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J Appl Physiol 84: 1359-1364, 1998;
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Vol. 84, Issue 4, 1359-1364, April 1998

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy

P. E. Mozdziak, M. L. Greaser, and E. Schultz

Department of Anatomy, University of Wisconsin Medical School, Madison, Wisconsin 53706

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3',5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

clenbuterol; 3,3',5-triiodo-L-thyronine; overload; myogenic regulatory factor; skeletal muscle

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE MYOGENIC REGULATORY FACTORS myogenin and MyoD are DNA-binding proteins that will cause mesenchymal cells to express muscle-specific markers (5, 33). Myogenin and MyoD expression is high during embryonic development, but their expression is low in mature muscle (7, 13, 31, 32). Previous studies have suggested that myogenin and MyoD may be involved in establishing and maintaining mature myofiber phenotype (slow or fast) because myogenin is expressed at higher levels than is MyoD in slow muscles, whereas the opposite is true for fast muscles (13, 31). Myogenin expression is associated with expression of the slow type I myosin heavy chain isoform and the fast type IIA myosin heavy chain isoform, which has the slowest contraction speed of the fast myosin heavy chain isoforms (3). Similarly, MyoD is associated with expression of the fast type IIX and IIB myosin heavy chain isoforms (12, 13).

Various experimental treatments can alter the myosin heavy chain isoform composition in skeletal muscle. For example, clenbuterol and thyroid hormone reduce the proportion of the slow type I myosin heavy chain isoform, and they will induce the appearance of the fast type IIX myosin heavy chain isoform in the rat soleus (4, 16), which contains predominantly the slow type I myosin heavy chain isoform. Changes in functional load can also change the proportion of each myosin heavy chain isoform in the rat soleus. Hindlimb unloading reduces the functional load on the rat soleus, resulting in a reduction of muscle mass, a reduction in the proportion of the slow type I myosin heavy chain isoform (17), and the appearance of the fast type IIX myosin heavy chain isoform (17, 29). Similarly, increasing the functional load on the soleus, by surgical ablation of the gastrocnemius and plantaris, increases muscle mass and drives the myosin heavy chain proportion toward the more slow type I myosin heavy chain isoform (17, 26).

Changes in myogenin and MyoD expression levels have been observed after treatments that alter myofiber phenotype, such as cross-reinnervation (13), hormone treatment (13), and denervation (31). However, these studies (13, 31) did not fully account for the effect of the experimental treatments on functional load or muscle mass. Similarly, others have shown changes in myogenin or MyoD expression after treatments that alter muscle mass by increasing functional load (18) or by ameliorating denervation- or immobilization-induced muscular atrophy (6, 19), but these authors (6, 18, 19) did not directly examine any potential myosin heavy chain isoform transitions. Similarly, Hughes et al. (13) showed that a combination of clenbuterol and thyroid hormone would induce the appearance of the fast type IIX myosin heavy chain isoform mRNA with a corresponding appearance of MyoD mRNA in the rat soleus. However, these workers did not investigate the effect of the clenbuterol and thyroid hormone treatment on soleus muscle mass, making it possible that the appearance of MyoD may have been correlated with increased muscle mass.

