Contractile function and low-intensity exercise effects of old dystrophic (mdx) mice

Oldmdx mice display a severe myopathyalmost identical to Duchenne's muscular dystrophy. This study examinedthe contractile properties of old mdxmuscles and investigated any effects of low-intensity exercise.Isometric contractile properties of the extensor digitorum longus (EDL)and soleus muscles were tested in adult (8-10 mo) and old (24 mo,split into sedentary and exercised groups)mdx mice. The EDL and soleus from oldmdx mice exhibited decreased absolutetwitch and tetanic forces, and the soleus exhibited a >50% decreasein relative forces (13.4 ± 0.4 vs. 6.0 ± 0.9 N/cm²) compared with adult mice.Old mdx muscles also showed longer contraction times and a higher percentage of type I fibers. Normal andmdx mice completed 10 wk of swimming,but mdx mice spent significantly lesstime swimming than normal animals (7.8 ± 0.4 vs. 15.8 ± 1.1 min, respectively). However, despite their severe dystrophy,mdx muscles responded positively tothe low-intensity exercise. Relative tetanic tensions were increased(~25% and ~45% for the EDL and soleus, respectively) after theswimming, although absolute forces were unaffected. Thus these resultsindicate that, even with a dystrophin-deficient myopathy,mdx muscles can still respond to low-intensity exercise. This study shows that the contractile functionof muscles of old mdx mice displaysmany similarities to that of human dystrophic patients and providesfurther evidence that the use of non-weight-bearing, low-intensityexercises, such as swimming, has no detrimental effect on dystrophicmuscle and could be a useful therapeutic aid for sufferers of musculardystrophy.

     

Programmed cell death in dystrophic (mdx) muscle is inhibited by IGF-II

The pathology of Duchenne Muscular Dystrophy (DMD) is characterised by unstable muscle fibres and by increased cell turnover due to the absence of functional dystrophin protein. We have used skeletal muscle, primary muscle stem cell cultures (Smith and Schofield, 1994; Smith et al., paper submitted) and clonal cell lines of the mouse DMD model (mdx) and its congenic control (C57BI) to demonstrate that programmed cell death (PCD) and apoptotic morphology is increased in dystrophic (mdx) muscle and in cultured muscle cells. We also show that the peptide growth factor (IGF-II), which is thought to play a role in mammalian myogenesis, reduces PCD in mammalian skeletal muscle myoblasts both in vivo and in vitro. This is the first time that apoptosis or PCD have been demonstrated in normal mammalian skeletal muscle. We discuss the potential of this system in determining the role of PCD in mammalian myogenesis and skeletal muscle maturation, its significance in dystrophic muscle, and suggest a novel therapeutic route whereby the pathology of DMD may be alleviated using the survival properties of IGF-II.     

Mofification of body composition by clenbuterol in normal and dystrophic (mdx) mice

Female dystrophic mice (mdx on C57 Black background) gained weight more rapidly than age-matched controls and had a higher body fat content (% body weight), a slightly lower protein content and a reduced mass of muscle. Chronic treatment (21 d) of the mice with the ₂-agonist clenbuterol stimulated weight gain in both genotypes without affecting energy intake. Clenbuterol increased the mass of the gastrocnemius and soleus muscle by 13% and 29% in normal and dystrophic mice, respectively, and raised body protein but depressed body fat. Body water and energy content were unaffected by clenbuterol, but the ratio of protein to fat in the carcasses was enhanced by 17% in normal and 56% in dystrophic mice following clenbuterol treatment. Thus, the ₂-agonist restored the body composition of dystrophic mice to normal and enhanced the protein to fat ratio in both these and normal mice.     

Extensive but coordinated reorganization of the membrane skeleton in myofibers of dystrophic (mdx) mice

We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. beta-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, beta-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, beta-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain beta-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with beta-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including alpha-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle.     

Dynamic responses of the glutathione system to acute oxidative stress in dystrophic mouse (mdx) muscles

The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to I/R exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles.     

Myotube phospholipid synthesis and sarcolemmal ATPase activity in dystrophic (mdx) mouse muscle.

Phospholipid incorporation of 32P by primary myotube cultures and the tissue activity of sarcolemmal Na+/K(+)-transporting ATPase were studied to determine whether the absence of dystrophin from dystrophic (mdx) muscle would affect membrane lipid synthesis and membrane function. The incorporation of 32P by phospholipid as a ratio with total protein was greater in cultured dystrophic cells compared with control cells. The mdx cells also incorporated more 32P than control cells into phosphatidylethanolamine, which is thought to increase prior to myoblast fusion, and less into phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. There was no difference in total protein content or [3H]leucine or 32P incorporation into the aqueous fraction of dystrophic and control cells, although dystrophic cells incorporated less [35S]methionine into protein than controls. Isolated sarcolemma from mdx skeletal muscle tissue demonstrated a consistently greater specific activity of ouabain-sensitive Na+/K(+)-transporting ATPase than sarcolemmal preparations from control skeletal muscle. These observations suggest that cytoskeletal changes such as dystrophin deficiency may alter the differentiation of membrane composition and function.     

