Pax7-positive Satellite Cells Research Articles

Mustn1 in Skeletal Muscle: A Novel Regulator?

Skeletal muscle is a complex organ essential for locomotion, posture, and metabolic health. This review explores our current knowledge of Mustn1, particularly in the development and function of skeletal muscle. Mustn1 expression originates from Pax7-positive satellite cells in skeletal muscle, peaks during around the third postnatal month, and is crucial for muscle fiber differentiation, fusion, growth, and regeneration. Clinically, Mustn1 expression is potentially linked to muscle-wasting conditions such as muscular dystrophies. Studies have illustrated that Mustn1 responds dynamically to injury and exercise. Notably, ablation of Mustn1 in skeletal muscle affects a broad spectrum of physiological aspects, including glucose metabolism, grip strength, gait, peak contractile strength, and myofiber composition. This review summarizes our current knowledge of Mustn1's role in skeletal muscle and proposes future research directions, with a goal of elucidating the molecular function of this regulatory gene.

Nystagmus Associated With the Absence of MYOD Expression Across the Lifespan in Extraocular and Limb Muscles.

The extraocular muscles (EOMs) undergo significant levels of continuous myonuclear turnover and myofiber remodeling throughout life, in contrast to limb skeletal muscles. Activation of the myogenic pathway in muscle precursor cells is controlled by myogenic transcription factors, such as MYOD. Limb muscles from MyoD-/- mice develop normally but have a regeneration defect, and these mice develop nystagmus. We examined MyoD-/- mice to determine if they have an aging phenotype. Eye movements of aging MyoD-/- mice and littermate controls (wild type) were examined using optokinetic nystagmus (OKN). We assessed limb muscle function, changes to myofiber number, mean cross-sectional area, and abundance of the PAX7 and PITX2 populations of myogenic precursor cells. Aging did not significantly affect limb muscle function despite decreased mean cross-sectional areas at 18+months. Aging wild type mice had normal OKN responses; all aging MyoD-/- mice had nystagmus. With OKN stimulus present, the MyoD-/- mice at all ages had shorter slow phase durations compared to wild type age matched controls. In the dark, the MyoD-/- mice had a shorter slow phase duration with age. This correlated with significantly decreased fiber numbers and cross-sectional areas. The EOM in MyoD-/- mice had increased numbers of PAX7-positive satellite cells and significantly decreased PITX2-positive myonuclei. The absence of MYOD expression in aging mice causes a decrease in on-going myofiber remodeling, EOM fiber size, and number, and is associated with the development of spontaneous nystagmus. These results suggest that muscle-specific mutations can result in nystagmus, with increasing aging-related changes in the MyoD-/- EOM.

Chemokine-like receptor 1 plays a critical role in modulating the regenerative and contractile properties of muscle tissue.

Musculoskeletal diseases are a leading contributor to mobility disability worldwide. Since the majority of patients with musculoskeletal diseases present with associated muscle weakness, treatment approaches typically comprise an element of resistance training to restore physical strength. The health-promoting effects of resistance exercise are mediated via complex, multifarious mechanisms including modulation of systemic and local inflammation. Here we investigated whether targeted inhibition of the chemerin pathway, which largely controls inflammatory processes via chemokine-like receptor 1 (CMKLR1), can improve skeletal muscle function. Using genetically modified mice, we demonstrate that blockade of CMKLR1 transiently increases maximal strength during growth, but lastingly decreases strength endurance. In-depth analyses of the underlying long-term adaptations revealed microscopic alterations in the number of Pax7-positive satellite cells, as well as molecular changes in genes governing myogenesis and calcium handling. Taken together, these data provide evidence of a critical role for CMKLR1 in regulating skeletal muscle function by modulating the regenerative and contractile properties of muscle tissue. CMKLR1 antagonists are increasingly viewed as therapeutic modalities for a variety of diseases (e.g., psoriasis, metabolic disorders, and multiple sclerosis). Our findings thus have implications for the development of novel drug substances that aim at targeting the chemerin pathway for musculoskeletal or other diseases.

