Immune pathways and TP53 missense mutations are associated with longer survival in canine osteosarcoma.

Osteosarcoma affects about 2.8% of dogs with cancer, with a one-year survival rate of approximately 45%. The purpose of this study was to characterize mutation and expression profiles of osteosarcoma and its association with outcome in dogs. The number of somatic variants identified across 26 samples ranged from 145 to 2,697 with top recurrent mutations observed in TP53 and SETD2. Additionally, 47 cancer genes were identified with copy number variations. Missense TP53 mutation status and low pre-treatment blood monocyte counts were associated with a longer disease-free interval (DFI). Patients with longer DFI also showed increased transcript levels of anti-tumor immune response genes. Although, T-cell and myeloid cell quantifications were not significantly associated with outcome; immune related genes, PDL-1 and CD160, were correlated with T-cell abundance. Overall, the association of gene expression and mutation profiles to outcome provides insights into pathogenesis and therapeutic interventions in osteosarcoma patients.

Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor.

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.

The Role of Innate and Adaptive Immune Cells in Skeletal Muscle Regeneration.

Skeletal muscle regeneration is highly dependent on the inflammatory response. A wide variety of innate and adaptive immune cells orchestrate the complex process of muscle repair. This review provides information about the various types of immune cells and biomolecules that have been shown to mediate muscle regeneration following injury and degenerative diseases. Recently developed cell and drug-based immunomodulatory strategies are highlighted. An improved understanding of the immune response to injured and diseased skeletal muscle will be essential for the development of therapeutic strategies.

A simple model of immune and muscle cell crosstalk during muscle regeneration.

Muscle injury during aging predisposes skeletal muscles to increased damage due to reduced regenerative capacity. Some of the common causes of muscle injury are strains, while other causes are more complex muscle myopathies and other illnesses, and even excessive exercise can lead to muscle damage. We develop a new mathematical model based on ordinary differential equations of muscle regeneration. It includes the interactions between the immune system, healthy and damaged myonuclei as well as satellite cells. Our new mathematical model expands beyond previous ones by accounting for 21 specific parameters, including those parameters that deal with the interactions between the damaged and dead myonuclei, the immune system, and the satellite cells. An important assumption of our model is the replacement of only damaged parts of the muscle fibers and the dead myonuclei. We conduce systematic sensitivity analysis to determine which parameters have larger effects on the model and therefore are more influential for the muscle regeneration process. We propose additional validation for these parameters. We further demonstrate that these simulations are species-, muscle-, and age-dependent. In addition, the knowledge of these parameters and their interactions, may suggest targeting or selecting these interactions for treatments that accelerate the muscle regeneration process.

Delineating the relationship between immune system aging and myogenesis in muscle repair.

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y-O) or old to old (O-O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL-10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y-O chimeras). Improved immune responses in Y-O chimeras were associated with greater satellite cell proliferation compared with O-O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age-related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.

miR-18a-3p and Its Target Protein HuR May Regulate Myogenic Differentiation in Immune-Mediated Necrotizing Myopathy.

Immune-mediated necrotizing myopathy (IMNM) is characterized by manifestation of myonecrosis and regeneration of muscle fibers; however, the underlying pathogenesis remains unclear. This study aimed to investigate the role and mechanism of miR-18a-3p and its target RNA-binding protein HuR in IMNM. HuR and miR-18a-3p levels were detected in the skeletal muscles of 18 patients with IMNM using quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) and western blotting analysis. Human myoblasts were transfected with small interfering RNA targeting HuR and miR-18a-3p mimic or inhibitor. Myogenic differentiation markers, myogenin and myosin heavy chain, were analyzed by qRT-PCR, western blotting analysis, and immunofluorescence staining. The results showed that miR-18a-3p was upregulated (p=0.0002), whereas HuR was downregulated (p=0.002) in the skeletal muscles of patients with IMNM. The expression of miR-18a-3p in patients with IMNM was negatively correlated with those of HuR (r = -0.512, p = 0.029). We also found that disease activity was positively correlated with HuR expression (r = 0.576, p = 0.012) but muscle activity was negatively correlated with miR-18a-3p expression (r = -0.550, p = 0.017). Besides, bioinformatics analysis and dual-luciferase reporter assays suggested that miR-18a-3p could directly target HuR. Cellular experiments showed that overexpression of miR-18a-3p inhibited myogenic differentiation by targeting HuR, whereas inhibition of miR-18a-3p led to opposite results. Therefore, miR-18a-3p and its target protein HuR may be responsible for modulating the myogenic process in IMNM and can thus be therapeutic targets for the same.

Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein ß antibody in differentiating myoblasts.

OBJECTIVE: CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPß expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPß, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPß, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPß, the band was present when C/EBPß was knocked down, confirming specificity for a protein other than C/EBPß. The objective of this work was to identify the contaminating reactivity. RESULTS: We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPß is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPß isoforms.

Differential Macrophage Subsets in Muscle Damage Induced by a K49-PLA2 from Bothrops jararacussu Venom Modulate the Time Course of the Regeneration Process.

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation's inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-α and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies.

Chronic infection stunts macrophage heterogeneity and disrupts immune-mediated myogenesis.

The robust regenerative potential of skeletal muscle is imperative for the maintenance of tissue function across a host of potential insults including exercise, infection, and trauma. The highly coordinated action of multiple immune populations, especially macrophages, plays an indispensable role in guiding this reparative program. However, it remains unclear how skeletal muscle repair proceeds in a chronically inflamed setting, such as infection, where an active immune response is already engaged. To address this question, we used a cardiotoxin injury model to challenge the reparative potential of chronically infected muscle. Compared with regenerating naive skeletal muscle, infected skeletal muscle exhibited multiple indicators of delayed muscle repair including a divergent morphologic response to injury and dysregulated expression of myogenic regulatory factors. Further, using both flow cytometric and single-cell RNA sequencing approaches, we show that reduced macrophage heterogeneity due to delayed emergence of restorative subsets underlies dysfunctional tissue repair during chronic infection. Our findings highlight how the preexisting inflammatory environment within tissue alters reparative immunity and ultimately the quality of tissue regeneration.

Biological scaffold-mediated delivery of myostatin inhibitor promotes a regenerative immune response in an animal model of Duchenne muscular dystrophy.

Recent studies have reported that the immune system significantly mediates skeletal muscle repair and regeneration. Additionally, biological scaffolds have been shown to play a role in polarizing the immune microenvironment toward pro-myogenic outcomes. Moreover, myostatin inhibitors are known to promote muscle regeneration and ameliorate fibrosis in animal models of Duchenne muscular dystrophy (DMD), a human disease characterized by chronic muscle degeneration. Biological scaffolds and myostatin inhibition can potentially influence immune-mediated regeneration in the dystrophic environment, but have not been evaluated together. Toward this end, here we created an injectable biological scaffold composed of hyaluronic acid and processed skeletal muscle extracellular matrix. This material formed a cytocompatible hydrogel at physiological temperatures in vitro When injected subfascially above the tibialis anterior muscles of both WT and dystrophic mdx-5Cv mice, a murine model of DMD, the hydrogel spreads across the entire muscle before completely degrading at 3 weeks in vivo We found that the hydrogel is associated with CD206+ pro-regenerative macrophage polarization and elevated anti-inflammatory cytokine expression in both WT and dystrophic mice. Co-injection of both hydrogel and myostatin inhibitor significantly increased FoxP3+ regulatory T cell modulation and Foxp3 gene expression in the scaffold immune microenvironment. Finally, delivery of myostatin inhibitor with the hydrogel increased its bioactivity in vivo, and transplantation of immortalized human myoblasts with the hydrogel promoted their survival in vivo This study identifies a key role for biological scaffolds and myostatin inhibitors in modulating a pro-regenerative immune microenvironment in dystrophic muscle.

CD4 T-cells regulate angiogenesis and myogenesis.

Ischemic diseases, such as peripheral artery disease, affect millions of people worldwide. While CD4+ T-cells regulate angiogenesis and myogenesis, it is not understood how the phenotype of these adaptive immune cells regulate these regenerative processes. The secreted factors from different types of CD4+ T-cells (Th1, Th2, Th17, and Treg) were utilized in a series of in vitro assays and delivered from an injectable alginate biomaterial into a murine model of ischemia to study their effects on vascular and skeletal muscle regeneration. Conditioned medium from Th2 and Th17  T-cells enhanced angiogenesis in vitro and in vivo, in part by directly stimulating endothelial sprouting. Th1 conditioned medium induced vascular regression in vitro and provided no benefit to angiogenesis in vivo. Th1, Th2, and Th17 conditioned medium, to varying extents, enhanced muscle precursor cell proliferation and inhibited their differentiation in vitro, and prolonged early stages of muscle regeneration in vivo. Treg conditioned medium had a moderate or no effect on these processes in vitro and no discernible effect in vivo. These findings suggest that Th2 and Th17 T-cells may enhance angiogenesis and myogenesis in ischemic injuries, which may be useful in the design of immunomodulatory biomaterials to treat these diseases.

