Troponin C isoform composition determines differences in Sr2+-activation characteristics between rat diaphragm fibers

Brett O'Connell,1 D. George Stephenson,2 Ronnie Blazev,1 and Gabriela M. M. Stephenson1

1School of Biomedical Sciences, Victoria University of Technology, Melbourne, Victoria 8001; and 2Department of Zoology, La Trobe University, Melbourne, Victoria 3086, Australia

Submitted 8 December 2003 ; accepted in final form 17 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Single fibers of rat diaphragm containing different naturally occurring combinations of myofibrillar protein isoforms were used to evaluate the contribution of troponin C (TnC) isoforms to fiber type-related differences with respect to sensitivity to Sr2+ of the contractile system. Mechanically skinned fibers were studied for their isometric force vs. Sr2+ concentration ([Sr2+]) relationships and then analyzed electrophoretically for myofibrillar protein isoform composition. Our data demonstrate that fiber-type differences in Sr2+ dependence of contractile activation processes are primarily determined by the TnC isoform composition, with the slow isoform conferring on average a sevenfold greater sensitivity to Sr2+ than the fast isoform. Moreover, the ratio of TnC isoforms determined functionally from the force-pSr (–log10 [Sr2+]) curves is tightly (r2 = 0.97) positively correlated with that estimated electrophoretically. Together, these results validate the use of Sr2+ activation characteristics to distinguish fibers containing different proportions of fast and slow TnC isoforms and to study the mechanisms by which divalent cations activate the contractile apparatus. We also found that the functionally and electrophoretically determined ratios of TnC isoforms present in a fiber display similar sigmoidal relationships with the ratio of myosin heavy chain (MHC) isoform types expressed. These relationships 1) offer further insight in the functional and molecular expression of TnC in relation to the molecular expression of MHC isoform types and 2) may provide the basis for predicting sensitivity to Sr2+, TnC, and MHC isoforms in pure and hybrid skeletal muscle fibers.

muscle contraction; skeletal muscle; myofibrillar proteins; single fiber; sensitivity to strontium; sensitivity to calcium


STRONTIUM ION (Sr2+) does not play a direct role in vertebrate skeletal muscle contractility in vivo yet has been used frequently in the laboratory as an activator of contractile processes in reconstituted or "synthetic" actomyosin, myosin B or "natural" actomyosin, actomyosin threads, myofibrils, glycerinated muscle, and mechanically skinned muscle fibers. The main reason for its use is that, as first reported by Ebashi et al. (6), cardiac/skeletal slow-twitch type and skeletal fast-twitch type muscle preparations differ markedly with respect to their sensitivity to Sr2+ of contractile activation. On the basis of this finding, Ebashi and colleagues (6) designed a series of elegant experiments which demonstrated that the regulatory effects of Ca2+, the physiological activator of muscle contraction, on contractile processes are mediated by troponin (Tn) via its divalent cation-binding subunit troponin C (TnC). Since this pioneering work of Ebashi et al., Sr2+ has been employed both by physiologists and biochemists as a tool for gaining further insights in the process of regulation of muscle contraction by divalent cations.

Fiber-type differences with respect to Sr2+ activation properties have been used by us (e.g., see Refs. 8 and 31) and others (5) as a physiological criterion for identifying slow- and fast-twitch fibers in studies of mammalian muscle contractility using single fiber preparations. Thus Sr2+ also has the potential to become a valuable tool in studies concerned with the functional diversity and plasticity of skeletal muscle fibers. This is particularly the case if the identity of the structure(s) responsible for the interfiber-type differences in Sr2+ sensitivity of contractile processes was precisely known. Here it is worth pointing out that, based on our knowledge to date, the Sr2+ sensitivity-based method of fiber typing does not appear to be fully correlated with the more widely used methods of fiber typing based on electrophoretically defined myosin heavy chain (MHC) isoform composition (2), which means that with some types of fibers one has to determine both Sr2+ activation characteristics and MHC isoform composition to obtain more complete functional and structural information.

In an earlier study, Yamamoto (34) showed that the sensitivity to Sr2+ of actomyosin ATPase regulated by a Tn complex containing skeletal troponin I (TnI) and troponin T (TnT) and cardiac TnC was higher than that of the enzyme regulated by another Tn complex containing cardiac TnI and TnT and skeletal fast-twitch TnC (TnC-f). This finding prompted Yamamoto and others (for review, see Ref. 21) to conclude that the difference in sensitivity to Sr2+ of contractile processes in cardiac and skeletal muscle preparations is "determined solely by the [molecular] species of TnC." Note that the same TnC isoform is expressed in cardiac and slow-twitch muscles (30). This idea is consistent with the results obtained from TnC extraction/reconstitution experiments by Morimoto and Ohtsuki (14) with rabbit skeletal muscle myofibrils, Babu et al. (1) with myocardial trabeculae of Syrian hamster, Hoar et al. (11) with rat cardiac fibers, and Sweeney et al. (28) with rabbit soleus fibers. In the study by Sweeney et al. (28), for example, the endogenous TnC was replaced with recombinant cardiac, native, or mutated TnC-f species.

Not all studies using reconstituted functional actomyosin systems support Yamamoto's conclusion regarding the contribution of the TnC molecular structure to the muscle type-specific relationship between Sr2+ concentration ([Sr2+]) and contractile activation parameters. Thus Kerrick et al. (12) reported that purified skeletal acto-heavy meromyosin systems regulated by the native Tn-tropomyosin (Tm) complex from either skeletal or cardiac muscle display the same [Sr2+] dependence of ATPase activation. Later, Kerrick et al. (13) also found that the relationship between isometric force and [Sr2+] for fast-twitch skinned muscle fibers was essentially the same before removal of endogenous TnC isoform and after its replacement with either cardiac or fast-twitch skeletal species. On the basis of these data, Kerrick and colleagues (13) argued that protein-protein interactions associated with the activation process, which strongly affect TnC affinity for Sr2+, rather than the molecular type of TnC, determine the difference in sensitivity to Sr2+ of contractile events in cardiac and fast-twitch skeletal muscle preparations.

