Neural Agrin Changes the Electrical Properties of Developing Human Skeletal Muscle Cells

Abstract

Recent investigations suggest that the effects of neural agrin might not be limited to neuromuscular junction formation and maintenance and that other aspects of muscle development might be promoted by agrin. Here we tested the hypothesis that agrin induces a change in the excitability properties in primary cultures of non-innervated human myotubes. Electrical membrane properties of human myotubes were recorded using the whole-cell patch-clamp technique. Cell incubation with recombinant chick neural agrin (1 nM) led to a more negative membrane resting potential. Addition of strophanthidin, a blocker of the Na(+)/K(+) ATPase, depolarized agrin-treated myotubes stronger than control, indicating, in the presence of agrin, a higher contribution of the Na(+)/K(+) ATPase in establishing the resting membrane potential. Indeed, larger amounts of both the alpha1 and the alpha2 isoforms of the Na(+)/K(+) ATPase protein were expressed in agrin-treated cells. A slight but significant down-regulation of functional apamin-sensitive K(+) channels was observed after agrin treatment. These results indicate that neural agrin might act as a trophic factor promoting the maturation of membrane electrical properties during differentiation, confirming the role of agrin as a general promoter of muscle development.

Acetylcholinesterase and agrin: Different functions, similar expression patterns, multiple roles

“…Common features of AChE and agrin extend to their potential to have, besides their respective cholinergic and synaptogenetic roles, also various other functions unrelated to NMJ. We have already demonstrated AChE involvement in myoblast apoptosis [18] and the stimulatory effects of agrin 8 on the maturation of e-c coupling [19] and membrane excitability [20]. The effects of agrin demonstrated in this study support the concept that agrin promotes myogenesis at various levels and that its otherwise undisputable synaptogenetic effect is just one in the spectrum of its myogenetic roles.…”

“…In accordance with the reports of Park et al [15], Zhang et al [16] and Toiber et al [17] we already demonstrated participation of AChE in myoblast apoptosis [18]. We also reported stimulatory actions of agrin on the maturation of the excitation-contraction (e-c) coupling mechanism [19] and membrane excitability [20] in human myotubes in vitro. Here, we report new results of the experiments in which we tested the influence of agrin on the maturation of the e-c coupling and on the maturation of the myosin isoforms.…”

“…Detailed description of the experimental techniques were provided before [19,20]. Briefly, Ca 2+ responses elicited by 40 mM caffeine and expression of voltage-dependent Ca 2+ channels (T-and L-type) was measured by patch-clamp.…”

Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system

“…Nevertheless, these systems have been utilized in multiple applications. For instance, they have been employed to study the functional integrity of MNs differentiated from human stem cells by testing their capability of innervating muscle [23, 27], to investigate the functional maturation process of SKMs derived from human SKM stem cells/progenitors in the co-culture with rat spinal explants [59-61], the mechanisms of NMJ formation on human SKMs [30-34, 55, 56, 59-63], and the pathogenesis of some spinal muscular diseases [64]. The system developed in this study, by the co-culture of human stem cell-derived MNs and SKMs, provides a model closer to the human condition that is capable of addressing not only all the above issues, but also those associated with neurological and/or muscular disease modeling, drug discovery and regenerative medicine for neuromuscular diseases or injuries.…”

Nestin- and Doublecortin-Positive Cells Reside in Adult Spinal Cord Meninges and Participate in Injury-Induced Parenchymal Reaction

“…Whole-cell patch-clamp recordings were performed in 15 cells after 30 days of in vitro neuronal differentiation as previously described [19]. For details, see Supporting Information.…”