Human Fetal Skeletal Muscle Cells Research Articles

The phosphodiesterase 5 inhibitor tadalafil regulates lipidic homeostasis in human skeletal muscle cell metabolism.

Tadalafil seems to ameliorate insulin resistance and glucose homeostasis in humans. We have previously reported that tadalafil targets human skeletal muscle cells with an insulin (I)-like effect. We aim to evaluate in human fetal skeletal muscle cells after tadalafil or I: (i) expression profile of I-regulated genes dedicated to cellular energy control, glycolitic activity or microtubule formation/vesicle transport, as GLUT4, PPARγ, HK2, IRS-1, KIF1C, and KIFAP3; (ii) GLUT4, Flotillin-1, and Caveolin-1 localization, all proteins involved in energy-dependent cell trafficking; (iii) activation of I-targeted paths, as IRS-1, PKB/AKT, mTOR, P70/S6K. Free fatty acids intracellular level was measured. Sildenafil or a cGMP synthetic analog were used for comparison; PDE5 and PDE11 gene expression was evaluated in human fetal skeletal muscle cells. RTq-PCR, PCR, western blot, free fatty acid assay commercial kit, and lipid stain non-fluorescent assay were used. Tadalafil upregulated I-targeted investigated genes with the same temporal pattern as I (GLUT4, PPARγ, and IRS-1 at 3 h; HK2, KIF1C, KIFAP3 at 12 h), re-localized GLUT4 in cell sites positively immune-decorated for Caveolin-1 and Flotillin-1, suggesting the involvement of lipid rafts, induced specific residue phosphorylation of IRS-1/AKT/mTOR complex in association with free fatty acid de novo synthesis. Sildenafil or GMP analog did not affect GLUT4 trafficking or free fatty acid levels. In human fetal skeletal muscle cells tadalafil likely favors energy storage by modulating lipid homeostasis via IRS-1-mediated mechanisms, involving activation of I-targeted genes and intracellular cascade related to metabolic control. Those data provide some biomolecular evidences explaining, in part, tadalafil-induced favorable control of human metabolism shown by clinical studies.

Testosterone insulin-like effects: an in vitro study on the short-term metabolic effects of testosterone in human skeletal muscle cells

PurposeTestosterone by promoting different metabolic pathways contributes to short-term homeostasis of skeletal muscle, the largest insulin-sensitive tissue and the primary site for insulin-stimulated glucose utilization. Despite evidences indicate a close relationship between testosterone and glucose metabolism, the molecular mechanisms responsible for a possible testosterone-mediated insulin-like effects on skeletal muscle are still unknown.MethodsHere we used undifferentiated proliferating or differentiated human fetal skeletal muscle cells (Hfsmc) to investigate the short-term effects of testosterone on the insulin-mediated biomolecular metabolic machinery. GLUT4 cell expression, localization and the phosphorylation/activation of AKT, ERK, mTOR and GSK3β insulin-related pathways at different time points after treatment with testosterone were analyzed.ResultsIndependently from cells differentiation status, testosterone, with an insulin-like effect, induced Glut4-mRNA expression, GLUT4 protein translocation to the cytoplasmic membrane, while no effect was observed on GLUT4 protein expression levels. Furthermore, testosterone treatment modulated the insulin-dependent signal transduction pathways inducing a rapid and persistent activation of AKT, ERK and mTOR, and a transient inhibition of GSK3β. T-related effects were shown to be androgen receptor dependent.ConclusionAll together our data indicate that testosterone through the activation of non-genomic pathways, participates in skeletal muscle glucose metabolism by inducing insulin-related effects.

Insulin-like effect of the phosphodiesterase type 5 inhibitor tadalafil onto male human skeletal muscle cells.