A close examination of the studies listed above reveals two kinds of methodological problems that may account for the conflicting data produced by the aforementioned studies. One kind relates to the inherent shortcomings of the TnC extraction/replacement strategy, well detailed in a review by Moss (17), and the other to the effectiveness of the protocols used for the separation, visualization, and identification of TnC isoform bands on electrophoretograms of TnC-extracted and TnC-reconstituted fibers. In the present study, we proposed to revisit the issue of the factors determining the fiber-type differences with respect to sensitivity to Sr2+ of contractile activation processes using an experimental strategy that avoids the two kinds of problems. Our strategy involves the use of pure and hybrid diaphragm fibers that contain different naturally occurring combinations of myofibrillar protein isoforms (2), in conjunction with a recently developed electrophoretic method for the unequivocal identification of TnC isoforms in single fiber segments (19).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats, aged 13 wk, were killed by deep halothane inhalation in accordance with the permits granted by the Animal Ethics Committee at Victoria University. The diaphragm muscle was rapidly and carefully dissected, blotted on filter paper, and pinned in a petri dish on a Sylgard 184 base under paraffin oil.

Preparation of single fibers. All operations described in this section were carried out at room temperature (RT; 21 ± 1°C), under oil. Single fibers were randomly isolated and mechanically skinned with fine jewelers forceps using a dissecting microscope, as previously described (27). The cross-sectional area (CSA) and volume of each skinned fiber segment, assumed to be cylindrical, were determined using the width (mean of 5 values) and length of the segment measured with the aid of a video camera monitor system (2). The CSA was necessary for calculating maximum Sr2+ (SrFmax)- or Ca2+ (CaFmax)-activated force per CSA, and the volume was necessary for electrophoretic analysis of protein isoforms in individual fibers. Each skinned fiber segment was finally mounted at slack length, between a sensitive force transducer (model 801; Sensonor, Horten, Norway) and a pair of fine Barcroft forceps, on the apparatus for mechanical measurements.

Solutions for force activation experiments. Details regarding the composition and preparation of the solutions used in these experiments can be found in a number of earlier studies (2, 31). Briefly, a set of 10 strongly buffered (with 50 mM total EGTA) Sr2+ solutions (pSr range of 3.6 to >9, where pSr = –log10[Sr2+]) and a set of 10 strongly buffered (with 50 mM EGTA) Ca2+ solutions (pCa range of 4.3 to >9, where pCa = –log10[Ca2+]) were prepared as previously described (2). In addition to the 50 mM Sr2+-EGTA or Ca2+-EGTA buffer, all solutions contained (in mM) 117 K+, 37 Na+, 1 Mg2+ (free), 90 HEPES (pH 7.10 ± 0.01), 8 total ATP, 10 creatine phosphate, and 1 N3. The apparent affinity constants for Sr2+ and Ca2+ binding to EGTA (1.53 x 104 M–1 and 4.78 x 106 M–1, respectively) used in this study were those measured earlier for the same conditions (26, 29). The pH, osmolality, and ionic strength of all solutions at RT were 7.10 ± 0.01, 295 ± 5 mosmol/kgH2O, and 234 ± 2 mM, respectively.

Protocol for isometric force activation experiments. The isometric force activation characteristics of individual fibers were determined at RT from the steady-state isometric force responses developed by the fiber segments in the strongly Sr2+- and Ca2+-buffered solutions. All these experiments were carried out with fiber segments at slack length, where the average sarcomere length is ~2.65 µm (2). Each preparation was initially equilibrated for 2 min in a relaxing solution (pCa, pSr > 9) and then was activated in a solution of pSr 5.3. On the basis of the size of the force response developed in this solution, we distinguished between fibers with fast twitch (no active force), fibers with slow twitch (almost maximal active force), and fibers with intermediate (intermediate active force) activation characteristics (e.g., see Refs. 2, 8, 31, 32). All fibers that developed forces >0.0025 mN, indicative of a slow-twitch activation component (2, 8, 31, 32), were sequentially activated in the Ca2+- and Sr2+-activating solutions as shown in Fig. 1A for a representative fiber. Of the fibers that did not develop force in the pSr 5.3 solution, five randomly selected fibers (used as reference for fast-twitch activation characteristics) were sequentially activated in the Ca2+ and Sr2+ solutions as shown in Fig. 1A, but the majority were activated only in the Sr2+ solutions. This protocol was consistent with the main thrust of this investigation of the relationship between myofibrillar protein isoform composition and fiber-type differences in Sr2+ activation characteristics. At the end of the activation protocol, each fiber segment was placed in SDS-PAGE solubilizing buffer (~1 µl/0.4 nl fiber volume) for electrophoretic analyses of myofibrillar protein isoform composition.



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Fig. 1. Sample of complete data collected for a single muscle fiber. Data were collected for one of the fibers from group 4 (see Table 1). A: continuous chart recording of force responses produced by placing the fiber in strongly Ca2+- and Sr2+-buffered solutions (pCa or pSr values indicated under arrowheads). The arrowheads indicate the moment when the fiber was transferred to a solution of given pSr or pCa. Note the small artifacts caused by the transfer of the preparation between solutions. B: steady-state force response (Pt)-pCa ({circ}) and Pt-pSr ({bullet}) curves generated from the trace shown in A using Eqs. 1 and 2 (see MATERIALS AND METHODS), respectively. The descriptors of the Pt-pSr and Pt-pCa curves are as follows: pSr50 value for the first Hill curve (pSr50/1) = 5.37, pSr50 value for the second Hill curve (pSr50/2) = 4.54, first Hill coefficient for Sr2+ (nSr1) = 4.63, second Hill coefficient for Sr2+ (nSr2) = 3.66, Ca2+ Hill coefficient (nCa) = 5.308, maximum Sr2+-activated force (SrFmax)/cross-sectional area (CSA) = 107 kN/m2, and maximum Ca2+-activated force (CaFmax)/CSA = 107 kN/m2. C: electrophoretogram showing the myosin heavy chain (MHC) isoform composition of the fiber. D: electrophoretogram showing some of the low-molecular-mass (less than ~30 kDa) protein isoforms detected in the fiber. Note that the troponin C (TnC) isoform bands can be readily identified by a characteristic upward curvature caused by the diffusion of EGTA applied to adjacent lane on left. MLC, myosin light chain; f, fast; s, slow.

 
Determination of Sr+ and Ca2+ activation characteristics of single fiber segments. For each fiber segment, the relationship between isometric force and [Sr2+] or Ca2+ concentration ([Ca2+]) was determined by plotting the steady-state force responses (Pt) developed by the fiber at different [Sr2+] or [Ca2+], expressed as a percentage of SrFmax or CaFmax, respectively (%maximum force), against the pSr or pCa values in solutions. To correct for the slight deterioration in force production associated with the repeated activation of skinned fiber preparations (see Fig. 1A), a simple interpolation protocol was used to estimate the values of SrFmax (or CaFmax) corresponding to the force responses obtained in a particular pSr (or pCa) solution (for details, see Ref. 24). The Pt-pSr and Pt-pCa data points were then best fitted by theoretical Hill curves using the nonlinear regression analysis protocols provided by GraphPad Prism software.

