E3-ubiquitin ligase TRIM6 aggravates myocardial ischemia/reperfusion injury via promoting STAT1-dependent cardiomyocyte apoptosis

Figure 1. TRIM6 expression is induced in the heart after MI/R injury. (A–D) Mice were subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At different time periods after reperfusion, the hearts were harvested for analyses. (A) The mRNA level of Trim6 in the heart was determined by qRT-PCR analysis. Actb was used as an endogenous control. Results relative to sham are shown. (B) The protein level of TRIM6 in the heart was determined by Western blotting analysis. β-Actin was used a loading control. The representative images (left) and band intensity relative to sham (right) are shown. (C) The expression of TRIM6 in the heart slices at 24 hrs after reperfusion was detected by immunohistochemistry (IHC). Scale bar, 100 μM. (D) The quantification of TRIM6 staining shown in (C) was shown as percentage of positive staining cardiomyocytes in the heart slices. Data are expressed as mean ± SD (n = 7). **, P < 0.01; *, P < 0.05 vs. sham group.

Figure 2. Cardiac TRIM6 promotes MI/R injury. (A–D) The mouse heart was pre-transfected in vivo with control siRNA (siCtrl) or siRNA targeting Trim6 (siTrim6) 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the heart and serum samples were harvested for analyses. (A) The protein level of TRIM6 in the heart was determined by Western blotting analysis. β-Actin was used a loading control. (B) The mid-myocardial cross sections of hearts were stained with TTC to show infarct size (IS). The representative images are shown. (C) The infarct severity in each group shown as in (B) is expressed as % of LV (IS/LV). (D) The level of serum creatine phosphokinase (CPK) from each group was measured (U/L). (E–H) The mouse heart was pre-infected in vivo with lentivirus expressing vector control (LV-vector) or Trim6 (LV-Trim6) 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the heart and serum samples were harvested for analyses. (E) The protein level of TRIM6 in the heart was determined by Western blotting analysis.…

Figure 3. TRIM6 promotes myocardial apoptosis after MI/R injury. (A–C) The mouse heart was pre-transfected in vivo with control siRNA (siCtrl) or siRNA targeting Trim6 (siTrim6) 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the hearts were harvested for analyses. (A) The myocardial apoptosis in the heart sections was identified by TUNEL staining. The quantitative analysis of TUNEL-positive cardiomyocytes is shown (%). (B) The caspase-3 activity of the heart was measured and expressed as nmol per hr per mg protein. (C) The protein expression of Bax and Bcl-2 was determined by Western blotting analysis. β-Actin was used a loading control. The representative images (left) and the Bax/Bcl-2 ratio relative to sham (right) are shown. (D–F) The mouse heart was pre-infected in vivo with lentivirus expressing vector control (LV-vector) or Trim6 (LV-Trim6) 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the hearts were harvested for analyses. The myocardial apoptosis (D), caspase-3 activity (E), and Bax/Bcl-2…

Figure 4. TRIM6 promotes myocardial apoptosis through inducing IKKε-mediated STAT-1 activation after MI/R injury. (A) The mouse heart was pre-transfected in vivo with control siRNA (siCtrl) or siRNA targeting Trim6 (siTrim6) 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the hearts were harvested for analyses. The protein expression of TRIM6, p-IKKε, IKKε, p-STAT1 and STAT1 in the heart was determined by Western blotting analysis. β-Actin was used a loading control. (B) The mouse heart was pre-infected in vivo with lentivirus expressing vector control (LV-vector) or Trim6 (LV-Trim6) 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the hearts were harvested for analyses. The protein expression of TRIM6, p-IKKε, IKKε, p-STAT1 and STAT1 in the heart was determined by Western blotting analysis. β-Actin was used a loading control. (C–F) The mouse heart was pre-infected in vivo with lentivirus expressing vector control (LV-vector) or Trim6 (LV-Trim6) in the presence or absence of CAY10576 or fludarabine 48…

Figure 5. Inhibition of IKKε/STAT-1 axis abrogates the promoted MI/R injury by TRIM6 overexpression. (A–B) The mouse heart was pre-infected in vivo with lentivirus expressing vector control (LV-vector) or Trim6 (LV-Trim6) in the presence or absence of CAY10576 or fludarabine 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the hearts were harvested for analyses. (A) The mid-myocardial cross sections of heart were stained with TTC, and the quantification of infarct size in each group (% of LV) is shown. (B) The level of serum creatine phosphokinase (CPK) from each group was measured. (C–F) The mouse heart was pre-infected in vivo with lentivirus expressing vector control (LV-vector), wild-type Trim6 (LV-Trim6-wt) or C15A RING mutant Trim6 (LV-Trim6-mut) 48 hrs before surgery. Mice were then subjected to sham surgery or experimental MI/R. Each group contained 7 mice. At 24 hrs after reperfusion, the hearts were harvested for analyses. (C) The lysates of heart tissues were co-immunoprecipitated (co-IP) by IKKε antibody. The expression of ubiquitin (K48) in the co-IP products and input samples was…

Figure 6. Proposed model of this study. Brief schematic diagram of the interplay between TRIM6 and MI/R injury. TRIM6 induces the activation of IKKε through K48-linked polyubiquitination and further activates p-STAT1, thus promoting myocardial apoptosis and MI/R injury, which in turn leads to the upregulation of TRIM6 via an unknown mechanism.