Identification of Cx43 variants predisposing to ventricular fibrillation in the acute phase of ST-elevation myocardial infarction

Figure 1. GJA1 variant selection. Schematic representation of study workflow. Cx43, connexin 43; FRAP, fluorescence recovery after photobleaching; STEMI, ST-elevation myocardial infarction; VF, ventricular fibrillation.

Figure 2. Identification of three novel GJA1 missense variants. Left: Schematic 2D structure of the four transmembrane segments of human connexin 43 (Cx43) with the location of the three GJA1 missense variants. Right: Multispecies protein sequence alignment illustrating the sequence conservation at the positions of the mutated residues.

Figure 3. Wild-type (WT) and mutated connexin 43 (Cx43) cellular localization. Pictures of cells expressing WT or mutated (V236I, H248R, and I327M) hCx43 and loaded for 30 min with calcein. Mutants and the WT hCx43 were expressed as fusions to enhanced cyan fluorescent protein. For each panel, the three images from left to right show hCx43, the cells, and a merged image. The left side illustrates cells at physiologic (7.4) pH and right side illustrates cells at acidic (6.5) pH. Scale bars represent 15 µm.

Figure 4. Functional properties of control and mutated hCx43 (V236I, H248R, and I327M) at physiological pH. (A) Mean normalized curves of fluorescence recovery after photobleaching are shown as a function of time (seconds), at physiological pH (7.4). Connected cells transfected with wild-type (WT) plasmids are shown in dark blue (circles). Connected cells transfected with mutated plasmids are shown in light blue (square: V236I, diamond: H248R, stars: I327M). Connected but not transfected cells are shown in grey and isolated cells are shown in black. Data are normalized using the WT condition. Data are shown as mean ± SEM. (B–F) Histograms showing focused results after 20 s (B), 60 s (C), 120 s (D), 200 s (E), and 300 s (F) after photobleaching. Histograms show the median (95% confidence interval). Statistical comparisons are made using a Kruskal–Wallis plus a Dunn’s test. Differences are deemed significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 5. Functional properties of control and mutated hCx43 (V236I, H248R, and I327M) at acidic pH. (A) Mean normalized curves of fluorescence recovery after photobleaching are shown as a function of time (seconds), at acidic pH (6.5). Connected cells transfected with WT plasmids are shown in dark blue (circles). Connected cells transfected with mutated plasmids are shown in light blue (square: V236I, diamond: H248R, stars: I327M). Connected but not transfected cells are shown in grey and isolated cells are shown in black. Data are normalized using the WT condition. Data are shown as mean ± SEM. (B–F) Histograms showing focused results after 20 s (B), 60 s (C), 120 s (D), 200 s (E), and 300 s (F) after photobleaching. Histograms show the median (95% confidence interval). Statistical comparisons were made using a Kruskal Wallis plus a Dunn’s test. Differences were deemed significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.