INTRODUCTION

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DIAPHRAGM MUSCLE (Dia_m) fibers express different myosin heavy chain (MHC) isoforms, which form the basis for fiber-type classification (11, 33, 36). Differences in muscle fiber contractile properties are associated with MHC isoform expression. For example, fibers expressing the MHC_slow isoform have a slower maximum shortening velocity (V_max) than fibers expressing fast MHC isoforms (MHC_2A, MHC_2X and MHC_2B/2X) (1, 6, 12, 30, 34, 35). Although controversial (8), it has been reported that fibers expressing fast MHC isoforms have a greater specific force (force per cross-sectional area) than fibers expressing the MHC_slow isoform (1, 6, 25, 34, 35).

The force generated by muscle fibers depends on myoplasmic calcium concentration ([Ca²⁺]; represented as pCa or log [Ca²⁺]), and Ca²⁺ sensitivity is often indexed as the pCa at which 50% of maximum force is generated (pCa₅₀). Generally, it has been reported that slow fibers have a higher pCa₅₀ than fast fibers (15, 17). However, with respect to Dia_m, an apparent controversy exists regarding fiber-type differences in Ca²⁺ sensitivity. For example, in the rabbit Dia_m, Hoar and Kerrick (15) found that fibers expressing the MHC_slow isoform (as determined electrophoretically) had greater Ca²⁺ sensitivity than fibers expressing fast MHC isoforms. In contrast, Eddinger and Moss (6) reported that in the rat Dia_m, the force-pCa curve for slow fibers (as determined histochemically and electrophoretically) was shifted rightward compared with that of fast fibers. In another study in the rat Dia_m, Danieli-Betto et al. (5) reported no significant differences in the pCa₅₀ of fibers expressing MHC_slow and MHC_2A isoforms (as determined electrophoretically). It is unclear why fiber-type differences in Ca²⁺ sensitivity should be unique for the rat Dia_m. Therefore, the purpose of the present study was to examine the force-pCa relationship of rat Dia_m fibers expressing different MHC isoforms. We hypothesize that, as in other skeletal muscles, the pCa₅₀ of Dia_m fibers expressing the MHC_slow isoform is higher than that of fibers expressing fast MHC isoforms, with no differences in Ca²⁺ sensitivity among fibers expressing MHC_2A, MHC_2X, and MHC_2B/2X isoforms.

It has been suggested that the presence of different isoforms of troponin C (TnC) may contribute to fiber-type differences in Ca²⁺ sensitivity (13, 23, 40). It has also been suggested that expression of TnT and TnI isoforms (12, 29, 32, 39) is important in determining fiber-type differences in Ca²⁺ sensitivity. Therefore, we further hypothesize that the greater Ca²⁺ sensitivity of Dia_m fibers expressing the MHC_slow isoform is associated with a unique combination of troponin (Tn) isoforms compared with fibers expressing fast MHC isoforms.

	METHODS

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Animal preparation and single-fiber dissection. Adult male Sprague-Dawley rats (body wt ~300 g) were injected intramuscularly with ketamine (60 mg/kg) and xylazine (2.5 mg/kg), and the right side of the Dia_m was excised. Muscle fiber bundles were stretched ~20% to optimal length, pinned on cork, and placed in a relaxing solution, at 5°C for 24 h, consisting of (in mM) 59.0 KAc; 6.7 MgAc₂; 5.6 Na-ATP; 10 EGTA, 2.0 1,4-dithiothreitol, 15.0 CrP, and 50 mM imidazole, as well 1 mg/ml creatine phosphokinase, for a total ionic strength of 200 mM at a pH of 7.0. The fiber bundles were then stored in relaxing solution containing 50% glycerol (vol/vol) for 2-3 wk.

Force measurements. Activating and relaxing solutions used for force measurements were determined by the computer program described by Fabiato and Fabiato (7) with stability constants listed by Godt and Lindley (10). The solutions contained the following (in mM): 10.0 EGTA, 1.0 free Mg²⁺, 5.0 MgATP, 15.0 creatine phosphate, 50.0 imidazole, and 2.0 1,4-dithiothreitol, as well as creatine phosphokinase at 1 mg/ml for a total ionic strength of 150 mM. The relaxing solution had a pCa of 9.0. In the activating solutions, pCa ranged from 7.0 to 4.0.

