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Departments of Pathology and * Neurosurgery, Chonnam National University Medical School and Research Institute of Medical Science, Gwangju, Korea
3To whom correspondence should be addressed. E-mail: mclee{at}chonnam.ac.kr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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KEY WORDS: • apoptosis • immunocytochemistry • myopathy • steroid • Western blot
Glucocorticoids are the most potent immunosuppressive and anti-inflammatory drugs and have been widely used in the treatment of autoimmune diseases. Glucocorticoid-associated adverse effects commonly develop and include hypertension (88%), Cushingoid features (66%), adrenal suppression (56%), myopathy (50%), osteopenia (46%), growth retardation (39%), obesity and hypercholesterolemia (30%), and cataracts (14%) (1). Several clinical studies have reported the occurrence of steroid-induced myopathy in intensive care unit patients after administration of high doses of glucocorticoids with or without neuromuscular junction blocking agents (2–4). Iatrogenic steroids, especially the 9-
-fluorinated ones like triamcinolone, betamethasone, or dexamethasone, can cause dose-dependent muscle wasting and weakness within weeks (5). This can be ameliorated by limitation of the steroid dose, alternate-day use, attention to exercise, and a high-protein diet. Electromyography shows myopathic features, whereas muscle biopsy reveals myopathy with selective loss of thick myosin filaments (rhabdomyolysis), necrosis, and atrophy of type II fibers (6,7).
In 3 previous studies (8–10), we investigated the development of steroid-induced myopathy of the extensor digitorum longus (EDL)4 and soleus in adult female rats treated with triamcinolone acetonide (TA, 5 mg · kg–1 · d–1 for 9 d). Rats treated with TA showed substantial loss of body and muscle weight. In the TA-treated group, cross-sectional areas of type II fibers of both EDL and soleus decreased in comparison with the controls. Necrotic changes occurred only in type II fibers of the soleus. Recovery from the weight loss with type II fiber atrophy was more pronounced in the exercise group (treadmill; speed 20 m/min, duration 30 min/d, 3 d/wk for 2 wk) than in the sedentary group, but not significantly different. The steroid-induced myopathy was partly amelorated by the exercise (8).
Moreover, to investigate whether apoptosis may contribute to the steroid-induced myopathy, rats treated with TA for 9 d were killed to detect apoptosis by in situ end labeling (ISEL) and electron microscopy in soleus muscle (9), as well as DNA electrophoresis (10). Immunohistochemical stainings of Fas antigen and p53 protein were performed to examine whether apoptosis-related proteins were present in the myopathy. Muscle-fiber necrosis and apoptotic myonuclei appeared in soleus muscle following administration of TA, whereas control muscle showed no evidence of apoptosis. Fas antigen was not detected in control muscle, but was expressed in soleus muscle with steroid-induced myopathy. Some of the Fas-antigen-expressing muscle fibers were positive for ISEL (Fig. 1). p53 Protein was not detected in any muscle fibers. These findings indicate that TA can induce apoptosis in differentiated skeletal muscles, and Fas antigen might be partly related to apoptotic muscle death in steroid-induced myopathy.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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Female Sprague-Dawley rats (n = 20) weighing 180 to 210 g were maintained under standard conditions. Rats were divided into 2 groups. Each group was given a daily intraperitoneal injection of either physiologic saline (n = 5) or TA (Dongkwang) at a dose of 5 mg/kg body wt for 9 d (n = 15). At d 10 after completion of the injections, the soleus muscles from both legs were taken under ether anesthesia. A 10-mm-long section was taken from the midbelly of the soleus and quick-frozen in liquid nitrogen (–196°C). The tissues were used for Western blot analysis.
Western blot analysis.
