INTRODUCTION

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Muscle weakness and atrophy are frequently encountered in patients under systemic corticosteroid therapy and are well-known side effects of such therapy. Peripheral as well as respiratory muscles are affected by corticosteroid treatment and myopathy develops during both acute and chronic administration of corticosteroids. Such effect is particularly detrimental in patients with chronic obstructive pulmonary disease (COPD) in which respiratory muscle weakness severely compromises respiratory function and may lead to respiratory failure. The mechanisms by which corticosteroids cause muscle atrophy are complex. Alterations in muscle protein content seem to be the major cause of muscle atrophy induced by corticosteroid treatment, suggesting therefore a potential role for the growth factors.

In this context, our interest was directed toward the potential role of the insulin-like growth factors (IGFs), IGF-1 and IGF-2, in the muscle atrophy seen after corticosteroid treatment. Both IGFs can stimulate division and fusion of skeletal muscle satellite cells (2); IGF-1 can also increase myonuclei number and myofiber size in tissue culture (3). Moreover, these growth factors have the particularity to act in an endocrine but also in an auto/paracrine manner (4, 5). Hepatic production of IGF-1 appears to account for the bulk of the circulating IGF-1 (6) but IGF-1, like IGF-2, is also produced in a variety of extrahepatic tissues including myoblasts from skeletal muscles (7). Local production of IGF-1 has been shown to be implicated in hypertrophic adaptation of muscle after mechanical loading (8) such that, on the contrary, it may be expected to play a role in muscle atrophy induced by corticosteroid treatment. Although IGF-2 is mainly important during development, its expression in adult rats has been shown to be upregulated during disuse atrophy after immobilization (9), denervation (9, 10) and also after muscle lesion and reinnervation (11) or downregulated in adipose tissue of obese adult rats (12). The relative contributions of systematically versus locally produced IGFs are not clear particularly because IGF actions are strongly dependent upon and modified by IGF-binding proteins.

Therefore, in order to determine whether IGFs were involved in muscle atrophy seen after acute corticosteroid treatment, we examined in an experimental model of acute steroid-induced muscle atrophy in rats the serum levels of IGF-1, and the IGF messenger RNA (mRNA) levels in the diaphragm, gastrocnemius, and liver. The model used in the present study was previously shown to induce a leftward shift of the diaphragm force-frequency curve after steroid treatment due to a diaphragmatic type IIx/b atrophy (1).

	METHODS

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Study Design

Thirty-two male Wistar rats (15 wk old, body weight 300 to 400 g) were randomized to receive during 5 d, daily intramuscular injection in the right hindlimb of either: saline 0.6 ml: control, C (n = 8); methylprednisolone, M (Solu-Medrol; Upjohn, Puurs, Belgium) 80 mg/kg (n = 8); triamcinolone, T (Albicort; Sanofi Winthrop, Brussels, Belgium) 80 mg/kg (n = 8); or saline 0.6 ml and same amount of food as triamcinolone-treated rats: pair-fed (PF) (n = 8). During the duration of the study, animals were kept in individual cages and body mass and food intake were measured daily. The study was approved by the animal experiments committee of the medical faculty.

Experimental Procedures

Twenty-four hours after the last injection, animals were anesthetized with sodium pentobarbital (Nembutal, 60 mg/kg, intraperitoneally), tracheostomized and mechanically ventilated (Harvard pump respirator, South Natick, MA), with an O₂-enriched gas mixture. A laparotomy was performed and the whole costal diaphragm was quickly removed while a blood sample was taken.

IGF-1 serum level determination. Blood samples were centrifuged at 3,000 cycles/min at room temperature for 10 min. The supernatant was collected and stored at 20° C. IGF-1 was subsequently measured in acid-ethanol extracted sera by radioimmunoassay as previously described (13) using a guinea pig polyclonal antiserum (Ciba-Geigy, Basel, Switzerland). The detection limit of this assay is 25 pg of IGF-1.

