Interrogation of skeletal muscle activation in an intact system via a myofilament incorporated FRET biosensor.

Abstract

It is important to understand the mechanisms involved in regulating skeletal muscle activation and contraction for advancing insights into acquired and inherited muscle disorders and for identifying potential targets for therapeutics. The field has elucidated many sarcomeric components needed for activation of contraction. We seek to expand this work using a fluorescent sarcomere biosensor that allows for monitoring of sarcomeric activation in live, intact skeletal muscles. This biosensor fuses fluorescent donor and acceptor proteins to the N- and C-terminals of fast skeletal troponin C protein, respectively. Using the biosensor, skeletal muscle activation is monitored in real time through Förster Resonance Energy Transfer (FRET) fluorescence. This system maintains all essential, physiological features of the muscle, including excitation-contraction coupling and load, which was not possible in reconstituted systems. Using a transgenic mouse line with the biosensor, we have demonstrated FRET fluorescence in intact muscles from transgenic animals, with the time to peak of the fluorescence preceding the force development and the biosensor kinetics uncoupled from intracellular calcium kinetics. Of note, the FRET signal has a prolonged return to baseline as compared to force in intact EDL muscles, something that is not observed in unloaded FDB myofibers, and which may be evidence of a “primed state” of myofilament activation. The “primed state” is uncoupled from Ca2+ and is modified using fast skeletal muscle specific small molecules that target either the thin filament or thick filament. As thick filament modulating small molecules could be detected by the TnC localized biosensor during single twitch contractions, this demonstrates intermyofilament signaling in live muscle. Additionally, the “primed state” duration can be impacted through varying stimulation protocols, including dual pulse stimulations. This unique phenomenon will be further investigated using mechanical and pharmacological manipulations.