Korean J Physiol Pharmacol.  2023 Mar;27(2):177-185. 10.4196/kjpp.2023.27.2.177.

KLF9 deficiency protects the heart from inflammatory injury triggered by myocardial infarction

• Chang Z
• Li H

• KMID: 2539572
• DOI: http://doi.org/10.4196/kjpp.2023.27.2.177

Abstract

The excessive inflammatory response induced by myocardial infarction exacerbates heart injury and leads to the development of heart failure. Recent studies have confirmed the involvement of multiple transcription factors in the modulation of cardiovascular disease processes. However, the role of KLF9 in the inflammatory response induced by cardiovascular diseases including myocardial infarction remains unclear. Here, we found that the expression of KLF9 significantly increased during myocardial infarction. Besides, we also detected high expression of KLF9 in infiltrated macrophages after myocardial infarction. Our functional studies revealed that KLF9 deficiency prevented cardiac function and adverse cardiac remodeling. Furthermore, the downregulation of KLF9 inhibited the activation of NF-κB and MAPK signaling, leading to the suppression of inflammatory responses of macrophages triggered by myocardial infarction. Mechanistically, KLF9 was directly bound to the TLR2 promoter to enhance its expression, subsequently promoting the activation of inflammation-related signaling pathways. Our results suggested that KLF9 is a pro-inflammatory transcription factor in macrophages and targeting KLF9 may be a novel therapeutic strategy for ischemic heart disease.

Keyword

Inflammation; KLF9; Myocardial infarction; Toll-like receptor 2; Transcription factors

Figure

• 

  Fig. 1 Upregulation of KLF9 is found in macrophages after myocardial infarction (MI). (A) Q-PCR analysis of Klf9 mRNA expression in myocardial tissues from wild-type mice after MI or sham operation. (B, C) Immunoblot analysis of KLF9 protein level in myocardial tissues from wild-type mice after MI or sham operation. (D) Q-PCR analysis of Klf9 mRNA expression in macrophages isolated from wild-type mice myocardium after MI or sham operation. (E, F) Immunoblot analysis of KLF9 protein level in macrophages isolated from wild-type mice myocardium after MI or sham operation. Data presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Unpaired Student’s t-test was performed.

• 

  Fig. 2 KLF9 knockout (KO) alleviates cardiac dysfunction and adverse cardiac remodeling after myocardial infarction (MI). (A) Q-PCR analysis of Klf9 mRNA expression in macrophages isolated KLF9 KO or littermate control wild-type (WT) mice. (B) Survival curves of Klf9 KO or littermate WT mice subjected to MI or sham operation. (C, D) Echocardiographic measurement of LVEF and LVFS of Klf9 KO or WT mice at baseline (day 0) and the indicated day after MI or sham operation. (E) Representative Masson’s Trichrome staining images in myocardial tissues from Klf9 KO or WT mice at day 28 after MI (×400). Data presented as mean ± SD. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. *p < 0.05; **p < 0.01; ***p < 0.001. Unpaired Student’s t-test or ANOVA was performed.

• 

  Fig. 3 Loss of KLF9 suppresses inflammatory response in myocardial tissues after myocardial infarction (MI). (A) Representative of H&E staining images in myocardial tissues from Klf9 KO or WT mice at day 7 after MI or sham operation (×200). (B) Q-PCR analysis of Il1, Il6 and Tnfa mRNA expression in myocardial tissues from Klf9 KO or WT mice at day 7 after MI. (C) ELISA of IL-1β, IL-6 and TNF-α production in myocardial tissue homogenates from Klf9 KO or WT mice at day 7 after MI. (D) Q-PCR analysis of Il1, Il6 and Tnfa mRNA expression in macrophages isolated from Klf9 KO or WT mice myocardial tissues at day 7 after MI. Data presented as mean ± SD. KO, knockout; WT, wild-type; IL, interleukin; TNF-α, tumor necrosis factor-α. ***p < 0.001. Unpaired Student’s t-test was performed.

• 

  Fig. 4 KLF9 promotes inflammatory responses triggered by high-mobility group box 1 (HMGB1) in macrophages. (A) ELISA of IL-1β, IL-6 and TNF-α production in BMDMs from Klf9 KO or WT mice after HMGB1 (100 ng/ml) treatment for 12 h. (B) ELISA of IL-1β, IL-6 and TNF-α production in supernatants of BMDMs transfection with Klf9 siRNA or control siRNA followed by treatment with HMGB1 (100 ng/ml) for 12 h. (C) Immunoblot analysis of phosphorylation levels of proteins in lysates of BMDMs from Klf9 KO or WT mice after HMGB1 (100 ng/ml) treatment. (D) Immunoblot analysis of phosphorylation levels of proteins in lysates of BMDMs transfected with the control siRNA or Klf9 siRNA after stimulated with HMGB1 (100 ng/ml). Data presented as mean ± SD. KO, knockout; WT, wild-type; IL, interleukin; TNF-α, tumor necrosis factor-α; BMDM, bone marrow derived macrophage. *p < 0.05; **p < 0.01; ***p < 0.001. Unpaired Student's t-test was performed.

• 

  Fig. 5 KLF9 promotes the expression of TLR2 in macrophages. (A) Q-PCR analysis of Tlr2 mRNA expression in myocardial tissues from WT mice after MI or sham operation. (B) Immunoblot analysis of TLR2 protein level in myocardial tissues from WT mice after MI or sham operation. (C) Q-PCR analysis of Tlr2 mRNA expression in myocardial tissues from Klf9 KO or WT mice at day 7 after MI. (D) Immunoblot analysis of TLR2 protein level in myocardial tissues from Klf9 KO or WT mice at day 7 after MI. (E) ChIP analysis of KLF9 enrichment level at the Tlr2 promoter in WT macrophages treated with HMGB1 (100 ng/ml) or control PBS (Ctrl) for 12 h. Data presented as mean ± SD. MI, myocardial infarction; KO, knockout; WT, wild-type; HMGB1, high-mobility group box 1. *p < 0.05; **p < 0.01; ***p < 0.001. Unpaired Student’s t-test or ANOVA was performed.

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