TRAF1 Exacerbates Myocardial Ischemia Reperfusion Injury via ASK1-JNK/p38 Signaling

Abstract

Background After acute myocardial infarction, the recovery of ischemic myocardial blood flow may cause myocardial reperfusion injury, which reduces the efficacy of myocardial reperfusion. Ways to reduce and prevent myocardial ischemia/reperfusion (I/R) injury are of great clinical significance in the treatment of patients with acute myocardial infarction. TRAF1 (tumor necrosis factor receptor-associated factor 1) is an important adapter protein that is implicated in molecular events regulating immunity, inflammation, and cell death. Little is known about the role and impact of TRAF1 in myocardial I/R injury. Methods and Results TRAF1 expression is markedly induced in wild-type mice and cardiomyocytes after I/R or hypoxia/reoxygenation stimulation. I/R models were established in TRAF1 knockout mice and wild type mice (n=10 per group). We demonstrated that TRAF1 deficiency protects against myocardial I/R-induced loss of heat function, inflammation, and cardiomyocyte death. In addition, overexpression of TRAF1 in primary cardiomyocytes promotes hypoxia/reoxygenation-induced inflammation and apoptosis in vitro. Mechanistically, TRAF1 promotes myocardial I/R injury through regulating ASK1 (apoptosis signal-regulating kinase 1)-mediated JNK/p38 (c-Jun N-terminal kinase/p38) MAPK (mitogen-activated protein kinase) cascades. Conclusions Our results indicated that TRAF1 aggravates the development of myocardial I/R injury by enhancing the activation of ASK1-mediated JNK/p38 cascades. Targeting the TRAF1-ASK1-JNK/p38 pathway provide feasible therapies for cardiac I/R injury.

Keywords: ASK1; TRAF1; apoptosis; cardiac ischemia reperfusion; inflammation.

Figure 1

TRAF1 expression is increased by reactive oxygen species during myocardial I/R. A, mRNA expression level of Traf 1 in heart samples of mice at indicated points after I/R (n=3 per group). B, TRAF1 protein expression level in heart tissues in the indicated groups (n=4 per group). C, TRAF1 protein expression level in neonatal rat primary cardiomyocytes exposed to H/R. D, TRAF1 protein expression level in neonatal rat primary cardiomyocytes exposed to H₂O₂. E, mRNA level of Traf 1 in neonatal rat primary cardiomyocytes in the indicated groups. F, TRAF1 protein expression level in neonatal rat primary cardiomyocytes in the indicated groups. Results shown are representative of 3 blots. For panels (B, C, D, and F), GAPDH served as loading control. For statistical analysis, 1‐way ANOVA was used for panels (A, B, D, and E); a 2‐tailed Student t test was used for panels (C and D). **P<0.01. H/R indicates hypoxia/reoxygenation; I/R, ischemia/reperfusion; NAC, N‐acetyl‐L‐cysteine; NS, not significant; TRAF1, tumor necrosis factor receptor–associated factor 1.

Figure 2

TRAF1 deficiency protected mouse hearts against I/R injury. A, Western blot of TRAF1 expression in KO and WT mice (n=3 per group). GAPDH served as loading control. B, Representative images of echocardiography in WT and TRAF1‐KO mice before (sham) and 24 hours after I/R. C and D, Echocardiographic (C) and hemodynamic (D) assessment of cardiac function in sham and I/R mice (n=10 per group). E, Images of myocardial tissues (Evans blue combination with TTC staining) from WT and TRAF1‐KO mice before (sham) and 24 hours after I/R. The ratios of AAR to left ventricle and infarction size to AAR were quantified by Image‐Pro Plus 6.0 (Media Cybernetics) in the WT and KO groups (n=5 per group). F and G, Quantitative results of serum CKP (F) and LDH (G) before (sham) and 24 hours after I/R (n=10 per group). For statistical analysis, 1‐way ANOVA was used for panels B, D, F, and G; a 2‐tailed Student t test was used for panel (E). **P<0.01. Red arrows represent the left ventricular end‐diastolic and end‐systolic dimension. AAR indicates area at risk; CPK, creatine phosphokinase; dP/dt max, decreased maximum rate of pressure increase; dP/dt min, decreased minimum rate of pressure increase; I/R, ischemia/reperfusion; KO, knockout; LDH, lactate dehydrogenase; LV, left ventricle; LVEF, left ventricular ejection fraction; NS, not significant; TRAF1, tumor necrosis factor receptor–associated factor 1; WT, wild type.

Figure 3

TRAF1 deficiency inhibited the I/R‐induced inflammatory response in heart. A, Immunofluorescence staining and the counted results of positive cells in each high‐power field of CD11b and LY6G in the I/R group (n=3 per group). Blue indicates nuclei; red indicates CD11b‐ and LY6G‐positive staining. B, Relative mRNA levels of cytokines, including TNF‐α, IFN‐γ, IL‐1β, Ccl2, ICAM1, IRF1, HIF‐1α, and PGE2 in heart tissues (n=4 per group). C, Western blot and quantitative results of NF‐κB signaling in sham and I/R groups. Results shown are representative of 3 blots, GAPDH served as loading control. For statistical analysis, a 2‐tailed Student t test was used for panels B and C. *0.01≤ P <0.05, **P<0.01. HPF, high‐power filed. Ccl2 indicates C‐C motif chemokine ligand 2; HIF‐1α, hypoxia inducible factor 1 subunit α; ICAM1, intercellular adhesion molecule 1; IFN‐γ, interferon γ; IL‐1β, interleukin 1β; I/R. ischemia/reperfusion; IRF1, interferon regulatory factor 1; KO, knockout; LY6G, lymphocyte antigen 6G; NF‐κB, nuclear factor κB; p‐, phosphorylated; PGE2, prostaglandin E2; TNF‐α, tumor necrosis factor α; TRAF1, tumor necrosis factor receptor–associated factor 1; WT, wild type.