FP.14 Dystrophin and satellite cell quantification in Duchenne and Becker muscular dystrophies

The dystrophinopathies are a group of muscular dystrophies stemming from mutations in the <i>DMD</i> gene. We have developed a series of sensitive and reproducible dystrophin and satellite cell quantification methods in order to better define associations between dystrophin expression and disease severity, as well as the molecular mechanisms of mutations in the N-terminal region of dystrophin. In a pilot study of 18 dystrophinopathy patient muscle biopsies, we employed western blot, capillary western immunoassay, and automated quantitative immunofluorescence analysis to quantify dystrophin expression and Pax7-positive satellite cells. Among 9 patients with a Duchenne muscular dystrophy phenotype, the mean dystrophin protein level by capillary western was 1.8% of normal. These biopsies displayed a mean of 6.8% immunofluorescence staining intensity of individual fibers and 26% dystrophin positive fibers (PDPF). This high PDPF reflects the use of a sensitive cutoff to improve the discriminating power of the assay at low expression levels. By contrast, patients with intermediate or Becker muscular dystrophy had 11% dystrophin expression by capillary western and 70% PDPF. Several truncating mutations in the first 4 exons of DMD were associated with milder phenotypes and significantly higher dystrophin expression (16% by capillary western and 79% PDPF), in keeping with activation of an internal ribosome entry site in exon 5. There was poor overall correlation between Pax7-positive satellite cell count and dystrophin expression. Analysis of individual muscle fibers however demonstrated that fibers with one or more adjacent Pax7-positive satellite cells had a lower mean dystrophin expression, indicating concentration of satellite cells around dystrophin-deficient fibers. This study lays the groundwork for a comprehensive characterization of genotype, phenotype, and molecular features in a broader cohort of dystrophinopathy patients.

In Vitro Model of Human Skeletal Muscle Tissue for the Study of Resident Macrophages and Stem Cells.

Simple SummaryThe skeletal muscle of younger adults has a remarkable regenerative capacity, which substantially declines with age. Despite many interspecies differences, animals have been used to study new treatments to promote muscle regeneration in humans. This study reports a novel human experimental model using human skeletal muscle tissue of older adults that was extracted during surgical procedures. We describe an optimal procedure for maintaining human skeletal muscle tissue under experimental conditions for 11 days. This experimental model allows the investigation of resident macrophages and stem cells, which mediate muscle regeneration.Findings from studies of muscle regeneration can significantly contribute to the treatment of age-related loss of skeletal muscle mass, which may predispose older adults to severe morbidities. We established a human experimental model using excised skeletal muscle tissues from reconstructive surgeries in eight older adults. Muscle samples from each participant were preserved immediately or maintained in agarose medium for the following 5, 9, or 11 days. Immunofluorescence analyses of the structural proteins, actin and desmin, confirmed the integrity of muscle fibers over 11 days of maintenance. Similarly, the numbers of CD80-positive M1 and CD163-positive M2 macrophages were stable over 11 days in vitro. However, the numbers of PAX7-positive satellite cells and MYOD-positive myoblasts changed in opposite ways, suggesting that satellite cells partially differentiated in vitro. Further experiments revealed that stimulation with unsaturated fatty acid C18[2]c (linoleic acid) increased resident M1 macrophages and satellite cells specifically. Thus, the use of human skeletal muscle tissue in vitro provides a direct experimental approach to study the regulation of muscle tissue regeneration by macrophages and stem cells and their responses to therapeutic compounds.

Human Skeletal Muscle Satellite Cells Co-Express Aldehyde Dehydrogenase Isoforms Aldh1A1 andamp; Aldh1A3

Satellite cells (SC) constitute the stem cell population of skeletal muscle and conduct myogenic growth and differentiation. Recently, aldehyde dehydrogenase 1 (ALDH1) has been identified as a novel myogenic factor in experimental models of SCs. ALDH1 constitutes a subfamily of the ALDH enzyme super family. The enzymatic functions of ALDH1 isoforms include both protection against oxidative stress products and regulation of differentiation as pacemaker enzyme in retinoic acid signaling. Although ALDH enzymatic activity has been demonstrated in SCs it is not clear which isoforms are important in human skeletal muscle. Here, we show that ALDH1A1 and ALDH1A3 are expressed in human SCs. Using antibodies directed against ALDH1 and its isoforms ALDH1A1 and ALDH1A3, respectively, we demonstrate immunohistochemical staining in peri-fascicular position matching the localization of SCs. Consistently, co-immunofluorescence reveals ALDH1 expression in CD56 positive stem cells and co-localization of the isoforms ALDH1A1 and ALDH1A3 in Pax7 positive SCs. Quantitative analysis of immunohistochemical staining showed no significant differences in the distribution of ALDH1 positive SCs in the skeletal muscle groups pectoralis, diaphragm and psoas that have been investigated in the present study. In conclusion, human SCs co-express the ALDH1 isoforms ALDH1A1 and ALDH1A3.

