MHC-based fiber type and E-C coupling characteristics in mechanically skinned muscle fibers of the rat

Craig Goodman1, Michael Patterson2, and Gabriela Stephenson1

1 Muscle Cell Biochemistry Laboratory, School of Biomedical Sciences, Victoria University, Melbourne City, Melbourne 8001; and 2 School of Zoology, Faculty of Science and Technology, La Trobe University, Bundoora, Victoria 3083, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we investigated whether the previously established differences between fast- and slow-twitch single skeletal muscle fibers of the rat, in terms of myosin heavy chain (MHC) isoform composition and contractile function, are also detectable in excitation-contraction (E-C) coupling. We compared the contractile responsiveness of electrophoretically typed, mechanically skinned single fibers from the soleus (Sol), the extensor digitorum longus (EDL), and the white region of the sternomastoid (SM) muscle to t-system depolarization-induced activation. The quantitative parameters assessed were the amplitude of the maximum depolarization-induced force response (DIFRmax; normalized to the maximum Ca2+-activated force in that fiber) and the number of responses elicited until the force declined by 75% of DIFRmax (R-D75%). The mean DIFRmax values for type IIB EDL and type IIB SM fibers were not statistically different, and both were greater than the mean DIFRmax for type I Sol fibers. The mean R-D75% for type IIB EDL fibers was greater than that for type I Sol fibers as well as type IIB SM fibers. These data suggest that E-C coupling characteristics of mechanically skinned rat single muscle fibers are related to MHC-based fiber type and the muscle of origin.

excitation-contraction coupling; myosin heavy chain; single muscle fiber; hybrid fiber


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that mammalian skeletal muscle is a heterogeneous tissue consisting of fibers of different functional and structural characteristics (10, 40, 44). The functional and structural diversity of skeletal muscle fibers has been related to interfiber-type differences in the relative proportions of some membrane systems [e.g., sarcoplasmic reticulum (SR) and t-tubular network (t system)] and to different amounts and molecular forms (isoforms) of proteins that play key roles in energy metabolism (e.g., glycolytic or oxidative enzymes) and excitation-contraction (E-C) coupling (e.g., myosin or Ca2+ regulatory proteins such as the Ca2+-ATPase pump). For the purposes of this study, E-C coupling refers to the whole sequence of events that occur in the twitch skeletal muscle fiber between the generation of an action potential and the activation of the contractile machinery (46).

The contractile protein myosin, which exists as a hexamer containing four myosin light chain subunits and two myosin heavy chain (MHC) subunits, accounts for almost half of the protein content in the myofibrillar compartment of skeletal muscle (21). The MHC subunit, a polymorphic protein, which is by far the most extensively studied of all the myofibrillar proteins, contains the enzyme (myosin ATPase) responsible for hydrolyzing ATP and the cross bridge responsible for the production of force and muscle shortening (1). Among the MHC isoforms expressed in mammalian skeletal muscle are four isoforms in adult mammalian muscle (slow-twitch isoform MHC I or MHCbeta /slow- and fast-twitch isoforms MHC IIa, MHC IId/x, and MHC IIb), two isoforms in developing and regenerating muscles (MHC-emb and MHC-neo), and five isoforms in highly specialized (extraocular and jaw muscles) muscles (for reviews, see Refs. 40 and 52).

In recent years, intense efforts have been directed toward the functional characterization of the contractile apparatus in single muscle fibers expressing specific MHC isoforms. These studies, in which MHC isoform analysis and determination of contractile parameters were carried out in the same fiber segment, have produced compelling evidence that the MHC isoform expressed in a single muscle fiber is a major determinant of the maximum shortening velocity (54), maximum power output (11), optimal velocity of shortening (9), rate of tension development (29), rate of ATP consumption (53), isometric force per cross-sectional area (26), and stretch activation properties (25) in skinned muscle fiber segments. Because of the central role of MHCs in muscle contractile performance and their relative abundance, MHC isoform expression (as detected by high-resolution PAGE or immunohistochemistry) has become the method of choice for classifying skeletal muscle fiber types into distinct functional groups (for reviews, see Refs. 5, 36, 52).

Very little is known about the E-C coupling characteristics (involving the t system and SR compartments) of different MHC-based fiber types. Qualitative molecular differences have been shown to exist between rodent extensor digitorum longus (EDL, containing predominantly fast-twitch fiber types) and soleus (Sol, containing predominantly slow-twitch fiber types) muscles: varying amounts of cardiac alpha 1C-dihydropyridine receptor (DHPR)/voltage sensor isoform (23, 39), slow calsequestrin isoform (16), and neonatal Ca2+ release channel/ryanodine receptor (RyR) type 3 (RyR3) isoform (3) are found in adult Sol, but not in EDL, muscles. Studies using whole muscles or untyped single fibers have demonstrated functional and structural differences between rat EDL and Sol muscles with respect to Ca2+ transients (17, 22), DHPR/voltage sensor density (33), number of SR RyR/Ca2+ release channels functionally coupled to t-tubular DHPR/voltage sensors (17), voltage dependence of DHPR/voltage sensor activation and inactivation (12), and SR volume (15). Given the diversity of MHC-based fiber types that make up most commonly used mammalian skeletal muscles (including the rat EDL and Sol) and the complexity of MHC isoform expression displayed by a large proportion of individual fibers (for review, see Ref. 52), the methodological approaches using whole muscles or single fibers of unknown MHC composition cannot determine whether E-C coupling characteristics differ between MHC-based fiber types. No studies have examined the E-C coupling characteristics and MHC isoform composition in the same fiber segment.

