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J Appl Physiol 82: 211-218, 1997;
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Journal of Applied Physiology
Vol. 82, No. 1, pp. 211-218, January 1997
EXERCISE AND MUSCLE

Modulation of myosin isoform expression by mechanical loading: role of stimulation frequency

Vincent J. Caiozzo, Michael J. Baker, and Kenneth M. Baldwin

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

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Caiozzo, Vincent J., Michael J. Baker, and Kenneth M. Baldwin. Modulation of myosin isoform expression by mechanical loading: role of stimulation frequency. J. Appl. Physiol. 82(1): 211-218, 1997.---This study tested the hypothesis that mechanical loading, not stimulation frequency per se, plays a key role in determining the plasticity of myosin heavy chain (MHC) protein isoform expression in muscle undergoing resistance training. Female Sprague-Dawley rats were randomly assigned to resistance-training programs that employed active 1) shortening (n = 7) or 2) lengthening contractions (n = 8). The medial gastrocnemius (MG) muscles in each group trained under loading conditions that approximated 90-95% of maximum isometric tetanic tension but were stimulated at frequencies of 100 and ~25 Hz, respectively. Lengthening and shortening contractions were produced by using a Cambridge ergometer system. The MG muscles trained every other day, performing a total of 16 training sessions. Both training programs produced significant (P < 0.01) and similar reductions in the fast type IIB MHC protein isoform in the white MG muscle, reducing its relative content to ~50% of the total MHC protein isoform pool. These changes were accompanied by increases in the relative content of the fast type IIX MHC protein isoform that were of similar magnitude for both groups. The results of this study clearly demonstrate that stimulation frequency does not play a key role in modulating MHC isoform alterations that result from high-resistance training.

heavy chains; slow type I; fast type IIA; fast type IIX; fast type IIB; electrophoresis; muscle injury; concentric; eccentric

INTRODUCTION

THE PHENOTYPIC EXPRESSION of skeletal muscle is thought to be regulated by a number of physiological factors, including innervation, hormonal status, and mechanical loading (3, 4, 17). To better delineate the potential influences of stimulation frequency and mechanical loading on muscle plasticity, we developed a unique rodent training model specifically designed to control both the mechanical loading conditions and activation patterns of skeletal muscle (6, 7). With the use of this model, we reported previously (6, 7) that a training program employing a high-loading condition [~90-95% maximum isometric tetanic tension (Po)] and a high stimulation frequency (100 Hz) downregulated the fast type IIB myosin heavy chain (MHC) isoform and concomitantly upregulated the expression of the slower fast type IIX MHC isoform in both the white and red regions of the medial gastrocnemius (MG) muscle. Presently, it is unclear whether these alterations were produced by the high-loading condition imposed on the muscle or alternatively by the high stimulation frequency that was used.

This issue was addressed by using the approach described in Fig. 1. At any given stimulation frequency, skeletal muscle can generate a greater amount of force during active lengthening (Actlength) as compared with active shortening (Actshort). As shown in Fig. 1, these different force-frequency relationships can be used to create conditions in which markedly different stimulation frequencies result in similar mechanical stresses on muscle. This approach was used to test the hypothesis that mechanical loading, not stimulation frequency per se, plays a key role in determining MHC protein isoform expression in muscle undergoing resistance training.

Fig. 1. Force-frequency relationships for a medial gastrocnemius muscle (MG) under isometric and active lengthening (Actlength) conditions. Isometric measurements were made at a length (Lo) where muscle produced maximal tetanic tension (Po). Actlength data were collected by using a positive strain rate of 0.12 Lo /s. This strain rate is slightly greater than strain rate that was used for training Actlength group (0.04 Lo /s). Importantly, both strain rates produce similar force-frequency curves. Measurements were centered around Lo and occurred in plateau region of length-tension relationship. Each data point represents peak force measured under a given condition. Note that under Actlength, a tension equivalent to 100% Po can be generated by using a stimulation frequency of ~25-30 Hz. This approach was used to alter the relationship between stimulation frequency and mechanical loading.
[View Larger Version of this Image (21K GIF file)]

The findings of this study demonstrate that training paradigms involving high-loading conditions (~90-95% Po) can produce alterations in MHC protein isoform composition that are independent of stimulation frequency (at least above ~25 Hz).

