Use of anabolic steroids to attenuate the effects of glucocorticoids on the rat diaphragm. (Research Reports).

Because of their potent anti-inflammatory effects, glucocorticoids are used to manage numerous clinical disorders, including asthma and chronic obstructive pulmonary disease. (1) However, one serious side effect of glucocorticoids is the catabolic effect of glucocorticoids on muscle tissue. (1) Glucocorticoids stimulate the breakdown of muscle into amino acids (2) and decrease protein synthesis rates, (3) both of which result in muscle atrophy. Because the diaphragm is the primary inspiratory muscle, the development of diaphragm muscle atrophy and the resulting loss of force production may have deleterious effects in patients with lung disease.

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 ([P.sub.o]), 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 types. Several investigators (11-13) 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 be of clinical benefit. Anabolic steroids may have the potential to antagonize glucocorticoid-induced effects on the diaphragm. Anabolic steroids increase the amount of protein in skeletal muscle by enhancing the rate of protein synthesis. (16) Because glucocorticoids decrease protein synthesis (3) 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 al (7) 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 [P.sub.o] 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 of 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 ([greater than or equal to] 1,000 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 [P.sub.o] commonly observed with glucocorticoids would be prevented in animals receiving both anabolic steroids and glucocorticoids.

Methods

Experimental Design

Eighty-eight adult (4-month-old) female Sprague-Dawley rats were housed individually, fed rat chow and water ad libitum, and maintained on a 12-hour light/dark photoperiod for 7 days prior to the beginning of the experiments. The animals were handled and sham injected daily to reduce contact stress during this 7-day period. At the end of the 7-day period, the animals were divided into 4 groups: (1) a control group that received daily sham saline injections for 13 days (CONT group, n=23), (2) a group that received daily prednisolone injections (0.5 mg/100 g) for 10 days (PRED group, n=23), (3) a group that received daily testosterone (19-nortestosterone/17-decanoate) injections (0.5 mg/100 g) for 13 days (TEST group, n=19), and (4) a group that received a combination of daily prednisolone (0.5 mg/100 g) and testosterone (0.5 mg/kg) injections for 10 and 13 days, respectively (COMBO group, n=23). Animals in the PRED group received sham saline injections for 3 days prior to beginning experimental injections so that animals in all groups received 13 days of injections. Testosterone was suspended in sesame oil, and prednisolone was suspended in 0.9% saline. The animals were injected subcutaneously for 13 days at approximately the same time every day. Animal weight was recorded daily, and drug doses were adjusted to reflect changes in body mass. The final weight was obtained 24 hours following the final injection. Prednisolone was chosen because it is prototypical of the nonfluorinated glucocorticoids used to manage disease in humans. The prednisolone dose used in this study is one that is commonly given to patients for the management of status asthmaticus. (21) The anabolic steroid 19-nortestosterone/17-decanoate was chosen because its effects are primarily anabolic as opposed to androgenic. (22) The dose of testosterone was chosen because a previous study by Prezant et al (23) showed an increase in diaphragm weight and contractility in rats given this dose for 2.5 weeks. Guidelines for animal use established by the American Physiological Society were followed.

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% [O.sub.2]/5% C[O.sub.2] 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 [micro]M 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% [O.sub.2]/5% C[O.sub.2]), and pH was maintained at 7.4. Temperature in the organ bath was maintained at 24[degrees]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 ([L.sub.o]) by stimulating the muscle strip along its entire length with platinum wire electrodes using a modified Grass Instruments S48 stimulator. ([dagger],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 [L.sub.o], 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 [L.sub.o] 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 ([double dagger])). 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 [L.sub.o] 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 [P.sub.o]. Muscle CSA was calculated by using the following formula (24):

CSA ([cm.sup.2]) = muscle mass (g) / [muscle length (cm)

x muscle density (g/[cm.sup.3]) ] and assuming muscle

density = 1.056 g/[cm.sup.3].

The remaining costal diaphragm was trimmed of fat and connective tissue, blotted dried, and weighed. By adding the weight of the muscle strip and the remaining costal diaphragm, total diaphragm weight was obtained.

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([section]) on a Gateway computer. ([parallel])

Results

Morphological Characteristics

At the beginning of the study, there were no differences in body weight among the 4 groups, At the conclusion of the study, there was an effect of treatment on rat body weight, with the body weights in the PRED group being different from the body weights in the CONT, TEST, and COMBO groups. Figure 1 illustrates the daily weight changes in animals from all 4 groups. By the end of the study, the body weights of the rats in the CONT group had increased by 5% (from 263.2 g [SD=45.9, range=214-273] to 277.9 g [SD=52.3, range=210-405]), and the body weights of the rats in the TEST group had increased by 16% (from 256.7 g [SD=37.5, range=212-361] to 305.5 g [SD=52.9, range=248-432]). Body weights of the rats in the PRED group decreased by 19% (from 255.9 g [SD=44.8, range=210-375] to 206.9 g [SD=45.7, range=151-306]), and those of the rats in the COMBO decreased by 6.6% (from 264.7 g [SD=41.2, range=216-392] to 247.1 g [SD=53.2, range=175-379]).

