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. 2020 Nov;24(21):12401-12410.
doi: 10.1111/jcmm.15748. Epub 2020 Sep 25.

Knockdown of circ_0060745 alleviates acute myocardial infarction by suppressing NF-κB activation

Changlin Zhai  1   2 , Gang Qian  1   2 , Huajun Wu  2 , Haihua Pan  2 , Shuoyin Xie  1 , Zhewei Sun  1 , Pingyang Shao  2 , Guanmin Tang  2 , Huilin Hu  1   2 , Song Zhang  3
Affiliations

Knockdown of circ_0060745 alleviates acute myocardial infarction by suppressing NF-κB activation

Changlin Zhai et al. J Cell Mol Med. 2020 Nov.

Abstract

It has been shown that circRNAs are involved in the development of heart diseases. However, few studies explored the role of circRNAs in acute myocardial infarction (AMI). The present study aims to investigate the role of circ_0060745 in the pathogenesis of AMI. We found that the expression of circ_0060745 was significantly increased in the myocardium of AMI mice and was mainly expressed in myocardial fibroblasts. The knockdown of circ_0060745 decreased myocardial infarct size and improved systolic cardiac functions after AMI. The knockdown of circ_0060745 in cardiac fibroblasts inhibited the migration of peritoneal macrophage, the apoptosis of cardiomyocytes and the expressions of IL-6, IL-12, IL-1β, TNF-α and NF-κB under hypoxia. Overexpression of circ_0060745 caused an increase in infarct size and worsened cardiac functions after AMI. In summary, our findings showed that knockdown of circ_0060745 mitigates AMI by suppressing cardiomyocyte apoptosis and inflammation. These protective effects could be attributed to inhibition of NF-κB activation.

Keywords: NF-κB signalling pathway; acute myocardial infarction; circ_0060745.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Relative circ_0060745 expression was up‐regulated after AMI. (A) Expression of circ_0060745 at 0, 2, 6, 12 and 24 h after AMI. (B) Expression of circ_0060745 in cardiomyocytes and cardiac fibroblasts at 24 h. (C) Expression of circ_0060745 in the myocardium of sham and AMI mice transfected with sh‐NC and sh‐Circ. (D) Area at risk (AAR) and infarct size (IS) at 3 d after AMI in mice transfected with sh‐NC and sh‐Circ. (E) Expression of circ_0060745 in the myocardium of sham and AMI mice transfected with vector plasmid and Circ. (F) Area at risk (AAR) and infarct size (IS) at 3 d after AMI in mice transfected with sh‐NC and sh‐Circ. Data are shown as means ± SD. *, P < 0.05
FIGURE 2
FIGURE 2
Circ_0060745 improved cardiac function after AMI. Cardiac function was assessed at 3 d after AMI by using metrics including (A) left ventricular ejection fraction (LVEF), (B) left ventricular fractional shortening (LVFS), (C, D) diastolic and systolic left ventricular internal diameter (LVID,d and LVID,s), (E) left ventricular (LV) maximum pressure and (F) dP/dt maximum rate. A‐F showed the effects of circ_0060745 knockdown by shRNA, while G‐L showed the results of circ_0060745 overexpression. Data are shown as means ± SD. *, P < 0.05
FIGURE 3
FIGURE 3
Knockdown of circ_0060745 inhibited apoptosis after AMI. (A) Cardiac cell apoptosis assessed by TUNEL staining. Scale bar: 50 μm. (B) The proportion of apoptotic cells calculated as the percentage of TUNEL‐positive cells. (C‐E) The protein expression of Bcl‐2 and Bax measured by Western blot at 3 d after AMI. Asterisks indicated statistical significance for AMI + sh‐NC vs Sham + sh‐NC and AMI + sh‐Circ vs AMI + sh‐NC. Data are shown as means ± SD. *, P < 0.05
FIGURE 4
FIGURE 4
Overexpression of circ_0060745 promoted apoptosis after AMI. (A) Cell apoptosis assessed by TUNEL staining. Scale bar: 50 μm. (B) The proportion of apoptotic cells calculated as the percentage of TUNEL‐positive cells. (C‐E) The protein expression of Bcl‐2 and Bax measured by Western blot at 3 d after AMI. Asterisks indicated statistical significance for AMI + sh‐NC vs Sham + sh‐NC and AMI + sh‐Circ vs AMI + sh‐NC. Data are shown as means ± SD. *, P < 0.05
FIGURE 5
FIGURE 5
Knockdown of circ_0060745 inhibited the migration of peritoneal macrophages. (A) The relative expression of circ_0060745 in nCFs cultured under hypoxia for 0, 2, 6, 12 and 24 h. (B) Sh‐Circ decreased the relative expression of circ_0060745 in nCFs cultured under hypoxic (H) or normoxic (N) for 12 h. (C, D) The migration of peritoneal macrophages co‐cultured with medium from nCFs transfected with sh‐NC or sh‐Circ under hypoxic (H) or normoxic (N) conditions for 12 h. (E‐H) The expressions of IL‐6, IL‐12, IL‐1β and TNF‐α. Data are shown as means ± SD. *, P < 0.05
FIGURE 6
FIGURE 6
Knockdown of circ_0060745 in cardiac fibroblasts inhibited the apoptosis of cardiomyocytes. (A, B) The apoptosis of cardiomyocytes assessed by flow cytometry. (C‐E) The protein expression of Bcl‐2 and Bax in cardiomyocytes co‐cultured with nCFs for 12 h. Data are shown as means ± SD. *, P < 0.05
FIGURE 7
FIGURE 7
Knockdown of circ_0060745 inhibited the translocation of NF‐κBp65. (A) The expression of NF‐κBp65, p‐IκBα and IκBα measured using Western blot. Data were normalized to the level of GAPDH. (B) The level of NF‐κBp65. (C) Ratio of p‐IκBα/IκBα. Data are shown as means ± SD. *, P < 0.05
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