Microscopic heat pulses activate cardiac thin filaments

Figures

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  Figure 1.. IR laser–based local heating and temperature imaging system. (A) Schematic illustration of the present experimental system. Reconstituted thin filaments (or F-actin) interacted with HMM attached to the glass surface. Reconstituted thin filaments and HMM are illustrated below. Temperature was directly increased by an IR laser beam (λ = 1,455 nm), which elicits thin filament movements in various directions (as shown by orange arrows), as a function of the distance from the heat source (distance shown by dashed gray circles from 0 to 80 µm, with temperatures denoted; cf. D). (B) Left: Fluorescence image of rhodamine-phalloidin–labeled F-actin filaments during heating. The IR laser was focused on a red point as indicated by “Heat source.” Right: Ratio image showing a change in FI of F-actin before and during heating. Color map on the right indicates the magnitude of change. F-actin at a concentration of ∼10 nM was applied to the coverslip. (C) Time course of changes in FI of F-actin filaments located at various distances from the heat source (distances indicated on right). Heating with a duration of 2 s was applied three times in a consecutive manner at an interval of 10 s. Blue,…

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  Figure 2.. Thermal activation of F-actin and reconstituted thin filaments at pCa 9. (A) Effects of IR laser irradiation on the motility of F-actin (top) or reconstituted thin filaments (bottom). Merged fluorescence images before (left), during (middle), and after (right) heating are shown. Heat source is indicated as a small white circle (denoted as “Heat source”) in the middle panels marked in pink. Images show 2 s before heating, at the onset of heating, and 2 s after heating, respectively, in the left, middle, and right panels (as indicated on the bottom in green). Images in red represent trajectories of the movement of filaments during 2 s (as indicated on bottom). Images in yellow represent immobile filaments. See Videos 1 and 2 for F-actin and reconstituted thin filaments, respectively. Scale bars, 20 μm. (B) Time courses of sliding velocity of F-actin (top) or reconstituted thin filaments (bottom) located at various distances from the heat source (see color map on right) before, during, and after heating. Heating was given for 2 s from 0 to 2 s (marked in pink). Note similar sliding velocities were obtained upon heating for F-actin and reconstituted thin filaments.

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  Figure 3.. Temperature dependence of sliding velocity of F-actin and reconstituted thin filaments at pCa 9. (A) Relationship between the distance from the heat source and sliding velocity for F-actin. (B) Same as in A for reconstituted thin filaments. (C) Sliding velocity plotted against temperature. Closed circles, F-actin; open circles, reconstituted thin filaments. Sliding velocities were compared at 25°C (baseline temperature) and at higher temperatures raised by IR laser irradiation. Data represent mean ± SEM for both the x and y axes. Sliding velocities at 25°C are 4.9 ± 0.1 µm/s (n = 513) and ∼0 µm/s for F-actin and reconstituted thin filaments, respectively. Those at higher temperatures are as follows: 14.5 ± 0.4 (n = 32) and 5.0 ± 1.0 µm/s (n = 6) at 34.4 ± 0.4 and 34.5 ± 1.0°C, 15.4 ± 0.4 (n = 65) and 8.3 ± 0.7 µm/s (n = 20) at 36.1 ± 0.4 and 36.2 ± 0.5°C, 16.7 ± 0.4 (n = 92) and 8.6 ± 0.5 µm/s (n = 26) at 37.9 ± 0.5 and 38.0 ± 0.7°C, 19.4 ± 0.4 (n = 120) and 14.5 ± 0.6 µm/s (n = 89) at 40.8 ± 0.7 and 40.8 ± 0.7°C, and 21.3 ± 0.8 µm/s (n = 39) and 21.5 ± 0.9 µm/s (n = 28) at 45.8 ± 1.4 and 45.6 ± 1.5°C for F-actin and reconstituted thin filaments, respectively. ***, P < 0.001 for…

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  Figure 4.. Temperature dependence of sliding velocity of F-actin and reconstituted thin filaments at pCa 5. (A) Relationship between the distance from the heat source and sliding velocity for F-actin. (B) Same as in A for reconstituted thin filaments. (C) Sliding velocity plotted against temperature. Closed circles, F-actin; open circles, reconstituted thin filaments. Sliding velocities were compared at 25°C (baseline temperature) and at higher temperatures raised by IR laser irradiation. Data represent mean ± SEM for both the x and y axes. Sliding velocities at 25°C are 4.5 ± 0.05 µm/s (n = 461) and 6.4 ± 0.1 µm/s (n = 352) for F-actin and reconstituted thin filaments, respectively. Those at higher temperatures are as follows: 12.6 ± 0.4 (n = 21) and 14.0 ± 0.3 µm/s (n = 32) at 34.2 ± 0.5 and 34.4 ± 0.4°C, 15.6 ± 0.3 (n = 69) and 15.4 ± 0.3 µm/s (n = 61) at 35.9 ± 0.3 and 36.1 ± 0.4°C, 18.0 ± 0.4 (n = 69) and 17.1 ± 0.3 µm/s (n = 72) at 37.9 ± 0.6 and 37.9 ± 0.6°C, 20.0 ± 0.3 (n = 119) and 17.9 ± 0.3 µm/s (n = 112) at 40.7 ± 0.7 and 41.0 ± 0.7°C, and 18.5 ± 0.7 (n = 45) and 17.4 ± 0.6 µm/s (n = 38) at 45.6 ± 1.7 and 44.9 ± 1.0°C for F-actin and reconstituted thin filaments, respectively.…