MHC polymorphism in rodent plantaris muscle: effects of mechanical overload and hypothyroidism

Vincent J. Caiozzo, Fadia Haddad, Mike Baker, Sam McCue, and Kenneth M. Baldwin

Departments of Physiology and Biophysics and Orthopaedics, College of Medicine, University of California, Irvine, California 92717


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study, it was shown that a combined treatment of hyperthyroidism and hindlimb suspension effectively converted the slow-twitch soleus muscle to a fast-twitch muscle. The objective of this study was to test the hypothesis that hypothyroidism [absence of triiodothyronine (-T3)] and mechanical overload (OV) would convert the plantaris (Plan) muscle from a fast- to a slow-twitch muscle. Single-fiber analyses demonstrated that the normal rodent Plan muscle was composed of ~13 different fiber types as defined by myosin heavy chain (MHC) isoform content. The largest proportion of fibers (~35%) coexpressed the fast type IIX and IIB MHC isoforms (i.e., type IIX/IIB fibers). In this context, the combined intervention of -T3 and OV produced a significant reduction in the relative proportion of the fast type IIB MHC isoform and a concomitant increase in the slow type I MHC isoform. These transitions were manifested by a large decrease in the proportion of type IIX/IIB fibers and a large increase in fibers coexpressing all four MHC protein isoforms. The mechanical consequences of these transitions, however, were modest, producing a 15% decrease in maximal shortening velocity. The findings of this study demonstrate that -T3 + OV does produce a partial shift toward a slower phenotype; however, the high degree of polymorphism found in the Plan muscle represents a unique design that appears to minimize the functional consequences of these significant MHC transitions.

single fiber; messenger RNA; rat; hybrid fibers; protein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MYOSIN HEAVY CHAIN (MHC) isoform composition in rodent skeletal muscle is controlled by a number of factors, including thyroid hormone (triiodothyronine, T3) and loading state (2, 3, 5, 7-9, 15, 18-21, 23, 25, 26). In general, hyperthyroidism (+T3) and hindlimb suspension (i.e., mechanical unloading; HS) upregulate the fast MHC protein isoform content while concomitantly downregulating the content of the slow MHC protein isoform (2). In contrast, hypothyroidism (-T3) and mechanical overload (OV) produce opposite effects (9, 25, 26).

In an attempt to more rigorously examine the commonality or lack thereof between the effects of +T3 and HS on MHC isoform composition, we recently performed extensive quantitative analyses comparing the effects of these two interventions on the MHC isoform composition of single fibers in the soleus (Sol) muscle (2, 3, 15). These analyses yielded two key findings that clearly differentiated the effects of +T3 from HS in the Sol muscle. First, +T3 affected a larger proportion of slow-twitch type I fibers than did HS. This finding suggests that there are subpopulations of slow-twitch type I fibers in the Sol muscle that differ in their sensitivity to thyroid vs. loading state. Second, although +T3 and HS produced significant pools of fibers that coexpressed various combinations of slow and fast MHC isoforms (i.e., so-called polymorphic or hybrid fibers), +T3 produced a profile of polymorphic fibers that differed substantially from that induced by HS (2, 3).

The comparison between the separate effects of +T3 and HS was complemented by an additive paradigm whereby both interventions were imposed simultaneously (2, 3, 15). +T3 + HS effectively converted the Sol muscle to a fast-twitch muscle, as evidenced by MHC protein isoform and contractile data. At the single-fiber level, this transformation was characterized by 1) the de novo expression of the fast type IIB MHC isoform and 2) a large pool of fibers (~60% of the total population) that coexpressed all four adult MHC isoforms (2, 3).

One possible interpretation of this additive/synergistic effect produced by +T3 + HS is that the effect of thyroid state on MHC isoform expression might be dependent on loading state. To further explore this possibility, the present study employed an approach analogous to that described above but contrasted, instead, the effects of -T3, mechanical overload (OV), and -T3 + OV on the MHC isoform expression in the fast-twitch plantaris (Plan) muscle. In a previous study (5), we noted that -T3 had little effect on the native myosin isoform composition of the fast-twitch Plan muscle. If loading state of the muscle modulates the effect of thyroid state, then we hypothesize that -T3 + OV should produce alterations in MHC isoform expression greater than those produced by -T3 alone.

The findings of this study demonstrate that the normal rodent Plan muscle contains a complex spectrum of muscle fiber types (i.e., ~13 different pools of fibers as defined by MHC isoforms) previously unrecognized. The largest proportion of fibers coexpressed the fast type IIX and IIB MHC isoforms (~35% of the total population). Although -T3 had little effect on the distribution of MHC isoforms, OV produced a significant reduction in the proportion of type IIX/IIB fibers and a concomitant increase in the proportions of type IIA/IIX and I/IIA/IIX fibers. -T3 + OV produced a further reduction in the proportion of type IIX/IIB fibers accompanied by a large proportion of fibers that coexpressed all four MHC isoforms (~30% of the total population of fibers). This latter finding is consistent with the hypothesis that the effect of thyroid state on MHC isoform expression is dependent on loading state. Of the three different interventions employed in this study, only the -T3 + OV group produced a significant reduction (-15%) in maximal shortening velocity (Vmax,muscle). In this context, however, the high degree of polymorphism found in the Plan muscle may represent a unique design feature that minimizes the functional consequences of large changes in MHC isoform composition.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care and experimental groups. All experiments described in this study were approved by our Institutional Animal Care and Use Committee before they were performed. Female Sprague-Dawley rats (~250-300 g) were randomly assigned to four groups designated 1) control (Con, n = 8), 2) hypothyroid (-T3, n = 8), 3) OV (n = 7), and 4) -T3 + OV (n = 7). All animals were housed individually and given food and water ad libitum. Animals were killed after 6 wk of a given treatment.

