Reduced sarcolemmal dystrophin distribution and upregulation of utrophin in the cardiac and skeletal muscles of CHF-146 dystrophic hamsters

Abnormalities in the dystrophic gene product, dystrophin, have been implicated in initiating the primary membrane defect and excessive intracellular calcium accumulation (EICA), which play fundamental pathogenetic roles in hereditary muscular dystrophy (HMD). Two other cytoskeletal proteins, spectrin and utrophin, bear remarkable structural and functional homologies to dystrophin. CHF-146 strain dystrophic hamsters (DH), like patients with Duchenne muscular dystrophy (DMD), die prematurely from cardiopulmonary insufficiency, focal myonecrosis, and progressive degeneration of the cardiac and skeletal muscles with EICA. Although DH present a suitable model for HMD, there are controversies concerning their dystrophin and utrophin status. Using immunocytochemistry and Western blotting, we studied dystrophin, spectrin, and utrophin anomalies in the cardiac and skeletal muscles of 6-mo-old male DH. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as controls. Sarcolemmal dystrophin staining was much weaker and interruptive in the DH. The densitometric analysis of the immunoblots revealed that dystrophin is reduced in DH by 83% in cardiac muscle (p<0.0001), and by 50% in skeletal muscle (p<0.0001). We conclude that sarcolemmal dystrophin distribution is markedly reduced and discontinuous in the cardiac and skeletal muscles of DH, with simultaneous upregulation of utrophin and a varied degree of spectrin labeling. This observation suggests that reduced sarcolemmal dystrophin is associated with membrane hyperpermeability, which leads to progressive muscle degeneration via EICA and segmental necrosis in DH. As in DMD, utrophin appears to play an important compensatory role in hamster dystrophinopathy.     

Reinnervation is followed by necrosis in previously denervated skeletal muscles of dystrophic hamsters

Hind leg muscles of dystrophic hamsters were continually denervated by multiple crushes of the sciatic nerve to as long as 93 days of age. In these muscles, the prevalence of centronucleated fibers which is a cumulative index of prior necrosis, remained very low. In control dystrophic muscles the prevalence of centronucleated fibers increased steadily to approximately 80% where it leveled off. By omitting further crushes in other groups of animals, previously denervated muscles became adequately reinnervated. In the reinnervated muscles the prevalence of centronucleated fibers steadily increased throughout the necrotic phase of dystrophy at a rate that was comparable to corresponding stages of the natural history of the disease. These experiments indicated that continued denervation was effective in negating skeletal muscle fiber necrosis throughout the necrotic phase and that the electromechanical activity of muscle fibers which allows muscle fiber necrosis was not a time-locked factor.     

Intracellular elemental content of cardiac and skeletal muscle of normal and dystrophic hamsters

To test the hypothesis that the genetic lesion causing muscular dystrophy might be reflected in an abnormal intracellular elemental content, the elemental content of individual cardiac and skeletal muscle fibers in 50-day-old male control and cardiomyopathic BIO 53.58 hamsters was determined. The technique of electron probe x-ray microanalysis of freeze-dried tissue was employed. No electrolyte content differences were found between control and diseased animals for nuclei, myofibrillar cytoplasm, or mitochondrially-enriched cytoplasm of cardiac myocytes. Sulfur was elevated in dystrophic cardiac myocytes and was the only element significantly different in heart tissue of control and cardiomyopathic animals. Sulfur was also elevated in dystrophic soleus muscle fibers. The pattern of electrolyte content of these cells reflected a mixture of normal cells and damaged cells with altered electrolyte content. In this hamster model, alteration of electrolyte content of myocytes appears to be a result of the disease process and not an inherent characteristic of muscular dystrophy. The elevated sulfur in dystrophic hamster myocytes reflects a biochemical lesion which deserves further study.     

Doxycycline ameliorates the dystrophic phenotype of skeletal and cardiac muscles in mdx mice

Introduction: We examined whether doxycycline, an antibiotic member of the tetracycline family, improves the histopathology and muscle function in mdx mice, the experimental model of DMD. Methods: Doxycycline was administered for 36 days (starting on postnatal day 0) and for 9 months (starting at 8 months of age) in drinking water. Histopathological, biochemical (creatine kinase), and functional (forelimb muscle grip strength) parameters were evaluated in limb, diaphragm, and cardiac muscle. Results: Doxycycline significantly minimized the dystrophic phenotype of skeletal and cardiac muscles and improved forelimb muscle strength. The drug protected muscle fibers against myonecrosis and reduced inflammation. Furthermore, it slowed the progression of myocardial fibrosis. Conclusions: This study provides evidence that doxycycline may be a potential therapeutic agent for DMD. Muscle Nerve 46: 400-406, 2012.     

