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Research Reports |
JM Eason, PT, PhD, is Associate Professor, Louisiana State University Health Scie Center, Department of Physical Therapy, 1900 Gravier St, New Orleans, LA 70112 (jeason{at}lsuhsc.edu). At the time this study was performed, she was a doctoral student in the Department of Exercise Science, University of Florida, Gainesville, Fla.
SL Dodd, PhD, FACSM, is Associate Professor, Department of Exercise and Sport University of Florida
SK Powers, EdD, PhD, FACSM, is Professor, Department of Exercise and Sport Sciences, University of Florida
Address correspondence to Dr Eason
Submitted December 27, 2001;
Accepted July 26, 2002
 | Abstract |
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Key Words: Anabolic steroids • Atrophy • Diaphragm muscle • Glucocorticoids • Rat
| Introduction |
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High doses of glucocorticoids for both short periods of time (5 days) and longer periods of time (8-14 days), as well as low doses for prolonged periods of time (6 months), result in decreases in diaphragm muscle mass from 15% to 28%.4–8 Concurrent with the muscle atrophy, there is a 12% to 26% decline in normalized maximal isometric tetanic tension (Po), or the amount of force generated per cross-sectional area of the rat diaphragm, depending on duration and dose of glucocorticoid administration.5,6,9,10 Furthermore, the muscle atrophy is fiber-type specific, with the slower fiber types being more resistant to atrophy than the faster fiber fiber types. Several investigators11–13 have shown that administration of glucocorticoids results in atrophy of type IIb and IIx fibers, but not type I or IIa fibers, in the rat diaphragm. Type IIx fibers are classified as fast fibers and possess biochemical and contractile characteristics intermediate between type IIa and IIb fibers.14,15
In light of the negative side effects of glucocorticoids, a pharmacological intervention that could antagonize the deleterious effects of glucocorticoids on the diaphragm could clinical benefit. Anabolic steroids may have the potential to antagonize glucocorticoid-induced effects on the diaphragm. Anabolic steroids increase the amount of protein in skele by enhancing the rate of protein synthesis.16 Because glucocorticoids decrease protein synthesis3 and stimulate proteolysis,2 it seemed reasonable to us to assume that anabolic steroids administered in conjunction with glucocorticoids may prevent the muscle wasting and contractile dysfunction observed with glucocorticoid treatment. Indeed, anabolic steroids have been used to reverse glucocorticoid-induced diaphragm contractile dysfunction in animals that received low doses of glucocorticoids for long periods of time.7,17 Van Balkom et al7 administered the anabolic steroid nandrolone decanoate during the last 3 months of long-term (9 months) administration of glucocorticoids (methylprednisone) in rats. The reduction in diaphragm PO observed in the animals that received only glucocorticoids was abolished in the animals that also received anabolic steroids (1 mg/kg/wk) during the last 3 months glucocorticoid treatment.7
Preventing the development of glucocorticoid-induced diaphragm contractile dysfunction following short-term administration of high doses of glucocorticoids may be more important than reversing the deleterious effects of glucorticoids. For example, acute diaphragm atrophy has been reported in people with asthma hospitalized with severe exacerbation of their disease and requiring high doses of glucocorticoids (
1,0000 mg/d) for short periods of time.18–20 To our knowledge, there are no human or animal studies that have attempted to prevent the development of glucocorticoid-induced contractile dysfunction with anabolic steroids in conjunction with short-term administration of high doses of glucocorticoids.
Based on the effects of anabolic steroids on muscle, we became interested in determining whether we could prevent the development of muscle atrophy and dysfunction as a result of glucocorticoid administration. We wanted to know whether administration of anabolic steroids several days prior to initiation of glucocorticoids and then continuing with simultaneous administration of anabolic steroids with glucocorticoids would prevent glucocorticoid-induced diaphragm muscle atrophy and contractile dysfunction in rats receiving glucocorticoid doses commonly used to manage status asthmaticus.21 Thus, the purpose of our study was to assess the effects of 3 days of testosterone injections followed by simultaneous administration of glucocorticoids and anabolic steroids for 10 days on the morphological and contractile properties of the rat diaphragm. We hypothesized that administration of anabolic steroids (0.5 mg/100 g/d) for 3 days followed by simultaneous administration of glucocorticoids (0.5 mg/100 g/d) would prevent the decrease in diaphragm and body weight commonly observed with glucocorticoids alone. Furthermore, we hypothesized that the decrease in diaphragm PO commonly observed with glucocorticoids would be prevented in animals receiving both anabolic steroids and glucocorticoids.