Hypothyroidism was induced by daily intraperitoneal injections of propylthiouracil (PTU; 12 mg/kg body wt). We have demonstrated, in previous studies, that heart weight and heart weight normalized to body mass decrease under hypothyroid conditions (5, 6, 9). Hence, the effectiveness of PTU in creating a hypothyroid condition in the present study was verified using heart weight and heart weight-to-body weight ratios.

OV of the left and right Plan muscles was accomplished by anesthetizing the animal with acepromazine (4.5 mg/kg) and ketamine (75 mg/kg) and then removing the Sol and gastrocnemius muscles. Previous studies by a number of investigators (11, 13, 17, 23, 25) have shown that this technique produces substantial hypertrophy of the Plan muscle.

After 6 wk of a given intervention, animals were anesthetized as described above, and then contractile measurements were made on the left Plan muscles. After these measurements, the left and right Plan muscles were harvested from each animal and weighed. The midportion of each left Plan muscle (~10-15 mm) was cut transversely and divided into two segments. One of these segments was used for determining the MHC isoform composition of single fibers. The second segment was placed into cooled glycerol and stored (-20°C) until it was used for determination of whole muscle MHC isoform composition. The right Plan muscle of each animal was quickly frozen by tongs cooled in liquid nitrogen. These samples were stored at -70°C until they were used for determination of the whole muscle MHC mRNA isoform distribution via the Northern blot technique.

Contractile measurements. Contractile measurements were made in accordance with methods described by us previously (2, 3, 5). The distal tendon of a Plan muscle was isolated and freed from all connective tissue along the medial and lateral borders. The nerve and vasculature were left intact. Contractions of the Plan muscle were produced by stimulating the sciatic nerve with a bipolar stainless steel electrode. The stimulation parameters were controlled using a stimulator with an isolation unit (model S-48; Grass Instruments, Quincy, MA). All muscles were stimulated using a pulse duration of 0.2 ms and a voltage that was two times greater than that needed to produce a maximal twitch.

All contractile measurements were made using a computer-controlled ergometer system (model 310; Cambridge Instruments, Watertown, MA). A computer and digital-to-analog board (model DAS-16G; MetraByte-Keithley, Taunton, MA) were used to control the afterload imposed on the muscle. Additionally, the computer and an analog-to-digital board (model DAC-06, MetraByte-Keithley) were used to collect force and muscle length data. Force production and muscle length data were each collected at a rate of 1,000 Hz.

The optimal muscle length was defined as the muscle length that yielded the greatest tetanic isometric tension. All subsequent measurements were made at this muscle length. After the determination of optimal muscle length, the isometric twitch properties of the muscle were determined. These properties included measurements of isometric twitch tension, time to peak tension, and one-half relaxation time.

The force-velocity relationship of a given muscle was determined using ~12-15 different afterload conditions that ranged from ~2 to 100% of maximal isometric tension (Po). The force-velocity data of a muscle were fitted mathematically using a linearized version of the Hill equation (2, 3, 5). This equation was also used to estimate maximal shortening velocity of the muscle (i.e., Vmax,muscle). The muscle was allowed 1 min of rest between each afterload condition. Immediately after the completion of contractile measurements, the Plan muscles were removed and processed as described above.

Hill's statistical model of the force-velocity relationship and the importance of MHC isoform distribution at the single-fiber level. Hill (16) developed a statistical model that described the dependence of the whole muscle force-velocity relationship on the force-velocity relationships of the individual fibers of that muscle. In this regard, we attempted to predict the effects of -T3 + OV on Vmax,muscle by using Hill's statistical model of the force-velocity relationship in conjunction with 1) force-velocity data of single fibers with known MHC isoform compositions (1) and 2) the distribution of MHC isoforms at the single-fiber level, as determined in the present study.

The Hill equation can be written as
(P + <IT>a</IT>)(<IT>V</IT> + <IT>b</IT>) = <IT>b</IT>(P<SUB>o</SUB> + <IT>a</IT>) (1)
where Po is maximal isometric tension, P is isotonic tension, V is shortening velocity, and a and b are constants with dimensions of force and velocity, respectively. The Hill equation (27) can be normalized relative to Po and Vmax as follows
(P/P<SUB>o</SUB> + <IT>a</IT>/P<SUB>o</SUB>)(<IT>V</IT>/<IT>V</IT><SUB>max</SUB> + <IT>b</IT>/<IT>V</IT><SUB>max</SUB>) = <IT>b</IT>/<IT>V</IT><SUB>max</SUB> (1 + <IT>a</IT>/P<SUB>o</SUB>)  (<IT>2</IT>)
By use of substitutions of P/Po = P', V/Vmax = V', and a/Po = b/Vmax = 1/G, Eq. 2 can be rewritten as follows
P′ = (1 − <IT>V</IT>′)/(1 + <IT>V</IT>′G) (3)
Whereas Hill used a statistical distribution of 82 fibers with 10 distinct populations differing in maximal shortening velocity (Vmax,fiber), the statistical distributions of fibers used in the present study were based on the actual MHC isoform distributions observed at the single-fiber level in the populations of Con and -T3 + OV fibers. The V' for any given pool of fibers can be determined by dividing the shortening velocity of the whole muscle (Vmuscle) by the maximal shortening velocity for that given group or pool of fibers (i.e., Vmax,fiber). The contribution of each pool of fibers to overall force production can be determined by multiplying the value obtained from Eq. 3 by the number of fibers in each pool. For the purposes of this model, the Vmax,fiber values of pure slow-twitch type I and fast-twitch type IIA, IIX, and IIB fibers were assumed to be 0.64, 1.40, 1.45, and 1.8 fiber lengths per second (1). The Vmax,fiber for any given fiber type was determined by
<IT>V</IT><SUB>max,fiber</SUB> = (f<SUB>I</SUB> * <IT>V</IT><SUB>max,I</SUB>) + (f<SUB>IIA</SUB> * <IT>V</IT><SUB>max,IIA</SUB>)

