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Issue Date: 14-Mar-2003
Publisher: Università degli studi di Trieste
Cell implantation has been proposed as a possible strategy for muscle regeneration and a good therapy for muscle diseases (Partridge et al, 1978). Primary cells and in particular muscle satellite cells are among the best candidates for transplantation. The satellite cells are quiescent precursor cells located beneath the basai lamina of mature muscle fibres; they can proliferate generating myoblasts that fuse into multinucleated fibres for muscle growth or in response to :muscle injury (for review Seale & Rudnicki, 2000). During differentiation, myoblasts express specific proteins as myosin, tropomyosin and different ionic channels. Voltagedependent sodium channels and acetylcholine receptors channels (AChRs) are among the frrst channels to be expressed during in uitro myogenesis: the former are already expressed in rat quiescent satellite cells (Bader et al., 1988), whereas functional AChRs appear only in proliferating myoblasts (Cossu et aJ., 1987). At this developmental stage sodium channels are responsible of action potential generation whereas AChRs are found to be involved in myoblasts fusion (Krause et aZ., 1995). Severallines of evidence demonstrated that there are ionic channels involved in neuromuscular diseases, for example Myotonic Dystrophy (MD). MD is the most common inherited neuromuscular disease in adults, with a global incidence of l in 8000 individuals. MD is an autosomal dominant, multisystemic disorder characterised primarily by myotonia and progressive muscle weakness. The genetic defect is a trinucleotide (CTG) repeat expansion within the 3'-untranslated region of a protein kinase gene (DMPK) located on chromosome 19ql3.3 (for review Tapscott & Thornton, 2001). Severallines of evidence demonstrate that altered modulation of Na+ channels may play an important role in the pathogenesis of MD. (Chahine et al., 1997; Mounsey et al., 2000). The aim of this work was to analyse the properties of voltage-dependent sod.ium channels and AChR during in uitro myogenesis of murine satellite cells. The properties of these channels have been investigated in human satellite cells coming from healthy and MD donors. The last part of the work was dealing with the same property on healthy donor of different age. Materials And Methods Cell culture. Cultures of i28 cells were established from mouse satellite cells (Irintchev et al, 1997) The cells have been expanded in HAM'S F-10 med.ium containing 20% fetal calf serum (FCS), L-glutamine (2mM), penicillin (l 00 units/ml) and streptomycin (100 uM/ml). To induce cell d.ifferentiation and myotube fusion, the medium was replaced, l day after plating, with DMEM supplemented with 2% horse serum and L-glutamine, penicillin, and streptomycin. Human myoblasts were grown in HAM'S F-10 medium supplemented with 50 ug/ml gentamycine and 20% FCS. For myoblast d.ifferentiation, growth medium was replaced by DMEM containing 10 uM/ml insulin and 100 ug/ml transferrin. Cell nuclea were counted using DAPI staining; fusion index was expressed (in percentage) as the ratio between the number of nuclei in multinucleated myotubes and total number of nuclea, for each considered field in the Petri dish. Human satellite cells have been coded as follows: DSQ satellite cells coming from 29 week-old DM foetus, BQ29 satellite cells coming from 29 week-old foetus, 30/00 coming from 2 year-old donor and STR13 satellite cells coming from 21 year-old don or, Electrophysiological recording. Patch-clamp recordings were performed, at room temperature, in the whole-cell and cell-attached con:figurations using borosilicate glass pipettes. ACh currents: ACh-induced total currents were measured in the whole-cell con:figuration in the voltage-clamp mode. Bath solution (NES) contained (mM): NaCl 140, KCl 2.8, CaCh 2, MgCh 2, glucose 10, HEPES-NaOH 10, pH 7,3. Pipettes (3-5 MQ) were filled with the following solution (mM): KC1120, CaCh l, MgCh 2, EGTA 11, HEPES-KOH 10, pH 7.3. ACh (10 J.LM) was applied by a gravity-driven perfusion system. In the cell-attached con:figuration, pipettes ( -8 MQ) were filled with NES containing ACh 100 nM. Na+ current: total Na+ currents were measured in the whole-cell con:figuration in the voltage-clamp mode. The extracellular solution was (mM): 40 TEA, 2.8 KCI, 100 NaCl, 2 CaCh, 2 MgCh, 10 HEPES, 10 glucose, pH=7.3. The pipette solution was (mM): 120 CsCl, 11 EGTA, l CaCh, 2 MgCh, 10 HEPES, pH=7,3. Currents were recorded using on Axopatch 200; signals were fùtered at 2kHz with a Bessel fùter (-3bB) and transferred to hard disk using the Digi.Data 1200 interface. Total and single-channel currents were analyzed using the pClamp 6 software (Axon Instruments). Results And Conclusion ACHR AND VOLTAGE-DEPENDENT SODIUM CHANNELS PROPERTIES IN MURINE SATELLITE CELLS Murine satellite cells started to express functional ACbRs wben they were stili undifferentiated. ACb-induced mean total current increased during differentiation; after 2 days in differentiation medium ali the cells were responsive to ACb application. The kinetics and the biopbysical properties of single ACb cbannels reveal that these cells express the foetal type of ACbRs, as observed in immature skeletal muscle and denervated adult muscles. Sodium currents in differentiated myoblasts were analysed plating the cell at low density in order to reduce the myoblasts fusion. In this condition only 20% of the total nuclea belonged to multinucleated myotubes, wbereas most of the cells were mononucleated. Tetrodotoxin (TTX)-insensitive and TTX-sensitive sodium current bave been found in mononucleated differentiated myoblasts. No significant differences bave been found in the kinetic properties of sodium currents in the presence or absence of TTX. Murine satellite cells maintained in cell culture fused in electrically excitable multinucleated myotubes whic, after 5-6 days in differentiation medium, contracted spontaneously. Spontaneous action potentials recorded in current-clamp configuration were blocked by TTX. Preliminary results sbow that a-bungarotoxin (a specific blocker for ACbR) could modulate the action potential frequency. ACHR AND VOLTAGE-DEPENDENT SODIUM CHANNELS PROPERTIES IN HUMAN SATELLITE CELLS For the frrst time the properties of AChRs in MD human satellite cells have been investigated. AChRs expressed during in vitro myogenesis by human healthy and MD satellite cells differed in single channel conductance which was significantly lower in pathological condition. Different subunits arrangement mechanism could be suggested to explain such differend single channel conductance: Kullberg et al ( 1990) showed that when AChR with only a.J3 and y subunits were expressed in Xenopus oocytes, the channels had a lower conductance than native receptors. We can conclude that a defect in AChR conductance, during in vitro differentiation of MD satellite cells, could be at least partially responsible of the reduced fusion already observed in MD myoblasts (Furling et al., 2001). ITX-sensitive and TTX-insensitive sodium channels were both present and the total current density was lower in DM cells. As previously observed (Rudel et al., 1989), sodium channels expressed in MD cells were activated at more positive membrane potentials. A difference in the kinetics of activation could be explained by phosphorilation processes induced by kinase like DMPK. Finally regarding the different age of the donors of satellite muscle cells, we have up to now analysed the byphysical properties of AChR. Single channels properties are not different and resemble those of the fetal type of AChR. The amplitude of total current was also not related of the age of donor increasing in the same way during the processes of myogenesis. Further experiments are needed to demonstrate if AChR induced current could be, at least in part, responsible for the different differentiation capability already observed.
Type: Doctoral
Appears in Collections:PREGRESSO

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