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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Zoology, La Trobe University, Melbourne; and 2Muscle Cell Biochemistry Laboratory, School of Biomedical Sciences, Victoria University, Melbourne, Victoria, Australia
Submitted 12 January 2006 ; accepted in final form 31 March 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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7.1, with EDL fibers displaying more consistent changes than SOL fibers; 3) decrease by 50% in specific force of all fiber types; 4) decrease in sensitivity to Ca2+, particularly for SOL fibers (by 40%); 5) decrease in the maximum steepness of the force-pCa curves, particularly for the hybrid I/IIA SOL fibers (by 35%); and 6) increased occurrence of biphasic behavior with respect to Sr2+ activation in SOL fibers, indicating the presence of both slow and fast troponin C isoforms. No fiber types common to the two muscles were detected at any time points (day 7, 21, and 50) after denervation. The results provide strong evidence that not only neural factors, but also the intrinsic properties of a muscle fiber, influence the structural and functional properties of a particular muscle cell and explain important functional changes induced by denervation at both whole muscle and single cell levels. mechanically skinned fibers; myosin heavy chain isoforms; lineage; sarcoplasmic reticulum; Ca2+; Sr2+ sensitivity; Long-Evans hooded rat
25 ms in 480-day cross-innervated EDL muscle vs. 35 ms in SOL muscle and 15 ms in EDL muscle; Refs. 5 and 6). This suggests that factors other than the neural input may contribute to the fiber phenotype.
Although it has been suggested that there are at least as many fiber types as there are motor units in the body of an animal (33, 34), skeletal muscle fibers are primarily classified based on the type of molecular motor protein isoform expressed in that fiber. The molecular motor protein in skeletal muscle is the myosin heavy chain (MHC) and according to the nomenclature used in our laboratories, the MHC isoform detected in a muscle or muscle fiber is identified by a Roman numeral and a lowercase letter (e.g., MHC IIa). A fiber containing only one MHC isoform is referred to as pure fiber and is identified by a Roman numeral and a capital letter (e.g., IIA fiber), whereas a fiber expressing more than one MHC isoform is referred to as a hybrid fiber and is identified by a composite symbol (e.g., I/IIA and IIB/D). Typically, the fastest/glycolytic to the slowest/oxidative rat limb muscle fibers express the MHC isoforms: IIb 
IIb/IId IId IId/IIa IIa IIa/I I, with fibers expressing type II MHC isoforms generally regarded as fast (fast-twitch) fibers and fibers expressing MHC I generally regarded as slow (slow-twitch) fibers.
Recent experiments on regenerated EDL and SOL muscles have revealed that denervated EDL and SOL stimulated with the same slow stimulus pattern expressed slow and fast MHC isoforms in different proportions, which led to the interpretation that the satellite cells in the EDL and SOL muscles from which the regenerated muscles originate, have intrinsically different properties (19). If the precursor cells from which the EDL and SOL muscles develop have different intrinsic properties with respect to factors that affect gene expression and the mechanism regulating transcription (see DISCUSSION), then one could hypothesize that on denervation, when the neural input is removed, the mature muscle cells from EDL and SOL muscles would converge to form different fiber phenotypes. To test this, we used a single fiber approach to examine fiber phenotypes from denervated EDL and SOL muscles not only with respect to the MHC isoform profile but also with respect to Ca2+-handling characteristics of the sarcoplasmic reticulum (SR) and contractile activation properties, which can also play an important role in determining how fast the fiber can be activated and relax (41).
In the adult Long-Evans hooded rat, EDL and SOL muscles do not overlap in their patterns of MHC expression and fiber type composition, with the EDL muscle containing only type IIB and IID fibers and a small fraction of type IIB/D hybrid fibers (14) and SOL containing only type I and IIA fibers and a small fraction of type I/IIA hybrid fibers (14, 35). In this study we used mechanically skinned muscle fiber preparations from Long-Evans hooded rats to investigate MHC isoform expression, SR properties, and contractile activation characteristics at the single fiber level. The results show that at 50 days post-denervation, there was still no overlap in the fiber types present in the EDL and SOL muscles, and that >75% of the fibers in both the EDL and SOL muscles became hybrid with respect to MHC composition, with the hybrid EDL fibers co-expressing MHC isoforms IIb and IId and the hybrid SOL fibers co-expressing MHC isoforms I and IIa. These hybrid fibers also displayed different properties with respect to Ca2+ activation of the contractile apparatus and SR Ca2+ handling, but not with respect to the maximum level of specific force produced. The results are important as they provide strong evidence that not only neural factors but also the intrinsic properties of the muscle fiber determine the functional properties of a particular muscle cell.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Two groups of male Long-Evans hooded rats aged 4–6 mo were used: 1) a control group of 6 nonoperated rats and 2) a denervated group of 17 right-side-denervated rats. Denervation of lower leg muscles was performed while the rats were under halothane anesthesia by removal of 3–5 mm of the sciatic nerve below the sciatic notch, as previously described (31). The contralateral (left) hindlimb muscles of denervated animals were not used as control muscles due to altered weight-bearing effects. Similarly, muscles from sham-operated animals were not used because altered muscle use in the sham-operated hindlimb was likely to have affected normal muscle functioning, particularly in the first week after the procedure. All work with animals was approved by La Trobe University’s Animal Ethics Committee.