MiR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.

miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

Regeneration competent satellite cell niches in rat engineered skeletal muscle.

Satellite cells reside in defined niches and are activated upon skeletal muscle injury to facilitate regeneration. Mechanistic studies of skeletal muscle regeneration are hampered by the inability to faithfully simulate satellite cell biology in vitro. We sought to overcome this limitation by developing tissue engineered skeletal muscle (ESM) with (1) satellite cell niches and (2) the capacity to regenerate after injury. ESMs contained quiescent Pax7‐positive satellite cells in morphologically defined niches. Satellite cells could be activated to repair (i) cardiotoxin and (ii) mechanical crush injuries. Activation of the Wnt‐pathway was essential for muscle regeneration. Finally, muscle progenitors from the engineered niche developed de novo ESM in vitro and regenerated skeletal muscle after cardiotoxin‐induced injury in vivo. We conclude that ESM with functional progenitor niches reminiscent of the in vivo satellite cell niches can be engineered in vitro. ESM may ultimately be exploited in disease modeling, drug screening, or muscle regeneration.

Sclerostin inhibition alleviates breast cancer-induced bone metastases and muscle weakness.

Breast cancer bone metastases often cause a debilitating non-curable condition with osteolytic lesions, muscle weakness and a high mortality. Current treatment comprises chemotherapy, irradiation, surgery and anti-resorptive drugs that restrict but do not revert bone destruction. In metastatic breast cancer cells, we determined the expression of sclerostin, a soluble Wnt inhibitor that represses osteoblast differentiation and bone formation. In mice with breast cancer bone metastases, pharmacological inhibition of sclerostin using an anti-sclerostin antibody (Scl-Ab) reduced metastases without tumor cell dissemination to other distant sites. Sclerostin inhibition prevented the cancer-induced bone destruction by augmenting osteoblast-mediated bone formation and reducing osteoclast-dependent bone resorption. During advanced disease, NF-κB and p38 signaling was increased in muscles in a TGF-β1-dependent manner, causing muscle fiber atrophy, muscle weakness and tissue regeneration with an increase in Pax7-positive satellite cells. Scl-Ab treatment restored NF-κB and p38 signaling, the abundance of Pax7-positive cells and ultimately muscle function. These effects improved the overall health condition and expanded the life span of cancer-bearing mice. Together, these results demonstrate that pharmacological inhibition of sclerostin reduces bone metastatic burden and muscle weakness with a prolongation of the survival time. This might provide novel options for treating musculoskeletal complications in breast cancer patients. .

SAT-560 Role of COUP-TFII and Thyroid Hormone Receptor α in Skeletal Muscle Loss

Myopathic changes including muscular dystrophy and weakness are commonly described in hypothyroid and hyperthyroid patients. The genomic actions of triiodothyronine (T3) are mediated by thyroid hormone nuclear receptors (TRs), which act as ligand-inducible transcription factors in almost all tissues, including skeletal muscle. Impaired skeletal muscle regeneration and sarcopenia with aging have been reported in a mouse model of Resistance to Thyroid Hormone (RTH) carrying a frame-shift mutation in the TRα gene (TRα1PV). One salient finding was a significantly smaller pool of PAX7-positive satellite cells (SCs) in the skeletal muscle of TRα1PV mice. Moreover, SC function during skeletal muscle injury was impaired four days after cardiotoxin (CTX)-induced skeletal muscle injury with a decreased activation of SC and a reduced proliferation of Myf5 expressing cells. TRα plays an important role in the maintenance of muscle mass and skeletal muscle regeneration after injury with aging, and loss of skeletal muscle mass with aging in TRα1PV mice was reported to be associated with loss of SC pool {1,2}. Interestingly, overexpression of the nuclear orphan receptor Chicken Ovalbumin Upstream Promoter-factor II (COUP-TFII or NR2F2) in murine satellite cells have been shown to induce a similar skeletal muscle phenotype, including skeletal muscle loss with aging, and inhibit myogenesis through modulation of Myf5 and MyoD expression {3}. We detected a higher expression of COUP-TFII in C2C12, a murine myoblast cell line, during proliferation and a decline during differentiation into myotube. Moreover, we analyzed the skeletal muscle of mice at different ages and we found a higher expression of COUP-TFII in the first 2 months of life followed by a decline with age. Thus, suggesting the important role of COUP-TFII in modulating post-natal myogenesis and onset of sarcopenia with aging. In proliferating C2C12 myoblasts and SCs from wild-type and TRα1PV mice, we demonstrated via co-immunoprecipitation that COUP-TFII and TRα interact. In addition, the skeletal muscle of TRα1PV mice showed significantly higher expression of COUP-TFII compared to their WT siblings, and in TRα1PV mice COUP-TFII expression remained higher with skeletal muscle aging. These results suggest a COUP-TFII role in skeletal muscle loss with aging and impaired skeletal muscle regeneration in TRαPV mice that may be mediated by COUP-TFII-TRα interaction. These new insights can provide a therapeutic target to prevent or treat myopathies, such as sarcopenia and Duchenne-like muscular dystrophy. 1. Milanesi A et al, Endocrinology 2015. 2. Milanesi A et al, Thyroid, 2017. 3. Lee HJ et al, Scientific Reports 2017. Sources of Research Support: NIH grants and VA MERIT GRANT.

