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Regulation of contraction kinetics in skinned skeletal muscle fibers by calcium and troponin C
ABB Archives of Biochemistry and Biophysics 456 (2006) 119–126 www.elsevier.com/locate/yabbi
Regulation of contraction kinetics in skinned skeletal muscle fibers by calcium and troponin C Ye Luo, Jack A. Rall
*
Department of Physiology and Cell Biology, Ohio State University, 1645 Neil Ave, Columbus, OH 43210-1218, USA Received 30 January 2006, and in revised form 6 April 2006 Available online 8 May 2006
Abstract The influences of [Ca2+] and Ca2+ dissociation rate from troponin C (TnC) on the kinetics of contraction (kCa) activated by photolysis of a caged Ca2+ compound in skinned fast-twitch psoas and slow-twitch soleus fibers from rabbits were investigated at 15 C. Increasing the amount of Ca2+ released increased the amount of force in psoas and soleus fibers and increased kCa in a curvilinear manner in psoas fibers 5-fold but did not alter kCa in soleus fibers. Reconstituting psoas fibers with mutants of TnC that in solution exhibited increased Ca2+ affinity and 2- to 5-fold decreased Ca2+ dissociation rate (M82Q TnC) or decreased Ca2+ affinity and 2-fold increased Ca2+ dissociation rate (NHdel TnC) did not affect maximal kCa. Thus the influence of [Ca2+] on kCa is fiber type dependent and the maximum kCa in psoas fibers is dominated by kinetics of cross-bridge cycling over kinetics of Ca2+ exchange with TnC. 2006 Elsevier Inc. All rights reserved. Keywords: Caged calcium compound; Troponin C mutants; Calcium dependence of force; Muscle contraction
The rate of isometric contraction in skinned psoas muscle fibers depends on the level of Ca2+ activation. This result has been shown in two different ways. First, at a fixed [Ca2+] the rate of force redevelopment (ktr) following a period of shortening with immediate re-stretch to the starting sarcomere length is Ca2+-dependent [1– 3]. Second, the rate of force development (kCa) when a muscle is activated from rest by the release of a fixed amount of Ca2+ through the photolysis of a caged Ca2+ compound is dependent on the amount of Ca2+ released [4–6]. In psoas muscle the ktr and kCa versus relative force relationships are curvilinear in which ktr and kCa do not change appreciably at lower forces but steeply increase at higher forces by up to 10-fold. A similar characteristic curve of ktr versus relative force but with less Ca2+ dependence has been observed in slow-twitch fibers in comparison with that in fast twitch fibers [3]. But a comparison of the Ca2+ dependence of kCa *
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between fast-twitch and slow-twitch skeletal muscle has not been made. ktr is thought to reflect the kinetics of cross-bridge transitions from detached states to the strongly bound, force-generating states but it may not include the process of Ca2+ binding with TnC and subsequent thin filament activation. In contrast kCa monitors force development from a relaxed state to the final steady-state level following a rapid Ca2+ release by photolysis of caged Ca2+ compounds. Thus, there are two coupled kinetic processes involved in kCa measurement: (1) Ca2+ binding and thin filament activation; (2) crossbridge attachment and transition to the force-generating states. The mechanism by which Ca2+ regulates the kinetics of skeletal muscle contraction remains unresolved, although two competing hypotheses have been proposed [7]. Ca2+ could regulate the rate of contraction by directly affecting the kinetics of cross-bridge transition from weakly to strongly bound, force-generating states independent of Ca2+ binding to TnC [1]. Alternatively, Ca2+ could exert a regulatory influence on the contractile kinetics via control
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of the level of the thin filament activation by binding to troponin C (TnC)1 without a direct effect on the kinetics of cross-bridge turnover per se [8,9]. This model couples the kinetics of Ca2+ exchange with TnC to the kinetics of cross-bridge transitions between weakly and strongly bound, force-generating states such that: k on
fapp
k off
gapp
Ca2þ -free; no force $ Ca2þ -bound; no force $ Ca2þ -bound; force:
This model is able to account for the Ca2+ dependence of ktr or kCa seen in skeletal muscle simply through Ca2+ exchange with TnC without requiring any Ca2+ sensitivity of the cross-bridge turnover rates. Ca2+ dissociation from TnC, a slow process in the exchange of Ca2+ with TnC, could be a primary determinant of the rate of muscle contraction since the kinetics of contraction would be proportional to koff + kon [Ca2+]. Support for this latter hypothesis comes from several lines of evidence. Varying the kinetics and amount of thin filament activation does influence the rate of contraction. When thin filaments were partially activated by N-ethylamaleimide-modified myosin subfragment 1 (NEM-S1), ktr increased at submaximal activation, producing a maximum rate at low [Ca2+] in rabbit skinned psoas fibers [10]. Substitution of a constitutively active form of TnC for the endogenous TnC increased ktr at submaximal activation levels [11]. When calmidazolium, which causes a decreased rate of Ca2+ dissociation from TnC in solution [12], was present in rabbit skinned psoas fibers, ktr decreased at maximum [Ca2+] but increased at submaximal [Ca2+] level [13]. These results indicate that changing the kinetics of thin filament activation can modulate the rate of contraction, especially at submaximal Ca2+ activation levels. However, this may not be the case at maximal Ca2+ activation. The Ca2+ association to the regulatory sites of TnC appears to be diffusion-dominated [12]. At high [Ca2+], such a rapid on-rate could overbalance the process of Ca2+ dissociation from TnC, making the equilibrium of Ca2+ exchange with TnC almost unidirectional, favoring the Ca2+ association side. Thus, Ca2+ dissociation from TnC may not be a primary determinant of the rate of contraction under maximum activation conditions. In maximally Ca2+ activated fibers, the kinetics of crossbridge cycling could be the primary determinant of the rate of contraction in skeletal muscle. Support for this hypothesis is given by the fact that maximal ktr correlates with the myosin isoforms present in fast-twitch and slow-twitch skeletal muscle [2,3]. Also maximal ktr corresponds closely to the changes in the kinetics of cross-bridge cycling. Increasing cross-bridge cycling rate by substituting ATP with 2-deoxy-ATP increases the maximal ktr, while decreasing cross-bridge cycling rate by lowering [ATP] decreases the maximal ktr [14]. Thus, the rate of maximum contrac-
1 Abbreviations used: TnC, troponin C; NEM-S1, N-ethylamaleimidemodified myosin subfragment 1; TFP, trifluoperazine dihydrochloride.
tion could be modulated primarily by the kinetics of cross-bridge cycling and be much less influenced by the thin filament activation. It is possible to directly assess the influence of Ca2+ exchange with TnC on the maximum rate of contraction by reconstituting skinned psoas fibers with mutants of TnC which exhibit varying Ca2+ off-rates (koff) and then maximally activating the fibers by photolysis of a caged Ca2+ compound to measure kCa. Mutants of TnC with decreased or increased Ca2+ off-rates but similar Ca2+ on-rates (kon) have been characterized in solution and in skinned psoas fibers [15]. The objectives of this investigation with mammalian skinned fibers were to utilize photolysis of caged Ca2+to determine: (a) Ca2+-dependence of kCa in psoas and soleus fibers and (b) the effect, if any, of variation of Ca2+ off-rate from TnC (koff) on maximum kCa in psoas fibers. Materials and methods Skinned fiber preparations and experimental apparatus Female New Zealand white rabbits were anaesthetized with ketamine (60 mg kg1 i.m.) and then euthanized with sodium pentobarbital (150 mg kg1 i.v.) before tissue harvest. The animal use protocol for these experiments was approved by the Institutional Laboratory Animal Care and Use Committee of the Ohio State University. Bundles of psoas and soleus muscle fibers were skinned and stored frozen for up to four weeks in a glycerinating solution containing leupeptin to prevent proteolysis. On the day of an experiment, single fibers of 2 mm in length were soaked in a dissecting solution containing 1% (v/v) Triton X-100 for 30 min to remove residual sarcoplasmic reticulum and sarcolemma. In order to minimize end compliance, the fiber ends were chemically fixed [16]. The fixed ends were then wrapped in aluminum foil T-clips in dissecting solution for attachment to hooks on the experimental apparatus. Fibers were maintained at 4 C in dissecting solution for up to 4 h. The single fiber experimental setup has been described [15]. The resting sarcomere length of the fiber was set at 2.6 lm using the first-order diffraction pattern from a He–Ne laser. Temperature was monitored continuously by a thermocouple placed near the fibers and maintained at 15 C unless otherwise noted. The procedures for activating fibers by flash photolysis of a caged Ca2+ compound have been described [6]. Briefly the UV flash for the photolysis of caged compounds was provided by a frequency-doubled ruby laser (model QSR2, Lumonics, Warwickshire, UK) which produced a 30 ns duration pulse at 347 nm with an energy of 300 mJ. Output energy was maintained at 100 mJ at the level of fiber by placing glass slides in the beam path to attenuate the laser output. The focused beam width was 2 mm and the beam length was controlled using an adjustable mask placed over the fiber such that the T-clips were not flashed but the fiber was illuminated over its entire length.