Muscle Dissection and Skinned Fiber Preparation
At the required time [day 0 (control), day 7, day 21, and day 50 post-denervation], the animals were euthanized by halothane overdose and the EDL and SOL muscles were dissected, blotted on filter paper, and placed under paraffin oil. Single fibers were isolated and mechanically skinned under a dissection microscope, as previously described (32). The mechanically skinned fibers were then attached between a sensitive force transducer (model AME875, Horten) and a pair of forceps fixed to a micromanipulator. The length and the average diameter of the fiber, measured at x40 magnification in three locations along the fiber, were then determined at slack length to allow calculation of the cross-sectional area (CSA) and volume of the preparation. The fiber was then placed in pre-release solution (Table 1).
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Solutions for skinned fiber experiments (Table 1) were prepared according to previously published methods (42). Solutions I and II were mixed in varying proportions to give activating solutions in the pCa (–log10 [Ca2+]) range of 7.2 to 4.0. Similarly, solutions I and SrII were mixed in varying proportions to give solutions in the pSr (–log10 [Sr2+]) range of 7.0 to 3.7. All other solutions in Table 1 were used in SR load-release experiments.
SR Load-Release Experiments
After being mounted, fibers were exposed for 2 min to pre-release solution and then transferred to the SR-release solution, which contained caffeine and low [Mg2+]. The resulting force response was indicative of the endogenous SR Ca2+ content and the fiber was kept in this solution for at least 1 min to thoroughly deplete the SR of Ca2+ (12). The SR Ca2+ loading properties of the fiber were then tested using a SR load-release protocol as described in the legend to Fig. 1. Briefly, the fiber underwent a 2-min exposure to a load solution buffered at either pCa 7.1 or 6.2, and thereafter to a full release by exposure to the SR-release solution. The sequence of solution changeovers was repeated such that each fiber underwent a load-release cycle at both pCa 7.1 and pCa 6.2.
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After the SR function experiments, preparations were first transferred to a relaxing solution I (Table 1) and then the Ca2+- and Sr2+-activation characteristics were determined by sequential exposure to a series of heavily buffered solutions of increasing [Ca2+] or [Sr2+] made from solution I and II and from solution I and SrII, respectively (1, 32).
For each mechanically skinned fiber segment, the following contractile activation parameters were determined: 1) maximum Ca2+-activated specific force (force per CSA: CaFmax/CSA, kN/m2) measured from the amplitude of the first force response at pCa 4.0 and from estimated fiber CSA measured in paraffin oil before exposure to aqueous solutions, 2) sensitivities of the contractile apparatus to Ca2+ (pCa50) and Sr2+ (pSr50), where [Ca50] and [Sr50] are the [Ca2+] and [Sr2+] respectively, corresponding to half-maximal activation of the force response and 3) the Hill coefficients associated with the force-pCa and force-pSr curves. The pCa50 and pSr50 values were obtained from fitting the relative force (Pr) produced by a fiber at each pCa or pSr by the simple Hill equation
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All Pr-pCa but not all Pr-pSr curves could be fitted by Eq. 1 with a correlation coefficient r2 
0.99. The Pr-pSr curves that could not be well fitted by the simple Hill Eq. 1 could be fitted with a correlation coefficient r2 0.99 by a "composite" Hill curve (consisting of the sum of two Hill curves; see also Refs. 1 and 29) described by the following equation:
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MHC Isoform Analysis in Single Fiber Segments and Whole Muscle Homogenates
After contractile experiments, the fiber segments (volume 0.6–4.0 nl) were placed in 12 µl SDS-PAGE solubilizing buffer, incubated at room temperature for 24 h, boiled for 3 min and stored at –84°C, for later analysis of MHC isoform composition as described in detail (1). For MHC isoform analyses of whole muscle homogenates, muscles were homogenized, after removal of fat and tendons, in 6 vol of solution I containing a mixture of protease inhibitors (1 µM pepstatin, 2 µM leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride). The protein concentration in muscle homogenates was measured with the Bradford protein assay (3) and each homogenate was diluted with solubilizing buffer to 1 mg protein/ml, boiled for 3 min, and finally stored at –84°C until the electrophoretic run.