Single-fiber gel electrophoresis. MHC isoform composition of single skeletal muscle fibers was identified by SDS-PAGE according to methods previously described (36). Single fibers were placed in 25 µl of SDS sample buffer containing 62.5 mM Tris · HCl, 2% (wt/vol) SDS, 10% (vol/vol) glycerol, and 0.001% (wt/vol) bromophenol blue at a pH of 6.8. The samples were denatured by boiling for 2 min. A modified procedure by Sugiura and Murakami (37) was used to prepare the gradient gels. The stacking gel contained a 3.5% acrylamide concentration (pH 6.8), and the separating gel contained 5-8% acrylamide (pH 8.8) with 25% glycerol (8 × 10 cm, 0.75 mm thick; Hoefer SE250). Control samples of Dia_m bundles in a 1:200 dilution (~9.0 ng/µl) were run on the gels for comparison of migration patterns of the MHC isoforms. The concentration of protein in Dia_m bundles was determined by the Lowry et al. method (18). Sample volumes of 10 µl were loaded per lane. The gels were stained with silver according to the procedure described by Oakley et al. (26).

Statistical analysis. One-way ANOVA was performed to compare the threshold pCa, pCa₅₀, and Hill coefficient values (n₁ and n₂) across fibers expressing different MHC isoforms. When appropriate, a Student's t-test with Bonferroni correction was used to compare between fiber types. A P < 0.05 was used to indicate statistical significance.

	RESULTS

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MHC isoform expression. In single dissected Dia_m fibers, MHC isoform expression was readily identified by SDS-PAGE and Western analysis (Fig. 1). The MHC isoform composition of 165 Dia_m fibers was evaluated in the present study. Most of these Dia_m fibers (~82%) expressed a single MHC isoform. In ~18% of all Dia_m fibers, coexpression of MHC isoforms was detected, with coexpression of the MHC_2B and MHC_2X isoforms predominating. In the present study, none of the dissected fibers displayed singular expression of MHC_2B. This is consistent with our previous observation of a relatively low incidence (~3-8%) of fibers expressing only MHC_2B in the rat Dia_m (36). Coexpression of the MHC_2A and MHC_2X isoforms was detected in one Dia_m fiber.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Myosin heavy chain (MHC) isoforms within rat diaphragm muscle (Diam) identified by SDS-PAGE.

Force-Ca²⁺ relationship. The dependency of force generation on myoplasmic [Ca²⁺] was systematically evaluated in 45 Dia_m fibers. The force-pCa relationships of Dia_m fibers expressing different MHC isoforms are shown in Fig. 2A. The force-pCa curve of fibers expressing the MHC_slow isoform was shifted leftward compared with curves for fibers expressing the fast MHC isoforms (MHC_2A, MHC_2X, and MHC_2B/2X). These fiber-type differences in Ca²⁺ sensitivity were reflected by differences in the pCa₅₀ (Table 1). Fibers expressing the MHC_slow isoform displayed significantly higher pCa₅₀ compared with fast fibers (Table 1, Fig. 2B). In fibers expressing MHC_2A, MHC_2X, and MHC_2B/2X isoforms, no significant differences in the pCa₅₀ values were found.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   A: development of force in single Diam fibers expressing different MHC isoforms depends on myoplasmic [Ca2+] (pCa). Values are means ± SE. , MHCslow; , MHC2A; , MHC2X; , MHC2B/2X. B: comparison of force-pCa relationship between fibers expressing MHCslow () isoform and fibers expressing fast MHC isoforms ().

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Hill plots of mean force-pCa data for rat Diam single fibers represent degree of cooperativity of Ca2+ binding to troponin C (TnC). P, tension; Po, maximum tension. Straight lines were drawn through data above and below pCa50 and fit with least squares regression analysis. Symbols are defined as in Fig. 2.

Tn isoform expression. The electrophoretic migration patterns of Tn isoforms expressed in different Dia_m fiber types are shown in Fig. 4. In a total of 111 Dia_m fibers expressing the MHC_2A, MHC_2X, and MHC_2B/2X isoforms, only the TnC-f isoform was expressed (Table 2). In 24 Dia_m fibers expressing the MHC_slow isoform, both the TnC-s and TnC-f isoforms were expressed, but in all cases, these isoforms were expressed singularly (Table 2). This sample is not meant to reflect the overall percentage of slow Dia_m fibers expressing the fast TnC isoform.

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Troponin (Tn) isoforms in rat muscles and single fibers identified by SDS-PAGE. Lanes 1 and 6, containing rat extensor digitorum longus (EDL) and soleus muscles (Sol), respectively, are present as controls for identification of fast and slow Tn isoforms in rat Diam single fibers (lanes 2-5). Tm, tropomyosin; mlc, myosin light chain.

