7 B). In fact, the increase in ktr varied with the extent of TnC extraction, i.e., fibers containing smaller amounts of residual TnC exhibited faster rates of force redevelopment during submaximal activation (Fig 10). Subsequent treatment of partially TnC extracted fibers with 3 µM NEM-S1 resulted in a ktrrelative force relationship similar to that observed with NEM-S1 treatment alone, although ktr at each level of activation was somewhat greater than the value obtained with NEM-S1 alone. However, the combination of partial TnC extraction and 6 µM NEM-S1 increased ktr at each level of activation to values greater than the maximum measured in control fibers and completely eliminated the activation dependence of ktr (Fig 7 B).
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DISCUSSION |
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The results of the present study show that strong binding of cross-bridges to actin increases the rate of force development in skeletal muscle and suggest that cooperativity in cross-bridge binding is an important determinant of activation kinetics. NEM-S1 increased the ktr measured during submaximal activations, with greatest effects at low levels of activation where NEM-S1 increased ktr to values greater than the maximum observed in control fibers at saturating [Ca2+]. From this result, it is evident that the rate of force development measured in control fibers at pCa 4.5 is not a true maximum. Although NEM-S1 dramatically accelerated ktr, strong binding of cross-bridges does not entirely account for the activation of cross-bridge kinetics, since NEM-S1 alone was insufficient at intermediate levels of activation to increase ktr to maximal or to completely eliminate the activation dependence of ktr. Instead, elimination of activation dependence required both NEM-S1 and partial extraction of TnC from the thin filament. Since partial extraction of TnC should disrupt near-neighbor communication between functional groups in the thin filament (
Models for Activation of Contraction
Ca2+ binding to TnC initiates muscle contraction, but complete activation of tension and the kinetics of tension development appears to involve cooperative effects due to cross-bridge binding to actin (for reviews see
The rate constant of force redevelopment (ktr) in steadily activated skeletal muscle preparations exhibits an 10-fold activation as [Ca2+] is increased from threshold to saturating levels, which was originally shown by
At least two models of activation might be used to explain the activation dependence of ktr.
Activation Dependence of Force and ktr Involves Activating Effects of Cross-bridge Binding and Near-neighbor Cooperativity within the Thin Filament
The results of our study provide support for models of regulation in which cross-bridge interaction kinetics are activated by cross-bridge binding to the thin filament. Experiments here show that force at submaximal [Ca2+] is increased due to activating effects of strongly bound cross-bridges, which confirms results from earlier studies from this and other laboratories (
Effects on Force and ktr due to Partial Extraction of Troponin C.
In this study, partial extraction of TnC was used to disrupt the spread of activation between adjacent functional groups in the thin filament, presumably because of constitutive inactivation of functional groups from which TnC was removed (
Whereas steady-state Ca2+-activated force was reduced after TnC extraction, the rate constant of force redevelopment (ktr) increased at each submaximal force, when compared with nonextracted fibers (Fig 7 B). Although not anticipated in
Effects on Force and ktr due to NEM-S1.