Before electrophoresis, homogenate- and single fiber-stock solutions were further diluted to a final concentration of 0.1 µg protein/ml and 0.05 nl fiber/µl, respectively. Analyses of MHC isoforms in small sample aliquots (4-µl homogenate sample/electrophoretic well or 6-µl single fiber sample/electrophoretic well) were performed using 0.75-mm-thick slab gels, the Hoefer Mighty Small gel apparatus and the alanine-SDS-PAGE protocol described by Goodman et al. (14). Gels were run for 26 h at 4–6°C at constant voltage (150 V) and stained with Bio-Rad Silver Stain Plus. MHC bands were analyzed with the use of a densitometer and ImageQuaNT software (version 4.1, Molecular Dynamics). Note that due to sample size limitations, the MHC isoform pattern was not obtained for a small number of fibers for which the contractile and SR characteristics were determined and that the small sizes of the fiber segments from the denervated muscles were too small for detection of troponin C (TnC) isoforms by SDS-PAGE.
Data Analysis
Results, expressed as means ± SE, were analyzed using Microsoft Excel 97 and Sigmaplot 4.01 (SPSS) software. Force-pCa (pSr) curves were constructed using Prism software (GraphPad Software). Statistical significance was tested at the P < 0.05 level using ANOVA, followed by the post hoc Newman-Keuls multiple-comparison test, or Student's t-test for unpaired samples, as appropriate.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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As expected, surgical removal of a section of the sciatic nerve (see MATERIALS AND METHODS) was accompanied by a decrease in the mass of both EDL and SOL muscles from male Long-Evans rats such that 50 days after denervation, the mass of both EDL and SOL muscles decreased by 80% (Fig. 2). Interestingly, at 7 days post-denervation, the drop in the EDL muscle mass was markedly smaller (–36%) than that recorded for the SOL muscle (–49%). A similar observation at 7 days post-denervation was made by Schulte et al. (39) who used female Wistar rats.
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Electrophoretic analyses of MHC isoform composition in whole muscle homogenates (see Fig. 3A) showed that in all rats used in this study (control and denervated), EDL muscles contained only MHC IIb and IId, whereas the SOL muscles contained only MHC I and IIa. The MHC ratios IIb/IId and I/IIa in the respective muscles from control and denervated rats at day 0, 7, 21, and 50 post-denervation are presented in Fig. 3B. The decrease in the respective MHC ratios post-denervation is graded in time and is statistically significant for both EDL and SOL muscles (one-way ANOVA; P < 0.002). In EDL muscles, denervation was associated with a gradual decrease in MHC IIb from 60% in controls to 33% at 50-day post-denervation and a concomitant increase in MHC IId from 40% to 67%. By comparison, in SOL muscles, denervation was associated with a gradual decrease in MHC I from 77% in control muscles to 61% in the 50-day-denervated muscles with a concomitant increase in MHC IIa from 23 to 39%.
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In a mechanically skinned fiber preparation, the relative levels of Ca2+ loading in the SR are indicated by the relative areas under the force responses elicited upon exposure of the preparation to the caffeine-and-low-Mg2+ SR Release solution (25). In a previous study from one of our laboratories (12), we showed that typically fast-twitch EDL muscle fibers and slow-twitch SOL fibers display quite different SR Ca2+-loading properties. When exposed to an intracellular Ca2+ concentration that is close to the endogenous level (pCa = 7.1), the Ca2+ loading of the SR is near capacity in slow-twitch SOL fibers, but is only at 40% of capacity in fast-twitch EDL fibers. Therefore, upon exposure to a higher [Ca2+] (pCa 6.2) the SR of typical fast-twitch EDL fibers, unlike that of typical slow-twitch SOL fibers, loads markedly more Ca2+ than it does at the endogenous pCa.
It should be noted that the relatively low level of SR Ca2+ loading in fast-twitch fibers facilitates the uptake of Ca2+ into the SR when myoplasmic Ca2+ rises, thus promoting fast relaxation. In contrast, if the SR is close to capacity, as is the case with the slow-twitch fibers, the rate of Ca2+ uptake will be markedly reduced, contributing to the slower time course of the twitch response. Thus the level of SR Ca2+ loading at physiological pCa (7.1) is an important factor in determining the time course of the force response (12).
In this study, the ratio between the SR Ca2+ content after 2-min loading at pCa 6.2 and at 7.10, estimated from the relative areas under the caffeine-induced force responses (see Fig. 5 and MATERIALS AND METHODS), has been used here as an indicator of the SR Ca2+-loading properties of individual fibers from control and denervated muscles.
