Isolation of neural precursor cells from skeletal muscle tissues and their differentiation into neuron-like cells

Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.     

Spatiotemporally controlled contraction of micropatterned skeletal muscle cells on a hydrogel sheet

We have developed gel sheet-supported C(2)C(12) myotube micropatterns and combined them with a microelectrode array chip to afford a skeletal muscle cell-based bioassay system. Myotube line patterns cultured on a glass substrate were transferred with 100% efficiency to the surface of fibrin gel sheets. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT). We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell.     

Fabrication of random and aligned electrospun nanofibers containing graphene oxide for skeletal muscle cells scaffold

Graphene oxide (GO)‐based materials have been explored in biomedical applications as active engineered materials for diagnosis and therapy. Although a large number of studies have been carried out in the last years, aspects involving the orientation and elongation of cells on GO immobilized on polymeric nanofibers are still scarce. We investigated the interactions between skeletal muscle cells and GO immobilized on random and aligned electrospun nanofibers of poly(caprolactone) (PCL), a biocompatible and biodegradable polymer. Oxygen plasma was employed to modify the nanofiber polymer surface to enhance the interactions between the PCL fibers and GO. Scanning electron microscopy and confocal microscopy revealed the morphology and orientation of skeletal muscle cells (C2C12 cells) on random and aligned GO/PCL nanofibers. The approach employed here is useful to investigate the interaction of skeletal muscle cells with biocompatible polymer nanofibers modified with GO intended for cell scaffolds and tissue engineering.     

Membrane dynamics of differentiating cultured embryonic chick skeletal muscle cells by fluorescence microscopy techniques

Changes in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FPR), with the use of a lipid-soluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.     

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.     

5-Hydroxytryptamino-induced calcium sparks in cultured rat stomach fundus smooth muscle cells

With the imaging fluorescence probe of Ca2+ (fluo-3) and a laser scanning confocal micro-scope, the spontaneous localized calcium release event was first discovered in resting rat cardiac myocytes by Cheng[1] in 1993. A mathematical simulation is developed with computer in order to reveal the effect, which is immediately suggested that these events are likely to reflect the local-ized release of Ca2+ from a small cluster of ryanodine-sensitive Ca2+ release channels in sar-coplasmic reticulum …     

Purification of rabbit skeletal muscle troponin C

Troponin C binds to phenyl-Sepharose in the presence of Ca2+ and can be eluted with EDTA. This property was used as an essential step in the purification of this protein from rabbit skeletal muscle. Troponin C was extracted with 6M urea from extensively washed ground muscle. The protein was bound to and eluted from DEAE-Sephadex, fractionated by size on Sephadex G75, and in a final step purified from UV-absorbing non-protein impurities on phenyl-Sepharose. The total yield of electrophoretically pure protein was 60 mg per 100 g of muscle, which is considerably higher than that previously obtained.     

Low-level laser therapy improves skeletal muscle performance, decreases skeletal muscle damage and modulates mRNA expression of COX-1 and COX-2 in a dose-dependent manner

We tested if modulation in mRNA expression of cyclooxygenase isoforms (COX-1 and COX-2) can be related to protective effects of phototherapy in skeletal muscle. Thirty male Wistar rats were divided into five groups receiving either one of four laser doses (0.1, 0.3, 1.0 and 3.0 J) or a no-treatment control group. Laser irradiation (904 nm, 15 mW average power) was performed immediately before the first contraction for treated groups. Electrical stimulation was used to induce six tetanic tibial anterior muscle contractions. Immediately after sixth contraction, blood samples were collected to evaluate creatine kinase activity and muscles were dissected and frozen in liquid nitrogen to evaluate mRNA expression of COX-1 and COX-2. The 1.0 and 3.0 J groups showed significant enhancement (P < 0.01) in total work performed in six tetanic contractions compared with control group. All laser groups, except the 3.0 J group, presented significantly lower post-exercise CK activity than control group. Additionally, 1.0 J group showed increased COX-1 and decreased COX-2 mRNA expression compared with control group and 0.1, 0.3 and 3.0 J laser groups (P < 0.01). We conclude that pre-exercise infrared laser irradiation with dose of 1.0 J enhances skeletal muscle performance and decreases post-exercise skeletal muscle damage and inflammation.     

Electrophoretic analysis of electrically trained skeletal muscle.

Sheep latissimus dorsi muscle was electrically trained, thereby inducing fast-to-slow fibre-type transformation. Using a combination of one- and two-dimensional gel electrophoresis techniques with computer analysis, we have analysed altered expression of contractile protein isoforms at protein and mRNA level over a time course of electrical training extending to 5 months. Myosin heavy chain and regulatory myosin light chain analysis showed predominant expression of their slow isoforms (86% and 92%, respectively) after 3 months of training. At the same time point, however, tropomyosin analysis revealed that the slow isoform of the alpha-subunit accounted for 64% of the total alpha expression. Troponin T isoform switching proceeded more slowly over the same time course than tropomyosin and the thick filament proteins studied. Troponin T analysis revealed 5 fast and 2 slow isoforms in the sheep, of which the second slow isoform only became clearly visible after 5 months' training. At this time point the two slow isoforms were more predominant than their fast counterparts. This suggests that a wide heterogeneity of fast and slow isoform combinations are possible in the thin filament of skeletal muscle.     