As reported earlier (
At submaximal levels of activation, NEM-S1 increased the rate of force redevelopment at each level of activation, but the increase was much greater at very low than at intermediate levels of activation. As the concentration of NEM-S1 was increased, ktr at low levels of activation was increased to maximal and supramaximal values. One possible way to explain this phenomenon is that at low levels of Ca2+, the increased force and maximal values of ktr are manifestations of preferential Ca2+ binding to functional groups in which NEM-S1 is also bound. The combined activating effects of Ca2+ and strongly bound cross-bridges within these functional groups would facilitate binding of endogenous cross-bridges and would accelerate the rate of binding. Although this is a plausible mechanism, it does not account for all of our results, since fibers activated with NEM-S1 in the absence of Ca2+ developed small forces but yielded ktr values that were maximal or supramaximal. This finding indicates that ktr measured in control fibers at pCa 4.5 is not the maximum value for this variable. Furthermore, it is possible to achieve maximal and even higher values of ktr in the absence of Ca2+ binding to TnC (i.e., at pCa 9.0) simply by increasing the numbers of strong-binding cross-bridges in the form of NEM-S1. We don't know the mechanism of this effect, but it is possible that such high values of ktr are due to a combination of NEM-S1 binding to a few discrete regions of the filament, and isolation of these regions from adjacent, inactive regions by intervening troponin complexes having no Ca2+ bound. The first condition would arise if the binding of NEM-S1 was not uniform along the thin filament, and would limit the number of endogenous cross-bridges that could bind to the thin filament, and thereby account for the small forces developed in the absence of Ca2+. The second condition would reduce or eliminate communication between adjacent functional groups, eliminate near-neighbor cooperative recruitment of cross-bridges from the noncycling pool, and thereby speed ktr. The fact that NEM-S1 does not further accelerate ktr in maximally Ca2+-activated fibers is consistent with both ideas, i.e., if NEM-S1 is nonuniformly distributed along the thin filament, then the maximum value of ktr would be limited by cooperative recruitment of cross-bridges from adjacent functional groups with less or no NEM-S1 bound.
At intermediate levels of activation (indexed by force or [Ca2+]), ktr measured in the presence of NEM-S1 was less than that observed at low and maximal activations, but was much faster than control values measured at similar levels of force or Ca2+ (Fig 7 A). The finding that ktr in the presence of NEM-S1 is not maximal at all levels of activation suggests that the thin filament is not saturated by Ca2+, cross-bridge binding, or by both. Again, it seems likely that NEM-S1 binding to the thin filament is not uniform, resulting in some regions with NEM-S1 bound and other regions with less or no NEM-S1. As discussed above, functional groups with NEM-S1 bound should be activated at lower levels of Ca2+, because of a greater Ca2+ binding affinity, and the rate of force development would be maximal because of the combined effects of Ca2+ and bound cross-bridges. By similar reasoning, those with less or no NEM-S1 would be recruited at higher (intermediate) levels of Ca2+ and will have fewer bound cross-bridges, i.e., cross-bridge activation of these functional groups is less, and force development is therefore slower. The fact that the records of force redevelopment at intermediate activation are well fit by a single rate constant suggests that the mix of variably activated functional groups confers a single level of activation to the entire thin filament. Thus, the effective size of a functional group appears to increase as [Ca2+] is increased to intermediate levels; the central region of the functional group has the greatest amounts of Ca2+ and NEM-S1 bound. Because of this, the rate of force development at intermediate levels of activation will be slowed because of cooperative recruitment of cross-bridges into the end regions of the functional group or adjacent functional groups. Ultimately, at high [Ca2+], fewer inactivated functional groups remain and the impact of cooperative interactions is reduced, but not totally eliminated since, in control fibers, ktr never achieves the maximum possible value. Correspondingly, the time course of force redevelopment becomes progressively faster, and ktr converges to the value obtained during maximal activation.
The effects of NEM-S1 to accelerate ktr appear to be significantly greater than the effects due to partial extraction of TnC (Fig 7 B). This result suggests that effects on kinetics due to cross-bridge binding within functional groups are greater than effects due to near-neighbor interactions between functional groups. Nevertheless, both mechanisms contribute to activation kinetics, since it was necessary to add NEM-S1 and partially extract TnC to eliminate the activation dependence of ktr.
Effects due to Partial Extraction of TnC and Treatment with NEM-S1. Partial extraction of TnC or application of NEM-S1 has been shown to independently alter the Ca2+ activation of force in skeletal muscle, effects that are additive or synergistic. When fibers were treated in either way, the steepness of the force-pCa relationship was reduced (Table 1), indicating a decrease in the apparent cooperativity of activation. Importantly, effects on steepness were much greater in fibers subjected to both interventions, i.e., extraction of 50% TnC and application of 6 µM NEM-S1, than with either alone. At present, we do not have a unique explanation for this result, but a simple model is one in which initial cross-bridge binding facilitates additional binding in the same and adjacent functional groups. In such a model, partial extraction of TnC would reduce steepness by disrupting near-neighbor interactions within the thin filament, and NEM-S1 would reduce steepness by cooperatively increasing the number of endogenous cross-bridges that bind within a functional group or in neighboring functional groups at each submaximal pCa.