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When only pure type II fibers from the EDL and type I fibers from SOL muscles are considered, the marked fiber type-related difference with respect to the ratio for SR Ca2+ loading at pCa 6.2 and pCa 7.1 observed in control muscles was maintained, with some exceptions as described below, throughout the 50-day denervation period (Fig. 5B). The pure type IIA fibers from the SOL muscles displayed a complex pattern with respect to the ratio of SR Ca2+ loading at pCa 6.2 and pCa 7.1, with one fiber from control muscle displaying a ratio of 1.07, one fiber from day 7 denervated muscle displaying a ratio of 0.74, and another fiber from day 21 denervated muscle displaying a ratio of 1.66. This finding is not surprising given that pure type IIA control SOL fibers have been previously reported (1) to also display hybrid characteristics with respect to the myosin light chain composition, containing both fast and slow myosin light chain isoforms. One pure SOL type I fiber from a 50-day denervated rat displayed a ratio of 2.46, which was typical of type II EDL fibers, indicating that the SR properties in that SOL fiber changed toward those characteristic of the type II EDL fibers before the fast MHC IIa isoform was expressed. In hybrid fibers of type I/IIA from denervated SOL muscles, there was also a large degree of variation with respect to the ratio of SR Ca2+ loading at pCa 6.2 and pCa 7.1, with values ranging between 1.01 and 1.75. Thus, it appears that in denervated SOL fibers, the SR characteristics of individual fibers are not tightly correlated with the MHC isoform(s) expressed, thus conferring unusual functional properties to the respective fibers.
Considering now the hybrid fibers from EDL (IIB/D) muscles, the ratio between the SR Ca2+ content at pCa 6.2 and 7.1 significantly (P < 0.05, double-sided t-test) decreased from 2.32 ± 0.03 (n = 3) in control muscles to 1.6 ± 0.2 (n = 6) in denervated muscles (7- to 50-day post-denervation). Because the percentage of the IIB/D hybrid fibers markedly increased in the denervated EDL muscle (Fig. 3A), these data indicate that the changes in the SR loading characteristics observed in the total fiber population of denervated EDL muscle result predominantly from changes in the SR characteristics of the hybrid (IIB/D) and not of the pure IIB and IID fibers.
Importantly, at day 50 post-denervation, there was a significant difference (P < 0.002, double-sided t-test) with respect to the ratio of SR Ca2+ loading at pCa 6.2 and pCa 7.1 between EDL type IIB/D hybrid fibers (1.71 ± 0.08; n = 4) and SOL type I/IIA hybrid fibers (1.21 ± 0.06; n = 5), indicating that the two predominant hybrid fiber populations of the day 50 denervated EDL and SOL muscles display dissimilar properties with respect to Ca2+ handling by the SR.
Contractile Properties
The Ca2+- and Sr2+-activation characteristics (maximum Ca2+-activated specific force, force-pCa parameters, force-pSr parameters) of individual fibers were determined by exposure of the skinned fiber preparation to a series of heavily buffered [Ca2+] or [Sr2+] solutions (see MATERIALS AND METHODS).
Maximum Ca2+-activated specific forces. The maximum Ca2+-activated specific forces (CaFmax/CSA) results for EDL and SOL fibers from normal and denervated muscles are shown in Fig. 6A. There was a marked decline in the maximal level of specific tension that could be developed in both EDL and SOL fibers post-denervation irrespective of fiber type (Fig. 6B). This is an important result because it shows that the marked reduction of whole muscle force following denervation is dependent not only on the reduction in muscle size post-denervation, but also on the reduced ability of individual muscle fibers to produce maximum specific force, probably because of the reduced fraction of the CSA occupied by the contractile apparatus in denervated fibers (46).
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There was also a statistically significant (t-test, P < 0.05) difference between pCa50 values of pure type II fibers from control EDL muscle and type I fibers from control SOL muscle, and a highly statistically significant (one-way ANOVA; P < 0.01) decrease in pCa50 for type I fibers from denervated muscles (6.23 ± 0.02, n = 6 for controls; 6.12 ± 0.04, n = 4 for 7-day denervated muscles; 5.95 ± 0.05, n = 2 for 21-day denervated muscle and 5.90 ± 0.07, n = 3 for day 50 denervated muscle). However, there was no statistically significant (P > 0.05) decrease in pCa50 values for pure type II EDL fibers in the denervated muscles.
