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Laboratoire de Plasticité Neuromusculaire, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Troponin C (TnC) plays a key role in the regulation of muscle contraction, thereby modulating the Ca2+-activation characteristics of skinned muscle fibers. This study was performed to assess the effects of a 15-day hindlimb unloading (HU) period on TnC expression and its functional behavior in the slow postural muscles of the rat. We investigated the TnC isoform expression in whole soleus muscles and in single fibers. The latter were also checked for their Ca2+ activation characteristics and sensitivity to bepridil, a Ca2+ sensitizer molecule. This drug has been described as exerting a differential effect on slow and fast fibers, depending on the TnC isoform. With regard to TnC expression, three populations were found in control muscle fibers: slow, hybrid slow, and hybrid fast fibers, with the TnC fast being always coexpressed with TnC slow. In the whole muscle, TnC fast expression increased after HU because of the increase in the proportion of hybrid fast fibers. The HU hybrid fast fibers had properties similar to those of control hybrid fast fibers. The fibers that remained slow after HU exhibited similar bepridil and Sr2+ properties as control slow fibers. Therefore, in these fibers, the changes could not be related to the TnC molecule.
regulatory proteins; calcium ion sensitizers; bepridil; simulated microgravity
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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TROPONIN C (TnC), a
subunit of the troponin complex, plays a key role in the regulation of
contraction, acting as a Ca2+ sensor to switch on tension
development when the Ca2+ concentration rises. TnC is a
dumbbell-shaped protein with two globular heads linked by a central
-helix (for review, see Ref. 7). Each globular head is
made up of two Ca2+ EF-hand domains: the two high-affinity
carboxy-terminal sites III and IV keep TnC bound to the thin filament
(32), whereas the two low-affinity amino-terminal sites I
and II regulate muscle contraction (21). This protein
exists in two isoforms, a fast isoform (TnCf) found in fast skeletal
muscles and a slow isoform (TnCs), lacking regulatory site I, found in
cardiac and slow skeletal muscles. Unloading conditions induced by real
or simulated microgravity have been demonstrated to cause not only a
clear atrophy of the slow antigravitational muscle fibers, but also a
shift in their functional and biochemical properties from a slow toward
a faster phenotype (9, 24). The changes in myosin
heavy chain (MHC) isoform expression have been well defined (4,
26), and transitions and coexpressions of MHC leading to hybrid
fibers are now well documented (25). However, few studies
have reported the effects of unloading on regulatory proteins: the
expression of troponins T and I was demonstrated to be modified after
hindlimb unloading (HU) (5), but no information is
actually available on the TnC isoform shown to be involved in the
Ca2+ dependence of tension development (1,
18).
The aim of our study was to examine the implication of TnC and the eventual transitions in the TnC isoform expression, in parallel with MHC and myosin light chain (MLC) transformations during changes that occurred in unloading conditions. The TnC content of single soleus muscle fiber was determined by immunoblotting, and its functional behavior was probed by using a pharmacological tool, bepridil (BPD). BPD is a Ca2+ sensitizer that targets the TnC molecule and stabilizes conformational changes that occur on Ca2+ fixation, causing an increase in the apparent Ca2+ affinity by decreasing the Ca2+ off rate (15). BPD has been shown to alter the affinity of the regulatory system differently in slow and fast fibers, with the modulation of the contractile response being directly dependent on the TnC isoform (12, 13).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and Muscle Preparation
Experiments were carried out on soleus and tibialis anterior muscles of adult male Wistar rats. The experiments as well as the maintenance conditions of the animals received authorization from the Ministry of Agriculture and the Ministry of Education (veterinary service of health and animal protection, authorization 03805). A first group was subjected to 15 days of HU by using the model of Morey (17). This model consisted of tail suspension of rats to mimic microgravity effects. The second group was composed of nonsuspended animals used as control. The two groups of animals were matched for age and weight.Soleus and tibialis muscles were removed from the control and suspended
rats, which were anesthetized with an intraperitoneal injection of
pentobarbital sodium (3 mg/kg). After removal, the muscles were either
homogenized (whole muscle results) or chemically skinned, as previously
described (31). The skinned muscles were stored at
20°C for up to 2 mo in a 50:50 glycerol-skinning solution (storage
solution), containing protease inhibitor leupeptin (1 µg/ml). Some
fibers were examined for calcium activation characteristics as well as
for TnC composition, whereas others were examined for TnC composition only.