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Solutions
TnC extraction and reconstitution
The caged compound solutions were prepared using the computer program of Fabiato [17]. The published values of Ca2+ and Mg2+ binding constants of NPEGTA [18] and DM-nitrophen [19] were adjusted for experimental temperature, ionic strength and pH such that they were able to produce solutions with anticipated [Ca2+] levels. A number of caged compound solutions were used for different test purposes, but the basic design was the same. The caged compound solutions contained (in mM): 35–53 KCl, 20 imidazole, 7 HDTA, 10 glutathione, 14.5 creatine phosphate and were adjusted to 180 mM ionic strength and pH 7.0 at 15 C except that various concentrations of caged compound (NP-EGTA 4–8 mM and DM-nitrophen 4–4.5 mM) were added. For the NP-EGTA solutions, the free [Mg2+] = 1 mM and the [MgÆATP] = 2.5 mM. Since DM-nitrophen binds Mg2+ strongly, to get adequate Ca2+ bound to induce full activation, the DM-nitrophen solutions contained low [Mg2+], i.e., the free [Mg2+] = 0.036 mM and the [MgÆATP] = 0.63 mM. To induce a desired level of Ca2+ activation at a certain total [NP-EGTA] or [DMnitrophen] and constant laser energy, the Fabiato program was used to calculate the necessary pre-photolysis pCa value to load a certain amount of Ca2+ onto the NP-EGTA or DM-nitrophen. Caged compound solutions were prepared from stocks containing all ingredients except the caged compound and were stored frozen in aliquots of 1 ml. Shortly before an experiment these pre-solutions were thawed and added to the caged compound in an appropriate amount to give a final caged compound solution. The final caged compound solutions were stored in a light proof container in the freezer and used within one or two days to avoid degradation of the caged compound. NP-EGTA was purchased from Molecular Probes, Eugene, OR and DM-nitrophen from Calbiochem, La Jolla, CA. Solutions for steady-state force measurements were prepared using a computer program developed by R. Godt (Medical College of Georgia) and have been described [15].
TnC extraction was performed at 20 C using an extraction solution that contained (in mM): 5 EDTA, 10 Hepes, and 0.5 trifluoperazine dihydrochloride (TFP) at pH 7.0 [20]. Fibers were stretched to a sarcomere length of 3.5 lm in pCa9.0 solution and then soaked in the extraction solution for 2 min. Fibers were then transferred back to pCa9.0 solution and released to a sarcomere length of 2.6 lm. After extraction, fibers were washed with pCa9.0 solution to remove TFP. Activation of fibers in pCa4.0 was used to assess the extent of TnC extraction. For skinned psoas fibers, a 2-min incubation in extraction solution was adequate to reduce average pCa4.0 force to less than 5% of the maximal isometric force before extraction. This procedure was unsuccessful for soleus fibers since only a 40–50% reduction in force at pCa4.0 occurred after greater than 30 min of extraction. Reconstitution of TnC in psoas fibers was achieved at 20 C by soaking TnC-extracted fibers in a pCa9.0 solution containing 16.7 lM purified TnC. Fibers were bathed in this solution for 20–60 s, then activated at pCa4.0 and returned to pCa9.0 solution with no TnC added. Fibers were cycled through this procedure until maximum force recovery was achieved, usually after 2 min incubation in purified TnC. Recombinant TnC Recombinant TnCs of chicken fast-twitch skeletal muscle that were utilized included wild-type (rTnC) and mutant TnCs (M82Q TnC, M46Q TnC and NHdel TnC [deletion of N terminal 1–11 amino acids]) that exhibited varying Ca2+ off-rates from their regulatory sites. Table 1 displays the Ca2+ binding and exchange properties of these TnC mutants in solution in the absence and presence of a TnI peptide, TnI96–148 [21,22]. The Ca2+ affinity of TnCÆTnI96–148 has been shown to be a better predictor of the Ca2+ sensitivity of muscle force production than the Ca2+ affinity of TnC alone [22]. Furthermore the Ca2+ off-rate from F29W TnCÆTnI96–148 (11 s1) at 15 C is similar to the Ca2+ off-rate observed in fully reconstituted thin filaments (actin/tropomyosin/troponin) in the presence of
Table 1 Properties of TnC mutants in solution in the absence and presence of TnI96–148 at 15 C Without TnI96–148 NHdel M82Q M46Q F29W
With TnI96–148
Kd (lM)
Off-rate (s1)
On-rate (·108 M1 s1)
Kd (lM)
Off-rate (s1)
On-rate (·107 M1 s1)
8.4 ± 0.05 0.7 ± 0.03 0.9 ± 0.03 3.2 ± 0.10
515 ± 2 76 ± 3 93 ± 2 340 ± 11
0.6 1.1 1.1 1.1
1304 ± 26 149 ± 0.05 288 ± 3.5 267 ± 1.5
16.6 ± 0.15 5.8 ± 0.05 12.6 ± 0.1 10.6 ± 0.1
1.3 3.9 4.4 4.0
Data without TnI96–148 [21] and with TnI96–148 [22]. NHdel = deletion of first 11 amino acids in N terminal of TnC (NHdel TnC data from J.P. Davis and S.B. Tikunova, unpublished). On-rates calculated assuming: Kd = koff/kon. The Ca2+ dissociation rates measured from F29W TnC and its mutants in the presence of TnI96–148 were similar to those measured in the presence of intact TnI [22]. Mutants of TnC were characterized in a F29W TnC construct in order to measure intrinsic fluorescence changes in response to Ca2+ binding and exchange. F29W TnC or rTnC reconstituted into psoas fibers resulted in a pCa50 of force similar to that observed in fibers containing endogenous TnC [15].
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myosin S1 (14 s1) (J. Davis and S. Tikunova, unpublished observations) and in isolated myofibrils in rigor (16 s1) [23]. Thus the Ca2+ off-rates for TnC in the presence of TnI96–148 are likely to be more similar to the off-rates that occur in muscle fibers than the rates observed in isolated TnC alone. Caged compound protocol To induce contraction, skinned fibers were first soaked in a caged compound solution containing NP-EGTA or DM-nitrophen for 2 min to allow for caged compound diffusion into the fibers. Fibers were then transferred into an empty chamber and flashed in air in order to lessen nonuniform photolysis of caged compound due to absorbance and attenuation of the laser light in solution. After contraction transients were recorded, fibers were transferred back to pCa9.0 solution. In order to avoid any changes in the condition of skinned fibers, all caged compound experiments were ‘one flash’. The protocol for study of the effects of rTnC or mutant TnCs on maximum contraction is illustrated in Fig. 1, including: (A) determine the maximum force in pCa4.0 (F4.0), (B) extract TnC until force in pCa4.0 was <5% of F4.0, (C) reconstitute with rTnC or mutant TnC until force in pCa4.0 was >90% of F4.0, (D) add DM-nitrophen and induce contraction by flash photolysis of DM-nitrophen. This example shows that the limitation of diffusional delay in skinned fibers can be overcome by the use of caged Ca2+ compounds. Also it has been shown that photolysis of DM-nitrophen produced a contraction in skinned frog fibers that is comparable in amplitude and rate to that observed in electrically stimulated intact fibers [6].