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.     

Myogenic NOS and endogenous NO production are defective in colon from dystrophic (mdx) mice

The aim of the present study was to evaluate whether alterations in the distribution and/or function of nitric oxide synthase (NOS) could be involved in the development of the spontaneous mechanical tone observed in colon from dystrophic (mdx) mice. By recording the intraluminal pressure of isolated colon from normal mice, we showed that N(omega)-nitro- L-arginine methyl ester (L-NAME) increased the tone, even in the presence of tetrodotoxin. The effect was prevented by L-arginine, nifedipine, or Ca(2+)-free solution. In colon from mdx mice, L-NAME was ineffective. Immunohistochemistry revealed that the presence and distribution of neuronal (nNOS), endothelial, and inducible NOS isoforms in smooth muscle cells and neurons of colon from mdx mice were the same as in controls. However, the expression of myogenic nNOS was markedly reduced in mdx mice. We conclude that there is a myogenic NOS in mouse colon that can tonically produce nitric oxide to limit influx of Ca(2+) through L-type voltage-dependent channels and modulate the mechanical tone. This mechanism appears to be defective in mdx mice.     

Ultrastructure of diaphragm from dystrophic alpha-sarcoglycan-null mice

alpha-Sarcoglycan is a 50 kDa single-pass transmembrane glycoprotein exclusively expressed in striated muscle that, together with beta-, gamma-, and delta-sarcoglycan, forms a sub-complex at the muscle fibre cell membrane. The sarcoglycans are components of the dystrophin-associated glycoprotein (DAG) complex which forms a mechanical link between the intracellular cytoskeleton and extracellular matrix. The DAG complex function is to protect the muscle membrane from the stress of contractile activity and as a structure for the docking of signalling proteins. Genetic defects of DAG components cause muscular dystrophies. A lack or defects of alpha-sarcoglycan causes the severe type 2D limb girdle muscular dystrophy. alpha-Sarcoglycan-null (Sgca-null) mice develop progressive muscular dystrophy similar to the human disorder. This animal model was used in the present work for an ultrastructural study of diaphragm muscle. Diaphragm from Sgca-null mouse presents a clear dystrophic phenotype, with necrosis, regeneration, fibre hypertrophy and splitting, excess of collagen and fatty infiltration. Some abnormalities were also observed, such as centrally located nuclei of abnormal shape, fibres containing inclusion bodies within the contractile structure, and fibres with electron-dense material dispersed over almost the entire cell. Additionally, unusual interstitial cells of uncertain identity were detected within muscle fibres. The abnormal ultrastructure of the diaphragm from Sgca-null mice is discussed.     

Satellite cells from dystrophic (mdx) mouse muscle are stimulated by fibroblast growth factor in vitro

Satellite cells cultured from dystrophic (mdx) and from control mouse hindlimb muscles grow and fuse to form muscle fibers within 4-5 days. Total cell number and muscle-fiber formation are stimulated by bovine fibroblast growth factor (FGF). At low FGF levels (0.02-0.20 ng/ml) control satellite cells as well as fibroblasts are unresponsive, while mdx satellite cells show three- to four-fold increases in growth. Control cells do not begin to respond until FGF levels reach 1-5 ng/ml. Heparin, a major constituent of muscle fiber basal lamina, inhibits myogenesis in these mouse muscle cultures. The heightened sensitivity of mdx satellite cells to FGF may permit high rates of new fiber formation in vivo without a parallel hyperplasia in the muscle fibroblast population. This finding may be important in explaining successful regeneration in mdx muscle in vivo and the fact that mdx animals escape the catastrophic symptoms seen in the related human Duchenne muscular dystrophy.     

Systemic administration of IGF-I enhances oxidative status and reduces contraction-induced injury in skeletal muscles of mdx dystrophic mice

The absence of dystrophin and resultant disruption of the dystrophin glycoprotein complex renders skeletal muscles of dystrophic patients and dystrophic mdx mice susceptible to contraction-induced injury. Strategies to reduce contraction-induced injury are of critical importance, because this mode of damage contributes to the etiology of myofiber breakdown in the dystrophic pathology. Transgenic overexpression of insulin-like growth factor-I (IGF-I) causes myofiber hypertrophy, increases force production, and can improve the dystrophic pathology in mdx mice. In contrast, the predominant effect of continuous exogenous administration of IGF-I to mdx mice at a low dose (1.0-1.5 mg.kg(-1).day(-1)) is a shift in muscle phenotype from fast glycolytic toward a more oxidative, fatigue-resistant, slow muscle without alterations in myofiber cross-sectional area, muscle mass, or maximum force-producing capacity. We found that exogenous administration of IGF-I to mdx mice increased myofiber succinate dehydrogenase activity, shifted the overall myosin heavy chain isoform composition toward a slower phenotype, and, most importantly, reduced contraction-induced damage in tibialis anterior muscles. The deficit in force-producing capacity after two damaging lengthening contractions was reduced significantly in tibialis anterior muscles of IGF-I-treated (53 +/- 4%) compared with untreated mdx mice (70 +/- 5%, P < 0.05). The results provide further evidence that IGF-I administration can enhance the functional properties of dystrophic skeletal muscle and, compared with results in transgenic mice or virus-mediated overexpression, highlight the disparities in different models of endocrine factor delivery.     