+ (f<SUB>IIX</SUB> * <IT>V</IT><SUB>max,IIX</SUB>) + (f<SUB>IIB</SUB> * <IT>V</IT><SUB>max,IIB</SUB>) (<IT>4</IT>)
where fx is the fraction of that given MHC isoform in the fiber and Vmax,x is the maximal shortening velocity associated with that given MHC isoform.

Isolation of single fibers. After removal, the muscle segment used for single-fiber MHC isoform analyses was placed immediately into a glycerol relaxing solution (50% glycerol, 2 mM EGTA, 1 mM MgCl2, 4 mM ATP, 10 mM imidazole, 100 mM KCl, pH 7.0). It was then quickly cut into small strips and stored in glycerol relaxing solution overnight at -20°C. To obtain single fibers, the muscle strips were placed into a small dissection chamber that contained the glycerol relaxing solution. Approximately 45 single fibers from each muscle (total 1,379, ~345 fibers/group) were then isolated using microsurgical forceps (Super Fine Dumont tweezers, Biomedical Research Instruments, Rockville, MD) and a dissection microscope (Technival 2, Jena, Germany) with backlighting. Each fiber was transferred to individual polypropylene microcentrifuge tubes (500 µl) that contained 30 µl of a running buffer [62.5 mM Tris (pH 6.8), 1.0% (wt/vol) SDS, 0.01% (wt/vol) bromphenol blue, 15.0% (vol/vol) glycerol, and 5.0% (vol/vol) beta -mercaptoethanol]. Each sample was heated (70°C for 2 min) and placed in a sonicator for 60 min. This method is a modification of that initially described by Giulian et al. (12) and published by us previously (2, 3). Approximately 15 µl of each sample were then loaded into the well of a gel, and electrophoresis was performed as described below.

Discontinuous PAGE separation of MHC isoforms. MHC protein isoforms were separated using techniques described previously (2, 3). The separating gel consisted of 8% acrylamide, 0.16% bis-acrylamide, 30% glycerol, 0.4% SDS, 0.2 M Tris (pH 8.8), and 0.1 M glycine. This solution was degassed for ~15 min. Polymerization was then initiated by addition of N,N,N',N'-tetramethylenediamine (0.05% final concentration) and ammonium persulfate (0.1% final concentration) to the separating gel solution. The separating gel was poured, layered with ethyl alcohol, and given ~30 min to polymerize. The stacking gel solution contained 4% acrylamide, 0.08% bis-acrylamide, 30% glycerol, 70 mM Tris (pH 6.7), 4 mM EDTA, and 0.4 % SDS. This solution was also degassed for 15 min before addition of N,N,N',N'-tetramethylenediamine (0.05% final concentration) and ammonium persulfate (0.1% final concentration). It was then layered onto the separating gel. The running buffer contained 0.1 M Tris, 0.15 M glycine, and 0.1% SDS. Myofibril samples obtained from the whole muscle segments were denatured in a sample buffer solution that contained 5% beta -mercaptoethanol, 100 mM Tris-base, 5% glycerol, 4% SDS, and bromphenol blue. Approximately 1 µg of protein was then loaded into each well. An SG-200 vertical slab gel system (CBS Scientific, Del Mar, CA) was used for electrophoresis. Gels were run for ~24 h and at 270 V. This method separated the fast type IIA, IIX, and IIB and the slow type I MHC isoforms (order of migration). MHC protein isoform bands obtained from the whole muscle segments were visualized using Coomassie blue G-250. Those obtained from single fibers were stained using a silver stain kit (Bio-Rad, Richmond, CA). A personal densitometer (Molecular Dynamics, Sunnyvale, CA) was used to scan and quantify the MHC isoform bands.

Northern blot analyses of MHC mRNA isoform content. The Northern blot analyses used in this study were similar to those described by us previously (4, 14, 15). Total cellular RNA was isolated using the RNAzol method (TEL-TEST, Friendswood, TX). Total RNA (5 µg) was electrophoretically fractionated, transferred to a nylon membrane, and ultraviolet fixed to immobilize the RNA on the membrane. The membrane was dried at 80°C for 30 min and then stored dry at 4°C until subsequent hybridization.