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Experimental Protocol
The methods used to determine the contractile properties in this experiment were similar to those previously described.8,9,11,13 These methods are described extensively in the literature and are standard measures used to determine contractile properties. Twenty-four hours following the final injection, animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg), and the entire diaphragm was removed and placed in a dissecting dish containing a Krebs-Hensleit solution equilibrated with a 95% O2/5% CO2 gas mixture.8,9,13 The animals then were sacrificed with an overdose of pentobarbital. A small strip of the costal diaphragm was carefully dissected with a portion of the central tendon on one end and the rib attachment on the other end. This diaphragm strip was used to determine in vitro contractile measurements.
The dissected muscle strip was suspended vertically between 2 plexiglass clamps in a jacketed tissue bath containing Krebs-Hensleit solution with 12 µM d-tubocurarine added to produce complete blockade at the neuromuscular junction and was connected to a force transducer (Cambridge Technology, model 300B*) with a force range of 0 to 100 g and a force signal resolution of 30 mg. The jacketed tissue bath was aerated with gas (95% O2/5% CO2), and pH was maintained at 7.4. Temperature in the organ bath was maintained at 24°C, and the osmolality of the bath was approximately 290 mOsm.
Following a 15-minute equilibration period, data were collected with the muscle strip at optimal length (Lo) by stimulating the muscle strip along its entire length with platinum wire electrodes using a modified Grass Instruments S48 stimulator
.8,9,13 Optimal length was defined as the length of the muscle at which maximal twitch tension was obtained when a square-wave, 2-millisecond pulse was delivered to the muscle. To obtain Lo, the muscle was lengthened with a micrometer while stimulating the muscle strip with 2-millisecond twitches at supramaximal voltage (140 V). The muscle was considered to be at Lo when maximal twitch tension was evoked.
The output from the transducer was amplified and differentiated by operational amplifiers and underwent analog-to-digital conversion at a sampling rate of 1,000 Hz for analysis using a computer-based data acquisition system (GW Instruments Series II
). Peak isometric tetanic tension was measured in triplicate and averaged, and the mean was used for statistical analysis. Peak isometric tetanic tension was obtained by applying a supramaximal stimulus train of 80 Hz and 330-millisecond duration to the muscle strip. In previous work in our laboratory, we determined that a stimulus applied in this manner would result in maximum force generation of the muscle strip.5
After the completion of contractile measurements, the weight and Lo of the muscle strip were measured for determination of muscle cross-sectional area (CSA) so that maximal tension generated during tetanic contractions could be normalized to muscle CSA. By expressing tension as the amount of force generated per CSA, the force outputs of muscle strips of different sizes can be compared. Once peak isometric tetanic tension is normalized to muscle CSA, this variable is referred to as the Po. Muscle CSA was calculated by using the following formula24:
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Data Analysis
Comparisons among the CONT, PRED, TEST, and COMBO groups was made by a 1 x 4 single-factor analysis of variance (ANOVA). To analyze the change in body weight, a mixed-ANOVA was used. Post hoc differences were determined with the Fisher least significant difference test. The alpha level was set at the .05 level of significance. Data were analyzed by the SPSS 10.0 statistical package
on a Gateway computer||.
| Results |
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| Discussion |
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Mechanisms of Action of Anabolic Steroids
The ability of anabolic steroids to counter the effects of glucocorticoids that were observed in this study could be due to several mechanisms. These mechanisms include antagonism of the muscle glucocorticoid receptor by anabolic steroids, a direct effect of anabolic steroids on the muscle, or a combination of these 2 mechanisms.
Several investigators25–27 have shown that anabolic steroids can preferentially bind to the muscle glucocorticoid receptors. Mayer and Rosen25 proposed a mechanism whereby anabolic steroids compete with glucocorticoids for binding to the muscle glucocorticoid receptors. The interaction of anabolic steroids with the glucocorticoid receptor would prevent binding of glucocorticoids to the receptor and therefore antagonize the catabolic activity of glucocorticoids on muscle tissue.