IGF mRNA determinationRNA extraction. The whole diaphragm and the right gastrocnemius were first dissected to remove fat and other tissue. Muscles were then blotted dry and weighed. Thereafter, they were immersed for a few minutes in liquid nitrogen and put in a small tube to be stored at 80° C. The same procedure was done for the liver. Total RNA was isolated using a modified guanidinium isothiocyanate-CsCl method (14). Approximately 0.2 g of tissue was homogenized using an Ultra-Turrax homogenizer (Janke & Kunnel, Staufen, Germany) in a solution containing 50% (wt/vol) guanidinium thiocyanate, 25 mM ethylenediaminetetraacetic acid (EDTA), 0.5% lauryl sarcosine, and 0.1 M 2-mercaptoethanol. The homogenate was layered on top of a solution containing 5.7 M CsCl, 25 mM sodium acetate (pH 5.0), and 10 mM EDTA. After ultracentrifugation at 20° C in a SW41 rotor (Beckman, München, Germany) at 32,000 rpm for approximately 16 h, the supernatant was removed, the RNA pellet was dissolved in water, the solution was adjusted to 0.3 M sodium acetate (pH 5.2), and the RNA was precipitated with 2.5 volumes of absolute ethanol. RNA was then rinsed in 70% ethanol, vacuum dried, and redissolved in water.

Quality and quantity of the RNA preparations were determined by measurement of absorbance at 260 nm and 280 nm and by Northern blot analysis.

Northern blotting. Samples of 20 µg of RNA from the diaphragm, the gastrocnemius, and the liver were separated on a 1% (wt/vol) agarose gel containing formaldehyde as described (15). After electrophoresis the RNA was transferred to Biotrans (+) membranes (ICN, Cleveland, OH) using a 20× saline sodium citrate (SSC) blotting buffer (3 M NaCl and 0.3 M trisodium citrate). After the transfer, the blot was exposed for 2 min to ultraviolet light and then baked at 80° C for 2 h to immobilize the RNA.

Northern blotting was used to ensure the position of the IGF probes. However, since with Northern blotting, the number of slots per gel was limited, dot blotting procedure was used in order to have the data of the whole study on one blot. Moreover, dot blotting has the advantage to allow measurement of all the transcripts of IGFs simultaneously. Thus, the data presented in the RESULTS section come from the analysis on dot blotting.

Dot blotting. Samples of 10 µg of RNA from the diaphragm, the gastrocnemius, and the liver were dissolved in SSC and 37% formaldehyde. Samples were subsequently spotted on blotting paper (Schleicher & Schuell, Dassel, Germany), humidified in 15× SSC, using a dot-blot apparatus (Minifold SCR 96; Schleicher & Schuell, Inc.). After the transfer, the blot was baked at 80° C for 2 h to immobilize the RNA.

Prehybridization and hybridization. Each blot was prehybridized in 50% (vol/vol) formamide, 50 mM sodium phosphate (pH 6.5), 5× SSC, 5× Denhardt's solution (100× Denhardt's: 2% [wt/vol] Ficoll 400, 2% [wt/vol] polyvinylpyrrolidone, 2% [wt/vol] bovine serum albumin fraction V), 250 µg/ml of boiled herring sperm DNA, 0.5% (wt/ vol) sodium dodecyl sulfate (SDS), and 1% (wt/vol) glycine at 42° C overnight.

The IGF 3' complementary DNA (cDNA) probes (obtained from Dr. Derek Leroith, Bethesda, MD) were excised from the pGEM3 vector with EcoRI and HindIII for IGF-1 and with PstI-BamHI for IGF-2. Each insert (376 bp for IGF-1 and 570 bp for IGF-2) was labeled with radioactive phosphorus-deoxycytidine triphosphate (³²P-dCTP) by random primed labeling (Boehringer Mannheim, Mannheim, Germany). Unincorporated nucleotides were removed by centrifugation of the reaction mixture through a Sephadex G50 column (Pharmacia, Uppsala, Sweden). The blot was incubated first with a radiolabeled IGF-1 probe at 1.5 × 10⁶ cpm/ml of hybridization buffer consisting of 50% (vol/vol) formamide, 20 mM sodium phosphate (pH 6.5), 5× SSC, 1× Denhardt's solution, 10 µg/ml of boiled herring sperm DNA, 0.5% (wt/vol) SDS, and 8% (wt/vol) dextran sulfate at 42° C for 16 to 24 h. After hybridization the blot was first washed twice (5 and 15 min) in 2× SSC, 0.1% (wt/vol) SDS at room temperature and then washed twice for 20 min at 65° C in 0.2× SSC, 0.1% (wt/vol) SDS. The blot was then exposed to the Phosphorimager (Molecular Dynamics, Sunnyvale, CA) for quantification and autoradiographed using intensifying screens. The probe was removed by placing the membrane in a boiling solution containing 0.1% (wt/vol) SDS and allowing the solution to cool down to room temperature. This allowed us to reuse the Northern and dot-blot for further hybridization with other probes such as IGF-2 and 18S probes. Equal loading of the samples was checked by hybridization with an 18S ribosomal RNA probe.