The most efficient strategy for probing events that occur between t-system depolarization and force production in a single fiber involves the use of a mechanically skinned fiber preparation, which retains functional coupling between the sealed t-tubular system and the SR (42), and the experimental protocol for t-system depolarization-induced activation of contraction developed by Lamb and Stephenson (30). Recently, we refined the alanine SDS-PAGE method developed by Nguyen and Stephenson (37, 38), which can be used to analyze MHC in rat single muscle fiber segments with a high degree of reproducibility. In this study, we combined these two methods to examine the E-C coupling characteristics and MHC isoform composition in single fiber segments from two predominantly fast-twitch, but functionally different, muscles [EDL and the white region of the sternomastoid (SM) muscle] and one predominantly slow-twitch (Sol) muscle.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Animals and Muscles

Adult Long Evans hooded rats, aged 20-21 wk, were killed by an overdose of halothane in accordance with Victoria University animal ethics procedures. EDL, Sol, and SM muscles were carefully removed, blotted dry on filter paper (Whatman no. 1), and placed under paraffin oil as previously described (7). Single fiber segments were randomly isolated under oil. The SM muscle has two distinct regions, red and white (18, 28). Both regions are considered fast twitch (35), but for comparison with the fast-twitch fibers isolated from the white EDL muscle, single SM fiber segments were isolated from the white region only.

Mechanically Skinned Single-Fiber Preparations

Mechanically skinned fiber segments (volume 1.0-8.8 nl) were prepared, and their dimensions were measured as described in detail by Bortolotto et al. (7). Each fiber was mounted between a force transducer (model AME801, SensoNor, Horten, Norway) and a pair of fine forceps (Bancroft) and stretched to 120% of its resting length, bringing the sarcomere length to 2.8-3.0 µm. The sarcomere length adjustment was performed on the basis of compelling evidence that 1) the plateau region for isometric force production in rat skeletal muscle occurs at longer sarcomere lengths (2.4-2.9 µm) (51), a characteristic that has been related to longer thin filaments (13), and 2) in vertebrate skeletal muscle, an increase in the sarcomere length was accompanied by an increase in the sensitivity of the contractile apparatus to Ca2+ (45). At sarcomere lengths of 2.8-3.0 µm, rat EDL muscle fibers produce close to maximum force. With the activation procedures used in this study, the sarcomere pattern and sarcomere homogeneity in small vertebrate skeletal muscle fiber segments are well preserved during submaximal activation (51). When they were not being used for single fiber isolation, muscles were stored under oil at 4°C. To minimize storage-related glycogen loss, each muscle was used within 3 h from dissection (27). All experiments were carried out at room temperature (22-25°C).

Depolarization-Induced Force Responses

The t tubules of mechanically skinned muscle fibers have previously been shown to reseal and repolarize in a K+-hexamethylenediaminetetraacetic acid (HDTA)-based repriming solution (see Solutions). This allows depolarization of the t tubules, which triggers a force response via the normal E-C coupling pathway, when the fiber is subsequently exposed to a solution mimicking the myoplasmic environment in which K+ is replaced with Na+ (30). Each fiber was placed immediately after skinning in the K+-HDTA solution for 2 min. Subsequently, the K+-HDTA solution was rapidly substituted with the Na+-HDTA solution, which causes the t-system membrane to depolarize and Ca2+ to be released from the SR via the normal E-C coupling mechanism. Each depolarization lasted for ~3-5 s; then the fiber was returned to the K+-HDTA solution for 1 min of repriming before the next depolarization. As discussed by Posterino et al. (43), this force response is at least one order of magnitude slower than an action potential-induced twitch and is not abolished by the inclusion of tetrodotoxin in the sealed t system. The typical pattern of t-system depolarization-induced force responses produced by a fiber subjected to this protocol includes 1) an initial "work-up" period, where the force generated gradually increases to a maximum value (14) and 2) a "run-down" period, where the force responses decrease until no more force responses can be elicited (30). The amplitude of the force response reached at the end of the work-up period may begin to decrease rapidly or may be maintained for several depolarization-repolarization cycles.

In this study, depolarizations were continued until the force response had run down by 75% of the largest depolarization-induced force response. To determine whether the rundown was due to depleted SR Ca2+ levels or to dysfunction of the Ca2+ release channels, each fiber was placed in a low-Mg2+ (0.015 mM) solution to release the endogenous Ca2+. Maximum Ca2+-activated force (CaFmax) was then obtained in a high-Ca2+ (pCa 4.5) solution. The parameters of E-C coupling recorded in this study were the amplitude of the maximum depolarization-induced force response (DIFRmax), normalized to CaFmax in the same fiber, and the number of DIFRs produced by the fiber until the force response was 75% lower than DIFRmax (R-D75%). Under our experimental conditions, accurate determination of the amplitude of the force response developed by a fiber was possible if the response was >= 12 µN (detection limit for 5 mV/scale).