Figure 4

TRAF1 deficiency decreased I/R‐induced apoptosis. A, Immunofluorescence staining and positive cell‐count results of TUNEL in heart tissues (n=3 per group). Blue indicates nuclei; green indicates TUNEL positive staining. B, Relative mRNA levels of apoptosis‐related molecules in heart tissues (n=4 per group). C, Western blot and quantitative results of apoptosis‐related protein expression levels in sham and I/R groups. Results shown are representative of 3 blots, GAPDH served as loading control. For statistical analysis, a 2‐tailed Student t test was used for panels B and C. **P<0.01. Bax indicates BCL2 associated X, apoptosis regulator; Bcl2, BCL2 apoptosis regulator; C‐Casp3, cleaved caspase 3; HPF, high‐power filed; I/R, ischemia/reperfusion; KO, knockout; TRAF1, tumor necrosis factor receptor–associated factor 1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling; WT, wild type.

Figure 5

TRAF1 overexpression exacerbates inflammation and apoptosis in cardiomyocytes after H/R stimulation in vitro. A, Western blot results of TRAF1 in AdTRAF1 and AdGFP control group. B, Relative mRNA levels of cytokines, including TNF‐α, IL‐1β, IL‐6, and IL‐10, in AdTRAF1‐ and AdGFP‐group cardiomyocytes exposed to H/R for 3 hours. C, Western blot and quantitative results of NF‐κB signaling in AdTRAF1 and AdGFP control group cardiomyocytes exposed to H/R for 3 hours or not. D, Relative mRNA levels of apoptosis‐related molecules in AdTRAF1‐ and AdGFP‐group cardiomyocytes exposed to H/R for 3 hours. E, Western blot and quantitative results of apoptosis‐related protein expression levels in AdTRAF1‐ and AdGFP‐group cardiomyocytes exposed to H/R for 3 hours or not. For panels B and D, results shown are representative of 3 independent experiments. For panels (A, C, and E), results shown are representative of 3 blots. GAPDH served as loading control. For statistical analysis, a 2‐tailed Student t test was used for panels (B through E). **P<0.01. Ad indicates adenoviral vector encoded; Bax, BCL2 associated X, apoptosis regulator; Bcl2, BCL2 apoptosis regulator; C‐Casp3, cleaved caspase 3; GFP, green fluorescent protein; H/R, hypoxia reoxygenation; IkBα, nuclear factor κB inhibitor α; Ikkβ, inhibitor of nuclear factor κB kinase subunit β; IL, interleukin; NF‐κB, nuclear factor κB; p‐, phosphorylated; TNF‐α, tumor necrosis factor α; TRAF1, tumor necrosis factor receptor–associated factor 1.

Figure 6

TRAF1 promotes activation of the ASK1–JNK/p38 signaling pathway after myocardial I/R. A, Western blot and quantitative results of phosphorylated and total ASK1, ERK, JNK, and p38 protein from heart tissue of mice in the indicated groups. B, Western blot and quantitative results of phosphorylated and total ASK1, ERK, JNK, and p38 protein in neonatal rat cardiomyocytes infected with AdGFP and AdTRAF1 exposed to H/R for 3 hours or not. Results shown are representative of 3 blots, and GAPDH served as loading control. For statistical analysis, a 2‐tailed Student t test was used for panels (B through E). **P<0.01. Ad indicates adenoviral vector encoded; ASK1, apoptosis signal‐regulating kinase 1; GFP, green fluorescent protein; H/R indicates hypoxia/reoxygenation; I/R, ischemia/reperfusion; JNK/p38, c‐Jun N‐terminal kinase/p38; KO, knockout; NS, not significant; p‐, phosphorylated; TRAF1, tumor necrosis factor receptor–associated factor 1; WT, wild type.

Figure 7

The effect of TRAF1 on cardiomyocyte inflammation and apoptosis is mediated by ASK1 activity. A, Western blot results and quantitative results of TRAF1, ASK1, and phosphorylated ASK1 in the indicated groups. B, Relative mRNA levels of cytokines, including TNF‐α, IFN‐γ, IL‐1β and IL‐6 in the indicated groups exposed to H/R for 3 hours. C, Western blot results and quantitative results of NF‐κB signaling in the indicated groups exposed to H/R for 3 hours. D, Relative mRNA levels of apoptosis‐related molecules in the indicated groups exposed to H/R for 3 hours. E, Western blot results and quantitative results of apoptosis‐related protein expression levels in the indicated groups exposed to H/R for 3 hours. For panels (A, C, and E), results shown are representative of 3 blots, and GAPDH served as loading control. For panels (B and D), results shown are representative of 3 independent experiments. For statistical analysis, 1‐way ANOVA was used for panels A through E. *0.01≤ P<0.05, **P<0.01. Ad indicates adenoviral vector encoded; ASK1, apoptosis signal‐regulating kinase 1; Bad, BCL2 associated agonist of cell death; Bax, BCL2 associated X, apoptosis regulator; Bcl2, BCL2 apoptosis regulator; C‐Casp3, cleaved caspase 3; GFP, green fluorescent protein; H/R, hypoxia/reoxygenation; IFN‐γ, interferon γ; IkBα, nuclear factor κB inhibitor α; Ikkβ, inhibitor of nuclear factor κB kinase subunit β; IL, interleukin; NF‐κB, nuclear factor κB; NS, not significant; p‐, phosphorylated; TNF‐α, tumor necrosis factor α; TRAF1, tumor necrosis factor receptor–associated factor 1.