effects of anabolic/androgenic steroids on regenerating skeletal muscles in the rat

Effects of anabolic/androgenic steroids on regenerating

skeletal muscles in the rat

A . F E R R Y , 1 , 2 P . N O I R E Z , 1 C . L E P A G E , 1 , 2 I . B E N S A L A H , 1 D . D A E G E L E N 3 and

M . R I E U 1

1 Laboratoire de Physiologie des Adaptations, FaculteÂ de MeÂdecine Cochin-Port Royal, UniversiteÂ ReneÂ Descartes, Paris, France

2 UniteÂ de formation et de Recherche en Sciences et Techniques des ActiviteÂs Physiques et Sportives, UniversiteÂ ReneÂ Descartes, Paris,

France

3 Institut National de la Recherche Scienti®que et MeÂdicale, UniteÂ 129, Paris, France

ABSTRACT

We have examined the effect of male sexual hormones on the regeneration of skeletal muscles.

Degeneration/regeneration of the left soleus and extensor digitorum longus muscles (EDL) of Wistar

male rats was induced by an injection of snake venom (2 lg, Notechis scutatus scutatus). During the

muscle regeneration (25 days), rats were treated with either oil (CON), nandrolone (NAN), NAN

combined with exercise (NAN + EXE) or were castrated (CAS). Muscle growth and myosin heavy

chain (MyHC) isoform content of regenerating muscles were studied. Castration altered the

concentrations of MyHC in venom-treated EDL (P < 0.01) and soleus (P < 0.05). NAN increased the

mass (P < 0.01) of regenerating soleus and decreased the relative amount of fast MyHC protein

(% of total, P < 0.05). The effect of NAN + EXE on the fast MyHC proteins of venom-treated soleus

was opposite (P < 0.05). NAN and NAN + EXE were without effect on the regenerating EDL

(P > 0.05). In conclusion, it is possible that male sexual hormones play a role in the growth (synthesis

of contractile proteins) of regenerating muscles in rat. In addition, contrary to NAN + EXE, NAN could

be bene®cial to soleus regeneration.

Keywords male sexual hormone, myogenesis, myosin heavy chain mRNA and protein, nandrolone,

skeletal muscle regeneration.

Received 16 October 1998, accepted 9 March 1999

Mature skeletal muscle is able to regenerate from muscle

precursor cells (mpc), i.e. satellite cells, which proliferate

and fuse together. This de novo formation of myo®bres

that reproduces ontogenetic development is under the

in¯uence of physiological factors that can be experi-

mentally decreased or increased. Recent studies have

demonstrated that denervation, hypodynamia±hypo-

kinesia (decreased mechanical load) and hypothyroidism

inhibit growth and maturation of regenerating muscle

compared with normal physiological conditions (D'Al-

bis et al. 1987, 1988, Whalen et al. 1990, Esser et al. 1993,

Esser & White 1995, Devor & White 1995, Bigard et al.

1996a, 1997). For instance, experimental hypothyroid-

ism (administration of an antithyroid drug) results in a

decrease in the mass of regenerating fast-twitch muscle

and in the expression of mature myosin or myosin heavy

chain (MyHC) proteins (D'Albis et al. 1987, Devor &

White 1995). These results suggest that normal inner-

vation, mechanical load and levels of thyroid hormones

are needed for a complete muscle regeneration.

However, whether a normal level of endogenous male

sexual hormones is also required for complete

myogenesis in vivo is not known, which also raises the

question of whether these are physiological determi-

nants of muscular regeneration. It was previously shown

that they can in¯uence myogenesis in vitro (see below).