When the DIFRmax values were compared between different fiber types, potential interexperimenter-related differences must be minimized so that any differences between fiber types can be attributed to a difference in E-C coupling structures/events. For example, some minor differences with respect to the rate of t-system depolarization may occur between experimenters if they introduce the mechanically skinned preparation into the depolarizing solution with different velocities, inasmuch as this will lead to interfiber differences with respect to the rate of diffusion of substances out of and into the fiber segment. In this study, the same experimenter performed the skinning, mounting, and depolarization protocol, using the same batch of solutions for all fibers. The sealing of the t system is assumed to be effective if a "cuff" (a skinning-generated structure indicating that the sarcolemma has been peeled back) appears during the skinning process (30).

Solutions

All chemicals were obtained from Sigma, unless otherwise stated. The K+-HDTA solution contained (in mM) 126 K+, 37 Na+, 50 HDTA2-, 8 ATP (total), 8.5 Mg2+ (total; Riedel-de Haen), 1 Mg2+ (free), 10 creatine phosphate, 0.05 EGTA, 1 NaN3, and 90 HEPES buffer at pH 7.10 ± 0.01 and pCa >7.0. The Na+-HDTA solution was identical to the K+-HDTA solution, except K+ was replaced by equimolar Na+. Between consecutive depolarizations, fibers were repolarized in the K+-HDTA solution. A K+-HDTA solution containing low free Mg2+ (0.015 mM) was used to release Ca2+ from the SR at the end of each single-fiber experiment. CaFmax was assessed using a solution that differed from the standard K+-HDTA solution, in that all HDTA was replaced with EGTA (50 mM) and the level of Ca2+ was higher (pCa 4.5). Each of the solutions was prepared as a batch and stored in individual aliquots at -85°C; the aliquots were brought to room temperature just before use.

MHC Analysis

After completion of the depolarization-induced activation protocol, single fiber segments (volume 1.0-8.8 nl) were placed in 12 µl of solubilizing buffer consisting of 62.5 mM Tris, 2.3% (wt/vol) SDS, 5% (vol/vol) beta -mercaptoethanol, 12.5% (vol/vol) glycerol, 13.6% (wt/vol) sucrose, 0.01% (wt/vol) bromphenol blue, 0.1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, and 1 µM pepstatin. Fiber segments were incubated at room temperature for 24 h and then boiled for 5 min. Myofibrillar proteins were separated by loading 6-µl sample aliquots, containing 0.05 nl fiber/µl, onto 0.75-mm-thick slab gels using the Hoefer Mighty Small gel apparatus. The alanine SDS-PAGE protocol used in this study is a slightly modified version of the protocol developed in our laboratory for separating MHC isoforms expressed in toad skeletal muscle (37, 38). The separating gel [total concentration of monomer (T) = 7.6%; concentration of cross-linker relative to T (C) = 1.2%] contained 425 mM Tris, pH 8.8, 30% (vol/vol) glycerol, 75 mM alanine, and 0.3% SDS (wt/vol) and was polymerized with 0.05% (wt/vol) ammonium persulfate and 0.075% (vol/vol) N,N,N',N',-tetramethylenediamine. The stacking gel (T = 4%; C = 2.6%) contained 125 mM Tris, pH 6.8, 40% glycerol (vol/vol), 4 mM EDTA, and 0.3% (wt/vol) SDS and was polymerized with 0.1% ammonium persulfate (wt/vol) and 0.05% (vol/vol) N,N,N',N',-tetramethylenediamine. The running buffer containing 175 mM alanine, 25 mM Tris, and 0.1% (wt/vol) SDS was freshly prepared and precooled at 4°C before use. Gels were run for 26 h at 4-6°C at constant voltage (150 V). The gels were stained with Bio-Rad Silver Stain Plus, and MHC bands were analyzed using a personal densitometer (Molecular Dynamics) and ImageQuaNT software (version 4.1, Molecular Dynamics). A fiber type is identified by a Roman numeral and a capital letter (e.g., IIA fiber); the MHC isoform expressed in the fiber is identified by a Roman numeral and the corresponding lowercase letter (e.g., IIa isoform).

Statistics

Values are means ± SE. Student's two-way t-test (GraphPad, Prism) was used for comparisons between two groups. Significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Profiles of t-System Depolarization-Induced Force Responses Developed by Electrophoretically Typed Single-Fiber Preparations From EDL, SM, and SOL Muscles

A total of 58 mechanically skinned single fibers from EDL (10), SM (18), and Sol (32) muscles were subjected to t-system depolarization triggered by the rapid substitution of K+ with Na+ in the depolarizing solution. Under our conditions, the fibers produced three types of t-system depolarization-induced force responses: 1) rapid transient responses (rtF), where force returned to a level equivalent to <10% of the peak within 2-3 s (Fig. 1), 2) prolonged responses (pF; Fig. 2A), where force remained >10% of the peak, even when the fiber was kept in the depolarizing solution for as long as 13 s (Fig. 2B), and 3) no detectable responses (nF; Fig. 3). On the basis of the profile of the maximum t-system depolarization-induced response developed by the fiber preparations, we identified three groups of fibers: 1) fibers producing rtF responses (rtF fibers), 2) fibers producing pF responses (pF fibers), and 3) fibers that did not develop detectable force responses, even after four to six successive depolarization-repolarization cycles (nF fibers).


