Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation

Figures

• 

  Figure 1.. Schematic illustration showing the concept of this study. (A) Explanative drawing of the concept of probing actomyosin conformation in a sarcomere by SHG anisotropy. Coupling with ATP hydrolysis, myosin changes conformation of its S1 region causing angular change of its S2 fragment, which is called mechanochemical coupling. The detected SHG anisotropy was altered by the attachment/detachment of myosin S1 to/from actin. (B) Explanative drawing of our sample preparation. (C) A microscopic photo of cardiomyocytes adhered on a line-and-space pattern substrate.

• 

  Figure S1.. Comparison of forward- and backward- scattered SHG images of cardiomyocytes. (A, B) A typical example of forward- and backward-scattered SHG intensity images of a hiPSC(253G1)-differentiated cardiomyocyte at the same field of view, whose contrast in the linear scale (A) and in the logarithmic scale (B). Scale bar in (A, B), 10 μm. (A, C) Histograms of pixel intensity for forward (left) and backward (right)-scattered SHG images shown in (A).

• 

  Figure S2.. Optical system of the present SHG polarization microscope. (A, B) A schematic drawing of the optical set up (A) and a photograph of its appearance (B) of the present microscope system. Laser, laser source (810 nm, 80 MHz, 200 fs pulsed laser); LPC, laser power controller; HWP, half-wave plate; P, linear polarizer; EOM, electro-optic modulator; XY, x-y galvanometer scanner; L, convex achromatic doublet lens; DM1, dichroic beam splitter (720 nm short-pass); DM2, dichroic beam splitter (532 nm notch); OL1, focusing objective lens (40× air-immersion, NA = 0.95); OL2, collecting objective lens for forward scattering light (60× oil-immersion, NA = 1.42 for cardiomyocyte; 60× air-immersion, NA = 0.8 for Drosophila prepupa); S, sample; TL, tube lens; SPF, short-pass filter (680 nm); BPF, band-pass filter (405 nm); PCM, photon-counting module; LED, green LED illumination.

• 

  Figure 2.. Probing structural conformation of actomyosin complex by SHG anisotropy. (A) Typical example of SHG image acquisition of cardiomyocytes on a patterned line. Top, bright field image; middle, fluorescent image stained with fluorescent phalloidin; bottom, SHG image. Arrows indicate the orthogonal axes of the incident polarization. (B) Incident polarization dependence of SHG emitted from a sarcomere in the absence (green, Rigor) or the presence of ATP (red, Relax). Pink circles and light green circles are typical 10 traces in the absence or the presence of ATP, respectively. Red circles and green circles are mean value of the 10 traces of them. Solid lines are fitting results with Equation (2). The value of SHG intensity is normalized by the intensity of averaged value of all obtained data for relax state at 90°. (C) Histograms of mean value of parameter γ in a field of view (FOV) in the absence (green, N = 60 FOVs) or the presence of ATP (red, N = 60 FOVs), and in the presence of AMPPNP (blue, N = 64 FOVs). (D) Violin plots of sarcomere length in the absence (green, N = 1,339 sarcomeres) or the presence of ATP (red, N = 1,420), and in the presence of AMPPNP (blue, N = 1,486).…

• 

  Figure 3.. Probing dynamic structural conformation of actomyosin complex in a living cardiomyocyte. (A) A typical example of SHG image acquisition of a living cardiomyocytes. Arrows indicate the orthogonal axes of the incident polarization. (B) A typical example of dynamic measurement of parameter γ with 80 ms time resolution at the area shown in green rectangle in (A). Top, the image kymograph based on the total SHG intensity; second top, sarcomere length SL; second bottom, parameter γ; bottom, sarcomere motion converted into numerals by calculating correlation with an image at relaxation state. Light magenta and orange in the back indicates contracting and resting phases, respectively. (C) Histogram of γ within a trace of 40 s. Black line is a fitting result with double Gaussian distributions. (D) Relationship between SL and γ obtained in data shown in (B). Cyan circles and lines are all raw data, and red ones are the average values shown in E, middle. (E) Average traces of SL (top), γ (middle), and sarcomere motion (correlation) (bottom) in 16 pulsations for 40 s. The time zero was adjusted at the time of the largest value of SL. Light colors are SD. (F) Cross-sections of the SHG image…