Satellite cells maintain regenerative capacity but fail to repair disease-associated muscle damage in mice with Pompe disease

Pompe disease is a metabolic myopathy that is caused by glycogen accumulation as a result of deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Previously, we showed that adult muscle stem cells termed satellite cells are present at normal levels in muscle from patients with Pompe disease, but that these are insufficiently activated to repair the severe muscle pathology. Here we characterized the muscle regenerative response during disease progression in a mouse model of Pompe disease and investigated the intrinsic capacity of Gaa−/− satellite cells to regenerate muscle damage. Gaa−/− mice showed progressive muscle pathology from 15 weeks of age as reflected by increased lysosomal size, decreased fiber diameter and reduced muscle wet weight. Only during the first 15 weeks of life but not thereafter, we detected a gradual increase in centrally nucleated fibers and proliferating satellite cells in Gaa−/− muscle, indicating a mild regenerative response. The levels of Pax7-positive satellite cells were increased in Gaa−/− mice at all ages, most likely as result of enhanced satellite cell activation in young Gaa−/− animals. Surprisingly, both young and old Gaa−/− mice regenerated experimentally-induced muscle injury efficiently as judged by rapid satellite cell activation and complete restoration of muscle histology. In response to serial injury, Gaa−/− mice also regenerated muscle efficiently and maintained the satellite cell pool. These findings suggest that, similar to human patients, Gaa−/− mice have insufficient satellite cell activation and muscle regeneration during disease progression. The initial endogenous satellite cell response in Gaa−/− mice may contribute to the delayed onset of muscle wasting compared to human patients. The rapid and efficient regeneration after experimental muscle injury suggest that Gaa−/− satellite cells are functional stem cells, opening avenues for developing muscle regenerative therapies for Pompe disease.

Loss of the adipokine lipocalin-2 impairs satellite cell activation and skeletal muscle regeneration.

Lipocalin-2 (LCN2) is an adipokine previously described for its contribution to numerous processes, including innate immunity and energy metabolism. LCN2 has also been demonstrated to be an extracellular matrix (ECM) regulator through its association with the ECM protease matrix metalloproteinase-9 (MMP-9). With the global rise in obesity and the associated comorbidities related to increasing adiposity, it is imperative to gain an understanding of the cross talk between adipose tissue and other metabolic tissues, such as skeletal muscle. Given the function of LCN2 on the ECM in other tissues and the importance of matrix remodeling in skeletal muscle regeneration, we examined the localization and expression of LCN2 in uninjured and regenerating wild-type skeletal muscle and assessed the impact of LCN2 deletion (LCN2-/-) on skeletal muscle repair following cardiotoxin injury. Though LCN2 was minimally present in uninjured skeletal muscle, its expression was increased significantly at 1 and 2 days postinjury, with expression present in Pax7-positive satellite cells. Although satellite cell content was unchanged, the ability of quiescent satellite cells to become activated was significantly impaired in LCN2-/- skeletal muscles. Skeletal muscle regeneration was also significantly compromised as evidenced by decreased embryonic myosin heavy chain expression and smaller regenerating myofiber areas. Consistent with a role for LCN2 in MMP-9 regulation, regenerating muscle also displayed a significant increase in fibrosis and lower ( P = 0.07) MMP-9 activity in LCN2-/- mice at 2 days postinjury. These data highlight a novel role for LCN2 in muscle regeneration and suggest that changes in adipokine expression can significantly impact skeletal muscle repair.

Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells

Satellite cells are muscle stem cells required for muscle regeneration upon damage. Of note, satellite cells are bipotent and have the capacity to differentiate not only into skeletal myocytes, but also into brown adipocytes. Epigenetic mechanisms regulating fate decision and differentiation of satellite cells during muscle regeneration are not yet fully understood. Here, we show that elevated levels of lysine-specific demethylase 1 (Kdm1a, also known as Lsd1) have a beneficial effect on muscle regeneration and recovery after injury, since Lsd1 directly regulates key myogenic transcription factor genes. Importantly, selective Lsd1 ablation or inhibition in Pax7-positive satellite cells, not only delays muscle regeneration, but changes cell fate towards brown adipocytes. Lsd1 prevents brown adipocyte differentiation of satellite cells by repressing expression of the novel pro-adipogenic transcription factor Glis1. Together, downregulation of Glis1 and upregulation of the muscle-specific transcription program ensure physiological muscle regeneration.

Abstract A16: Fusion-negative rhabdomyosarcoma originating from endothelial progenitors

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood, and despite rigorous clinical trials the survival for children with high-risk RMS has not changed for three decades. RMS is subdivided into two major classes, fusion-positive (FP) and fusion-negative RMS (FN-RMS), based on the presence or absence of the PAX3-FOXO1 or PAX7-FOXO1 gene fusions. RMS occurs at locations throughout the body with nearly 40% of tumors occurring in the head and neck. Tumor location and fusion status are key prognostic factors. RMS resembles developing skeletal muscle and has been speculated to originate from genetically compromised skeletal muscle progenitors. However, the genes that control RMS development and specify location remain elusive. RMS also occurs in tissues devoid of skeletal muscle such as the urinary bladder, prostate, and biliary tree, suggesting the possibility of origins outside of the skeletal muscle lineage. Currently, the cell of origin and the factors that specify RMS location and thus prognosis are unknown. Previously, we showed that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human FN-RMS. In this model, tumorigenesis occurs with high penetrance (~80%), is early onset (by 2 months of age), and is anatomically restricted to the head and neck. Also, unlike previous RMS models, this model requires no additional background mutations, such as inactivation of p53, and drives only FN-RMS neoplasia. We illustrated that the transcriptome of the aP2-Cre;SmoM2 tumors recapitulates both other mouse FN-RMS models as well as human FN-RMS. With the short latency and anatomic restricted tumor location, we sought to leverage this model to explore the cell of origin of FN-RMS. Here we use genetic fate mapping with fluorescent reporter mice to interrogate the cell of origin of FN-RMS in the aP2-Cre;SmoM2 model. Tracing the aP2-Cre labeled cells with reporter mice illustrated labeled cells in both brown and white adipose tissue as well as a discrete population of cells lying between skeletal muscle fibers but not beneath the laminin sheath. These aP2-Cre labeled cells are distinct from Pax7-positive skeletal muscle stem cells or satellite cells and do not contribute to myotube formation in vitro or in vivo. Gene profiling of tomato positive cells isolated by FACS from the sternocleidomastoid (SCM) of aP2-Cre;R26-Tom and aP2-Cre;R26-Tom;SmoM2 revealed that these aP2-Cre labeled cells in muscle interstitium are endothelial cells. In SCM sections, aP2-Cre labeled cells colocalize with Pecam1. When compared to aP2-Cre;R26-Tom mice, the addition of oncogenic SmoM2 (aP2-Cre;R26-Tom;SmoM2) results in embryonic expansion of the aP2-labeled muscle interstitial cells and formation of FN-RMS. These expansions became positive for the myogenic regulatory factor MyoD1 and did not express Pecam1 at embryonic day 17.5. Subsequent gene expression analysis revealed that both myogenic regulatory factors and endothelial genes are upregulated in tumor cells. Skeletal muscle and endothelial cells share a common progenitor, and together these results suggest that aberrant Sonic Hedgehog signaling promotes a myogenic fate switch in aP2-Cre expressing endothelial progenitor cells that results in FN-RMS formation. Citation Format: Catherine J. Drummond, Jason A. Hanna, Matthew R. Garcia, Daniel Devine, Jennifer Peters, Victoria Frohlich, David Finkelstein, Mark E. Hatley. Fusion-negative rhabdomyosarcoma originating from endothelial progenitors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A16.

Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration

Salamanders exhibit extensive regenerative capacities and serve as a unique model in regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl limb regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl regeneration.

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.

Abstract 1036: Non-myogenic origin of embryonal rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy and surgery, clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS includes two histopathologic subtypes: alveolar RMS, driven by the fusion protein PAX3/7-FOXO1, and embryonal RMS (ERMS), which is genetically heterogeneous. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle such as the prostate, bladder, biliary tree and the omentum. Previously, we showed that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human ERMS. In this model, tumorigenesis occurs with high penetrance (~80%), is early onset (by 2 months of age), and is restricted to the head and neck. Also, unlike previous RMS models, this model requires no additional background mutations, such as inactivation of p53, and results in only ERMS neoplasia. We illustrated that the gene expression signature of the aP2-Cre;SmoM2 tumors recapitulates both other mouse ERMS models as well as human ERMS. With the short latency and anatomic restricted tumor location, we sought to leverage this model to explore the cell of origin. Here, we use genetic fate mapping of aP2-cre with reporter mice to determine the effect of constitutive hedgehog activation on the identity and localization of aP2-Cre expressing cells. aP2-cre expressing cells are found in both brown and white adipose and to be localized within the muscle intersitium but not beneath the laminin sheath of the muscle fibers. These aP2-cre expressing cells are distinct from Pax7-positive skeletal muscle stem cells or satellite cells and do not contribute to myofiber formation. Instead these cells were confirmed to be endothelial by gene expression analysis. Colocalization between aP2-cre expressing cells and CD31 was also observed. When compared to aP2-Cre;R26-Tom mice, the addition of oncogenic SmoM2 (aP2-Cre;R26-Tom;SmoM2) resulted in embryonic expansion of these aP2-lineage interstitial muscle cells and formation of ERMS. Our findings suggest that non-skeletal muscle progenitors are a cell of origin for Sonic Hedgehog-driven ERMS. Citation Format: Catherine J. Drummond, Matthew R. Garcia, Daniel J. Devine, Jennifer Peters, Victoria Frohlich, David Finkelstein, Mark E. Hatley. Non-myogenic origin of embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1036. doi:10.1158/1538-7445.AM2017-1036

Extracorporeal Shock Wave Therapy Accelerates Regeneration After Acute Skeletal Muscle Injury

Background: Muscle injuries are among the most common sports-related lesions in athletes; however, optimal treatment remains obscure. Extracorporeal shock wave therapy (ESWT) may be a promising approach in this context, because it has gained increasing importance in tissue regeneration in various medical fields. Hypothesis: ESWT stimulates and accelerates regenerative processes of acute muscle injuries. Study Design: Controlled laboratory study. Methods: Adult Sprague-Dawley rats were divided into 4 experimental groups (2 ESWT+ groups and 2 ESWT– groups) as well as an uninjured control group (n ≥ 6 in each group). An acute cardiotoxin-induced injury was set into the quadriceps femoris muscle of rats in the experimental groups. A single ESWT session was administered to injured muscles of the ESWT+ groups 1 day after injury, whereas ESWT– groups received no further treatment. At 4 and 7 days after injury, 1 each of the ESWT+ and ESWT– groups was euthanized. Regenerating lesions were excised and analyzed by histomorphometry and immunohistochemistry to assess fiber size, myonuclear content, and recruitment of satellite cells. Results: The size and myonuclear content of regenerating fibers in ESWT+ muscle was significantly increased compared with ESWT– muscle fibers at both 4 and 7 days after injury. Similarly, at both time points, ESWT+ muscles exhibited significantly higher contents of pax7-positive satellite cells, mitotically active H3P+ cells, and, of cells expressing the myogenic regulatory factors, myoD and myogenin, indicating enhanced proliferation and differentiation rates of satellite cells after ESWT. Mitotic activity at 4 days after injury was doubled in ESWT+ compared with ESWT– muscles. Conclusion: ESWT stimulates regeneration of skeletal muscle tissue and accelerates repair processes. Clinical Relevance: We provide evidence for accelerated regeneration of damaged skeletal muscle after ESWT. Although further studies are necessary, our findings support the view that ESWT is an effective method to improve muscle healing, with special relevance to sports injuries.