Electrophoresis
Muscles.
Soleus muscles from control and HU animals were homogenized in EDTA
buffer (6.35 mM, pH 7.0) and centrifuged at 13,000 rpm for 10 min at
4°C. The pellet was washed twice in a 50 mM KCl buffer and dissolved
in SDS sample buffer without 5% 
-mercaptoethanol [62.5 mM
Tris · HCl, 10% glycerol (vol/vol), 2% SDS (wt/vol), 0.02 bromophenol blue (wt/vol), pH 6.8]. The protein concentration was
determined by using the Lowry protein estimation method
(14) before the addition of 5% -mercaptoethanol
(vol/vol) to the sample buffer and was stored at 80°C.
Fibers.
Fibers were randomly removed from either control or HU skinned soleus
and tibialis muscles. pCa-tension relationships (pCa/T; tension vs.
Ca2+ concentrations) were established on some of these
fibers. All fibers were then dissolved in 20 µl SDS sample buffer,
heated at 90°C for 3 min, and stored at 80°C until
electrophoretic analysis.
Immunoblotting
Electrotransfer was performed on a 0.2-µm nitrocellulose sheet (Schleicher & Schuell, Dassel, Germany). The membranes were blocked with a PBS solution (pH 7.4) containing 5% nonfat dry milk and 0.2% sodium azide. The TnC isoforms were localized by a cardiac mouse monoclonal antibody (NCL Trop-C, Novocastra, UK) that was incubated overnight. This antibody recognized slow skeletal TnC isoform but cross-reacted with fast skeletal TnC (29). TnC antibodies were detected by an extravidin-biotin peroxidase staining kit and were visualized by an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ) to ensure optimal protein detection. Signal intensities were evaluated by densitometry (GS-700 Imaging Densitometer, Bio-Rad, Ivry sur Seine, France).The profile of TnC expression was determined by measuring the relative proportions of the TnC signals of fast and slow isoforms. In the whole study, fiber type was based on TnC content of the fibers. The fibers considered as hybrid for the TnC expression were classified as hybrid slow (HS) or hybrid fast (HF). However, it should be stated that the antibody recognized preferentially the slow isoform. Fibers showing a TnCf signal higher than the TnCs signal were thus unambiguously classified as HF fibers. To avoid an overestimation of HS fibers because of the preferential recognition of the slow isoform, we checked the MLC expression for the fibers that expressed a signal ratio of TnCs to TnCf >1; fibers expressing predominantly slow MLC isoforms were kept and classified as HS, whereas fibers that expressed preferentially fast MLC isoforms were discarded. However, discrepancy between TnC and MLC predominant isoform expressions did not exceed 5% of the fibers.
Force Measurements and Recording
The experiments were carried out in a thermostatically controlled room (19 ± 1°C). A 2- to 2.5-mm fiber segment was connected in an experimental chamber to a force transducer (Fort 10, WPI, Aston, UK) under constant stirring. The sarcomere length was determined by diffraction by using a helium-neon laser (Spectra Physics, Carlsbad, CA). To allow maximal isometric tension development on ionic activation, the fiber was stretched to 2.60 µm; this sarcomere length was readjusted when necessary during the experiment. To eliminate a potential effect of the sarcoplasmic reticulum on the tension developed by the myofilaments, each fiber was bathed for 20 min in a Brij solution made up of 2% Brij 58 (polyoxyethylene 20 cetyl ether) in relaxing solution.Experimental Protocol
The experimental sequence was defined as described previously (13). Briefly, the fiber was activated to a tension level (P) in a given pCa (with pCa =log[Ca2+], where
brackets denote concentration) solution, immediately followed by a
maximal contraction (P0) ensured by the application of a
pCa 4.2 solution. This procedure allowed the calculation of the
relative tension P/P0 from pCa 7.0-4.2 with a step
equal to 0.2 and to the establishment of the pCa/T curves. The
steepness of the pCa/T curve was determined by the Hill coefficients
(nH), either n1 or
n2, calculated according to the equation
(2)
![P<IT>/</IT>P<SUB><IT>0</IT></SUB><IT>=</IT>([Ca<SUP><IT>2+</IT></SUP>]<IT>/K</IT>)<SUP><IT>n</IT><SUB>H</SUB></SUP><IT>/</IT>{<IT>1+</IT>([Ca<SUP><IT>2+</IT></SUP>]<IT>/K</IT>)<SUP><IT>n</IT><SUB>H</SUB></SUP>}](/content/vol90/issue3/fulltext/1095/img001.gif)
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log K = pCa50, where Ca50
is the calcium concentration producing half-maximal activation). The n1 corresponded to P/P0 >50%, and
n2 to P/P0 <50% (19).