At day 50 post-denervation, the hybrid fibers of type I/IIA from the SOL muscle showed a statistically significant (P < 0.015) decrease in pCa50 values compared with the hybrid fibers from SOL controls (5.96 ± 0.03, n = 9 vs. 6.14 ± 0.05, n = 3). No statistically significant difference associated with denervation was observed for IIB/D hybrid fibers from EDL muscle, with the IIB/D hybrid fibers from the day 50 denervated EDL muscle displaying a similar sensitivity to Ca2+ as I/IIA hybrid fibers from the day 50 denervated SOL muscle. Taken together, these results indicate that, while all fiber types from the SOL muscle become markedly less sensitive to Ca2+ soon after denervation, there is a smaller change in the sensitivity to Ca2+ in EDL fibers. Furthermore, the hybrid fibers from the day 50 denervated EDL and SOL muscles, which are predominant in these muscles, display similar sensitivities to Ca2+ even though they contain different MHC isoforms.
nCa significantly (determined via one-way ANOVA) decreased for fibers from the EDL and SOL muscles post-denervation, indicating a reduction in the steepness of the force-pCa curves (Fig. 8B). Notably, the nCa values stabilized after 21 days of denervation for EDL and SOL fibers and were significantly smaller than those for controls (Newman-Keuls post hoc multiple test, P < 0.05 for EDL; P < 0.01 for SOL). There was no significant difference between pure type II and hybrid IIB/D fibers from control EDL muscles (4.40 ± 0.42, n = 7 vs. 4.62 ± 0.98, n = 3), but there was a significant difference between type I and I/IIA fibers from control SOL muscles, with the nCa for type I fibers being significantly lower than the nCa for hybrid I/IIA fibers (2.81 ± 0.15, n = 6, vs. 4.34 ± 0.7, n = 3; P < 0.03, double-sided t-test). Denervation did not cause a significant decrease in nCa values for pure type I SOL fibers (2.81 ± 0.15, n = 6 for control rats vs. 2.70 ± 0.29, n = 4 for day 50 denervated rats), but did for the hybrid type I/IIA fibers (4.34 ± 0.70, n = 3 for control rats vs. 2.82 ± 0.23, n = 9, for day 50 denervated rats) such that the Hill coefficients for the two fiber types reached similar values in day 50 denervated SOL muscles. The nCa values for the IIB/D hybrid fibers from the day 50 denervated EDL muscle (3.43 ± 0.45; n = 7) also significantly decreased compared with those from control EDL muscle but were not statistically significantly different from those for the I/IIA hybrid fibers from the 50-day denervated SOL muscle. Thus, the main contributors to the overall decrease in the steepness of the Ca2+-activation curves post-denervation were the hybrid fibers, which displayed significantly smaller Hill coefficients in the denervated muscle.
Given the simple relationship between the threshold for contraction (pCa10, pCa50, and nH; see Eq. 2), it follows that the decrease in the value of the Hill coefficient contributes to an increase in pCa10 value at constant pCa50. Thus the decrease in pCa50 observed with different fiber types is compensated by the decrease in the value of nH such that the threshold values change less than the pCa50 values in the denervated fibers. For example, the decrease in pCa50 values by 0.11 and 0.24 pCa units for the EDL and SOL fibers from day 50 denervated muscles (Fig. 8A) would correspond to decreases in the pCa10 values by only 0.06 and 0.17 pCa units, respectively.
Sr2+ Activation Curves
Recent results from our laboratories (29) showed unequivocally that TnC isoform expression determines fiber type differences with respect to Sr2+ sensitivity (pSr50) and to the relative sensitivity of the contractile activation process to Ca2+ and Sr2+ (pCa50-pSr50). Examples of Ca2+- and Sr2+-activation curves for representative fiber types are shown in Fig. 7. If only the TnC slow isoform (TnC-s) is present, then the fiber is relatively sensitive to Sr2+ and pCa50-pSr50 < 0.7. In contrast, if only TnC fast isoform (TnC-f) is expressed, then the fiber is 10-fold less sensitive to Sr2+ and pCa50-pSr50 > 1.2. When both fast and slow TnC isoforms are expressed in the same fiber, the isometric force-pSr activation curve appears biphasic (see Fig. 7C), displaying a plateau region at submaximal levels of activation whose position on the ordinate is closely related to the proportion of the functional TnC isoforms present and is well fitted by the composite Hill equation described by Eq. 3 in MATERIALS AND METHODS.
After denervation, the sensitivity to Sr2+ remained essentially the same (pSr50 4.62–4.66) in EDL fibers, whereas the trend was for it to decrease in SOL fibers (Fig. 9). There was little change in sensitivity to Sr2+ after denervation of pure EDL fibers, but there was a marked and significant decrease in the sensitivity to Sr2+ of pure type I SOL fibers up to 21-day post-denervation, when it stabilized. Thus, 50 days post-denervation, the sensitivity to Sr2+ of type I SOL fibers decreased by a factor of 3 (pSr50 : 5.20 ± 0.12, n = 3 compared with 5.73 ± 0.05; n = 6 for controls).