	DISCUSSION

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The results of the present study clearly indicate that rat Dia_m fibers expressing the MHC_slow isoform have greater Ca²⁺ sensitivity of force generation than fibers expressing fast MHC isoforms. These results are in general agreement with several previous studies in limb skeletal muscles (5, 12, 17) as well as in the rabbit Dia_m (15). Furthermore, the results of the present study provide new information demonstrating that Ca²⁺ sensitivity does not vary across Dia_m fibers expressing MHC_2A, MHC_2X, and MHC_2B/2X isoforms. The greater Ca²⁺ sensitivity of Dia_m fibers expressing the MHC_slow isoform was associated with the expression of slow isoforms of the Tn complex. However, a small proportion of slow Dia_m fibers expressed the fast isoform of TnC, indicating that TnC isoform composition is not the only determinant of Ca²⁺ sensitivity in Dia_m fibers.

Previous studies examining Ca²⁺ sensitivity of rat Dia_m fibers have been equivocal. For example, Eddinger and Moss (6) showed that the force-pCa curve of slow fibers was shifted to the right compared with that of fast fibers [cf. Fig. 5A in Eddinger and Moss (6)]. However, these investigators stated that fast Dia_m fibers were less sensitive to Ca²⁺ than slow fibers. Because pCa₅₀ values were not reported, the basis for this stated fiber-type difference in Ca²⁺ sensitivity was unclear. In another study, Danieli-Betto et al. (5) reported no significant difference in Ca²⁺ sensitivity between fast and slow fibers in the rat Dia_m. However, in this study only two slow Dia_m fibers were compared with only five fast fibers, which were all classified as type 2A.

The results of the present study demonstrated that Ca²⁺ sensitivity was comparable across rat Dia_m fibers expressing MHC_2A, MHC_2X, and MHC_2B/2X isoforms. These results contrast with those of Eddinger and Moss (6), who reported that histochemically classified type IIa fibers were less sensitive to Ca²⁺ than type IIb fibers. It is likely that most of the fibers histochemically classified as type IIb in the rat Dia_m actually express the MHC_2X isoform (11, 33, 36). For example, we previously reported that only ~3-8% of all fibers in the rat Dia_m singularly express the MHC_2B isoform, whereas ~12% coexpress MHC_2X and MHC_2B isoforms (36). However, it is unlikely that the ambiguity of histochemical subclassification of fast fiber types accounted for the apparent discrepancy between the results of the present study and those of Eddinger and Moss (6). It is of interest that although Dia_m fibers expressing different fast MHC isoforms do not vary in the Ca²⁺ sensitivity of force generation they do vary with respect to maximum shortening velocity and maximum specific force (34, 35), consistent with observations in several other skeletal muscles (1, 6, 25).

It has been suggested that the presence of different isoforms of TnC may contribute to fiber-type differences in Ca²⁺ sensitivity (13, 23). The slow or cardiac TnC isoform (TnC-s) has only one low-affinity Ca²⁺-binding site, yet this binding site has a higher affinity for Ca²⁺ than the two low-affinity binding sites present on the TnC-f isoform found in fast skeletal muscle fibers (27, 40.) The observation in the present study that the onset of force in Dia_m fibers expressing the MHC_slow isoform occurred at lower myoplasmic Ca²⁺ concentrations (higher pCa values) is consistent with a higher Ca²⁺-binding affinity of the TnC-s isoform. This suggests a possible role for fiber-type specific isoforms of TnC in conferring Ca²⁺ sensitivity. However, experiments that substitute native TnC in skeletal muscle fibers with the opposite TnC isoform have produced controversial results. For example, in an initial study by Moss et al. (23), it was shown that substitution of the TnC-s isoform into fast fibers resulted in a reduction of Ca²⁺ sensitivity. However, in a subsequent study, these investigators concluded that this result was the consequence of incomplete reconstitution of TnC rather than a shift in the force-pCa relationship (21, 24) because partial extraction of TnC has been shown to cause a reduction in Ca²⁺ sensitivity (22). The role of TnC isoforms in conferring Ca²⁺ sensitivity is also challenged by the observation of the present study that a small population of Dia_m fibers expressing the MHC_slow isoform contained the TnC-f isoform. This observation is consistent with the previous report of Danieli-Betto et al. (5), who also found that slow fibers of the rat Dia_m expressed both TnC-s and TnC-f. Together, these results indicate that the expression of different TnC isoforms is not the sole determinant of fiber-type differences in Ca²⁺ sensitivity.

