Toll-like receptor 4 contributes to a myofibroblast phenotype in cardiac fibroblasts and is associated with autophagy after myocardial infarction in a mouse model.

BACKGROUND AND AIMS

Cardiac fibrosis after myocardial infarction (MI) is involved in fibroblast transforming and differentiating into myofibroblast phenoconversion, however, the underlying mechanisms are poorly understood. Toll-like receptor 4 (TLR4)-mediated pathogen-associated molecular patterns are key factors that deteriorate cardiac remodelling after MI. Moreover, autophagy has dual roles in cell survival in myocardial tissues after MI. We evaluated the relationship between TLR4 signalling and cardiac myofibroblast transformation-differentiation after MI in vivo and in vitro and analysed the role of autophagy.

METHODS

We reproduced a model of MI by the permanent ligation of the left anterior descending coronary artery of Tlr4-knockout (Tlr4) and wild-type (WT) male mice. We evaluated scar formation, myofibroblast phenoconversion, LC3 dot formation, autophagy related proteins and α-smooth muscle actin (SMA) in cardiac tissues, 7, 14, and 28 days after myocardial infarction. Cardiac fibroblasts were cultured from Tlr4 or WT mice. Vimentin, α-SMA, bilayer membrane vesicle structures of autophagosomes, and autophagy related proteins were observed after treatment with lipopolysaccharide (LPS) or 3-methyladenine (3-MA) at 24 h.

RESULTS

After MI on 7, 14, and 28 days, Tlr4 mice showed that heart tissue fibrosis and expression of α-SMA, a marker of myofibroblasts, were decreased compared to WT mice. Additionally, levels of LC3II, Atg5, Atg7, and Beclin-1, which are involved in autophagy, were lower than those in WT mice. Further, p62 expression, which is negatively correlated with autophagy levels, was higher in Tlr4 mice. Moreover, LC3-labelled autophagosomes in cardiac tissues were reduced in these animals. In vitro, LPS, a ligand of TLR4, stimulated α-SMA expression in cardiac fibroblasts, enhanced autophagic flux, and increased autophagosome numbers. In contrast, these effects were not obvious in Tlr4 cardiac fibroblasts. LC3II, Atg5, Atg7, and Beclin-1 were upregulated, and p62 was downregulated in cardiac fibroblasts of WT mice stimulated with LPS. However, these effects were blocked by 3-methyladenine, an inhibitor of autophagy.

CONCLUSIONS

These results suggest that TLR4 signalling executes the development of a myofibroblast phenotype after MI via autophagy and could be therapeutically exploited to improve outcome after myocardial injury.