Abstract
We assessed the feasibility of using light-activated channels and pumps to modulate the excitability of adult skeletal muscle fibers. Fibers were enzymatically isolated from FDB muscles of the mouse and studied under current- or voltage-clamp conditions using a two-microelectrode amplifier. Fibers were kept in Tyrode at a resting (or holding) potential of −90mV. Contraction was arrested by adding 40mM EGTA to the pipettes' solution. Plasmids coding channelrhodopsin-2-EYFP [CHR2; Addgene] and archaerhodopsin-3-ECFP [ARCH; subcloned from pFCK-ARCH-CFP (Addgene) into pECFPN1], were transfected into FDB fibers via in vivo electroporation. Expression was evaluated 7-10 days later. CHR2 and ARCH were excited with the 488nm and 514nm laser lines from an argon laser. Light power was controlled with an acousto-optic modulator, and focused onto selected areas (10-20μm) of the fibers via optic fibers. TPLSM imaging showed that CHR2 and ARCH were targeted to t-tubules and surface membranes. In fibers expressing CHR2, 1-3ms light pulses of increasing power elicited graded transient depolarizations until an action potential (AP) was triggered. The amplitude and FDHM of light-triggered APs were similar to those in response to current pulses. At all powers used, illuminating non-transfected fibers had no effect. Repetitive (<10Hz) pulsed illuminations elicited trains of APs with no failures. Failures at higher frequencies could be reduced by increasing the muscle fiber's input resistance (e.g. by blocking the chloride conductance with 9-AC), and/or increasing the illumination power. In voltage-clamped fibers, 40ms light pulses elicited slowly decaying inward currents with amplitudes graded with the applied illumination power. When fibers expressing ARCH were illuminated (at 514nm) otherwise supra-threshold current pulses became ineffective to trigger APs. Our results demonstrate that optogenetic tools can be used to excite or depress the excitability of adult skeletal muscle fibers.Supported by NIH/NIAMS grants AR047664, AR041802, and AR054816.
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