In this study, a proportion of the Pt-pSr curves and all Pt-pCa curves could be fitted to the data points with a correlation coefficient, r2 ≥ 0.9990, using a simple Hill equation:

(1)
where nH is the associated Hill coefficient, x is pSr or pCa, and x50 is the pSr or pCa value where 50% of SrFmax (pSr50) or CaFmax (pCa50) was reached. When the Pt-pSr curves could not be fitted by the simple Hill equation with a correlation coefficient r2 ≥ 0.9990, then the data points were fitted by a "composite" Hill curve (consisting of the sum of 2 Hill curves; see also Ref. 2):

(2)
where w1 and w2 are normalized weighting factors (w1 + w2 = 1) referring to the slow (w1) and fast (w2) Sr2+ sensitivity components, nSr1 and nSr2 are the corresponding Hill coefficients, and pSr50/1 and pSr50/2 are the corresponding pSr50 values for the two Hill curves. The above composite Hill curves fitted all Pt-pSr data point sets with a correlation coefficient r2 ≥ 0.9990. In Fig. 1B are shown Pt-pCa and Pt-pSr curves that have been fitted to the data points obtained from Fig. 1A using Eqs. 1 and 2, respectively.

The following Sr2+ and, where applicable, Ca2+ activation parameters were determined for the individual skinned fiber segments: 1) maximum Sr2+- or Ca2+-activated force per CSA (SrFmax/CSA; CaFmax/CSA; kN/m2), 2) pSr50 or pCa50 (sensitivity to Sr2+/Ca2+) if the data points were well fitted by a simple Hill equation, or pSr50/1 and pSr50/2 if the data points were well fitted by the composite Hill equation, and 3) nSr or nCa (minimum number of cooperating Sr2+- or Ca2+-binding sites) if the data points were well fitted by a simple Hill equation or nSr1 and nSr2 if the data points were well fitted by the composite Hill equation. SrFmax/CSA and CaFmax/CSA were determined from the amplitude of the force responses developed by the mechanically skinned fiber segment in the maximally Sr2+- or Ca2+-activating solutions at the end of the first pSr (or pCa) staircase run (Fig. 1A) and from its estimated CSA measured in paraffin oil before exposure to aqueous solutions.

SDS-PAGE analysis of MHC isoforms. In this study, the SDS-PAGE solubilization buffer contained 80 mM Tris·HCl (pH 6.8), 2.3% wt/vol SDS, 710 mM {beta}-mercaptoethanol, 10 mM DTT, 12.5% vol/vol glycerol, 13.6% wt/vol sucrose, 0.01% wt/vol bromophenol blue, 0.1 mM PMSF, 0.002 mM leupeptin, and 0.001 mM pepstatin. The samples were left overnight at RT and boiled for 3 min the following day. The alanine-SDS-PAGE protocol was used to separate MHC isoforms, as previously described (9). Briefly, samples (4 µl, containing ~1.6 nl fiber) were run on 0.75-mm-thick slab gels using the Hoefer Mighty Small gel apparatus. The separating gel (T = 7.6%, C = 1.2%) contained 425 mM Tris·HCl, pH 8.8, 75 mM alanine, 40% vol/vol glycerol, 0.3% wt/vol SDS, 0.05% wt/vol ammonium persulfate, and 0.1% vol/vol N,N,N',N'-tetramethylethylethylenediamine (TEMED), and the stacking gel (T = 4%, C = 2.6%) contained 125 mM Tris·HCl, pH 6.8, 4 mM EDTA, 40% vol/vol glycerol, 0.3% wt/vol SDS, 0.1% wt/vol ammonium persulfate, and 0.05% vol/vol TEMED. Electrophoresis was carried out at constant voltage (150 volts) for 28 h at 4°C with a running buffer containing 0.1% wt/vol SDS, 25 mM Tris, and 175 mM alanine. The gels were stained with silver according to the Bio-Rad method, scanned using a Molecular Dynamics Personal Densitometer, and analyzed with ImageQuant software. In Fig. 1C is shown the electrophoretogram of MHC isoforms for the fiber segment used to obtain the force responses displayed in Fig. 1A. The MHC isoform composition of single fibers was established using an MHC laboratory marker containing all four MHC isoforms known to be expressed in skeletal muscles of adult rats. The identification of the protein bands in this MHC marker was described in detail by Bortolotto et al. (3).

SDS-PAGE analysis of TnC isoforms. TnC isoform composition in single fiber segments was unambiguously identified electrophoretically using purified rat TnC isoforms (20) as markers and an EGTA concentration gradient created across the sample lane to cause a distinctive upward curve of TnC bands on the side of the EGTA lane, as previously described (19). In brief, samples (10 µl containing ~4 nl fiber) were run on 0.75-mm-thick slab glycine-SDS gels using the Hoefer Mighty Small gel apparatus. The separating gel (T = 16%, C = 2.6%) contained 750 mM Tris·HCl, pH 9.3, 10% vol/vol glycerol, 0.1% wt/vol SDS, 0.04% wt/vol ammonium persulfate, and 0.116% vol/vol TEMED, and the stacking gel (T = 4%, C = 4.76%) contained 125 mM Tris·HCl, pH 6.8, 10% vol/vol glycerol, 0.1% wt/vol SDS, 0.1% wt/vol ammonium persulfate, and 0.1% vol/vol TEMED. Electrophoresis was carried out at constant current (10 mA/gel) for 4.25 h at RT. The samples were loaded in such a way that each single fiber sample was adjacent to one lane of solubilization buffer (5 µl) containing 10 mM EGTA (i.e., 50 nmol EGTA/well). The gels were stained with silver according to the Hoefer method, scanned using a Molecular Dynamics Personal Densitometer, and analyzed with ImageQuant software. In Fig. 1D is displayed the electrophoretogram of the low-molecular-mass (<30 kDa) protein isoforms (note curvature in the TnC bands induced by the diffusion of EGTA from the adjacent lane on left) for the fiber segment used to obtain the force responses shown in Fig. 1A. Because it was not possible to perform a full densitometric analysis of the curved TnC bands, the following procedure was adopted to quantify the percentage of the two TnC isoforms present in the preparation: 1) if only one curved band was visible on the gel then only one TnC species was ascribed to that fiber segment, 2) if one curved band was clearly more intense than the other, then a 75:25% ratio for the two TnC isoforms expressed was given, and 3) if the curved bands displayed similar intensities (like in Fig. 1D), then a 50:50% ratio for the two TnC isoforms was given. Note that the limit of detection of any of the two TnC isoform bands was 5% of the total TnC present in the fiber segments used in this study (19).