• 

  Figure S3.. Examples of measurement of sarcomere length SL during myocardial contraction. (A) Two typical examples of kymograph indicating dynamic change of SHG intensity distribution along the myofibril axis in hiPSC-derived cardiomyocytes in the case that the image contrast has remarkable decrease by defocusing because of muscle contraction. The vertical axis is the myofibril axis and the horizontal axis is the elapsed time. Scale bar, 2 μm. (B, C) Line profiles along the myofibril axis of the averaged images within the resting state (B) or within the contracting state (C). When living cardiomyocytes are the object of observation, such as in this study, a sarcomere is occasionally out of the confocal position because of muscle contraction, causing the degradation of SHG intensity during measurement. Periodicity of a sarcomere could be confirmed by averaging, even in images with such the low contrast ratio, enabling measurement of SLcont.

• 

  Figure 4.. Effect of drugs on γ-value during cardiomyocyte beating. (A) A typical trace of γ-value after 10 μm BS treatment (upper) and further after inactivation of BS efficacy by blue light irradiation (lower). Red arrows indicate each pulsation confirmed by eye inspection of the video. (B) A typical trace of γ-value after 10 μm OM treatment. (C) Violin plots of γ in the case of no chemical treatment (red, N = 295 traces), 10 μm BS treatment (magenta, N = 176), after inactivation of 10 μm BS treatment (blue, N = 143), and 10 μm OM treatment (green, N = 141). Asterisks indicate less than 0.01 of the P-value in the t test.

• 

  Figure 5.. SHG anisotropy measurement of cardiomyocytes differentiated from genetic disease patient-derived hiPSCs. (A) A schematic drawing of the experimental procedure. (B) A typical example of dynamic measurement of the sarcomere movement of parameter γ with 80 ms time resolution in Mybpc3t/+ (top), Mybpc3t/t (middle), and Mybpc3+/+ cells (bottom). Right panels are histograms of γ-value in each trace. Solid lines in the right panels are fitting results with double Gaussian distributions. Magenta lines indicate γcont and orange lines indicate γrest in each trace obtained by the fitting. Asterisk indicates the occasional change of the beating cycle. (C) Violin plots of γrest (left) and γcont (right) for Mybpc3t/+ (red, N = 21 FOVs), Mybpc3t/t (blue, N = 36), and Mybpc3+/+ (green, N = 28). (D) Violin plots of dγave = γcont - γrest in Mybpc3t/+ (red, N = 21), Mybpc3t/t (blue, N = 36), and Mybpc3+/+ (green, N = 28). (E) Bar graphs of SL in the resting state (filled) and in the contracting state (opened) (upper) and contraction yield (lower). Error bars are SD. (F) Relationship between SLrest or SLcont and γrest or γcont for Mybpc3t/+ (top), Mybpc3t/t (middle) and Mybpc3+/+ (bottom). Filled…