Data Analysis

Statistical analysis was performed using the SAS Statistical package (SAS Institute, Cary, NC). Comparisons between groups were performed using one-way analysis of variance. Differences between means were assessed using Gabriel's test for all pairwise comparisons. Data are expressed as means ± standard deviation (SD).

	RESULTS

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Body Mass, Diaphragm Weight, and Food Intake

Starting body weight was similar between the four groups (Table 1). At the end of treatment, compared with C, body weight was significantly decreased by 14% in PF animals and by 19 and 21% after M and T treatment, respectively (p < 0.05 versus C). Similarly, compared with C, diaphragm mass was significantly reduced in PF rats (10%) and even more so after M (15%) and T (23%) treatment (p < 0.05 versus C) (Table 1). A previous study showed that this diaphragm wasting was associated with mainly a type IIx/b fiber atrophy (type IIx/b cross-sectional area PF: 10%, M: 22%, and T: 30%, p < 0.05 versus C) and also with a type IIa atrophy in PF (13%, not significant [NS]) and T (19%, p < 0.05 versus C) groups (1). Compared with respective initial values, food intake was similarly decreased in PF and T-treated rats, whereas it was also reduced in M-treated rats but to a lesser extent and remained unchanged in control animals (Table 1).

IGF-1 Serum Levels

Compared with C (845 ± 128 ng/ml), IGF-1 serum levels were significantly reduced in PF animals (505 ± 33 ng/ml, p < 0.001 versus C and M), after M treatment (699 ± 90 ng/ml, p < 0.001 versus C), and particularly after T treatment (273 ± 134 ng/ml, p < 0.001 versus other groups).

IGF mRNA Study

After hybridization with a rat cDNA probe for IGF-1, Northern blotting revealed the presence of three IGF-1 transcripts, with an apparent size of 7 to 7.8, 1.6 to 2.1, and 0.8 to 1.2 kilobases, respectively (Figure 1). Because the transcript at 7 to 7.8 kilobases was the most prominent, only this transcript was used for quantification of IGF-1 mRNA with the Phosphorimager. However, these data normalized with the data obtained after hybridization with an 18S ribosomal ribunucleic acid (rRNA) probe were similar to the data obtained on dot blotting. When normalized, dot blotting data showed that IGF-1 relative expression in liver was decreased by 19% (p < 0.001 versus T) and 74% (p < 0.001 versus others) after M and T treatment, respectively, and by 35% (p < 0.001 versus C) in PF animals (Figure 2, upper panel ). For the diaphragm, IGF-1 mRNA content was reduced by 44% (p < 0.0001 versus C) and 69% (p < 0.0001 versus C) in M- and T-treated rats, respectively. By contrast, IGF-1 relative expression was unchanged in PF animals (+13%, NS versus C) and was significantly greater compared with M and T groups (p < 0.0001) (Figure 3). Finally, IGF-1 relative expression in gastrocnemius was also smaller after corticosteroid treatment (51% and 59% after M and T, respectively, p < 0.001 versus C) as well as in PF animals (40%, p < 0.001 versus C) (Figure 4, upper panel ).

View larger version (54K): [in this window] [in a new window]   Figure 1.   Northern blot autoradiography of the liver in C, M-treated, T-treated, and PF animals after hybridization with a 32P IGF-1 3' cDNA probe. Each lane represents data from different animals.