It has also been reported that experimental increases

in the mechanical load (running on a treadmill) and in

the levels of thyroid hormones (administration of thy-

roid hormones) affect muscular regeneration (D'Albis

et al. 1987, Devor & White 1995, Esser & White 1995,

Bigard et al. 1996b, Ferry et al. 1997). For instance,

administration of thyroid hormone (hyperthyroidism)

markedly increases the expression of MyHC-2x/d and

Correspondence: Prof. Arnaud Ferry, Laboratoire de Physiologie des Adaptations, FaculteÂ de MeÂdecine Cochin-Port-Royal, UniversiteÂ ReneÂ

Descartes, 24, rue du Faubourg Saint-Jacques 75014 Paris, France.

Acta Physiol Scand 1999, 166, 105±110

Ó 1999 Scandinavian Physiological Society 105

MyHC-2b proteins (fast MyHC) of regenerating soleus

(Devor & White 1995). MyHC phenotype of regener-

ating EDL is not so extensively affected by thyroid

hormone administration (Devor & White 1995).

However, the effects of other hormonal treatments

such as the administration of male sexual hormones on

regenerating muscles are not known. Could they im-

prove muscle regeneration in vivo? It has been previ-

ously observed that steroids could in¯uence

myogenesis in vitro (Powers & Florini 1975, Doumit

et al. 1996). Indeed, anabolic/androgenic steroids in-

crease the proliferation of satellite cells in vitro (Powers

& Florini 1975) but this effect is not always consistent

(Doumit et al. 1996). The effect of these steroids on the

maturation of myotubes is not obvious, as a decrease in

differentiation (Doumit et al. 1996) or no effect

(Thomson et al. 1989) have been reported.

This study is the ®rst to determine the effects of

castration (decreased levels of male sexual hormones)

on the regeneration of male rat soleus and EDL. We

investigate whether endogenous anabolic/androgenic

sexual steroids play a role in skeletal muscle regenera-

tion. In the context of rehabilitation after muscle de-

generation, we also wanted to see whether a treatment

by nandrolone (nortestosterone), nandrolone adminis-

tration combined with muscular exercise (treadmill

running) could in¯uence growth and maturation of

regenerating muscle. It has been suggested that physical

exercise potentiates the effect of anabolic/androgenic

treatment on muscle (Eggerton 1987). Muscular de-

generation was induced by necrosis of all ®bres after

injury by snake venom (Notechis scutatus scutatus), fol-

lowed by a relatively rapid (3 weeks) and complete re-

generative process (D'Albis et al. 1987, 1988, Whalen

et al. 1990, Bigard et al. 1996a, b, 1997, Ferry et al. 1997).

At the end of the experimental period (25 days after

venom treatment), we measured some aspects of

muscle growth (muscle mass, protein and MyHC con-

centrations) and maturation (relative amounts of

MyHC) of the regenerating muscles in the different

treatments.

MATERIALS AND METHODS

Animals

Normal and castrated male Wistar rats, ranging in body

weight from 130 to 160 g (aged 6±7 weeks) were pur-

chased from Iffa-Credo (Les oncins, France). The ani-

mals were cared for according to the Helsinki

agreement for human treatment of animals during ex-

perimentation. Rats were housed in a thermoneutral

environment (21 °C) and were allowed free access to

food and water. After 5±6 days of acclimatization, the

animals were anaesthetized with chloral hydrate

(0.4 g kg±1) and the left soleus and EDL muscles were

injected with (2 lg) of snake venom (Notechis scutatus

scutatus, V-0251, Sigma, La VerpillieÁre, France).

Rats were assigned to four groups (eight rats per

group): (1) normal control (CON), (2) castrated (CAS),

(3) treated by nandrolone (NAN), (4) treated by nan-

drolone and endurance exercised (NAN + EXE).

Nandrolone decanoate (im, 2 mg kg±1; Sigma, La Ve-

rpillieÁre, France) or nandrolone vehicle (oil) was ad-

ministered to NAN or CON and CAS rats 1 day after

the venom injection, on a weekly basis, until the rats

were killed. The exercise programme began 1 day post-

injection and stopped 1 day before the rats were killed.

