Sarcomeric network analysis of ex vivo cultivated human atrial appendage tissue using super-resolution microscopy

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  Figure 1. Time-dependent analysis of cellular metabolic activity in LAA slices using MTT assay. (a) Representative image of a LAA slice in a petri dish that actively metabolized the applied MTT salt into formazan, thereby causing a dark violet appearance of the tissue. (b) Representative enlarged bright-field microscopic image of LAA slice illustrates accumulated formazan in highly viable MTT-stained (left) vs. unstained (right) tissue. Scale bar = 100 μm. (c) The metabolic activity of LAA slices obtained from multiple patients was evaluated at six different time points. Using spectrophotometric measurement, the optical absorbance of dissolved formazan was defined for each slice and normalized to its weight. The analysis indicated a constantly rising metabolic activity within the first week of cultivation, which promptly dropped thereafter. Statistical significance was determined using one-way ANOVA followed by Tukey post hoc test, n = 4 slices (from 4 independent LAA samples) for each time point, *p \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek}…

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  Figure 2. Time-dependent SIM-based analysis of sarcomeric network in LAA cardiomyocytes. (a) Panels demonstrate representative SIM images of LAA cryosections immunolabeled at D0, D5 and D10 with an antibody directed against \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha $$\end{document}α-actinin (red), WGA conjugate to visualize cell membrane (green), DAPI staining for cell nuclei (blue) as well as the overlay of all three stainings (merged). Fluorescence images show clear disorganization of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha $$\end{document}α-actinin in cardiomyocytes over the course of cultivation. Scale bar = 20 μm. (b) Binary images of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs}…

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  Figure 3. Time-dependent SIM-based evaluation of sarcomere orientation in LAA cardiomyocytes. (a) Representative SIM images of antibody-labeled \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha $$\end{document}α-actinin filaments taken at D0, D5 and D10 served as a source for qualitative orientation analysis. The direction of the sarcomeric network was colored according to the presented orientation map. The prevalent orientation in 70\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ $$\end{document}∘–90\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ $$\end{document}∘ is largely colored by red, pink and yellow. The multicolored sarcomere…

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  Figure 4. Relative expression of cardiac-specific structural and functional proteins in LAA cultivated over time analyzed by qRT-PCR. Levels of the transcripts encoded by (a) ACTN2, (b) TNNT2, (c) MYH6, (d) MYL7, (e) MYL2, (f) CX43, (g) RYR2 and (h) KCNJ3 genes were evaluated in cultured tissue at different time points. Except CX43, all targets demonstrated strongly decreased expression over time. Gene expression was normalized to the internal reference gene GAPDH in each sample and presented on a log scale as fold change. Statistical analysis of the data was performed using one-way ANOVA followed by Tukey post hoc test, n = 4 slices (from 4 independent LAA samples) per each time point, n = 3 slices (from 3 independent LAA samples) for D0 MYL7 and D10 TNNT2, CX43 and RYR2. *p \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$< 0.05$$\end{document}<0.05.

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  Figure 5. Preparation of left atrial appendage slices. (a) Representative image of excluded LAA that was cut open with scissors along the site marked by a dashed line. (b) The muscle-rich region in LAA with low trabeculation (marked by a dashed triangle) was used for further procedures. (c, d) A typical LAA specimen in a petri-dish with the endocardium layer on the top, which was carefully removed. (e) LAA slices with 250 μm thickness were prepared using a vibratome. (f) The slices were placed on culture inserts, transferred into a 6-well plate filled with 1 ml medium and cultured via ALI method for 10 days.