Myogenic Differentiation of ASCs Using Biochemical and Biophysical Induction.

Adipose-derived stem/stromal cells (ASCs) constitute a very promising source for cell therapy and tissue engineering approaches as they can be easily obtained in large quantities with comparatively minimal patient discomfort. Moreover, ASCs have multilineage differentiation capacity. Among these, differentiation capacity along the myogenic lineage is of particular interest since myogenic precursors are scarce and obtaining a large number of cells from skeletal muscle biopsies is difficult. Here, we describe a method to effectively induce ASC myogenesis through the combination of biochemical (cocktail including 5-azacytidine and horse serum) and biophysical (dynamic culture via uniaxial cyclic strain) stimulation. This method results in multinucleated cells that are positive in myogenic markers including Pax 3/7, desmin, myoD, and myosin heavy chain.

Inflammation during skeletal muscle regeneration and tissue remodeling: application to exercise-induced muscle damage management.

Increase in the practice of sport by more and more numerous people in the Western countries is associated with an increase in muscle injuries, and in demand for improving muscle function and acceleration of muscle recovery after damage. Most of the treatments used target inflammation. Indeed, several lines of experimental evidence in animal models that are supported by human studies identify inflammatory cells, and particularly macrophages, as essential players in skeletal muscle regeneration. Macrophages act not only through their immune functions, but also control myogenesis and extracellular matrix remodeling by directly acting on myogenic precursors and fibro-adipogenic precursors. In light of these recent biological advances, the question of early treatment aiming at blunting inflammation after exercise-induced muscle injury is discussed.

Evaluation of interferon response induced by anti-myostatin shRNA constructs in goat (Capra hircus) fetal fibroblasts by quantitative real time-polymerase chain reaction.

RNAi is an evolutionary conserved, highly efficient, and cost effective technique of gene silencing. It holds considerable promise and success has been achieved both in vitro and in vivo experiments. However, it is not devoid of undesirable side effects as dsRNA can trigger the immune response and can also cause non-specific off-target gene silencing. In the present study, silencing of myostatin gene, a negative regulator of myogenesis, was evaluated in caprine fetal fibroblasts using three different shRNA constructs. Out of these three constructs, two constructs sh1 and sh2 showed, 72% and 50% reduction (p<0.05) of myostatin mRNA, respectively. Efficient suppression (42-86%) of MSTN gene (p<0.05) was achieved even by reducing the concentration of shRNA constructs. The induction of classical interferon stimulated gene (Oligoandenylate Synthetase-1, OAS-1) was studied to analyze the immune response against shRNAs. Notably, a reduction in the potency of shRNAs to induce interferon response was observed at lower concentration for OAS1 gene. The results obtained in the study would be helpful in the abrogation of the bystander effects of RNAi for long term stable expression of anti-MSTN expression constructs in the muscle.

Macrophage-specific expression of urokinase-type plasminogen activator promotes skeletal muscle regeneration.

Macrophages (Mp) and the plasminogen system play important roles in tissue repair following injury. We hypothesized that Mp-specific expression of urokinase-type plasminogen activator (uPA) is sufficient for Mp to migrate into damaged muscle and for efficient muscle regeneration. We generated transgenic mice expressing uPA only in Mp, and we assessed the ability of these mice to repair muscle injury. Mp-only uPA expression was sufficient to induce wild-type levels of Mp accumulation, angiogenesis, and new muscle fiber formation. In mice with wild-type uPA expression, Mp-specific overexpression further increased Mp accumulation and enhanced muscle fiber regeneration. Furthermore, Mp expression of uPA regulated the level of active hepatocyte growth factor, which is required for muscle fiber regeneration, in damaged muscle. In vitro studies demonstrated that uPA promotes Mp migration through proteolytic and nonproteolytic mechanisms, including proteolytic activation of hepatocyte growth factor. In summary, Mp-derived uPA promotes muscle regeneration by inducing Mp migration, angiogenesis, and myogenesis.

Generation of a monoclonal antibody reactive to prefusion myocytes.

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion-incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell-cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.

Skeletal muscle diseases, inflammation, and NF-kappaB signaling: insights and opportunities for therapeutic intervention.

