Restoration of dystrophin-associated proteins in skeletal muscle of mdx mice transgenic for dystrophin gene

Duchenne muscular dystrophy (DMD) patients and mdx mice are characterized by the absence of dystrophin, a membrane cytoskeletal protein. Dystrophin is associated with a large oligomeric complex of sarcolemmal glycoproteins, including dystroglycan which provides a linkage to the extarcellular matrix component, laminin. The finding that all of the dystrophin-associated proteins (DAPs) are drastically reduced in DMD and mdx skeletal muscle supports the primary function of dystrophin as an anchor of the sarcolemmal glycoprotein complex to the subsarcolemmal cytoskeleton. These findings indicate that the efficacy of dystrophin gene therapy will depend not only on replacing dystrophin but also on restoring all of the DAPs in the sarcolemma. Here we have investigated the status of the DAPs in the skeletal muscle of mdx mice transgenic for the dystrophin gene. Our results demonstrate that transfer of dystrophin gene restores all of the DAPs together with dystrophin, suggesting that dystrophin gene therapy should be effective in restoring the entire dystrophin-glycoprotein complex.     

Developmental regulation of mechanosensitive calcium channels in skeletal muscle from normal and mdx mice.

Single-channel activity was recorded from cell-attached membrane patches on flexor digitorum brevis fibres acutely isolated from normal and mdx mice at different stages of postnatal development. Recordings from cell-attached patches on both normal and mdx fibres were dominated by the activity of mechanosensitive ion channels with a conductance of approximately 17 pS with 110 mM Ba2+ in the patch electrode. In a small fraction of the patches on mdx fibres from young mice, channels showed very high levels of activity. Channel activity recorded from mdx fibres from older mice was higher than in age-matched normal fibres and the level of activity decreased during development. Channel density decreased in normal fibres, whereas it remained relatively constant in mdx fibres, as if channels are down-regulated in normal, but not mdx, fibres during postnatal development. An early step in the dystrophic process may be an alteration of the mechanisms that regulate the expression of functional channels.     

Satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice

To determine whether muscle satellite cells and utrophin are correlated with the degree of damage in mdx skeletal muscles, we measured the area of the degenerative region as an indicator of myofiber degeneration in the masseter, gastrocnemius, soleus, and diaphragm muscles of mdx mice. Furthermore, we analyzed the expression levels of the paired box homeotic gene 7 (pax7), m-cadherin (the makers of muscle satellite cells), and utrophin mRNA. We also investigated the immunolocalization of m-cadherin and utrophin proteins in the muscles of normal C57BL/10J (B10) and mdx mice. The expression level of pax7 mRNA and the percentage of m-cadherin-positive cells among the total number of cell nuclei in the muscle tissues in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there was no significant correlation between muscle damage and expression level for pax7 mRNA (R = –0.140), nor was there a correlation between muscle damage and the percentage of satellite cells among the total number of cell nuclei (R = –0.411) in the mdx mice. The expression level of utrophin mRNA and the intensity of immunostaining for utrophin in all four muscles studied were greater in the mdx mice than in the B10 mice. However, there also was not a significant correlation between muscle damage and expression level of utrophin mRNA (R = 0.231) in the mdx mice, although upregulated utrophin was incorporated into the sarcolemma. These results suggest that satellite cells and utrophin are not directly correlated with the degree of skeletal muscle damage in mdx mice. dystrophy; pax7; m-cadherin; dystrophin-related proteins     

Intramembrane charge movement and L-type calcium current in skeletal muscle fibers isolated from control and mdx mice

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 +/- 2 nC/ micro F, -37 +/- 3 mV, and 13 +/- 1 mV (n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (tau approximately 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 +/- 15 S/F, 65.5 +/- 2.9 mV, -0.76 +/- 1.2 mV, and 6.2 +/- 0.5 mV (n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (-6.2 +/- 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.     

Abnormalities in structure and function of limb skeletal muscle fibres of dystrophic mdx mice.

In this study we have shown that the skeletal muscle fibres from adult (older than 26 weeks) mdx mice have gross structural deformities. We have characterized the onset and age dependence of this feature in mdx mice. The three dimensional structure of these deformities has been visualized in isolated fibres and the orientation of these deformities was determined within the muscle by confocal laser scanning microscopy. We have also shown that the occurrence of morphologically abnormal fibres is greater in muscles with longer fibres (extensor digitorum longus (EDL) and soleus, 6-7.3 mm long), than in muscles with shorter fibres (flexor digitorum brevis (FDB), 0.3-0.4 mm long). A population of post-degenerative fibres, with both central and peripheral nuclei coexistent along the length of the fibre, has also been identified in the muscles studied. We showed that a mild protocol of lengthening (eccentric) contractions (the muscle was stretched by 12% during a tetanic contraction) caused a major reduction in the maximal tetanic force subsequently produced by mdx EDL muscle. In contrast, maximal tetanic force production in normal soleus, normal EDL and mdx soleus muscles was not altered by this protocol. We suggest that the deformed fast glycolytic fibres which are found in adult mdx EDL but not in adult mdx soleus muscles are the population of fibres damaged by the lengthening protocol.     