Fig. 1. Protocol for study of effects of rTnC or mutant TnC on maximum contraction in skinned psoas fibers. (A) Time course of contraction in pCa4.0. (B) Force developed in pCa4.0 after extraction of endogenous TnC. (C) Force developed in pCa4.0 after reconstitution of NHdel TnC. (D) Force development induced by photolysis of DM-nitrophen in the reconstituted fibers. (E) Same trace as (D) but on an expanded time scale.
Data analysis The steady-state forces developed upon photolysis of NP-EGTA or DM-nitrophen were normalized to the maximal force in the preceding pCa4.0 contraction. The force transients following photolysis were normalized to the extent of contraction (DF). The resulting traces were fit to a single exponential equation. Results are presented as means ± SEM. Results Characterization of caged Ca2+ induced contraction in psoas and soleus fibers Photolysis of DM-nitrophen generated 96 ± 1% of the force produced in a pCa4.0 solution and NP-EGTA generated 110 ± 2% of the force produced in a pCa4.0 solution. Fig. 2A shows typical normalized maximum contractions in psoas fibers induced by photolysis of DM-nitrophen or
Fig. 2. Time courses of maximal force development in skinned psoas fibers due to photolysis of NP-EGTA or DM-nitrophen (A) and comparison of time course of maximum contraction in psoas and soleus fibers induced by photolysis of NP-EGTA (B) at 15 C.
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NP-EGTA. On average, the rates of contraction, kCa, in response to photolysis of DM-nitrophen (20.4 ± 1.3 s1, n = 5) or NP-EGTA (16.0 ± 2.5 s1, n = 5) were not significantly different (Fig. 1). The fact that photolysis of NPEGTA and DM-nitrophen elicited full force and similar force transients indicates that the free [Mg2+] in the range of 0.036–1.0 mM has little effect on the maximum rate of contraction and that both caged compounds are suitable for examining the kinetics of contraction. Fig. 2B compares traces of maximum contraction in soleus and psoas fibers induced by photolysis of NP-EGTA. The time course of force development in soleus was significantly slower than in the psoas. The average rate of contraction was 25-fold slower in soleus fibers: kCa of 0.61 ± 0.02 s1 versus 16.0 ± 2.5 s1. To determine if this slow rise of force was a result of fiber evaporation in air, we examined the time courses of force development in skinned soleus fibers at 15 and 12 C (the dew-point). Similar results were observed except for slightly slower traces at 12 C than at 15 C. Effects of altered [Ca2+] on contraction amplitude and rate in psoas and soleus fibers To achieve various levels of [Ca2+] after photolysis at constant free [Mg2+], the extent of Ca2+ loading of NPEGTA was altered such that different levels of steady-state force were produced following photolysis but without significant force before photolysis. Fig. 3 shows examples of force development in psoas fibers at various levels of Ca2+ activation. Force development was examined over a wide range of [Ca2+]s that supported post-photolytic steady-state forces from 0.1 to 1.2 F/F4.0. As shown in Fig. 3, it is clear that lowering [Ca2+] not only decreased steady-state force (Fig. 3A) but also dramatically decreased the rate of force development (Fig. 3B). A plot of kCa versus relative force for psoas fibers is shown in Fig. 4A where relative force is taken as a measure of extent of Ca2+ activation representing the steady-state force following photolysis normalized to the force in an adjacent pCa4.0 contraction (F/F4.0). The relationship between kCa and relative force shows that kCa increased by 5-fold in a curvilinear manner from low to full Ca2+ activation. Fig. 4B shows the kCaversus relative force relationship in soleus fibers. The results for soleus fibers indicate that the rate of force development is independent of the level of Ca2+ activation. The average kCa for all contractions in the soleus fibers was 0.61 ± 0.02 s1 (n = 20). Thus, there is a dramatic difference between the Ca2+ sensitivity of contraction rate in psoas and soleus muscle fibers. Effects of altered Ca2+ dissociation rate from TnC on maximum contraction rate in psoas fibers Effects of varying Ca2+ dissociation rate from TnC on contraction were determined in fibers that contained mutant TnCs that exhibited varying Ca2+ off-rates and
Fig. 3. Effect of Ca2+ on the rate of force development in skinned psoas fibers induced by photolysis of NP-EGTA at 15 C. (A) Force produced following photolysis relative to the adjacent maximal force in pCa4.0. (B) Traces normalized to maximal force development induced by NP-EGTA.
compared to fibers containing endogenous TnC or rTnC. M82Q TnC and M46Q TnC both exhibited an increased Ca2+ affinity and an 5-fold slower Ca2+ dissociation rate from isolated TnC compared to F29W TnC ([21] and Table 1). But Ca2+ affinity and the dissociation rate from M46Q TnCÆTnI96–148 are similar to the F29W TnCÆTnI96–148 control whereas the Ca2+ affinity is increased and the Ca2+ dissociation rate is decreased 2fold for M82Q TnCÆTnI96–148 ([22] and Table 1). M82Q TnC produced a corresponding leftward shift in the force versus pCa relation in psoas fibers compared to endogenous TnC, rTnC or F29W TnC [15] but M46Q TnC did not [22]. On the other hand, NHdel TnC in the absence and presence of TnI96–148 exhibited a decreased Ca2+ affinity, an 2-fold faster Ca2+ off-rate and a rightward shift in the force versus pCa relation. Thus, the rate of Ca2+ dissociation from TnC in the reconstituted fibers was expected to be slower for M82Q TnC, similar for M46Q TnC and faster for NHdel TnC when compared to that with rTnC. Fig. 5 shows examples of maximum contractions in fibers reconstituted with rTnC, M82Q, or NHdel TnC
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and all results are summarized in Table 2. During the application of each solution, a full Ca2+ activation was produced upon photolysis of DM-nitrophen with no significant force before photolysis. Fibers containing rTnC or mutant TnC supported force well in pCa4.0 (Frecon/ Fendo = 0.91–1.01). Average force produced by DM-nitrophen photolysis ranged from 0.92 to 0.97 of force in adjacent pCa4.0 contraction after rTnC or mutant TnC reconstitution. The rate of maximum force development in control fibers with rTnC (22.5 ± 2.9 s1, n = 4) was similar to that observed in the unextracted fibers (20.4 ± 1.3 s1, n = 5). These results indicate that there is little effect on the functionality of the reconstituted fibers due to the extraction/reconstitution procedure. Despite differences in Ca2+ binding and exchange properties in the absence and presence of TnI96–148, the rate of maximum force development in the presence of M82Q TnC was not significantly different than that observed in the presence of M46Q TnC and thus the results were pooled together. Compared with rTnC, neither the introduction of M82Q TnC or M46Q TnC (pooled average 21.8 ± 6.1 s1, n = 5) or NHdel TnC (20.8 ± 1.0 s1, n = 6) had a significant effect on rate of maximal contraction. Thus, the rate of maximum force development was insensitive to either a decrease or increase in Ca2+ dissociation rate from TnC. Discussion
Fig. 4. Rate of contraction induced by photolysis of NP-EGTA (kCa) versus relative force in rabbit psoas and soleus fibers at 15 C. (A) Results for psoas fibers fitted to kCa = A · exp(B · F/F4.0) + C, where A = 0.015 s1, B = 5.82 and C = 5.52 s1. (B) Results for soleus fibers. Data obtained from 20 soleus fibers by ‘one flash’ of NP-EGTA.
The present results demonstrate that in rabbit skinned psoas fibers the rate of force development (kCa) induced by photolysis of a caged Ca2+ compound is dependent on the level of Ca2+ activation. When the [Ca2+] was elevated, kCa increased along with the steady-state post-photolytic force in a curvilinear relation in which kCa increased mainly at concentrations of Ca2+ yielding activation greater than half-maximal. In dramatic contrast, in soleus fibers the rate of force development was independent of the level of Ca2+ activation. At maximal Ca2+ activation in psoas fibers, kCa was insensitive to either an increase or a decrease in the rate of Ca2+ dissociation from TnC. Regulation of contraction by Ca2+ in psoas and soleus fibers
Fig. 5. Examples of time course of maximum force development in presence of rTnC or mutants of TnC in rabbit psoas fibers at 15 C. Mutants of TnC were characterized in a F29W TnC construct. F29W TnC reconstituted into psoas fibers resulted in a pCa50 of force similar to that observed in fibers containing endogenous or rTnC [15].
