Fig 2: Sepsis reduces myocardial Kcnh2 expression. For LPS-induced rat model (A) and CLP rat model (B), haematoxylin–eosin staining (H&E staining) was performed to detect heart injury. Scale bar: 50 µm. After stimulation for 6 h, KCNH2 expression was immunoblotted in rat heart of (C) LPS model (n = 6) and (D) CLP model (n = 5). (E) The expression of KCNH2 was showed in cultured cardiomyocytes and (F) cardiac fibroblasts by Western blot at 12 h after either saline or LPS treatment at different concentration (0, 0.1, 0.2, 0.5, 1.0, 2.0 µg/mL), (n = 4). (These results were performed as mean ± SD of at least 4 independent experiments. Statistical analysis was performed with one-way ANOVA followed by Tukey's test. *, P < .05; **, P < .01; ***, P < .001 and ****, P < .0001)

Fig 3: Kcnh2 defect promotes the heart damage induced by sepsis. The levels of (A) mRNA of Kcnh2 and (B-C) protein of KCNH2 in WT or Kcnh2+/- cardiac extracts, (n = 6). (D) Survival status of Kcnh2+/- and WT rats after CLP surgery (n = 8) or (E) after LPS stimulation (n = 8). The change of heart function in Kcnh2+/- and WT rats at 6 h after LPS challenge, (F-G) left ventricular ejection fraction (FS %) and fractional shortening (EF %) measured with echocardiography, (n = 8); (H) H&E staining for detecting the heart tissue damage, (n = 5). (I) The cardiac apoptosis determined by TUNEL assay (n = 5), scale bar: 75 µm. (J-K) Western blot and quantitative analysis for FOXO3A, BCL-2, BIM and PUMA in cardiac extracts (n = 6). (All the results were performed as mean ± SD of at least 6 independent experiments. Statistical analysis was performed with one-way ANOVA followed by Tukey's test. *, P < .05; **, P < .01; ***, P < .001 and ****, P < .0001)

Fig 4: Schematic representation shows the mechanisms of Kcnh2-modulated cardiac dysfunction following sepsis stimulus. Sepsis-induced decrease of Kcnh2 resulted in inhibition of AKT in cardiomyocytes. Attenuation of AKT of cardiomyocytes mediated by Kcnh2 upregulated FOXO3A expression, which initiated the transcription of BIM/PUMA genes. Finally, enhanced BIM/PUMA caused the cardiomyocyte apoptosis, leading to cardiac dysfunction

Fig 5: FOXO3A mediates the modulation of Kcnh2 on sepsis‐induced apoptosis. (A) The immunofluorescence was performed to detect the express and location of FOXO3A in Kcnh2+/‐ and WT rats at 6 h after LPS challenge. (n = 4). Scale bar: 50 μm. (B) The apoptosis image of Kcnh2+/‐ or WT NRCMs after knockdown of FOXO3A by si‐RNA in presence of LPS, (n = 4). Scale bar: 50 μm. (C) Western blot and quantitative analysis for the expression of FOXO3A, BCL‐2, BIM and PUMA in NRCMs after inhibition of FOXO3A under LPS treatment, (n = 4). (All the results were performed as mean ± SD of at least 4 independent experiments. Statistical analysis was performed with one‐way ANOVA followed by Tukey's test. *, P < .05; **, P < .01; ***, P < .001 and ****, P < .0001)