Phosphodiesterase type 5 inhibitors (PDE5i), widely used to treat male erectile dysfunction, seem to counteract insulin resistance (IR) in animals and humans. IR, primarily manifest in peripheral tissues and particularly in skeletal muscle, is due to impaired insulin signal transduction. Investigators have been focusing onto intracellular defects responsible for IR to identify suitable pharmacological tools targeted toward the specific defects. Albeit some effects of PDE5i have been reported onto animal muscular tissues or cells, whether and how they might affect metabolic processes directly in human skeletal muscle still remains unclear. We aimed to investigate in human fetal skeletal muscle cells (Hfsmc) the effect of tadalafil, one of PDE5i, onto some intracellular factors involved in response to insulin, such as ras-raf mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt), glycogen synthase kinase 3β (GSK-3β), and the transcriptional factor c-Myc; proliferation rate; lactate (lact) and free fatty acid (ffa) release; activity of citrate synthase (CS) and succinate dehydrogenase (SDH), both enzymes of Kreb's cycle; PDE5 gene expression. Western blot analysis, enzyme-linked immunosorbent assay, enzymatic assays, cell count, MTT assay and Real Time PCR were performed in Hfsmc with and without tadalafil. In Hfsmc tadalafil affected the insulin-related intracellular cascade, by increasing MAPK, PKB/Akt, GSK-3β phosphorylation and c-Myc expression. ffa release and CS activity also significantly increased, with no changes in SDH activity and lact release. Tadalafil, like insulin, targeted part of the machinery dedicated to energy management and metabolic control in human skeletal muscle cells.

Inflammatory response in human skeletal muscle cells: CXCL10 as a potential therapeutic target

Inflammatory myopathies (IMs) are systemic diseases characterized by a T helper (Th) 1 type inflammatory response and cell infiltrates within skeletal muscles. The mainstay of treatment is drugs aimed at suppressing the immune system – corticosteroids and immunosuppressants. About 25% of patients are non-responders. Skeletal muscle cells seem actively involved in the immune-inflammatory response and not only a target; understanding the molecular bases of IMs might help drug development strategies. Within muscles the interaction between the chemokine interferon (IFN)γ inducible 10 kDa protein, CXCL10 or IP-10, and its specific receptor CXCR3, present on Th1 type infiltrating cells, likely plays a pivotal role, potentially offering the opportunity for therapeutic intervention. We aimed to clarify the involvement of human skeletal muscle cells in inflammatory processes in terms of CXCL10 secretion, to elucidate the engaged molecular mechanism(s) and, finally, to evaluate muscular cell responses, if any, to some immunosuppressants routinely used in IM treatment, such as methylprednisolone, methotrexate, cyclosporin A and Infliximab. We first isolated and characterized human fetal skeletal muscle cells (Hfsmc), which expressed the specific lineage markers and showed the competence to react in the context of an in vitro alloresponse. CXCL10 protein secretion by Hfsmc was similarly induced by the inflammatory cytokines interferon (IFN)γ and tumor necrosis factor (TNF)α, above undetectable control levels, through the activation of Stat1 and NF-kB pathways, respectively; CXCL10 secretion was significantly magnified by cytokine combination, and this synergy was associated to a significant up-regulation of TNFαRII; cytokine-induced CXCL10 secretion was considerably affected only by Infliximab. Our data suggested that human skeletal muscle cells might actively self-promote muscular inflammation by eliciting CXCL10 secretion, which is known to amplify Th1 cell tissue infiltration in vivo. In conclusion, we sustain that pharmacological targeting of CXCL10 within muscular cells might contribute to keep in control pro-Th1 polarization of the immune/inflammatory response.

Human muscular fetal cells: a potential cell source for muscular therapies

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.

Immunocytochemical colocalization of desmin and vimentin in human fetal skeletal muscle cells.