The exercised animals ran 23 times (once a day) on a

motorized treadmill (10% slope) for 60 min (the bouts

of exercise were not exhausting). The intensity of ex-

ercise was progressively increased (10±42 m min±1). At

the 23rd bout of exercise, the rats ran 2370 m (15 min

at 32 m min±1 and 45 min at 42 m min±1).

Tissue processing

Twenty-®ve days after the venom injection, the

animals were weighed (CON � 351.9 � 27.9 g,

CAS � 320.0 � 24.2 g, NAN � 359.4 � 21.3 g, NAN

+ EXE � 316.2 � 14.3 g) anaesthetized (chloral hy-

drate, 0.4 g kg±1) and the left (venom-treated) soleus

and EDL muscles were excised and cleaned of adipose

and connective tissues. Muscles were rapidly weighed.

Each sample was frozen in liquid nitrogen and stored at

±80 °C until assayed.

MyHC protein analysis (electrophoresis)

Samples of the individual muscles were homogenized in

50 mM K2HPO4, 0.1 M KH2P4, and 0.3 M KCl buffer

solution. After stirring for 15 min on ice, the homog-

enates were centrifuged at 1000 g for 15 min (4 °C).

Aliquots of the supernatant fractions were combined

with an equal volume of glycerol and then stored at

±20 °C until electrophoresis of MyHC proteins.

Vertical sodium dodecyl sulphate electrophoresis

was performed using the mini-protean II electropho-

resis cell (Biorad). The homogenates were combined

with an equal volume of denaturing buffer (Laemmli

buffer, Biorad) and boiled for 3 min. Separation of

MyHC proteins (2a, 2x/d, 2b, 1) was performed ac-

cording to Talmadge & Roy (1993). For each sample,

10 lg of protein were loaded per lane. The gels con-

tained 30% glycerol. The stacking and separation gels

contained, respectively, 4 and 8% acrylamide±bis. The

temperature was maintained below 10 °C for the du-

ration of the electrophoretic run (2 h at 70 V and then

22 h at 100 V). Moreover, determination of the con-

centration of total MyHC (MyHC-all; the MyHCs

Male steroids and muscle regeneration � A Ferry et al. Acta Physiol Scand 1999, 166, 105±110

106 Ó 1999 Scandinavian Physiological Society

proteins were not separated under these conditions)

was performed using stacking and separating gels

containing 5 and 10% acrylamide±bis. The gels were

stained with 0.25% (w/v) Commassie blue (R250) in

50% (v/v) methanol and 10% (v/v) acetic acid. The

concentration of MHC-all (densitometric arbitrary

units ´ 5 lg±1 protein) or the relative amounts of

MyHC proteins (% of total; the wells were loaded with

10 lg of protein) were determined by densitometry

(Biopro®l, Vilber Lourmat, Marne la ValleÂe, France).

MyHC mRNA analysis (Northern blotting)

Total RNA was extracted from individual frozen

muscles (Chomczynski & Sacchii 1987). RNA samples

were stored frozen at ±80 °C until Northern blotting.

The DNA probes for MyHC-1 (256 bp, a gift from

M. Buckingham, Institut Pasteur, Paris, France) and

MyHC-2a (140 bp, a gift from S. Schiaf®no, University

of Padova, Italy), which correspond to the 3¢ non-

translated portion of the two mRNA, were previously

described (DeNardi et al. 1993). These speci®c probes

were isolated from the vectors and labelled by the

random priming method using 32P dCTP before hy-

bridization (DeNardi et al. 1993). Labelled DNA was

separated from free nucleotides on a Sephadex G50

column.

Extracted RNA (15 lg) for each muscle was elec-

trophoresed in a 1.2% agarose denaturing (formalde-

hyde) gel by electrophoresis (5 h at 100 V). The RNA

was then transferred to a nylon membrane (Hybond-

N+, Amersham) by the capillary method using 20´saline-sodium citrate (SSC) as a transfer buffer.