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With regard to the differential sensitivity of the contractile activation process to Ca2+ and Sr2+, pCa50 - pSr50, there was a statistically significant increase by 0.3 log units (ANOVA; P < 0.01) for type I SOL fibers after 21- and 50-day of denervation, but a clear-cut difference in this parameter between type II and type I fibers was maintained in denervated fibers.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The study provides strong evidence that the intrinsic properties of a particular muscle play a major role in determining the fiber phenotype after the removal of neural input.
Distinct Fiber Phenotypes in Denervated EDL and SOL Muscles
The fibers in the denervated EDL and SOL muscles of Long-Evans hooded rats converged to form two distinct hybrid fiber phenotypes specific to the EDL and SOL muscles, respectively. Thus, 50 days post-denervation, >75% of the fibers in EDL and SOL muscles were hybrid with respect to MHC content (IIB/D in EDL and I/IIA in SOL). Most of the fibers present in the 50-day denervated muscles must have been preexisting fibers rather than new fibers originating from proliferating satellite cells since it is established that, in the absence of innervation, regenerating fibers express only type IIb and IId MHCs (9, 18, 19), and there was no trace of MHC isoforms IIb and/or IId in any of the denervated SOL muscles. It is also important to point out that the rapid and marked atrophy exhibited by the rat muscles over 2 mo post-denervation is due to a massive reduction in the cytoplasmic fiber volume of individual fibers and not to a significant decrease in the number of muscle fibers (46). Consequently, the most likely scenario is that upon denervation, the muscle fibers of the SOL and EDL muscles atrophy and gradually undergo molecular transformation, converging to two different hybrid phenotypes: IIB/D in the EDL muscle and I/IIA in the SOL muscle. The complete lack of overlap between fiber types or MHC present in the denervated muscle 50 days post-denervation, together with the emergence of the two predominant and distinct hybrid fiber phenotypes in the EDL and SOL muscles support the idea that these two fiber phenotypes represent stable fiber populations in the denervated muscles with potential to transform back, upon reinnervation, into one of the two major pure fiber phenotypes found in the normally innervated muscles: types IIB and IID in the EDL and types I and IIA in the SOL. The two hybrid fiber types display distinct characteristics not only with respect to MHC isoform composition but also with respect to SR Ca2+ handling and contractile activation. One likely explanation for the different fiber phenotypes in long-term denervated SOL and EDL muscles may lie in the different myoblast lineages of the two muscles, conferring on the fibers different intrinsic properties with respect to factors that affect gene expression and the mechanism regulating transcription. For example in rodents, the SOL muscle fibers develop predominantly from primary myotubes, whereas the EDL fibers develop mainly from secondary myotubes (30) and there is evidence that myotubes derived from primary and secondary myoblasts have distinct myogenic programs of MHC expression (40). The development of SOL and EDL fibers from two different populations of myoblasts with distinct myogenic programs can directly explain the observation made here that upon removing the external neural input, the fibers in the two muscles converge to form two distinct hybrid fiber phenotypes specific to the myonuclei lineages in the respective muscles. Conversely, the results from this study provide support to the hypothesis that myonuclei from primary and secondary myotubes have distinct myogenic programs. This interpretation also helps explain the restricted adaptive range of individual muscle fibers, as initially suggested by Stockdale (43) for chicken muscle, that the extent of muscle fiber transformation in response to changes in the pattern of neural stimulation will also depend on the distinct myogenic programs of the nuclei present in that particular fiber.
This work also highlights that the fiber type composition of the normally innervated EDL and SOL muscles in the Long-Evans hooded male rats is different from that in Wistar-Kyoto rats. In Wistar-Kyoto rats, the EDL muscles contain in addition to the predominant IIb and IId MHC isoforms/type IIB and type IID fibers, 8–20% IIa MHC/IIA fibers, and small amounts of MHCI/type I fibers (13, 17, 47). In a previous study (2), we also found that type IIA and IIA/IID hybrid fibers were present in sizeable proportions in both EDL (9.6 and 17.3%) and SOL (7.5 and 8.2%) muscles of Wistar-Kyoto rats. This was not the case with the Long-Evans rats, where the fiber types present in the EDL muscle do not overlap with the fiber types in the SOL muscle.
