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Abstract
Excitation-contraction coupling in muscle cells is initiated by a restricted membrane depolarization delimited within the neuromuscular junction. This targeted depolarization triggers an action potential that propagates and induces a global cellular calcium response and a consequent contraction. To date, numerous studies have investigated this excitation-calcium response coupling by using different techniques to depolarize muscle cells. However, none of these techniques mimic the temporal and spatial resolution of membrane depolarization observed in the neuromuscular junction. By using optogenetics in C2C12 muscle cells, we developed a technique to study the calcium response following membrane depolarization induced by photostimulations of membrane surface similar or narrower than the neuromuscular junction area. These stimulations coupled to confocal calcium imaging generate a global cellular calcium response that is the consequence of a membrane depolarization propagation. In this context, this technique provides an interesting, contactless and relatively easy way of investigation of calcium increase/release as well as calcium decrease/re-uptake triggered by a propagated membrane depolarization.
Highlights
Motor neuron inputs trigger electrical activity of muscle fibers through the neuromuscular junction[1,2,3]
Excitation/calcium release coupling, which eventually leads to contraction, is a central mechanism of muscular function and is widely studied in the physiological and physiopathological context
C2C12 myotubes have previously been investigated as a model for muscle excitation-contraction coupling in several studies[24, 25]
Summary
Motor neuron inputs trigger electrical activity of muscle fibers through the neuromuscular junction[1,2,3]. Several methods are commonly used to investigate excitation/calcium release coupling in vitro: patch-clamp technique, stimulation by means of an electric field using external electrodes[13,14,15], superfusion of a depolarizing high-concentrated potassium solution[11, 12, 16] and focal pressure application (puff) of acetycholine through a glass capillarie[17,18,19] Most of these techniques display some weaknesses in their spatial and temporal resolution of cell depolarization properties. Stimulation with a high extracellular potassium solution depends on the superfusion kinetics (and spatial diffusion), which is very difficult to control and provides longer depolarization as compared to action potentials generated through the activation of a motor end-plate This low temporal and spatial resolutions do not allow studying precisely both intracellular calcium rise and decrease initiated by membrane depolarization. The relationship between optical stimulation and calcium homeostasis in these cells has never been investigated yet
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