Northern blots were ®xed by ultraviolet light and pre-

hybridized (2 h, 65 °C) in Denhardt's solution, 10%

poly ethylene-glycol, 1% sodium dodecyl sulphate

containing denatured salmon sperm DNA

(100 lg mL±1). Hybridization to MyHC-1 or MyHC-2a

labelled probes was performed overnight (65 °C) with a

probe concentration of 1 ´ 106±2 ´ 106 cpm mL±1.

Membranes were washed in 2´ SSC and 1% SDS

(30 min, 65 °C) twice, followed by 1´ SSC and 1%

SDS, followed by 0.5´ SSC and 1% SDS, and ®nally

0.2´ SSC and 1% SDS (10±20 min, 65 °C). The MyHC

probe was washed off the membranes by boiling for

10±15 min in 0.1´ SSC and 1% SDS (two times) before

rehybridization. The blots were ®nally rehybridized

with the probe for 18S rRNA. Blots were exposed to an

X-ray ®lm (X-OMAT, Kodak) with intensifying screen

at ±80 °C for 2 h (MyHC-1 probe), 10 days (MyHC-2a)

or 8 min (18S rRNA probe). Bands for every probe

were quanti®ed by densitometry. The values of MyHC

mRNAs were normalized to their corresponding 18S

rRNA.

Statistical analysis

Data were analysed using an analysis of variance (AN-

OVA). Mean comparisons of the signi®cant effect of the

treatments (castration, nandrolone, nandrolone com-

bined with exercise) were determined by a post hoc test

(Scheffe). The software Statistica 4 was used.

RESULTS

Effect of castration (CAS vs. CON muscles)

There was no effect of castration on muscle mass,

protein concentration (Tables 1 and 2), relative

amounts of MyHC proteins (Tables 3 and 4) in re-

generating EDL or soleus, and amounts of MyHC

mRNA in venom-treated soleus (Fig. 1)(P > 0.05).

Castration only altered MyHC-all protein concentration

in regenerating EDL (P < 0.01) and soleus (P < 0.05)

(Tables 1 and 2).

Effect of NAN treatment (NAN vs. CON muscles)

Contrary to EDL, soleus muscle was affected by NAN

treatment. The masses (mg and mg g±1 body weight) of

venom-treated soleus of NAN rats were increased

(+63%) compared with CON rats (Table 2, P < 0.01).

Moreover, NAN treatment increased the relative

amount of MyHC-1 protein (+13%, P < 0.05) and

reduced the relative amount of MyHC-2a protein

(±85%, P < 0.05), in NAN venom-treated soleus

(Table 4). In contrast, the amounts of MyHC-1 and

Table 1 Masses (mg and mg kg±1 body weight), protein (lg protein mg±1 muscle) and MyHC-all protein (arbitrary unit) concentrations of

venom-treated EDL

Mass Mass/body Protein MyHC-all

CON (n = 8) 198.0 � 46.7 0.562 � 0.129 23.0 � 8.3 64.8 � 13.1

CAS (n = 8) 163.1 � 13.6 0.510 � 0.042 23.8 � 11.1 152.4 � 56.9*

NAN (n = 8) 218.4 � 62.9 0.611 � 0.189 26.4 � 11.1 85.3 � 15.7

NAN + EXE (n = 8) 164.6 � 15.3 0.521 � 0.052 30.1 � 15.0 108.4 � 30.4

Values are means � SD.

* signi®cantly different from CON (P < 0.01, effect of castration).


Acta Physiol Scand 1999, 166, 105±110 A Ferry et al. � Male steroids and muscle regeneration

MyHC-2a mRNA in venom-treated soleus were not

signi®cantly altered by NAN treatment (Fig. 1,

P > 0.05).

Effect of NAN + EXE treatment (NAN + EXE vs.