METHODS

Animal care and experimental groups. Female Sprague-Dawley rats (~250-300 g) were randomly assigned to the various experimental groups shown in Table 1. The experiments reported in this study were conducted in two phases. Phase 1 examined the MHC isoform alterations produced by Actshort and Actlength training programs that employed similar loading conditions but markedly different stimulation frequencies. Experiments conducted in phase 2 were designed to determine whether the responses observed in phase 1 might be attributable to muscle injury occurring at early stages of the training program. All animals were housed individually and given access to food and water ad libitum. All procedures involving animal welfare were approved by our institutional review board before these experiments were conducted.

Table 1. Summary of studies conducted, types of analyses, and training programs

Phase Objective Analysis n Training Program
Type of contraction Training sessions Sets/ session Contractions/ set
1 Employ Actshort and Actlength contractions to explore importance of mechanical loading on MHC isoform expression Hematoxylin and eosin Myofibrillar protein concentration MHC isoforms 7 8  Actshort Actlength 16 16  4 4  10 10 
2 Determine whether muscle injury is produced by Actshort and/or Actlength contractions Native myosin isoforms Hematoxylin and eosin BrdU labeling of activated satellite cells 3 3 3 3  Actshort Actshort Actlength Actlength 1 2 1 2  4 4 4 4  10 10 10 10
n = No. of animals; Actshort, active shortening; Actlength, active lengthening; MHC, myosin heavy chain; BrdU, 5-bromo-2'-deoxyuridine.

Surgical implantation of stimulation electrodes and training programs. Each animal was anesthetized (acepromazine = 4.5 mg/kg; ketamine = 75 mg/kg), and stimulating electrodes were surgically implanted in the left hindlimb of the animal so that the activation pattern of the MG muscle could be controlled. A sham operation was performed on the right leg, which served as the control.

After 1 wk of recovery, the two groups of animals in phase 1 began their respective training programs (see Table 1). Approximately 20 min before each training session, the animals were lightly anesthetized with acepromazine (5 mg/kg) and ketamine hydrochloride (20 mg/kg). Shortening or lengthening contractions were produced by using a computer-controlled Cambridge ergometer system (6, 7). The major components of this system include 1) a Cambridge ergometer (model 310, Cambridge Instruments, Watertown, MA) that was used to control the mechanical loading conditions imposed on the target muscle during training; 2) a computer that activates the stimulator and controls the parameters of the ergometer; and 3) a training platform that translates the moment at the ankle into a linear force. In the present study, the ergometer was used to impose a slow constant angular velocity of 12°/s that caused the MG muscles to train under loading conditions that briefly approximated 90-95% Po. Each Actshort contraction began with the ankle in a neutral position (i.e., 90° relative to the tibia). In contrast, each Actlength contraction began with the ankle at 48° of plantar flexion relative to the neutral position (see Fig. 2). The combination of these conditions (i.e., angular velocity and range of motion) resulted in negative/positive strain rates that approximated 0.04 muscle length (Lo)/s. It should be noted that a positive strain rate of 0.04 Lo /s is very similar to that used to produce the Actlength data shown in Fig. 1. Muscles in the Actshort and Actlength groups were stimulated at frequencies of 100 and ~25 Hz, respectively. Each contraction was 4 s in duration and was followed by a 6-s rest interval. Each group of animals performed 4 sets of 10 contractions during a training session and trained every other day for 4 wk (see Table 1). Twelve hours after the last training session, the left and right MG muscles were removed from each animal, weighed, and separated into regions designated as red and white (7). Once the muscles had been removed, the animal was killed by using a lethal injection of pentabarbitol sodium. The types of analyses that were performed on each muscle group are reported in Table 1. The portion of each region that was used for myofibrillar and MHC protein isoform analyses was placed into cooled glycerol and stored at -20°C until analyzed. Those sections that were used for histological analyses were frozen in isopentane cooled by liquid nitrogen and stored at -70°C until sectioned.
Fig. 2. Forces produced under active shortening (Actshort; A; solid line; 100 Hz) and Actlength (B; dotted line; 27 Hz) conditions. C: y-axis, angular position (°) of ankle during each contraction and subsequent relaxation phase, with 0 as ankle in a neutral position (i.e., 90° relative to tibia). Muscles in each group were activated throughout entire Actshort or Actlength phase (i.e., 4 s) (see METHODS). Note that muscles were required to generate a contraction every 10 s.
[View Larger Version of this Image (13K GIF file)]

