Akiyoshi Uezumi

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.

Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo

Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein–positive CSPs were intravenously infused into adult rats, many more (∼12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts.

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.

Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice

Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin‐related proteins, and myostatin antibodies. Although follistatin shows potent myostatin‐inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (Kd) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (Kd) of a follistatin‐derived myostatin inhibitor, designated FS I‐I, to activin and myostatin are 64.3 μM and 46.8 nM, respectively. Transgenic mice expressing FS I‐I, under the control of a skeletal muscle‐specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I‐I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I‐I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I‐I mice. These results indicate that myostatin blockade by FS I‐I has a therapeutic potential for muscular dystrophy.—Nakatani, M., Takehara, Y., Sugino, H., Matsumoto, M., Hashimoto, O., Hasegawa, Y., Murakami, T., Uezumi, A., Takeda, S., Noji, S., Sunada, Y., Tsuchida, K. Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice. FASEB J. 22, 477–487 (2008)

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.

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.

Activin signaling as an emerging target for therapeutic interventions

After the initial discovery of activins as important regulators of reproduction, novel and diverse roles have been unraveled for them. Activins are expressed in various tissues and have a broad range of activities including the regulation of gonadal function, hormonal homeostasis, growth and differentiation of musculoskeletal tissues, regulation of growth and metastasis of cancer cells, proliferation and differentiation of embryonic stem cells, and even higher brain functions. Activins signal through a combination of type I and II transmembrane serine/threonine kinase receptors. Activin receptors are shared by multiple transforming growth factor-β (TGF-β) ligands such as myostatin, growth and differentiation factor-11 and nodal. Thus, although the activity of each ligand is distinct, they are also redundant, both physiologically and pathologically in vivo. Activin receptors activated by ligands phosphorylate the receptor-regulated Smads for TGF-β, Smad2 and 3. The Smad proteins then undergo multimerization with the co-mediator Smad4, and translocate into the nucleus to regulate the transcription of target genes in cooperation with nuclear cofactors. Signaling through receptors and Smads is controlled by multiple mechanisms including phosphorylation and other posttranslational modifications such as sumoylation, which affect potein localization, stability and transcriptional activity. Non-Smad signaling also plays an important role in activin signaling. Extracellularly, follistatin and related proteins bind to activins and related TGF-β ligands, and control the signaling and availability of ligands.The functions of activins through activin receptors are pleiotrophic, cell type-specific and contextual, and they are involved in the etiology and pathogenesis of a variety of diseases. Accordingly, activin signaling may be a target for therapeutic interventions. In this review, we summarize the current knowledge on activin signaling and discuss the potential roles of this pathway as a molecular target of therapy for metabolic diseases, musculoskeletal disorders, cancers and neural damages.

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.

Muscle CD31(-) CD45(-) side population cells promote muscle regeneration by stimulating proliferation and migration of myoblasts

CD31(-) CD45(-) side population (SP) cells are a minor SP subfraction that have mesenchymal stem cell-like properties in uninjured skeletal muscle but that can expand on muscle injury. To clarify the role of these SP cells in muscle regeneration, we injected green fluorescent protein (GFP)-positive myoblasts with or without CD31(-) CD45(-) SP cells into the tibialis anterior muscles of immunodeficient NOD/scid mice or dystrophin-deficient mdx mice. More GFP-positive fibers were formed after co-transplantation than after transplantation of GFP-positive myoblasts alone in both mdx and NOD/scid muscles. Moreover, grafted myoblasts were more widely distributed after co-transplantation than after transplantation of myoblasts alone. Immunohistochemistry with anti-phosphorylated histone H3 antibody revealed that CD31(-) CD45(-) SP cells stimulated cell division of co-grafted myoblasts. Genome-wide gene expression analyses showed that these SP cells specifically express a variety of extracellular matrix proteins, membrane proteins, and cytokines. We also found that they express high levels of matrix metalloproteinase-2 mRNA and gelatinase activity. Furthermore, matrix metalloproteinase-2 derived from CD31(-) CD45(-) SP cells promoted migration of myoblasts in vivo. Our results suggest that CD31(-) CD45(-) SP cells support muscle regeneration by promoting proliferation and migration of myoblasts. Future studies to further define the molecular and cellular mechanisms of muscle regeneration will aid in the development of cell therapies for muscular dystrophy.