TRF2 Recovers Ischemic Postconditioning Cardioprotection in Aged Myocardiocytes by Regulating CSNK2A2 Localization and FUNDC1 Dephosphorylation

Figures

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  FIGURE 1. Failure to improve injury during ischemic postconditioning in elderly mice is associated with decreased expression of TRF2. (A) Echocardiography shows LVEF% and FS% 12 h after the operation (n = 5). (B) CK‐MB expression detected by ELISA of venous blood of mice (n = 5). (C) IL‐6 concentrations detected by ELISA of venous blood in mice (n = 5). (D) TERT mRNA expression by qRT‐PCR (n = 5). (E) Telomere length detected by RT‐PCR (n = 5). (F) TUNEL staining of myocardial apoptosis in each treatment group (n = 3). (G) Immunohistochemistry of TRF2 protein expression in myocardial tissue (n = 3). (H) Western blot analysis of TRF2, Bcl2, Bax, and P53 protein expression in the myocardium of adult aged mice in the sham and I/Post groups (n = 5).

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  FIGURE 2. TRF2 reduces I/Post injury in aged mice. (A) Schema of mouse experiments using the AAV model. (B) Western blot analysis of TRF2, Bcl‐2, bax, and P53 protein expression in the myocardium of aged mice after I/Post (n = 5). (C) Immunohistochemistry of TRF2 protein expression in the myocardium in each treatment group (n = 3). (D) TUNEL staining of myocardial apoptosis in each treatment group (n = 3). (E) CK‐MB concentrations by ELASA (n = 5). (F) IL‐6 concentrations by ELISA. (G) TERT mRNA expression by qRT‐PCR (n = 5). (H) Telomere length by qRT‐PCR (n = 5). (I) Echocardiography shows the LVEF% and FS% (n = 5).

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  FIGURE 3. H/Post of aging cardiomyocytes does not improve H/R injury, which is related to decreased TRF2 protein expression. (A) HL‐1 cells were treated with 0, 75, 150, 300, and 600 μM H2O2 for 4 h, and the CCK‐8 assay was used to detect myocardial cell activity after 24, 48, and 72 h (F time = 705.3, p < 0.0001; F concentration = 417.9, p < 0.0001). (B) TERT mRNA expression levels of HL‐1 cells treated with 0, 75, 150, 300, and 600 μM for 4 h, after 24 h measured by qRT‐PCR (F = 4550.4, p < 0.0001). (C) Telomere length by qRT‐PCR (F = 1143.7, p = 0.0047). (D) SA‐β‐gal staining of HL‐1 cells treated with 0, 75, 150, 300, and 600 μM after 24 h (*p < 0.05 vs. 0 μM group). (E) HL‐1 cells were treated with different concentrations of H2O2 for 4 h, and after 24 h, protein was collected. Western blot analysis of P53, TRF2, Bcl2, and Bax relative protein expression levels is shown (*p < 0.05 vs. 0 μM group). (F) Western blot analysis of P53, TRF2, Bcl2, and Bax relative protein expression levels in HL‐1 cells treated after different periods of hypoxia (*p < 0.05 vs. 0 h group). (G) Western blot analysis of P53, TRF2, Bcl2, and Bax protein expression after H/R and H/Post in HL‐1 cells treated…

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  FIGURE 4. Knockdown of TRF2 increases H/Post injury of aged cardiomyocytes. The HL‐1 cell line was treated with 150 μM H2O2 for 4 h. After 24 h, the cells were transfected with control vector or shTRF2, followed by the H/Post experiment after another 24 h. (A) Western blot analysis of TRF2, P53, Bcl‐2/Bax, IL‐6, SIRT1, and PCG‐1α protein expression in each treatment group. (B) Flow cytometry of cellular apoptosis and the necrosis ratio. (C) Mitochondrial tracing staining to indicate mitochondrial morphology. (D) Immunofluorescence of γ‐H2AX staining in each treatment group. (E) Fluorescence of the JC‐1 probe as observed by fluorescence microscopy. (F) The degree of cellular senescence shown by SA‐β‐gal dye. (G) TERT mRNA expression levels by qRT‐PCR（*p < 0.05）. (H) Telomere length by qRT‐PCR (*p < 0.05).

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  FIGURE 5. TRF2 inhibits cGAS/STING pathway activation by promoting mitochondrial autophagy following H/Post. (A) HL‐1 cells were treated with 150 μM H2O2 for 4 h, and H/Post was performed after 24 h. Western blot analysis of LC3BII/I, P62, cGAS, STING, and p‐NF‐κB protein expression is shown. (B) Hl‐1 cells were treated with 150 μM H2O2 and then transfected with pcDNA or pcDNA‐TRF2. Twenty‐four or 48 h after H/Post, protein expression was analyzed by western blotting. (C–F) A mitochondrial lysosomal immunofluorescence co‐staining experiment and electron microscopic showed the occurrence of mitophagy.

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  FIGURE 6. TRF2 interacts with CSNK2A2 to regulate the dephosphorylation of FUNDC1. (A) After transient pcDNA‐TRF2 or empty vector transfection in the HL‐1 cell line, western blotting was performed. CSNK2A2, FUNDC1, and p‐FUNDC1 protein expression is shown. (B) CSNK2A2, FUNDC1, and p‐FUNDC1 protein expression after transfection of shTRF2 or control vector into the HL‐1 cell line as shown by a western blot. (C) Using HL‐1 cells, endogenous immunoprecipitation showed an interaction between TRF2 and CSNK2A2. (D) HL‐1 cells were co‐transfected with Flag‐TRF2 and Myc‐CSNK2A2, and then cells were lysed and immunoprecipitated with anti‐Flag antibody 24 h after transfection. (E) Immunofluorescence images show that the changes in expression and positioning of CSNK2A2 protein after overexpressing TRF2. (F) CSNK2A2, FUNDC1, and p‐FUNDC1 protein expression in the nucleus, cytoplasm, and mitochondria as shown by a western blot.

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  FIGURE 7. TRF2 improves H/Post injury of aging cardiomyocytes by regulating CSNK2A2/FUNDC1. (A) Western blot of CSNK2A2, FUNDC1, and p‐FUNDC1 protein expression with overexpression of CSNK2A2. (B) Western blot analysis of LC3B, P62, cGAS/STING, Bcl‐2/Bax, and p‐NF‐κB protein expression after transfection of CSNK2A2 and co‐transfection of CSNK2A2 and TRF2 in aged cardiomyocytes following H/Post. (C) Western blot of FUNDC1 and p‐FUNDC1 protein expression with overexpression of FUNDC1ser17A. (D) Western blot analysis of LC3B, P62, cGAS/STING, Bcl‐2/Bax, and p‐NF‐κB protein expression after transfection of FUNDC1ser17A and co‐transfection of FUNDC1ser17A and shTRF2 in aged cardiomyocytes following H/Post.

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  FIGURE 8. Schematic diagram showing the mechanism by which TRF2 alleviates myocardial I/Post injury in aged myocardiocytes.