SL changes during the cardiac cycle in vivo. (A) Data showing the time course of changes in SL during the cardiac cycle: i.e., diastole (i), systole (ii), and diastole (iii). Five consecutive sarcomeres in the yellow rectangular outline in the myocyte in B (at [i] 151, [ii] 271, and [iii] 351 ms, as indicated by arrows in the graph) were analyzed. The sarcomeres analyzed in systole differed from those in diastole (hence indicated by different numbers, i.e., 6–10), because of movements of the myocyte associated with heartbeat. Blue triangles indicate average values. Error bars represent mean ± SD (see Materials and methods). Systolic LVP was 49 mmHg, and HR was 251 bpm (both average values from 5 heartbeats before and after the SL data in graph). (B; Top left) Confocal image of the myocyte in diastole (at 151 ms; [i] in A). Sarcomeres numbered from 1 to 5 were analyzed. (Bottom left) Plot profile along the longitudinal axis of the myocyte in top. SL, 2.00 ± 0.11 µm. (Middle top) Confocal image of the myocyte in systole (at 271 ms; [ii] in A). Sarcomeres numbered from 6 to 10 were analyzed. (Middle bottom) Plot profile along the longitudinal axis of the myocyte in top. SL, 1.72 ± 0.07 µm (P < 0.05 compared with the value in [i]). (Top right) Confocal image of the myocyte in diastole (at 351 ms; [iii] in A). Sarcomeres numbered from 1 to 5 were analyzed below. Bar, 10 µm. (Bottom right) Plot profile along the longitudinal axis of the myocyte in top. SL, 1.97 ± 0.10 µm (P < 0.05 compared with the value in [ii]; P > 0.05 compared with the value in [i]). Five consecutive sarcomeres were analyzed in the same myocyte by the multi-peak Gaussian fitting (see Materials and methods in the supplemental text). Length of the myocyte in the image plane, >35 µm throughout the cardiac cycle. Note that the image in the middle (i.e., during systole) was defocused because of movement of the focal point upon cardiac contraction (hence, different numbers were used for sarcomeres). Bar, 10 µm. See Video 1. (C) Typical raw data showing the time course of changes in SL during cardiac cycles. Small black circles indicate the average values of five consecutive sarcomeres at various time points. Data from 29 cardiac cycles obtained from the animal used for A, (B) are shown (bar, data used for the analysis in A; i.e., 151–351 ms). SL values were not analyzed at the time points that the images were out of focus because of movement of the heart (as indicated by the gaps between symbols). (D) Frequency distribution plot for SL (mean of five sarcomeres). The maximal (diastolic) and minimal (systolic) SL values were quantified by fitting the distribution with the combination of two Gaussian functions (see Materials and methods in the supplemental text) (i.e., 1.93 and 1.73 µm for diastolic and systolic SL values, respectively), from 29 cardiac cycles in this animal. Note that the diastolic SL value is slightly different from that obtained in the analysis for B, as a result of fluctuation of diastolic SL (see C). Solid line, combination of two Gaussian functions; dashed line, individual Gaussian functions. (E) Diastolic and systolic SL values obtained by using the two-peak Gaussian fitting in (D) from 13 animals. Average SL values (closed circles) were 1.90 ± 0.06 and 1.68 ± 0.06 µm (mean ± SEM) in diastole and systole, respectively (P < 0.05). Thin lines with open circles indicate individual data. Gray symbols indicate the data obtained from the animal in A–D. Error bars represent mean ± SEM.