Abstract IA03: Insights into the origins and pathogenesis of embryonal rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite aggressive chemotherapy, radiotherapy and surgery, clinical outcomes for RMS have not improved for three decades, emphasizing the need to uncover the molecular underpinnings of the disease. RMS is an aggressive skeletal muscle-lineage tumor composed of malignant myoblasts that fail to exit the cell cycle and are blocked from fusing into syncytial muscle. RMS includes two histolopathologic subtypes: alveolar RMS, driven by the fusion protein PAX3/7-FOXO1, and embryonal RMS (ERMS), which is genetically heterogeneous. RMS has been presumed to originate from derailed muscle progenitors based on the histologic appearance and gene expression pattern of the tumors. However, an origin restricted to skeletal muscle does not explain RMS occurring in tissues devoid of skeletal muscle such as the prostate, bladder, biliary tree and the omentum. Previously, we showed that activation of Sonic Hedgehog signaling through expression of a conditional, constitutively active Smoothened allele, SmoM2, under control of an adipocyte-restricted adipose protein 2 (aP2)-Cre recombinase transgene in mice gives rise to aggressive skeletal muscle tumors that display the histologic and molecular characteristics of human ERMS. In this model, tumorigenesis occurs with high penetrance (~80%), is early onset (by 2 months of age), and is restricted to the head and neck. Also, unlike previous RMS models, this model requires no additional background mutations, such as inactivation of p53, and drives only ERMS neoplasia. We illustrated that the transcriptome of the aP2-Cre;SmoM2 tumors recapitulates both other mouse ERMS models as well as human ERMS. With the short latency and anatomic restricted tumor location, we sought to leverage this model to explore the cell of origin. Lineage tracing the aP2-Cre with reporter mice illustrated aP2-Cre expression in both brown and white adipose tissue as well as a discrete population of cells lying between skeletal muscle fibers but not beneath the laminin sheath of the muscle fibers. These aP2-lineage cells are distinct from Pax7-positive skeletal muscle stem cells or satellite cells. The aP2-lineage cells do not contribute to myotube formation in vitro. When compared to aP2-Cre;R26-Tom mice, the addition of oncogenic SmoM2 (aP2-Cre;R26-Tom;SmoM2) results in embryonic expansion of the aP2-lineage interstitial muscle cells. Our findings suggest that non-skeletal muscle progenitors are a potential cell of origin for Sonic Hedgehog-driven ERMS. Citation Format: Catherine J. Drummond, Daniel Devine, Matthew R. Garcia, Mark E. Hatley. Insights into the origins and pathogenesis of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA03.

Customized platelet-rich plasma with transforming growth factor β1 neutralization antibody to reduce fibrosis in skeletal muscle

The formation of fibrous tissue during the healing of skeletal muscle injuries leads to incomplete recovery of the injured muscle. Platelet-rich-plasma (PRP) contains beneficial growth factors for skeletal muscle repair; however, it also contains deleterious cytokines and growth factors, such as TGF-β1, that can cause fibrosis and inhibit optimal muscle healing. ObjectiveTo test if neutralizing TGF-β1's action within PRP, through neutralization antibodies, could improve PRP's beneficial effect on skeletal muscle repair. MethodsPRP was isolated from in-bred Fisher rats. TGF-β1 neutralization antibody (Ab) was used to block the TGF-β1 within the PRP prior to injection. The effects of customized PRP (TGF-β1 neutralized PRP) on muscle healing was tested on a cardiotoxin (CTX) induced muscle injury model. ResultsA significant increase in the numbers of regenerative myofibers was observed in the PRP and customized PRP groups compared to the untreated control. A significant decrease in collagen deposition was observed in customized PRP groups when compared to the control and PRP groups. Significantly enhanced angiogenesis and more Pax-7 positive satellite cells were found in the PRP and customized PRP groups compared to the control group. Macrophage infiltration was increased in the customized PRP groups when compared with the PRP group. More M2 macrophages were recruited to the injury site in the customized PRP groups when compared with the PRP and control groups. ConclusionNeutralizing TGF-β1 within PRP significantly promotes muscle regeneration while significantly reducing fibrosis. Not only did the neutralization reduce fibrosis, it enhanced angiogenesis, prolonged satellite cell activation, and recruited a greater number of M2 macrophages to the injury site which also contributed to the efficacy that the customized PRP had on muscle healing.