For the pCa/T + BPD relationship determination, 100 µM BPD were
added in each pCa solution, as previously described (13).
To quantify the shift that occurred when BPD was added to the
activating solution, we defined a 
n, which
represented the shift expressed in pCa units at n% of
P/P0. In our analyses, we measured 10, 50, and 90. The 10-90
was representative of the extent of cooperativity alteration in the
presence of the drug. Half-maximal activation in the presence of
strontium ions (pSr50) was also determined to establish the
' parameter, which corresponds to the difference between
pCa50 and pSr50 values. The ' criterion allowed a functional identification of the fiber (either slow or fast
phenotype), because fast muscle fibers are less sensitive to
Sr2+ than are slow fibers (11).
Fibers were rejected if force declined during a sustained contraction or decreased by >20% during the whole experiment and if the pCa/T series (with and without BPD) was not completely achieved. The proportion of fibers tested for their Ca2+ activation characteristics does not reflect the fiber-type composition at the whole muscle level.
Solutions
All reagents without indications were provided by Sigma Chemical (St Louis, MO). The composition of all the solutions was calculated by the Fabiato computer program (6). The program calculation was used with stability constants listed for Ca2+ (20) and for Sr2+ (16) to keep final ionic strength at 200 mM. pH was adjusted to 7.0, and ATP at 2.5 mM was added to each solution. The skinning solution was made up of MOPS (10 mM), potassium propionate (170 mM), magnesium acetate (MgAc, 2.5 mM), and EGTA (5 mM). The following solutions were used for the T/pCa curve determination: a washing solution composed of MOPS (10 mM), potassium propionate (185 mM), and MgAc (2.5 mM); a relaxing solution identical to the skinning solution; and pCa-activating solutions consisting of washing solution plus various concentrations of free Ca2+ from CaCO3 buffered with EGTA and added in proportions to obtain the different pCa values (7.0-4.2). The pSr solutions were similar to the pCa solutions except for free Sr2+ from SrCl2. BPD was prepared fresh each day and used as a 20 mM stock solution in absolute ethanol. At concentrations used, ethanol itself had no effect on the developed tensions (data not shown).Statistical Analysis
All the data are reported as means ± SE. The statistical significance of the difference between means was determined by using the Student's t-test or paired t-test, when data were obtained from the same fiber in different experimental conditions. Differences
95% confidence level were considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Structural Analysis
TnC and MHC changes at the whole muscle level.
Figure 1A shows the expression
of slow and fast MHC and TnC proteins in whole soleus control
muscles (n = 3; average weights, 0.47 ± 0.001 mg/g animal wt). The slow isoforms of these proteins were largely
predominant, with only small amounts of MHC IIa (9.9 ± 4.7% of
the total MHC expression) and TnCf (12.2 ± 2.9% of the total TnC
signal). Fig. 1B illustrates modifications in the expression of MHC and TnC proteins in soleus muscles submitted to unloading conditions (n = 3). These muscles displayed
significantly lower average weights (0.21 ± 0.004 mg/g animal
wt). Fast MHC isoforms were more abundant in these muscles compared
with control conditions, with an increase in MHC IIa up to 23.9 ± 4.4% and the appearance of MHC IId/x and IIb (15.5 ± 4.5 and
4.9 ± 1.9%, respectively). TnC expression was changed by HU,
with the TnCf signal rising up to 22.5 ± 1.9%. This represented
an approximately twofold increase in the TnCf isoform signal compared
with control conditions.
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TnC and MLC expressions at the single-fiber level.
Distributions of the MLC and TnC isoforms, analyzed on the same gel for
each single fiber, are represented in Fig.
2. As indicated in METHODS,
the MLC profiles were used for a clear discrimination between HS and HF
fibers. In control conditions, we showed either an expression of TnCs
alone (fibers classified as slow, lane 2) or a coexpression
of both slow and fast isoforms in fibers classified as HS (predominant
expression of TnCs and slow MLC, lane 3) or HF (predominant
expression of TnCf and fast MLC, lane 4). The proportions of
these slow, HS, and HF fibers in the control muscles were 74, 10, and
16%, respectively (n = 80 of 3 different muscles). Fibers expressing TnCf alone were never found in these control muscles.