The objectives of this study were to examine any potential relationships between muscle mass, functional load, myosin heavy chain isoform plasticity, myogenin expression, and MyoD expression. The rationale was to separate increases in muscle mass (hypertrophy) from changes in myosin heavy chain isoform composition to determine the variables (mass, functional load, or myosin heavy chain isoform composition) most closely associated with changes in myogenin or MyoD expression. A combination of clenbuterol and 3, 3',5-triiodo-L-thyronine (CT) treatment was utilized to increase muscle mass and increase the proportion of fast myosin heavy chain isoform in the rat soleus without altering functional load. The contralateral soleus muscles were also overloaded in CT-treated rats to increase muscle mass pharmacologically and mechanically. It was expected that increased functional load would partially counteract the CT-induced transition to a faster myosin heavy chain isoform profile and stimulate an increase in muscle mass greater than would CT treatment alone. The soleus was overloaded in non-CT-treated rats to increase muscle mass without significantly changing the myosin heavy chain isoform profile. This paradigm would show whether changes in myogenin or MyoD expression were associated with increases in muscle mass. Alternatively, if myogenin or MyoD expression were more closely associated with myofiber phenotype, then alterations in their expression would be most closely associated with CT treatment.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rats. Eighteen 3-mo-old female Charles-Dawley rats (Charles River Laboratories, Wilmington, MA) were randomly split into two groups. The first group (CT; n = 9) was supplemented with clenbuterol (10 parts/million) in their drinking water (34), and they were given subcutaneous injections of 3,3',5-triiodo-L-thyronine (350 µg/kg body wt; 27) every 48 h for 4 wk. The second group (n = 9) served as non-CT-treated controls. The right soleus muscles of all rats (n = 18) were functionally overloaded by removing the distal two-thirds of the gastrocnemius and plantaris muscles. Neither the blood nor the nervous supply to the soleus was disturbed during the surgical procedures. All surgeries took place while the rats were deeply anaesthetized (90 mg/kg body wt ketamine, 9 mg/kg body wt xylazine), and all surgical procedures were approved by the University of Wisconsin Animal Care Committee. The left soleus muscles served as nonoperated controls. Four weeks after functional overload, the rats were killed by an overdose of Beuthanasia-D (Schering-Plough Animal Health, Kenilworth, NJ; 0.25 ml/kg body wt), and both soleus muscles were removed from all rats. All muscles were weighed and frozen in liquid nitrogen.

Northern analysis. Total RNA was isolated from CT-treated (n = 4) and non-CT-treated (n = 5) rats by using Trizol (GIBCO, Grand Island, NY). RNA concentration was determined by measuring the optical density at 260 nm. Total RNA (30 µg) was fractionated through a 1% agarose-formaldehyde gel. After removal of residual formaldehyde, RNA was transferred by capillary blotting to a nylon membrane (Zetabind, Cuno Life Sciences, Meriden, CT), and it was fixed to the membrane by baking at 80°C for 2 h in a vacuum oven. Membranes were prehybridized overnight with 5× Denhardt's solution (Amresco, Solon, OH), 5× saline-sodium phosphate EDTA [SSPE; containing (in M) 0.9 NaCl, 0.05 Na2HPO4, and 0.005 EDTA, pH 7.4], 0.1% sodium dodecyl sulfate (SDS), 50% formamide, and 0.15 mg/ml denatured tRNA at 42°C. Membranes were sequentially hybridized with 32P-labeled probes synthesized from myogenin (1.4 kb; 33) and MyoD (1.8 kb; 5) cDNAs by using a random primed DNA labeling kit (Boehringer Mannheim, Indianapolis, IN). Last, membranes were hybridized with a 32P-riboprobe (316 base pairs) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ambion, Austin, TX) that was generated by using the T7 polymerase (Promega, Madison, WI). In all cases, hybridization took place overnight in 2× Denhardt's solution, 5× SSPE, 0.1% SDS, 10% dextran sulfate, 50% formamide, and 0.15 mg/ml denatured tRNA at 42°C. After hybridization, membranes were washed twice with 2× SSPE and 0.1% SDS, followed by three washes with 0.1× SSPE and 0.1% SDS. Membranes were exposed to X-ray film at -80°C, and images of the autoradiograms were acquired with a scanner (Leaf Scan 45, Leaf Systems, Southboro, MA) for densitometric evaluation. Myogenin and MyoD mRNA levels were expressed relative to GAPDH mRNA levels to account for any variations in RNA loading. The results of Tsika et al. (30) and McCarthy et al. (20) suggest that GAPDH may be under the control of elements affecting myofiber type. However, these authors did not provide any statistical analysis of their GAPDH data, making it possible that GAPDH mRNA levels were not statistically different between their treatments. In the present study, there was no difference (P > 0.05) in raw GAPDH levels between any of the treatments, suggesting that GAPDH was a suitable internal control.

Myosin heavy chain isoform separation. Myofibrils were isolated from soleus muscles of CT-treated (n = 5) and non-CT-treated (n = 4) rats by homogenizing the muscles in rigor buffer [containing (in mM) 5 KH2PO4, 75 KCl, 2 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 2 MgCl2, and 2 NaN3, pH 7.2], followed by centrifugation at 1,000 g for 10 min and resuspension in rigor buffer. The myofibrils were boiled in sample buffer [8 M urea, 2 M thiourea, 0.05 M tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 6.8), 75 mM DL-dithiothreitol, 3% SDS, and 0.05% bromophenol blue; (8)] for 3 min at a final protein concentration of 0.125 mg/ml. Total protein was determined by using the bicinchoninic acid protein assay (Sigma Chemical, St. Louis, MO).

