Expression of Trisk 51, agrin and nicotinic-acetycholine receptor epsilon-subunit during muscle development in a novel three-dimensional muscle-neuronal co-culture system.

Abstract

The purpose of our study was to create functional muscle tissue in vitro and to investigate the influence of organotypic neuronal slice cultures from rat spinal cord on the differentiation and function of primary rat myoblasts in a novel three-dimensional culture system. Three-dimensional muscle-neuronal cultures were established by co-cultivating primary rat skeletal muscle cells of newborn rats with organotypic slice cultures of the spinal cord prepared from isogenic rats in a fibrin matrix. These constructs were cultured for up to 4 weeks. Differentiation and fusion of the myoblasts to myofibers was evaluated by analyzing the expression pattern and localization of muscle- and neuron-specific markers. The fibrin matrix provided a suitable environment for three-dimensional myoblast culture. Co-culturing of organotypic spinal cord slices with myoblasts induced the formation of spontaneously contracting multinuclear and parallel-aligned myofibers. Pharmacological tests suggested the formation of neuromuscular junctions. The analysis of neural agrin expression and myogenic desmin, myogenin, MyoD, Trisk 51, and nicotinic-acetycholine receptor (nACh-receptor) epsilon-subunit expression revealed the differentiation of the myoblasts to myofibers. The presented novel three-dimensional co-culture system allows the in vitro investigation of myoblast differentiation and neuron-myoblast interaction. Our results suggest the existence of an alternative pathway for the maturation of the nAChR gamma-subunit to the epsilon-subunit without neural agrin activity.