Increased calcium-activated neutral protease activity in muscles of dystrophic hamsters and mice

A Ca²⁺-activated neutral protease activity was examined in muscles of normal and dystrophic hamsters and mice. Light grey and golden brown strains of normal and B10 14.6 strain of dystrophic hamsters were used. Normal and dystrophic mice were of the Bar Harbor 129 ReJ strain. Enzyme activity was measured in the post myofibrillar fraction (homogenate) and in the 75,000 × g pellet (particulate fraction) and supernatant using purified myofibrils.

In normal and dystrophic hamsters or mice, the Ca²⁺-activated neutral protease was most active in the supernatant followed by the homogenate and particulate fractions. As compared to fractions from normal muscle, enzyme activity was significantly elevated in all 3 fractions from dystrophic muscles of hamsters and mice. Both homogenate and supernatant fractions from muscles of normal hamsters had significantly higher enzyme activity than those of normal mice. Enzyme activity was similar in the particulate fraction. Similarly enzyme activity in the 3 fractions from dystrophic hamster and mouse muscles showed no significant difference.

It is suggested that the Ca²⁺-activated neutral protease may be involved in muscle fibre necrosis in muscular dystrophy.     

骨骼肌兴奋收缩偶联与细胞内的钙稳态

背景：在骨骼肌胞浆内,Ca2+是神经兴奋和肌肉收缩之间的重要偶联因子,控制着肌肉收缩的启动和舒张的终止,对骨骼肌的兴奋收缩偶联起着不可或缺的作用,骨骼肌的兴奋收缩偶联与细胞内钙稳态的变化紧密相关。目的：研究近年来骨骼肌细胞内钙调控机制的研究进展及钙稳态与运动之关系的进展。方法：电子检索CNKI数据库、读秀学术搜索等中文数据库和Elsevier SD,Springer Link等英文数据库1980/2010收录的骨骼肌兴奋收缩偶联和钙稳态的相关综述和实验研究报告,分析骨骼肌细胞内钙调控机制的研究进展及钙稳态与运动之关系的研究进展。结果与结论：共纳入骨骼肌兴奋收缩偶联与钙稳态相关文献43篇。骨骼肌的兴奋收缩偶联与细胞内钙稳态之间有着密不可分的关系,但骨骼肌内钙稳态的调控机制还不甚明朗,尚期待大量研究。对运动与肌细胞内钙稳态的关系的研究也仍需要进一步努力,探索适宜适当的运动,探索运动性疲劳的发病机制,有效预防和对抗运动性疲劳及肌肉损伤仍将是研究者的工作重点。     

Force and stiffness of old dystrophic (mdx) mouse skeletal muscles

It has recently been suggested, based on studies of tissue pathology, that the limb muscles of old mdx mice may be a good model for the muscular changes seen in human Duchenne muscular dystrophy. To test this hypothesis, we measured force and stiffness of soleus and extensor digitorum longus (EDL) muscles of old (20–21 months) mdx mice and age-matched controls. The mdx and control muscles generated similar twitch, tetanic, and eccentric forces. They were also equally stiff. The results show that the mechanics of aged mdx limb muscles differ greatly from Duchenne muscular dystrophy in humans, and disagree with the hypothesis. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:536–539, 1998.     

The isometric responses of fast and slow twitch muscles from normal and genetically dystrophic hamsters

There has been a need for some time to examine the effects of the dystrophic process upon the mechanical properties of the limb muscles of the dystrophic hamster. This paper reports the findings of such a study by using an in vivo technique to record the isometric twitch characteristics of the extensor digitorum longus and soleus muscles. Normal and dystrophic animals have been examined at various ages as it has been reported that the disease is progressive. Normal muscle characteristics fall in line with those already published for small mammals. Dystrophic muscles show little difference from normal muscles with respect to their twitch characteristics. Only at the 60 day age point was the time to half relaxation of dystrophic muscles longer than its normal counterpart. Differences were found between normal and dystrophic muscle weights and between the ratio of muscle weight to body weight. It is proposed that these result probably from increased hydration of the muscles caused secondarily to the cardiomyopathy.     

