Interaction between satellite cells and skeletal muscle fibers

Single myofibers with attached satellite cells isolated from adult rats were used to study the influence of the mature myofiber on the proliferation of satellite cells. The satellite cells remain quiescent when cultured in serum containing medium but proliferate when exposed to mitogen from an extract of crushed adult muscle. The response of satellite cells to mitogen was measured under three situations with respect to cell contact: (1) in contact with a viable myofiber and its basal lamina, (2) detached from the myofiber by centrifugal force and deposited on the substratum and (3) beneath the basal lamina of a Marcaine killed myofiber. The results show that satellite cells in contact with the plasmalemma of a viable myofiber have reduced mitogenic response. Since inhibiting growth may induce differentiation, I tested whether satellite cells proliferating on the surface of a myofiber would fuse. Although the satellite cell progeny were fusion competent, they did not fuse with the myofiber. To determine whether fusion competence of the myofiber changes with time in culture, embryonic myoblasts were challenged to fuse with myofibers that had been stripped of satellite cells and cultured for several days. The myoblasts fused with pseudopodial sprouts growing from the ends of the myofiber, but did not fuse with the original myofiber surface. These results indicate that contact with the surface of a mature myofiber suppresses proliferation of myogenic cells but the cells do not fuse with the myofiber.     

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.     

Skeletal muscle fibers in suspension: a new approach to the study of oxidative and glycolytic metabolism in differentiated muscle

A preparation of suspended fibers from m. flexor digitorum brevis of the rat was characterized with respect to morphological features, and its relevance for the study of muscular metabolism investigated. The activities of oxidative (palmitate and pyruvate oxidation) and glycolytic (lactate formation) pathways were enhanced in myofiber suspensions when compared to intact whole muscle. The rate of glycolysis was stimulated about two-fold by insulin in both the myofiber suspensions and intact muscle. Parameters of oxidative metabolism responded similarly to metabolic effectors in the myofiber suspensions and in intact muscle. It is concluded that the myofiber suspensions have distinct advantages over intact muscle for biochemical and pharmacological studies.     

Double gene deletion reveals the lack of cooperation between PPARalpha and PPARbeta in skeletal muscle

The peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of most of the pathways linked to lipid metabolism. PPARalpha and PPARbeta isotypes are known to regulate muscle fatty acid oxidation and a reciprocal compensation of their function has been proposed. Herein, we investigated muscle contractile and metabolic phenotypes in PPARalpha-/-, PPARbeta-/-, and double PPARalpha-/- beta-/- mice. Heart and soleus muscle analyses show that the deletion of PPARalpha induces a decrease of the HAD activity (beta-oxidation) while soleus contractile phenotype remains unchanged. A PPARbeta deletion alone has no effect. However, these mild phenotypes are not due to a reciprocal compensation of PPARbeta and PPARalpha functions since double gene deletion PPARalpha-PPARbeta mostly reproduces the null PPARalpha-mediated reduced beta-oxidation, in addition to a shift from fast to slow fibers. In conclusion, PPARbeta is not required for maintaining skeletal muscle metabolic activity and does not compensate the lack of PPARalpha in PPARalpha null mice.     

Systemic administration of IGF-I enhances oxidative status and reduces contraction-induced injury in skeletal muscles of mdx dystrophic mice

The absence of dystrophin and resultant disruption of the dystrophin glycoprotein complex renders skeletal muscles of dystrophic patients and dystrophic mdx mice susceptible to contraction-induced injury. Strategies to reduce contraction-induced injury are of critical importance, because this mode of damage contributes to the etiology of myofiber breakdown in the dystrophic pathology. Transgenic overexpression of insulin-like growth factor-I (IGF-I) causes myofiber hypertrophy, increases force production, and can improve the dystrophic pathology in mdx mice. In contrast, the predominant effect of continuous exogenous administration of IGF-I to mdx mice at a low dose (1.0-1.5 mg.kg(-1).day(-1)) is a shift in muscle phenotype from fast glycolytic toward a more oxidative, fatigue-resistant, slow muscle without alterations in myofiber cross-sectional area, muscle mass, or maximum force-producing capacity. We found that exogenous administration of IGF-I to mdx mice increased myofiber succinate dehydrogenase activity, shifted the overall myosin heavy chain isoform composition toward a slower phenotype, and, most importantly, reduced contraction-induced damage in tibialis anterior muscles. The deficit in force-producing capacity after two damaging lengthening contractions was reduced significantly in tibialis anterior muscles of IGF-I-treated (53 +/- 4%) compared with untreated mdx mice (70 +/- 5%, P < 0.05). The results provide further evidence that IGF-I administration can enhance the functional properties of dystrophic skeletal muscle and, compared with results in transgenic mice or virus-mediated overexpression, highlight the disparities in different models of endocrine factor delivery.     

Fetal muscle contains different CD34+ cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages

We have previously demonstrated that CD34(+) cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34(+) cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34(+) subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34(+) population comprised a CD31(+)/CD45(-) cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34(+)/CD31(-)/CD45(-)/Sca1(+) subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34(+)/CD31(-)/CD45(-)/Sca1(-) cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34(+) cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.     

Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche

Satellite cells are situated beneath the basal lamina that surrounds each myofiber and function as myogenic precursors for muscle growth and repair. The source of satellite cell renewal is controversial and has been suggested to be a separate circulating or interstitial stem cell population. Here, we transplant single intact myofibers into radiation-ablated muscles and demonstrate that satellite cells are self-sufficient as a source of regeneration. As few as seven satellite cells associated with one transplanted myofiber can generate over 100 new myofibers containing thousands of myonuclei. Moreover, the transplanted satellite cells vigorously self-renew, expanding in number and repopulating the host muscle with new satellite cells. Following experimental injury, these cells proliferate extensively and regenerate large compact clusters of myofibers. Thus, within a normally stable tissue, the satellite cell exhibits archetypal stem cell properties and is competent to form the basal origin of adult muscle regeneration.     

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.     

The transcriptional corepressor RIP140 regulates oxidative metabolism in skeletal muscle

Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.     

Developmental behavior of embryonic myogenic progenitors transplanted into adult muscle as revealed by desmin LacZ recombinant gene.

We studied the behavior of myogenic progenitors from donor desmin(+/-) LacZ embryos after implantation into tibialis anterior muscle of 2-month-old mouse hosts. Myogenic progenitors were collected from 10-day post-coital mouse embryo somite dermomyotomes (DMs), forelimb buds (LBs), and trunks. The replacement of desmin by the LacZ coding sequence allowed specific monitoring of beta-galactosidase expression in donor myogenic cells. Immunostaining for myosin heavy chain and laminin expression was performed together with acetylcholine receptor histochemistry on sections of implanted muscle. Myogenic progenitors generated from DM, LB, and trunk were able to proliferate and adopt a myogenic pathway after transplantation into adult mouse muscle. Although their development appeared to be limited for DM and LB cell transplantation, the differentiation of myogenic progenitors occurred readily with trunk cell injection, suggesting that cell types associated with DM cells were involved in long-term myofiber differentiation (21 day). When neural tube/notochord (NTN) or sclerotomal (S) cells were co-transplanted with DM cells, myogenic nuclei were produced, indicating that both NTN and S are required for the differentiation of DMs grafted into adult muscle. These data are consistent with the differentiation of neural tissues and bone from NTN and S, respectively, and with the development of anatomic relations among all in vivo-differentiated tissues. These results suggest that embryonic trunk cells can be used to repair different types of injured tissues (especially skeletal muscle) under appropriate environmental conditions.