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. 2022 Oct 21;146(21):1610–1626. doi: 10.1161/CIRCULATIONAHA.121.057329

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© 2022 The Authors.

Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited.

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Figure 4. — Disrupted ICD structure in DSG2 (desmoglein-2)–W2A mutant mice. A, Representative z-stack reconstruction of segmented intercalated discs (ICDs) in top and side view acquired with structured illumination microscopy. Overlay of analyzed ICD volume is shown in gray. ICDs are marked by DSP (desmoplakin; magenta). Scheme on top presents segmented area and pictograms on the right display the respective angle of view. Corresponding analysis of ICD volume and width of ICD between adjacent cardiomyocytes below. Scale bar, 5 µm. *P<0.05, Mann-Whitney test. Each dot represents the value of 1 ICD from 4 mice per genotype. B, Representative images of DSG2 (magenta) and filamentary actin (f-actin; white) z-stacks acquired by structured illumination microscopy and presented as maximum intensity projection. Lower row shows color-coded height projection of DSG2 signals in z-stack after signal thresholding as performed for analysis. Related analysis of DSG2 signal volume and number per ICD length is shown below. Pictograms on the right display the respective angle of view. Scale bar, 5 µm. *P<0.05, unpaired Student t test, with Welch correction. Each dot represents the value of 1 ICD from in total 3 mice per genotype. C, Fluorescence recovery after photobleaching analysis of DSG2–wild type (WT) and DSG2-W2A-mGFP fusion proteins at the cell–cell junction of neonatal cardiomyocytes with representative intensity kymographs of bleached areas on the left. Time point 0 = bleach as indicated by the black arrow. Analysis of the mobile fraction of the indicated mGFP-fusion proteins is shown on the right. *P<0.05, unpaired Student t test, with Welch correction. Each dot represents the mean value of 1 heart from in total 3 isolations. D, Representative triton-X-100 assay immunoblot with separation of a soluble (sol), noncytoskeletal bound protein fraction from a nonsoluble (non-sol), cytoskeletal-anchored fraction and corresponding analysis shown below. PG (plakoglobin), PKP2 (plakophilin-2), and N-cadherin (NCAD) were analyzed. Intensity of proteins was normalized to the total amount of protein detected by ponceau staining. GAPDH and DSP served as separation control. *P<0.05 or as indicated, unpaired Student t test (PG, NCAD, PKP2) or Mann-Whitney test (DSP). Each dot represents the result from 1 mouse. E, Immunostaining of connexin-43 (CX43; red in overlay) in DSG2-W2A hearts. NCAD (yellow) marks ICDs, DAPI (blue) nuclei, and F-actin (green) the sarcomere system. Orange arrowheads mark ICD, pink arrowheads highlight lateralization of CX43 staining. Scale bars, 25 µm. Images representative for 5 mice per genotype. Box with color indications of respective groups applies to the entire figure.