View larger version (18K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   Figure 2.   IGF-1 (upper panel ) and IGF-2 (lower panel ) expression (in arbitrary units, AU) in the liver of C, M-treated, T-treated, and PF animals after hybridization with a 32P IGF-1 3' cDNA probe. Values are means ± SD. #p < 0.001 versus others, *p < 0.001 versus C.

View larger version (76K): [in this window] [in a new window]   View larger version (71K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 3.   (A) Dot-blot autoradiography of diaphragm in C, M-treated, T-treated, and PF animals after hybridization with a 32P- IGF-1 3' cDNA probe. (B) Same as in A but after hybridization with 32P-18S cDNA probe. (C ) IGF-1 expression (in AU) in diaphragm of C, M-treated, T-treated, and PF animals. Values are means ± SD of the animal data from Figure 3A corrected with data from Figure 3B. *p < 0.0001 versus C, **p < 0.0001 versus M and T.

View larger version (19K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Figure 4.   IGF-1 (upper panel ) and IGF-2 (lower panel ) expression (in AU) in the gastrocnemius of C, M-treated, T-treated, and PF animals after hybridization with a 32P IGF 3' cDNA probe. Values are means ± SD. *p < 0.001 versus C, **p < 0.0001 versus M and T.

Hybridization of Northern blots with a rat cDNA probe for IGF-2 revealed the presence of two detectable transcripts at 4.7 and 3.8 kilobases (Figure 5) particularly visible in muscles. In fact, IGF-2 mRNA in the liver was used as an index of IGF-2 serum levels because no other techniques were available to quantify serum levels. Dot-blot data, after normalization, showed that IGF-2 mRNA content in liver remained unchanged whatever the treatment (Figure 2, lower panel ). In the diaphragm, however, IGF-2 mRNA was depressed by 36% and 37% after M and T treatment, respectively, these decreases being however not significant (Figure 6). By contrast, IGF-2 relative expression was unchanged in the diaphragm of PF animals (+34%, NS versus C), being increased compared with M and T (p < 0.0003) (Figure 6). A similar pattern was present in the gastrocnemius where IGF-2 mRNA was depressed after M (28%, NS) and T (39%, NS), whereas it remained unaltered in PF animals (+19%, NS versus C), but was significantly greater compared with M and T groups (p < 0.001) (Figure 4, lower panel ).

View larger version (67K): [in this window] [in a new window]   Figure 5.   Northern blot autoradiography of the diaphragm in C, M-treated, T-treated, and PF animals after hybridization with a 32P IGF-2 3' cDNA probe. Each lane represents data from different animals.

View larger version (58K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 6.   (A) Dot-blot autoradiography of diaphragm in C, M-treated, T-treated, and PF animals after hybridization with a 32P IGF-2 3' cDNA probe. See Figure 3B for 18S values. (B) IGF-2 expression (in AU) in diaphragm of C, M-treated, T-treated, and PF animals. Values are means ± SD of the animal data from Figure 6A corrected with data from Figure 3B. *p < 0.0003 versus M and T.

	DISCUSSION

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The present study showed that: (1) IGF-1 and IGF-2 are expressed in the diaphragm and the gastrocnemius of adult rats; (2) corticosteroid treatment was associated with a decrease in IGF-1 serum levels particularly after T treatment, which resulted from a decrease in IGF-1 expression in the liver. A reduction in both IGF-1 and to a lesser extent in IGF-2 expression was observed after corticosteroid treatment in the diaphragm and the gastrocnemius, whereas IGF-2 mRNA content in the liver remained unchanged; (3) malnutrition reduced IGF-1 serum levels and IGF-1 expression in the liver but to a lesser extent than T treatment whereas it significantly depressed the expression of IGF-1 in the gastrocnemius and did not alter IGF-2 expression in the muscles.