Oligonucleotides were purchased from Chemgene (Waltham, MA). The 5'-ends of the oligonucleotide probes were labeled with [gamma -32P]ATP (1-2 × 109 cpm/µg). Northern blots were prehybridized (~2 h, 5°C less than the melting temperature of the probe) in 10× Denhardt's solution, 6× saline-sodium-phosphate-EDTA, 1% SDS, sonicated salmon sperm DNA (50 µg/ml), and yeast tRNA (50 µg/ml). Hybridization was performed overnight at the prehybridization temperature in 6× saline-sodium phosphate-EDTA and 1% SDS at a probe concentration of 1-2 × 106 cpm/ml. The blots were then washed and exposed to an X-omat autoradiographic film (Kodak) with intensifying screen (DuPont) at -70°C for 1-3 days, depending on the signal intensity. After signal detection by autoradiography, the probes were washed off the blots by boiling for 10-15 min in 1% SDS. The Northern blot membranes were rehybridized with an excess of a 32P end-labeled 18S oligoprobe that hybridizes to 18S rRNA. Band intensities on the autoradiogram were quantitated using a laser scanning densitometer (Molecular Dynamics), and each specific MHC absorbance was normalized to its corresponding 18S signal on the Northern blots.

Statistical analyses. All statistical analyses were performed using a computer program (Systat, Evanston, IL). The protein MHC, mRNA MHC, and contractile data were analyzed using a two-way ANOVA. The data for each separate MHC isoform were analyzed independent of the other isoforms. If a significant F ratio was obtained, Tukey's honestly significant difference test was employed to determine significant differences between groups. Differences between distributions of fiber types (e.g., HA distributions are dependent on T3 and HS status) were determined using chi 2 analysis. Only those chi 2 tests that were statistically significant are reported in RESULTS. Statistical tests were considered significant when P <=  0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Physical characteristics and muscle mass. The heart weights (absolute and relative to body mass) of the animals assigned to the -T3 and -T3 + OV groups were significantly less than those of the animals assigned to the Con and OV groups (Table 1). This finding suggests that the administration of PTU was effective in creating a -T3 condition.

                              
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Table 1.   Physical characteristics of animals and muscles

Muscle weights are reported in Table 1. The weights of the Plan muscles in the OV (+63%) and -T3 + OV (+39%) groups were significantly greater than those of the Con group. This finding demonstrates that synergistic ablation was very effective in producing compensatory hypertrophy in the OV and -T3 + OV groups.

Whole muscle MHC protein and mRNA isoforms. The whole muscle MHC protein isoform data are shown in Fig. 1. The predominant MHC protein isoforms found in the Con Plan muscles were the fast type IIX (~37% total MHC pool) and IIB MHC isoforms (~45% total MHC pool). Hypothyroidism had little effect on the distribution of MHC protein isoforms. In contrast, OV and -T3 + OV produced substantial changes. OV significantly reduced the fast type IIB MHC isoform and concomitantly upregulated the expression of the slow type I and fast type IIA MHC isoforms. -T3 + OV produced an MHC isoform profile that was slower than that produced by OV alone. This slower profile was characterized by a larger pool (~20% total MHC pool) of the slow type I MHC protein isoform.


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Fig. 1.   Effect of absence of triiodothyronine (-T3), mechanical overload (OV), and -T3 + OV on whole muscle myosin heavy chain (MHC) protein isoform composition of plantaris (Plan) muscles. A: type I; B: type IIA; C: type IIX; D: type IIB. Values are means ± SE. OV and -T3 + OV produced substantial decreases in the fast type IIB MHC protein isoform content. OV and -T3 + OV produced significant increases in slow type I and fast type IIA MHC protein isoforms. -T3 + OV produced a larger increase in slow type I MHC isoform than did OV. a P < 0.05 compared with control (Con). b P < 0.05 compared with -T3. C P < 0.05 compared with OV.

The whole muscle MHC mRNA data are shown in Fig. 2. The trends observed at the mRNA level for the slow type I, fast type IIA, and fast type IIB MHC isoforms were fairly consistent with that seen at the protein level. For instance, OV and -T3 + OV produced significant reductions in the fast type IIB MHC mRNA isoform signal, as was observed at the protein level. However, there was a discrepancy between the mRNA and protein data for the fast type IIX MHC isoform.


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Fig. 2.   Effects of -T3, OV, and -T3 + OV on whole muscle MHC mRNA isoform signals. Values are means ± SE. OV and -T3 + OV produced substantial reductions in fast type IIB MHC mRNA signals. -T3 + OV also produced a substantial increase in slow type I MHC mRNA signal. a P < 0.05 compared with Con. b P < 0.05 compared with -T3. C P < 0.05 compared with OV. A: type I; B: type IIA; C: type IIX; D: type IIB.

Single-fiber MHC protein isoform composition. As indicated previously (2, 3), single-fiber analyses of MHC isoform composition can be considered valid only if two criteria are met: 1) random sampling and 2) adequate number of observations. With respect to this latter issue, ~1,300 single fibers were analyzed in this study. The regression analyses between single-fiber and whole muscle analyses yielded a coefficient of determination of 0.93 (Fig. 3). Importantly, the slope approximated 1.0 and the y-intercept was close to 0. Another approach that could be used to validate the single-fiber methods used in this study would be to simply compare distributions obtained from several different studies. Within this context, we recently examined the developmental time course of MHC isoform expression in the Plan muscle (10). The patterns of single-fiber polymorphism observed at 40 days postpartum are remarkably similar to those shown for the Con Plan muscles in Fig. 4. Collectively, these findings provide a high degree of confidence in the single-fiber MHC isoform distributions shown in Fig. 4.