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Fig. 1.   Rapid transient force (rtF) response developed by a type IIB extensor digitorum longus (EDL) muscle fiber. Time scale, 30 s, except during Na+ depolarizations and low-Mg2+ (LMg2+) responses (2 s). CaFmax, maximum Ca2+-activated force; DIFRmax, maximum t-system depolarization-induced force response; R-D75%, number of DIFRs to 75% rundown.



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Fig. 2.   A: prolonged force (pF) responses developed by type I soleus (Sol) fibers. B: pF response developed by a Sol fiber subjected to prolonged (~13 s) depolarization. C: force response developed by a Sol fiber in transition from pF to rtF during work-up period. Time scale, 30 s, except during Na+ depolarizations and low-Mg2+ responses (2 s).



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Fig. 3.   Trace from a Sol fiber that produced no force (nF) in response to t-system depolarization. Time scale, 30 s, except during Na+ depolarizations and low-Mg2+ responses (2 s).

The fiber type (based on MHC composition) populations dissected from EDL, SM, and Sol muscles and the profiles of the force responses produced by t-system depolarization in these fibers are summarized in Table 1. Also shown in Table 1 are the low Mg2+-induced force responses developed by rtF and pF fibers after rundown and by nF fibers after failure to respond to four to six successive depolarization-repolarization cycles. The pF profile was displayed by a total of 11 Sol fibers, a large enough number to validate treatment as a separate group. However, because only the data collected for three of these fibers were complete, we subjected the fibers to quantitative analyses and reported only these fibers. Three Sol fibers (2 type I and 1 type I/IIA) initially developed pF-type force responses (Fig. 2C), but, by the end of the work-up period, these responses had changed to rtF-type responses. In this study, only fibers displaying rtF and pF responses were subjected to analyses of quantitative E-C coupling characteristics. Representative electrophoretograms of the four fiber types examined in this study are shown in Fig. 4.

                              
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Table 1.   t-System depolarization-induced force responses and magnitude of low-Mg2+-induced force responses



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Fig. 4.   Representative electrophoretogram of EDL, sternomastoid (SM), and Sol muscle fibers. Myosin heavy chain (MHC) isoforms were identified by comparison with a marker prepared from muscle homogenates containing all 4 rat hindlimb MHC isoforms.

Relation Between Quantitative E-C Coupling Characteristics

The two quantitative E-C coupling characteristics examined in this study are DIFRmax and R-D75%. The data show a poor correlation between DIFRmax and R-D75% for pure type IIB fibers from EDL (r2 = 0.19, n = 8; Fig. 5A) and SM (r2 = 0.20, n = 13; Fig. 5B) and for pure type I fibers from Sol (r2 = 0.02, n = 8; Fig. 5C). This suggests that in these fibers the intracellular process(es) responsible for the use-dependent loss of contractile responsiveness to t-system depolarization is/are in large part independent of the factors determining the size of DIFRmax. We did not include fiber type groups IIB/D (SM; n = 3) and I/IIA (Sol; n = 4), which consisted of fewer than eight fibers.


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Fig. 5.   Relation between DIFRmax and R-D75% for type IIB EDL fibers (r2 = 0.19; A), type IIB SM fibers (r2 = 0.20; B), and type I Sol fibers (r2 = 0.02; C).

Quantitative E-C Coupling Characteristics in Electrophoretically Typed Single Fibers From EDL, SM, and Sol Muscles

In this study, DIFRmax and R-D75% were compared in 1) fibers of the same type from different muscles (type IIB EDL fibers vs. type IIB SM fibers), 2) different fiber types from different muscles (type IIB EDL fibers and type IIB SM fibers vs. type I Sol fibers), 3) different fiber types (hybrid vs. pure) from the same muscle (type IIB/D SM fibers vs. type IIB SM fibers; type I/IIA Sol fibers vs. type I Sol fibers), and 4) fibers of the same type from the same muscle (type I Sol fibers).

Type IIB EDL fibers vs. type IIB SM fibers. The mean DIFRmax for type IIB fibers from EDL (84.0 ± 6.0%) was not different from that for IIB SM fibers (78.8 ± 2.9%; Fig. 6A). By comparison, the mean R-D75% for type IIB SM fibers (20.0 ± 2.9) was significantly lower than that for type IIB EDL fibers (98.6 ± 20.1; Fig. 6B). The interfiber variability (coefficient of variation) with respect to the two E-C coupling characteristics considered was 20.2% (DIFRmax) and 57.6% (R-D75%) for type IIB fibers from EDL muscle and 26.7% (DIFRmax) and 53.5% (R-D75%) for type IIB fibers from SM muscle.


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Fig. 6.   A: amplitude of DIFRmax (expressed as percentage of maximum Ca2+-activated force) for type IIB EDL, type IIB SM, and type I Sol mechanically skinned muscle fibers. #Significantly different from EDL and SM (P < 0.001). B: R-D75% in type IIB EDL, type IIB SM, and type I Sol mechanically skinned muscle fibers. * Significantly different from SM (P < 0.01) and Sol (P < 0.001).