The purpose of the experiments in phase 2 was to determine whether injury was induced at early time points in the Actlength training program. Animals in this phase were trained under identical conditions, except that they performed either one or two training sessions (see Table 1). Muscles from these groups were removed ~24 h after the last training session. Tissues were stored as described above for phase 1.

Myofibrillar protein content. Purified myofibril preparations were extracted by using techniques described previously (5, 7). This included homogenization of the muscle in a solution (solution A; pH 6.8) containing (in mM) 250 sucrose, 100 KCl, 20 tris(hydroxymethyl)aminomethane (Tris), and 5 EDTA. The homogenate was centrifuged at 1,000 g for 10 min at 4°C. The resulting pellet was resuspended in a solution (solution B; pH 7.0) containing 175 mM KCl and 20 mM Tris and centrifuged as described above. The resulting pellet was again suspended in solution B and adjusted to a protein concentration of 6 mg/ml by using the biuret technique. Myofibrils were then stored at 1 mg/ml and at -20°C in a solution containing 50% glycerol, 50 mM Na4P2O7, 2.5 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1 mM 2-mercaptoethanol (pH 8.8).

Electrophoretic separation of MHC isoforms. MHC protein isoforms were separated by using techniques described previously (5, 6, 20). The separating gel solution contained 8% acrylamide, 0.16% bis-acrylamide, 30% glycerol, 0.4% sodium dodecyl sulfate (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 adding N, N, N', N'-tetramethylethylenediamine (TEMED; 0.05% final concentration) and ammonium persulfate (0.1% final concentration) to the separating gel solution. After the separating gel had been poured, it was layered with ethyl alcohol and allowed ~30 min to polymerize. Once the separating gel was polymerized, the stacking gel was poured. The composition of the stacking gel was 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 TEMED (0.05% final concentration) and ammonium persulfate (0.1% final concentration). The composition of the running buffer was 0.1 M Tris, 0.15 M glycine, and 0.1% SDS. Myofibril samples were denatured by using a sample buffer solution containing 5% beta -mercaptoethanol, 100 mM Tris-base, 5% glycerol, 4% SDS, and bromophenol blue. Approximately 1 µg of protein was loaded into each well. Electrophoresis was performed by using a SG-200 vertical-slab gel system (CBS Scientific, Del Mar, CA). Gels were run by using a constant voltage of 275 V for ~24 h. This method separated the fast type IIA, fast type IIX, fast type IIB, and slow type I MHC isoforms (order of migration). MHC protein isoform bands were stained by using Coomassie blue G-250. The MHC protein isoform bands were scanned and quantified by using a Molecular Dynamics Personal Densitometer (Molecular Dynamics, Sunnyvale, CA).

Electrophoretic separation of native myosin isoforms. Skeletal muscle injury can lead to the de novo expression of neonatal native myosin isoforms (8, 9, 11, 15). Consequently, muscle samples from the phase 2 experiments were analyzed for the presence of neonatal myosin isoforms by using nondissociating electrophoretic procedures described previously (7). Briefly, ~5-10 µg of myofibrillar protein were loaded onto tube gels (60 × 5 mm). The tube gels were composed of 4.17% acrylamide, 2.6% bis-acrylamide, 20 mM Na4P2O7, 10% glycerol, and 0.2% TEMED. This solution was adjusted to a pH of 8.8. The running buffer contained 20 mM Na4P2O7, 10% glycerol, and 0.2 mM cysteine and was adjusted to a pH of 8.8. Electrophoresis was performed for 22 h by using a constant voltage of 90 V. After the electrophoresis, the gels were stained by using a solution containing 0.1% Coomassie blue R-250. The migration pattern and distance of adult and neonatal native myosin isoforms were determined by using a plantaris muscle obtained from a 15-day-old rat.

