Progressive fibrosis of muscles in Duchenne muscular dystrophy impairs muscle function and contributes to premature death of patients. The use of many potentially-therapeutic molecules is limited by an inability to target their delivery specifically to sites of active muscle pathology. In this dissertation research, we tested the hypothesis that inflammatory cells can function as vectors to deliver therapeutic molecules to sites of active pathology in dystrophic muscle. We designed a transgene in which expression of leukemia inhibitory factor (LIF) is driven by the promoter for CD11b, a surface marker highly expressed by mature macrophages. Expression of the transgene (CD11b/LIF) in the mdx mouse model of Duchenne muscular dystrophy increased LIF protein content at inflammatory muscle lesions without off-target expression. Transplantation of transgenic bone marrow cells to non-transgenic, mdx mice showed that a single intervention can provide long-term benefits to dystrophic muscles. The primary benefit of the transgene was reduced fibrosis. Transgenic LIF inhibited fibrogenesis by reducing transforming growth factor-β expression and reducing the numbers of fibro/adipogenic progenitor cells in mdx muscles. CD11b/LIF expression also biased macrophages away from a CD163+/CD206+, pro-fibrotic phenotype and reduced their intramuscular numbers. Reduced chemotactic response of CD11b/LIF+ macrophages to C-C motif chemokine ligand-2 contributed to the reduced macrophage numbers. The dispersion of cytolytic, CD68+ macrophages was impaired in transgenic muscles early in the pathology. Localized accumulation of CD68+ macrophages increased the numbers of injured fibers. At later stages of the pathology, the transgene did not affect macrophage distribution but did reduce muscle damage. Collectively, our observations show that targeted expression of the CD11b/LIF transgene improves dystrophic muscle health. More importantly, we have shown that genetically-modified macrophages can be used as vectors for the delivery of therapeutic molecules to diseased tissues with a significant inflammatory component.

The transition of muscle stem cells, known as satellite cells (SCs), from a quiescent state to an active, proliferative, and myogenic state facilitates muscle repair following injury. We tested the hypothesis that hemizygous expression of Jmjd3, a H3K27me3 histone demethylase, in SCs following acute injury would affect various stages of muscle regeneration. Our findings show there is an increased proportion of H3K27me2/3+/Pax7+ cells out of total Pax7+ cells in acutely injured Jmjd3 hemizygous mice, suggesting the mutation reduced H3K27me2/3 demethylation. We next investigated muscle fiber growth and myonuclei per muscle fiber to determine myocyte fusion. Our data indicate the Jmjd3 mutation diminished the average cross-sectional area (CSA) of muscle fibers after injury, while having no effect on myonuclei per muscle fiber suggesting myocyte fusion was not affected. We then investigated myocyte proliferation and found the Jmjd3 hemizygous mutation reduced the proportion of Ki67+/Pax7+ cells out of total Pax7+ cells, suggesting a reduction in injured muscle compared to Jmjd3 control mice. Our results show attenuation of H3K27me3 histone demethylation, muscle fiber growth, and SC proliferation when SC Jmjd3 expression is reduced, thus affecting muscle regeneration.

Skeletal muscle undergoes progressive mass loss, termed sarcopenia, and increased accumulation of fibrotic tissue during aging, which reduces the life quality of the elderly and causes significant economic burden on healthcare services of society. The importance of the immune system, especially myeloid cells, in modulating muscle growth and regeneration following injury suggests that myeloid cells may also have significant influences on sarcopenia and muscle fibrosis during aging. In this investigation, we studied the regulatory interactions between myeloid cells and skeletal muscle during aging. We found that muscle aging is associated with increased number of anti-inflammatory M2a macrophages that can increase muscle fibrosis. Expression of a muscle-specific transgene of nNOS prevented the age-related increases in M2a macrophage and reduced fibrosis in aging muscle. We then tested whether aging of myeloid cells contributes to the age-related increases in M2a macrophages and the associated increases in fibrosis. Transplantation of young bone marrow cells into old mice resulted in fewer M2a macrophages and less accumulation of collagen compared to age-matched, non-transplant mice. We also found that transplantation of young bone marrow cells into old mice prevented sarcopenia. On the other hand, muscles of young mice receiving old bone marrow cells showed decreased numbers of muscle stem cells, called satellite cells, and increased numbers of fibrogenic-converted satellite cells. In vitro, media conditioned by young, but not old, bone marrow-derived macrophages increased muscle cell proliferation. These data suggest that aging of myeloid cells promotes the shift of satellites cell from a myogenic lineage to a more fibrogenic lineage during aging, and contributes to sarcopenia and muscle fibrosis. However, both the nNOS transgene and the heterochronic bone marrow transplantation can also affect muscle aging through unknown mechanisms other than changes in myeloid cells. To specifically manipulate the myeloid cell population, we designed a mouse line with a myeloid-cell-specific mutation of transcription factor Sfpi1 which specifically reduced the number of M2 macrophages in muscle. Myeloid-cell-specific mutation of Sfpi1 prevented sarcopenia and age-related muscle fibrosis, strongly suggesting that muscle aging is at least partly attributable to myeloid cells. Previous studies in our lab and by other groups suggested that tumor necrotic factor-alpha (TNFα) is a macrophage-derived factor that may contribute significantly to muscle aging. We found that systemic TNFα knockout increased satellite cell fusion into muscle fibers in old muscle and prevented sarcopenia. Furthermore, we observed that transplantation of wild-type bone marrow cells into TNFα knockout mice induced sarcopenia and reduced muscle cell fusion, indicating that TNFα secreted by myeloid cells contributes significantly to the reduction of satellite cell myogenic capacity during aging and causes sarcopenia. Overall, our findings provide insight into mechanisms of muscle aging and the regulatory interactions between myeloid cells and skeletal muscle during aging.