Whereas the contractile mechanism has been widely investigated in fast-twitch skeletal muscle, fewer studies have been carried out in slow-twitch skeletal muscle. Despite overall similarity, there are a number of quantitative and qualitative differences in the mechanical properties of fast- and slow-twitch fibers. Compared with psoas fast fibers, the rate of force development induced by photolysis of a caged Ca2+ compound (kCa) and the Ca2+ dependence of kCa were greatly reduced in soleus fibers, which is consistent with the previous reports with ktr measurements [2,24]. These differences between fiber types may be related to the unique protein expression patterns in the fast- and slowtwitch muscle contractile machinery, such as different isoforms of myosin, TnC and TnI [2,25]. More specifically,
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Table 2 Kinetics of maximal force development in skinned psoas fibers induced by photolysis of DM-nitrophen at 15 C in the presence of rTnC or mutant TnC N
TnC
Fext/F4.0
Frecon/Fendo
FDM/F4.0
kCa (s1)
5 4 5 6
Endogenous rTnC M82Q and M46Q NHdel
0.01 0.01 0.01
1.01 ± 0.01 0.93 ± 0.01 0.91 ± 0.02
0.96 ± 0.01 0.97 ± 0.02 0.92 ± 0.07 0.92 ± 0.02
20.4 ± 1.3 22.5 ± 2.9 21.8 ± 6.1 20.8 ± 1.0
Fext/F4.0 = force in pCa4.0 after extraction divided force in pCa4.0 before extraction. Frecon/Fendo = force in pCa4.0 after rTnC or mutant TnC reconstitution divided by force in pCa4.0 before extraction. FDM/F4.0 = steady-state force following photolysis of DM-nitrophen divided by force in adjacent pCa4.0 contraction. Conditions ([DM-nitrophen] in mM): (1) M82Q TnC: pCa7.4/4 mM, (2) wild-type TnC: pCa7.1/4 mM, (3) endogenous TnC: pCa6.5/4 mM, (4) NHdel TnC: pCa6.5–6.7/3–4.5 mM.
they may be due to kinetic differences in some transitions in the cross-bridge cycle. The basic mechanism of force generation is thought to be similar in both fiber types, presumably involving a two-step mechanism for Pi release coupled with force generation [26,27]. But the corresponding rate constants appear to be different in fast- and slowtwitch fibers. It has been suggested that the isomerization step preceding the force development is significantly slower in soleus than in psoas muscle [26,27]. The differences in the rate constants of specific cross-bridge transitions may be responsible for the observed differences between fast- and slow-twitch fibers in the magnitude and the Ca2+ dependence of contraction rates. The slowing of both forward and reverse rate constants of the isomerization step would lead to a slow contraction, as well as less Ca2+ dependence of contraction rate in slow twitch muscle. Effect of altered Ca2+ dissociation rate from TnC on maximum contraction rate The maximal kCa in muscle fibers was not affected by mutants of TnC that exhibited, in solution, varying rates of Ca2+ dissociation from TnC (up to threefold in the TnCÆTnI96–148 constructs, Table 1). Thus, the maximum rate of contraction is independent of Ca2+ exchange with TnC and depends solely on the intrinsic rate of cross-bridge force development. In contrast M82Q TnC, which slows Ca2+ dissociation from TnC in solution, dramatically slowed relaxation in skinned psoas fibers by two-fold [15]. But NHdel TnC, which accelerates Ca2+ dissociation from TnC in solution, did not alter the rate of relaxation. These results suggest that the Ca2+ dissociation rate from TnC and the cross-bridge detachment rate are similar to each other and together limit the rate of relaxation. In this case, slowing Ca2+ dissociation from TnC would slow relaxation but accelerating Ca2+ dissociation from TnC would have little effect on relaxation because cross-bridge detachment would become rate limiting. Taken together the data suggests that the mechanisms that govern the maximum rate of contraction and rate of relaxation are not the same. Instead of having a direct effect on the cross-bridge kinetics, Ca2+ could, in principle, regulate kCa by the modulation of thin filament activation via the kinetics of Ca2+ binding with TnC and the subsequent interactions within the thin filament. At high [Ca2+], the on-rate dominates
Ca2+ association to TnC [12] such that the kinetics of cross-bridge cycling becomes the only potential determinant of the rate of contraction. The present results showing that maximal kCa was insensitive to changes in the rate of Ca2+ dissociation from TnC confirm these predictions at maximal Ca2+ activation level. During submaximal Ca2+ activation, the rate of Ca2+ binding to TnC is relatively slow because the Ca2+ on-rate is proportional to [Ca2+]. At low [Ca2+] Ca2+ dissociation from TnC could be an important factor by affecting the activated time of individual thin filament regulatory units to control strong crossbridge attachment and force generation even though the Ca2+ association to TnC is thought to be too rapid to limit the rate of muscle contraction. Nonetheless little or no effect was found on ktr at submaximal Ca2+ activation levels in the presence of NHdel TnC [28]. This lack of a further inhibition of ktr was explained by suggesting that the rate of Ca2+ dissociation from TnC is maximally tuned such that increasing the Ca2+ off-rate can not inhibit ktr further, only slowing down Ca2+ dissociation from TnC can speed up ktr. Another possibility is that since ktr measurements are made at constant [Ca2+] any slow kinetic steps involved in the Ca2+ exchange with TnC and subsequent thin filament activation may not be detected with this approach. In contrast, kCa measurement appears to be a better model from this point of view. Therefore, further investigation of the role of Ca2+ dissociation from TnC in determining the rate of skeletal muscle contraction should be expanded to include submaximal Ca2+ activation levels. In conclusion, the rate of force development (kCa) induced by photolysis of a caged Ca2+ compound accelerates as the level of Ca2+ activation is increased in rabbit skinned psoas fibers but is independent of Ca2+ activation in soleus fibers. Differences in the rate constants of specific cross-bridge transitions may be responsible for the differences between fast- and slow-twitch fibers in the magnitude and Ca2+ dependence of contraction rates. At maximal activation kCa in psoas fibers is determined by the cross-bridge kinetics instead of the rate of Ca2+ dissociation from TnC. Thus, the present results are consistent with the notion that the rate of contraction is dominated by the kinetics of cross-bridge cycling over the kinetics of Ca2+ binding with TnC in maximally activated fast twitch skeletal muscle.
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Acknowledgments The authors thank J.P. Davis and S.B. Tikunova for sharing unpublished data on reconstituted thin filaments and critically reading the manuscript. NIH AR020792 to J.A.R. supported this work. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
B. Brenner, Proc. Natl. Acad. Sci. USA 85 (1988) 3265–3269. J.M. Metzger, R.L. Moss, Science 247 (1990) 1088–1090. N.C. Millar, E. Homsher, J. Biol. Chem. 265 (1990) 20234–20240. C.C. Ashley, I.P. Mulligan, T.J. Lea, Q. Rev. Biophys. 24 (1991) 1–73. A. Araujo, J.W. Walker, Am. J. Physiol. Heart Circ. Physiol. 267 (1994) H1643–H1653. P.A. Wahr, J.A. Rall, Am. J. Physiol. Cell Physiol. 272 (1997) C1664– C1671. A.M. Gordon, E. Homsher, M. Regnier, Physiol. Rev. 80 (2000) 853– 924. A. Landesberg, S. Sideman, Am. J. Physiol. Heart Circ. Physiol. 266 (1994) H1260–H1271. W.O. Hancock, L.L. Huntsman, A.M. Gordon, J. Muscle Res. Cell Motil. 18 (1997) 671–681. D.R. Swartz, R.L. Moss, J. Biol. Chem. 267 (1991) 20497–20506. P.B. Chase, D.A. Martyn, J.D. Hannon, Biophys. J. 67 (1994) 1994– 2001. J.D. Johnson, R.J. Nakkula, C. Vasulka, L.B. Smillie, J. Biol. Chem. 269 (1994) 8919–8923.