Histological and immunohistochemical analyses of muscle injury. Muscle injury can lead to the activation and incorporation of satellite cells into myofibers (26, 28). These satellite cells can be identified by using 5-bromo-2'-deoxyuridine (BrdU), which is a thymidine analogue. Previous reports (16) have shown that muscle injury can be detected as early as 8-12 h after mechanical overload of skeletal muscle. Additionally, satellite cell activation has been detected 24 h after injury to the rat soleus muscle and peaks ~3 days after the onset of injury (18). Animals in phase 2 were injected with BrdU (100 µg/100 g body weight ip) 12 h after each training session. Relative to the time course of satellite cell activation described above for the rat soleus muscle, the labeling scheme used in the present study included early (i.e., 12 h after the onset of training) and peak time points (i.e., 21/2 days after the onset of training). At the time of death, a section of intestine and the midportion of the red and white regions of the trained and contralateral control muscles were frozen in isopentane cooled by liquid nitrogen. The tissue was then sectioned (10 µm) in a cryostat. Approximately 50 µl of solution containing a primary monoclonal antibody specific for BrdU (1:12; Becton-Dickinson) was placed on the tissue sections for 1 h at 37°C. The tissue sections were rinsed for 5 min in a phosphate-buffered saline (PBS) solution. The rinsing was repeated a second time. After these steps, the tissue sections were treated with a PBS-blocking solution containing 0.5% bovine serum albumin (wt/vol) and 1% Tween-20 (vol/vol). Once this step was completed, the tissue sections were incubated with a rhodamine-labeled second monoclonal antibody (1:40) for 1 h at 37°C. The tissue was then rinsed three times (5 min each) in PBS solution. After these rinses, the tissue sections were exposed for ~10 s to 100 µl of a solution containing 1% Hoechst 33342 (Molecular Probes). The tissue sections were rinsed a final time in PBS solution (~30 s) and mounted by using 100% glycerol. The tissue sections were then analyzed by using a charge-coupled device camera (XC-77, Sony) attached to a Nikon fluorescent microscope. Images of the tissue sections were captured and analyzed by using the public-domain National Institutes of Health Image program (written by Wayne Rasband). The number of BrdU-positive nuclei was expressed relative to the number of muscle fibers. In addition to the BrdU labeling, all muscle samples underwent hematoxylin and eosin staining.

Statistical analyses. All statistical analyses were performed by using a computer program (Systat, Evanston, IL). The myofibril and MHC data were analyzed by using a two-way analysis of variance. The data for each separate MHC isoform were analyzed independently of the other isoforms. Statistical comparisons were considered significant when P < 0.05.

RESULTS

Muscle weight and myofibrillar protein. The data for muscle weight and myofibrillar protein concentration are reported in Table 2. Both the Actshort and Actlength training programs produced significant increases in muscle mass that were similar in magnitude (i.e., +11%). Neither of the training programs altered the myofibrillar protein concentration in either the white or red regions of the MG muscle.

Table 2. Muscle weight and myofibrillar protein concentration

Group Muscle Wt, mg Myofibrillar Protein Concentration, mg/g muscle
White MG Red MG
Actshort
  Con 917 ± 88  105 ± 11  99 ± 9 
  Trn 1,021 ± 98* 107 ± 13  100 ± 11 
Actlength
  Con 856 ± 56  106 ± 10  98 ± 12 
  Trn 952 ± 80* 101 ± 11  92 ± 7
Values are means ± SD. MG, Medial gastrocnemius; Con, contralateral side that underwent sham electrode placement; Trn, trained. * Significantly different, P < 0.001.