Type IIB EDL and type IIB SM fibers vs. type I Sol fibers. Figure 6 also shows DIFRmax (Fig. 6A) and R-D75% (Fig. 6B) values for type I Sol fibers. These fibers displayed significantly lower values than pure type IIB EDL fibers for DIFRmax (Fig. 6A) and R-D75% (Fig. 6B). Pure type I Sol fibers also produced significantly lower maximum depolarization-induced force responses (Fig. 6A) than pure type IIB SM fibers. However, although R-D75% developed by type I Sol fibers was lower (1.7 times) than that developed by type IIB SM fibers, this difference just failed to reach statistical significance (P = 0.0529; Fig. 6B). The coefficients of variation for the two E-C coupling characteristics in Sol fibers were 81.2% (DIFRmax) and 20.7% (R-D75%).

Type IIB/D vs. type IIB SM fibers and type I/IIA vs. type I Sol fibers. A comparison of the small group (n = 3) of hybrid type IIB/D SM fibers with the pure type IIB SM fibers (n = 13) showed no significant difference with respect to DIFRmax (78.8 ± 5.8 vs. 95.1 ± 3.9%) or R-D75% (20.0 ± 2.9 vs. 26.0 ± 8.1). Taken together with the data presented above (see Type IIB EDL and type IIB SM fibers vs. type I Sol fibers), this finding could indicate that, under the conditions used in this study, mechanically skinned rat muscle fibers containing MHC II isoforms are more responsive to t-system depolarization than fibers expressing the MHC I isoform. Contrary to this conclusion, the four type I/IIA hybrid Sol fibers examined in this study, all of which contained a larger proportion (mean 72.5%) of MHC IIa isoforms, were not significantly different from the pure type I Sol fibers (n = 8) with respect to DIFRmax (23.1 ± 12.8 vs. 29.8 ± 8.5%) or R-D75% (10.3 ± 0.6 vs. 12.1 ± 0.9).

Type I Sol fibers. As mentioned above, five pure type I Sol fibers produced rapid transient force responses, and three gave prolonged responses to t-system depolarization. A comparison of the E-C coupling characteristics in the two groups of type I Sol fibers showed, however, that rtF fibers did not differ significantly from pF fibers with respect to DIFRmax (34.0 ± 8.5 vs. 22.9 ± 19.9%) or R-D75% (13.2 ± 0.8 vs. 10.3 ± 1.7). To determine whether the prolonged elevated force responses developed by the pF fibers were artifacts due to contaminating Ca2+ in the depolarizing solution that would directly activate the contractile apparatus, pF fibers were incubated in a 2% Triton-K+-HDTA solution (to disrupt the t-tubular and SR membrane systems and abolish E-C coupling) for 10 min after maximum Ca2+-activated force was obtained, washed in a Triton-free K+-HDTA solution, and then exposed to the depolarizing solution. Under these conditions, no force was developed by any of the pF fibers, indicating that contaminating Ca2+ was not responsible for the prolonged elevated forces (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first systematic investigation, at the single-fiber level, of the relation between functional parameters of the t system and SR compartments and MHC-based fiber type in mammalian skeletal muscle. More specifically, this study was undertaken to address whether E-C coupling characteristics, measured in a mechanically skinned fiber preparation, are correlated to the MHC isoform(s) expressed in the same fiber segment. Our experimental strategy involved the use of single muscle fibers from one predominantly slow-twitch (Sol) and two predominantly fast-twitch, but functionally distinct, muscles (EDL and the white region of SM) of the adult rat.

E-C Coupling Parameters

In mechanically skinned muscle fiber preparations, DIFRmax is ultimately determined by the amount of Ca2+ released from the SR, the rate of Ca2+ release/uptake, and the sensitivity of the contractile apparatus to Ca2+. Several non-experimenter-related events involving the t system and SR can impact the amount of Ca2+ released and the rate of Ca2+ release/uptake by the SR compartment (Table 2). Generally, the size of a DIFR is influenced by the number of voltage sensors activated by the depolarizing stimulus, the number of SR Ca2+ release channels opened as the result of the cross talk between voltage sensors and the SR channels, and the SR luminal Ca2+ concentration. It is assumed that if Ca2+ loss from the intramyofibrillar space into the aqueous bathing solution is minimal (30), full coupling of the two compartments will result in release of enough Ca2+ from the SR to elicit a near-maximal contractile response compared with the CaFmax (30).

