Adrenergic Signaling-Induced Ultrastructural Strengthening of Intercalated Discs via Plakoglobin Is Crucial for Positive Adhesiotropy in Murine Cardiomyocytes

FIGURE 1. Adrenergic signaling caused ultrastructural changes in ICDs of murine cardiomyocytes and leads to positive adhesiotropy. (A) Transmission electron microscopy was performed from cardiac slices derived from the hearts of 12-week-old Jup WT and KO mice (for the sake of terminology here, we used PG gene name “Jup” for labeling) treated with F/R and Iso for 1 h and representative images were displayed. Scale bar: 375 nm. * indicates gap junction and the yellow arrow indicates the area composita. n = 3 mice per condition. (B) Exemplar images of how the analysis of junctional plaque thickness and length of area composita were obtained (as explained in section “Materials and Methods”). (C) Bar graphs of plaque thickness and length of area composita measured corresponding to A. Every dot corresponds to one ICD, mean ± SEM. (D) Dissociation assays performed in cardiac slices derived from Jup WT and KO mice treated with F/R and Iso for 1 h showing an increase of cell adhesion upon adrenergic signaling treatment in Jup WT mice which was completely absent in Jup KO mice. n = 4 mice per condition. Two-way repeated measure ANOVA with Tukey’s post hoc test was performed. **p < 0.005, ***p <…

FIGURE 2. cAMP enhancement in murine cardiac slices leads to changes in the distribution of Dsg2 and N-cad. STED microscopy analysis was performed on cardiac slices derived from hearts of 12-week-old Jup WT and KO mice treated with F/R for 1 h. STED images of ICDs of (A) Jup WT and (B) Jup KO mice treated with and without F/R for 1 h and stained for Dsg2 (red) and N-cad (green) (n = 4 mice per condition). Scale bar: 2 μm. Zoomed image of Dsg2 and N-cad representing the inset was shown in a separate panel.

FIGURE 3. Altered cardiac myocyte cohesion after depletion of Dsg2 and N-cad. Confluent HL-1 cardiac myocyte monolayers were treated with and without F/R for 1 h to increase cAMP. (A) Depletion of Dsg2 and N-cad was done by using a siRNA approach, and Western blots were performed to confirm the knockdown of respective proteins. α-Tubulin (α-Tub) was used as a loading control, and representative blots are shown. (B) Dissociation assays were performed to determine cell–cell adhesion after depletion of either Dsg2 or N-cad after treating the cells with and without F/R for 1 h, N = 3–6 experiments performed in triplicates. Multiple t-test with Holm–Sidak multiple comparisons was performed. * is p < 0.0001 compared to si NT, #p < 0.0001 compared to si NT+F/R. (C) Distribution of the desmosomal proteins DSG2 (green) and N-cad (red) was analyzed by immunostaining. n = 3, scale bar: 10 μm.

FIGURE 4. F/R-mediated phosphorylation of PG at serine 665. (A) Western blot analysis of ventricular cardiac slices from Jup WT and KO mice treated with F/R for 1 h to analyze phosphorylation state of PG at serine 665. (B) Bar graphs depict the mean band intensity by densitometric quantification in WT mice cardiac slices compared to the total protein or α-tubulin (α-tub) and normalized as fold of control ± SEM, n = 5 mice per condition. Unpaired student’s t-test was performed. * is p < 0.05.