Decreases in IGF-1 serum levels have been previously reported after corticosteroid administration in patients with various diseases (Cushing's syndrome, pituitary disease, nephrosis) (16). So far, IGF-1 serum levels have never been measured in animals treated with corticosteroids. Our data obtained in rats, however, agree with studies performed in humans. In addition, changes in IGF-1 serum levels observed after corticosteroid treatment or undernutrition in our study were in agreement with the alterations observed in liver IGF-1 expression. Our results show that although undernutrition also depressed IGF-1 serum levels, this effect was less pronounced than after triamcinolone treatment. Indeed, IGF-1 plasma concentration is known to be regulated by nutritional status as previously reported in malnourished humans (19) and in patients (20, 21) as well as in rats with dietary protein restriction (22, 23), or in young growing rats under acute nutritional deprivation (24). Thus, in animals, a diet deficient in energy and/or protein resulted in decreased plasma IGF-1 levels (19, 25) by a mechanism which has not been elucidated yet. In rats, it has been suggested that the effect of undernutrition on circulating IGF-1 resulted from a decrease in hepatic production by enhancing the growth hormone resistant state (26, 27). Indeed, fasting has been shown to decrease hepatic growth hormone binding (19), and the response of IGF-1 to growth hormone injection is blunted in protein-malnourished and fasted rats (25, 28). Whether a similar mechanism is involved in the reduction seen with corticosteroid treatment still needs to be determined.

Quantification of IGF-2 expression in the liver after Northern or dot blotting was not easy probably because IGF-2 expression in liver of adult rats is lower than in skeletal muscle (29). Our data show that IGF-2 expression in liver was not altered by corticosteroid treatment or by undernutrition. The effects of corticosteroid treatment on IGF-2 serum levels have never been studied in adult rats. However, in normal male volunteers, treatment with 2 mg dexamethasone twice daily has been shown not to affect IGF-2 serum concentrations (30). Besides, in adult rats, no data on the effect of undernutrition on serum IGF-2 are available. Studies have mainly been directed toward the effects of maternal fasting and its consequences for fetal growth. But data in normal subjects have shown that IGF-2 serum concentrations remained unchanged after 60 h (31), 4 d (32), or 5 d (33) of fasting.

Although IGF-1 expression has already been demonstrated in rat skeletal muscles and also in the diaphragm (34), the effects of malnutrition (as induced in our study) on IGF-1 mRNA in rat skeletal muscles have never been investigated. Only the effects of fasting or protein-deficient diet on IGF-1 expression in muscle have been examined (27, 34, 35). Unfortunately, the study of VandeHaar and coworkers (35) was performed on growing female rats 4 wk of age in which the decrease in IGF-1 mRNA levels was particularly pronounced (37%) 1 wk after a low protein diet, leading to a decreased body weight gain (44%). However, it has recently been shown that IGF-1 mRNA levels in rat skeletal muscle change considerably during maturation (36), such that comparison with our data obtained in adult rats seems to be difficult. Fasting has been shown to decrease IGF-1 expression in rat skeletal muscle (27) including the diaphragm (34). In our study, however, undernutrition did not reduce IGF-1 mRNA in the diaphragm because IGF-1 expression remained unchanged compared with control animals whereas it increased by 13% compared with the corticosteroid-treated groups. These data show that the effects of undernutrition on IGF-1 mRNA are different from those induced by corticosteroid treatment at least for the diaphragm. The discrepant results obtained after malnutrition and fasting (27, 34) may be related to several factors such as animal age, knowing that young, growing rats are more sensitive to fasting (24). The type of muscle may also be of importance, as shown by our data, where the same reduction in food intake resulted in decreased IGF-1 expression in the gastrocnemius but not in the diaphragm. The fact that the diaphragm is a muscle which is continuously active may exert a protection against protein wasting. Finally, the type of nutritional impairment is more likely to be responsible for discrepancies with previous reports as suggested by the data of Bornfeldt and coworkers (34). Indeed, they showed that the effects of fasting and protein-reduced diet on IGF-1 expression were totally different among different tissues including the liver and the diaphragm (34). Unfortunately, the reasons why different nutritional impairments distinctly affect IGF-1 expression in muscle are not known and are beyond the scope of the present study. Obviously, muscle wasting induced by corticosteroid treatment and undernutrition seems to result from distinct mechanisms that cannot be unraveled by the present study.

Similarly to IGF-1, IGF-2 expression has been demonstrated in rat skeletal muscles such as the gastrocnemius (5), the soleus and the plantaris (4) but not yet in the diaphragm except for the present study. In addition, our study is the first report showing that 5 d undernutrition did not alter IGF-2 expression in the diaphragm and gastrocnemius of the rat. As previously stated for IGF-1 regulation, the reasons why corticosteroid treatment and undernutrition differently affect IGF-2 expression in muscles are not known.