[13] M. Regnier, D.A. Martyn, P.B. Chase, Biophys. J. 71 (1996) 2786– 2794. [14] M. Regnier, D.A. Martyn, P.B. Chase, Biophys. J. 74 (1998) 2005– 2015. [15] Y. Luo, J.P. Davis, L.B. Smillie, J.A. Rall, J. Physiol. 545 (2002) 887– 901. [16] P.B. Chase, M.J. Kushmerick, Biophys. J. 53 (1988) 935–946. [17] A. Fabiato, Methods Enzymol. 157 (1988) 378–417. [18] G.C.R. Ellis-Davies, J.H. Kaplan, Proc. Natl. Acad. Sci. USA 91 (1994) 187–191. [19] J.H. Kaplan, G.C. Ellis-Davies, Proc. Natl. Acad. Sci. USA 85 (1988) 6571–6575. [20] J.M. Metzger, M.L. Greaser, R.L. Moss, J. Gen. Physiol. 93 (1989) 855–883. [21] S.B. Tikunova, J.A. Rall, J.P. Davis, Biochemistry 41 (2002) 6697– 6705. [22] J.P. Davis, J.A. Rall, C. Alionte, S.B. Tikunova, J. Biol. Chem. 279 (2004) 17348–17360. [23] J.P. Davis, S.B. Tikunova, D.R. Swartz, J.A. Rall, Biophys. J. 86 (2004) A218. [24] C. Tesi, F. Colomo, S. Nencini, N. Piroddi, C. Poggesi, Biophys. J. 78 (2000) 3081–3092. [25] P.J. Reiser, R.L. Moss, G.G. Giulian, M.L. Greaser, J. Biol. Chem. 260 (1985) 9077–9080. [26] N.C. Millar, E. Homsher, Am. J. Physiol. Cell Physiol. 262 (1992) C1239–C1245. [27] G. Wang, M. Kawai, Biophys. J. 73 (1997) 878–894. [28] M. Regnier, A.J. Rivera, P.B. Chase, L.B. Smillie, M.M. Sorenson, Biophys. J. 76 (1999) 2664–2672.
Regulation of contraction kinetics in skinned skeletal muscle fibers by calcium and troponin C Ye Luo, Jack A. Rall
*
Department of Physiology and Cell Biology, Ohio State University, 1645 Neil Ave, Columbus, OH 43210-1218, USA Received 30 January 2006, and in revised form 6 April 2006 Available online 8 May 2006
Abstract The influences of [Ca2+] and Ca2+ dissociation rate from troponin C (TnC) on the kinetics of contraction (kCa) activated by photolysis of a caged Ca2+ compound in skinned fast-twitch psoas and slow-twitch soleus fibers from rabbits were investigated at 15 C. Increasing the amount of Ca2+ released increased the amount of force in psoas and soleus fibers and increased kCa in a curvilinear manner in psoas fibers 5-fold but did not alter kCa in soleus fibers. Reconstituting psoas fibers with mutants of TnC that in solution exhibited increased Ca2+ affinity and 2- to 5-fold decreased Ca2+ dissociation rate (M82Q TnC) or decreased Ca2+ affinity and 2-fold increased Ca2+ dissociation rate (NHdel TnC) did not affect maximal kCa. Thus the influence of [Ca2+] on kCa is fiber type dependent and the maximum kCa in psoas fibers is dominated by kinetics of cross-bridge cycling over kinetics of Ca2+ exchange with TnC. 2006 Elsevier Inc. All rights reserved. Keywords: Caged calcium compound; Troponin C mutants; Calcium dependence of force; Muscle contraction
The rate of isometric contraction in skinned psoas muscle fibers depends on the level of Ca2+ activation. This result has been shown in two different ways. First, at a fixed [Ca2+] the rate of force redevelopment (ktr) following a period of shortening with immediate re-stretch to the starting sarcomere length is Ca2+-dependent [1– 3]. Second, the rate of force development (kCa) when a muscle is activated from rest by the release of a fixed amount of Ca2+ through the photolysis of a caged Ca2+ compound is dependent on the amount of Ca2+ released [4–6]. In psoas muscle the ktr and kCa versus relative force relationships are curvilinear in which ktr and kCa do not change appreciably at lower forces but steeply increase at higher forces by up to 10-fold. A similar characteristic curve of ktr versus relative force but with less Ca2+ dependence has been observed in slow-twitch fibers in comparison with that in fast twitch fibers [3]. But a comparison of the Ca2+ dependence of kCa *
Corresponding author. Fax: +1 614 292 4888. E-mail address: [email protected] (J.A. Rall).
0003-9861/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.04.014
between fast-twitch and slow-twitch skeletal muscle has not been made. ktr is thought to reflect the kinetics of cross-bridge transitions from detached states to the strongly bound, force-generating states but it may not include the process of Ca2+ binding with TnC and subsequent thin filament activation. In contrast kCa monitors force development from a relaxed state to the final steady-state level following a rapid Ca2+ release by photolysis of caged Ca2+ compounds. Thus, there are two coupled kinetic processes involved in kCa measurement: (1) Ca2+ binding and thin filament activation; (2) crossbridge attachment and transition to the force-generating states. The mechanism by which Ca2+ regulates the kinetics of skeletal muscle contraction remains unresolved, although two competing hypotheses have been proposed [7]. Ca2+ could regulate the rate of contraction by directly affecting the kinetics of cross-bridge transition from weakly to strongly bound, force-generating states independent of Ca2+ binding to TnC [1]. Alternatively, Ca2+ could exert a regulatory influence on the contractile kinetics via control
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of the level of the thin filament activation by binding to troponin C (TnC)1 without a direct effect on the kinetics of cross-bridge turnover per se [8,9]. This model couples the kinetics of Ca2+ exchange with TnC to the kinetics of cross-bridge transitions between weakly and strongly bound, force-generating states such that: k on
fapp
k off
gapp
Ca2þ -free; no force $ Ca2þ -bound; no force $ Ca2þ -bound; force:
This model is able to account for the Ca2+ dependence of ktr or kCa seen in skeletal muscle simply through Ca2+ exchange with TnC without requiring any Ca2+ sensitivity of the cross-bridge turnover rates. Ca2+ dissociation from TnC, a slow process in the exchange of Ca2+ with TnC, could be a primary determinant of the rate of muscle contraction since the kinetics of contraction would be proportional to koff + kon [Ca2+]. Support for this latter hypothesis comes from several lines of evidence. Varying the kinetics and amount of thin filament activation does influence the rate of contraction. When thin filaments were partially activated by N-ethylamaleimide-modified myosin subfragment 1 (NEM-S1), ktr increased at submaximal activation, producing a maximum rate at low [Ca2+] in rabbit skinned psoas fibers [10]. Substitution of a constitutively active form of TnC for the endogenous TnC increased ktr at submaximal activation levels [11]. When calmidazolium, which causes a decreased rate of Ca2+ dissociation from TnC in solution [12], was present in rabbit skinned psoas fibers, ktr decreased at maximum [Ca2+] but increased at submaximal [Ca2+] level [13]. These results indicate that changing the kinetics of thin filament activation can modulate the rate of contraction, especially at submaximal Ca2+ activation levels. However, this may not be the case at maximal Ca2+ activation. The Ca2+ association to the regulatory sites of TnC appears to be diffusion-dominated [12]. At high [Ca2+], such a rapid on-rate could overbalance the process of Ca2+ dissociation from TnC, making the equilibrium of Ca2+ exchange with TnC almost unidirectional, favoring the Ca2+ association side. Thus, Ca2+ dissociation from TnC may not be a primary determinant of the rate of contraction under maximum activation conditions. In maximally Ca2+ activated fibers, the kinetics of crossbridge cycling could be the primary determinant of the rate of contraction in skeletal muscle. Support for this hypothesis is given by the fact that maximal ktr correlates with the myosin isoforms present in fast-twitch and slow-twitch skeletal muscle [2,3]. Also maximal ktr corresponds closely to the changes in the kinetics of cross-bridge cycling. Increasing cross-bridge cycling rate by substituting ATP with 2-deoxy-ATP increases the maximal ktr, while decreasing cross-bridge cycling rate by lowering [ATP] decreases the maximal ktr [14]. Thus, the rate of maximum contrac-
1 Abbreviations used: TnC, troponin C; NEM-S1, N-ethylamaleimidemodified myosin subfragment 1; TFP, trifluoperazine dihydrochloride.