Signaling through nuclear factor-kappa B (NF-kappaB) is emerging as an important regulator of muscle development, maintenance, and regeneration. Classic signaling modulates early muscle development by enhancing proliferation and inhibiting differentiation, and alternative signaling promotes myofiber maintenance and metabolism. Likewise, NF-kappaB signaling is critical for the development of immunity. Although these processes occur normally, dysregulation of NF-kappaB signaling has prohibitive effects on muscle growth and regeneration and can perpetuate inflammation in muscle diseases. Aberrant NF-kappaB signaling from immune and muscle cells has been detected and implicated in the pathologic progression of numerous dystrophies and myopathies, indicating that targeted NF-kappaB inhibitors may prove clinically beneficial.

Integrating the immune system with the regulation of growth and efficiency.

Muscle growth in meat animals is a complex process governed by integrated signals emanating from multiple endocrine and immune cells. A generalized phenomenon among meat animal industries is that animals commonly fail to meet their genetic potential for growth in commercial production settings. Recent evidence indicates that adipocytes and myofibers are equipped with functional pattern recognition receptors and are capable of responding directly to the corresponding pathogens and other receptor ligands. Thus, these cells are active participants in the innate immune response and, as such, produce a number of immune and metabolic regulators, including proinflammatory cytokines and adiponectin. Specifically, the transcription factor, nuclear factor kappa B, is activated in adipocytes and muscle cells by bacterial lipopolysaccharide and certain saturated fatty acids, which are potent agonists for the Toll-like receptor-4 pattern recognition receptor. Receptor activation results in the local production of interleukin-6 and tumor necrosis factor-alpha, and creates a local environment by which these cytokines regulate both metabolic and immunological pathways. However, adipocytes are also the predominant source of the antiinflammatory hormone, adiponectin, which suppresses the activation of nuclear factor kappa B and the production of proinflammatory cytokines. The molecular ability to recognize antigens and produce regulatory molecules strategically positions adipocytes and myofibers to regulate growth locally and to reciprocally regulate metabolism in peripheral tissues.

Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.

Macrophages (MPs) are important for skeletal muscle regeneration in vivo and may exert beneficial effects on myogenic cell growth through mitogenic and antiapoptotic activities in vitro. However, MPs are highly versatile and may exert various, and even opposite, functions depending on their activation state. We studied monocyte (MO)/MP phenotypes and functions during skeletal muscle repair. Selective labeling of circulating MOs by latex beads in CX3CR1(GFP/+) mice showed that injured muscle recruited only CX3CR1(lo)/Ly-6C(+) MOs from blood that exhibited a nondividing, F4/80(lo), proinflammatory profile. Then, within muscle, these cells switched their phenotype to become proliferating antiinflammatory CX3CR1(hi)/Ly-6C(-) cells that further differentiated into F4/80(hi) MPs. In vitro, phagocytosis of muscle cell debris induced a switch of proinflammatory MPs toward an antiinflammatory phenotype releasing transforming growth factor beta1. In co-cultures, inflammatory MPs stimulated myogenic cell proliferation, whereas antiinflammatory MPs exhibited differentiating activity, assessed by both myogenin expression and fusion into myotubes. Finally, depletion of circulating MOs in CD11b-diphtheria toxin receptor mice at the time of injury totally prevented muscle regeneration, whereas depletion of intramuscular F4/80(hi) MPs at later stages reduced the diameter of regenerating fibers. In conclusion, injured skeletal muscle recruits MOs exhibiting inflammatory profiles that operate phagocytosis and rapidly convert to antiinflammatory MPs that stimulate myogenesis and fiber growth.

Adipocytes, myofibers, and cytokine biology: new horizons in the regulation of growth and body composition.

Muscle growth in meat animals is a complex process governed by integrated signals emanating from multiple endocrine and immune cells. A generalized phenomenon among meat animal industries is that animals commonly fail to meet their genetic potential for growth in commercial production settings. Therefore, understanding the impact of stress and disease on muscle growth is essential to improving production efficiency. The adipocyte in particular seems to be well positioned as an interface between energy status and immune function, and may thus influence nutrient partitioning and growth through a combination of signals that influence fat metabolism, glucose uptake, and insulin sensitivity. Adipocytes and myofibers are active participants in the innate immune response, and as such, produce a number of metabolic regulators, including leptin, adiponectin, and proinflammatory cytokines. Specifically, adipocytes and muscle cells respond directly to bacterial lipopolysaccharide (LPS) by producing interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFalpha). However, adipocytes are also the predominant source of the antiinflammatory hormone adiponectin, which regulates the nuclear factor kappa-B transcription factor. The ability to recognize antigens and produce regulatory molecules strategically positions adipocytes and myofibers to regulate growth locally, and to reciprocally regulate metabolism peripherally.