4E-BP inhibition ameliorates heart failure through translational upregulation of SERCA2a and modulation of mitochondrial redox signaling in cardiomyocytes

Figures

• 

  Fig. 1. 4E-BP1/2 double knockout (DKO) conferred protection to mice against transverse aortic constriction (TAC)-induced mortality, cardiac remodeling, and LV dysfunction. (A) Mortality rates elevated by TAC were attenuated in 4E-BP1/2 DKO mice. (B-E) Following TAC, 4E-BP1/2 DKO had no impact on the ratio of left ventricular (LV) weight to body weight; however, it mitigated TAC-triggered increases in the ratios of left atrial (LA) weight, lung weight, and right ventricular (RV) weight to body weight. (F–H) Echocardiography and a 1.2F pressure transducer were utilized to evaluate LV ejection fraction, as well as LV dP/dtmax and dP/dtmin, in wild-type (WT) and 4E-BP1/2 DKO mice, respectively. (I) Masson's trichrome and wheat germ agglutinin (WGA) staining were performed on representative heart sections, and the relative collagenous area of the LV and cross-sectional area of myocytes were quantified. Scale bar = 50 μm. (J) Relative mRNA levels of genes associated with hypertrophy and fibrosis were measured by qPCR. All values are expressed as mean ± SEM. ∗ represents a significance level of p < 0.05; ∗∗ represents a significance level of p < 0.01; ∗∗∗ represents a significance level of p <…

• 

  Fig. 2. 4E-BP1/2 DKO attenuated cardiac remodeling and LV dysfunction early after TAC. (A) 4E-BP1/2 DKO did not affect LV hypertrophy after 2 weeks of TAC. (B and C) 4E-BP1/2 DKO significantly attenuated TAC-induced LV hypertrophy and pulmonary congestion. (D) TAC for 2 weeks had no effect on RV hypertrophy in either WT or 4E-BP1/2 DKO mice. (E and F) After TAC for 2 weeks, 4E-BP1/2 DKO mice had lower LV end diastolic diameter and LV end systolic diameter. (G, H and I) TAC caused significantly less LV dysfunction in 4E-BP1/2 DKO mice, as demonstrated by a higher ejection fraction, a higher LV dP/dtmax, and a lesser decline of LV dP/dtmin. The values are presented as means ± SEMs. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01 and ∗∗∗ indicates p < 0.001.

• 

  Fig. 3. 4E-BP1/2 DKO modulated TAC-induced alterations in gene expression profiles and the enrichment of related pathways. (A) Volcano plots were used to visualize the fold changes of differentially expressed genes (DEGs) in the LV of WT and 4E-BP1/2 DKO mice at 4 weeks post sham or TAC surgery. DEGs were defined using a threshold of adjusted P value ≤ 0.001 and an absolute log2 fold change (|log2FC|) ≥1. (B) A Venn diagram illustrated the number of unique and overlapping DEGs across different groups. (C) Gene set enrichment analysis (GSEA) was conducted on DEGs from the WT-sham vs. WT-TAC and WT-TAC vs. DKO-TAC comparisons. Forest plots displayed high-ranking GSEA pathways that were either suppressed or activated, based on the normalized enrichment score (NES), in the two groups. (D) Enrichment plots showed representative pathways that were activated or suppressed in the WT-TAC and DKO-TAC groups. (E) A heatmap depicted the expression of genes involved in the electron transfer chain pathway. (F) qPCR was performed to determine the relative mRNA levels of selected genes in the mitochondrial electron transfer chain pathway. All values are expressed as mean ± SEM. ∗ signifies p < 0.05; ∗∗…