Reinnervation of dystrophic muscles

OBJECTIVE: To analyse the regenerating capability of the peripheral nerve fibers and the capability of the muscle fibers to accept the regenerating and new nerve sprouts in myotonic dystrophy (MD). MATERIAL AND METHODS: One male, aged 58 years, diagnosed of MD at the age of 30 years, suffered neuralgic amyotrophy in the right shoulder girdle 4 weeks before admission. Needle EMG and nerve conduction studies were performed on admission and 6, 12, and 18 months later. RESULTS: On admission there were atrophy and absence of voluntary contraction in deltoids, supra- and infraspinatus muscles. EMG showed abundant fibrillations, positive sharp waves and myotonic bursts in these muscles without voluntary activity, consistent with axonal neuropathy of both axillary and suprascapular nerves. The follow-up showed signs of reinnervation 6 months later and slight loss of long duration and high amplitude MUPs at 18 months of evolution, with good clinical recovery. This is compatible with chronic neurogenic atrophy, presumably as an expression of type grouping. CONCLUSIONS: The reinnervation capability of the nerve fibers and the capability of muscle fibers membrane to accept regenerating and new nerve sprout remain in MD. Myotonic bursts persist after total denervation.     

The effects of diltiazem in dystrophic hamsters

Calcium accumulates in muscles of dystrophic hamsters (DH) and patients with Duchenne muscular dystrophy. Various Ca antagonists were beneficial to the cardiomyopathy of DH, but had only minor effects on skeletal muscle. We administered a new Ca antagonist, diltiazem, 25 mg/kg/day orally to normal and dystrophic hamsters from ages 37 to 92 days. We observed a marked reduction in muscle Ca in DH treated with diltiazem: 73% in the heart, 61% in the diaphragm, and 48% in the rectus femoris. Plasma CK was significantly lower (by 37%) in treated DH, while the elevated rate of noncollagen protein synthesis in the diaphragm was not diminished. Histologically, the most important change was a reduction in Ca deposits in the heart. Diltiazem was well-tolerated by all animals and did not modify Ca content in normal hamsters. This study suggests that diltizem may have therapeutic value in those conditions that are accompanied by excessive accumulation of Ca in tissues, such as muscular dystrophy.     

Decreased chemiluminescence in thymocytes of dystrophic hamsters

When thymocytes are stimulated they generate reactive oxygen species, which under appropriate conditions produce chemiluminescence (CL). The reactions occur near the cell surface. Since genetically determined muscular dystrophies are currently considered “membrane diseases”, we tested the CL of thymocytes from dystrophic hamsters, strain BIO 14.6, in comparison with control animals of the Rb-strain. CL of 33 × 10⁶ thymocytes each was monitored in a liquid scintillation counter at 32 C. Dystrophic cells stimulated with concanavalin A (Con A) reached only 60% of the normal peak CL. When stimulated with the calcium ionophore A23187, dystrophic cells exhibited only 40% of the peak CL of control cells. The thymus weight of dystrophic hamsters was significantly reduced. It is not known yet whether these alterations of the thymus are secondary to extrathymic factors or whether they are intrinsic to the thymocyte. If the latter is true, it would be an indication that the genetic defect of dystrophic hamsters is also expressed in the thymus.     

An experimental morphometric study of neutral lipid accumulation in skeletal muscles

The neutral lipid content of skeletal muscle fibers was determined by computing the lipid accumulation index (LAI) on transverse cryostat sections stained with Oil Red O. The LAI was defined as: (total area of neutral lipid droplets in a fiber) × 100/(total cross-sectional area of a fiber). The biceps, diaphragm, and soleus muscles were studied in 3 groups of guinea pigs: normal animals, animals fasted for 48 hours, and animals subjected to muscle denervation and then fasted for 48 hours. and animals, subjected to muscle denervation and then fasted for 48 hours. In normal animals, the highest mean LAI was found in the diaphragm (4.93) in comparison with lower values in the biceps (2.25) and soleus (2.09). After fasting, these values were markedly increased; there was also a concomitant increase in plasma free fatty acid (FFA) concentration. Prior denervation further increased the LAI in biceps and soleus but reduced it in the diaphragm. Type 2A fibers tended to show high lipid accumulation when the plasma FFA concentration was high. Type 2B fibers never accumulated much lipid under any circumstances. Type 1 fibers varied in their response in the different muscles.     