Analysis of low-molecular-mass myofibrillar proteins (other than TnC) on SDS-polyacrylamide gels. Under our conditions, the isoforms of myosin light chains (MLCs) and TnI were resolved and visualized as shown in Figs. 2 and 3. The identity of the protein bands was established on the basis of their apparent electrophoretic mobility using as reference 1) low-molecular-mass markers, 2) purified rat skeletal muscle myosin for MLC bands, 3) relevant data reported in the literature for TnI isoforms (25), and 4) typically pure fast-twitch and pure slow-twitch fibers from the extensor digitorum longus and soleus muscles of the rat. Note that the apparent molecular mass of the rat TnI isoforms as determined from the 16% gels in this study (~26 kDa for the slow skeletal and ~25 kDa for the fast skeletal isoforms) were close to those previously reported on 12% gels (~25 kDa for the slow skeletal and ~24 kDa for the fast skeletal isoforms; see Ref. 25). Tm and TnT isoforms were visualized and identified, not individually, but as a group of bands (Tm + TnT; see Fig. 3) located in the molecular mass range ~29–39 kDa, as predicted from the data of Muroya et al. (18) and Mortola and Naso (16).



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Fig. 2. Representative electrophoretograms showing the MHC and TnC isoforms detected in the single muscle fibers examined. The fibers were classified according to their MHC and TnC isoform composition into 1 of 5 groups as follows: lane 1, group 1 fibers displayed 1 fast MHC isoform and TnC-f only; lane 2, group 2 fibers displayed two or more fast MHC isoforms and TnC-f only; lane 3, group 3 fibers displayed both fast and slow MHC isoforms but TnC-f only; lane 4, group 4 fibers displayed both fast and slow MHC isoforms and both TnC-f and TnC-s; lane 5, group 5 consisted of 1 fiber that contained the slow MHC and TnC-s only. The identity of the MHC isoforms present in the fibers is shown under each lane.

 


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Fig. 3. Representative electrophoretograms showing typical regulatory protein isoform composition for each group. Lane 1, group 1 fiber; lane 2, group 2 fiber; lane 3, group 3 fiber; lane 4, group 4 fiber; lane 5, the slow group 5 fiber. See MATERIALS AND METHODS for details of protein band identification. Note that Tm and troponin T (TnT) isoforms were visualized and identified, not individually, but rather as a group of bands (Tm + TnT) located in the molecular mass range 29–39 kDa. The minor band (*) was detected in fibers from groups 3 and 4 but not in fibers from groups 1 and 2. The identity of the MHC isoforms present in the fibers shown is shown under each lane. TnI, troponin I.

 
Statistics. Results are expressed as means ± SE. Two-tailed Student's t-test and ANOVA, followed by Bonferroni posttest were used, as appropriate, to determine the statistical significance of differences between fiber types with respect to contractile parameters.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we determined contractile activation characteristics and myofibrillar protein isoform composition in the same single fiber segments of rat diaphragm muscle (see MATERIALS AND METHODS). A representative set of data obtained from 1 of the 59 fiber segments examined is presented in Fig. 1, A-D. The isometric force responses developed by the fiber segment in strongly Ca2+- and Sr2+-buffered solutions are presented in Fig. 1A. The amplitudes of these force responses were subsequently used to generate the Pt-pCa and Pt-pSr curves shown in Fig. 1B. At the end of the activation protocol, the fiber segment was placed in SDS-PAGE solubilizing buffer for electrophoretic analyses of myofibrillar protein isoform composition. In Fig. 1, C and D, are shown the electrophoretograms of the MHC and some low-molecular-mass (less than ~30 kDa) myofibrillar protein isoforms, respectively. Note the EGTA-induced curvature of the TnC isoform bands (see MATERIALS AND METHODS), with the slow TnC isoform (TnC-s) displaying a higher electrophoretic mobility than the TnC-f.

Myofibrillar protein isoform combinations in single fibers of rat diaphragm. Fibers were classified into five groups according to their electrophoretically determined MHC and TnC isoform composition (see Table 1): group 1 (n = 41), composed of fast-twitch fibers displaying a single fast MHC isoform and only TnC-f; group 2 (n = 8), composed of hybrid fast-twitch fibers displaying more than one (in this case 2) fast MHC isoform and only TnC-f; group 3 (n = 4), composed of hybrid fast- and slow-twitch fibers displaying fast and slow MHC isoforms but only TnC-f; group 4 (n = 5), composed of hybrid fast- and slow-twitch fibers displaying fast and slow MHC isoforms and both TnC-f and TnC-s; and group 5 (n = 1), composed of one pure slow-twitch fiber displaying only slow MHC and TnC-s. Seven of the fibers examined in this study contained "atypical" (23) MHC isoform combinations [I + IId + IIb (2 fibers); I + IId (5 fibers)]. Such deviations from the "nearest neighbor rule," while rare in nontransforming muscles, have been found by us also in rat sternomastoid muscle fibers (19) and by Wu et al. (33) in dog thyroarytenoid muscle fibers. Note that, in agreement with our previous report (19), no fibers displaying two TnC isoforms and only one MHC isoform type were found in this study. The relative proportions of slow and fast MHC isoform types in fibers from groups 3 and 4 were determined by densitometric analysis (3) and are indicated in parentheses in column 2 in Table 1.


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Table 1. MLC, TnI, TnT + Tm isoform composition, and Sr2+ activation characteristics of fibers classified by MHC and TnC isoform composition

 
Representative electrophoretograms showing the MHC, MLC, and TnC isoform compositions of a fiber from each group are presented in Fig. 2, with the gel regions containing the other low-molecular-mass myofibrillar proteins from the same fibers shown in Fig. 3. The majority of fibers in group 1 were of type IID, and there was only one type IIA fiber. All fibers in group 2 were type IID + IIB fibers. All the fibers in groups 1 and 2 (lanes 1 and 2) appeared to contain only fast MLC isoforms and both fast and slow TnI isoforms. All fibers belonging to these two groups displayed the same pattern of Tm + TnT bands. The fibers in groups 3 and 4 (lanes 3 and 4) appeared to contain both fast and slow MLC and TnI isoforms. The patterns of Tm + TnT bands detected in fibers from these two groups were very similar but different from those of the fibers in groups 1 and 2 (note the minor band indicated by an asterisk in Fig. 3, which can be seen in lanes 3 and 4 but not in lanes 1 and 2). The type I fiber from group 5 (lane 5) displayed only slow MLC and TnI isoforms and a Tm + TnT pattern that differed from that of the other groups, particularly in the region of the three slowest migrating bands. When both TnC isoforms were detected in one fiber (as it was the case for group 4 fibers; see Fig. 2, lane 4), their relative abundance was estimated visually by comparing the staining intensity of the TnC bands. As stated in MATERIALS AND METHODS, the presence of a population of TnC isoforms (fast or slow) could be routinely detected if it amounted to ≥5% of the total TnC normally present in the fiber (19).