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Ca2+ Activation Properties
Control fibers.
Slow, HS, and HF fibers had no significant differences in their
diameter and tensions normalized to cross-sectional area (Fig. 3). As previously described, BPD had no
effect on maximal tensions (data not shown). Ca2+
activation parameters of all fiber types are reported in Table 1.
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10-90 parameter was equal to 0.36 ± 0.04 for
slow fibers and 0.37 ± 0.01 for HS fibers (Table
2).
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10-90 (Table 2), with the shift being less
important compared with that for the slow or HS fibers (0.14 ± 0.01 pCa units in the linear part of the curve vs. 0.24 ± 0.02 pCa units for the slow fibers). Moreover, BPD reactivity appeared
similar to that seen in fast fibers from tibialis anterior (expressing
only TnCf), which is represented in Fig. 4D.
HU fibers. Slow, HS, and HF HU fibers were found to develop lower normalized tensions than their respective controls, with the averaged diameter being significantly decreased after the HU period. No change in maximal tension appeared in the presence of BPD (data not shown). The pCa/T relationships are reported in Fig. 4. No differences appeared in HF fibers between control and HU conditions, neither in the absence nor in the presence of BPD (Fig. 4C and Table 2). The pCa/T relationship of slow HU fibers was different from the pCa/T of control fibers: the pCa threshold and pCa50 values were lowered, and n1 (nH corresponding to P/P0 >50%) was largely increased. In the presence of BPD, the pCa/T relationship merged with that obtained in control conditions for slow fibers (Fig. 4A). Ca2+ activation characteristics of HS muscle fibers were modified toward a faster phenotype after HU: pCa threshold and pCa50 values were lowered, whereas the nH values increased. Thus the pCa/T curve of HS fibers after HU could be considered as intermediate between the curves of slow and HF soleus fibers. It is noteworthy that the pCa/T curve established in the presence of BPD was not significantly different from the control HS curve, and thus not different from the control slow fibers.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study reports, for the first time, the distribution of TnC isoforms in soleus muscles at the single-fiber level in control and HU conditions and shows that HU induced changes in the TnC expression. A major point raised by our data is the limited transitions in the TnC expression in soleus fibers, which acquired typical fast Ca2+ activation properties.
TnC Expression
In homogenized, slow postural soleus muscles, TnCf is expressed in a low amount (Fig. 1). To define the distribution of TnCf among fibers, we achieved a detailed study at the single muscle fiber level. We showed that TnCs was either expressed alone (slow fibers) or coexpressed with TnCf in hybrid fibers (HS or HF fibers, depending on the predominant isoform).In good agreement with the literature, we report changes in the MHC content of the atrophied soleus muscle after HU, with an increase in MHC IIa and the appearance of MHC IId/x and IIb. The modifications in MHC expression pattern and averaged weights after HU attested that the phenotypic transformations had occurred. Moreover, we showed modifications in the TnC expression, with the TnCf signal being almost twofold higher after HU. This increase in fast isoform expression of TnC might be attributed to either a slow-to-fast switch of TnC expression, leading to an expression of TnCf alone in transformed fibers, or an increase in the number of hybrid fibers, or both. The study at the single-fiber level showed an increase in the proportions of HF fibers: 45% of the soleus fibers coexpressed TnCf with TnCs. It was previously reported that soleus fibers never expressed only fast MHC (23): in the same way, we never found TnCf expressed alone in soleus fibers. Consequently, the low quantity of TnCf shown in homogenized control muscles must come exclusively from the hybrid fibers. In contrast, pure fast fibers (2%, based on the MHC content) have been observed after a 14-day HU period (3). Similarly, after HU, an expression of the troponin T fast isoform alone was found in soleus muscle (5). Therefore, our data suggest that the TnC transitions were more moderated or appeared with slower kinetics than those of other contractile proteins such as MHC and troponin T.