Rapamycin ameliorates dystrophic phenotype in mdx mouse skeletal muscle

Duchenne muscular dystrophy (DMD) is an X-linked, lethal, degenerative disease that results from mutations in the dystrophin gene, causing necrosis and inflammation in skeletal muscle tissue. Treatments that reduce muscle fiber destruction and immune cell infiltration can ameliorate DMD pathology. We treated the mdx mouse, a model for DMD, with the immunosuppressant drug rapamycin (RAPA) both locally and systemically to examine its effects on dystrophic mdx muscles. We observed a significant reduction of muscle fiber necrosis in treated mdx mouse tibialis anterior (TA) and diaphragm (Dia) muscles 6 wks post-treatment. This effect was associated with a significant reduction in infiltration of effector CD4(+) and CD8(+) T cells in skeletal muscle tissue, while Foxp3(+) regulatory T cells were preserved. Because RAPA exerts its effects through the mammalian target of RAPA (mTOR), we studied the activation of mTOR in mdx TA and Dia with and without RAPA treatment. Surprisingly, mTOR activation levels in mdx TA were not different from control C57BL/10 (B10). However, mTOR activation was different in Dia between mdx and B10; mTOR activation levels did not rise between 6 and 12 wks of age in mdx Dia muscle, whereas a rise in mTOR activation level was observed in B10 Dia muscle. Furthermore, mdx Dia, but not TA, muscle mTOR activation was responsive to RAPA treatment.     

Myogenin regulates exercise capacity but is dispensable for skeletal muscle regeneration in adult mdx mice

Duchenne muscular dystrophy (DMD) is the most prevalent inherited childhood muscle disorder in humans. mdx mice exhibit a similar pathophysiology to the human disorder allowing for an in-depth investigation of DMD. Myogenin, a myogenic regulatory factor, is best known for its role in embryonic myogenesis, but its role in adult muscle maintenance and regeneration is still poorly understood. Here, we generated an mdx:Myog(flox/flox) mouse harboring a tamoxifen-inducible Cre recombinase transgene, which was used to conditionally delete Myog during adult life. After tamoxifen treatment, three groups of mice were created to study the effects of Myog deletion: mdx:Myog(flox/flox) mice (mdx), Myog(flox/flox) mice (wild-type), and mdx:Myog(floxΔ/floxΔ):Cre-ER mice (mdx:Myog-deleted). mdx:Myog-deleted mice exhibited no adverse phenotype and behaved normally. When run to exhaustion, mdx:Myog-deleted mice demonstrated an enhanced capacity for exercise compared to mdx mice, running nearly as far as wild-type mice. Moreover, these mice showed the same signature characteristics of muscle regeneration as mdx mice. Unexpectedly, we found that myogenin was dispensable for muscle regeneration. Factors associated with muscle fatigue, metabolism, and proteolysis were significantly altered in mdx:Myog-deleted mice, and this might contribute to their increased exercise capacity. Our results reveal novel functions for myogenin in adult muscle and suggest that reducing Myog expression in other muscle disease models may partially restore muscle function.     

Curcumin alleviates dystrophic muscle pathology in mdx mice

Abnormal activation of nuclear factor kappa B (NF-kappaB) probably plays an important role in the pathogenesis of Duchenne's muscular dystrophy (DMD). In this report, we evaluated the efficacy of curcumin, a potent NF-kappaB inhibitor, in mdx mice, a mouse model of DMD. We found that it improved sarcolemmic integrity and enhanced muscle strength after intraperitoneal (i.p.) injection. Histological analysis revealed that the structural defects of myofibrils were reduced, and biochemical analysis showed that creatine kinase (CK) activity was decreased. We also found that levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and inducible nitric oxide synthase (iNOS) in the mdx mice were decreased by curcumin administration. EMSA analysis showed that NF-kappaB activity was also inhibited. We thus conclude that curcumin is effective in the therapy of muscular dystrophy in mdx mice, and that the mechanism may involve inhibition of NF-kappaB activity. Since curcumin is a non-toxic compound derived from plants, we propose that it may be useful for DMD therapy.     