Sr2+ activation characteristics. Shown in Fig. 4 are representative Sr2+ activation curves (Pt-pSr) for all five groups of fibers investigated. The sensitivity to Sr2+ was indicated by the pSr50 descriptor (see Eq. 1 in MATERIALS AND METHODS) if the data were fitted by a simple Hill curve, as it was the case for all fibers in group 1 (Fig. 4A), group 2 (Fig. 4B), group 3 (Fig. 4C), and group 5 (Fig. 4E). If the data points were fitted by a composite Hill curve, as it was the case for all fibers in group 4 (Fig. 4D), then the sensitivity to Sr2+ was indicated by two pSr descriptors, pSr50/1 and pSr50/2 (see Eq. 2 in MATERIALS AND METHODS). In the latter case, we also apportioned the fraction of the two functional components (w1 and w2) characterized by pSr50/1 and pSr50/2, respectively. The values for pSr50 or pSr50/1/pSr50/2 and the proportions of w1 and w2 are summarized in the last column of Table 1.



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Fig. 4. Representative Pt-pSr curves of one fiber from each of the five groups. A: group 1 (pure fast), pSr50 = 4.55, nSr = 6.51, r2 = 1.000. B: group 2 (fast-fast hybrid), pSr50 = 4.54, nSr =8.91, r2 =0.9998. C: group 3 (fast-slow hybrid containing only TnC-f), pSr50 = 4.59, nSr = 10.81, r2 = 1.000. D: group 4 (fast-slow hybrid containing both TnC-f and TnC-s), pSr50/1 = 5.37, pSr50/2 = 4.54, nSr1 = 3.79, nSr2 = 3.39. Note that the data points for group 4 fibers could not be fitted to Eq. 1 (see MATERIALS AND METHODS) with r2 > 0.9990, however, with Eq. 2 (see MATERIALS AND METHODS) the curve fit the data points of this representative fiber with r2 = 0.9998. E: group 5 (pure slow) pSr50 = 5.35, nSr = 3.67, r2 = 0.9998. The solid lines are the Hill curves fitted through the experimental data points. The identity of the MHC isoforms present in the fibers shown is shown on the respective panel.

 
Note that all fibers expressing only fast MHC isoforms (groups 1 and 2) had pSr50 values within a tight range around 4.50, characteristic of typical fast-twitch fibers (see Ref. 2), even though all appeared to contain both fast and slow TnI isoforms. Also, fibers in group 3 that expressed a combination of fast and slow MHC and MLC isoforms had pSr50 values close to 4.50. Taken together, these data indicate that a value of pSr50 close to 4.50 was not tightly correlated with the MHC, MLC, or TnI isoform composition of the fibers.

We previously showed that the sensitivity to Sr2+ of typically slow-twitch fibers is characterized by pSr50 values that are 0.7–0.9 log units greater than those of fast-twitch fibers (2). Consistent with these data, the pSr50 value determined for the only typical slow-twitch fiber detected among the 59 diaphragm fibers examined in this study was 5.35. (Note that all myofibrillar protein isoforms in this fiber appear to be of the slow type.) This value is very close to the average value for pSr50 (5.36 ± 0.10) of a group of four pure type I rat diaphragm fibers comprising the fiber presented in Table 1, a fiber described by us in the study of Bortolotto et al. (2), and two other fibers originating from different strains of rats (Zucker obese and Zucker lean; unpublished data).

A further look at the results obtained for groups 3 and 4 indicates that the only difference between these two fiber groups, with respect to the myofibrillar protein isoform composition, relates to the TnC isoforms, with fibers in group 3 expressing only TnC-f and fibers in group 4 expressing both TnC-f and TnC-s. The presence of both TnC isoforms in group 4 fibers was tightly associated with the occurrence of "composite Sr2+ activation curves" characterized by the pSr50/1 and pSr50/2 descriptors, with the values of the pSr50/1 being close to the pSr50 value of the fiber that expressed only the TnC-s (group 5) and the values of the pSr50/2 being in the vicinity of the pSr50 value typical of fibers that expressed only TnC-f (groups 1–3). Thus the data in Table 1 unequivocally show that the lower sensitivity to Sr2+ (pSr ~4.5) is directly correlated with the presence of TnC-f isoform, whereas the higher sensitivity to Sr2+ (pSr ~5.4) is directly correlated with the presence of TnC-s isoform. Indeed, the relative abundance of the TnC-s and TnC-f isoforms detected in group 4 fibers (Table 1, column 3) is well reflected in the proportion of the "slow-type" and "fast-type" Sr2+ sensitivity components (w1/w2; last column in Table 1) in the composite Sr2+ activation curves of the fibers. This is shown in Fig. 5 where the relative proportion of the slow-type Sr2+ sensitivity component (w1, functional descriptor of TnC isoform, y-axis) for all fibers was plotted against the estimated proportion of TnC-s isoform (biochemical descriptor of TnC isoform, x-axis) detected in the respective fibers. The line of best fit in Fig. 5 passes in very close proximity to the origin on the graph axes, has a slope of 1.01 ± 0.02, and displays a very high correlation coefficient (r2 = 0.97), suggesting that both the functional and biochemical parameters describe the same population of TnC in the fiber. We argue that this population of TnC is most likely to be assembled in the myofilaments, because 1) only TnC fully assembled in the myofilaments would render the contractile response sensitive to divalent cations, and 2) only TnC tightly associated with, or most likely fully assembled in, the myofilament structure (i.e., "unwashable" TnC) would be detectable by electrophoretic analysis of skinned fiber segments that had been exposed to aqueous solutions for ~10 min before incubation in the SDS-PAGE solubilizing buffer. Note that, under our conditions, one could routinely detect the presence of a functional TnC-s isoform when its functional contribution was >5%, as demonstrated by the finding that theoretical composite curves with a slow-type component (w1) ≥0.04 could not be fitted by a simple Hill equation (Eq. 1) with r2 ≥ 0.9990 and therefore required fitting with Eq. 2.



