Effect of anabolic/androgenic steroids on myosin heavy chain expression in hindlimb muscles of male rats. Accepted: 27 July 1999. Abstract We have examined ...
Eur J Appl Physiol (2000) 81: 155±158
Ó Springer-Verlag 2000
SHORT COMMUNICATION
Philippe Noirez á Arnaud Ferry
Effect of anabolic/androgenic steroids on myosin heavy chain expression in hindlimb muscles of male rats
Accepted: 27 July 1999
Abstract We have examined the eect of male sex hormones on the myosin heavy chain (MHC) expression of the soleus and extensor digitorum longus (EDL) muscles. Young male adult Wistar rats were treated over a 25-day period with either oil (CON, n = 8), nandrolone (nortestosterone decanoate, NAN, n = 8), nandrolone combined with endurance exercise (treadmill running, NAN+EXE, n = 8), or were castrated (CAS, n = 8). The MHC composition of the soleus and EDL muscles was measured by electrophoresis. Castration and treatment with nandrolone had no eect (P > 0.05) on the relative levels of MHC in the soleus and EDL. In contrast, in NAN+EXE rats, the relative level of MHC-1 increased [slow isoform; mean (SD) 97.6 (4.7)% in NAN+EXE rats, versus 86.5 (7.5)% in CON rats] and MHC-2a decreased [fast isoform; 2.4 (4.7)% in NAN+EXE, versus 13.5 (7.5)% in CON rats], only in the soleus (P < 0.05). In conclusion, it appears that endogenous anabolic/androgenic steroids are not essential for the maintenance of the MHC expression of fast- and slow-twitch muscles in the young adult male rat. In addition, nandrolone combined with endurance exercise induced a shift from a fast to a slower MHC phenotype of the slow-twitch muscle. Key words Exercise á Male sex hormones á Muscle á Myosin heavy chain á Nandrolone
P. Noirez á A. Ferry (&) Laboratoire de Physiologie des Adaptations, Faculte de MeÂdecine Cochin-Port Royal, Universite Rene Descartes, 24 Rue du Faubaurg Saint-Jacques, F-75014, Paris, France Fax: +33-1-44412318 A. Ferry Unite de Formation et de Recherche en Sciences et Techniques des ActiviteÂs Physiques et Sportives, Universite Rene Descartes, Paris, France
Introduction Unlike thyroid hormones (reviewed in D'Albis and Butler-Browne 1993; Vigneron et al. 1989), the in¯uence of male sex hormones on the phenotype (e.g. ®bretype distribution and protein isoform composition) of hindlimb muscles in young adult subjects is not clear. Much attention has focused on the eects of anabolicandrogenic steroids (AAS) on muscle growth. In the case of female sex steroids, it was found very recently that ovariectomy did not alter the percentage of type I ®bres in the plantaris (a fast-twitch muscle) of female rats, but increased that of the soleus (a slow-twitch muscle, Fisher et al. 1998). Chronic AAS administration did not alter the myosin expression in the plantaris and soleus of female rats (Tsika et al. 1987). However, some studies have indicated that this treatment modulated the distribution of ®bre types in the extensor digitorum longus (EDL, a fast-twitch muscle), soleus and red gastrocnemius of female rats (Egginton 1987; Holmang et al. 1990), but this eect is not always consistent (Bisschop et al. 1997), Thus, there is a need for further inquiry into the eects of decreased and increased levels of AAS on the phenotype of hindlimb muscles in the adult animal. To our knowledge, no study had analysed the eects of AAS (deprivation and administration) on the myosin heavy chain (MHC) expression in both fast- and slow-twitch muscles of the young adult male rat. In the present study, we determined the eect of castration on the expression of the dierent MHC isoforms in the EDL and soleus of young adult male rats. Are endogenous male sex hormones essential for the maintenance of the MHC phenotype in hindlimb muscles, as is the case in sexually dimorphic muscles and the heart (D'Albis and Butler-Browne 1993; Lengsfeld et al. 1988; Lyons et al. 1986; Scheuer et al. 1987; Vigneron et al. 1989)? Some gender dierences have been reported concerning the ®bre-type distribution of hindlimb muscles of young adult animals (Bisschop et al. 1997;
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and reviewed in Vigneron et al. 1989) and humans (Simoneau and Bouchard 1989). For instance, the proportion of type IIa ®bres in the gastrocnemius was greater in male compared with female rats (Bisschop et al. 1997). It was also found that the proportion of type I ®bres in the vastus lateralis was lower in men than in women (Simoneau and Bouchard 1989). We also studied the eect of treatment with nandrolone (nortestosterone), and nandrolone administration combined with physical exercise (treadmill running) on MHC phenotype. We wanted to examine whether AAS administration could have some impact on the MHC expression in sedentary or exercised intact (uncastrated) animals. AAS can be used as a medication or drug with the intention of improving physical performance (e.g. in athletes). Electrophoretic measurement of MHC provided a sensitive assessment of muscle phenotype.
