Targeted Optogenetic Activation of Calcium Transients in Developing Skeletal Muscle Cells

Activation of calcium transients through the electrical stimulation of myotubes is a pre-requisite for advanced differentiation in developing skeletal muscle. This stimulation occurs early in the muscle development with the appearance of the neuromuscular junction (NMJ) that allows to depolarize cell membrane and to induce post-synaptic potentials. Consequent generated action potentials activate excitation-calcium release coupling mechanism also known to participate in the calcium-dependent differentiation pathways for the maturation of striated muscle cells. To date, depolarization-evoked intracellular calcium increases are mainly investigated in vitro using electrical field stimulation or high potassium solution perfusion. However, these methods are not representative of a spatiotemporal NMJ stimulation. Moreover, calcium signaling kinetics and local calcium increases depend on the stimulation approaches. Here we have devised a non-invasive experimental approach to enable genetically targeted photostimulation of developing skeletal muscle cells with fine temporal and spatial resolution. We applied optical stimulation to C2C12 myotubes, genetically engineered to express ChR2-GFP to investigate depolarization-dependent calcium increases initiated from a very small and localized area of light stimulation. We found that the optical stimulation of membrane area smaller than 1 µm2 allowed the depolarization of the whole cell membrane and activated depolarization-induced calcium increases in ChR2-expressing myotubes. Optical stimulation conditions (area, surface and light power), associated with the use of pharmacological blockers, were investigated to understand the required conditions for obtaining voltage-dependent calcium transients. Moreover, the analysis of localized calcium increases demonstrates very different kinetics and amplitude of increases in different subcellular compartments. These results suggest that local optical stimulation, that mimics motor neurons inputs, allows to investigate finely the consequent calcium increases and provides new results about calcium homeostasis in subcellular compartments.