tion could be modulated primarily by the kinetics of cross-bridge cycling and be much less influenced by the thin filament activation. It is possible to directly assess the influence of Ca2+ exchange with TnC on the maximum rate of contraction by reconstituting skinned psoas fibers with mutants of TnC which exhibit varying Ca2+ off-rates (koff) and then maximally activating the fibers by photolysis of a caged Ca2+ compound to measure kCa. Mutants of TnC with decreased or increased Ca2+ off-rates but similar Ca2+ on-rates (kon) have been characterized in solution and in skinned psoas fibers [15]. The objectives of this investigation with mammalian skinned fibers were to utilize photolysis of caged Ca2+to determine: (a) Ca2+-dependence of kCa in psoas and soleus fibers and (b) the effect, if any, of variation of Ca2+ off-rate from TnC (koff) on maximum kCa in psoas fibers. Materials and methods Skinned fiber preparations and experimental apparatus Female New Zealand white rabbits were anaesthetized with ketamine (60 mg kg1 i.m.) and then euthanized with sodium pentobarbital (150 mg kg1 i.v.) before tissue harvest. The animal use protocol for these experiments was approved by the Institutional Laboratory Animal Care and Use Committee of the Ohio State University. Bundles of psoas and soleus muscle fibers were skinned and stored frozen for up to four weeks in a glycerinating solution containing leupeptin to prevent proteolysis. On the day of an experiment, single fibers of 2 mm in length were soaked in a dissecting solution containing 1% (v/v) Triton X-100 for 30 min to remove residual sarcoplasmic reticulum and sarcolemma. In order to minimize end compliance, the fiber ends were chemically fixed [16]. The fixed ends were then wrapped in aluminum foil T-clips in dissecting solution for attachment to hooks on the experimental apparatus. Fibers were maintained at 4 C in dissecting solution for up to 4 h. The single fiber experimental setup has been described [15]. The resting sarcomere length of the fiber was set at 2.6 lm using the first-order diffraction pattern from a He–Ne laser. Temperature was monitored continuously by a thermocouple placed near the fibers and maintained at 15 C unless otherwise noted. The procedures for activating fibers by flash photolysis of a caged Ca2+ compound have been described [6]. Briefly the UV flash for the photolysis of caged compounds was provided by a frequency-doubled ruby laser (model QSR2, Lumonics, Warwickshire, UK) which produced a 30 ns duration pulse at 347 nm with an energy of 300 mJ. Output energy was maintained at 100 mJ at the level of fiber by placing glass slides in the beam path to attenuate the laser output. The focused beam width was 2 mm and the beam length was controlled using an adjustable mask placed over the fiber such that the T-clips were not flashed but the fiber was illuminated over its entire length.
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Solutions
TnC extraction and reconstitution
The caged compound solutions were prepared using the computer program of Fabiato [17]. The published values of Ca2+ and Mg2+ binding constants of NPEGTA [18] and DM-nitrophen [19] were adjusted for experimental temperature, ionic strength and pH such that they were able to produce solutions with anticipated [Ca2+] levels. A number of caged compound solutions were used for different test purposes, but the basic design was the same. The caged compound solutions contained (in mM): 35–53 KCl, 20 imidazole, 7 HDTA, 10 glutathione, 14.5 creatine phosphate and were adjusted to 180 mM ionic strength and pH 7.0 at 15 C except that various concentrations of caged compound (NP-EGTA 4–8 mM and DM-nitrophen 4–4.5 mM) were added. For the NP-EGTA solutions, the free [Mg2+] = 1 mM and the [MgÆATP] = 2.5 mM. Since DM-nitrophen binds Mg2+ strongly, to get adequate Ca2+ bound to induce full activation, the DM-nitrophen solutions contained low [Mg2+], i.e., the free [Mg2+] = 0.036 mM and the [MgÆATP] = 0.63 mM. To induce a desired level of Ca2+ activation at a certain total [NP-EGTA] or [DMnitrophen] and constant laser energy, the Fabiato program was used to calculate the necessary pre-photolysis pCa value to load a certain amount of Ca2+ onto the NP-EGTA or DM-nitrophen. Caged compound solutions were prepared from stocks containing all ingredients except the caged compound and were stored frozen in aliquots of 1 ml. Shortly before an experiment these pre-solutions were thawed and added to the caged compound in an appropriate amount to give a final caged compound solution. The final caged compound solutions were stored in a light proof container in the freezer and used within one or two days to avoid degradation of the caged compound. NP-EGTA was purchased from Molecular Probes, Eugene, OR and DM-nitrophen from Calbiochem, La Jolla, CA. Solutions for steady-state force measurements were prepared using a computer program developed by R. Godt (Medical College of Georgia) and have been described [15].
TnC extraction was performed at 20 C using an extraction solution that contained (in mM): 5 EDTA, 10 Hepes, and 0.5 trifluoperazine dihydrochloride (TFP) at pH 7.0 [20]. Fibers were stretched to a sarcomere length of 3.5 lm in pCa9.0 solution and then soaked in the extraction solution for 2 min. Fibers were then transferred back to pCa9.0 solution and released to a sarcomere length of 2.6 lm. After extraction, fibers were washed with pCa9.0 solution to remove TFP. Activation of fibers in pCa4.0 was used to assess the extent of TnC extraction. For skinned psoas fibers, a 2-min incubation in extraction solution was adequate to reduce average pCa4.0 force to less than 5% of the maximal isometric force before extraction. This procedure was unsuccessful for soleus fibers since only a 40–50% reduction in force at pCa4.0 occurred after greater than 30 min of extraction. Reconstitution of TnC in psoas fibers was achieved at 20 C by soaking TnC-extracted fibers in a pCa9.0 solution containing 16.7 lM purified TnC. Fibers were bathed in this solution for 20–60 s, then activated at pCa4.0 and returned to pCa9.0 solution with no TnC added. Fibers were cycled through this procedure until maximum force recovery was achieved, usually after 2 min incubation in purified TnC. Recombinant TnC Recombinant TnCs of chicken fast-twitch skeletal muscle that were utilized included wild-type (rTnC) and mutant TnCs (M82Q TnC, M46Q TnC and NHdel TnC [deletion of N terminal 1–11 amino acids]) that exhibited varying Ca2+ off-rates from their regulatory sites. Table 1 displays the Ca2+ binding and exchange properties of these TnC mutants in solution in the absence and presence of a TnI peptide, TnI96–148 [21,22]. The Ca2+ affinity of TnCÆTnI96–148 has been shown to be a better predictor of the Ca2+ sensitivity of muscle force production than the Ca2+ affinity of TnC alone [22]. Furthermore the Ca2+ off-rate from F29W TnCÆTnI96–148 (11 s1) at 15 C is similar to the Ca2+ off-rate observed in fully reconstituted thin filaments (actin/tropomyosin/troponin) in the presence of
Table 1 Properties of TnC mutants in solution in the absence and presence of TnI96–148 at 15 C Without TnI96–148 NHdel M82Q M46Q F29W
With TnI96–148
Kd (lM)
Off-rate (s1)
On-rate (·108 M1 s1)
Kd (lM)
Off-rate (s1)
On-rate (·107 M1 s1)
8.4 ± 0.05 0.7 ± 0.03 0.9 ± 0.03 3.2 ± 0.10
515 ± 2 76 ± 3 93 ± 2 340 ± 11
0.6 1.1 1.1 1.1
1304 ± 26 149 ± 0.05 288 ± 3.5 267 ± 1.5
16.6 ± 0.15 5.8 ± 0.05 12.6 ± 0.1 10.6 ± 0.1
1.3 3.9 4.4 4.0
Data without TnI96–148 [21] and with TnI96–148 [22]. NHdel = deletion of first 11 amino acids in N terminal of TnC (NHdel TnC data from J.P. Davis and S.B. Tikunova, unpublished). On-rates calculated assuming: Kd = koff/kon. The Ca2+ dissociation rates measured from F29W TnC and its mutants in the presence of TnI96–148 were similar to those measured in the presence of intact TnI [22]. Mutants of TnC were characterized in a F29W TnC construct in order to measure intrinsic fluorescence changes in response to Ca2+ binding and exchange. F29W TnC or rTnC reconstituted into psoas fibers resulted in a pCa50 of force similar to that observed in fibers containing endogenous TnC [15].