Methods Experimental groups Normal and castrated young adult male Wistar rats (body mass: 40±160 g, age 6±7 weeks) were purchased from Ia-Credo (Les oncins, France). The animals were cared for according to the Helsinki agreement for the human treatment of animals during experimentation. Rats were assigned to one of four groups (eight rats/group): (1) normal control (CON), (2) castrated (CAS), (3) treated with nandrolone (NAN), and (4) treated with nandrolone and endurance exercised (NAN+EXE). Gonadectomy was performed 1 week before the beginning of the experiment. At the time of animal sacri®ce, the adequacy of the gonadectomy was determined grossly according to the absence of testicular tissue. Nandrolone decanoate (given intramuscularly 2 mg á kg)1, Sigma; Bisschop et al. 1997) or nandrolone vehicle (oil) was administered to NAN or CON and CAS rats, respectively, on a weekly basis until the rats were sacri®ced (25 days after the ®rst injection of oil or NAN). The exercise program (endurance) began 1 day postinjection and stopped 1 day before the rats were sacri®ed. The exercised animals ran on a motorized treadmill (10% slope) for
Table 1 Relative levels of myosin heavy chain (MHC) isoforms in the extensor digitorum longus (EDL; percentage of the total) of rats treated with oil (controls, CON), castration (CAS), nandrolone (NAN) or NAN combined with endurance exercise (NAN+EXE)
Table 2 Relative levels of MHC subunits in the soleus (percentage of the total) of rats treated with oil (CON), CAS, NAN, or NAN + EXE
60 min á day)1. The intensity of exercise was increased progressively (10±42 m á min)1). MHC protein analysis (electrophoresis) Twenty-®ve days after the ®rst injection, the animals were weighed, anaesthetized (chloral hydrate, 0.4 g á kg)1), and the left soleus and EDL muscles were exercised and cleaned of adipose and connective tissues. The muscles were rapidly weighed. Each sample was frozen in liquid nitrogen and stored at )80°C until assayed. Samples of the individual muscles were homogenized in 50 mmol á l)1 K2HPO4, 0.1 mol á l)1 KH2P4, and 0.3 mol á l)1 KCl buer solution. After stirring for 15 min on ice, the homogenates were centrifuged at 1000 g for 15 min at 4°C. Aliquots of the supernatant fractions were combined with an equal volume of glycerol and then stored at )20°C until electrophoresis of MHC proteins. Vertical sodium dodecyl sulfate and polyacrylamide gel electrophoresis was performed using the mini-protean II electrophoresis cell (Biorad). The homogenates were combined with an equal volume of denaturing buer (Laemmli buer, Biorad) and boiled for 3 min. Separation of the dierent isoforms of MHC (types: 2a, 2x/d, 2b and 1) was performed according to Talmadge and Roy (1993). For each sample, 10 lg of protein (Biorad, protein assay) were loaded per lane. The gels contained 30% glycerol. The stacking and separation gels contained 4% and 8% acrylamide-bis, respectively. The temperature was maintained below 10°C for the duration of the electrophoretic run (2 h at 70 V and then 22 h at 100 V). The gels were stained with 0.25% (w/v) Coomassie blue (R250) in 50% (v/v) methanol and 10% (v/v) acetic acid. The relative amounts of MHC proteins (percentage of the total) were determined by densitometry (Biopro®l, Vilber Lourmat, Marne la ValleÂe, France). Stastistical analysis Data were analysed using a one-way analysis of variance. Mean comparisons were determined by a post-hoc test (Schee).
Results There was no signi®cant dierence between CAS and CON rats with respect to the MHC expression in both the EDL and soleus muscles (Tables 1 and 2; P > 0.05).
