Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.

M1 macrophages play a major role in worsening muscle injury in the mdx mouse model of Duchenne muscular dystrophy. However, mdx muscle also contains M2c macrophages that can promote tissue repair, indicating that factors regulating the balance between M1 and M2c phenotypes could influence the severity of the disease. Because interleukin-10 (IL-10) modulates macrophage activation in vitro and its expression is elevated in mdx muscles, we tested whether IL-10 influenced the macrophage phenotype in mdx muscle and whether changes in IL-10 expression affected the pathology of muscular dystrophy. Ablation of IL-10 expression in mdx mice increased muscle damage in vivo and reduced mouse strength. Treating mdx muscle macrophages with IL-10 reduced activation of the M1 phenotype, assessed by iNOS expression, and macrophages from IL-10 null mutant mice were more cytolytic than macrophages isolated from wild-type mice. Our data also showed that muscle cells in mdx muscle expressed the IL-10 receptor, suggesting that IL-10 could have direct effects on muscle cells. We assayed whether ablation of IL-10 in mdx mice affected satellite cell numbers, using Pax7 expression as an index, but found no effect. However, IL-10 mutation significantly increased myogenin expression in vivo during the acute and the regenerative phase of mdx pathology. Together, the results show that IL-10 plays a significant regulatory role in muscular dystrophy that may be caused by reducing M1 macrophage activation and cytotoxicity, increasing M2c macrophage activation and modulating muscle differentiation.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

Duchenne muscular dystrophy is a degenerative disorder that leads to death by the third decade of life. Previous investigations have shown that macrophages that invade dystrophic muscle are a heterogeneous population consisting of M1 and M2 macrophages that promote injury and repair, respectively. In the present investigation, we tested whether interferon-γ (IFNγ) worsens the severity of mdx dystrophy by activating macrophages to a cytolytic, M1 phenotype and by suppressing the activation of pro-regenerative macrophages to a M2 phenotype. IFNγ is a strong inducer of the M1 phenotype and is elevated in mdx dystrophy. Contrary to our expectations, null mutation of IFNγ caused no reduction of cytotoxicity of macrophages isolated from mdx muscle and did not reduce muscle fiber damage in vivo or improve gross motor function of mdx mice at the early, acute peak of pathology. In contrast, ablation of IFNγ reduced muscle damage in vivo during the regenerative stage of the disease and increased activation of the M2 phenotype and improved motor function of mdx mice at that later stage of the disease. IFNγ also inhibited muscle cell proliferation and differentiation in vitro and IFNγ mutation increased MyoD expression in mdx muscle in vivo, showing that IFNγ can have direct effects on muscle cells that could impair repair. Together, the findings show that suppression of IFNγ signaling in muscular dystrophy reduces muscle damage and improves motor performance by promoting the M2 macrophage phenotype and by direct actions on muscle cells.

This study was aimed to achieve a definitive and unambiguous identification of fiber types in canine skeletal muscles and of myosin isoforms that are expressed therein. Correspondence of canine myosin isoforms with orthologs in other species as assessed by base sequence comparison was the basis for primer preparation and for expression analysis with RT-PCR. Expression was confirmed at protein level with histochemistry, immunohistochemistry, and SDS-PAGE combined together and showed that limb and trunk muscles of the dog express myosin heavy chain (MHC) type 1, 2A, and 2X isoforms and the so-called "type 2dog" fibers express the MHC-2X isoform. MHC-2A was found to be the most abundant isoform in the trunk and limb muscle. MHC-2X was expressed in most but not all muscles and more frequently in hybrid 2A-2X fibers than in pure 2X fibers. MHC-2B was restricted to specialized extraocular and laryngeal muscles, although 2B mRNA, but not 2B protein, was occasionally detected in the semimembranosus muscle. Isometric tension (P(o)) and maximum shortening velocity (V(o)) were measured in single fibers classified on the basis of their MHC isoform composition. Purified myosin isoforms were extracted from single muscle fibers and characterized by the speed (V(f)) of actin filament sliding on myosin in an in vitro motility assay. A close proportionality between V(o) and V(f) indicated that the diversity in V(o) was due to the different myosin isoform composition. V(o) increased progressively in the order 1/slow < 2A < 2X < 2B, thus confirming the identification of the myosin isoforms and providing their first functional characterization of canine muscle fibers.