The combination of TnC extraction and application of NEM-S1 also had greater effects on the activation dependence of ktr than either treatment alone. In fact, addition of 6 µM NEM-S1 to partially TnC-extracted fibers increased ktr to greater than maximal values at all levels of activation and completely eliminated the activation dependence of ktr (Fig 7 B). These results strongly suggest that the slowing of ktr observed in untreated fibers at low and intermediate levels of activation is due to cooperative binding of cross-bridges within the same and neighboring functional groups, and the acceleration of ktr at high levels of activation reflects a decrease in the importance of such cooperation. This conclusion is consistent with
Consideration of Possible Artifacts in Experimental Measurements
Most measurements of ktr in the present study were done without sarcomere length control, which has been shown previously to result in underestimation of the rate constant by as much as 50% because of mechanical effects of end compliance at the points of attachment to the fiber (14 s-1 was
80% of the value [
18 s-1] obtained in an earlier study using sarcomere length control;
Another possibility is that NEM-S1 actually stiffens the fiber, thereby increasing ktr at each level of activation. We regard this as highly unlikely for a couple of reasons. First, in our previous measurement of ktr (1 s-1. This is much lower than the values of ktr measured in the present study in the presence of NEM-S1 (
16 s-1) and with both NEM-S1 treatment and partial extraction of TnC (
19 s-1). Second, treatment with NEM-S1 alone had no effect on ktr in maximally activated fibers (ktr =
14 s-1), but NEM-S1 plus partial extraction of TnC increased ktr to
19 s-1.
Implications of Results for Regulation of Force and the Kinetics of Force Development under Physiological Conditions
Our results support the idea that Ca2+ activation of isometric force involves significant contributions due to cooperativity in cross-bridge binding (for most recent review see
Our results also indicate that the Ca2+ dependence of the kinetics of force development can be completely eliminated by a combination of strong-binding cross-bridges (treatment with NEM-S1) and disruption of near-neighbor cooperativity in the thin filament (partial extraction of TnC). Although it is tempting to conclude from these results that cross-bridge binding is the primary activator of cross-bridge kinetics, our results do not exclude the possibility that other mechanisms are operative under physiological conditions, where the number of strongly bound cross-bridges is certainly less than in our experiments with NEM-S1. In support of the this idea, ktr in control fibers did not increase substantially until Ca2+-activated isometric forces were greater than half-maximal (Fig 7 A). Thus, it is possible in our experiments that strong binding cross-bridges in the form of NEM-S1 are such potent activators of the thin filament that other activating processes involving the effects of Ca2+ binding to potential regulatory sites, such as regulatory light chain (
We also observed that the effects on ktr because of NEM-S1 alone were greater than the effects of TnC extraction alone. At first glance, this suggests that cooperativity in the activation of contraction is predominantly due to cross-bridge binding within functional groups, with much lesser contributions due to near-neighbor cooperation between adjacent functional groups. However, it must again be recognized that the addition of NEM-S1 in our experiments substantially increases the number of strong-binding cross-bridges above that normally found in skeletal muscle fibers under physiological conditions. Thus, it is likely that strong-binding cross-bridges are not as dominant in activating contraction in living muscles, and that near-neighbor mechanisms play a proportionately greater role than implied by our present results with NEM-S1. Our finding that partial extraction of TnC was required to completely eliminate the activation dependence of ktr is consistent with this conclusion.
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Footnotes |
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1 Abbreviations used in this paper: NEM, N-ethylmaleimide; NEM-S1, NEM-modified myosin subfragment-1; TnC, troponin C.
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Acknowledgements |
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The authors thank Chad Warren for the preparation of NEM-S1 and Dr. James Graham for SDS-PAGE analysis of the muscle fibers.
This study was supported by grant HL-54581 from the National Institutes of Health to R.L. Moss.
Submitted: 29 March 2000
Revised: 18 December 2000
Accepted: 20 December 2000
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References |
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