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Fig. 3.   Regression analyses between percentage of whole muscle myosin MHC isoform composition predicted by single-fiber (total n = 1,379 fibers) analyses and actual whole muscle analyses. Mean percentage of a given MHC isoform determined from single-fiber analyses within a given group (e.g., Con group) was calculated as follows: fraction of a given MHC isoform (fMHC,fiber type) within a given fiber type was multiplied by fraction of that pool of fibers (ffibers). Resulting value was then multiplied by cross-sectional area for a given fiber type (xsectfiber type), and product for a given MHC isoform was summed across all fiber types (e.g., sumMHC I). This value was then divided by total sum (i.e., sumMHC I + sumMHC IIA + sumMHC IIX + sumMHC IIB) and multiplied by 100. This yielded an estimate of relative proportion of a given MHC isoform. , Relative amount of slow type I MHC isoform from 4 different groups; , relative amount of fast type IIA MHC isoform from 4 different groups; black-triangle, relative amount of fast type IIX MHC isoform from 4 different groups; black-down-triangle , relative amount of fast type IIB MHC isoform from 4 different groups.



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Fig. 4.   Single-fiber MHC protein isoform composition of Plan muscles. All possible combinations of MHC isoform expression are shown along x-axis. Each bar represents proportion of a given population relative to total population of fibers examined for that group. Solid, dark gray, light gray, and open bars represent relative amounts of slow type I, fast type IIA, fast type IIX, and fast type IIB MHC protein isoforms, respectively, found within any given fiber type. Con Plan muscles contained a large number of polymorphic fibers with predominant population coexpressing fast type IIX and IIB MHC protein isoforms. -T3 + OV produced a significant reduction in proportion of type IIX/IIB fibers and a large increase in fibers coexpressing all 4 MHC protein isoforms. A: Con; B: -T3; C: OV; D: T3 + OV.

The single-fiber MHC isoform data are shown in Fig. 4. With respect to the single-fiber distribution of the Con Plan muscles, there are four key findings. First, only ~33% of the fibers exclusively expressed one type of MHC isoform. In this context, it was surprising to observe that fibers expressing solely the fast type IIB MHC isoform represented only 10-12% of the total population of fibers. Second, the majority of fibers (i.e., ~66% of the total population) exhibited various forms of polymorphism. Third, collectively the Con Plan muscles contained ~13 different types of fibers. Finally, the fast-twitch type IIX/IIB fibers represented the largest proportion of fibers (~33% of the total population) in the Con Plan muscles.

As discussed previously, the whole muscle analyses (Fig. 1) suggest that -T3 had little effect on the relative distribution of MHC protein isoforms. The single-fiber analyses are largely consistent with this perspective (Fig. 4); however, there were subtle differences between the population distributions of the Con and -T3 groups.

The whole muscle analyses demonstrated that OV produced a significant reduction in the relative content of the fast type IIB MHC protein isoform (Fig. 1). This was manifested in two different ways at the single-fiber level: 1) there was a selective reduction in the relative proportion of the fast type IIB MHC isoform within the IIX/IIB pool of fibers (Fig. 4), and 2) there was a reduction in the proportion of fast-twitch type IIX/IIB fibers (Fig. 4). In addition to these alterations, the OV Plan muscles contained larger proportions of type IIA/IIX and I/IIA/IIX fibers than did the Con or -T3 Plan muscles (Fig. 4).

The whole muscle MHC protein isoform analyses demonstrated that -T3 + OV produced substantial decreases in the relative amount of the fast type IIB MHC protein isoform and a concomitant increase in the slow type I MHC protein isoform. As shown at the single-fiber level (Fig. 4), the reduction in the relative amount of the fast type IIB MHC protein isoform occurred primarily as a result of the loss of type IIB and IIX/IIB fibers. The upregulation of the slow type I MHC protein isoform resulted from increases in the proportions of types I/IIA, I/IIA/IIX, and I/IIA/IIX/IIB fibers.

Contractile data. The contractile data are reported in Table 2. Consistent with the muscle mass data (Table 1), OV and -T3 + OV produced significant increases in Po. Neither -T3 nor OV had an effect on Vmax,muscle. In contrast, -T3 + OV did produce a significant reduction (~15%) in Vmax,muscle. Consistent with this observation, the Hill statistical model predicted an ~11% decrease in Vmax,muscle (see Fig. 6) for the -T3 + OV group. In accordance with the transition to a slower phenotype, -T3 + OV also produced significant increases in time to peak tension and one-half relaxation time.

                              
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Table 2.   Contractile properties of muscles exposed to 6 wk of intervention


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Four unique findings of this study are highly relevant to the field of muscle plasticity. First, the single-fiber analyses demonstrate that the normal Plan muscle contains a large proportion of polymorphic fibers (i.e., fibers coexpressing MHC isoforms), with the largest proportion (~35% of the total population) of these coexpressing the fast type IIX and IIB MHC isoforms. Second, the findings of this study provide further evidence to suggest that loading state plays a more significant role than thyroid state in modulating the MHC isoform composition of fast-twitch skeletal muscle. Third, the interactive effects of -T3 and OV suggest that the response of skeletal muscle to altered thyroid state is dependent on the loading state of the muscle. Finally, the high degree of polymorphism found in the Plan muscle may represent a unique strategy for minimizing the functional impact of myosin isoform transitions.

Polymorphic expression of MHC protein isoforms in single fibers of the rodent Plan muscle. In previous studies (2-4), we examined the MHC composition of single fibers from rodent Sol and white medial gastrocnemius muscles. Under normal conditions, the majority of fibers in both of these muscles express only one MHC protein isoform. In this regard, these two muscles represent the extremes of a spectrum of possible combinations of MHC protein isoform expression. We have also shown (2-4), however, that perturbations of the Sol and white medial gastrocnemius muscles, via manipulation of thyroid hormone level or mechanical loading, lead to dramatic increases in the number of fibers coexpressing various combinations of MHC protein isoforms (i.e., polymorphic fibers).