MHC-2b
CON (n = 8) CAS (n = 8) NAN (n = 8) NAN+EXE (n = 8)
MHC-2a
MHC-1
Mean
SD
Mean
SD
Mean
SD
Mean
SD
55.7 62.6 57.7 52.0
6.7 15.0 7.8 15.0
26.8 25.7 25.6 28.4
5.7 10.5 6.6 5.4
12.0 9.3 12.6 15.5
4.1 5.4 1.8 5.0
5.5 2.4 4.2 4.0
1.9 3.4 6.6 9.5
MHC-2b
CON (n = 8) CAS (n = 8) NAN (n = 8) NAN+EXE (n = 8)
MHC-2x/2d
MHC-2x/2d
MHC-2a
MHC-1
Mean
SD
Mean
SD
Mean
SD
Mean
SD
0.0 0.0 0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0 0.7 0.0
0.0 0.0 1.9 0.0
13.5 12.2 15.3 2.4
7.5 11.7 8.7 4.7*,**
86.5 87.8 84.0 97.6
7.5 11.7 8.7 4.7*,**
* Signi®cantly dierent from CON rats (P < 0.05) ** Signi®cantly dierent from NAN rats (P < 0.01)
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Moreover, nandrolone treatment had no eect on the expression of MHC either the EDL or soleus (P > 0.05; Tables 1 and 2). MHC expression in the EDL (Table 1) was not changed in the NAN+EXE rats (P > 0.05). In contrast, in NAN+EXE rats the expression of MHC in the soleus was altered (Table 2). In fact, the relative levels of type 2a MHC (MHC-2a) and MHC-1 were, respectively, decreased and increased in the NAN+EXE soleus compared to the CON soleus (P < 0.05). Moreover, the relative levels of MHC-1 and MHC-2a were dierent between the NAN+EXE soleus and NAN soleus (P < 0.01; Table 2).
Discussion In the case of well-known sexually dimorphic muscles and the heart, it has been established that male sex steroids modulate muscle phenotype in pre- and postpubertal animals (D'Albis and Butler-Browne 1993; Lengsfeld et al. 1988; Lyons et al. 1986; Scheuer et al. 1987; Vigneron et al. 1989). In contrast, to our knowledge, information is lacking concerning the eect of decreased and increased levels of AAS on the expression of the dierent isoforms of MHC in the hindlimb muscles of young adult male rats. The ®rst signi®cant ®nding of the present study was that castration did not in¯uence the MHC composition of the EDL and soleus. Concerning the fast-twitch muscle (EDL), our observations agree well with the results of the few studies in which phenotypic changes of hindlimb muscles of young adult animals to gonadectomy were investigated (Boissonneault et al. 1989; Fisher et al. 1998). In fact, gonadectomy did not alter the expression of type 1s myosin light chain in the plantaris of male rats (Boissonneault et al. 1989) or the ®bre-type distribution in the plantaris of female rats (Fisher et al. 1998). Castration is known to lower the circulating levels of testosterone (Boissonneault et al. 1989; Lengsfeld et al. 1988). The results of the present and previous studies (Boissonneault et al. 1989; Fisher et al. 1998) suggest that normal levels of endogenous male or female sex steroids are not essential for the maintenance of the phenotype of hindlimb fast-twitch muscles of young adult male or female rats. One can also note that castration at the end of weaning did not modify the ®bre-type distribution in the EDL of male guinea pigs (Beaune et al. 1994). However, it was found that prebubertal castration caused a decrease in the proportion of type IIa ®bres in the white gastrocnemius of male rats (Krotkiewsky et al. 1980). Concerning the slow-twitch muscle (soleus), Fisher et al. (1998) found that ovariectomy increased the percentage of type I ®bres from 88% to 94±95% (in the present study we found no signi®cant eect of castration). It is possible that male and female steroids have dierent eects of muscle phenotype. The determination
of ®bre type in that study (ATPase-staining) could raise some questions since, in the same study, Fisher et al. (1998) did not demonstrate the usual decreased percentage of type I ®bres induced by hindlimb unloading (decreased mechanical loading). However, the increased percentage of type I ®bres (which express MHC-1) agrees with the result showing that castration increases the expression of cardiac MHC-b (MHC-1) or type V3 myosin (b-homodimer) in male rats (Lengsfeld et al. 1988; Scheuer et al. 1987). The results of the present study indicate that treatment with a high therapeutic dose (2 mg á kg)1 á week)1, Bisschop et al. 1997) of nortestosterone (nandrolone decanoate) over a 25-day period did not modify muscle phenotype. This result agrees well with those of Tsika et al. (1987) and Bisschop et al. (1997). Taken together, these observations suggest that nandrolone treatment does not alter the ®bre-type distribution (Bisschop et al. 1997), myosin (Tsika et al. 1987) and MHC expression (the present study) in the fast- and slow-twitch muscles of the male (the present