CON muscles)

NAN + EXE treatment was without effect on regen-

erating EDL. This treatment only altered the MyHC

protein phenotype of venom-treated soleus. The relative

amount of MyHC-2x/d protein was increased in venom-

treated soleus (´ 18; P < 0.05, Table 4). In contrast, there

was no signi®cant difference between NAN + EXE and

CON rats concerning the amounts of MyHC-1 and

MyHC-2a mRNAs in venom-treated soleus (Fig. 1).

DISCUSSIONS

Role of male hormones during regeneration

To our knowledge, the effect of a withdrawal of male

sexual hormones on muscle regeneration has not been

previously investigated. Castration is known to lower

the circulating levels of testosterone (Boissoneault et al.

1989). We found that the relative amounts of MyHC

proteins and the amounts of MyHC mRNA of regen-

erating EDL and soleus from CAS rats do not signi®-

cantly differ from those of CON rats. Therefore, in

contrast to normal muscular innervation, mechanical

load and thyroid hormone levels (see Introduction), a

normal level of endogenous male sexual steroids is not

necessary to muscle maturation (development of a

mature MyHC phenotype) during regeneration. How-

ever, it is possible that male sexual hormones play a role

in the growth of regenerating muscles. In the present

study, castration increased or decreased MyHC-all

concentrations (levels of contractile proteins) in,

respectively, regenerating EDL and soleus (without

altering muscle mass and protein concentration). In

order to conclude that male sexual hormones are nec-

essary for complete regeneration, it would be important

to also study mechanical properties (maximal isometric

tension, contraction and relaxation times) of regener-

ated muscles in castrated rats.

Effects of nandrolone administrations on muscle regeneration

The present study examined whether an increased level

of male sexual hormones (NAN: nortestosterone) could

improve muscular in vivo regeneration. We demonstrated

that NAN (nortestosterone) treatment did not alter EDL

regeneration but exerted a marked in¯uence on both

growth and maturation of regenerating soleus. Indeed,

with NAN treatment the regenerating soleus hypertro-

phied without altering its protein and MyHC-all protein

concentrations (no evidence of oedema and ®brosis).

This effect of NAN treatment on regenerating soleus

growth is similar to what was observed in the case of

increased physical activity (Ferry et al. 1997). It remains

to be determined whether NAN increases proliferation

of mpc and/or myotube growth as the results from

previous studies concerning myogenesis in vitro are not

obvious (see Introduction). As the peak force generated

by a muscle (not measured) is related to its mass (and

amount of contractile machinery in parallel), our results

suggest that NAN treatment could increase the maxi-

mum force of the venom-treated soleus.

Administration of NAN also induced a greater

maturation/specialization of this slow regenerating

muscle (increased relative amount of MyHC-1 protein).

It is also likely that this slower regenerating muscle is

Figure 1 Levels (arbitrary units) of MyHC mRNAs in venom-treated

soleus (n � 8 per group). Values are means � SD. j, CON; ,

CAS; h, NAN; , NAN +EXE.

Table 2 Masses (mg and mg g±1

body weigth), protein (lg pro-

tein mg±1 muscle) and MyHC-all

protein (arbitrary unit) concentra-

tions of venom-treated soleus

Mass Mass/body Protein MyHC-all

CON (n = 8) 126.1 � 17.5 0.358 � 0.047 30.2 � 15.1 52.3 � 34.0

CAS (n = 8) 114.0 � 19.9 0.356 � 0.061 37.1 � 10.3 40.2 � 24.4**

NAN (n = 8) 205.2 � 64.0* 0.575 � 0.191* 25.3 � 19.8 53.8 � 33.3

NAN + EXE (n = 8) 147.0 � 18.3 0.465 � 0.062 29.9 � 13.6 76.0 � 28.0

Values are means � SD.

* signi®cantly different from CON (P < 0.01, effect of nandrolone).