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myosin S1 (14 s1) (J. Davis and S. Tikunova, unpublished observations) and in isolated myofibrils in rigor (16 s1) [23]. Thus the Ca2+ off-rates for TnC in the presence of TnI96–148 are likely to be more similar to the off-rates that occur in muscle fibers than the rates observed in isolated TnC alone. Caged compound protocol To induce contraction, skinned fibers were first soaked in a caged compound solution containing NP-EGTA or DM-nitrophen for 2 min to allow for caged compound diffusion into the fibers. Fibers were then transferred into an empty chamber and flashed in air in order to lessen nonuniform photolysis of caged compound due to absorbance and attenuation of the laser light in solution. After contraction transients were recorded, fibers were transferred back to pCa9.0 solution. In order to avoid any changes in the condition of skinned fibers, all caged compound experiments were ‘one flash’. The protocol for study of the effects of rTnC or mutant TnCs on maximum contraction is illustrated in Fig. 1, including: (A) determine the maximum force in pCa4.0 (F4.0), (B) extract TnC until force in pCa4.0 was <5% of F4.0, (C) reconstitute with rTnC or mutant TnC until force in pCa4.0 was >90% of F4.0, (D) add DM-nitrophen and induce contraction by flash photolysis of DM-nitrophen. This example shows that the limitation of diffusional delay in skinned fibers can be overcome by the use of caged Ca2+ compounds. Also it has been shown that photolysis of DM-nitrophen produced a contraction in skinned frog fibers that is comparable in amplitude and rate to that observed in electrically stimulated intact fibers [6].
Fig. 1. Protocol for study of effects of rTnC or mutant TnC on maximum contraction in skinned psoas fibers. (A) Time course of contraction in pCa4.0. (B) Force developed in pCa4.0 after extraction of endogenous TnC. (C) Force developed in pCa4.0 after reconstitution of NHdel TnC. (D) Force development induced by photolysis of DM-nitrophen in the reconstituted fibers. (E) Same trace as (D) but on an expanded time scale.
Data analysis The steady-state forces developed upon photolysis of NP-EGTA or DM-nitrophen were normalized to the maximal force in the preceding pCa4.0 contraction. The force transients following photolysis were normalized to the extent of contraction (DF). The resulting traces were fit to a single exponential equation. Results are presented as means ± SEM. Results Characterization of caged Ca2+ induced contraction in psoas and soleus fibers Photolysis of DM-nitrophen generated 96 ± 1% of the force produced in a pCa4.0 solution and NP-EGTA generated 110 ± 2% of the force produced in a pCa4.0 solution. Fig. 2A shows typical normalized maximum contractions in psoas fibers induced by photolysis of DM-nitrophen or
Fig. 2. Time courses of maximal force development in skinned psoas fibers due to photolysis of NP-EGTA or DM-nitrophen (A) and comparison of time course of maximum contraction in psoas and soleus fibers induced by photolysis of NP-EGTA (B) at 15 C.
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NP-EGTA. On average, the rates of contraction, kCa, in response to photolysis of DM-nitrophen (20.4 ± 1.3 s1, n = 5) or NP-EGTA (16.0 ± 2.5 s1, n = 5) were not significantly different (Fig. 1). The fact that photolysis of NPEGTA and DM-nitrophen elicited full force and similar force transients indicates that the free [Mg2+] in the range of 0.036–1.0 mM has little effect on the maximum rate of contraction and that both caged compounds are suitable for examining the kinetics of contraction. Fig. 2B compares traces of maximum contraction in soleus and psoas fibers induced by photolysis of NP-EGTA. The time course of force development in soleus was significantly slower than in the psoas. The average rate of contraction was 25-fold slower in soleus fibers: kCa of 0.61 ± 0.02 s1 versus 16.0 ± 2.5 s1. To determine if this slow rise of force was a result of fiber evaporation in air, we examined the time courses of force development in skinned soleus fibers at 15 and 12 C (the dew-point). Similar results were observed except for slightly slower traces at 12 C than at 15 C. Effects of altered [Ca2+] on contraction amplitude and rate in psoas and soleus fibers To achieve various levels of [Ca2+] after photolysis at constant free [Mg2+], the extent of Ca2+ loading of NPEGTA was altered such that different levels of steady-state force were produced following photolysis but without significant force before photolysis. Fig. 3 shows examples of force development in psoas fibers at various levels of Ca2+ activation. Force development was examined over a wide range of [Ca2+]s that supported post-photolytic steady-state forces from 0.1 to 1.2 F/F4.0. As shown in Fig. 3, it is clear that lowering [Ca2+] not only decreased steady-state force (Fig. 3A) but also dramatically decreased the rate of force development (Fig. 3B). A plot of kCa versus relative force for psoas fibers is shown in Fig. 4A where relative force is taken as a measure of extent of Ca2+ activation representing the steady-state force following photolysis normalized to the force in an adjacent pCa4.0 contraction (F/F4.0). The relationship between kCa and relative force shows that kCa increased by 5-fold in a curvilinear manner from low to full Ca2+ activation. Fig. 4B shows the kCaversus relative force relationship in soleus fibers. The results for soleus fibers indicate that the rate of force development is independent of the level of Ca2+ activation. The average kCa for all contractions in the soleus fibers was 0.61 ± 0.02 s1 (n = 20). Thus, there is a dramatic difference between the Ca2+ sensitivity of contraction rate in psoas and soleus muscle fibers. Effects of altered Ca2+ dissociation rate from TnC on maximum contraction rate in psoas fibers Effects of varying Ca2+ dissociation rate from TnC on contraction were determined in fibers that contained mutant TnCs that exhibited varying Ca2+ off-rates and
Fig. 3. Effect of Ca2+ on the rate of force development in skinned psoas fibers induced by photolysis of NP-EGTA at 15 C. (A) Force produced following photolysis relative to the adjacent maximal force in pCa4.0. (B) Traces normalized to maximal force development induced by NP-EGTA.
compared to fibers containing endogenous TnC or rTnC. M82Q TnC and M46Q TnC both exhibited an increased Ca2+ affinity and an 5-fold slower Ca2+ dissociation rate from isolated TnC compared to F29W TnC ([21] and Table 1). But Ca2+ affinity and the dissociation rate from M46Q TnCÆTnI96–148 are similar to the F29W TnCÆTnI96–148 control whereas the Ca2+ affinity is increased and the Ca2+ dissociation rate is decreased 2fold for M82Q TnCÆTnI96–148 ([22] and Table 1). M82Q TnC produced a corresponding leftward shift in the force versus pCa relation in psoas fibers compared to endogenous TnC, rTnC or F29W TnC [15] but M46Q TnC did not [22]. On the other hand, NHdel TnC in the absence and presence of TnI96–148 exhibited a decreased Ca2+ affinity, an 2-fold faster Ca2+ off-rate and a rightward shift in the force versus pCa relation. Thus, the rate of Ca2+ dissociation from TnC in the reconstituted fibers was expected to be slower for M82Q TnC, similar for M46Q TnC and faster for NHdel TnC when compared to that with rTnC. Fig. 5 shows examples of maximum contractions in fibers reconstituted with rTnC, M82Q, or NHdel TnC
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and all results are summarized in Table 2. During the application of each solution, a full Ca2+ activation was produced upon photolysis of DM-nitrophen with no significant force before photolysis. Fibers containing rTnC or mutant TnC supported force well in pCa4.0 (Frecon/ Fendo = 0.91–1.01). Average force produced by DM-nitrophen photolysis ranged from 0.92 to 0.97 of force in adjacent pCa4.0 contraction after rTnC or mutant TnC reconstitution. The rate of maximum force development in control fibers with rTnC (22.5 ± 2.9 s1, n = 4) was similar to that observed in the unextracted fibers (20.4 ± 1.3 s1, n = 5). These results indicate that there is little effect on the functionality of the reconstituted fibers due to the extraction/reconstitution procedure. Despite differences in Ca2+ binding and exchange properties in the absence and presence of TnI96–148, the rate of maximum force development in the presence of M82Q TnC was not significantly different than that observed in the presence of M46Q TnC and thus the results were pooled together. Compared with rTnC, neither the introduction of M82Q TnC or M46Q TnC (pooled average 21.8 ± 6.1 s1, n = 5) or NHdel TnC (20.8 ± 1.0 s1, n = 6) had a significant effect on rate of maximal contraction. Thus, the rate of maximum force development was insensitive to either a decrease or increase in Ca2+ dissociation rate from TnC. Discussion
Fig. 4. Rate of contraction induced by photolysis of NP-EGTA (kCa) versus relative force in rabbit psoas and soleus fibers at 15 C. (A) Results for psoas fibers fitted to kCa = A · exp(B · F/F4.0) + C, where A = 0.015 s1, B = 5.82 and C = 5.52 s1. (B) Results for soleus fibers. Data obtained from 20 soleus fibers by ‘one flash’ of NP-EGTA.