• 

  Figure 6.. SHG anisotropy measurement of acquired force generation dysfunction. (A) A schematic drawing of the experimental procedure. (B) Confocal fluorescent microscope image of cardiomyocytes stained with anti-α-actinin (green) and DAPI (blue). Cardiomyocytes derived from hiPSCs were irradiated by UV light (254 nm) at energies of 0.00 (left), 31.25 (center), and 125.00 mJ/cm2 (right). The insets are enlarged images shown in a white rectangle in each image. (C) Kymographs of the SHG image of sarcomeres in cardiomyocytes derived from hiPSCs irradiated at 0.00 (top), 31.25 (second top), 62.5 (second bottom), and 125.00 mJ/cm2 (bottom) by UV. (D) Violin plots of γave in a sarcomere in cardiomyocytes derived from hiPSCs irradiated at 0.00 (red, N = 13 FOVs), 31.25 (magenta, N = 13), 62.5 (blue, N = 13), and 125.00 mJ/cm2 (green, N = 15) by UV. Asterisks indicate less than 0.01 of theP-value by the Mann–Whitney’s U-test. (E) Correlation plots between SLave and γave for 0.00 (red), 31.25 (magenta), 62.5 (blue), and 125.00 mJ/cm2 (green). Solid line is a fitting result of data with a linear function. Overlapped gray marks and lines in the middle and bottom panels are those for 0.00 mJ/cm2. (F)…

• 

  Figure S4.. Immunofluorescence confocal images of Sox2 and Oct4 in UV-irradiated hiPSCs. Confocal images of DAPI (4′,6-diamidino-2-phenylindole), showing AT region of double stranded DNA (left), immunostaining of Sox2 with Alexa594 (second left) and Oct4 with Alexa488 (second right), and merged of them (right) for 0 (top), 31.25 (second top), 62.5 (second bottom), and 125 (bottom) mJ/cm2 irradiation, respectively. The images obtained with a commercialized confocal microscope system (Zeiss LSM780 inverted microscope with 20× objective, N.A. = 0.8). Scale bar, 50 μm. The protocol for fixation and immunostaining was descried in the Materials and Methods section. The primary antibodies were anti-Sox2 mouse monoclonal antibody (1:50, clone E-4, sc-365823; Santa Cruz Biotechnology) and anti-Oct4 rabbit polyclonal antibody (1:100, 09-0023; Stemgent), and secondary antibodies were Alexa Fluor 594 conjugated anti-mouse IgG (1:500, A11032; Molecular probe) and Alexa Fluor 488 conjugated goat anti-rabbit IgG (1:500, A11034; Molecular probe), respectively.

• 

  Figure S5.. Fast Fourier transform (FFT) analysis of sarcomere images in cardiomyocytes differentiated from UV-irradiated hiPSCs. (A) A typical example of a pair of SHG image (left) and FFT image (right). Scale bar, 5 μm. (A, B) The line profile on the axis along average myofibril orientation (as indicated in a yellow line in (A)). Arrows correspond to the period of sarcomere repetition. (C) Average traces of the FFT line profile for 0.0 (red, N = 13), 31.25 (magenta, N = 10), 62.5 (blue, N = 7), and 125 mJ/cm2 (green, N = 10). Error bars are SD. There was no obvious sarcomere length shortening in UV-irradiated samples compared with control (0 mJ/cm2) as indicated by dashed lines.

• 

  Figure 7.. SHG anisotropy measurement in a Drosophila model of Barth syndrome. (A) A schematic drawing of the experimental procedure. (B) Photograph (left) and micrograph (right) of an example of mounted Drosophila prepupa between a coverslip and a transparent adhesive tape. (C) Typical SHG image of body wall muscles in a prepupa of WT, control strain, w1118 (upper), and tafazzin deficiency, TAZ−/− (lower). (D) Typical two examples of image kymograph for a SHG intensity (top), time courses of SL (middle), and parameter γ (bottom) with 500 ms time resolution for 60 s in a control prepupa. Gray in the back indicates the time when sarcomeres were not detectable any more. Light magenta and orange in the back indicates contracting and resting phases that could be classified, respectively. (E) Correlation plots between SL and γ in typical three FOVs for control (left) and TAZ−/− (right). Overlapped gray marks are those for the other. (F) Correlation plots between SLave and γave in a FOV for w1118 (red) and TAZ−/− (blue). Light colors are all data for each time point. Data suspected to be outliers are surrounded by purple broken lines. (G) Histograms of parameter γ in a prepupa of control (red, N…