As stated above, it has become clear that a number of perturbations can produce transitions that give rise to the polymorphic expression of MHC isoforms within a given muscle fiber. However, the prevailing impression has been that muscles under steady-state conditions contain few polymorphic fibers. Clearly, on the basis of the findings of previous investigators (22, 24) and the findings shown in Fig. 4, it is clear that this view needs to be modified. The normal Plan muscles, in the present study, contained at least eight different populations of polymorphic fibers, with the largest proportion of fibers coexpressing the fast type IIX and IIB MHC isoforms.

Control of MHC isoform expression by thyroid hormone and loading state: is one more dominant than the other? Two different approaches have been used to compare the relative importance of thyroid hormone and loading state in regulating MHC isoform expression. The first approach (2, 3, 15) simply has contrasted the separate effects of each intervention. The second approach (9, 17, 25) has employed a competitive strategy whereby one manipulation (e.g., hyperthyroidism) is used to counteract the effects of the other (e.g., mechanical loading).

As shown in this and other studies (17, 25), the loading state imposed on the Plan muscle appears to play a more important role than thyroid state (e.g., hypothyroidism) in modulating MHC protein and mRNA isoform profiles (Figs. 1 and 4). In the present study we found that -T3 produced only minor changes in the single-fiber distribution of MHC isoforms. OV, in contrast, produced a significant reduction in the relative amount of the fast type IIB MHC protein isoform that was manifested by 1) a reduction in the proportion of fast-twitch type IIX/IIB fibers and 2) a selective reduction in the relative amount of the fast type IIB MHC isoform within the type IIX/IIB fibers. Consistent with the concept that loading state exerts a stronger influence over MHC expression in the rodent fast-twitch Plan muscle, Swoap et al. (25) reported that OV significantly attenuated the effects of hyperthyroidism on the fast type IIB MHC isoform.

Interestingly, these findings cannot be generalized to include rodent slow-twitch skeletal muscle. In the rodent Sol muscle, the data (2, 3, 9, 15) suggest that thyroid state exerts stronger control over MHC isoform expression than loading state. The findings of competitive experiments are also consistent with this interpretation (9).

The competitive effects of thyroid state (+T3/-T3) and increased neuromuscular activity have also been examined. Kirschbaum et al. (19) examined the antagonistic effects of chronic low-frequency stimulation (CLFS) and thyroid state on MHC expression in rodent tibialis anterior and extensor digitorum muscles. Their findings suggest that thyroid state is only partially effective in blunting the effects of CLFS.

Collectively, the studies noted above suggest that, from a comparative perspective (i.e., thyroid vs. loading state), 1) loading state plays a more significant role in controlling the expression of MHC isoforms in fast-twitch skeletal muscle, 2) thyroid state is a more powerful modulator of MHC isoform expression in slow-twitch skeletal muscle, and 3) the antagonistic effects of thyroid state are dependent on the competitor (e.g., loading state or CLFS). In making these generalizations, however, it must be noted that the dominance of one intervention (i.e., thyroid vs. loading state) over the other appears to be isoform specific and time dependent. For instance, in the present study, the major difference between the effects of -T3 and OV was the greater repression of the fast type IIB MHC isoform induced by OV. With respect to time dependence, we recently observed (2) that +T3 had a greater impact than HS on the expression of the slow type I and fast type IIA MHC isoforms after 4 wk of intervention, whereas very little difference was found at earlier time points of 1 and 2 wk.

Interactive effects between thyroid hormone and loading states appear to be isoform specific. As shown in Figs. 1 and 2, the interactive effects of -T3 and OV, at the whole muscle level, were isoform specific and appeared to be restricted primarily to the slow type I MHC isoform. Importantly, however, this finding supports the concept that the effect of the thyroid hormone is dependent on the loading state of the muscle. -T3 + OV produced an increase in the slow type I MHC isoform signal that appeared to be the sum of the individual effects of -T3 and OV (i.e., an additive effect). Analyses at the single-fiber level, however, revealed a much more complex interaction between these two stimuli. Specifically, the reduction in the proportion of type IIX/IIB fibers and the concomitant increase in the proportion of type I/IIA/IIX/IIB fibers produced by -T3 + OV were much greater than that predicted from the individual effects of -T3 and OV. Despite these synergistic effects on the proportions of type IIX/IIB and type I/IIA/IIX/IIB fibers, -T3 + OV was only partly successful in converting the fast-twitch Plan muscle to a slow-twitch muscle. This interactive effect is in dramatic contrast to the effect of +T3 + HS on the slow-twitch Sol muscle, whereby the slow-twitch Sol muscle was effectively converted to a fast-twitch muscle (2, 3).

In previous studies (11, 23) employing immunohistochemical techniques, it has been noted that OV produces a two- to threefold increase in the percentage of fibers exclusively expressing the slow type I MHC isoform. Although we did not observe a similar increase in the percentage of such fibers, we did find that OV produced an ~2.5-fold increase in the proportion of fibers (monomorphic + polymorphic) expressing the slow type I MHC isoform (Con ~16%, OV ~40%). This finding raises the possibility that many of the fibers classified as pure type I fibers in previous studies (11, 23) may have, in fact, been polymorphic fibers that expressed the slow type I MHC isoform in various combinations with other MHC isoforms. As noted above, the interactive effect of OV and -T3 on the expression of the slow type I MHC isoform was complex. Within this context, it should be noted that ~70% of the fibers in the -T3 + OV group expressed some level of the slow type I MHC isoform as identified by electrophoretic analyses. This finding is consistent with immunohistochemical analyses performed using a monoclonal antibody (BAD5) specific for the slow type I MHC isoform (Fig. 5). Hence, although -T3 + OV did not produce dramatic increases in the relative proportion of the slow type I MHC isoform at the whole muscle level (compared with OV alone), it significantly expanded the pool of fibers that expressed this MHC isoform.


