Abstract Body: The sarcomere is the functional unit of striated muscle contraction. Previous studies
have provided valuable information about the sarcomeric proteins and modulators that
are necessary for activation of contraction through inter-myofilament signaling. This
work has revealed key differences in contraction kinetics and paradigms between
cardiac, slow skeletal, and fast skeletal muscle. We seek here to expand these studies
through the use of myofilament incorporated Förster Resonance Energy Transfer
(FRET) fluorescent biosensor in live, intact muscle. This biosensor fuses fluorescent
donor and acceptor proteins to the N- and C-terminals of fast skeletal and cardiac (slow)
troponin C, respectively, and allows for the monitoring in real-time of the stepwise
physiological mechanism of muscle sarcomere activation in an intact system which
preserves significant features of the muscle, including excitation-contraction coupling
and load. This biosensor has been designed and validated in both cardiac and skeletal
muscle and using intact muscles from transgenic animals we have demonstrated FRET
fluorescence in cardiac papillaries, extensor digitorum longus, and soleus muscles. This
biosensor has shown unique sarcomere activation properties between cardiac and
skeletal muscle, notably the emergence of a prolonged return to baseline of the FRET
signal in loaded fast and slow skeletal muscle, which is evidence of a “primed state” of
myofilament activation unique to skeletal muscles under load. Elucidation of the “primed
state” of sarcomere activation is evidence of a transient memory of recent activity
enabling the enhanced contraction properties of skeletal muscle. This finding advances
previous understanding of skeletal muscle activation, including summation and tetanic
contraction, which is not possible in cardiac muscle. The implementation of this
biosensor provides a new approach to interrogate the essential physiological features of
muscle sarcomere activation and contraction and adds a valuable new tool for small
molecule and gene-based discoveries for muscle disease remediation.