Phase-targeting rapid cryofixation of the beating heart and histological analysis unveil contractile state-dependent sarcomere dynamics

Figures

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  Fig. 1. Diagram of the rapid-freezing system for the perfused rat heart. (A) Overall view of the system. (B) Photos showing the main body in the system composed of the cryogen-ejection system and sample stage (upper left panel), the sample chamber on the sample stage (upper right panel), and a top-down view of the heart situated in the chamber with the ring-shaped electrode on the left ventricular surface (bottom panel). (C) Schematic illustrations of the system before (left panel) and during (right panel) the rapid freezing of the heart. The gauge pressure of 0.05 MPa was applied into the cryogen tank. ECG: electrocardiogram.

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  Fig. 2. Side views of the heart during rapid freezing. (A-i) Sequential images captured 7-ms before, immediately upon (0 ms), during (7 ms and 14 ms), and after the heart was exposed to the cryogen during peak systole. (A-ii) Side view of the heart (left panel: at end diastole) and image showing the vertical shift of the ventricular surface (ring electrode) over time during the cardiac phase (right panel) with the corresponding traces of electrocardiogram (ECG) and electrical stimulation (ES: 0.5 Hz) below. (A-iii) Rapid freezing of the heart during peak systole. Side-view image of the heart (left panel: at end diastole) before cryogen exposure and an image of the vertical shift of the ventricular surface with rapid cryogen exposure during peak systole. Note that the contact of the heart surface (the ring-shaped electrode) was recognized as an abrupt disappearance of the ventricular surface with large deflections of the ECG trace (a timepoint indicated by the arrowhead). (B) Rapid freezing of the heart during end diastole. Side-view image of the heart (left panel: at end diastole) before cryogen exposure and an image of the vertical shift of the ventricular surface with rapid cryogen…

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  Fig. 3. Gross and microscopy images of the rapidly-frozen heart. (A) Gross appearance of the rapidly-frozen hearts, immediately after rapid-freezing (left panel) and after subsequent freeze-substitution (right panel). (B) Schematic illustration showing directions of slicing of thin sections for HE-staining. (C) Cross-sections underneath the cryogen-exposed surface (inside the ring of the ring-shaped electrode on the left ventricle) and nonexposed surface (underneath the ring-shaped electrode). The corresponding binarized images are shown to emphasize the presence of small vacuoles. For the cryogen-exposed surface, enlarged images of the binarized image are shown up to an approximately 50 μm depth from the surface (a) and that depth is greater than approximately 50 μm (b). (D) The en face plane near the cryogen-exposed ventricle surface (< 30 μm) of the heart during peak systole and end diastole and the nonrapidly-frozen heart fixed by PFA perfusion. Thickness of the sections: 4 μm.

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  Fig. 4. Confocal fluorescence images of the subcellular myocyte structures of the cryofixed hearts that were rapidly frozen during peak systole (“RF-systole”) and end diastole (“RF-diastole”). (A) Fluorescence images of the Z-lines, M-lines, thin filaments (F-actin), and cell membranes labeled with immunofluorescence staining for α-actinin and titin, and fluorescence labeling with phalloidin and wheat germ agglutinin (WGA), respectively. The images in the 2nd to 4th columns from the left are cropped and enlarged from the white square region in the leftmost images. (B) Averaged intensity line-profiles of the Z-line (green), M-line (red), and thin filament (yellow) extracted along with the white-dotted lines (a) and (b) in the images in the fourth column of (A). The profiles were averaged from 3 adjacent line profiles.

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  Fig. 5. Quantitative results for the myocyte sarcomere length (SL) in the cryofixed heart during systole and diastole based on a spatial frequency analysis using fast Fourier transform (FFT). (A-i) Fluorescence images of α-actinin, N-cadherin, and the cell membrane of rapidly-frozen hearts during peak systole (“RF-systole”) and end diastole (“RF-diastole”), as measured with a confocal laser-scanning microscope. Scale bars: 50 μm. (A-ii) Representative intensity profiles obtained along the lines labeled as (a) in the fluorescence images in A-i. (A-iii) Amplitude spectra of each line profile in the fluorescence images, calculated using FFT. The peak positions are adopted as the representative SL at the sampled myocytes. (B, C) Statistical results for the extracted sarcomere length per heart (B: n = 3–5 hearts) and per myocyte (C: n = 20–25 cells/heart). **p < 0.01, ***p < 0.001.

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  Fig. 6. Heatmaps depicting the distributions of local sarcomere lengths (SL), based on the fluorescence images of Z-lines (immunostaining of α-actinin) in the hearts fixed under each condition: rapid-freezing during peak systole (A: “RF-systole”) and end diastole (B: “RF-diastole”), rapid freezing under BDM treatment (C: “RF-diastole (BDM)”), fixation by PFA-perfusion alone (D: “PFA”), and rapid-freezing after PFA fixation (E: “PFA + RF”). The three different heatmaps were obtained from three different hearts. Please note that the areas of nonmyocyte or intra-myocyte SL out of the range of 1.0 to 2.6 μm are colored by black. Scale bar: 50 μm.

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  Fig. 7. Heatmaps showing the local sarcomere length (SL) in the heart under ventricular fibrillation (VF) fixed with rapid freezing (A: “RF-VF”, n = 2 hearts) and PFA perfusion (B: “PFA-VF”, n = 1 heart). Please note that the areas of nonmyocyte or intra-myocyte SL out of the range of 1.0 to 2.6 μm are colored by black. Scale bar: 50 μm.