• 

  Fig. 4. 4E-BP1/2 DKO alleviated TAC-induced mitochondrial dysfunction in the heart. (A) Substrate uncoupler inhibitor titration protocols were applied to measure the oxygen consumption rate (OCR) in saponin-permeabilized LV tissues. Abbreviations: G + M + P (glutamate, malate, pyruvate), CytC (cytochrome C), Suc (succinate), CCCP (carbonyl cyanide m-chlorophenylhydrazone), OXPHOS (oxidative phosphorylation), ETSmax (maximal capacity of the electron transport system), ROX (residual oxygen consumption). (B) Western blotting was performed to analyze LV lysates with a total OXPHOS rodent antibody cocktail. The standard in Lane 1 represents the positive control (rat heart mitochondria). (C) Myocardial ATP content was determined in WT and 4E-BP1/2 DKO mice at 4 weeks post sham or TAC surgery. (D) Relative mitochondrial DNA (mtDNA) levels were determined by calculating the ratio of mtDNA to nuclear DNA (nDNA) in the LV of WT and 4E-BP1/2 DKO mice. (E) Transmission electron microscopy (TEM) was used to randomly examine representative ultrathin heart sections (upper panel; scale bar = 500 nm). MitoSOX staining was conducted on representative frozen heart sections (lower panel; scale bar = 50 μm).…

• 

  Fig. 5. 4E-BP1/2 DKO preserved the mRNA levels of transcriptional nuclear factors related to mitochondrial energy metabolism and their downstream targets after TAC. LV samples collected from 4E-BP1/2 DKO mice and wild type mice 4 weeks after surgery were used for determining transcriptional nuclear factors related to mitochondrial biogenesis and their downstream targets. The values are presented as means ± SEMs. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01 and ∗∗∗ indicates p < 0.001.

• 

  Fig. 6. Knockdown of 4E-BP1 mitigated TAC-induced LV dysfunction, fibrosis, and mitochondrial impairment. (A) The experimental design is outlined as follows: one week after TAC, WT BALB/c mice received tail vein injections of AAV9-GFP or AAV9-sh4ebp1, followed by a 3-week maintenance period. (B) Mortality rates in each group were recorded after injection. (C-E) At the end of the experiment, the ratios of LV weight and lung weight to body weight, as well as LV EF values, were measured. (F) 4E-BP1 knockdown led to a reduction in the mRNA levels of genes related to hypertrophy and fibrosis. (G) Masson's trichrome, WGA, and MitoSOX staining were performed on representative heart sections. The relative collagenous area, myocyte cross-sectional area, and mitochondrial superoxide levels were quantified. (H) The levels of core proteins in OXPHOS complexes in LV lysates were determined by Western blotting analysis. The sample loaded in Lane 1 (standard) serves as the positive control, which consists of rat heart mitochondria. (I, J) The ATP content and mtDNA/nDNA ratio in the LV of pressure-overloaded mice were measured. (K) LV lysates were analyzed via Western blotting. All values are expressed…

• 

  Fig. 7. Overexpression of 4E-BP1 aggravated TAC-induced LV dysfunction, fibrosis, and mitochondrial impairment. (A) The experimental workflow is shown: one week after TAC, WT BALB/c mice were injected with AAV9-GFP or AAV9-cTnT-4ebp1 via the tail vein and then maintained for an additional 2 weeks. (B-D) At the end of the experiment, the ratios of LV weight and lung weight to body weight, along with LV ejection fraction values, were determined. (E) Representative heart sections were stained with Masson's trichrome, WGA, and MitoSOX. The relative collagenous area, myocyte cross-sectional area, and mitochondrial superoxide levels were quantified. (F) The OCR in saponin-permeabilized LV was measured. (G) The expression of core proteins in OXPHOS complexes in the LV was assessed by Western blotting. (H, I) The LV ATP content and mtDNA/nDNA ratio were measured. (J) The levels of target proteins in the LV lysates were determined by Western blotting. All values are expressed as mean ± SEM. ∗ signifies p < 0.05; ∗∗ signifies p < 0.01; and ∗∗∗ signifies p < 0.001.

