Proteomic mapping of atrial and ventricular heart tissue in patients with aortic valve stenosis

Figures

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  Figure 1. Azan staining of atrial and diseased ventricular tissue. Cardiomyocytes (CMs) are stained orange to pink; connective tissue is visualized in blue. (a) Atrium with endocardial tissue surrounding islets of myocardial tissue. Myocardial tissue is interspersed with interstitial tissue. (b) Hypertrophic myocardium containing enlarged CMs (cell diameter approx. 35 µm) and scant interstitial tissue without additional signs for decompensation. (c) Severe contraction banding. (d) Atrophic CMs. (e) Replacement fibrosis. (f) Interstitial fibrosis. (g) Perivascular fibrosis. Thin blue arrow: interstitial tissue, thin orange arrow: CMs (longitudinal section is marked perpendicular; cross-section is marked horizontal), thin red arrow: arterioles, black arrows: normal cross striations, thin green arrows: myofibroblasts, thick purple arrow: pericardium, asterix: cavity, thick orange arrow: contraction banding, thick green arrow: atrophic CMs, thick blue arrow: fibrosis. Dotted line surrounds altered areas. Endocardium (thick blue arrow), CMs (thick orange arrow, cross section horizontal, longitudinal perpendicular), Enlarged cell diameter (black line), Cavity (Asterix), Interstitial tissue (blue…

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  Figure 2. Experimental workflow. (a) Atrial and left ventricular samples were taken during aortic valve replacement and Morrow resection. (b) iPSC-atrial and ventricular cardiomyocytes were labeled with heavy amino acids and the isolated proteins of three separated replicates of differentiation of iPSCs into atrial and ventricular cardiomyocytes were mixed to establish the protein quantification standard. (c) After cell lysis, tissue samples and quantification standard were mixed 1:1 and underwent filter aided sample preparation, followed by high-resolution mass spectrometric analysis. Graphical artwork for this figure was modified from versions provided by Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License (www. smart.servier.com).

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  Figure 3. Evaluation of proteomic data and assessment of SILAC standard. (a) Ranked abundance plot of all 4,268 proteins detected in the study. Proteins are ranked by their mean log10 MS1 intensities across all experiments, indicating close to 5 order of magnitude linear abundance range. (b) Visualization of protein detection completeness. Protein quantitation events are grouped by their percentages of quantifiable values in either SILAC standard (yellow), tissue (purple) or both (checkered) SILAC channels. Protein detections without any quantifiable values are displayed in grey. Less than 1% of protein quantitation events (purple) are not covered by the used SILAC standard. (c) Histogram showing the normalized log2 ratio distribution of ratiometric SILAC quantitation values. Values show a normal distribution. (d) Frequency of protein detections in the standard versus FFPE tissue with select proteins detected predominantly in tissue or standard.

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  Figure 4. Differentially expressed proteins between atria and ventricles. (a) Principal component analysis of atrial (blue) and ventricular (red) protein expression. Shown are the first two principal components. (b) Violin plot showing the distribution of the number of quantified proteins per sample, separated into atria and ventricles. While the mean is almost identical between the groups, there is small variation in the range. (c) Histogram showing the frequency in which proteins of certain Log2(FC) ratio ranges were measured in atria versus ventricle tissue. (d) Volcano plot: adjusted p-values are plotted against the log2 fold change ratios between atria and ventricles. Red (higher expression in ventricles) and blue (higher expression in atria) dots indicate significantly regulated proteins (adj. p < 0.05) with a fold change over 1 or − 1 respectively. (e–h) Immunhistology, (antibody signal brown, nuclei purple) for MLC2a (e, f) and NPPA (g, h) in atrium (e, g) and ventricle (f, h).

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  Figure 5. Assessing potentially disease related differences (a) Scatterplot comparing the fold change ratios between atria and ventricles for each protein to the fold change ratios published for healthy hearts by Doll et al. Each point represents a protein. Diagonal line: Deming (Model II) linear regression calculated in GraphPad Prism 9 (Y = 0.9427* X—0.09166). Large dots: Proteins with a distance to the regression line of more than 2 in Y direction. Black: proteins significantly regulated (adj. p < 0.05) in both studies. Pink: significantly regulated in Doll et al.17 but not in this study. Blue: significantly regulated in this study but not in the study of Doll et al. Negative values indicate an upregulation in this study, positive value a regulation towards healthy tissue. (b) Overrepresentation analysis of significantly regulated proteins of this and the study of Doll et al. White: adj. p value > 0.05, light green: adj. p value > 1 × 10–15, green: adj. p value > 1 × 10–50, dark green: adj. p value < 1 × 10–50.