                              
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Table 2.   Essential positive contributors to upstroke and downstroke of t-system depolarization-induced force responses in mechanically skinned fiber preparations

The determinants of R-D75% are unknown. According to Lamb and Stephenson (30), rundown is not related to the length of exposure of a fiber to aqueous solutions, nor is it related to the depletion of SR Ca2+ content, inasmuch as exposure to low Mg2+ concentration (0.015 mM) produces relatively large force responses in rundown fibers, indicative of adequate SR free Ca2+ and functional RyR/Ca2+ release channels (32). Rundown in mechanically skinned muscle fibers is unlikely to be related to energy depletion, inasmuch as experiments are carried out in the presence of high ATP (8 mM) and creatine phosphate (10 mM) concentrations, which maintain the energetic potential of the fiber. One possibility is that the decrease in depolarization-induced force responses in mechanically skinned fiber preparations is related to a use-dependent loss of some factor such as glycogen located near the triad region (49). Ca2+-dependent or phosphorylation/dephosphorylation-dependent processes leading to a gradual inactivation of t-tubule voltage sensors or disruption of the coupling between the DHPR/voltage sensor and RyR/Ca2+ release channel may also contribute to the rundown (for review, see Ref. 48). Regardless of the cellular mechanism involved, the rundown phenomenon described by R-D75% indicates a use-dependent loss of functional E-C coupling.

DIFRmax and MHC-Based Fiber Type

The DIFRmax values found in the present study for mechanically skinned type IIB fibers from rat EDL muscle (84.0%) are similar to those reported for rat EDL fibers in previous investigations (70-90%) in which the same kind of preparation and experimental protocol were used (31). In the present study, we have also determined DIFRmax in fast-twitch fiber preparations from the white region of rat SM (35), a predominantly fast-twitch skeletal muscle not used in previous investigations of E-C coupling processes in mechanically skinned fiber segments. Under the same conditions of t-system depolarization, type IIB SM fibers also produced high force responses (78.8%) that were not statistically different from those produced by type IIB EDL fibers. Although we have not measured the membrane potential of any of the single fibers examined in this study, the relatively large depolarization-induced force responses (compared with CaFmax) elicited in type IIB EDL and type IIB SM fibers suggest that, in these preparations, rapid replacement of K+ with Na+ achieved near-maximal t-system depolarization (30).

Slow-twitch fibers are known to be more sensitive than fast-twitch fibers to membrane depolarization (19) and to have a higher SR luminal Ca2+ concentration (24). Furthermore, the contractile apparatus is more sensitive to activating levels of Ca2+ in slow- than in fast-twitch fibers (50). Despite these factors, which favor the production of force, mechanically skinned pure type I slow-twitch Sol fibers gave consistently smaller responses (29.8 ± 8.5% of CaFmax, n = 8) to t-tubule membrane depolarization than the type IIB fast-twitch fibers from EDL and SM. This result is in agreement with the preliminary data of Stephenson et al. (47) and with two studies in which mechanically skinned rat Sol fibers were used to investigate the effects of clenbuterol (2) and hydrogen peroxide (41) on E-C coupling parameters. Our value for the DIFRmax in Sol fibers (29.8%) is lower than that reported by Plant et al. (41) (47 ± 5%, n = 5) but higher than that reported by Stephenson et al. (47) (10 ± 4.5%, n = 8), although Bakker et al. (2) did not report the relative size of the Sol force responses.

The smaller responses of the type I slow-twitch fibers to t-system depolarization could be partly explained by the lower proportion of DHPRs functionally coupled to SR Ca2+ release channels [resulting in less Ca2+ released on depolarization (17)] and by the slower rate of Ca2+ release (22) previously reported for Sol fibers. Other potential contributors include incomplete sealing of the t tubules after they are skinned and ineffective depolarization/repolarization of the t system. From the data obtained from an extensive series of experiments performed on skinned fiber preparations from toad and rat, in which fluorescent dyes were trapped within the t system, Launikonis and Stephenson (34, and personal communication) concluded that, in all fibers (fast and slow twitch) mechanically skinned by the procedure used in our study, the t system is invariably and uniformly sealed. This finding supports the possibility that, under our conditions, t-system depolarization and/or repolarization is less effective in slow-twitch type I fibers than in fast-twitch type IIB fibers. This may be due to subtle fiber type-specific differences in structure/function of the molecular components of the t system, such as Na+-K+-ATPase. No studies have quantified the number, density, or activity of Na+-K+ pumps located in the t-tubule compartment of electrophoretically typed single fibers from slow- or fast-twitch muscle.

R-D75% and MHC-Based Fiber Type

This study is the first to compare the number of DIFRs to rundown in fibers expressing the same MHC isoform from two different muscles. Our data show that t-system depolarization-induced force responses developed by type IIB fibers from the SM muscle declined 4.9 times faster than those developed by type IIB EDL fibers. This result is surprising, given the similarity displayed by type IIB EDL and type IIB SM fibers with respect to DIFRmax.

We have shown also that the t-system depolarization-induced force responses developed by fibers expressing the MHC I isoform from the Sol muscle decline faster than those developed by fibers expressing MHC IIb isoform from EDL (8.1 times) and SM (1.7 times) muscles. This result is in agreement with the data of Plant et al. (41), who reported that Sol fibers, classified as slow-twitch fibers on the basis of the sensitivity of the contractile apparatus to Sr2+, produced 2.9 times fewer responses to rundown than fast-twitch EDL fibers.

Within the framework of the previously discussed mechanisms involved in the rundown phenomenon (30), the differences in R-D75% between type IIB fibers from EDL and SM muscles and between the type I (Sol) and type IIB (EDL and SM) fibers indicate that the processes associated with the use-dependent loss of E-C coupling in mechanically skinned fiber preparations may be specific for muscle of origin and muscle fiber type. It is unlikely that rundown in either of these fiber groups was related to SR dysfunction or depleted SR Ca2+, as indicated by the relatively large (>70% of CaFmax) responses to low Mg2+ produced by these fibers (Table 1).

