MITF Regulates CFTR Expression to Participate in Myocardial Ischemia–Reperfusion Injury

Figures

• 

  FIGURE 1. Disease target screening for MIRI. (A) Standardized sample distribution of the GSE6381 dataset, showing balanced expression profiles across subjects. (B) Heatmap of DEGs in GSE6381, illustrating distinct transcriptional patterns between groups. (C) Volcano plot highlighting significantly upregulated and downregulated genes. (D) GSEA pathways enriched in the MIRI group. (E) Distribution of MIRI‐related disease targets across database sources. (F) PPI network revealing major interacting proteins associated with MIRI. “Control” refers to myocardial samples from AMI patients without reperfusion injury.

• 

  FIGURE 2. WGCNA of core genes. (A) Determination of soft‐threshold power for scale‐free topology (β = 9). (B) Clustering analysis showing no outlier samples. (C) Gene dendrogram identifying co‐expression modules. (D) Module‐trait correlations, with the light‐yellow module most strongly associated with MIRI. (E) Heatmap displaying module‐specific expression patterns. (F) Intersection of WGCNA‐derived genes and disease‐related targets, yielding 42 core genes. Analyses were performed on eight samples in the GSE6381 dataset.

• 

  FIGURE 3. External dataset validation and enrichment analysis. (A,B) Core gene expression patterns across samples and corresponding box plots. (C) ROC curves demonstrating diagnostic value of several core genes. (D) Immune infiltration profiles comparing MIRI and control samples. (E,F) GO functional enrichment showing involvement in ion transport and cellular structure. (G) KEGG pathway enrichment featuring arginine biosynthesis and related processes. (H) Mapping of identified genes onto the arginine biosynthesis pathway.

• 

  FIGURE 4. CFTR‐transcription factor regulatory network and expression validation. (A) Regulatory network of transcription factors associated with the seven core genes. (B) CFTR‐centered transcription factor network showing MITF, TP53, and STAT3. (C) DO enrichment analysis of CFTR‐related transcription factor targets. (D) GO enrichment analysis of CFTR‐related transcription factor targets. (E) KEGG pathway enrichment analysis of CFTR‐related transcription factor targets. F. CFTR protein expression in AC16 cells at 1, 3, and 6 h after reoxygenation following OGD, with the untreated control shown for comparison. (G) mRNA expression of CFTR, STAT3, MITF, and TP53 in control and OGD/OGR‐treated AC16 cells. (H) Protein expression of CFTR, p‐STAT3/STAT3, MITF, and TP53 in control and OGD/OGR‐treated AC16 cells. I. ChIP‐qPCR analysis of MITF, TP53, and STAT3 at two predicted regions of the CFTR promoter in AC16 cells. For panels F–I, all cell experiments were performed with three biological replicates. Data are presented as mean ± SD and analyzed by one‐way ANOVA or two‐way ANOVA (I).

• 

  FIGURE 5. Knockdown of MITF exacerbated cell injury in the OGD/OGR model. (A,B) MITF knockdown efficiency confirmed by RT‐qPCR and Western blot. (C,D) RT‐qPCR and Western blot analysis of MITF and CFTR expression after MITF knockdown and OGD/OGR treatment. The CFTR band shown corresponds to the mature band (C). MITF and CFTR levels were reduced in MITF‐silenced cells under OGD/OGR conditions. (E) Reduced cell viability following MITF knockdown by CCK‐8. (F) Impaired proliferation shown by EdU staining. (G) Increased apoptotic rate detected by flow cytometry. (H) NO, L‐arginine, and citrulline levels in AC16 cells under four conditions: NC, siMITF, NC+OGD/OGR, and siMITF+OGD/OGR. Data are shown as mean ± SD from three biological replicates.

• 

  FIGURE 6. MITF may alleviate OGD/OGR‐induced injury by regulating CFTR expression. (A) RT‐qPCR analysis showing successful overexpression of CFTR in AC16 cells transfected with CFTR plasmid (NC vs. oeCFTR). (B) Western blot analysis of CFTR expression after CFTR overexpression. Then NC and NC+OGD/OGR served as baseline and model controls, while siMITF, siCFTR, and siMITF+CFTR were used as loss‐of‐function and rescue controls. (C) Cell viability across experimental groups, with CFTR rescue showing partial improvement. (D) EdU staining indicating restored proliferation after CFTR re‐expression. (E) Apoptosis analysis showing attenuation of MITF knockdown‐induced apoptosis by CFTR restoration. (F) Predicted MITF binding regions within the CFTR promoter. (G) Dual‐luciferase assay showing MITF‐dependent activation of the CFTR promoter. (H) NO, L‐arginine, and citrulline levels measured in AC16 cells under NC, OGD/OGR, siMITF+OGD/OGR, and siMITF+oeCFTR+OGD/OGR conditions. Each experiment included three biological replicates. Data shown as mean ± SD; one‐way ANOVA.

• 

  FIGURE 7. Effects of CFTR knockdown and ARG1 inhibition on AC16 cells under OGD/OGR conditions. (A) RT‐qPCR detection of CFTR mRNA expression in AC16 cells transfected with NC or three CFTR‐targeting siRNAs (siCFTR‐1, siCFTR‐2, and siCFTR‐3). (B) Western blot analysis of CFTR protein levels in the same groups. Cell viability assessed by CCK‐8 in NC, NC+OGD/OGR, siCFTR+OGD/OGR, and siCFTR+OGD/OGR+ARG1 inhibitor (CB‐1158) groups. (D) EdU staining for cell proliferation in the same groups. (E) Flow cytometry analysis of apoptosis in the same groups. All experiments included three biological replicates. Data are presented as mean ± SD and analyzed by one‐way ANOVA.

• 

  FIGURE 8. Effects of MITF knockdown and CFTR overexpression on myocardial I/R injury in rats. (A) Echocardiographic assessment showing worsened systolic function after MITF knockdown and partial recovery following CFTR overexpression. (B) H&E staining demonstrating increased tissue damage with MITF silencing and improved morphology with CFTR rescue. (C) Elisa analysis of the CK and CK‐MB levels. (D) Western blot analysis of CFTR (The CFTR band shown corresponds to the mature band C), MITF, and cleaved‐caspase‐3 protein levels. (E) NO, L‐arginine, and citrulline levels measured in myocardial tissue of rats under sham, I/R, shMITF+I/R, and shMITF+oeCFTR+I/R conditions. Each group included six rats. Data presented as mean ± SD and analyzed by one‐way ANOVA.