Despite advancements in our understanding of the immunobiology of Duchenne muscular dystrophy (DMD), previous findings were not translated into improvements in clinical practice. The standard clinical treatment for DMD includes administration of non-specific anti-inflammatory corticosteroids that provide limited benefits for patients and produce non-target effects that impair muscle regeneration. Thus, the search for an efficacious immunotherapy without adverse effects remains imperative. Because DMD pathology is exacerbated by fibrotic mechanisms mediated by myeloid and fibrogenic cells, we examined the effect of an immune suppressing fusion protein consisting of cytotoxic T-lymphocyte-associated protein4 and immunoglobulin G (CTLA4-Ig) in the mdx murine model of DMD. We found that CTLA4-Ig ameliorates pathology, via the disruption of fibrotic mechanisms, as treatment reduced collagen accumulation and expression in dystrophic muscle. These findings indicate that CTLA4-Ig treatment has inhibitory effects on profibrotic cells that exacerbate DMD pathology and could potentially serve as a promising treatment option for patients with DMD.

Duchenne muscular dystrophy (DMD) is an X-linked disease characterized by muscle membrane damage and muscle wasting that eventually causes death of the afflicted. Klotho is a powerful longevity protein that has been linked to lifespan and kidney function; in recent years, Klotho's role in muscle growth has been reported. Previous findings in our lab showed that Klotho expression is suppressed in DMD patients and mdx mice, an animal model of muscular dystrophy. In this study, we investigate the epigenetic mechanisms that regulate Klotho expression in dystrophic muscle. Our data suggest DNA hypermethylation and histone 3 lysine 9 dimethylation at the Klotho transcriptional start site are involved in the transcriptional suppression under oxidative stress. Furthermore, our data also show disrupted demethylation in oxidatively stressed muscle cells during differentiation that may also contribute to the hypermethylation seen in muscle.

Skeletal muscle function can be compromised by injury or trauma. Muscle regeneration requires the activation and proliferation of satellite cells. The anti-aging protein Klotho (KL) has been shown to enhance regeneration of dystrophic muscle by stimulating satellite cell production. However, it is not known whether exogenous KL treatment can be used to improve muscle regeneration after acute injury. Using C57BL/6 mice that received intraperitoneal injections of recombinant KL prior to muscle injury and throughout recovery, we found that KL did not affect muscle regeneration or expression of inflammatory and fibrogenic transcripts. We also tested whether KL affected Wnt signaling in myogenic cells. Our investigation confirms that KL is an antagonist of Wnt signaling. Although our findings do not support the therapeutic use of KL in young muscle after acute injury, our results suggest that KL modulates Wnt signaling and therefore could be therapeutic in conditions of aberrant Wnt-signaling in muscle.