Regarding the second possible mechanism, it is known that independent of glucocorticoids, anabolic steroids exert an effect on normal skeletal muscle.17 Anabolic steroids have been shown to decrease catabolism of amino acids, promote incorporation of amino acids, and increase nitrogen retention, all of which result in tissue growth.25 Increasing protein synthesis in muscle would result in an increase of myosin and other myofibrillar proteins. Because glucocorticoids decrease protein synthesis,3 the increase in protein synthesis produced by anabolic steroids could protect against glucocorticoid-induced muscle atrophy. Furthermore, the direct effect of anabolic steroids is enhanced in fast muscle fibers.28
Morphological Changes
The 19% decline in body weight observed in the PRED group as compared with the CONT group is similar to declines in body weight found other studies.4,6,8,9,29 The loss in body weight is primarily due to loss in muscle weight secondary to glucocorticoids, although it is known that glucocorticoid-treated animals experience a reduction in food intake concurrent with administration of glucocorticoids.9
One way to determine whether the decline in body weight observed in glucocorticoid-treated animals is due to a drug effect and not malnutrition is to include a pair-fed group of animals in the study. Several investigators have utilized pair-fed animals to test the notion that the changes in body and muscle weight as well as impairment in contractile properties observed in glucocorticoid-treated animals are not due solely to a reduction in caloric intake, but also may be due to the use of glucocorticoids. Moore et al8 and Gardiner et al30 showed that pair-fed animals did not lose body weight as compared with glucocorticoid-treated animals and concluded that the loss of body and diaphragm weight observed in their studies was due to the effect of glucocorticoids and not a reduction in caloric intake. Moore et al8 and Gardiner et al30 showed that pair-fed animals actually gained weight by the end of their studies, whereas the glucocorticoid-treated animals lost weight.
In a different version of pair feeding, van Balkom et al13 included a group of animals that were food restricted to keep their body weights the same as those in a glucocorticoid-treated group. At the conclusion of the study, despite similarities in body weight between the 2 groups, the authors found differences in diaphragm muscle area and contractile properties and concluded that the differences were not simply the result of decreased nutrition. We did not measure food intake in our study, and thus we acknowledge that caloric deficit may have played a role in the weight loss of the glucocorticoid-treated animals. However, based on the results of previous studies,8,9,13,30 we believe that the changes in morphological and contractile characteristics observed in our study are due primarily to the catabolic effect of glucocorticoids on skeletal muscle and not a caloric deficit.
The 23% decrease in diaphragm weight in the PRED group as compared with the CONT group is similar to the decreases in diaphragm weight found in previous studies.4,6,8,9 The use of glucocorticoids results in preferential atrophy of type IIx and type IIb fibers,4,11,13,31 and because the diaphragm is composed primarily of type IIx fibers and a smaller proportion of type IIb fibers, it is likely that the decrease in diaphragm mass observed in our study was due to atrophy of type IIx and type IIb fibers.
The decreases in diaphragm weights with glucocorticoid treatment were partially attenuated with testosterone. Diaphragm weights were greater in the COMBO group than in the PRED group, but they were not maintained at the same level as in the CONT group. Although we did not directly measure fiber type dimensions, it is likely that the addition of anabolic steroids to glucocorticoid treatment decreased the degree of muscle fiber atrophy that is normally observed in glucocorticoid-treated animals. Indeed, Bisschop et al32 showed an increase in type IIx and type IIb fiber dimensions in the diaphragms of rats treated with 5 weekly injections of 7.5 mg/kg of nandrolone decanoate. Because the use of glucocorticoids results in preferential atrophy of type IIx and type IIb fiber types,11–13 it is likely that simultaneous injections of testosterone with glucocorticoids in the COMBO group were responsible for preventing atrophy of type IIx and type IIb fibers and resulted in an attenuation of body weight and diaphragm weight. However, despite the high doses of anabolic steroids used in our study and the fact that animals in the COMBO group also received testosterone injections for 3 days prior to administration of glucocorticoids, we were unable to completely prevent a loss of diaphragm weight in the COMBO group. The cellular and molecular mechanisms are not fully known, and discussion of these mechanisms is beyond the scope of this study.