[FIGURE 1 OMITTED]

The differences among groups in diaphragm weight were somewhat similar to the decreases in body weight (Fig. 2). The diaphragm weights of the rats in the PRED group were less than those of the rats in the CONT, TEST, and COMBO groups (487.3 mg [SD=109.5, range=363-683] versus 625.9 mg [SD=115.7, range=426-916], 661.8 mg [SD=97.5, range-504-846], and 550.9 mg [SD=106.5, range=380-787], respectively). The diaphragm weights of the rats in the COMBO group were less than those of the rats in the CONT and TEST groups (550.9 mg [SD=106.5, range=380-787] versus 625.9 mg [SD=115.7, range=426-916] and 661.8 mg [SD=97.5, range=504-846], respectively). There was no difference in diaphragm weights between the CONT and TEST groups.

[FIGURE 2 OMITTED]

Normalized Maximal Isometric Tetanic Tension

Normalized maximal isometric tetanic tension was less in the PRED group than in the CONT, TEST, and COMBO groups (16.8 N/[cm.sup.-2] [SD=2.7, range=11.5-22.7] versus 18.9 N/[cm.sup.-2] [SD=2.5, range=15.3-24.9], 18.9 N/[cm.sup.-2] [SD=3.0, range=13.2-24.5], and 19.4 N/[cm.sup.-2] [SD=3.2, range=13.9-26.6], respectively) (Fig. 3). There was no difference in [P.sub.o] among the CONT, TEST, and COMBO groups.

[FIGURE 3 OMITTED]

Discussion

Our results support our hypothesis that simultaneous administration of anabolic steroids with glucocorticoids prevents all of the decrease in [P.sub.o] that is commonly observed with glucocorticoid treatment alone. Our results also show that some of the decline in body weight can be prevented in animals treated with both glucocorticoids and anabolic steroids, whereas the decline in diaphragm weight is partially ameliorated with the combination of drugs.

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 investigators (25-27) have shown that anabolic steroids can preferentially bind to the muscle glucocorticoid receptors. Mayer and Rosen (25) 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 al (8) and Gardiner et also 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 al (8) and Gardiner et al (30) 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 al (13) 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 al (32) 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 [P.sub.o] was 10% less in the PRED group than in the CONT group. This result is in agreement with other studies (6,9,13,31,33) that have shown similar declines in [P.sub.o], depending on duration and dose of glucocorticoid administration. The glucocorticoid-induced reduction in [P.sub.o] 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 [P.sub.o] that is normally observed with glucocorticoid treatment only. Kayali et al (34) 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 al (29) 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 [P.sub.o] 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.

Conclusion

The results of our study indicate that glucocorticoids cause decreases in body weight and diaphragm weight as well as a decrease in the force generation of the diaphragm. Concomitant administration of anabolic steroids prevents the development of contractile dysfunction and body weight loss while partially preventing the muscle weight decline observed when glucocorticoids are administered alone. Further research in the potential use of anabolic steroids in the medical treatment of patients with pulmonary disease is warranted.

* Cambridge Technology Inc, 109 Smith Pl, Cambridge, MA 02138.

([dagger]) Grass Instruments, 600 E Greenwich Ave, West Warwick, RI 02893.

([double dagger]) GW Instruments, 35 Medford St, Somerville, MA 02143.

([section]) SPSS Inc, 233 S Wacker Dr, Chicago, IL 60606.

([parallel]) Gateway Computer, 4545 Town Centre Ct, San Diego, CA 92121.

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JM Eason, PT, PhD, is Associate Professor, Louisiana State University Health Sciences Center, Department of Physical Therapy, 1900 Gravier St, New Orleans, LA 70112 (USA) ([e-mail]). At the time this study was performed, she was a doctoral student in the Department of Exercise Science, University of Florida, Gainesville, Fla. Address all correspondence to Dr Eason.

SL Dodd, PhD, FACSM, is Associate Professor, Department of Exercise and Sport Sciences, University of Florida.

SK Powers, EdD, PhD, FACSM, is Professor, Department of Exercise and Sport Sciences, University of Florida.

All authors provided concept/idea/research design, data analysis, fund procurement, and consultation (including review of manuscript before submission). Dr Eason provided writing, data collection, project management, and subjects. Dr Dodd and Dr Powers provided facilities/equipment.

This project was approved by the University of Florida Institutional Animal Care and Use 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.

This article was submitted December 27, 2001, and was accepted July 26, 2002.