Preparation and analysis of heart and skeletal muscle specimens with LAMMA

Summary A technique is described to obtain shockfrozen tissue specimens from a beating heart in situ using liquid propane as cooling agent. The method allows to take up to 8 samples from the ischemic area of the left ventricle of a pig heart following acute coronary artery occlusion.A freeze-drying apparatus was developed in which, under a vacuum of 10^–6 mm Hg, the specimens remain for 10 days with the temperature slowly rising from initially –100 C to room temperature. Freezing damages in the tissues samples due to the formation of ice crystals either during shock-freezing or freeze-drying are usually below the spatial resolution of the microscope system employed in the LAMMA instrument. Already after a few minutes of ischemia, a considerable decrease of the intracellular K/Na relation was measured. LAMMA spectra from skeletal muscle were taken to check for possible ion redistribution which had eventually occurred during the various steps of the preparation procedure employed.Supported by Deutsche Forschungsgemeinschaft, SFB 68, A 10     

Calsequestrin: a well-known but curious protein in skeletal muscle

Calsequestrin (CASQ) was discovered in rabbit skeletal muscle tissues in 1971 and has been considered simply a passive Ca²⁺-buffering protein in the sarcoplasmic reticulum (SR) that provides Ca²⁺ ions for various Ca²⁺ signals. For the past three decades, physiologists, biochemists, and structural biologists have examined the roles of the skeletal muscle type of CASQ (CASQ1) in skeletal muscle and revealed that CASQ1 has various important functions as (1) a major Ca²⁺-buffering protein to maintain the SR with a suitable amount of Ca²⁺ at each moment, (2) a dynamic Ca²⁺ sensor in the SR that regulates Ca²⁺ release from the SR to the cytosol, (3) a structural regulator for the proper formation of terminal cisternae, (4) a reverse-directional regulator of extracellular Ca²⁺ entries, and (5) a cause of human skeletal muscle diseases. This review is focused on understanding these functions of CASQ1 in the physiological or pathophysiological status of skeletal muscle.Subject terms: Physiology, Calcium and vitamin D, Diseases     

Novel nitric oxide microsensor and its application to the study of smooth muscle cells

A novel sensitive, selective and stable nitric oxide (NO) microsensor is described, which is modified by nano Au colloid and Nafion. As determined by atomic forced microscopy (AFM), the diameter of Au colloid particles is from 7 to 14 nm. The detection of NO is based on the nano Au particles catalysis of NO oxidation at an anodic potential of +0.74 V (versus saturated calomel electrode (SCE)). The microsensor showed a low detection limit, high selectivity and sensitivity for NO determination. The oxidation current (measured by differential pulse amperometric technique) was linear with NO concentration ranging from 1.0×10⁻⁷ to 4.0×10⁻⁵ mol/l with a calculated detection limit of 5.0×10⁻⁸ mol/l (S/N=3). Using the microsensor, the direct real time production of NO in the smooth muscle cells was continuously measured, which showed the NO levels was increased by stimulating with l-arginine (l-Arg), acetylcholine (Ach) and a self-made flavonoid medicine.     

Effect of phalloidin on the skeletal muscle ADP-actin filaments

The effect of phalloidin on the thermal stability of skeletal actin filaments polymerized from ADP-binding monomers was investigated with the method of differential scanning calorimetry. Phalloidin shifted the melting temperature of the ADP-F-actin from 59.1±1.0 to 80.0±1.2°C. The stabilizing effect of phalloidin propagated cooperatively along the filament. The cooperativity factor according to the applied model was 1.07±0.11. With these measurements it was possible to demonstrate that the binding of phalloidin has lower influence on the adjacent protomers in ADP- (k=1) than in ATP-actin filaments (k=3).     

Exercise type and muscle fiber specific induction of caveolin-1 expression for insulin sensitivity of skeletal muscle

It is well known that exercise can have beneficial effects on insulin resistance by activation of glucose transporter. Following up our previous report that caveolin-1 plays an important role in glucose uptake in L6 skeletal muscle cells, we examined whether exercise alters the expression of caveolin-1, and whether exercise-caused changes are muscle fiber and exercise type specific. Fifty week-old Sprague Dawley (SD) rats were trained to climb a ladder and treadmill for 8 weeks and their soleus muscles (SOL) and extensor digitorum longus muscles (EDL) were removed after the last bout of exercise and compared with those from non-exercised animals. We found that the expression of insulin related proteins and caveolins did not change in SOL muscles after exercise. However, in EDL muscles, the expression of insulin receptor beta (IR beta) and glucose transporter-4 (GLUT-4) as well as phosphorylation of AKT and AMPK increased with resistance exercise but not with aerobic exercise. Also, caveolin-1 and caveolin-3 increased along with insulin related proteins only in EDL muscles by resistance exercise. These results suggest that upregulation of caveolin-1 in the skeletal muscle is fiber specific and exercise type specific, implicating the requirement of the specific mode of exercise to improve insulin sensitivity.     

Rab5 in the regulation of cell motility and invasion

Cellular invasion requires careful regulation of the cell migration and apoptotic signaling cascades, allowing cell movement and survival of the emigrating populations. Components of the endosomal machinery are involved in these processes, and in particular the role of small GTPases of the Rab family has become appreciated. Rab5 is best known for its role in regulating the trafficking of early endosomes, however, it has recently been appreciated to associate with and regulate the routing of complexes containing integrins, the primary cellular receptors for the extracellular matrix. The association regulates the spatio temporal activation of signals of downstream growth factors and integrins. Rab proteins have also been linked to apoptosis mediated by cell surface death receptors, which elicit the activation of the death cascade via activation of caspase 8. Recently, the link between trafficking, apoptosis and cell migration was strengthened, as Rab5 was determined to work in conjunction with caspase 8 in promoting tumor cell motility and metastasis by regulating β1 integrin traffic. The capacity to connect and regulate these pathways identifies Rab5 as a key player in future studies of cell migration and tumor dissemination.