IGF-I treatment improves the functional properties of fast- and slow-twitch skeletal muscles from dystrophic mice

Although insulin-like growth factor-I (IGF-I) has been proposed for use by patients suffering from muscle wasting conditions, few studies have investigated the functional properties of dystrophic skeletal muscle following IGF-I treatment. 129P1 ReJ-Lama2(dy) (129 ReJ dy/dy) dystrophic mice suffer from a deficiency in the structural protein, laminin, and exhibit severe muscle wasting and weakness. We tested the hypothesis that 4 weeks of IGF-I treatment ( approximately 2 mg/kg body mass, 50 g/h via mini-osmotic pump, subcutaneously) would increase the mass and force producing capacity of skeletal muscles from dystrophic mice. IGF-I treatment increased the mass of the extensor digitorum longus (EDL) and soleus muscles of dystrophic mice by 20 and 29%, respectively, compared with untreated dystrophic mice (administered saline-vehicle only). Absolute maximum force (P(o)) of the EDL and soleus muscle was increased by 40 and 32%, respectively, following IGF-I treatment. Specific P(o) (sP(o)) was increased by 23% in the EDL muscles of treated compared with untreated mice, but in the soleus muscle sP(o) was unchanged. IGF-I treatment increased the proportion of type IIB and type IIA fibres and decreased the proportion of type I fibres in the EDL muscles of dystrophic mice. In the soleus muscles of dystrophic mice, IGF-I treatment increased the proportion of type IIA fibres and decreased the proportion of type I fibres. Average fibre cross-sectional area was increased in the EDL and soleus muscles of treated compared with untreated mice. We conclude that IGF-I treatment ameliorates muscle wasting and improves the functional properties of skeletal muscles of dystrophic mice. The findings have important implications for the role of IGF-I in ameliorating muscle wasting associated with the muscular dystrophies.     

3H]Ouabain binding in normal and dystrophic mouse skeletal muscles and the effect of age

The numbers of Na+-K+ ATPase sites in skeletal muscles of normal and dystrophic mice between 3 and 17 months of age have been estimated using [3H]ouabain binding assays. In normal mice, at all ages, slow twitch muscle, soleus (SOL), bound significantly more [3H]ouabain than fast-twitch muscle, extensor digitorum longus (EDL). [3H]Ouabain binding did not alter in either SOL or EDL from normal mice over the age range studied. The numbers of Na+-K+ ATPase sites did alter in muscles taken from dystrophic mice (C57BL/6J dy2J/dy2J). In EDL there was an increase and in SOL a decrease in [3H]ouabain binding. This may be related to a change in muscle fibre metabolism from glycolytic to oxidative or to an altered activity pattern. Increasing age resulted in a progressive reduction in [3H]ouabain binding of both SOL and EDL from dystrophic mice. Part of this reduction may be only apparent and due to an increase in connective tissue composition of dystrophic muscles. A limited study of muscles from neonate dystrophic mice indicated that abnormal [3H]ouabain binding was not present in EDL before two weeks of age.     

Reversal of impaired oxidative phosphorylation and calcium overloading in the skeletal muscle mitochondria of CHF-146 dystrophic hamsters

