A, Representative single cardiac myocyte traces of simultaneous sarcomere length, and the Clover, and mRuby2 flurorescence transients, with Clover fluorescence decreasing and simultaneously mRuby2 fluorescence increasing, indicating FRET. Scale bar represents a 5% change from baseline. B, Amplitude normalized Clover (green) and sarcomere length (black) dynamics from ensemble averaged myocytes (transients normalized from 0 to 100% to compare kinetics) show TnC biosensor Clover transient preceding sarcomere length shortening, inset shows the same data, highlighting the peak timing differences. C, Summary statistics from sarcomere length dynamics and Clover intensity show that peak activation of the TnC biosensor precedes sarcomere length change, and biosensor inactivation follows relaxation of the myocyte. Myocytes are unloaded and measured at 37°C with 0.2Hz stimulation, n=14 myocytes in each group. Mean ± S.E.M. are presented, unpaired two-tailed t-test: *P < 0.05.