In Wistar rats, denervation of the EDL muscle results in fiber transformation such that 75% of fibers are of type IIA at day 60 post denervation, rising to 90% by day 130 post-denervation (47). Thus, the pattern of fiber transformation upon denervation differs not only between EDL and SOL muscles from the same rat, but also between EDL muscles of different rat strains that have a different fiber type composition before denervation. The pattern of denervation-induced changes in MHC isoform composition in the EDL muscles nevertheless follows the general pattern of denervation-induced changes in MHC composition for fast-twitch muscles: from faster to slower MHC isoforms (34), although it appears from this study and others (13, 17, 47) that denervated EDL muscle does not express new MHC isoforms that were not expressed in the muscle before denervation.
The most direct explanation as to why the fibers from denervated EDL and SOL muscles of Long-Evans and Wistar rats do not converge toward the same phenotypes is that the SOL and EDL muscles of the Long-Evans rats originate from close to pure lineages of primary and secondary myoblasts, respectively, whereas the lineage of the SOL and EDL fibers of Wistar rats from primary and secondary myoblasts, respectively, is less well defined. This interpretation would also readily explain the incomplete fiber transformation revealed, but not further explored, by cross-innervation SOL and EDL experiments (5, 6).
Common Trends Induced by Denervation
It is important to point out that although the emphasis in this study was on showing that the intrinsic properties of a particular muscle play a major role in determining the fiber phenotype after the removal of neural input, this study also shows that there are several common trends induced by denervation irrespective of the muscle or the fiber types present. Thus, upon denervation (day 50), both EDL and SOL muscles atrophy markedly and contain mainly hybrid fibers. Furthermore, the maximum Ca2+-activated specific force declines by a similar factor irrespective of the muscle or fiber type. These results can be directly explained by the massive reduction in the cytoplasmic (and myofibrillar) fiber volume of individual fibers (46). Another common trend from this study is that the sensitivity to Ca2+ described by the parameter pCa50 converges to a similar value (5.95) for fibers in both muscles, irrespective of the MHC or TnC isoforms expressed. The Hill coefficient nCa describing the steepness of the force-pCa curves also significantly decreases in hybrid fibers from both EDL (type IIB/D) and SOL (type I/IIA) muscles such that at 50-day post-denervation, when the two types of fibers are predominant in the two muscles, the nCa values are not significantly different between the two fiber types. These results indicate that these Ca2+-activation parameters are not simply determined by the MHC and TnC isoforms present.
Denervation and EDL Muscle
Changes in SR properties. In relation to SR Ca2+-loading properties, pure type IIB and type IID EDL fibers from denervated muscles maintained control-like SR loading properties. However, type IIB/D EDL hybrids displayed SR Ca2+ loading properties that were shifted toward the slow phenotype, suggesting that the SR Ca2+ loading at pCa 7.1 was closer to capacity. Evidence of increased SR Ca2+ loading in fibers from denervated EDL muscles was also reported for day 7 denervated Wistar rat (13) and day 14 denervated rabbit (44) EDL muscles. Because the time course of the twitch response is slower and the height of the twitch response is greater when the SR of EDL fibers is loaded with Ca2+ above endogenous levels (36), the presence in denervated EDL muscles of a large population of hybrid IIB/D fibers that are more loaded with Ca2+ explains why the EDL twitch becomes slower and the twitch to tetanus ratio rises post-denervation (13, 22). In relation to SR loading properties, Schulte et al. (39) showed that denervation caused a marked decrease in the density of the fast isoform of the sarco(endo)plasmic reticulum Ca2+-adenosinetriphosphatase (SERCA1) in EDL muscles of Wistar rats 14 days after denervation. The slow SERCA isoform (SERCA2a) was not detected in denervated EDL muscles for up to 28 days post-denervation. Reduced pump density and activity in denervated EDL fibers (24) could also contribute to prolongation of the twitch time course and increased peak response. Results obtained in this study also explain the previously reported increased sensitivity to caffeine of denervated EDL muscle (15) because a higher level of SR Ca2+ loading is known to increase the SR sensitivity to caffeine (2, 21). Although an increased level of SR Ca2+ loading may increase the level of Ca2+ released from the SR for a certain stimulus intensity, the marked shift in the voltage dependence of K+ contractures to more negative membrane potentials is more likely due to the shift of voltage sensor charge movements to more negative potentials (8). In summary, it appears that changes in the properties of surface and SR membranes in the EDL muscle post denervation markedly affect its function.
Changes in properties of contractile apparatus.