Myosin heavy chain isoforms were separated by using procedures modified from Talmadge and Roy (28). The stacking gels were composed of 2.56% acrylamide, 0.45% N, N'-diallyltartardiamide, 10% glycerol, 0.1 M Tris · HCl (pH 6.8), 0.1% SDS, 0.05% ammonium persulfate, and 0.5% N, N, N', N'-tetramethylethylenediamine (8). The acrylamide stock solution for the separating gels was composed of 29.4% acrylamide and 0.6% N, N'-methylenebisacrylamide. The separating gels were composed of 30% glycerol, 8% total acrylamide, 0.2 M Tris (pH 8.8), 0.1 M glycine, 0.4% SDS, 0.03% ammonium persulfate, and 0.1% N, N, N', N'-tetramethylenediamine. The upper running buffer consisted of 0.1 M Tris (base), 150 mM glycine, 0.1% SDS, and 7.5 mM beta -mercaptoethanol. The lower running buffer was composed of 0.05 M Tris (base), 75 mM glycine, and 0.05% SDS. Each lane was loaded with 0.5 µg of protein, and the gels were run in a Hoefer SE280 electrophoresis unit (San Francisco, CA) at 70 V (constant voltage) in a cold room (4°C) for 24 h.

After electrophoresis, the gels were silver stained (9), scanned with an imaging densitometer (model GS-670, Bio-Rad, Hercules, CA), and evaluated by integrating the area under each myosin heavy chain isoform peak. The area under each peak was expressed as a percentage of the total area under all myosin heavy chain isoform peaks.

Statistical analysis. Data were analyzed by using the general linear models procedure of SAS (25). Least squares means were separated on the basis of least significant differences (23). If population variances were found to be unequal, a logarithmic transformation was performed on the data before analysis. The lowest level of significance accepted was P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Body and muscle weights. CT- and non-CT-treated rats weighed the same on the day of surgery, but CT-treated rats weighed significantly more than did non-CT-treated rats at the conclusion of the experimental treatment (Table 1), indicating that CT treatment enhanced growth. Similarly, soleus muscles from CT-treated rats weighed significantly more than did soleus muscles from non-CT-treated rats (Table 2), indicating that CT promoted skeletal muscle growth. Functional overload also promoted skeletal muscle growth in the soleus because overloaded muscles weighed significantly more than those from the contralateral side (Table 2).

                              
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  		Table 1.   Body weights of CT-treated and non-CT-treated rats at beginning and end of 4-wk CT treatment period

                              
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  		Table 2.   OV and NO soleus muscle weights for CT-treated and non-CT-treated rats

Myogenin and MyoD expression. Functional overload of the soleus did not alter myogenin (Figs. 1 and 2) or MyoD expression (Figs. 1 and 3), but it increased muscle mass (Table 2). MyoD was never detected in soleus muscles from non-CT-treated rats. However, CT treatment induced detectable levels of MyoD, and it significantly reduced myogenin expression (expressed relative to GAPDH levels; Figs. 1 and 2).


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  		Fig. 1.   Myogenin and MyoD expression in clenbuterol and 3,3',5-triiodo-L-thyronine (CT)- treated and in non-CT-treated rats. Representative Northern for myogenin, MyoD, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Lane 1, CT + nonoverload; lane 2, CT + overload; lane 3, nonoverload; lane 4, overload.


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  		Fig. 2.   Myogenin expression relative to GAPDH expression in soleus muscles from CT-treated (n = 4) and non-CT-treated (n = 5) rats. Values are means ± SE.


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  		Fig. 3.   MyoD expression relative to GAPDH expression in soleus muscles from CT-treated (n = 4) rats. Values are means ± SE.