Duchenne muscular dystrophy (DMD) involves a complex pathophysiology that is not easily explained by the loss of the protein dystrophin, the primary defect in DMD. Instead, many features of the pathology are attributable to the secondary loss of neuronal nitric oxide synthase (nNOS) from dystrophin-deficient muscle. In this investigation, we tested whether the loss of nNOS contributes to the increased fatigability of mdx mice, a model of DMD. Our findings show that the expression of a muscle-specific, nNOS transgene increases the endurance of mdx mice and enhances glycogen metabolism during treadmill-running, but did not affect vascular perfusion of muscles. We also find that the specific activity of phosphofructokinase (PFK; the rate limiting enzyme in glycolysis) is positively affected by nNOS in muscle; PFK-specific activity is significantly reduced in mdx muscles and the muscles of nNOS null mutants, but significantly increased in nNOS transgenic muscles and muscles from mdx mice that express the nNOS transgene. PFK activity measured under allosteric conditions was significantly increased by nNOS, but unaffected by endothelial NOS or inducible NOS. The specific domain of nNOS that positively regulates PFK activity was assayed by cloning and expressing different domains of nNOS and assaying their effects on PFK activity. This approach yielded a polypeptide that included the flavin adenine dinucleotide (FAD)-binding domain of nNOS as the region of the molecule that promotes PFK activity. Smaller peptides in this domain were then synthesized and used in activity assays that showed a 36-amino acid peptide in the FAD-binding domain in which most of the positive allosteric activity of nNOS for PFK resides. Mapping this peptide onto the structure of nNOS shows that the peptide is exposed on the surface, readily available for binding. Collectively, these findings indicate that defects in glycolytic metabolism and increased fatigability in dystrophic muscle may be caused in part by the loss of positive allosteric interactions between nNOS and PFK.

Duchenne muscular dystrophy (DMD) is a muscle wasting disease in which inflammation influences the severity of pathology. We found that the onset of muscle inflammation in the mdx mouse model of DMD coincides with large increases in expression of pro-inflammatory cytokines [tumor necrosis factor-α (TNFα); interferon gamma (IFNγ)] and dramatic reductions of the pro-myogenic protein Klotho in muscle cells and large increases of Klotho in pro-regenerative, CD206+ macrophages. Furthermore, TNFα and IFNγ treatments reduced Klotho in muscle cells and increased Klotho in macrophages. Because CD206+/Klotho+ macrophages were concentrated at sites of muscle regeneration, we tested whether macrophage-derived Klotho promotes myogenesis. Klotho transgenic macrophages had a pro-proliferative influence on muscle cells that was ablated by neutralizing antibodies to Klotho and conditioned media from Klotho mutant macrophages did not increase muscle cell proliferation in vitro. In addition, transplantation of bone marrow cells from Klotho transgenic mice into mdx recipients increased numbers of myogenic cells and increased the size of muscle fibers. Klotho also acted directly on macrophages, stimulating their secretion of TNFα. Because TNFα is a muscle mitogen, we tested whether the pro-proliferative effects of Klotho on muscle cells were mediated by TNFα and found that increased proliferation caused by Klotho was reduced by anti-TNFα. Collectively, these data show that pro-inflammatory cytokines contribute to silencing of Klotho in dystrophic muscle, but increase Klotho expression by macrophages. Our findings also show that macrophage-derived Klotho can promote muscle regeneration by expanding populations of muscle stem cells and increasing muscle fiber growth in dystrophic muscle.

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