The pCa50 of EDL fibers decreased by 0.1 pCa units after day 50 denervation and the steepness of the force-pCa curve also decreased (nCa decreased), requiring a larger pCa range to fully activate the contractile machinery. Evidence was also provided that only the TnC-f isoform was functional in all EDL fibers examined from control and denervated muscles, indicating that the decrease in sensitivity to Ca2+ and nCa must be due to denervation-induced changes in myofibrillar components other than TnC. Germinario et al. (13) also reported a small decrease in Ca2+ sensitivity (pCa50) at 7-day post-denervation in Wistar EDL fibers. These results rule out the possibility that the marked shift to more negative membrane potentials of the contraction threshold (31) and of the curve relating the strength of the K+ contractures to the membrane potential in intact denervated EDL muscle is due to an increased sensitivity of the contractile apparatus to Ca2+ (8). The marked decrease of maximally Ca2+-activated specific force in fibers from denervated EDL muscles after 21 days of denervation explains to a large extent the decrease in specific maximum tetanic force in long-term denervated rat EDL muscle previously reported by Finol et al. (11).
Denervation and SOL Muscle
Changes in SR properties. In relation to the SR Ca2+-loading properties, the hybrid I/IIA fibers that become prevalent in SOL muscle after day 50 of denervation displayed properties that are intermediate between typical type I and type II fibers with some type I fibers exhibiting properties similar to those of typical type II EDL fibers. This trend is consistent with the results of Schulte et al. (39) showing that after day 28 of denervation the ratio between SERCA1 and SERCA2a in SOL muscle increases due to a decrease in SERCA2a expression. The SR Ca2+ loading capacity also increases in fibers from denervated SOL muscle (27). This is also consistent with a shift toward the SR characteristics of fast-twitch fibers, which have a severalfold greater SR Ca2+ loading capacity compared with slow-twitch fibers (12). After 14 and 28 days of denervation, there is also a net decrease in the total SERCA protein per total muscle protein (39), implying a decrease in the density of SR Ca2+ pumps in denervated SOL muscles. These changes in SR properties would promote increased Ca2+ release due to increased SR Ca2+ capacity and slower reuptake due to reduced SR Ca2+ pump capacity leading to a prolongation of the twitch time course and increased twitch peak in the denervated rat SOL muscle as reported in several studies (11, 23, 27). Unlike the denervated EDL fibers, the denervated SOL fibers do not display marked changes in the asymmetrical charge movements associated with the voltage sensors (8).
Changes in the properties of contractile apparatus. MHC isoform changes observed in this study for SOL are consistent with the general pattern of denervation-induced changes in MHC composition in slow-twitch muscles reported in other studies showing a transition from slower to faster MHC isoforms (33). However, the 50-day denervated muscles did not contain a larger proportion of pure type IIA fibers, but a marked increase in the proportion of hybrid I/IIA fibers co-expressing both TnC-s and TnC-f isoforms. Denervation of the SOL muscle gave rise to a decrease in sensitivity to Ca2+ that was more marked and occurred earlier in SOL than in EDL fibers, such that the significant difference in pCa50 values seen for fibers from control SOL and EDL muscles completely disappeared for fibers from the 50-day denervated preparations. The increase in the proportion of fibers expressing both TnC isoforms in denervated SOL fibers explains the more accentuated decrease in pCa50 in SOL fibers than in EDL fibers and the marked decrease of nCa values for hybrid I/IIA fibers from denervated SOL muscles. There was also a more pronounced decrease in specific maximum Ca2+-activated force after denervation in SOL than in EDL fibers (Fig. 6), in agreement with previous results reported by Midrio et al. (27) for denervated SOL muscle from Wistar rats. The results from this study can fully explain the drop in specific tetanic tension reported by Finol et al. (11) for 42-day denervated rat SOL muscle. Because the contractile apparatus is the major contributor to the muscle mass, the larger decrease in total muscle mass in SOL than in EDL muscle 7-day post denervation (Fig. 2), is consistent with the more marked changes in the properties of the contractile apparatus and Ca2+ regulatory system in the SOL than in the EDL muscle at this point in time after denervation. Thus it appears that denervation of the SOL muscle causes more marked changes in the contractile apparatus and the regulatory system than in the membrane components of the denervated fibers.
In conclusion, the different MHC isoform profiles, SR loading properties, and contractile activation characteristics of single fibers from 7- to 50-day-denervated EDL and SOL muscles provide strong evidence that the intrinsic properties of a particular muscle play a major role in determining the fiber phenotype after the removal of neural input. The single fiber results reported here can also be used to explain a number of important functional changes induced by denervation at the whole muscle level.
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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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) and Sr2+- (force-pSr, 
) activation curves for type II EDL (A), type I SOL (B), and type I/IIA SOL (C) fibers from 7-day (A, B) and 50-day (C) denervated muscles. All force-pCa data points in A–C and force-pSr points in A and B were well fitted (r2 > 0.99) by a simple Hill curve (
