PLXND1-mediated calcium dyshomeostasis impairs endocardial endothelial autophagy in atrial fibrillation

FIGURE 1. Histological and cellular changes in the hearts of atrial fibrillation mouse models. (A), Experimental schema of AF mice model. Male Tbx5 +/− mice (Tbx5 fl/+ ; CMV-Cre) and CMV-Cre mice were used as SR and AF mice respectively. (B) ECG tracing of SR mice (upper) and AF mice (lower); rapid and irregular atrial waves are indicated by red arrows. (C) Histological images of hearts from SR and AF mice obtained by Masson’s trichrome staining. Scale bar: 100 μm (right panel). (D) The degree of fibrosis was evaluated and calculated by measuring the blue regions (collagen) relative to the total tissue area in sections, as shown in (C). (E) Representative images stained with WGA (red) to delineate the sarcolemma, and DAPI (blue). Scale bar, 20 μm. (F) Quantification of the CSAs of atrial cardiomyocytes. (G–I) Statistics of plasma concentrations of BNP, TnI, and CK-MB in mice in two groups. AF, atrial fibrillation; SR, sinus rhythm; ECG, electrocardiogram; WGA, wheat germ agglutinin; CSA, cross-sectional area; BNP, brain natriuretic peptide; TnI, troponin I; CK-MB, creatine kinase MB.

FIGURE 2. Atrial fibrillation promotes PLXND1 expression and inhibits autophagy in endocardial endothelial cells. (A) Diagram of the EEC isolation from SR and AF mice. (B) Verification with specific markers (NPR3 and CDH11) in EECs compared to those in HUVECs and HPMECs by WB data. (C) Verification with specific markers (NPR3 and CDH11) in EECs compared to those in HUVECs and HPMECs by FACS. (D) Immunostaining data verifying the colocalization of EEC marker NPR3 (red) and CDH11 (green) in EECs. (E) Representative WB and quantification of PLXND1 in EECs isolated from SR and AF mice. (G–I) Representative WB and quantification of autophagy-related protein levels of EECs subjected to BAFA1 (100 nM) treatment or no treatment. (J) EECs from SR and AF mice were infected with a tandem GFP-mRFP-LC3 adenovirus for 24 h. Representative images show formation of puncta in different groups. Scale bar: 10 μm. (K) Quantitative analysis of yellow and free red puncta in merged images. The number of both yellow and free red puncta decreased in the EECs of AF mice compared to that in SR mice. (n = 10 cells per group; cells were isolated from three mice for one experiment and three independent experiments were…

FIGURE 3. PLXND1 inhibits autophagy via intracellular calcium signaling. Mouse EECs were treated with NC, LV-Plxnd1, and shPlxnd1. (A) Representative WB of PLXND1 in EECs subjected to the indicated treatments. (B) Quantitative analysis of the efficacy of overexpression and knockdown of Plxnd1. (C) Representative western blots of autophagic markers in EECs subjected to the indicated treatments. (D–E) Quantitative analysis indicates that PLXND1 significantly affects the level of autophagy. (F) Immunofluorescence staining was used to detect MAP1LC3B in EECs. Scale bar: 20 μm. (G) Intracellular calcium was marked by the Fluo-3-AM probe and measured continuously for 8 min (H) EECs were incubated with the calcium probe Fluo-3-AM. EGTA + BAPTA-AM was used to obtain the minimum fluorescence intensity (Fmin), whereas ionomycin + calcium was used to obtain the maximum fluorescence intensity (Fmax). Scale bar: 20 μm. Quantitative analysis showed the fluorescence intensity in the different groups. (I-J) EECs were infected with a tandem GFP-mRFP-LC3 adenovirus for 24 h. Representative merged images (I) and quantitative analysis (J) of yellow and free red puncta in merged images of different treatment…

FIGURE 4. PLXND1 binds to ORAI1 in endocardial endothelial cells to regulate intracellular calcium levels. (A–B) Structure of predicted PLXND1 (A) and ORAI1(B). The elements of secondary structure (α-helices and β-sheets) are represented by different colors. (C) Schematic visualization of the PLXND1-ORAI1 complex, and the coupling regions were marked by red and blue solid circle respectively. (D)Three-dimensional structure showing PLXND1-ORAI1 in (C). PLXND1 and ORAI1 are represented as the grey and blue ribbon respectively. A view rotated 180° about the y axis is shown with the enlargement of interact surface in red solid circle (right). (E) Immunofluorescent staining was applied to mark PLXND1 (red) and ORAI1 (green) in EECs. White arrows represent their colocalization on the EECs. Scale bar: 20 μm. (F) Lysates from EECs were subjected to IP with IgG, anti-PLXND1, or anti-ORAI1 antibody, followed by western blotting for PLXND1 and ORAI1. PLXND1, plexinD1; EECs, endocardial endothelial cells; IP, immunoprecipitation; IB, immunoblotting; NC, negative control; WB, western blot.

FIGURE 5. Decrease in CAMK2 phosphorylation is associated with PLXND1-mediated autophagic inhibition and endocardial endothelial cell dysfunction. (A–B) Representative WB and quantification of PLXND1-mediated CAMK2 activity in cells subjected to treatment with STO-609 or no treatment. (C–E) Representative WB and quantification of PLXND1-mediated autophagic marker levels in cells subjected to treatment with STO-609 or no treatment. (F–H) Representative WB and quantification of PLXND1-mediated autophagic marker levels in cells subjected to treatment with BAPTA or no treatment. (I–J) EECs were infected with a tandem GFP-mRFP-LC3 adenovirus for 24 h. Representative merged images (I) and quantitative analysis (J) of yellow and free red puncta in merged images in the treated groups. Scale bar: 10 μm. (K) EECs from SR and AF mice were seeded in E-plates, and the normalized cell index was recorded in real time for different treatment groups. CAMK2, calmodulin-dependent protein kinase II; PLXND1, plexinD1; WB, western blot; AF, atrial fibrillation; SR; sinus rhythm.

FIGURE 6. Schematic figure. The expression of the mechanosensitive protein PLXND1 was increased in the cytomembranes of EECs during AF, which served as a scaffold protein to bind with ORAI1 and decreased ORAI1-mediated calcium influx. Decreased calcium influx-mediated phosphorylation of CAMK2 is associated with the inhibition of autophagy and results in EEC dysfunction in AF.