EDL muscle fibers and the t-system depolarization/repolarization protocol described here have been used in several studies for determining the effects of particular compounds on E-C coupling (2, 4, 20). After the work-up period, test responses produced by the fiber preparations, when exposed to depolarization/repolarization solutions containing the compound of interest, are preceded and followed ("bracketed") by control responses (i.e., responses produced in the absence of the compound of interest). The sensitivity of this method is determined in large part by the number and amplitude of successive responses of similar height developed by a fiber between the end of the work-up process and the onset of the rundown process. Given the relatively small number of total responses (12.1) to rundown found by us in mechanically skinned type I Sol fibers, it is reasonable to suggest that, in its present form, the protocol developed by Lamb and Stephenson (30) may not be suitable for accurately determining the magnitude and reversibility of effects of particular compounds on E-C coupling characteristics in slow-twitch fiber preparations.

Profile of t-System Depolarization-Induced Force Responses Produced by Mechanically Skinned Fibers From EDL, SM, and Sol Muscles

All previous studies using the t-system depolarization method of Lamb and Stephenson (30) and mechanically skinned single-fiber segments have reported only rtF-type depolarization-induced force responses for rat EDL (30, 41) and Sol fibers (2, 41). Under the same conditions, however, the fibers examined in the present study produced two different types of responses, rtF and pF, or did not respond, even after several depolarization-repolarization cycles. The lowest variability with respect to the profile of force responses induced by t-system depolarization was found among type IIB EDL fibers, all of which developed only rtF responses. By comparison, although the majority of type IIB SM fibers also produced rtF responses, a small proportion failed to respond. The highest variability with regard to this parameter was found in the population of type I Sol fibers, which contained a small proportion of fibers producing typical rtF responses, a small proportion of fibers producing pF responses, and a large proportion of fibers that did not develop any force (nF). Taken together, these data suggest that, under our conditions, the profile of t-system depolarization-induced force responses produced by mechanically skinned muscle fiber preparations is related to fiber type, muscle of origin, and even individual fiber characteristics.

Our finding that some Sol muscle fibers developed rapid transient forces (indicating complete inactivation) while others developed prolonged forces (indicating incomplete inactivation) in response to t-system depolarization provides the strongest evidence for the "voltage window" model put forward in an earlier study by Chua and Dulhunty (12). According to this model, the activation and inactivation curves for a given muscle preparation overlap to some extent, thereby creating a "window"-like area, the size of which is related to the dependence of the activation and inactivation processes on the membrane potential at the peak of depolarization. Because the sensitivity of the voltage sensor to membrane potential is higher (activation) and lower (inactivation) in the Sol than in the EDL muscle, the size of the voltage window is expected to be larger for Sol than for EDL muscle preparations. Chua and Dulhunty used the voltage window model to explain the incomplete inactivation of K+ contractures ("pedestal tensions") observed in bundles of intact Sol fibers and argued against the possibility that the profile of these contractures could have been due to a subpopulation of Sol fibers that did not inactivate. The single-fiber study presented here revealed the presence in the rat Sol muscle of rtF and pF fibers; their responsiveness to t-system depolarization can be easily explained if one considers that, at the peak of depolarization, the membrane potential reached a value that was more positive (rtF) than the limit of the voltage window or was within it (pF). Given the steepness of the activation and inactivation curves constructed for Sol muscles (see Fig. 6, A and B, in Ref. 12), we suggest that, despite the marked difference in the profile of the rtF and pF force responses, the actual difference between the membrane potentials of rtF and pF fibers could be very small.

The forces produced by a small number (2 type I and 1 type I/IIA) of rtF Sol fibers during the work-up period displayed a pF-type profile, with the pedestal component becoming smaller with successive depolarization-repolarization cycles, such that the DIFRmax was of the rtF type. The work-up phenomenon has been related to the presence in the sealed t system of Cl- ions, which exert a polarizing effect on the membrane potential, thereby reducing the effectiveness of the depolarization step (14). With successive depolarizations, Cl- gradually diffuses out of the t system, the membrane potential reaches more positive values, more DHPR/voltage sensors become activated, and the fiber produces force responses of greater magnitude. Within this context, the transition of the aforementioned force responses from pF to rtF suggests that the membrane potential reached in these fibers in the depolarizing solution at the beginning of an experiment may be within the voltage window and that successive depolarization-repolarization cycles (and subsequent loss of t-system Cl-) cause the membrane potential reached during depolarization to shift out of the voltage window closer to zero.

An interesting finding of this study is that 11.1% of type IIB SM fibers and >50% of type I Sol fibers did not respond to t-system depolarization. The large proportion of nonresponding fibers to t-system depolarization in Sol muscle of the rat makes studies on E-C coupling in type I fibers using the skinned fiber preparation and the depolarization protocol of Lamb and Stephenson (30) a difficult task. It is unlikely that the nF fibers could have been damaged, because all single fibers used in this study were dissected, skinned, and mounted by the same investigator. Furthermore, all the nF fibers produced large force responses when exposed to a low-Mg2+ solution (Table 1), indicating that the SR compartment was not depleted of Ca2+ and that the RyR/Ca2+ release channels were functional. On the basis of these data, we suggest that in nF fibers the DHPR/voltage sensors may have been irreversibly inactivated and/or the coupling mechanism between the DHPR/voltage sensors and the RyR/Ca2+ release channels may have been disrupted by some unknown causes.