MHC isoforms. The MHC protein isoform alterations produced by the different training programs are shown in Figs. 3-5. With respect to the white region of the MG muscle (see Fig. 4), the fast type IIB MHC protein isoform comprised ~75-80% of the total MHC protein isoform pool in each of the control groups. Importantly, both the Actshort and Actlength training programs produced similar reductions in the fast type IIB MHC protein isoform, reducing the relative content in each group to ~50% of the total MHC protein isoform pool. The fast type IIX MHC protein isoform contents of the white MG control groups were ~20-25% of the total MHC protein isoform pool. Both training programs produced an approximately twofold increase in the relative content of the fast type IIX MHC protein isoform.
Fig. 3. Electrophoretic gels of control and trained white MG muscles. Lanes 2 and 4 in A and B are taken from control white MG muscles. Trained contralateral muscles are shown in lanes 1 and 3. Note that both Actshort (A) and Actlength (B) training downregulated fast type IIB mysoin heavy chain (MHC) isoform and a concomitantly upregulated relative amount of fast type IIX MHC isoform. Actlength MG muscles trained at a stimulation frequency of 25 Hz.
[View Larger Version of this Image (33K GIF file)]

Fig. 4. Effects of Actshort and Actlength training programs on MHC isoform composition of white MG. White MG only expressed fast type IIX (A) and IIB MHC (B) isoforms, hence absence of any data related to slow type I and fast type IIA MHC isoforms. Data are means ± SD. Note that both training programs had similar effects on fast type IIX and IIB MHC isoforms while similar loading conditions but very different stimulation frequencies were used. A two-way analysis of variance demonstrated that differences between control (open bars) and trained muscles (filled bars) were significant at P < 0.001 for both fast type IIX and IIB MHC isoforms. Group (Actshort, Actlength)-training (control, trained) interactions were not significant for fast type IIX and type IIB MHC isoforms, demonstrating that training had a similar effect on both Actshort and Actlength groups.
[View Larger Version of this Image (13K GIF file)]

Fig. 5. Effects of Actshort and Actlength training programs on MHC composition of red region of MG muscle. Data are means ± SD. Note that neither training program affected slow type I (A) or fast type IIA (B) MHC isoform content. However, both training programs produced similar changes in fast type IIX (C) and IIB (D) MHC isoform contents. These results are consistent with those observed for white region of MG muscle. Differences between control (open bars) and trained (filled bars) muscles for fast type IIX and type IIB MHC isoforms were significant at P < 0.001. Group (Actshort, Actlength)-training (control, trained) interactions were not significant for fast type IIX and type IIB MHC isoforms, demonstrating that training had a similar effect on both Actshort and Actlength groups.
[View Larger Version of this Image (12K GIF file)]

In contrast to the white MG region, the control Actshort and Actlength red MG regions contained all four MHC protein isoforms (see Fig. 5). Neither training program influenced the slow type I and fast type IIA MHC protein isoform contents of the red MG region. However, consistent with the data from the white MG region, both the Actshort and Actlength training programs produced similar increases in the fast type IIX MHC protein isoform content of the red MG regions, increasing the fast type IIX MHC protein isoform content from ~35 to 55% of the total MHC protein isoform pool. Concomitantly, the Actshort and Actlength training programs reduced the fast type IIB MHC protein isoform content to a similar degree.

Muscle injury. The potential presence of muscle injury in the Actlength group at early time points of the training program was evaluated in phase 2 by using three criteria: 1) the presence of neonatal native isoforms; 2) the histological presence of inflammatory cells, degenerating muscle fibers, and internal nuclei; and 3) positive staining of nuclei for BrdU. As shown in Fig. 6, the muscles from the phase 2 Actshort and Actlength groups only expressed adult native myosin isoforms. Consistent with these data, necrotic or degenerating fibers were rarely evident in either the phase 2 Actshort or Actlength groups (see Fig. 7). Finally, there were no differences between the percentage of BrdU-labeled nuclei in a comparison of the trained phase 2 Actshort and Actlength muscles with their respective control samples (see Table 3, Fig. 8). As shown in Table 3, very few BrdU-labeled cells were found in both the trained and control muscles. Typically, ~0.15% of fibers examined contained BrdU-labeled cells. If the BrdU-labeled cells were expressed relative to the total number of nuclei, then the importance of the BrdU-labeled cells would be diminished further, given that there were ~4-5 nuclei/fiber. Collectively, these data provide strong evidence to suggest that the MHC alterations produced by the Actlength training program in phase 1 were not the result of muscle fiber degeneration/regeneration induced by injury.
Fig. 6. Native gel of myosin isoform contents of control and trained white MG muscles. A: Actshort group. B: Actlength group. Far left lane in A and B is from plantaris (PL) muscle taken from a 15-day-old rat. Note presence of native neonatal isoform (Neo) in this sample. Lanes 1 and 3 in A and B are from control white MG muscles of animals that trained for 1 and 2 days, respectively. Lanes 2 and 4 in A and B are from white regions of MG muscles of animals that trained for 1 and 2 days, respectively. Note absence of Neo in these samples. SM, slow myosin; FM, fast myosin; IM, intermediate myosin.
[View Larger Version of this Image (48K GIF file)]