The results of the present study suggest that the expression of other Tn isoforms (TnT, TnI) or tropomyosin (Tm) isoforms may be important in determining fiber-type differences in Ca²⁺ sensitivity. In the present study, distinct isoforms of TnT were found in fibers expressing the MHC_slow isoform. The presence of two slow TnT isoforms in these fibers is in agreement with Schachat et al. (31), who found that slow fibers from the rabbit Dia_m express TnT_1s and TnT_2s isoforms. These same investigators found the fast TnT isoform, TnT_1f, in fast fibers from the rabbit Dia_m, although they did not indicate the MHC isoform of these fibers. In the study by Schachat et al. (32), differences in Ca²⁺ sensitivity were attributable to the combination of TnT and Tm isoforms. These investigators reported that among rabbit fast fibers from a variety of muscles, those that expressed a combination of the TnT_2f and ₂Tm isoforms had greater Ca²⁺ sensitivity than fast fibers expressing a combination of the TnT_1f and Tm/Tm isoforms. This is in agreement with the results of the present study in which fibers expressing different fast MHC isoforms but the same TnT and Tm isoforms did not have a significant difference in Ca²⁺ sensitivity.

Similar differences in TnT isoforms have been found in other skeletal muscles when fast and slow fibers were compared. For example, Greaser et al. (12) reported that rabbit plantaris muscle fibers expressing the MHC_slow isoform contained a slow TnT isoform, whereas plantaris fibers expressing fast MHC isoforms contained a fast TnT isoform. These investigators concluded that this difference in TnT isoform composition accounted for the greater Ca²⁺ sensitivity of slow vs. fast plantaris muscle fibers. Furthermore, they found that among fast plantaris muscle fibers several TnT isoforms were expressed and that Ca²⁺ sensitivity was dependent on the molecular weight of these fast TnT isoforms. Reiser et al. (29) also found that variations in TnT isoforms in adult chicken skeletal muscles corresponded with variations in Ca²⁺ sensitivity among fast muscle fibers. However, a comparison between fast and slow fibers in this study was conflicting. Slow fibers from the anterior latissimus dorsi muscle, expressing only the TnT slow isoform, had higher pCa₅₀ values than fast fibers from the posterior latissimus dorsi muscle but lower pCa₅₀ values than fast fibers from the pectoralis major muscle (29). In this study, the fast posterior latissimus dorsi and the pectoralis major muscles expressed variable amounts of fast TnT isoforms.

In addition to TnT and TnC, a role for TnI in conferring Ca²⁺ sensitivity has also been considered. For example, Van Eyk et al. (39) examined a variety of TnI mutants and fragments with different affinities for actin-Tm and the TnT-TnC complex. Results of this study indicate a possible role for TnI residues 96-148 in determining Ca²⁺ sensitivity of the thin filament. However, TnI recombinants used in this study were from fast rabbit and chicken muscle, and a comparison with slow TnI was not made. Results from the present study indicate differences in TnI isoforms between fast and slow fibers in the rat Dia_m, but future studies are needed to determine whether TnI isoforms are directly involved in conferring fiber-type differences in Ca²⁺ sensitivity.

In the biphasic force-pCa relationship, molecular cooperativity occurs predominantly in the activation of force up to half-maximal with little cooperativity present at higher Ca²⁺ concentrations (20). Therefore, the discussion of cooperative processes will be limited to conditions of low Ca²⁺ concentration (n₁). In the present study, rat Dia_m fibers expressing the MHC_2A, MHC_2X, and MHC_2B/2X isoforms displayed significantly higher n₁ values than did fibers expressing the MHC_slow isoform. Previous studies in the rat Dia_m (6) as well as in limb skeletal muscles (12, 23) have also reported that fast fibers have higher n₁ values than slow fibers. Furthermore, Eddinger and Moss (6) reported that, in the rat Dia_m, fibers histochemically classified as IIb had higher n₁ values than fibers classified as IIa. In the present study fibers expressing the MHC_2X and MHC_2B/2X isoforms did not have significantly greater n₁ values than fibers expressing the MHC_2A isoform (Table 1). In limb muscles, differences in n₁ between fibers expressing the MHC_2A isoform and other fast MHC isoforms were not explored. Therefore, it appears that fibers expressing fast MHC isoforms have greater cooperativity of force development than fibers expressing the MHC_slow isoform.