A significant reduction of the diaphragm in mdx:MyoD-/-(9th) embryos suggests a role for MyoD in the diaphragm development

To further investigate the role of MyoD during skeletal myogenesis, we backcrossed mdx mutant mice (lacking dystrophin) with MyoD knock-out mice to obtain viable mice with MyoD allele on a pure mdx background. However, after nine generations of backcrossing, it was not possible to obtain a viable mdx:MyoD-/- phenotype (designated as: mdx:MyoD-/-(9th)). The compound-mutant embryos were examined just before birth. Essentially normal Myf5-dependent and most of the MyoD-dependent musculature was observed. By contrast, the skeletal muscle compartment of the diaphragm was significantly reduced. The mesenchymal compartment of the diaphragm was intact and no herniations were observed. Other examined organs (e.g., liver, kidney, brain, etc.) showed no histological abnormalities. Pulmonary hypoplasia was determined as the cause of neonatal death. Therefore, using a different approach, our new data supplement our previous findings and suggest an essential role for MyoD in development of skeletal muscle of the diaphragm. The failure of mdx:MyoD-/-(9th) diaphragm to develop normally is not caused by a reduced number of satellite cells, but from the inability of stem cells to progress through the myogenic program. Our data also suggest that functions of MyoD and Myf5 (and the respective muscle precursor cell sub-populations) are not entirely redundant by term, as previously suggested, since Myf5 is not capable of fully substituting for MyoD in the diaphragm development.     

Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles

Mdx mice uniquely recover from degenerative dystrophic lesions by an intense myoproliferative (regenerative) response. To investigate a potential role of endogenous basic fibroblast growth factor (bFGF) in injury-repair processes, we investigated its localization in several striated muscles of mdx and control mice using immunofluorescence labeling with specific antibodies. Basic FGF was localized consistently to the myofiber periphery and nuclei of intact myofibers, as well as in single, dystrophin-positive cells in close association with the myofibers (potential myosatellite cells). In mdx mice, actively degenerating skeletal or cardiac muscle fibers presented intense cytoplasmic anti-bFGF staining prior to mononuclear infiltration. Small regenerating fibers in mdx skeletal muscle exhibited greater bFGF accumulation than adjacent larger myofibers. Strong nuclear anti-bFGF immunolabeling was frequently observed in mdx cardiac myocytes at the borders of necrotic regions. In agreement with differences in intensity of immunolabeling, extracts from slow-twitch muscles contained higher levels of bFGF compared to those from fast-twitch muscles, in both control and mdx mice. In addition, bFGF levels were consistently higher in extracts from all mdx tissues compared to those derived from their control counterparts. Our data suggest that bFGF participates in the degenerative and regenerative responses of striated muscle to dystrophic injury and also indicate a potential involvement of this factor with the physiology of different striated muscles.     

Reversal by relaxin of altered ileal spontaneous contractions in dystrophic (mdx) mice through a nitric oxide-mediated mechanism

Altered nitric oxide (NO) production/release is involved in gastrointestinal motor disorders occurring in dystrophic (mdx) mice. Since the hormone relaxin (RLX) can upregulate NO biosynthesis, its effects on spontaneous motility and NO synthase (NOS) expression in the ileum of dystrophic (mdx) mice were investigated. Mechanical responses of ileal preparations were recorded in vitro via force-displacement transducers. Evaluation of the expression of NOS isoforms was performed by immunohistochemistry and Western blot. Normal and mdx mice were distributed into three groups: untreated, RLX pretreated, and vehicle pretreated. Ileal preparations from the untreated animals showed spontaneous muscular contractions whose amplitude was significantly higher in mdx than in normal mice. Addition of RLX, alone or together with l-arginine, to the bath medium depressed the amplitude of the contractions in the mdx mice, thus reestablishing a motility pattern typical of the normal mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) or the guanylate cyclase inhibitor ODQ reversed the effects of RLX. In RLX-pretreated mdx mice, the amplitude of spontaneous motility was reduced, thus resembling that of the normal mice, and NOS II expression in the muscle coat was increased in respect to the vehicle-pretreated mdx animals. These results indicate that RLX can reverse the altered ileal motility of mdx mice to a normal pattern, likely by upregulating NOS II expression and NO biosynthesis in the ileal smooth muscle.