Fig. 7. Hematoxylin and eosin staining of Actshort and Actlength trained (Trn) white MG muscles after 2 and 16 training sessions (total of 4 and 16 days total, respectively). Note that none of muscles exhibits signs of muscle injury.
[View Larger Version of this Image (139K GIF file)]

Table 3. BrdU labeling of activated satellite cells in white MG muscles that performed 2 training sessions

Group Muscle No. of BrdU-Labeled Cells No. of Muscle Fibers Counted %Fibers With BrdU-Labeled Cells
Actshort
  Con 1 10 3,724 0.27
2 4 3,682 0.11
3 42 2,873 1.46
  Trn 1 12 4,470 0.27
2 5 5,944 0.08
3 4 4,479 0.09
Actlength
  Con 1 10 7,881 0.13
2 8 7,407 0.11
3 4 6,237 0.06
  Trn 1 12 8,117 0.15
2 10 6,838 0.15
3 10 6,864 0.15
Data for red region and muscles that performed 1 training session are not included because they appeared similar to those for muscles presented herein.

Fig. 8. Tissue sections stained with monoclonal antibody specific for BrdU. A: intestinal section showing uptake of BrdU by nuclei (arrow). B and C: Actlength control (B) and trained (C) white MG muscle after 2 training sessions (total of 4 days). Note absence of satellite cells staining positive for BrdU in B and C.
[View Larger Version of this Image (97K GIF file)]

DISCUSSION

There are two key findings in this study. First, both the Actshort and Actlength training programs produced similar alterations in the MHC isoform expression that were characterized by a downregulation of the fast type IIB MHC isoform and a concomitant upregulation of the fast type IIX MHC isoform. This finding suggests that previous results (6, 7) were not due to the high stimulation frequency employed in these earlier studies. Second, the Actlength training paradigm, as used in the present study, did not produce signs of muscle injury. Hence, the similarity in MHC isoform alterations produced by the Actlength and Actshort training programs cannot be ascribed to MHC isoform plasticity linked to muscle fiber degeneration/regeneration.

Mechanical loading of skeletal muscle has been shown to play an important role in the modulation of MHC isoform composition. A large amount of information has been obtained (17) from three key experimental manipulations: 1) hindlimb suspension; 2) exposure to microgravity; and 3) compensatory overload. Studies employing either hindlimb suspension or microgravity to unload skeletal muscles have reported an increased expression of fast MHC isoforms at both the protein and mRNA levels (5, 10). In contrast, increased mechanical loading, as induced by compensatory overload, upregulates the slower forms of MHC protein and mRNA isoforms (19).

The downregulation of the fast type IIB MHC isoform and the concomitant upregulation of the fast type IIX MHC isoform observed in the present study are consistent with the hypothesis implicating mechanical loading as a key factor modulating MHC gene plasticity. The most likely explanation of our training data is that the alterations in MHC isoform composition induced by the two different training programs were invoked by similar mechanical factors (e.g., stress) acting on similar cellular mechanisms that act to reciprocally regulate fast type IIB and IIX MHC isoform genes. Consistent with the suggestion that our training programs affected MHC protein isoform expression by altering MHC gene expression, we have observed previously (6) that the Actshort training program used in this study produces substantial alterations in both fast type IIB and IIX MHC mRNA isoform content.