Although there is no cure for Duchenne muscular dystrophy (DMD), patients are commonly prescribed corticosteroids, typically prednisone, to slow disease progression and dampen the immune response. Prednisone promotes transcription of anti-inflammatory genes and reduces transcription of pro-inflammatory genes, leading to downregulation of prostaglandins that recruit immune cells. Because the immune system modulates the severity of pathology in DMD and in mdx mice (a genetic model for DMD), immunotherapy may also be beneficial. Cytotoxic T- lymphocyte-associated protein-4 (CTLA-4) is a negative regulator of the immune system that inhibits activation of cytotoxic T-cells. CTLA4-Ig is a fusion protein that blocks the activation and invasion of macrophages and cytotoxic T-cells in other inflammatory and autoimmune diseases, which suggests that it may also positively affect the pathology of muscular dystrophy. Our lab previously demonstrated that CTLA4-Ig treatment alone is effective in reducing many aspects of mdx pathology. The objective of this study is to determine if the beneficial effects of utilizing CTLA4-Ig are affected by co-administration with prednisone in an mdx mouse model. At the peak of muscular pathology, we found that the combined treatment of prednisone and CTLA4-Ig reduced inflammation, necrosis, and fibrosis in 4-week mdx quadriceps. Because of these promising outcomes, the combination of immunotherapy and corticosteroids may serve as a potential treatment for patients with DMD.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease characterized by chronic muscle degeneration. Because the immune system modulates the severity of DMD, it provides a potential therapeutic target for DMD. Previous investigations have shown that cytotoxic T-lymphocyte-associated protein 4 immunoglobulin (CTLA-4Ig) fusion protein inhibits T cell activation and reduces migration of macrophages. The objective of this study was to assess CTLA-4Ig as a potential DMD immunotherapy. Mdx mice, a mouse model of DMD, were administered CTLA-4Ig and euthanized at 4-week or 3-months old. At the peak of muscle inflammation, CTLA-4Ig-treated 4-week mice exhibited significantly reduced necrosis and reduced numbers of CD68+ M1 macrophages and CD163+ M2 macrophages in muscle and in individual injured myofibers. At 3-months, we saw little treatment effects indicating that the influence of CTLA-4Ig is transient. Due to the reduction in necrosis and inflammatory cells in the dystrophic muscles, CTLA-4Ig is a promising immunotherapy for DMD.

Skeletal muscle is one of the most abundant tissues in the body because it supports fundamental processes of lifelike movement and breathing. Muscle stem cells, called satellite cells, support muscle development and life-long muscle repair through the activation of the b-catenin-mediated canonical Wnt pathway. Therefore, any changes in the number of satellite cells or in the activation of canonical Wnt signaling can impair myogenesis throughout life. In this dissertation research, we examined the role of the age-related molecule Klotho during early postnatal muscle development by testing the hypothesis that Klotho influences myogenesis through the epigenetic regulation of key genes required for satellite cell-mediated myogenesis. Regulatory pathways that influence muscle growth and repair, like Wnt/b-catenin signaling, are particularly interesting in the context of this work because activation of the Wnt/b-catenin pathway is the limiting step in myogenic differentiation. Through this work we identified a novel pathway that showed Klotho influenced myogenesis through the epigenetic regulation of canonical Wnt genes mediated by the histone 3 lysine 27 (H3K27) demethylase Jmjd3. We showed muscle cells treated with Klotho had reduced Jmjd3 expression and increased gene repressive H3K27 methylation (H3K27m2/3). Subsequent experiments revealed a reduction of canonical Wnt gene expression in muscle cells treated with Klotho and in whole muscles of mice that continuously express elevated klotho. We showed satellite cells are particularly responsive to Klotho during early postnatal development, a characteristic that did not occur in adult muscles. Our data revealed muscle cells treated with Klotho and siRNA targeting Jmjd3 did not have additive effects on Wnt gene expression indicating that Klotho and Jmjd3 operate in a similar pathway. Immunohistological analysis showed Klotho reduced the proportion of satellite cells with active b-catenin, suggesting Klotho’s effects on myogenesis may be caused by reduced Wnt/b-catenin signaling. Next, we developed a mouse model with a satellite cell-specific mutation in the Jmjd3 gene to test whether knocking down Jmjd3 in satellite cells of developing muscle would mimic the effects of Klotho on myogenesis and the canonical Wnt pathway. We were surprised to find that muscle-specific Jmjd3 is essential for neonatal survival. Although Jmjd3 mutant mice died within hours after birth, we confirmed Jmjd3 mutant mice muscles had reduced expression of the canonical Wnt genes identified in prior experiments and immunohistological work showed the proportion of satellite cells with active b-catenin was reduced in the absence of Jmjd3. Collectively, this work showed elevated Klotho and reduced Jmjd3 modulated myogenesis through a similar epigenetic regulatory pathway that influenced b-catenin-mediated canonical Wnt signaling. Of note, our work revealed expression of muscle-specific Jmjd3 is essential for survival and therefore should continue to be investigated in processes affecting myogenesis.