Desmin and vimentin are the major intermediate filaments in muscle. They have been extensively studied in animal experiments. This study is the first to identify the distribution and to analyse the correlation of desmin and vimentin in human fetal skeletal muscle. Vimentin might be replaced by or transformed into desmin during myogenesis in chick embryo, although the precise process remains to be elucidated. The aim of this report is to evaluate the ratio of desmin to vimentin in human fetal muscle. By double-labeling immunoelectron microscopy, desmin and vimentin intermediate filaments were localized in developing skeletal muscles of 20-29-week-old human fetuses. The ratio of desmin and vimentin was analyzed statistically. Two sizes of colloidal gold particles, 5 nm (vimentin) and 10 nm (desmin), were distributed along the intermediate filaments. The commonest distance between gold particles was approximately 40-50 nm. Desmin and vimentin labeled with gold particles were arranged very close together in the same intermediate filament. The ratio of vimentin to desmin varied but the amount of vimentin decreased progressively from the undifferentiated myoblast to the differentiated myocytes. As the fetuses developed, desmin increased and vimentin decreased. Desmin and vimentin intermediate filaments were identified in the intermyofibrils of differentiated myocytes, in subsarcolemmal space, and in myoblast. Desmin and vimentin were colocalized in the same intermediate filaments. More vimentin existed in the less differentiated myocytes, although a small number of desmin filaments were already found in undifferentiated myoblasts. These intermediate filaments may not only connect myofibril bundles, cell organelles, and cell membrane but also provide a basis for myofibrillogenesis that is similar to relation between connective fibers and parenchymal cells.

Analysis of Fibronectin and Vitronectin Receptors on Human Fetal Skeletal Muscle Cells upon Differentiation

The role of fibronectin (FN) and vitronectin (VN) receptors for cell adhesion and matrix assembly was analyzed during human fetal myogenesis in vivo and in vitro. In human fetal muscle at 10 weeks gestational age FN and laminin are present in the extracellular matrix. Analysis of the integrin repertoire at this developmental stage reveals that the differentiated muscle cells in vivo express α5 and α6 integrins, but not αv, α1, and α3 integrins. However, in vitro cultured myoblasts (G6) isolated from the same gestational age express αv, α1, and α3 integrins in addition to α5 and α6 integrins. A more detailed analysis of FN and VN receptors in vitro shows that the localization of different αv heterodimers into focal contacts is differently regulated. αvβ1 and αvβ3 are present at focal contacts throughout in vitro myogenesis whereas αvβ5 appears to depend on an endogenously produced factor to localize to focal contacts. The αvβ1, αvβ5, and α3β1 heterodimers, often reported not to focalize, did form focal contacts in G6 cells, indicating that these myoblasts possess components facilitating the formation of cytoskeletal linkages containing these integrins. α5β1 colocalized with FN in myoblast cultures, whereas myotubes lacked both FN and α5β1 on the cell surface. In summary, we show that concomitant with in vitro differentiation of G6 cells, FN matrix contacts are abolished, but vitronectin receptors continue to fulfill an anchoring function during the differentiation process in vitro. Further studies are needed to assess the relative importance of the FN and VN binding integrins for the differentiation process in comparison with the laminin-binding integrins α6 and α7, also present on these cells.

Colocalization of Nestin and Vimentin/Desmin in Skeletal Muscle Cells Demonstrated by Three-Dimensional Fluorescence Digital Imaging Microscopy

During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggests that charged amino acid residues in the α-helical rod domain may play a role in the interaction.

Golgi apparatus: distinct structure of acid phosphatase localization in regenerating human skeletal muscle fiber.

The localization of acid phosphatase activity has been investigated ultrastructurally in regenerating human fetal skeletal muscle cells. The heavy reaction product was observed in the cisternae of Golgi apparatus and lysosomal vesicles, whereas the slight reaction product was also occasionally found in the nuclear envelope, endoplasmic reticulum and sarcoplasmic reticulum of the regenerating myocytes. The enzymatic basis for these deposits was confirmed by their absence in the incubation of the specimen in substrate-free medium and the enzyme inactivation of the specimen by exposure to NaF or heat.