Contraction-induced injury to single permeabilized muscle fibers from mdx, transgenic mdx, and control mice

Muscle fibers of mdx mice that lack dystrophin are moresusceptible to contraction-induced injury, particularly when stretched. In contrast, transgenic mdx (tg-mdx) mice, whichoverexpress dystrophin, show no morphological or functional signs ofdystrophy. Permeabilization disrupts the sarcolemma of fibers frommuscles of mdx, tg-mdx, and control mice. Wetested the null hypothesis stating that, after single stretches ofmaximally activated single permeabilized fibers, force deficits do notdiffer among fibers from extensor digitorum longus muscles ofmdx, tg-mdx, or control mice. Fibers weremaximally activated by Ca²⁺ (pCa 4.5) and then stretchedthrough strains of 10%, 20%, or 30% of fiber length(L_f) at a velocity of 0.5 L_f/s. Immediately after each strain, theforce deficits were not different for fibers from each of the threegroups of mice. When collated with studies of membrane-intact fibers inwhole muscles of mdx, tg-mdx, and control mice,these results indicate that dystrophic symptoms do not arise fromfactors within myofibrils but, rather, from disruption of thesarcolemmal integrity that normally provides protection fromcontraction-induced injury.

     

Synthesis of proteoglycans is augmented in dystrophic mdx mouse skeletal muscle

Mdx mice uniquely recover from degenerative dystrophic lesions through an intense myoproliferative response. The onset and progression of this process are controlled by a complex set of interactions between myoblasts and their environment. The presence of the extracellular matrix is essential for normal myogenesis. Proteoglycans are abundant components of the extracellular matrix. The synthesis of proteoglycans in mdx mice during skeletal muscle regeneration was evaluated. Incorporation of radioactive sulfate demonstrated a significant increase in the synthesis of several types of proteoglycans in mdx animals compared to age-matched controls. The size and charge of proteoglycans synthesized by the mdx mice remained unchanged. In particular, one of the up-regulated proteoglycans, the small chondroitin/dermatan sulfate proteoglycan decorin which is known to bind and to sequester transforming growth factor-beta, was investigated. Immunocytolocalization and in situ hybridization studies showed that decorin mainly accumulated in the endomysium, i.e. around individual skeletal muscle fibers from M. tibialis anterior and diaphragm.     

Human skeletal muscle proteins

Summary The primary structure of the major component of human skeletal muscle troponin C has been established. The troponin C was purified by ammonium sulphate and isoelectric fractionation, followed by two chromatographic steps on DEAE Sephadex. The sequence was determined from the different overlapping enzymic peptides and by dansyl-Edman degradation. The only difference between rabbit skeletal muscle troponin C and the major component of human skeletal troponin C was found at position 112: Ala (rabbit), Pro (human). The partial amino acid sequence of the first 86 residues of the minor component of human skeletal troponin C was found to resemble the troponin C from bovine cardiac muscle. The only difference between them, has tentatively been located at position 62: Glu (human), Asp (bovine). These similarities suggest that troponin C is, from the point of view of molecular evolution, one of the most conservative proteins so far studied.     

Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle

Nebulin is a giant modular sarcomeric protein that has been proposed to play critical roles in myofibrillogenesis, thin filament length regulation, and muscle contraction. To investigate the functional role of nebulin in vivo, we generated nebulin-deficient mice by using a Cre knock-in strategy. Lineage studies utilizing this mouse model demonstrated that nebulin is expressed uniformly in all skeletal muscles. Nebulin-deficient mice die within 8-11 d after birth, with symptoms including decreased milk intake and muscle weakness. Although myofibrillogenesis had occurred, skeletal muscle thin filament lengths were up to 25% shorter compared with wild type, and thin filaments were uniform in length both within and between muscle types. Ultrastructural studies also demonstrated a critical role for nebulin in the maintenance of sarcomeric structure in skeletal muscle. The functional importance of nebulin in skeletal muscle function was revealed by isometric contractility assays, which demonstrated a dramatic reduction in force production in nebulin-deficient skeletal muscle.     

Differentiation of original and regenerated skeletal muscle fibres in mdx dystrophic muscles

The differentiation of both original muscle fibres and the regenerated muscle fibres following necrosis in mdx muscles was investigated using immunoblotting and immunocytochemical procedures. Before the onset of necrosis, postnatal skeletal muscles in mdx mouse differentiated well with only a slight delay in differentiation indicated by the level of developmental isoforms of troponin T. Prior to the onset of apparent myopathic change, both fast and slow skeletal muscle fibre types in mdx leg muscles also differentiated well when investigated by analysis of specific myosin heavy chain expression pattern. While the original muscle fibres in mdx leg muscles developed well, the differentiation of regenerated myotubes into both slow and distinct fast muscle fibre types, however, was markedly delayed or inhibited as indicated by several clusters of homogeneously staining fibres even at 14 weeks of age. The number of slow myosin heavy chain-positive myotubes amongst the regenerated muscle clusters was quite small even in soleus. This study thus established that while muscle fibres initially develop normally with only a slight delay in the differentiation process, the differentiation of regenerated myotubes in mdx muscles is markedly compromised and consequently delayed.