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Fig. 5.   Transverse section (10 µm thick) of -T3 + OV Plan muscle. Section was probed using a monoclonal antibody (BAD5) specific for slow type I MHC isoform. Note large percentage of fibers (~65%) that stained positive for presence of slow type I MHC isoform. On basis of single-fiber electrophoretic analyses presented in Fig. 4, it is clear that most of these fibers coexpressed slow type I MHC isoform in various combinations with other MHC isoforms.

Single-fiber MHC polymorphism may represent a unique design for minimizing the functional impact of MHC isoform transitions. The intrinsic shortening speed of a muscle is often described by measuring its maximal shortening velocity (i.e., Vmax,muscle). Of the three different interventions employed in this study, only -T3 + OV produced a statistically significant reduction in Vmax,muscle. This decrease, however, was modest (~15%), despite a substantial reduction in the relative proportion of the fast type IIB MHC isoform (45% Con right-arrow 18% -T3 + OV) and a concomitant increase in the slow type I MHC isoform (3% Con right-arrow 21% -T3 + OV). As noted by us previously (3), the Vmax,muscle is determined by the force-velocity relationships of each fiber or group of fibers making up the whole muscle. In this regard, the Vmax,muscle is largely dependent on the interaction of two key factors: 1) the MHC isoform composition of the muscle and 2) the distribution of the isoforms at the single-fiber level. In this context, alterations in Vmax,muscle must be dependent on 1) the specific isoforms affected by a given intervention and 2) the pattern of these alterations within individual fibers. To better appreciate the potential impact of the transitions in the slow type I and fast type IIB MHC isoforms induced by -T3 + OV, Hill's statistical model of the force-velocity relationship was utilized in conjunction with 1) force-velocity data of single fibers published previously (1) and 2) actual distributions of MHC isoforms observed at the single-fiber level (see -T3 + OV data in Fig. 4). Tables 3 and 4 list the proportions of various fiber types and their corresponding Vmax,fiber values. By using Eq. 3 and multiplying by the proportion of any group of fibers, it is possible to determine the contribution of each group of fibers to total force production at any given shortening velocity (Vmuscle). As shown in Fig. 6, this integrated model predicted a decrease in Vmax,muscle of ~11%, which is consistent with the 15% decline actually observed. Collectively, the actual measurements of Vmax,muscle and the theoretical estimates of Vmax,muscle strongly suggest that the highly polymorphic nature of the Plan muscle represents a design feature that minimizes the effects of substantial transitions in MHC isoform composition on the intrinsic speed of a muscle (e.g., Vmax,muscle). This then minimizes the effect of MHC transitions on the ability of the Plan muscle to produce power and mechanical work under any given loading condition. In also considering the hypertrophic response, it is clear that -T3 + OV dramatically increased the ability of the Plan muscle to produce power and mechanical work. This was achieved by expanding the force-generating capacity of the muscle (i.e., hypertrophic response) without drastically compromising its speed properties.

                              
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Table 3.   Distribution of fiber types from Con group and corresponding Vmax,fiber for each fiber type


                              
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Table 4.   Distribution of fiber types from -T3 + OV group and corresponding Vmax,fiber for each fiber type



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Fig. 6.   Theoretical force-velocity curves determined from integration of Hill's statistical model with single-fiber MHC data and previously published maximal fiber velocity (Vmax,fiber) data from Bottinelli et al. (1). Single-fiber data used to generate these relationships are from distributions shown in Fig. 4. FL, fiber length; P, isotonic tension; Po, maximal isometric tension.

Summary. Collectively, the single-fiber data reported in this study offer new and exciting perspectives on muscle plasticity. The high degree of MHC polymorphism and the large number of different fiber types found in the Plan muscle are clearly surprising in light of present perspectives about fibers expressing only one MHC isoform under steady-state conditions. In this regard, it will be interesting to survey a broad group of muscles to determine the applicability of these findings. OV clearly exerts an effect on the single-fiber MHC isoform distribution that is greater than that induced by -T3. This effect was primarily constrained, however, to three types of fibers (IIA/IIX, IIX/IIB, and I/IIA/IIX). Finally, the distribution of MHC isoforms across a large number of fiber types (~13) represents a unique design criterion that appears to minimize, at the whole muscle level, the functional consequences of significant MHC isoform transitions.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-30346.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: V. J. Caiozzo, Medical Sciences I B-152, Dept. of Orthopaedics, College of Medicine, University of California, Irvine, CA 92717 (E-mail: vjcaiozz{at}uci.edu).

Received 11 February 1999; accepted in final form 16 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bottinelli, R, Schiaffino S, and Reggiani C. Force-velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. J Physiol (Lond) 437: 655-672, 1991[Abstract].

2.   Caiozzo, VJ, Baker MJ, and Baldwin KM. Novel transitions in myosin isoforms: separate and combined effects of thyroid hormone and mechanical unloading. J Appl Physiol 85: 2237-2248, 1998[Abstract/Free Full Text].

