So-ichiro Fukada

Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle

Ectopic fat deposition in skeletal muscle is closely associated with several disorders, however, the origin of these adipocytes is not clear, nor is the mechanism of their formation. Satellite cells function as adult muscle stem cells but are proposed to possess multipotency. Here, we prospectively identify PDGFRα+ mesenchymal progenitors as being distinct from satellite cells and located in the muscle interstitium. We show that, of the muscle-derived cell populations, only PDGFRα+ cells show efficient adipogenic differentiation both in vitro and in vivo. Reciprocal transplantations between regenerating and degenerating muscles, and co-culture experiments revealed that adipogenesis of PDGFRα+ cells is strongly inhibited by the presence of satellite cell-derived myofibres. These results suggest that PDGFRα+ mesenchymal progenitors are the major contributor to ectopic fat cell formation in skeletal muscle, and emphasize that interaction between muscle cells and PDGFRα+ mesenchymal progenitors, not the fate decision of satellite cells, has a considerable impact on muscle homeostasis.

Molecular Signature of Quiescent Satellite Cells in Adult Skeletal Muscle

Skeletal muscle satellite cells play key roles in postnatal muscle growth and regeneration. To study molecular regulation of satellite cells, we directly prepared satellite cells from 8‐ to 12‐week‐old C57BL/6 mice and performed genome‐wide gene expression analysis. Compared with activated/cycling satellite cells, 507 genes were highly upregulated in quiescent satellite cells. These included negative regulators of cell cycle and myogenic inhibitors. Gene set enrichment analysis revealed that quiescent satellite cells preferentially express the genes involved in cell‐cell adhesion, regulation of cell growth, formation of extracellular matrix, copper and iron homeostasis, and lipid transportation. Furthermore, reverse transcription‐polymerase chain reaction on differentially expressed genes confirmed that calcitonin receptor (CTR) was exclusively expressed in dormant satellite cells but not in activated satellite cells. In addition, CTR mRNA is hardly detected in nonmyogenic cells. Therefore, we next examined the expression of CTR in vivo. CTR was specifically expressed on quiescent satellite cells, but the expression was not found on activated/proliferating satellite cells during muscle regeneration. CTR‐positive cells reappeared at the rim of regenerating myofibers in later stages of muscle regeneration. Calcitonin stimulation delayed the activation of quiescent satellite cells. Our data provide roles of CTR in quiescent satellite cells and a solid scaffold to further dissect molecular regulation of satellite cells.

Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle

Accumulation of adipocytes and collagen type-I-producing cells (fibrosis) is observed in muscular dystrophies. The origin of these cells had been largely unknown, but recently we identified mesenchymal progenitors positive for platelet-derived growth factor receptor alpha (PDGFRα) as the origin of adipocytes in skeletal muscle. However, the origin of muscle fibrosis remains largely unknown. In this study, clonal analyses show that PDGFRα+ cells also differentiate into collagen type-I-producing cells. In fact, PDGFRα+ cells accumulated in fibrotic areas of the diaphragm in the mdx mouse, a model of Duchenne muscular dystrophy. Furthermore, mRNA of fibrosis markers was expressed exclusively in the PDGFRα+ cell fraction in the mdx diaphragm. Importantly, TGF-β isoforms, known as potent profibrotic cytokines, induced expression of markers of fibrosis in PDGFRα+ cells but not in myogenic cells. Transplantation studies revealed that fibrogenic PDGFRα+ cells mainly derived from pre-existing PDGFRα+ cells and that the contribution of PDGFRα− cells and circulating cells was limited. These results indicate that mesenchymal progenitors are the main origin of not only fat accumulation but also fibrosis in skeletal muscle.

NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS

Forkhead box O (Foxo) transcription factors induce muscle atrophy by upregulating the muscle-specific E3 ubiquitin ligases MuRF-1 and atrogin-1/MAFbx, but other than Akt, the upstream regulators of Foxos during muscle atrophy are largely unknown. To examine the involvement of the dystrophin glycoprotein complex (DGC) in regulation of Foxo activities and muscle atrophy, we analyzed the expression of DGC members during tail suspension, a model of unloading-induced muscle atrophy. Among several DGC members, only neuronal NOS (nNOS) quickly dislocated from the sarcolemma to the cytoplasm during tail suspension. Electron paramagnetic resonance spectrometry revealed production of NO in atrophying muscle. nNOS-null mice showed much milder muscle atrophy after tail suspension than did wild-type mice. Importantly, nuclear accumulation of dephosphorylated Foxo3a was not evident in nNOS-null muscle, and neither MuRF-1 nor atrogin-1/MAFbx were upregulated during tail suspension. Furthermore, an nNOS-specific inhibitor, 7-nitroindazole, significantly prevented suspension-induced muscle atrophy. The NF-kappaB pathway was activated in both wild-type and nNOS-null muscle during tail suspension. We also show that nNOS was involved in the mechanism of denervation-induced atrophy. We conclude that nNOS/NO mediates muscle atrophy via regulation of Foxo transcription factors and is a new therapeutic target for disuse-induced muscle atrophy.

Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells

Induced pluripotent stem (iPS) cells, which are a type of pluripotent stem cell generated from reprogrammed somatic cells, are expected to have potential for patient‐oriented disease investigation, drug screening, toxicity tests, and transplantation therapies. Here, we demonstrated that murine iPS cells have the potential to develop in vitro into skeletal muscle stem/progenitor cells, which are almost equivalent to murine embryonic stem cells. Cells with strong in vitro myogenic potential effectively were enriched by fluorescence‐activated cell sorting using the anti‐satellite cell antibody SM/C‐2.6. Furthermore, on transplantation into mdx mice, SM/C‐2.6+ cells exerted sustained myogenic lineage differentiation in injured muscles, while providing long‐lived muscle stem cell support. Our data suggest that iPS cells have the potential to be used in clinical treatment of muscular dystrophies.—Mizuno, Y., Chang, H., Umeda, K., Niwa, A, Iwasa, T., Awaya, T., Fukada, S., Yamamoto, H., Yamanaka, S., Nakahata, T., Heike, T. Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells. FASEB J. 24, 2245–2253 (2010). www.fasebj.org

Suppression of macrophage functions impairs skeletal muscle regeneration with severe fibrosis

When damaged, skeletal muscle regenerates. In the early phases of regeneration, inflammatory cells such as neutrophils/granulocytes and macrophages infiltrate damaged muscle tissue. To reveal the roles of macrophages during skeletal muscle regeneration, we injected an antibody, AFS98 that blocks the binding of M-CSF to its receptor into normal mice that received muscle damages. Anti-M-CSF receptor administration suppressed macrophage but not neutrophil infiltration. Histological study indicated that suppression of macrophages function leads to the incomplete muscle regeneration. In addition FACS and immunohistochemical study showed that the acute lack of macrophages delayed proliferation and differentiation of muscle satellite cells in vivo. Furthermore, mice injected with the anti-M-CSF receptor antibody exhibited not only adipogenesis, but also significant collagen deposition, i.e., fibrosis and continuous high expression of connective tissue growth factor. Finally we indicate that these fibrosis markers were strongly enriched in CD90(+) cells that do not include myogenic cells. These results indicate that macrophages directly affect satellite cell proliferation and that a macrophage deficiency severely impairs skeletal muscle regeneration and causes fibrosis.

Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers

Satellite cells, which are skeletal muscle stem cells, divide to provide new myonuclei to growing muscle fibers during postnatal development, and then are maintained in an undifferentiated quiescent state in adult skeletal muscle. This state is considered to be essential for the maintenance of satellite cells, but their molecular regulation is unknown. We show that Hesr1 (Hey1) and Hesr3 (Heyl) (which are known Notch target genes) are expressed simultaneously in skeletal muscle only in satellite cells. In Hesr1 and Hesr3 single-knockout mice, no obvious abnormalities of satellite cells or muscle regenerative potentials are observed. However, the generation of undifferentiated quiescent satellite cells is impaired during postnatal development in Hesr1/3 double-knockout mice. As a result, myogenic (MyoD and myogenin) and proliferative (Ki67) proteins are expressed in adult satellite cells. Consistent with the in vivo results, Hesr1/3-null myoblasts generate very few Pax7+ MyoD– undifferentiated cells in vitro. Furthermore, the satellite cell number gradually decreases in Hesr1/3 double-knockout mice even after it has stabilized in control mice, and an age-dependent regeneration defect is observed. In vivo results suggest that premature differentiation, but not cell death, is the reason for the reduced number of satellite cells in Hesr1/3 double-knockout mice. These results indicate that Hesr1 and Hesr3 are essential for the generation of adult satellite cells and for the maintenance of skeletal muscle homeostasis.