• 

  Fig. 8. 4E-BP1/2 DKO enhanced cardiac SERCA2a protein expression by selectively improving its translation efficiency. (A) In mice subjected to sham or TAC surgery, 4E-BP1/2 DKO increased LV SERCA2a expression. Western blotting and qPCR were employed to assess SERCA2a protein expression and mRNA levels, respectively. (B) 4E-BP1 knockout elevated LV SERCA2a levels without influencing SERCA2a mRNA levels. (C) In pressure-overloaded mice, 4E-BP1 knockdown or overexpression exerted an effect on cardiac SERCA2a protein expression. (D) 4E-BP1/2 DKO promoted the recruitment of SERCA2a mRNA to the eIF4F complex in LV tissues. LV lysates were immunoprecipitated using an anti-eIF4GI antibody. Western blotting was used to determine the protein input (LV lysates) and the amount of proteins associated with eIF4GI. qPCR was employed to quantify SERCA2a mRNA pulled down in the immunocomplex. (E) Adult cardiomyocytes were incubated with 35S-Met/35S-Leu to label newly synthesized proteins. SERCA2a was immunoprecipitated from cell lysates and separated via SDS-PAGE. (F) Adult rat cardiomyocytes were infected with adenovirus carrying a construct encoding GFP, WT HA-4E-BP1, or the constitutively active,…

• 

  Fig. 9. Serca2a knockdown in 4E-BP1/2 DKO mice aggravated TAC-induced LV dysfunction, fibrosis, and mitochondrial impairment. (A) The experimental protocol is summarized: one week after TAC, 4E-BP1/2 DKO mice were administered AAV9-GFP or AAV9-shSerca2a via tail vein injection and then maintained for an additional 3 weeks. (B-D) At the end of the experiment, the ratios of LV weight and lung weight to body weight, as well as LV ejection fraction values, were measured. (E) Serca2a knockdown resulted in an increase in the mRNA levels of genes related to hypertrophy and fibrosis. (F) Masson's trichrome, WGA, and MitoSOX staining were performed on representative heart sections (scale bar = 50 μm). The relative collagenous area, myocyte cross-sectional area, and mitochondrial superoxide levels were quantified. (G) The OCR in saponin-permeabilized LV was measured. (H) Serca2a knockdown reduced the expression of core proteins in OXPHOS complexes in the LV of 4E-BP1/2 DKO mice after TAC. (H, I) The LV ATP content and mtDNA/nDNA ratio were assessed in 4E-BP1/2 DKO mice injected with AAV9-GFP or AAV9-shSerca2a following TAC. (J) LV lysates were analyzed via Western blotting. All values are…

• 

  Fig. 10. 4E-BP1 regulated mitochondrial respiration and oxidative stress in H9c2 cardiomyocytes. After infection with adenovirus carrying sh4ebp1 or scramble shRNA (shScr), H9c2 cells were treated with 50 μM phenylephrine (PE) for 48 h. (A) Western blotting was used to analyze cell lysates and assess the protein expression of 4E-BP1 and SERCA2a. (B) After treatment, cells were stained with FITC-labeled phalloidin plus DAPI (upper panel; scale bar = 50 μm), mitochondrial tracker green (middle panel; scale bar = 5 μm), and MitoSOX (lower panel; scale bar = 50 μm). (C) The protein expression of mitochondrial complexes was assessed by Western blotting. (D) 4E-BP1 knockdown increased the OCR in PE-treated H9c2 cells. (E-G) H9c2 cells were infected with Ad-4E-BP1 or empty vector (Ad-empty) and then treated with PE for 48 h. Cell lysates were analyzed via Western blotting (E). 4E-BP1 overexpression decreased the OCR (F) and increased intracellular superoxide levels (G) in PE-treated H9c2 cells. (H-J) H9c2 cells were infected with adenoviruses encoding sh4ebp1 plus shSerca2a or shScr, and subsequently treated with PE for 48 h. Cell lysates were analyzed via Western blotting (H). After PE…