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Fig. 5. Relationship between the estimated amount of TnC-s in single pure and hybrid fiber segments (n = 59) and the proportion of the slow-type (w1) Sr2+ sensitivity component of the force-pSr curve produced by the fibers.

 
With respect to SrFmax/CSA, there were no statistically significant differences (ANOVA, P > 0.05) between fibers in group 1 (274 ± 13 kN/m2), group 2 (225 ± 18 kN/m2), and group 3 (290 ± 72 kN/m2). However, fibers in group 4 produced significantly lower maximum Sr2+-activated specific forces (156 ± 15 kN/m2) than fibers in group 1 (ANOVA, P < 0.05), as did the fiber in group 5 (112 kN/m2). The Hill coefficients for the force-pSr curves were 10. 68 ± 1.50 (group 1, n = 33), 6.50 ± 0.32 (group 2; n = 8), 8.50 ± 1.59 (group 3, n = 4), 5.56 ± 2.11 (fast component) and 3.76 ± 0.21 (slow component) (group 4, n = 5), and 3.69 (group 5, n = 1). A two-tailed Student's t-test found that the nSr values for groups 1–3 and the fast-type Sr2+ sensitivity component of group 4 fibers were not significantly different from one another, but all were significantly higher than the nSr values for both the slow-type Sr2+ sensitivity component of group 4 fibers and the pure slow fiber in group 5 (P < 0.05).

Other contractile activation characteristics. Regarding the descriptors of force-pCa curves, the pCa50 (5.68 ± 0.04, n = 5) and Hill coefficient values (4.79 ± 0.46, n = 5) for fibers containing both TnC-f and TnC-s isoforms (group 4) were significantly smaller (P < 0.05; Student's 2-tailed t-test) than the corresponding pCa50 (5.78 ± 0.02, n = 5) and Hill coefficient values (6.14 ± 0.49, n = 5) for fibers containing only TnC-f (e.g., fibers from group 1). The only pure slow-twitch fiber in group 5 had a pCa50 value of 5.68 and a Hill coefficient of 3.72. For all fibers that were also maximally activated in Ca2+ solutions (5 group 1 fibers, all group 4 fibers, and 1 group 5 fiber; see MATERIALS AND METHODS), the ratio SrFmax/CaFmax was close to 1.00 (0.95 ± 0.01, n = 5 for group 1; 0.96 ± 0.03, n = 5 for group 4; and 1.05 for the fiber in group 5).


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This study has unequivocally shown that, in isometrically contracting fibers of the rat diaphragm muscle, the presence of the TnC-f isoform confers a much lower sensitivity to Sr2+ (by a factor of ~7) than the presence of the TnC-s isoform. Thus 1) all fibers that displayed only TnC-f (groups 1–3) presented a consistent pSr50 value of ~4.5 ([Sr2+] = 3.15 x 10–5 M) regardless of the MHC, MLC, TnI, and Tm + TnT isoform composition, 2) the fiber in group 5 that displayed only the TnC-s isoform, as well as three other pure type I rat diaphragm fibers (see RESULTS), had an average pSr50 value of 5.36 ([Sr2+] = 4.37 x 10–6 M), and 3) all fibers that displayed both TnC isoforms (group 4) produced composite force-pSr activation curves made up of two functional components, one characterized by high sensitivity to Sr2+ (pSr50/1 value around 5.36) and the other by low sensitivity to Sr2+ (pSr50/2 values around 4.5). Moreover, the proportion of one of the two functional components (the slow component, w1) derived from fitting the composite curves to the data points was directly proportional to the relative proportion of the respective TnC isoform (TnC-s) detected electrophoretically in the individual fibers (Table 1 and Fig. 5).

As mentioned in the introduction, previous TnC isoform substitution studies have led to conflicting conclusions with respect to the role of TnC in determining sensitivity to Sr2+ of contractile activation processes. In our view, the disagreement between the various groups concerning the major determinant of Sr2+ sensitivity differences in mammalian skeletal muscle is because of the inherent problems of the extraction-reconstitution experiments and the inadequacy of the protocols used to resolve, visualize, and identify the TnC isoforms in TnC extracted/reconstituted fibers. In the present study, we used single skeletal muscle fibers displaying a wide range of naturally occurring combinations of myofibrillar protein isoforms, thus avoiding the inherent drawbacks of the extraction/reconstitution experiments, and a rapid electrophoretic method that allows unequivocal identification of TnC isoforms in single fiber segments. The results obtained lead to the clear-cut conclusion that skeletal muscle fiber-type differences in sensitivity to Sr2+ of contractile activation processes are determined primarily by the difference in TnC isoform composition.

The results of this study provide us with further insight into the functional and molecular expression of TnC isoforms in relation to the molecular expression of MHC isoform type. In Fig. 6 are plotted the percentage of the slow-type Sr2+ sensitivity component (w1, the functional indicator of TnC-s isoform) and the proportion of the TnC-s isoform, estimated visually, against the proportion of the slow-type MHC isoform, determined densitometrically using data points collected from all 59 single fiber segments examined in this study. The sigmoidal curve shown in Fig. 6 highlights the trend followed by the experimental data. Several conclusions can be drawn from this graph. First, the data show that the levels of TnC isoform expression of a fiber, as measured by either functional or electrophoretic means, display a similar relationship to the MHC isoform type composition of the fiber. This further supports our earlier argument (see RESULTS) that the functional and biochemical analyses performed in this study define the same population of TnC, that is, the TnC assembled in the myofilaments. Second, the profile of the sigmoidal curve shown in Fig. 6 (which reflects the absence of fibers containing both TnC isoforms in combination with only 1 MHC isoform type; see RESULTS) suggests that the assembly of TnC in the myofilaments does not occur before that of the MHC protein. Furthermore, from the inflection points of the curve shown in Fig. 6, one can deduce that a TnC isoform (in this case TnC-s) is assembled in myofibrils, thus becoming functional, only when the proportion of the matching MHC isoform type (in this case the slow MHC I) reaches values higher than ~20%.



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Fig. 6. Relationship between the proportion of TnC-s, estimated electrophoretically (*, y-axis on right), the proportion of the slow-type Sr2+ sensitivity component (w1) of the force-pSr curve ({circ}, y-axis on left), and the proportion of slow MHC isoform present in each fiber (x-axis). The sigmoidal curve indicating the trend followed by the data was generated using the nonlinear regression (variable slope) option provided by Graphpad Prism.