Hypertension and dysregulated proinflammatory cytokine production in receptor activity-modifying protein 1-deficient mice

Calcitonin gene-related peptide (CGRP) is thought to be a prominent neuropeptide in cardiovascular regulation and neuroimmune modulation. There are two isoforms of CGRP (αCGRP and βCGRP), and the main CGRP receptors are probably composed of a calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP)1. However, the physiological functions of CGRP that are mediated through the CLR/RAMP1 receptors remain to be clarified. For an improved understanding of the functions, we generated mice deficient in RAMP1, a specific subunit of CGRP receptors, by a conditional gene-targeting technique. The RAMP1-deficient mice (RAMP1−/−) exhibited high blood pressure, with no changes in heart rate. αCGRP was found to have a potent vascular relaxant activity compared with βCGRP in the artery of the WT (RAMP1+/+) mice. The activities of both CGRP isoforms were remarkably suppressed in the arteries of the RAMP1−/− mice. The LPS-induced inflammatory responses of the RAMP1−/− mice revealed a transient and significant increase in the serum CGRP levels and high serum levels of proinflammatory cytokines compared with the RAMP1+/+ mice. αCGRP and βCGRP equally suppressed the production of TNF-α and IL-12 in bone marrow-derived dendritic cells stimulated with lipopolysaccharide. Their inhibitory effects were not observed in the bone marrow-derived dendritic cells of the RAMP1−/− mice. These results indicate that CGRP signaling through CLR/RAMP1 receptors plays a crucial role in the regulation of both blood pressure by vascular relaxation and proinflammatory cytokine production from dendritic cells.

Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells

Satellite cells are myogenic stem cells responsible for the postnatal regeneration of skeletal muscle. Here we report the successful in vitro induction of Pax7‐positive satellite‐like cells from mouse embryonic stem (mES) cells. Embryoid bodies were generated from mES cells and cultured on Matrigel‐coated dishes with Dulbeccos modified Eagle medium containing fetal bovine serum and horse serum. Pax7‐positive satellite‐like cells were enriched by fluorescence‐activated cell sorting using a novel anti‐satellite cell antibody, SM/C‐2.6. SM/C‐2.6‐positive cells efficiently differentiate into skeletal muscle fibers both in vitro and in vivo. Furthermore, the cells demonstrate satellite cell characteristics such as extensive self‐renewal capacity in subsequent muscle injury model, long‐term engraft‐ment up to 24 wk, and the ability to be secondarily transplanted with remarkably high engraftment efficiency compared to myoblast transplantation. This is the first report of transplantable, functional satellite‐like cells derived from mES cells and will provide a foundation for new therapies for degenerative muscle disor‐ders.—Chang, H.,Yoshimoto, M., Umeda, K., Iwasa, T., Mizuno, Y., Fukada, S., Yamamoto, H., Motohashi, N., Yuko‐Miyagoe‐Suzuki, Takeda, S., Heike, T., Nakahata, T. Generation of transplantable, functional satellite‐like cells from mouse embryonic stem cells. FASEB J. 23, 1907–1919 (2009)

Genetic Background Affects Properties of Satellite Cells and mdx Phenotypes

Duchenne muscular dystrophy (DMD) is the most common lethal genetic disorder of children. The mdx (C57BL/10 background, C57BL/10-mdx) mouse is a widely used model of DMD, but the histopathological hallmarks of DMD, such as the smaller number of myofibers, accumulation of fat and fibrosis, and insufficient regeneration of myofibers, are not observed in adult C57BL/10-mdx except for in the diaphragm. In this study, we showed that DBA/2 mice exhibited decreased muscle weight, as well as lower myofiber numbers after repeated degeneration-regeneration cycles. Furthermore, the self-renewal efficiency of satellite cells of DBA/2 is lower than that of C57BL/6. Therefore, we produced a DBA/2-mdx strain by crossing DBA/2 and C57BL/10-mdx. The hind limb muscles of DBA/2-mdx mice exhibited lower muscle weight, fewer myofibers, and increased fat and fibrosis, in comparison with C57BL/10-mdx. Moreover, remarkable muscle weakness was observed in DBA/2-mdx. These results indicate that the DBA/2-mdx mouse is a more suitable model for DMD studies, and the efficient satellite cell self-renewal ability of C57BL/10-mdx might explain the difference in pathologies between humans and mice.