KLF13 overexpression protects sepsis-induced myocardial injury and LPS-induced inflammation and apoptosis

Abstract

Sepsis remains a worldwide public health problem. This study aims to explore the role and mechanism of transcriptional factors (TFs) in sepsis-induced myocardial injury. Firstly, TF KLF13 was selected to explore its role in sepsis-induced myocardial injury. The caecal ligation and puncture (CLP) -induced sepsis mouse model was established and the septic mice were examined using standard histopathological methods. KLF13 expression was detected in the septic mouse heart and was also seen in a lipoploysaccharide (LPS) -induced cellular inflammation model. To explore this further both pro-apoptotic cleaved-caspase3/caspase3 and Bax levels and anti-apoptotic Bcl2 levels were examined, also in both models, In addition inflammatory cytokine (IL-1β, TNF-α, IL-8 and MCP-1) production and IκB-α protein level and p65 phosphorylation were examined in both septic mice and LPS-induced cells. Thus three parameters - cardiomyocyte apoptosis, inflammatory response and NF-κB pathway activation were evaluated under similar conditions. The septic mice showed significant oedema, disordered myofilament arrangement and degradation and necrosis to varying degrees in the myocardial cells. KLF13 was downregulated in both the septic mouse heart and the LPS-induced cellular inflammation model. Furthermore, both models showed abnormally increased cardiomyocyte apoptosis (increased cleaved-caspase3/caspase and Bax protein levels and decreased Bcl2 level), elevated inflammation (increased production of inflammatory cytokines) and the activated NF-κB pathway (increased p65 phosphorylation and decreased IκB-α protein level). KLF13 overexpression notably ameliorated sepsis-induced myocardial injury in vivo and in vitro. KLF13 overexpression protected against sepsis-induced myocardial injury and LPS-induced cellular inflammation and apoptosis via inhibiting the inflammatory pathways (especially NF-κB signalling) and cardiomyocyte apoptosis.

Keywords: KLF13; cardiomyocyte apoptosis; inflammation; myocardial injury; sepsis; transcriptional factors.

FIGURE 1

Selecting transcriptional factor (TF) correlated to sepsis‐induced myocardial injury. (A‐B) The TF list (GO:0003700, TF activity) was extracted from Ensembl (http://asia.ensembl.org/), and 1351 TF genes were obtained. Expression of these TFs was analysed in normal and septic hearts based on GSE171546. The top 10 most significantly differentially expressed TFs were shown in the volcano plots and hierarchical clustering. (C) The expression of KLF13 in normal and septic hearts is based on data from GSE171546 and GSE79962. (D) A total of 419 genes correlated to KLF13 were applied for Gene Ontology (GO) functional enrichment annotation in metascape (https://metascape.org/)

FIGURE 2

KLF13 is downregulated in septic mouse hearts. Cecal ligation and puncture (CLP)‐induced sepsis model was established in mice and examined for histopathological changes of normal and septic mouse hearts by H&E staining (A); the levels and distribution of KLF13 in normal and septic mouse hearts by immunohistochemical (IHC) staining (B); (C); mRNA expression levels of KLF13 in normal and septic mouse heart by qRT‐PCR (D); the protein levels of cleaved‐caspase3, caspase3, Bax and Bcl2 in normal and septic mouse heart by Immunoblotting

FIGURE 3

In vivo effects of KLF13 on septic mouse heart. (A‐B) Mice were injected with lentivirus overexpressing KLF13 (Lv‐KLF13) and then received CLP 5 days later. KLF13 overexpression in cardiac tissues was confirmed by qRT‐PCR and immunoblotting. Mice were assigned to four groups: sham, sepsis model, sham injected with Lv‐KLF13 and sepsis model injected with Lv‐KLF13. Mice in each group received the injection, underwent surgery accordingly and examined for the protein levels of cleaved‐caspase3, caspase3, Bax and Bcl2 in normal and septic mouse heart by immunoblotting (C); levels of cytokines including IL‐1β, TNF‐α, IL‐8 and MCP‐1 in normal and septic mouse heart by ELISA (D); protein levels of IκB‐α, p‐p65 and p65 by Immunoblotting (E)

FIGURE 4

In vitro effects of KLF13 on LPS‐induced cellular inflammation model. HL‐1 mouse cardiomyocytes were transfected or non‐transfected with KLF13‐overexpressing vector, treated or non‐treated with 10 μg/mL LPS for 48 h and examined for KLF13 mRNA expression by qRT‐PCR and protein levels by Immunoblotting (A,B); cell viability by CCK‐8 assay (C); cell apoptosis by flow cytometry (D); levels of cytokines including IL‐1β, TNF‐α, IL‐8 and MCP‐1 in normal and septic mouse heart by ELISA (E); protein levels of IκB‐α, p‐p65, p65, Bax and Bcl2 by immunoblotting (F)