Muscles frozen in liquid nitrogen were homogenized in a lysis buffer solution containing 100 mmol/L Tris-HCl (pH 7.4), 10 mmol/L EDTA, 50 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L leupeptin, and 20% glycerol. The homogenates were centrifuged at room temperature for 5 min at 5000 x g, and the supernatants were collected. The protein concentration in each sample was measured with a spectrophotometer using a detergent-compatible protein assay kit (Bio-Rad). An equal amount of protein from each sample was mixed with loading buffer (300 µg/lane), analyzed on 12% SDS-PAGE under reducing conditions, and then transferred onto Immobilon-P membranes (Millipore). After transfer, the Immobilon-P membranes were blocked in buffer containing 5% dry milk in PBS–0.05% Tween 20 and then incubated overnight at 4°C with primary antibodies (Table 1). Immunodetection was performed by means of peroxidase-conjugated goat antimouse, antirat, and antirabbit antibodies (SantaCruz Biotech) matched to sources of primary antibodies and visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech). Each blot was scanned into a computer, and images were stored in JPG format.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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Apoptosis is the endpoint of an energy-dependent cascade of molecular events, initiated by physiologic or harmful stimuli and consisting of 4 separable but overlapping components: signaling pathways, control and integration, common-execution phase, and removal of dead cells (11–14). Fas-Fas ligand was a positive transmembrane signal determinant of apoptosis in previous studies (9,10). The Fas molecule, synonymously referred to as APO-1, contains a cytoplasmic "death domain" shared with the type I tumor necrosis factor receptor (15). To date, apoptosis induced by Fas antigen has been extensively investigated in the lymphoid systems (16,17). Immunohistochemical studies have demonstrated that Fas antigen is expressed on muscle fibers from patients with various muscle wasting diseases, but not in normal muscle cells (18,19).
The control and integration stage is performed by specific proteins that connect death signals to the execution program. There are commonly 2 broad schemes for this stage, which are not mutually exclusive. One involves the direct transmission of signals by specific adapter proteins to the execution mechanism, as described for the Fas-Fas ligand model and target cell killing by cytotoxic T lymphocytes (15,20,21). Fas-Fas ligand interacts with 3 death domains, consisting of FADD, RAIDD, and Daxx (22,23). Initiator caspase molecules, caspases 2 and 8, potentiate the process (24). The second scheme involves members of the Bcl-2 family of proteins, which play major and ubiquitous roles in apoptotic regulation largely by regulating mitochondrial function (25,26).
In the present study, the levels of proapoptotic proteins (caspase 8, Bax, Bad, and Bid) were higher in the steroid-induced myopathy group than in the control group, whereas antiapoptotic proteins (Bcl-2, Bcl-XL) did not change between the 2 groups. Akt protein levels did not change; however, phosphorylated Akt levels decreased slightly. These elevated apoptotic signals result in mitochondrial permeability changes (27) with cytochrome c release from the mitochondria into the cytosol (28,29). Other proteins, such as p53 protein and viral protease inhibitor proteins, are also involved in apoptotic regulation (30,31). However, p53 protein was not involved in steroid-induced myopathy in the previous study (9).
The execution phase is the final pathway of the actual apoptotic death program and is accomplished largely by the caspase family of proteases, consisting of caspase 9 and caspases 3, 6, 7 (29,32). These execution caspases disrupt the cytoskeleton by cleavage of cytoskeletal and nuclear matrix proteins (33). Caspase 9 was slightly elevated in the steroid-induced myopathy group. Apoptotic cells and their fragments have marker molecules on their surface, which facilitates early recognition by adjacent cells or phagocytic cells for phagocytic uptake and disposal. These overall processes of apoptosis might be involved in skeletal muscle myopathies induced by prolonged treatment with glucocorticosteroids.
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CONCLUSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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FOOTNOTES |
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2 Supported in part by the National Space Biomedical Research Institute through Cooperative Agreement NCC 9–58 with NASA.
4 Abbreviations used: EDL, extensor digitorum longus; FADD, Fas-associated death domain; ISEL in situ end labeling; TA, triamcinolone acetonide.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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