Functional maturation of nicotinic acetylcholine receptors as an indicator of murine muscular differentiation in a new nerve-muscle co-culture system

Abstract

Under normal conditions in situ, muscle fibers and motoneurons, the main partners of motor units, are strongly dependent on each other. This interdependence hinders ex vivo studies of neuromuscular disorders where nervous or muscular components are considered separately. To allow in vitro access to complex nerve-muscle relationships, we developed a novel nerve-muscle co-culture system where mouse muscle innervation is assured by rat spinal cord explants. The degree of muscular maturation during co-culture was evaluated using the distribution of nicotinic acetylcholine receptors (AChRs) and their electrophysiological characteristics before and after innervation. In myotubes from non-innervated cultures, AChRs were diffusely distributed over the entire myotube surface. Their single-channel conductance (33.5+/-0.6 pS) and mean open time (8.1+/-0.7 ms) are characteristic of AChRs described in embryonic or denervated skeletal muscles. In innervated muscle fibers from co-cultures, AChRs appear as discrete aggregates and co-localize with synaptotagmin. In addition to the embryonic type currents, in innervated fibers AChR currents having high conductance (53.3+/-5.9 pS) and short mean open time (2.6+/-0.1 ms), characteristic of AChRs at mature neuromuscular junctions, were observed. Our data support the use of this new nerve-muscle co-culture system as a reliable model for the study of murine muscular differentiation and function.

Regeneration of skeletal muscle

“…Furthermore, Gregorevic et al (2004) have demonstrated that these regenerating fibers adopt their fast and slow twitch properties as they regenerate (Gregorevic et al 2004). Studies investigating the innervation of skeletal muscle have, to date, been limited to in vitro co-cultures of myoblasts, satellite cells and neurons (Larkin et al 2006;Das et al 2007;Mars and Martincic 2001;Mehrke et al 1984;Wagner et al 2003). These studies have revealed that nerve-muscle constructs generate greater contractile force compared with muscle-only constructs.…”

Current opportunities and challenges in skeletal muscle tissue engineering

“…On muscle cells AChRs accumulate and are surrounded by neural extensions that harbour neurofilaments. These nerve-muscle constructs show spontaneous contractions and can also be stimulated (Wagner et al, 2003). Larkin et al showed in vitro that stimulated nerve-muscle constructs had better contractility characteristics than muscle-only constructs: muscle-only constructs produced a twitch force of 40 µN and a tetanic force of 95 µN, while stimulated nerve-muscle constructs produced a twitch force of 100 µN and a tetanic force of 200 µN (Larkin et al, 2006).…”

Inhibition of iPLA2β and of stretch‐activated channels by doxorubicin alters dystrophic muscle function

“…Myotubes were prepared from EDL‐MDX‐2 myoblasts as described previously (Wagner et al ., ; Basset et al ., ). Briefly, myoblasts were plated on collagen‐treated Petri dishes (Falcon; Becton Dickinson, Heidelberg, Germany) in a proliferation medium containing a 1:1 mixture of Dulbecco's modified minimal essential medium (DMEM; Invitrogen, Karlsruhe, Germany) with 4.5 g·L −1 glucose and MCBD202 (supplemented with NaHCO 3 and 1% v/v Glutamax II x100; Gibco, Paisley, UK), 10% FBS (Invitrogen, Zug Switzerland), 1% Ultroser SF (BioSepra, Saint‐Christophe, Cergy, France) and 10 μg·mL −1 ciprofloxacin (Bayer, Leverkusen, Germany).…”