Search

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.

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.

The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.