From a historical perspective, the cross-innervation study by Buller et al. (4) has provided a strong argument that some aspect of neural input plays an important role in determining skeletal muscle phenotype. Buller et al. (4) proposed a number of hypotheses (e.g., frequency hypothesis, aggregate hypothesis) to explain the results of their cross-innervation experiments. The "frequency hypothesis" stipulates that muscles stimulated at low frequencies develop phenotypic properties similar to those of slow skeletal muscle, whereas those muscles stimulated at high frequencies exhibit phenotypic properties similar to those of fast skeletal muscle. Although Buller et al. (4) specifically rejected this hypothesis, Lomo et al. (14) reported that denervated soleus muscles stimulated at 100 Hz developed fast contractile properties, whereas those that were stimulated at 10 Hz developed slow contractile properties. More recently, it has been reported that high stimulation frequencies caused the soleus (2, 12) and the extensor digitorum longus (2) muscles to upregulate their fast MHC isoform content. An important distinction needs to be made regarding the approach used in these earlier studies (2, 12, 14) and that used in the present study. These previous studies (2, 12, 14) employed a chronic type of electrical stimulation, causing the target muscle to contract thousands of times per day. In the present study, the muscles produced 40 contractions every other day. Hence, although stimulation frequency might be an important modulator of muscle phenotype when the stimulation pattern is applied in a chronic fashion, under the conditions employed in the present study, stimulation frequency does not appear to play a key role in modulating MHC isoform expression.

Previous studies have shown that, under specific cirsumstances, active lengthening can produce skeletal muscle injury (13, 21) and that this injury initiates developmental programs of myosin isoform expression (8, 9, 11, 15). Although it is doubtful that such a program could explain the alterations produced by the Actlength training program used in the present study and the similarity of these changes to those produced by the Actshort training program, both groups of trained muscles were examined for the presence of muscle injury. On the basis of the collective criteria used in the present study, the muscles that trained under Actlength conditions did not exhibit any signs of muscle injury. Hence, the alterations produced by the Actlength training program are not obfuscated by issues related to degeneration/regeneration.

From an applied perspective, it has been shown that human subjects performing high-resistance training programs downregulate the expression of the fast type IIB/IIX MHC isoform while reciprocally upregulating the slower fast type IIA MHC isoform (1). The similarity between this response and that seen in the present study suggests that the rodent high-resistance training model employed in the present study represents a powerful tool for exploring important issues related to humans performing resistance training. In this respect, the cellular/molecular training response of skeletal muscle to Actlength contractions has rarely been studied. In the present study, we observed that when mechanical loading was held constant, both the Actshort and Actlength training programs produced identical responses with respect to muscle mass and MHC isoform expression. Hence, when mechanical loading conditions are similar, it does not appear as though one type of training is more effective than the other in modulating MHC isoform composition.

In summary, the findings of this study clearly indicate that stimulation frequency is not responsible for the MHC isoform alterations observed by us previously, and they refute the concept of a frequency hypothesis as it applies to training conditions used in this study. Although these results are consistent with the mechanical loading hypothesis, further experimentation will be required to better delineate the putative role of mechanical factors in modulating MHC isoform expression and the cellular/molecular mechanisms they invoke.

ACKNOWLEDGEMENTS

This work was supported in part by Orthopedic Research and Education Foundation Grant 92-019 and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-30346 and AR-43126.

FOOTNOTES

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

Received 21 February 1996; accepted in final form 17 September 1996.