** signi®cantly different from CON venom-treated (P < 0.05, effect of castration).



more fatigue-resistant as the MyHC-1 is the only

MyHC isoform found in the fatigue-resistant type I

®bres (Andersen & Schiaf®no 1997). This effect of

NAN on slow muscle ®bres could also have some

consequences for athletes using and abusing anabolic-

androgenic steroids as physical training could induce

repeated muscle damages. If the increased maturation/

specialization induced by NAN treatment is also the

case in human muscle, it is possible to expect a slower

MyHC phenotype in these athletes.

Together, these results suggest that NAN treatment

could be bene®cial to a regenerating slow muscle.

However, it can be noted that NAN treatment

increases myo®bre damage in mdx (dystrophy) mouse

muscle (Krahn & Anderson 1994). In contrast to

NAN treatment, NAN + EXE treatment appears to

have a detrimental effect on soleus regeneration. In-

deed, the regenerating soleus of NAN + EXE rats

expressed a higher relative amount of fast MyHC

protein (MyHC-2x/d) compared with CON rats. This

result suggests that NAN + EXE diminished the

maturation of the venom-treated soleus. It could not be

explained by the detrimental effect of NAN (see

above) or endurance exercise as exercise did not alter

soleus maturation of CON rats (Ferry et al. 1997). One

explanation of this observation could be that the

alterations induced by the two combined stimuli

exhaust the plasticity of the regenerating soleus. It is

possible that another exercise programme (decreased

duration/intensity of each bout of exercise, delayed

onset of exercise) could have given another result in

combination with NAN.

Venom-treated EDL was not sensitive to NAN and

NAN + EXE treatments. For instance, contrary to the

venom-treated soleus, the relative amounts of MyHC

isoforms in regenerating EDL was not affected by our

treatments (NAN, NAN + EXE). This restriction of

plasticity of regenerating EDL in response to treat-

ments could be owing to intrinsic properties of its mpc

(mpc from EDL would be unable to express in variable

amounts the MyHC). However, it was recently reported

that regenerating EDL could be completely trans-

formed into a slow muscle with regard to expression of

MyHC (Snoj-Cvetko et al. 1996).

In the present study, we also studied the amounts of

MyHC-1 and MyHC-2a mRNAs to determine whether

pre-transduction mechanisms could explain the changes

observed at the level of their corresponding proteins

(polypeptide chain) in regenerating soleus (the only

plastic muscle studied). This could not to be the case

for NAN treatment as the amount of MyHC-2a or

MyHC-1 mRNAs in the regenerating muscle was not

signi®cantly decreased or increased, contrary to the

relative amount of MyHC-2a or MyHC-1 protein.

However, interpretation of these results could take into

account that: (1) we did not determine absolute

amounts of MyHC proteins (but a percentage of the

total); (2) there is a more rapid turnover of MyHC

mRNA compared with its corresponding protein.

During phenotype transition (as during regeneration), it

has been recently demonstrated that muscle ®bres

could express at a given time a MyHC protein but not

its corresponding mRNA (and vice versa)(Andersen &

Schiaf®no 1997).

In conclusion, we demonstrated that male sexual

steroids are not necessary to hindlimb muscle matura-

tion in the male rat, contrary to innervation, physical

activity and thyroid hormones. However, they seem to

in¯uence the concentration of contractile protein

(MyHC) in the regenerating muscles. Moreover, in

contrast to nandrolone combined with exercise, nan-

drolone treatment seems bene®cial to a slow-twitch

muscle regeneration.

We thank G.S. Butler-Browne (Centre National de la Recherche

Scienti®que, URA 2115, Paris France) for critical reading, M. Buck-

ingham (Institut Pasteur, Paris, France) and S. Schiaf®no (University

of Padova, Italy) for the gifts of MyHC probes and Catherine Lacroix

for revision of the English manuscript. This work was supported by

the Association FrancËaise contre les Myopathies (AFM).

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Table 3 Relative amounts of MyHC proteins in venom-treated EDL

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effects of anabolic/androgenic steroids on regenerating skeletal muscles in the rat

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