Disrupting the interaction between connexin 43 and calmodulin restores gap junction function and mitigates reperfusion arrhythmias

Fig. 1. H/R treatment impaired GJ functionality and downregulated Cx43 expression in cardiomyocytes. (A) The H9c2 cells were treated in normoxia and hypoxia for the indicated times, and the distance of LY dye spread was measured to evaluate the GJ functionality. Scale bar = 300 μm. (B) Statistical assay of relative LY dye diffusion distance. (C, D) The H9c2 cells were treated in normoxia and hypoxia for the indicated times, and the Cx43 expression was detected by Western blot assay and quantitatively analyzed by ImageJ software. (E) After H/R treatment, intracellular ROS levels in H9c2 cells were assessed using DCFH-DA. Scale bar = 50 μm (F) After H/R treatment, the cell viabilities of H9c2 cells were detected using CCK-8 assay. (G-I) After H/R treatment, the levels of oxidative stress indicators (MDA, SOD, and LDH) were determined using commercial kits. In Fig. 1B and D, data are expressed as the mean ± SD. Statistical differences were determined using one-way ANOVA, followed by post hoc Tukey’s test, *P < 0.05 and ***P < 0.001 vs. NC group. In Figs. 1F-I, data are expressed as the mean ± SD. Statistical differences were determined using an unpaired two-tailed student’s t-test, *P <…

Fig. 2. H/R treatment enhanced CaM expression and interaction between Cx43 and CaM in cardiomyocytes. (A) The volcano plot showing upregulated and downregulated DEGs identified by the GEO2R online analysis tool. (B) GO-MF enrichment analysis of the DEGs. (C, D) After H/R treatment, the CaM expression was detected by Western blot assay and quantitatively analyzed by ImageJ software. (E, F) After H/R treatment, the co-IP experiment determined the binding between Cx43 and CaM. Data are expressed as the mean ± SD. Statistical differences were determined using an unpaired two-tailed student’s t-test, *P < 0.05 vs. NC group.

Fig. 3. Knockdown of CaM attenuated the impairment of GJ functionality induced by H/R treatment. (A, B) H9c2 cells were transfected with LV - CaM or LV - SCR, and then the CaM expression was detected by Western blot assay and quantitatively analyzed by ImageJ software. (C) The GJ functionality was evaluated using scrape loading/dye transfer assay. Scale bar = 300 μm. (D) Statistical assay of relative LY dye diffusion distance. Data are expressed as the mean ± SD. Statistical differences were determined using one-way ANOVA, followed by post hoc Tukey’s test, *P < 0.05 vs. NC group. #P < 0.05 and ###P < 0.001 vs. Vehicle group.

Fig. 4. Interfering with the combination of Cx43 and CaM mitigated the impact of H/R on GJ functionality. (A) Cx43 mimetic peptide (SP15) was designed using the CL domain of Cx43 (blue) and then conjugated with CPP. (B, C) After treatment with SP15 (50 µM) for 24 h, the expression levels of Cx43 and CaM in H9c2 cells were determined using Western blot assay. (D, E) The effect of SP15 on Cx43-CaM interaction was evaluated by co-IP assay. (F) The H/R-treated H9c2 cells were cultured in a culture medium containing SP15 (50 µM) for 24 h, and the distance of LY dye spread was measured to evaluate the GJ functionality. Scale bar = 300 μm. (G) Statistical assay of relative LY dye diffusion distance. Data are expressed as the mean ± SD. Statistical differences were determined using one-way ANOVA, followed by post hoc Tukey’s test, *P < 0.05 vs. NC group. #P < 0.05 vs. Vehicle group.

Fig. 5. The establishment of isolated rat heart IR injury model. (A) Schematic representation of the experimental protocol of isolated rat heart IR injury model. (B) Representative TTC staining images showing myocardial infarct size after I/R injury. White areas indicate infarcted myocardium, while red areas represent viable (non-infarcted) myocardium. (C) Quantitative analysis of myocardial infarct size expressed as a percentage of the total myocardial area. (D) HE staining was performed to observe the I/R-induced histological changes. (E) Typical arrhythmias in the I/R group, the red arrows indicate premature electrical events, while the blue arrows indicate ST-segment elevation.

Fig. 6. The effects of CaM knockdown and SP15 treatment on the interaction between Cx43 and CaM in an isolated rat heart IR injury model. (A, B) After treatment of CaM knockdown or SP15, the Cx43-CaM interaction was evaluated by co-IP assay. (C) Representative immunofluorescence images of cardiac tissues staining with Cx43 (red) and CaM (green). Scale bar = 25 μm.

Fig. 7. Interfering with the interaction between Cx43 and CaM improved myocardial electrophysiological parameters and reduced arrhythmia scores. (A) Recorded representative epicardial activation mapping of left ventricular. (B-D) Quantification of conduction velocity, absolute inhomogeneity, and inhomogeneity index of left ventricular. (E) Quantification of arrhythmia score in different groups. Data are expressed as the mean ± SD (n = 6). Statistical differences were determined using one-way ANOVA, followed by post hoc Tukey’s test. ***P < 0.001 I/R group vs. control group; #P < 0.05 and ##P < 0.01 IR + sh-CaM#1 group vs. I/R group; &P < 0.05, &&P < 0.01 and &&&P < 0.001 IR + SP15 group vs. I/R group.