Myosin heavy chain isoform composition. Functional overload did not alter myosin heavy chain isoform composition in non-CT-treated rats (Figs. 4 and 5). Soleus muscles from CT-treated rats had a significantly smaller proportion of the slow type I myosin heavy chain isoform than did similarly treated (overloaded or nonoverloaded) muscles from non-CT-treated rats (Figs. 4 and 5). Similarly, the fast type IIX myosin heavy chain isoform was detected in soleus muscles from CT-treated rats but not in soleus muscles from non-CT-treated rats. Functionally overloaded soleus muscles from CT-treated rats had a significantly higher proportion of slow type I myosin heavy chain isoform than nonoverloaded contralateral control muscles (Figs. 4 and 5). The proportion of the slow type I myosin heavy chain isoform in overloaded soleus muscles from CT-treated rats was the same as in the nonoverloaded soleus muscles from non-CT-treated rats.


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  		Fig. 4.   Myosin heavy chain isoform distribution in soleus muscles from CT- treated and non-CT-treated rats. Representative SDS polyacrylamide gel illustrates myosin heavy chain isoform distribution (I, IIA, IIX) in soleus muscles from CT-treated rats (lane 1, nonoverload; lane 2, overload) and non-CT-treated (lane 3, nonoverload; lane 4, overload) rats. Samples from muscles containing detectable levels of the fast type IIB myosin heavy chain isoform (diaphragm) produced a band that migrated an approximately equal distance between type IIX and type I bands (data not shown).


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  		Fig. 5.   Myosin heavy chain (MHC) isoform distribution in nonoverloaded (NO) and overloaded (OV) soleus muscles from CT-treated (n = 5) and non-CT-treated (n = 4) rats. Amount of each myosin heavy chain isoform is expressed as percentage of total MHC. Values are means ± SE. Bars within each MHC isoform group (type I, IIA, IIX) with different superscript are significantly different (P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Clenbuterol and thyroid hormone affect skeletal muscle growth through alterations in protein degradation pathways (1, 2, 24). Clenbuterol depresses rates of protein degradation to promote protein accretion in skeletal muscle (2, 24), whereas thyroid hormone increases rates of protein degradation without altering rates of protein synthesis to cause muscular atrophy (1). It has been postulated that thyroid hormone administration counteracts the anabolic effects of clenbuterol (13). However, CT had an anabolic effect on soleus muscles (Table 2) that likely occurred through a decrease in the rate of protein degradation. Similarly, functional overload had an anabolic effect on soleus muscles (Table 2) that likely occurred through an increase in the protein synthetic rate (22).

Myogenin and MyoD expression in mature skeletal muscle may help regulate protein synthesis and degradation because they have been suggested to be factors potentially influencing mature muscle size (18). If myogenin or MyoD were involved in regulating net protein synthesis or degradation pathways, they would exhibit expression levels that were proportional to the altered muscle mass after experimental manipulations, such as increased or decreased functional load. However, in this study, it appears that myogenin and MyoD did not play an active role in governing mature muscle mass, because nonoverloaded soleus muscles from CT-treated rats weighed the same (Table 2) as did overloaded muscles from non-CT-treated rats, but each group had different levels of myogenin and MyoD mRNA (Figs. 1 and 2). The soleus muscle weight-to-body weight ratio was higher for overloaded muscles from non-CT-treated rats than for nonoverloaded muscles from CT-treated rats (Table 2), but the higher ratio was not associated with any change in myogenin or MyoD levels. Increasing functional load on the soleus had no effect on myogenin (Figs. 1 and 2) or MyoD (Figs. 1 and 3) expression because overloaded muscles had the same levels of myogenin and MyoD as did nonoverloaded muscles. Thus myogenin and MyoD mRNA levels seem to have little association with alterations in net rates of protein synthesis or degradation because alterations in their expression were not correlated with changes in muscle weight.

Myogenin and MyoD mRNA levels may be associated with the regulation of the synthesis of specific proteins. Myogenin is expressed at higher levels than is MyoD in predominantly slow muscles of mature animals, whereas MyoD is expressed at higher levels than is myogenin in predominantly fast muscles of mature animals (13, 31), suggesting that myogenin and MyoD mRNA may be related to the synthesis of proteins related to myofiber type (fast or slow; 10). For example, myogenin expression is associated with myofibers containing the slow type I and fast type IIA myosin heavy chain isoforms, whereas MyoD expression is associated with myofibers containing the fast type IIX and IIB myosin heavy chain isoforms (12, 13). Clenbuterol and thyroid hormone singly (4, 16) or in combination (Figs. 4 and 5) decrease the proportion of the slow type I myosin heavy chain isoform and induce the appearance of the fast type IIX myosin heavy chain isoform in the rat soleus, suggesting that they promote the synthesis of the fast type IIX myosin heavy chain isoform without increasing the overall rate of protein synthesis (2, 24).