Membrane-mediated excessive intracellular calcium accumulation (EICA) and diminished cellular energy production are the hallmarks of dystrophic pathobiology in Duchenne and Becker muscular dystrophies. We reported reversal of respiratory damage and Ca²⁺-overloading in the in vitro cardiac mitochondria from CHF-146 dystrophic hamsters (DH) with hereditary muscular dystrophy (Bhattacharya et al., 1993). Here we studied respiratory dysfunctions in the skeletal muscle mitochondria from young and old DH, and whether these abnormalities can be reversed by reducing [Ca²⁺] in the isolation medium, thereby lowering intramitochondrial Ca²⁺-overloading. Age- and sex-matched CHF-148 albino normal hamsters (NH) served as controls. As an index of EICA and cellular degeneration, Ca and Mg levels were assayed in the skeletal muscle and mitochondria. Mitochondria from young and old DH, isolated without EDTA (B_o medium), revealed poor coupling of oxidative phosphorylation, diminished stimulated oxygen consumption rate, and lower respiratory control ratio and ADP/O ratios, compared to NH. Incorporation of 10 mM EDTA (B_E medium) in the isolation medium restored mitochondrial functions of the dystrophic organelles to a near-normal level, and reduced Ca²⁺-overloading. The mitochondrial Ca level in DH was significantly higher than in NH, irrespective of the medium. However, compared to B_o medium, the dystrophic organelles isolated in B_E medium had lower Ca levels and markedly improved oxidative phosphorylation as seen in NH. Muscle Ca contents in the young and old DH were elevated relative to NH, showing a positive correlation with the increased mitochondrial Ca²⁺-sequestration. Dystrophic muscle also revealed Ca deposition with an abundance of Ca²⁺-positive and necrotic myofibers by light microscopy, and intramitochondrial Ca²⁺-overloading by electron microscopy, respectively. However, Mg levels in the muscle and mitochondria did not alter with age or dystrophy. These data parallel our observations in the heart, and suggest that functional impairments and Ca²⁺-overloading also occur in the skeletal muscle mitochondria of DH, and are indeed reversible if EICA is regulated by slow Ca²⁺-channel blocker therapy (Johnson and Bhattacharya, 1993).     

ZnT-1 expression in astroglial cells protects against zinc toxicity and slows the accumulation of intracellular zinc

Zinc ions are emerging as an important factor in the etiology of neurodegenerative disorders and in brain damage resulting from ischemia or seizure activity. High intracellular levels of zinc are toxic not only to neurons but also to astrocytes, the major population of glial cells in the brain. In the present study, the role of ZnT-1 in reducing zinc-dependent cell damage in astrocytes was assessed. Zinc-dependent cell damage was apparent within 2 h of exposure to zinc, and occurred within a narrow range of approximately 200 microM. Pretreatment with sublethal concentrations of zinc rendered astrocytes less sensitive to toxic zinc levels, indicating that preconditioning protects astrocytes from zinc toxicity. Fluorescence cell imaging revealed a steep reduction in intracellular zinc accumulation for the zinc-pretreated cells mediated by L-type calcium channels. Heterologous expression of ZnT-1 had similar effects; intracellular zinc accumulation was slowed down and the sensitivity of astrocytes to toxic zinc levels was reduced, indicating that this is specifically mediated by ZnT-1 expression. Immunohistochemical analysis demonstrated endogenous ZnT-1 expression in cultured astroglia, microglia, and oligodendrocytes. Pretreatment with zinc induced a 4-fold increase in the expression of the putative zinc transporter ZnT-1 in astroglia as shown by immunoblot analysis. The elevated ZnT-1 expression following zinc priming or after heterologous expression of ZnT-1 may explain the reduced zinc accumulation and the subsequent reduction in sensitivity toward toxic zinc levels. Induction of ZnT-1 may play a protective role when mild episodes of stroke or seizures are followed by a massive brain insult.     

Administration of insulin-like growth factor-I improves fatigue resistance of skeletal muscles from dystrophic mdx mice

Muscle fatigue occurs in many neuromuscular diseases, including the muscular dystrophies, and it contributes to a loss of functional capacity and reduced quality of life for affected patients. An improvement in fatigue resistance has been observed in diaphragm muscles of mdx mice following insulin-like growth factor-I (IGF-I) administration. Whether similar treatment can improve locomotor muscle function in mdx mice is not known. We examined the efficacy of IGF-I administration (1 mg/kg daily s.c. for 8 weeks) on structural, metabolic, and functional properties of extensor digitorum longus (EDL) and soleus muscles of mdx mice, and tested the hypothesis that IGF-I treatment would improve function in these muscles. After treatment, muscles were more resistant to fatigue during repeated maximal contractions than muscles from untreated mice, an improvement associated with increased muscle fiber succinate dehydrogenase activity in the absence of changes in cellular (single-fiber) contractile activation characteristics. The findings have important clinical implications, not just for the dystrophinopathies, but for all neuromuscular pathologies where fatigue of locomotor muscles limits functional capacity and decreases quality of life.