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
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ABSTRACT |
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muscle contraction; skeletal muscle; myofibrillar proteins; single fiber; sensitivity to strontium; sensitivity to calcium
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).
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MATERIALS AND METHODS |
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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 M1 and 4.78 x 106 M1, 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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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:
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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 -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
2939 kDa, as predicted from the data of Muroya et al. (18) and Mortola and Naso (16).
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RESULTS |
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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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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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We previously showed that the sensitivity to Sr2+ of typically slow-twitch fibers is characterized by pSr50 values that are 0.70.9 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 13). 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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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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DISCUSSION |
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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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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 13, 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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GRANTS |
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FOOTNOTES |
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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.
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