 
On the basis of the relationships between sensitivity to Sr2+, TnC isoform, and MHC isoform type composition established from the graph in Fig. 6, one can predict that MHC hybrid fibers that display slow to fast MHC isoform ratios in the range 0.2–0.8 are also likely to display hybrid TnC profiles and composite Sr2+ activation curves. Furthermore, these relationships provide the basis for predicting two of the above three parameters when the third is determined experimentally in pure and hybrid fibers. Here it is important to stress that, from data on TnC isoform expression obtained by either the functional or the biochemical protocols described in this study, one is able to predict only the type of MHC present but not the number or identity of the individual MHC isoforms.

The mechanism responsible for the higher Sr2+ sensitivity of the contractile apparatus in fibers expressing the TnC-s isoform than in fibers expressing the TnC-f isoform is most likely related to differences with respect to the affinity for Sr2+ of the two TnC isoforms. A corollary of this is that the affinity of the regulatory site II for Sr2+ (but not Ca2+) is much greater if the regulatory site I is inactive (as is the case for the slow/cardiac TnC isoform) than when site I is functional (as is the case with the TnC-f isoform). This idea is consistent with the findings reported by others (e.g., see Refs. 28 and 22). The loss of site I in TnC-s is also most likely to be responsible for the significantly smaller Hill coefficient associated with the slow Pt-pSr curves or slow force components.

It is worth noting then that, within a given fiber, the affinities of the Ca2+ (and likely Sr2+)-binding sites for divalent cations are also affected by the incorporation of TnC in the Tn complex, the incorporation of the Tn complex in the thin filament structure (35), and the interactions between myosin and actin filaments (4, 15). One should also keep in mind that there are many other factors that can alter the sensitivity of the contractile apparatus for Ca2+ and Sr2+ without altering the binding properties of TnC for the activator (10). Therefore, in absolute terms, the sensitivity to Sr2+ and Ca2+ of the contractile activation process in an individual fiber is ultimately dependent not only on the molecular species of TnC but also on the complex interactions between TnC and other molecular entities. Indeed, the only typical slow-twitch fiber in group 5, expressing only TnC-s, appeared to be less rather than more sensitive to Ca2+ than any of the fast-twitch fibers in group 1, a result contrary to previous observations from this laboratory using similar activating solutions (e.g., see Ref. 2), but consistent with another study on diaphragm muscle (7). Moreover, all composite fibers in group 4, which expressed both TnC isoforms, displayed a significantly reduced sensitivity to Ca2+ compared with the fast-twitch fibers in group 1. This result suggests that an increased apparent sensitivity to Sr2+ conferred by the expression of the TnC-s isoform is not also translated into an equivalent increase in Ca2+ sensitivity. If so, the conformational changes induced by Sr2+ binding to the TnC isoforms in the Ca2+ regulatory system may be different from those induced by the binding of Ca2+ to the same isoforms. This is not surprising considering that in some skeletal muscle fibers Sr2+ is unable to induce the full activation of the contractile apparatus (26, 29).

Notwithstanding the contribution of the various interactions between TnC and other myofibrillar molecular entities to the precise sensitivity of the contractile apparatus to activating divalent cations, the very marked difference between the affinities of the TnC-s and TnC-f isoforms for Sr2+ (but not for Ca2+) is translated into clear-cut differences in the relative sensitivities to Sr2+ (but not to Ca2+) of the contractile apparatus, irrespective of the other interactions between myofibrillar components.

The composite fibers in group 4, expressing both TnC-f and TnC-s isoforms, also produced significantly lower maximal Sr2+- and Ca2+-activated specific force responses compared with the other fibers in groups 1–3, which expressed only the TnC-f isoform. The SrFmax/CSA and CaFmax/CSA values in the composite fibers in group 4 were, however, similar to the respective values of two slow (type I MHC) diaphragm fibers (1 in group 5 of this study and 1 in an earlier study on diaphragm muscle form this laboratory; see Ref. 2). Therefore, the reduced force level produced in these fibers cannot be simply ascribed to the coexistence of the two TnC isoforms. Regarding the steepness of the Pt-pCa curves, the group 4 fibers displayed Hill coefficients that were intermediate between the Hill coefficients associated with the pure fast fibers (group 1) and the two slow diaphragm fibers mentioned above, suggesting that these fibers would be active over a broader range of [Ca2+] than the most abundant group of fibers (IID) commonly detected in the rat diaphragm muscle.

In conclusion, the results obtained in this study with pure and hybrid rat diaphragm muscle fibers show clearly that differences with respect to Sr2+ dependence of contractile activation processes between MHC-based fiber types are determined by the molecular species of TnC protein present in the fibers, with the presence of TnC-s isoform conferring on average an approximately sevenfold greater sensitivity to Sr2+ than the TnC-f isoform. These results also have direct practical importance because they validate the use of differential sensitivities to Sr2+ to distinguish between fibers containing different proportions of TnC-f and TnC-s isoforms and provide the basis for predicting either sensitivity to Sr2+, TnC isoform composition, or MHC isoform type composition of pure and hybrid fibers when only one of these parameters is determined experimentally.


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This work was supported by the Australian Research Council and by the National Health and Medical Research Council (Australia).


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. M. Stephenson, School of Biomedical Sciences, Victoria Univ. of Technology, PO Box 14428, MCMC, Melbourne, Victoria 8001, Australia (E-mail: gabriela.stephenson{at}vu.edu.au).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Babu A, Scordilis SP, Sonnenblick EH, and Gulati J. The control of myocardial contraction with skeletal fast muscle troponin C. J Biol Chem 262: 5815–5822, 1987.[Abstract/Free Full Text]

2. Bortolotto SK, Cellini M, Stephenson DG, and Stephenson GMM. MHC isoform composition and Ca2+- or Sr2+-activation properties of rat skeletal muscle fibers. Am J Physiol Cell Physiol 279: C1564–C1577, 2000.[Abstract/Free Full Text]

3. Bortolotto SK, Stephenson DG, and Stephenson GMM. Fiber type populations and Ca2+-activation properties of single fibers in soleus muscles from SHR and WKY rats. Am J Physiol Cell Physiol 276: C628–C637, 1999.[Abstract/Free Full Text]

4. Bremel RD and Weber A. Cooperation within actin filament in vertebrate skeletal muscle. Nat New Biol 238: 97–101, 1972.[ISI][Medline]

5. Cordonnier C, Stevens L, Picquet F, and Mounier Y. Structure-function relationship of soleus muscle fibres from the rhesus monkey. Pflügers Arch 430: 19–25, 1985.