REFERENCES

1. Adams, G. R., B. M. Hathers, K. M. Baldwin, and G. A. Dudley. Skeletal muscle myosin heavy chain composition and resistance training. J. Appl. Physiol. 74: 911-915, 1993. [Abstract/Free Full Text]
2. Ausoni, L., L. Gorza, S. Schiaffino, K. Gunderson, and T. Lomo. Expression of myosin heavy chain isoforms in stimulated fast and slow rat muscles. J. Neurosci. 10: 153-160, 1990. [Abstract]
3. Booth, F. W., and K. M. Baldwin. Muscle plasticity: energy demanding and supply processes. Handb. Physiol. 45: 1075-1123, 1996.
4. Buller, A. J., J. C. Eccles, and R. M. Eccles. Interactions between motoneurons and muscles in respect of the characteristic speeds of their responses. J. Physiol. Lond. 150: 417-439, 1960.
5. Caiozzo, V. J., M. J. Baker, R. E. Herrick, and K. M. Baldwin. Effect of spaceflight on skeletal muscle: mechanical properties and myosin isoform content of a slow antigravity muscle. J. Appl. Physiol. 76: 1764-1773, 1994. [Abstract/Free Full Text]
6. Caiozzo, V. J., F. Haddad, M. J. Baker, and K. M. Baldwin. 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]
7. Caiozzo, V. J., E. Ma, S. McCue, E. Smith, R. E. Herrick, and K. M. Baldwin. A new animal model for modulating myosin isoform expression by altered mechanical activity. J. Appl. Physiol. 73: 1432-1440, 1992. [Abstract/Free Full Text]
8. Carraro, U., L. D. Libera, and C. Catani. Myosin light and heavy chains in muscle regenerating in absence of the nerve: transient appearance of embryonic light chain. Exp. Neurol. 79: 106-117, 1983. [Medline]
9. D'Albis, A., R. Couteauz, C. Janmot, A. Roulet, and J. Mira. Regeneration after cardiotoxin injury of innervated and denervated slow and fast muscles of mammals. Eur. J. Biochem. 174: 103-110, 1988. [Medline]
10. Diffee, G. M., F. Haddad, R. E. Herrick, and K. M. Baldwin. Control of myosin heavy chain expression: interaction of hypothyroidism and hindlimb suspension. Am. J. Physiol. 261 (Cell Physiol. 30): C1099-C1106, 1991. [Abstract/Free Full Text]
11. Esser, K. A., and T. P. White. Mechanical load affects growth and maturation of skeletal muscle grafts. J. Appl. Physiol. 78: 30-37, 1995. [Abstract/Free Full Text]
12. Gorza, L., K. Gunderson, T. Lomo, and S. Schiaffino. Slow-to-fast transformation of denervated soleus muscles by chronic high frequency stimulation in the rat. J. Physiol. Lond. 402: 627-649, 1988. [Abstract/Free Full Text]
13. Lieber, R. L., M. C. Schmitz, D. K. Mishra, and J. Friden. Contractile and cellular remodeling in rabbit skeletal muscle after cyclic active lengthening contractions. J. Appl. Physiol. 77: 1926-1934, 1994. [Abstract/Free Full Text]
14. Lomo, T., R. H. Westgaard, and H. A. Dahl. Contractile properties of muscle: control by pattern of muscle activity in the rat. Proc. R. Soc. Lond. Ser. B Biol. Sci. 187: 99-103, 1974. [Medline]
15. Marechal, G, K. Schwartz, G. Beckers-Bleukx, and E. Ghins. Isozymes of myosin in growing and regenerating rat muscles. Eur. J. Biochem. 138: 421-428, 1984. [Medline]
16. Rantanen, J., T. Hurme, R. Lukka, J. Heino, and H. Kalimo. Satellite cell proliferation and the expression of myogenin and desmin in regenerating skeletal muscle: evidence for two different populations of satellite cells. Lab. Invest. 72: 341-347, 1995. [Medline]
17. Roy, R. R., K. M. Baldwin, and V. R. Edgerton. The plasticity of skeletal muscle: effects of neuromuscular activity. Exercise Sports Sci. Rev. 19: 269-312, 1991. [Medline]
18. Saito, Y., and I. Nonaka. Initiation of satellite cell replication in bupivacaine-induced myonecrosis. Acta Neuropathol. 88: 252-257, 1994. [Medline]
19. Swoap, S. J., F. Haddad, V. J. Caiozzo, R. E. Herrick, S. A. McCue, and K. M. Baldwin. Interaction of thyroid hormone and functional overload on skeletal muscle isomyosin expression. J. Appl. Physiol. 77: 621-629, 1994. [Abstract/Free Full Text]
20. Talmadge, R. J., and R. R. Roy. Electrophoretic separation of rat skeletal muscle myosin heavy chain isoforms. J. Appl. Physiol. 75: 2337-2340, 1993. [Abstract/Free Full Text]
21. Warren, G. L., D. A. Hayes, D. A. Lowe, and R. B. Armstrong. Mechanical factors in the initiation of active lengthening contraction-induced injury in rat soleus muscle. J. Physiol. Lond. 464: 457-475, 1993. [Abstract/Free Full Text]
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