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 PDGFRalpha(+) mesenchymal progenitors as being distinct from satellite cells and located in the muscle interstitium. We show that, of the muscle-derived cell populations, only PDGFRalpha(+) 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 PDGFRalpha(+) cells is strongly inhibited by the presence of satellite cell-derived myofibres. These results suggest that PDGFRalpha(+) mesenchymal progenitors are the major contributor to ectopic fat cell formation in skeletal muscle, and emphasize that interaction between muscle cells and PDGFRalpha(+) mesenchymal progenitors, not the fate decision of satellite cells, has a considerable impact on muscle homeostasis.

SummaryAccumulation 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 (PDGFRa) as the origin of adipocytes in skeletal muscle. However, the origin of muscle fibrosis remains largely unknown. In this study, clonal analyses show that PDGFRa + cells also differentiate into collagen type-I-producing cells. In fact, PDGFRa + 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 PDGFRa + cell fraction in the mdx diaphragm. Importantly, TGF-b isoforms, known as potent profibrotic cytokines, induced expression of markers of fibrosis in PDGFRa + cells but not in myogenic cells. Transplantation studies revealed that fibrogenic PDGFRa + cells mainly derived from pre-existing PDGFRa + cells and that the contribution of PDGFRa 2 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.

ABSTRACT

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Objective Duchenne muscular dystrophy (DMD) is caused by the inability to produce dystrophin protein at the myofiber membrane. A method to rescue dystrophin production by antisense oligonucleotides, termed `exon-skipping', has been reported for the mdx mouse and in four DMD patients by local intramuscular injection. We sought to test efficacy and toxicity of intravenous oligonucleotide (morpholino) induced exon skipping in the DMD dog model. Methods We tested a series of antisense drugs singly and as cocktails, both in primary cell culture, and two in vivo delivery methods (intramuscular injection, and systemic intravenous injection). The efficiency and efficacy of multi-exon skipping (exons 6-9) was tested at the mRNA, protein, histological, and clinical levels. Results Weekly or biweekly systemic intravenous injections with a three morpholino cocktail over the course of 5–22 weeks induced therapeutic levels of dystrophin expression throughout the body, with an average of about 26% normal levels. This was accompanied by reduced inflammatory signals examined by MRI and histology, improved or stabilized timed running tests and clinical symptoms. Blood tests indicated no evidence of toxicity. Interpretation This is the first report of widespread rescue of dystrophin expression to therapeutic levels in the dog model of DMD. This study also provides a proof of concept for systemic multi-exon skipping therapy. Use of cocktails of morpholinos, as shown here, allows broader application of this approach to a greater proportion of DMD patients (90%), and also offers the prospect of selecting deletions that optimize the functionality of the dystrophin protein.

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-κB pathway was activated in both wildtype 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.

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.

Using the gene targeting technique, we have generated a new mouse model of congenital muscular dystrophy (CMD), a null mutant for the laminin oc2 chain. These homozygous mice, designated dy 3K ldy 3K , are characterized by growth retardation and severe muscular dystrophic symptoms and die by 5 weeks of age. Light microscopy revealed that muscle fiber degeneration in these mice begins no later than postnatal day 9. In degenerating muscles, considerable amounts of TUNEL positive nuclei were detected as well as DNA laddering, suggesting increased apoptotic cell death was involved in the process of muscle fiber degeneration.