The present results demonstrate that in rabbit skinned psoas fibers the rate of force development (kCa) induced by photolysis of a caged Ca2+ compound is dependent on the level of Ca2+ activation. When the [Ca2+] was elevated, kCa increased along with the steady-state post-photolytic force in a curvilinear relation in which kCa increased mainly at concentrations of Ca2+ yielding activation greater than half-maximal. In dramatic contrast, in soleus fibers the rate of force development was independent of the level of Ca2+ activation. At maximal Ca2+ activation in psoas fibers, kCa was insensitive to either an increase or a decrease in the rate of Ca2+ dissociation from TnC. Regulation of contraction by Ca2+ in psoas and soleus fibers
Fig. 5. Examples of time course of maximum force development in presence of rTnC or mutants of TnC in rabbit psoas fibers at 15 C. Mutants of TnC were characterized in a F29W TnC construct. F29W TnC reconstituted into psoas fibers resulted in a pCa50 of force similar to that observed in fibers containing endogenous or rTnC [15].
Whereas the contractile mechanism has been widely investigated in fast-twitch skeletal muscle, fewer studies have been carried out in slow-twitch skeletal muscle. Despite overall similarity, there are a number of quantitative and qualitative differences in the mechanical properties of fast- and slow-twitch fibers. Compared with psoas fast fibers, the rate of force development induced by photolysis of a caged Ca2+ compound (kCa) and the Ca2+ dependence of kCa were greatly reduced in soleus fibers, which is consistent with the previous reports with ktr measurements [2,24]. These differences between fiber types may be related to the unique protein expression patterns in the fast- and slowtwitch muscle contractile machinery, such as different isoforms of myosin, TnC and TnI [2,25]. More specifically,
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Table 2 Kinetics of maximal force development in skinned psoas fibers induced by photolysis of DM-nitrophen at 15 C in the presence of rTnC or mutant TnC N
TnC
Fext/F4.0
Frecon/Fendo
FDM/F4.0
kCa (s1)
5 4 5 6
Endogenous rTnC M82Q and M46Q NHdel
0.01 0.01 0.01
1.01 ± 0.01 0.93 ± 0.01 0.91 ± 0.02
0.96 ± 0.01 0.97 ± 0.02 0.92 ± 0.07 0.92 ± 0.02
20.4 ± 1.3 22.5 ± 2.9 21.8 ± 6.1 20.8 ± 1.0
Fext/F4.0 = force in pCa4.0 after extraction divided force in pCa4.0 before extraction. Frecon/Fendo = force in pCa4.0 after rTnC or mutant TnC reconstitution divided by force in pCa4.0 before extraction. FDM/F4.0 = steady-state force following photolysis of DM-nitrophen divided by force in adjacent pCa4.0 contraction. Conditions ([DM-nitrophen] in mM): (1) M82Q TnC: pCa7.4/4 mM, (2) wild-type TnC: pCa7.1/4 mM, (3) endogenous TnC: pCa6.5/4 mM, (4) NHdel TnC: pCa6.5–6.7/3–4.5 mM.
they may be due to kinetic differences in some transitions in the cross-bridge cycle. The basic mechanism of force generation is thought to be similar in both fiber types, presumably involving a two-step mechanism for Pi release coupled with force generation [26,27]. But the corresponding rate constants appear to be different in fast- and slowtwitch fibers. It has been suggested that the isomerization step preceding the force development is significantly slower in soleus than in psoas muscle [26,27]. The differences in the rate constants of specific cross-bridge transitions may be responsible for the observed differences between fast- and slow-twitch fibers in the magnitude and the Ca2+ dependence of contraction rates. The slowing of both forward and reverse rate constants of the isomerization step would lead to a slow contraction, as well as less Ca2+ dependence of contraction rate in slow twitch muscle. Effect of altered Ca2+ dissociation rate from TnC on maximum contraction rate The maximal kCa in muscle fibers was not affected by mutants of TnC that exhibited, in solution, varying rates of Ca2+ dissociation from TnC (up to threefold in the TnCÆTnI96–148 constructs, Table 1). Thus, the maximum rate of contraction is independent of Ca2+ exchange with TnC and depends solely on the intrinsic rate of cross-bridge force development. In contrast M82Q TnC, which slows Ca2+ dissociation from TnC in solution, dramatically slowed relaxation in skinned psoas fibers by two-fold [15]. But NHdel TnC, which accelerates Ca2+ dissociation from TnC in solution, did not alter the rate of relaxation. These results suggest that the Ca2+ dissociation rate from TnC and the cross-bridge detachment rate are similar to each other and together limit the rate of relaxation. In this case, slowing Ca2+ dissociation from TnC would slow relaxation but accelerating Ca2+ dissociation from TnC would have little effect on relaxation because cross-bridge detachment would become rate limiting. Taken together the data suggests that the mechanisms that govern the maximum rate of contraction and rate of relaxation are not the same. Instead of having a direct effect on the cross-bridge kinetics, Ca2+ could, in principle, regulate kCa by the modulation of thin filament activation via the kinetics of Ca2+ binding with TnC and the subsequent interactions within the thin filament. At high [Ca2+], the on-rate dominates
Ca2+ association to TnC [12] such that the kinetics of cross-bridge cycling becomes the only potential determinant of the rate of contraction. The present results showing that maximal kCa was insensitive to changes in the rate of Ca2+ dissociation from TnC confirm these predictions at maximal Ca2+ activation level. During submaximal Ca2+ activation, the rate of Ca2+ binding to TnC is relatively slow because the Ca2+ on-rate is proportional to [Ca2+]. At low [Ca2+] Ca2+ dissociation from TnC could be an important factor by affecting the activated time of individual thin filament regulatory units to control strong crossbridge attachment and force generation even though the Ca2+ association to TnC is thought to be too rapid to limit the rate of muscle contraction. Nonetheless little or no effect was found on ktr at submaximal Ca2+ activation levels in the presence of NHdel TnC [28]. This lack of a further inhibition of ktr was explained by suggesting that the rate of Ca2+ dissociation from TnC is maximally tuned such that increasing the Ca2+ off-rate can not inhibit ktr further, only slowing down Ca2+ dissociation from TnC can speed up ktr. Another possibility is that since ktr measurements are made at constant [Ca2+] any slow kinetic steps involved in the Ca2+ exchange with TnC and subsequent thin filament activation may not be detected with this approach. In contrast, kCa measurement appears to be a better model from this point of view. Therefore, further investigation of the role of Ca2+ dissociation from TnC in determining the rate of skeletal muscle contraction should be expanded to include submaximal Ca2+ activation levels. In conclusion, the rate of force development (kCa) induced by photolysis of a caged Ca2+ compound accelerates as the level of Ca2+ activation is increased in rabbit skinned psoas fibers but is independent of Ca2+ activation in soleus fibers. Differences in the rate constants of specific cross-bridge transitions may be responsible for the differences between fast- and slow-twitch fibers in the magnitude and Ca2+ dependence of contraction rates. At maximal activation kCa in psoas fibers is determined by the cross-bridge kinetics instead of the rate of Ca2+ dissociation from TnC. Thus, the present results are consistent with the notion that the rate of contraction is dominated by the kinetics of cross-bridge cycling over the kinetics of Ca2+ binding with TnC in maximally activated fast twitch skeletal muscle.
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