3.   Caiozzo, VJ, Baker MJ, McCue SA, and Baldwin KM. Single fiber and whole muscle analyses of MyHC isoform plasticity: an interaction between thyroid hormone and mechanical unloading. Am J Physiol Cell Physiol 273: C944-C952, 1997[Abstract/Free Full Text].

4.   Caiozzo, VJ, Haddad F, Baker MJ, and Baldwin KM. The influence of mechanical loading upon myosin heavy chain protein and mRNA isoform expression. J Appl Physiol 80: 1503-1512, 1996[Abstract/Free Full Text].

5.   Caiozzo, VJ, Herrick RE, and Baldwin KM. Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms. Am J Physiol Cell Physiol 263: C86-C94, 1992[Abstract/Free Full Text].

6.   Caiozzo, VJ, Swoap S, Tao M, Menzel D, and Baldwin KM. Single fiber analyses of type IIA myosin heavy chain distribution in hyper- and hypothyroid soleus. Am J Physiol Cell Physiol 265: C842-C850, 1993[Abstract/Free Full Text].

7.   Devor, ST, and White TP. Myosin heavy chain of immature soleus muscle grafts adapts to hyperthyroidism more than to physical activity. J Appl Physiol 80: 789-794, 1996[Abstract/Free Full Text].

8.   Devor, ST, and White TP. Myosin heavy chain phenotype in regenerating skeletal muscle is affected by thyroid hormone. Med Sci Sports Exerc 27: 674-681, 1995[ISI][Medline].

9.   Diffee, GM, Haddad F, Herrick RE, and Baldwin KM. Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb suspension. Am J Physiol Cell Physiol 261: C1099-C1106, 1991[Abstract/Free Full Text].

10.   Di Maso, NA, Caiozzo VJ, and Baldwin KM. Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism. Am J Physiol Regulatory Integrative Comp Physiol 278: R1099-R1106, 2000[Abstract/Free Full Text].

11.   Dunn, SE, and Michel RN. Coordinated expression of myosin heavy chain isoforms and metabolic enzymes within overloaded rat muscle fibers. Am J Physiol Cell Physiol 273: C371-C383, 1997[Abstract/Free Full Text].

12.   Giulian, GG, Moss RL, and Greaser M. Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels. Anal Biochem 129: 277-287, 1983[ISI][Medline].

13.   Gregory, P, Gagnon J, Essig DA, Reid SK, Prior G, and Zak R. Differential regulation of actin and myosin isoenzyme synthesis in functionally overloaded skeletal muscle. Biochem J 265: 525-532, 1990[ISI][Medline].

14.   Haddad, F, Arnold C, Zeng M, and Baldwin K. Interaction of thyroid state and denervation on skeletal myosin heavy chain expression. Muscle Nerve 20: 1487-1496, 1997[ISI][Medline].

15.   Haddad, F, Qin AX, Zeng M, McCue SA, and Baldwin KM. Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression. J Appl Physiol 85: 2227-2236, 1998[Abstract/Free Full Text].

16.   Hill, AV. First and Last Experiments in Muscle Mechanics. Cambridge, UK: Cambridge University Press, 1970.

17.   Ianuzzo, CD, Hamilton N, and Li B. Competitive control of myosin expression: hypertrophy vs. hyperthyroidism. J Appl Physiol 70: 2328-2330, 1991[Abstract/Free Full Text].

18.   Izumo, S, Nadal-Ginard B, and Mahdavi V. All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science 231: 597-600, 1986[ISI][Medline].

19.   Kirschbaum, BJ, Kucher HB, Termin A, Kelly AM, and Pette D. Antagonistic effects of chronic low-frequency stimulation and thyroid hormone on myosin expression in rat fast-twitch muscle. J Biol Chem 265: 13974-13980, 1990[Abstract/Free Full Text].

20.   Larsson, L, Li X, Teresi A, and Salviati G. Effects of thyroid hormone on fast- and slow-twitch skeletal muscles in young and old rats. J Physiol (Lond) 481: 149-161, 1994[Abstract].

21.   Larsson, L, Meuller U, Li X, and Schiaffino S. Thyroid hormone regulation of myosin heavy chain isoform composition in young and old rats, with special reference to IIX myosin. Acta Physiol Scand 153: 109-116, 1995[ISI][Medline].

22.   Peuker, H, and Pette D. Quantitative analyses of myosin heavy chain mRNA and protein isoforms in single fibers reveal a pronounced fiber heterogeneity in normal rabbit muscles. Eur J Biochem 271: 30-36, 1997.

23.   Roy, RR, Talmadge RJ, Fox K, Lee M, Ishihara A, and Edgerton VR. Modulation of MHC isoforms in functionally overloaded and exercised rat plantaris fibers. J Appl Physiol 83: 280-290, 1997[Abstract/Free Full Text].

24.   Staron, RS, and Pette D. The continuum of pure and hybrid myosin heavy chain-based fibre types in rat skeletal muscle. Histochemistry 100: 149-153, 1993[ISI][Medline].

25.   Swoap, SJ, Haddad F, Caiozzo VJ, Herrick RE, McCue SA, and Baldwin KM. Interaction of thyroid hormone and functional overload on skeletal muscle isomyosin expression. J Appl Physiol 77: 621-629, 1994[Abstract/Free Full Text].

26.   Tsika, RW, Herrick RE, and Baldwin KM. Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression. J Appl Physiol 63: 2111-2121, 1987[Abstract/Free Full Text].

27.   Woledge, RC, Curtin NA, and Homsher E. Energetic Aspects of Muscle Contraction. New York: Academic, 1985.


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