In the present study, myogenin expression decreased and MyoD mRNA appeared after CT treatment. The alterations in myogenin and MyoD expression were coincident with the appearance of the fast type IIX myosin heavy chain isoform; this suggests that myogenin and MyoD mRNA may play a role in governing the synthesis of proteins specific to myofiber type. The present studies confirm the findings of Hughes et al. (13), who showed that CT induced the expression of the fast type IIX myosin heavy chain isoform with a corresponding increase in MyoD expression. However, Hughes et al. did not find any alterations in slow type I myosin heavy chain isoform expression or myogenin expression. The discrepancies between these studies could be related to the more potent CT treatment used in the present study. The present findings extend the studies of Hughes et al. because myogenin or MyoD expression was not associated with alterations in functional load or muscle size but only with myosin heavy chain isoform plasticity.

A simple relationship between myogenin and MyoD expression and expression of a specific myosin heavy chain isoform does not fully account for all changes observed in this study. Functionally overloaded soleus muscles from CT-treated rats had significantly more slow type I myosin heavy chain than did contralateral control muscles; this suggests that overload partially counteracted the effect of CT on myosin heavy chain isoform transitions. However, there were no differences in myogenin or MyoD expression between overloaded and nonoverloaded soleus muscles from CT-treated rats. Furthermore, overloaded muscles from CT-treated rats had the same amount of slow type I myosin heavy chain as did nonoverloaded soleus muscles from non-CT-treated rats, but there were differing levels of myogenin between the groups. It would be expected that slow type I myosin heavy chain isoform levels would be correlated with changes in myogenin expression, but it is possible that the decreased myogenin expression may be related to the appearance of the fast type IIX myosin heavy chain isoform in soleus muscles from rats treated with CT.

The appearance of MyoD was correlated with the appearance of the fast type IIX myosin heavy chain isoform in the CT-treated rats. Similarly, the lower myogenin expression levels in CT-treated rats compared with non-CT-treated rats was also correlated with the appearance of the fast type IIX myosin heavy chain isoform, suggesting that there may not be a simple relationship between elevated MyoD expression levels and the appearance of the fast type IIX myosin heavy chain isoform. Similarly, other authors have suggested that myogenin and MyoD expression may not have a simple relationship to myofiber type (11, 14, 15, 21). It is possible that ratios of various myogenic regulatory factors are more important in modulating myofiber type (myosin heavy chain isoform distribution) in a mature muscle than are changes in a single myogenic regulatory factor.

It is clear from the present study that CT increased soleus muscle mass, induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced the appearance of MyoD. Similarly, it is clear that overload increased soleus muscle mass but did not change myogenin or MyoD expression or induce the appearance of the fast type IIX myosin heavy chain isoform. Thus it appears that myogenin and MyoD mRNA levels are more associated with alterations in myosin heavy chain isoform composition than alterations in muscle mass or functional load. However, the data indicate that further study is needed concerning the interactive roles of myogenin and MyoD in myosin heavy chain isoform plasticity.

    ACKNOWLEDGEMENTS

The authors thank Dr. James Ervasti for use of the imaging densitometer.

    FOOTNOTES

This work was supported by the National Aeronautics and Space Administration (NASA) Space Biology Research Associates program (P. E. Mozdziak), the Charles River Laboratories Animal Gift Program (P. E. Mozdziak), US Dept. of Agriculture/National Research Initiative Competitive Grants Program Grant 96-35206-3524 (P. E. Mozdziak), and NASA Grant NAG2-671 (E. Schultz).

Address for reprint requests: P. E. Mozdziak, Dept. of Anatomy, 1300 Univ. Ave., Madison, WI 53706 (E-mail: pemozdzi{at}facstaff.wisc.edu).

Received 17 September 1997; accepted in final form 19 December 1997.

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Abstract
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Materials & Methods
Results
Discussion
References

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J APPL PHYSIOL 84(4):1359-1364
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