Simultaneous measurement of single SL and [Ca²⁺]_i in neonatal myocytes under electric field stimulation. (A, top) Epi-illumination image of an AcGFP-expressing myocyte loaded with Fluo-4 (image taken at rest); clip from Videos 8 and 9. Sarcomeres in the yellow outlined rectangle with numbers from 1 to 4 were used for the analysis. (middle) Enlarged view of the region of interest in the top image at peak shortening and peak lengthening with 1-Hz stimulation. (bottom) Plot profiles of the four sarcomeres at peak shortening and peak lengthening. Sarcomere numbers are indicated. (B) Time-dependent changes in SL and Fluo-4–dependent FI in an AcGFP-expressing neonatal cardiomyocyte. Sarcomere 3 in A was used for the analysis. The arrow indicates the onset of stimulation. (C) Relationship of stimulation frequency versus shortening time (top), lengthening time (middle), and SL amplitude (bottom). Data obtained at 1–5 Hz are summarized. Asterisks in red in the middle graph indicate a significant difference (P < 0.05) compared with the corresponding value in the top graph. (D) Relationship of stimulation frequency versus diastolic FI/systolic FI. Gray line, diastolic FI/systolic FI = 0. (C and D) *, P < 0.05; #, P < 0.05; †, P < 0.05; and ‡, P < 0.05 compared with the corresponding values at 1, 2, 3, and 4 Hz, respectively. Data taken from 10 myocytes, with one plot obtained from sequentially connecting three sarcomeres. Error bars indicate means ± SEM. a.u., arbitrary units.