Right ventricular pressure overload disturbs T-tubule maturation via MEF2D’s transcriptional regulation of BIN1

Figures

• 

  Figure 1. T-tubule maturation deficit in pediatric patients with RVPO. (A) Representative echocardiographic images from patients with ventricular septal defect (VSD, nonoverload group) and tetralogy of Fallot (TOF, pressure-overload group). (B) Quantification of Right Ventricular fractional area change (RV FAC) and transvalvular pressure gradient (TPG) measured by transthoracic echocardiography (n = 5 patients per group). (C) Representative images of T-tubules in the VSD and TOF groups. Scale bar: 25 μm. Zoomed-in view indicates red arrows that show T-elements, blue arrows indicate L-elements, and yellow lines denote the sarcolemma. (D) Quantification of T-element density, T-tubule regularity, and T-tubule integrity index (n = 6 rats per group, with 30 cardiomyocytes from 5 independent sections per rat). Data are presented as mean ± SD. P values were calculated by 2-tailed Student’s t test. **P < 0.01, ***P < 0.001, ****P < 0.0001.

• 

  Figure 2. RVPO impairs T-tubule maturation in neonatal rats. (A) Representative T-tubule images from sham and PAB groups. Scale bar: 10 μm. Zoomed-in panels show green arrows that indicate T-elements, purple arrows that indicate L-elements, and yellow arrows that denote the sarcolemma. (B) Quantification of T-element density, T-tubule regularity, and T-tubule integrity index (n = 6 rats per group, with 30 cardiomyocytes from 5 independent sections per rat). (C) Upper panel: representative calcium transient traces from sham group cardiomyocytes. Lower panel: representative traces from PAB group cardiomyocytes. Note the elevated basal calcium signal intensity in PAB cardiomyocytes (~1.4) compared with sham (~1.0). (D) Quantification of calcium transient amplitude and time to peak (n = 15 cardiomyocytes per group; Mann-Whitney U test). (E) Principal component analysis of transcriptional profiles from sham and PAB samples. (F) Volcano plot of differentially expressed genes in PAB versus sham (adjusted P < 0.05, |log2FC| > 1). (G) Gene Ontology enrichment analysis of downregulated genes in PAB RVs. (H) Heatmap of downregulated genes in PAB-influenced RVs. Data are presented as mean ± SD. P…

• 

  Figure 3. RVPO alters chromatin accessibility of T-tubule–associated genes. (A) Heatmap of shared and differentially accessible ATAC-seq peaks and associated genes. (B) Summary of differential gene expression and chromatin accessibility. “Up-regulated” and “Down-regulated” refer to RNA-seq results; “More accessible” and “Less accessible” refer to ATAC-seq results. (C) Enrichment analysis of genes with reduced chromatin accessibility in PAB RVs. (D) Chromatin accessibility profile around the transcription start site (TSS) of the Bin1 gene. (E) Representative BIN1 Western blot in sham and PAB groups. (F) Quantification of pBIN1 protein expression (n = 6 rats per group). (G) Quantification of cBIN1 protein expression (n = 6 rats per group). (H) Representative BIN1 Western blot in VSD and TOF patient samples. (I) Quantification of pBIN1 expression in human samples (n = 5 patients per group). (J) Quantification of cBIN1 expression in human samples (n = 5 patients per group). Data are presented as mean ± SD. P values were calculated by 2-tailed Student’s t test. **P < 0.01, ***P < 0.001.

• 

  Figure 4. BIN1 overexpression rescues T-tubule maturation in neonatal RVPO rats. (A) Schematic of the BIN1 overexpression strategy. (B and C) AAV9 infection efficiency. (D) Representative BIN1 WES immunoblot from isolated RV cardiomyocytes. (E) Quantification of pBIN1 expression (n = 6 rats). (F) Quantification of cBIN1 expression (n = 6 rats). (G) Right ventricular fractional area change (RV FAC, %) of rats at P21. (H) Left ventricular ejection fraction (LVEF, %). (I) Representative T-tubule images from BIN1-overexpressing (BIN1-OE) and control cardiomyocytes in PAB RVs. Scale bar: 25 μm. Zoomed-in panels show green arrows that indicate T-elements, purple arrows that indicate L-elements, and yellow arrows that denote the sarcolemma. (J–L) Quantification of T-element density (J), T-tubule regularity (K), and T-tubule integrity index (L) (n = 6 rats per group, with 30 cardiomyocytes per rat). Data are presented as mean ± SD. P values were calculated by 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

• 

  Figure 5. Mef2d motif is highly enriched in PAB rat RVs. (A) Dot plot showing enrichment of known transcription factor motifs. (B) Comparison of Mef2a, Mef2b, and Mef2d motif enrichment between sham and PAB RVs. (C) Sequence logo of the Mef2d motif. (D) Chromatin accessibility profile around the Mef2d TSS. Data are presented as mean ± SD. P values were calculated by 2-tailed Student’s t test. *P < 0.05.

• 

  Figure 6. MEF2D knockdown rescues T-tubule maturation in neonatal RVPO rats. (A) Schematic of the MEF2D knockdown strategy. (B and C) AAV9 infection efficiency. (D) Representative MEF2D WES immunoblot from isolated RV cardiomyocytes. (E) Quantification of MEF2D expression (n = 6 rats). (F) Right ventricular fractional area change (RV FAC, %) of rats at P21. (G) Left ventricular ejection fraction (LVEF, %). (H) Representative BIN1 WES immunoblot from isolated cardiomyocytes. (I) Quantification of pBIN1 expression (n = 6 rats). (J) Quantification of cBIN1 expression (n = 6 rats). (K) Representative T-tubule images from MEF2D-knockdown (MEF2D-KD) and control cardiomyocytes in PAB RVs. Scale bar: 25 μm. Green arrows indicate T-elements, purple arrows indicate L-elements, and yellow arrows denote the sarcolemma. (L–N) Quantification of T-element density (L), T-tubule regularity (M), and T-tubule integrity index (N) (n = 6 rats per group, with 30 cardiomyocytes per rat). Data are presented as mean ± SD. P values were calculated by 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001.

• 

  Figure 7. MEF2D inhibits Bin1 promoter activity. (A) Schematic of the dual-luciferase reporter assay. (B) Luciferase activity assays evaluating MEF2D regulation of the Bin1 promoter. Data were analyzed by Kruskal-Wallis test with post hoc Dunn’s test. **P < 0.01, ***P < 0.001. (C) Graphical summary of the RVPO/MEF2D/BIN1/T-tubule pathway identified in this study.