Changes in Force Generation
The Po was 10% less in the PRED group than in the CONT group. This result is in agreement with other studies6,9,13,31,33 that have shown similar declines in Po, depending on duration and dose of glucocorticoid administration. The glucocorticoid-induced reduction in Po that we observed was completely prevented with the addition of testosterone to glucocorticoid treatment. Although the effects of anabolic steroids alone on muscle contractile properties are inconsistent,17 our results were likely due to an antagonizing action of testosterone on the muscle glucocorticoid receptor as well as a direct effect on the muscle. The direct effect of increasing protein synthesis and thus increasing myosin and other myofibrillar protein content would prevent the diaphragm atrophy and the reduction in Po that is normally observed with glucocorticoid treatment only. Kayali et al34 showed that glucocorticoid treatment (corticosterone at a dose of 10 mg/kg/d) in rats for 10 days results in a selective loss of myofibrillar proteins from the plantaris muscle. Furthermore, Lieu et al29 reported that 10 days of glucocorticoid treatment at the same dose used in our study resulted in a reduction of myofibrillar protein concentration in the rat diaphragm, and they postulated that the change in protein concentration could result in a decrease in the number of crossbridges for muscle contraction. Therefore, although we did not measure myofibrillar protein concentration in the diaphragm, it appears likely that the reduction in Po in the PRED group was due a reduction in myofibrillar protein concentration and that coadministration of anabolic steroids with glucocorticoids prevented the loss of myofibrillar protein.
Critique of Experimental Model
Because our study was conducted using healthy rats, the results are not readily generalizable to humans. In addition, many patients with pulmonary disease have concomitant medical problems such as malnutrition, hypoxemia, hyperinflation, and cardiac failure that may contribute to diaphragm dysfunction. Thus, it may have been more useful to conduct these studies using an animal model of pulmonary disease. Researchers, therefore, should examine the effect of coadministration of glucocorticoids and anabolic steroids on the morphological and contractile properties of the diaphragm in an animal model of pulmonary disease. The results of our study, however, provide a basis for future research in this area.
We used a dose of glucocorticoids that typically would be prescribed for a patient with status asthmaticus.22 Problems may occur even when such doses of glucocorticoids are administered. In general, these problems are related to differences in metabolism of the drug among individuals. Furthermore, it is often difficult to separate the effects of the disease from the iatrogenic effects of the drug. Future research in this area will need to take these factors into consideration.
We recognize that initiating administration of anabolic steroids prior to glucocorticoid treatment would be unlikely in a clinical setting. Our rationale for this approach was that we hoped that anabolic steroid injections prior to glucocorticoid administration would result in an increase in body weight (and presumably diaphragm weight) in the animals and thus would help prevent any morphological changes in the diaphragm that occur with the administration of glucocorticoids. However, the increase in body weight (and presumably the diaphragm) did not occur in either the TEST group or the COMBO group following 3 days of anabolic steroid injections. We believe that concurrent administration of anabolic steroids with glucocorticoids was clinically relevant, because declines in body weight and Po were prevented in the COMBO group as compared with the PRED group. Furthermore, the decrease in diaphragm weight in the COMBO group was partially ameliorated as compared with the PRED group.
We used what we would consider a supraphysiological dose of anabolic steroids. Some of the adverse side effects of 19-nortestosterone/17-decanoate include liver failure, liver tumors, and blood lipid changes,23 and future studies should include titrating the dose of anabolic steroids to a dose that could still yield beneficial effects without adverse side effects.
Clinical Implications
Although physical therapists cannot prescribe pharmacologic therapy, the results of our study may be useful to physical therapists in several ways. First, physical therapists should be aware of the role that glucocorticoids may play in the development of diaphragm dysfunction in patients with pulmonary diagnoses. Second, understanding the underlying mechanisms of glucocorticoid-induced muscle dysfunction may be helpful in developing appropriate physical therapy interventions. Third, physical therapists should have a broad understanding of interactions of classes of drugs and how these interactions may affect the medical condition of patients, and ultimately how these interactions may determine the development of appropriate physical therapy strategies.
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| Footnotes |
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This project was approved by the University of Florida Institutional Animal Care and Committee.
This study was supported by a grant from the Foundation for Physical Therapy to Dr Eason and a grant from the Division of Sponsored Research, University of Florida, to Dr Dodd.
* Cambridge Technology Inc, 109 Smith Pl, Cambridge, MA 02138. 
Grass Instruments, 600 E Greenwich Ave, West Warwick, RI 02893.
GW Instruments, 35 Medford St, Somerville, MA 02143.
SPSS Inc, 233 S Wacker Dr, Chicago, IL 60606.
|| Gateway Computer, 4545 Town Centre Ct, San Diego, CA 92121.
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