study) and female rat (Bisschop et al. 1997; Tsika et al. 1987) hindlimbs. Once more, hindlimb muscles appear to be less sensitive to AAS compared to sexually dimorphic muscle or the heart (D'Albis and Butler-Browne 1996; Lengsfeld et al. 1988; Lyons et al. 1986; Morano et al. 1990; Scheuer et al. 1987; Vigneron et al. 1989). The reasons underlying the discrepancy between these observations (the present study; Bisschop et al. 1997; Tsika et al. 1987) and those of other previous studies (Egginton 1987; Holmang et al. 1990) showing that AAS treatment modulated the ®bre-type distribution in hindlimb muscles are not known. Egginton (1987) and Holmang et al. (1990), respectively, indicated that the percentages of slow/oxidative-type ®bres (type I ®bres) in the EDL and soleus were lower in nandrolone-treated female (intact) rats (versus untreated rats), and the percentage of type II ®bres in the red gastrocnemius and soleus were increased by testosterone in (oophorectomized) female rats. The discrepancy between the relative levels of protein isoforms in muscle homogenate (the present study; Tsika et al. 1987) and ®bre-type distribution (Egginton 1987; Holmang et al. 1990) does not seem to be attributable to the fact that type I or slow/oxidative ®bres hypertrophied more than type II ®bres with AAS treatment, since this was not the case (Bisschop et al. 1997; Egginton 1989). It should be noted that the results of Egginton (1987) and Holmang et al. (1990) suggest that the expression of hindlimb muscle MHC-1 decreases with AAS. This suggestion agrees with the results showing that testosterone treatment of castrated male rats favours the appearance of ventricular MHC-a at the expense of MHC-b (MHC-1): pre-translational regulation (Morano et al. 1990). The second important ®nding of this study was that, contrary to nandrolone treatment alone, MHC expression in the soleus is altered by nandrolone treatment combined with endurance exercise in NAN+EXE
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rats, the relative level of MHC-1 increased (+13%) and that of MHC-2a decreased ()82.2%). Moreover, the relative amounts of MHC-1 and MHC-2a were increased or decreased, respectively, in the soleus of NAN+EXE rats compared to NAN rats (Table 2). Since nandrolone (the present study; Bisschop et al. 1997; Tsika et al. 1987) or exercise alone (Sullivan et al. 1995) were without eect on the phenotype of the soleus, the observations suggest that nandrolone ``potentiated'' the eect of exercise, or that there is an eect of nandrolone only in the exercised soleus. However, one group of rats is missing in the present study (exercised rats without nandrolone treatment), which is required in order to conclude whether exercise and nandrolone treatment have any additive role. It is possible that the EDL was less aected by this treatment (versus the soleus) because it is recruited less during treadmill running. The increased relative expression of MHC-1 in the soleus of NAN+EXE rats could explain the result that AAS administration increased running endurance in rats (Van Zyl et al. 1995), since slow-twitch ®bres (expressing MHC-1) are more fatigue resistant. In conclusion, the present and previous studies have demonstrated that the eects of AAS on hindlimb muscle phenotype are variable. Bisschop et al. (1997) emphasized that comparison of the results in the literature is rather dicult because the eects of AAS are in¯uenced by a number of dierent parameters (e.g. age, androgen dose, mode of administration, and duration of treatment). This is also our opinion. The data of the present study do not suggest a role for endogenous male sex hormones in the maintenance of MHC phenotype in the hindlimb muscles of the young adult male rat. Moreover, only nortestosterone combined with endurance exercise altered the MHC phenotype of the slowtwitch muscle. The eects of long-term treatments and higher doses of nandrolone remain to be determined. Globally, we noted that AAS deprivation and administration had a limited in¯uence on the MHC expression of the skeletal muscles of young adult rats compared to that of well-known sexually dimorphic muscles and the heart. It would be interesting to investigate this musclespeci®c regulation of the MHC gene expression induced by male sex hormones. Acknowledgements We thank Christine Le Page (University of Paris V) for critical reading and Imed Ben Salah (University of Paris V) for help throughout the study.
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