Are Functional Characteristics of the t-System and SR Compartments Related to MHC-Based Fiber Type?

MHCs are the most widely used molecular markers of fiber-type differentiation, specialization, and adaptation; yet the extent to which the functional properties of the t system and SR in different fiber types are related to MHC-based fiber type is far from being understood. One of our findings is that, regardless of the muscle of origin (EDL or SM), pure fast-twitch fiber preparations expressing the MHC IIb isoform produce larger t-system depolarization-induced responses than pure slow-twitch fiber preparations expressing the MHC I isoform. On the basis of this finding, which suggests a close relation between MHC isoform expression and the amplitude of the responses, one would predict that DIFRs produced by pure muscle fibers expressing MHC IIa (type IIA fibers) or MHC IId/x isoforms (type IID/X fibers) and by hybrid I/II fibers expressing a larger proportion of MHC II isoform should be larger than the DIFRs produced by fibers expressing only MHC I or a combination of MHC I and MHC II, with MHC I being the major component. Surprisingly, the force responses produced by four type I/IIA Sol fibers, expressing predominantly the MHC IIa isoform (mean 73%), were not different from those produced by pure type I Sol fibers. This result may indicate that, in the rat, type IIA fibers resemble type I, rather than type IIB, fibers with respect to the intracellular factors/events determining the amplitude of DIFR. Unfortunately, we could not test this possibility, because the pool of fibers used in this study contained only one pure type IIA fiber, which failed to respond to t-system-induced depolarization. Moreover, there is no published information on the E-C coupling phenotype of mechanically skinned fiber preparations expressing the MHC IIa isoform. The similarity between the DIFRmax values for type I/IIA and type I Sol fibers may indicate, however, that the relation between MHC isoform expression and the factors determining this parameter (Table 2) is stronger for MHC I than for MHC IIa. This possibility seems unlikely given that, for the population of type I Sol fibers, the interfiber variability (as indicated by coefficient of variation) with respect to DIFRmax was four times higher than that for type IIB EDL and type IIB SM fibers. A third possible explanation for the lower DIFRs developed by the type I/IIA Sol fibers expressing predominantly the MHC IIa isoform is that the factors determining DIFRmax relate more to the muscle of origin (Sol) and its specific physiological role than to the MHC isoform (IIa) expressed in the fiber. In this case, one would expect that, if present in Sol muscle, even pure type IIB fibers would produce lower force responses to t-system depolarization, comparable in height to those produced by the pure type I and type I/IIA Sol fibers. Again, the absence of type IIB fibers from the pool of rat Sol muscle fibers examined in this study made it impossible to validate/reject this point.

The possibility that responsiveness to t-system depolarization in a mechanically skinned fiber preparation relates to MHC-based fiber type as well as to the muscle from which the fiber was obtained is supported also by the R-D75% data. Type I Sol fibers produced fewer force responses to rundown than type IIB fibers from EDL and SM (in agreement with a close relation between E-C coupling characteristics and fiber type), and type IIB SM fibers produced fewer force responses to rundown than type IIB EDL fibers (in agreement with a close relation between E-C coupling characteristics and the muscle of origin). Recently, we reported another example of functional/structural differences between fibers expressing the same MHC isoform but originating from two different rat muscles. In this case, in which significantly higher sensitivity of the contractile apparatus to Ca2+ was displayed by type IIA Sol than by type IIA EDL fibers (6), the intermuscle difference involved components of the myofibrillar compartment (such as troponin C). Taken together with the results presented in this study, these data suggest that the optimum contractile function of a skeletal muscle fiber type is related to its MHC isoform composition and the innervation pattern and functional role of the muscle of origin (8).

Concluding Remarks

Our study provides the first evidence that E-C coupling characteristics of mechanically skinned, single fibers from adult rat skeletal muscles are related to MHC-based fiber type and the muscle from which the fiber originated. This finding supports the emerging idea that the functional phenotype of a muscle fiber may be related to the muscle of origin. This study also provides the first evidence that the voltage window created by the overlap between activation and inactivation curves, reported by Chua and Dulhunty (12) using bundles of Sol fibers, is also detectable in single type I Sol fiber preparations. This finding strongly supports the model of the overlap between voltage sensor activation and inactivation curves in rat slow-twitch Sol fibers. Our study also provides strong evidence that, in its present form, the protocol developed by Lamb and Stephenson (30) may not be suitable for accurately determining the magnitude and reversibility of effects of particular compounds on E-C coupling characteristics in slow-twitch fiber preparations.


    ACKNOWLEDGEMENTS

This work was supported by the National Health and Medical Research Council of Australia.


    FOOTNOTES

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

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 29, 2003;10.1152/ajpcell.00569.2002

Received 6 December 2002; accepted in final form 25 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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