A possible role for TnC isoforms in conferring fiber-type specific differences in the steepness of the force-pCa relationship has also been considered. However, stochiometric substitution of TnC isoforms in skeletal muscle did not affect the slopes of the force-pCa relationship in fast and slow fibers (24). Expression of different TnT isoforms has been associated with differences in cooperativity of muscle fibers. In the study by Schachat et al. (32), the greatest Hill coefficient value in a variety of fast rabbit muscles was found in fibers expressing the greatest fraction of both TnT_2f and ₂Tm. Another theory proposed to explain thin-filament cooperativity involves differences in the response of the thin filament to strong-binding myosin cross bridges. In these experiments, use of a myosin derivative with strong binding properties, N-ethylmaleimide S1, increased Ca²⁺ sensitivity and reduced the slope of the tension-pCa curve to a greater extent in slow muscles than in fast muscles, indicating that slow muscles respond more readily to strongly bound cross bridges (41). Thus, on the basis of these results, it would appear the need for cooperativity with thin-filament activation is less prominent in slow fibers.

Modulation of Ca²⁺ sensitivity and the slope of the force-pCa relationship by cross-bridge cycling kinetics should also be considered in this discussion. On the basis of Brenner's model (2) for regulation of muscle contraction, the transition of cross bridges from a non-force-generating state to a force-generating state is described by the apparent rate constant f_app; the reverse transition from a force-generating state to a non-force-generating state is described by the apparent rate constant g_app. Estimates of these rate constants, and thus cross-bridge cycling kinetics, can be obtained from measuring the rate of tension redevelopment, k_tr, after a quick release and restretch where k_tr = f_app + g_app. Brenner reported that f_app is Ca²⁺ sensitive and that an increase in the ratio of f_app to g_app (f_app/g_app) would be expected to shift the force-pCa curve to the left, with a concomitant increase in the slope at low Ca²⁺ levels (2). Thus Brenner concluded that differences in Ca²⁺ sensitivity are not necessarily attributed solely to the binding affinity and cooperative properties of TnC but that cross-bridge cycling kinetics could also play a role.

However, additional studies indicate the molecular mechanisms regulating Ca²⁺ sensitivity may depend on the level of activation. For example, at maximal Ca²⁺ activation the rate of tension redevelopment is determined by cross-bridge cycling kinetics and depends on MHC isoform expression in fast and slow mammalian skeletal muscle (19). In contrast, more recent studies reveal that k_tr varies with the kinetics of thin-filament activation at submaximal Ca²⁺ activation. For example, in a study by Regnier and colleagues (28), an increase in the Ca²⁺ affinity of TnC with calmidazolium results in an increase in k_tr at submaximal Ca²⁺ levels with no change in k_tr at maximal Ca²⁺ activation. In addition, when native TnC is replaced with cardiac TnC or an activated form of TnC (aTnC), reducing the Ca²⁺-binding affinity of TnC, the Ca²⁺ sensitivity of k_tr is reduced or eliminated, respectively (4). On the basis of these experiments, the Ca²⁺-binding kinetics of the TnC isoform present determine the degree to which changes in submaximal Ca²⁺ can affect cross-bridge cycling kinetics. These studies make cross-bridge cycling rates an unlikely mechanism for the Ca²⁺ sensitivity of force development at submaximal [Ca²⁺].

In conclusion, results from this study indicate that fibers expressing the MHC_slow isoform have greater Ca²⁺ sensitivity than fibers expressing fast MHC isoforms. Furthermore, the Ca²⁺ sensitivity and cooperativity of force generation were not different across Dia_m fibers expressing MHC_2A, MHC_2X, and MHC_2B/2X isoforms. This fiber-type difference in Ca²⁺ sensitivity may be attributable to the isoforms of the Tn complex present. Slow fibers typically express slow Tn isoforms, although results from this study reveal that both fast and slow TnC isoforms can be singularly expressed in these fibers. Because the population of slow fibers expressing the fast TnC isoform in this study was small, and in light of new experiments indicating that the Ca²⁺-binding kinetics of TnC isoforms affect the rate of tension redevelopment, TnC isoforms cannot be ruled out as a determinant of Ca²⁺ sensitivity. However, the results of TnC substitution studies have been inconclusive, and a predominant role for TnC isoforms in determining Ca²⁺ sensitivity has not been clearly demonstrated. Although it is likely that thin-filament activation affects the Ca²⁺ sensitivity of force development, future studies are needed to further define the integrative roles of TnC, TnT, and TnI isoforms in this regulation.