6. Ebashi S, Kodama A, and Ebashi F. Troponin I. Preparation and physiological function. J Biochem (Tokyo) 64: 465–477, 1968.[ISI][Medline]

7. Eddinger TJ and Moss RL. Mechanical properties of skinned single fibers of identified types from rat diaphragm. Am J Physiol Cell Physiol 253: C210–C218, 1987.[Abstract/Free Full Text]

8. Fink RH, Stephenson DG, and Williams DA. Calcium and strontium activation of single skinned muscle fibres of normal and dystrophic mice. J Physiol 373: 513–525, 1986.[Abstract]

9. Goodman C, Patterson M, and Stephenson G. MHC-based fiber type and E-C coupling characteristics in mechanically skinned muscle fibers of the rat. Am J Physiol Cell Physiol 284: C1448–C1459, 2003.[Abstract/Free Full Text]

10. Gordon AM, Homsher E, and Regnier M. Regulation of contraction in striated muscle. Physiol Rev 80: 853–924, 2000.[Abstract/Free Full Text]

11. Hoar PE, Potter JD, and Kerrick WGL. Skinned ventricular fibres: troponin C extraction is species-dependent and its replacement with skeletal troponin C changes Sr2+ activation properties. J Muscle Res Cell Motil 9: 165–173, 1988.[ISI][Medline]

12. Kerrick WGL, Malencik DA, Hoar PE, Potter JD, Coby RL, Pocinwong S, and Fischer EH. Ca2+ and Sr2+ activation: comparison of cardiac and skeletal muscle contraction models. Pflügers Arch 386: 207–213, 1980.[ISI][Medline]

13. Kerrick WGL, Zot HG, Hoar PE, and Potter JD. Evidence that the Sr2+ activation properties of cardiac Troponin C are altered when substituted into skinned skeletal muscle fibers. J Biol Chem 260: 15687–15693, 1985.[Abstract/Free Full Text]

14. Morimoto S and Ohtsuki I. Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: Effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins. J Biochem (Tokyo) 101: 291–301, 1987.[Abstract]

15. Morris CA, Tobacman LS, and Homsher E. Modulation of contractile activation in skeletal muscle by a calcium-insensitive Troponin C mutant. J Biol Chem 276: 20245–20251, 2003.

16. Mortola JP and Naso L. Electrophoretic analysis of contractile proteins of the diaphragm in chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 269: L371–L376, 1995.[Abstract/Free Full Text]

17. Moss RL. Ca2+ regulation of mechanical properties of striated muscle. Mechanistic studies using extraction and replacement of regulatory proteins. Circ Res 70: 865–884, 1992.[Abstract]

18. Muroya S, Nakajima I, and Chikuni K. Amino acid sequences of multiple fast and slow troponin T isoforms expressed in adult bovine skeletal muscles. J Anim Sci 81: 1185–1192, 2003.[Abstract/Free Full Text]

19. O'Connell B, Nguyen LT, and Stephenson GMM. A single fibre study of the relationship between MHC and TnC isoform composition in rat skeletal muscle. Biochem J 378: 269–274, 2004.[CrossRef][ISI][Medline]

20. O'Connell B and Stephenson GMM. Purification of Troponin C isoforms from EDL and Soleus muscles of the rat. J Muscle Res Cell Motil 24: 555–559, 2003.[CrossRef][ISI][Medline]

21. Ohtsuki I, Maruyama K, and Ebashi S. Regulatory and cytoskeletal proteins of vertebrate skeletal muscle. Adv Protein Chem 18: 1–67, 1986.

22. Pearlstone JR, Chandra M, Sorenson MM, and Smillie LB. Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal Troponin C are impaired by invariant site I or II Glu mutations. J Biol Chem 275: 35106–35115.

23. Pette D, Peuker H, and Staron RS. The impact of biochemical methods for single muscle fibre analysis. Acta Physiol Scand 166: 261–277, 1999.[CrossRef][ISI][Medline]

24. Rees BB and Stephenson DG. Thermal dependence of maximum Ca2+ activated force in skinned muscle fibres of the toad Bufo marinus acclimated at different temperatures. J Exp Biol 129: 309–327, 1987.[Abstract]

25. Simpson JA, Labugger R, Hesketh GG, D'Arsigny C, O'Donnell D, Matsumoto N, Collier CP, Iscoe S, and Van Eyk JE. Differential detection of skeletal Troponin I isoforms in serum of a patient with Rhabdomyolysis: markers of muscle injury? Clin Chem 48: 1112–1114, 2002.[Free Full Text]

26. Stephenson DG and Williams DA. Activation of skinned arthropod muscle fibres by Ca2+ and Sr2+. J Muscle Res Cell Motil 1: 73–87, 1980.[Medline]

27. Stephenson DG and Williams DA. Calcium activated force responses in fast- and slow-twitch skinned muscle fibres of the rat at different temperatures. J Physiol 317: 281–302, 1981.[Abstract]

28. Sweeney HL, Brito RMM, Rosevear PR, and Putkey JA. The low affinity Ca2+ binding sites in cardiac/slow skeletal muscle troponin C perform distinct functions: site I alone cannot trigger contraction. Proc Natl Acad Sci USA 87: 9538–9542, 1990.[Abstract]

29. West JM and Stephenson DG. Ca2+ and Sr2+ activation properties of skinned muscle fibres with different regulatory systems from crustacea and rat. J Physiol 462: 579–596, 1993.[Abstract]

30. Wilkinson JM. Troponin C from rabbit slow skeletal and cardiac muscle is the product of a single gene. Eur J Biochem 103: 179–188, 1980.[Abstract]

31. Wilson GJ and Stephenson DG. Calcium and strontium activation characteristics of skeletal muscle fibres from the small marsupial Sminthopsis macroura. J Muscle Res Cell Motil 11: 12–24, 1990.[ISI][Medline]

32. Woolley PA, Patterson MF, Stephenson GM, and Stephenson DG. The ilio-marsupialis muscle in the dasyurid marsupial Sminthopsis douglasi: form, function and fiber-type profiles in females with and without suckling young. J Exp Biol 205: 3775–3781, 2002.[ISI][Medline]

33. Wu YZ, Baker MJ, Crumley RL, Blanks RH, and Caiozzo VJ. A new concept in laryngeal muscle: multiple myosin isoforms types in single muscle fibers of the lateral cricoarytenoid. Otolyrngeal Head Neck Surg 188: 86–94, 1998.

34. Yamamoto K. Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins. J Biochem (Tokyo) 93: 1061–1069, 1984.[ISI]

35. Zot HG, Guth K, and Potter JD. Fast skeletal muscle skinned fibers and myofibrils reconstituted with N-terminal fluorescent analogues of troponin C. J Biol Chem 261: 15883–15890, 1986.[Abstract/Free Full Text]