Functional Behavior of TnC in Both Experimental Conditions
Similar relative Ca2+ and Sr2+ affinities and similar sensitivity to BPD were found for slow and HS fibers. This indicated that, in HS fibers, the presence of TnCf at a lower level than TnCs did not influence the Ca2+ activation characteristics. They were identical to those found in pure slow fibers (with TnCs alone), a result which suggested that functional properties related to TnC are dependent on the TnC isoform predominantly expressed.After HU, both diameter and normalized P0 decreased in slow and fast fibers, as previously described, and illustrated the altered protein turnover in unweighted muscles that led to protein loss (for review, see Ref. 28) and that occurred preferentially in muscles involved in weight bearing (8, 9, 27). Our data show that the pCa/T relationships of slow fibers were modified: the sensitivity (pCa threshold) and the affinity (pCa50) were lowered, whereas the cooperativity (nH) increased. Activation properties (TnC behavior) of these fibers were found to be identical to those obtained in control conditions (HU pCa/T + BPD and control pCa/T + BPD curves merged); it is, therefore, unlikely that TnC could account for the changes in Ca2+ activation characteristics shown in the absence of BPD. Because neither TnC expression nor TnC behavior was altered, other regulatory proteins may be involved in these functional changes, troponin T for instance (5, 10, 22).
For the HF fibers, which are mainly transformed fibers (16% in control vs. 45% in HU conditions), the Ca2+ activation properties became identical to those of control fast fibers, because the pCa/T relationships and the Sr2+ and BPD sensitivities of control and HU fast fibers were not different. Therefore, we gave evidence that the effects of HU consisted of a transition from a slow type (TnCs only and pCa/T relationship typical of control slow fibers) to a fast type (TnCf coexpressed in a predominant proportion with TnCs and pCa/T relationship typical of control fast fibers).
To conclude, we showed a modification in TnC expression in HU muscles. The presence of TnCf was always associated with a coexpression of TnCs in both control and HU fibers, but the proportions of HF fibers increased after HU. These fibers displayed functional properties similar to those of control fast fibers. The remaining slow fibers, on the contrary, presented modifications that could not be attributed to TnC. Thus further studies will be necessary to determine the implication of other regulatory proteins in the functional changes that occurred after HU.
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ACKNOWLEDGEMENTS |
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This work was supported by the Centre National d'Etudes Spatiales (Grant 993027), the Fonds Européen de Développement Régional F007, and the Nord Pas-de-Calais Regional Council.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Kischel, Université des Sciences et Technologies de Lille, Laboratoire de Plasticité Neuromusculaire, Bât. SN4, 59655 Villeneuve d'Ascq Cedex, France (E-mail: Philippe.Kischel{at}univ-lille1.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 July 2000; accepted in final form 5 October 2000.
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METHODS
RESULTS
DISCUSSION
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  J. Hedou, C. Cieniewski-Bernard, Y. Leroy, J.-C. Michalski, Y. Mounier, and B. Bastide O-Linked N-Acetylglucosaminylation Is Involved in the Ca2+ Activation Properties of Rat Skeletal Muscle J. Biol. Chem., April 6, 2007; 282(14): 10360 - 10369. [Abstract] [Full Text] [PDF]
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 Z. B. Yu, F. Gao, H. Z. Feng, and J.-P. Jin Differential regulation of myofilament protein isoforms underlying the contractility changes in skeletal muscle unloading Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1192 - C1203. [Abstract] [Full Text] [PDF]
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 C. Bozzo, L. Stevens, V. Bouet, V. Montel, F. Picquet, M. Falempin, M. Lacour, and Y. Mounier Hypergravity from conception to adult stage: effects on contractile properties and skeletal muscle phenotype J. Exp. Biol., July 15, 2004; 207(16): 2793 - 2802. [Abstract] [Full Text] [PDF]
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 L. Stevens, C. Bozzo, T. Nemirovskaya, V. Montel, M. Falempin, and Y. Mounier Contractile properties of rat single muscle fibers and myosin and troponin isoform expression after hypergravity J Appl Physiol, June 1, 2003; 94(6): 2398 - 2405. [Abstract] [Full Text] [PDF]
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L. Stevens, B. Bastide, P. Kischel, D. Pette, and Y. Mounier Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle Am J Physiol Cell Physiol, May 1, 2002; 282(5): C1025 - C1030. [Abstract] [Full Text] [PDF]
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, pCa; 
, pCa + bepridil (BPD). For the HU fibers: 
, pCa;
, pCa + BPD. Curves are fitted using the Hill
coefficients n1 and n2
(see METHODS).