To the best of our knowledge, no data on the effects of corticosteroids on IGF expression in skeletal muscle are available. Our data show that deficiency of both systemic IGF-1 and local production of IGF-1 and 2 may contribute to the muscle atrophy previously shown after acute administration of corticosteroids (1). After malnutrition, the decline in serum IGF-1 concentration was less pronounced than after triamcinolone treatment and IGF-1 mRNA in the gastrocnemius was decreased whereas it remained unchanged in the diaphragm. This is, in fact, in accordance with the less pronounced muscle wasting and atrophy observed after malnutrition alone compared with corticosteroid treatment (Table 1). Except for the decreased IGF-1 expression observed after undernutrition in the gastrocnemius, it is interesting to mention that the effects of corticosteroids on the diaphragm and the gastrocnemius were similar.

Interestingly, the decrease in systemic and local IGF-1 production may be used as a potential marker of the atrophy induced by corticosteroid treatment. Indeed, in other models where muscle atrophy was present, the changes in IGF-1 were different (37, 38). It is, however, of value to mention that the doses of corticosteroids used in our rat model were 5 or 6 times higher than the maximal doses given in patients. This model was originally developed to examine in rats whether treatment with high doses of corticosteroids would induce acute myopathy as described in patients. Indeed, a potential role of corticosteroids in acute myopathy development has been suggested in patients treated acutely with high doses of systemic corticosteroids (39, 40), a treatment regimen used to treat status asthmaticus or to prevent rejection after lung transplantation. Nevertheless, it seems rather unlikely that different conclusions would be obtained with lower doses of corticosteroids if these doses resulted in muscle atrophy. Indeed, there is no clear reason to expect that the mechanisms by which corticosteroids would induce atrophy will depend on the dose administered. In fact, it may be postulated that if atrophy is present after corticosteroid treatment then changes in IGF expression are likely to occur. With lower doses of corticosteroids leading, however, to muscle wasting, the limiting factor to quantify these changes would be to find an appropriate technique sensitive enough to result in quantitative rather than qualitative measurements. This issue is under study. Whether IGF-1 serum concentrations and expression in muscle is changed in COPD patients treated with high doses of corticosteroids is not yet known. If so, then this observation may provide a specific diagnostic tool for steroid-induced myopathy in patients and it may lead to more specific therapy of steroid-induced myopathy. However, further clinical studies are needed to address these issues.

It remains to be determined whether the changes in IGF expression we observed in our rat model were associated with concomitant and/or similar changes at the protein levels. However, it is important to mention that basal IGF-1 protein level is already very low in control muscle (around 10 ng/g wet weight). Thus, if a decrease in IGF-1 protein is expected to occur in our model because IGF-1 expression is reduced in muscle, quantification of IGF-1 peptide might be a problem in the sense that it might fall below the detection limit of the radioimmunoassay. Furthermore, because IGF-1 concentration is at least 100 times higher in serum than in muscle, contamination from blood during IGF-1 extraction from muscle is difficult to exclude. For IGF-2, an efficient extraction method and a sensitive radioimmunoassay have only recently been developed by Lee and coworkers (29) but unfortunately, these methods are not yet widely available.

Finally, to better understand the mechanisms by which corticosteroids may influence IGF-1 production, regulation of IGF binding proteins (IGFBPs) should be taken into account because they may modulate IGF actions. Further investigations are, however, required in order to examine the potential role of the different IGFBPs in modulating the effects of IGF-1 expression observed after corticosteroid treatment in our rat model.

In conclusion, the present study demonstrated that treatment with massive doses of corticosteroids led to a decrease in IGF-1 serum levels resulting from a decrease in IGF-1 expression in the liver. Steroid treatment was also associated with a reduction in both IGF-1 and IGF-2 expression in the diaphragm and the gastrocnemius. These changes in IGF mRNA levels appear to be specific to corticosteroid treatment and they may contribute, at least in part, to the muscle alterations previously observed under such treatment (1). Whether these changes are also present in patients treated with corticosteroids and developing steroid myopathy needs to be determined.