Ruhui Yang1, Zhaoxia Song2, Songquan Wu3, Zhe Wei3, Yini Xu4, Xiangchun Shen5. 1. The Department of Pharmacology of Materia Medica (the State Key Laboratory of Functions and Applications of Medicinal Plants, The High Efficacy Application of Natural Medicinal Resources Engineering Center of Guizhou Province, The Key Laboratory of Optimal Utilizaiton of Natural Medicine Resources), School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guian New District, Guizhou, 550025, China; Department of Pharmacology of College of Medicine and Health, Lishui University, Lishui, 323000, China. 2. Department of Obstetrics, Lishui Maternal and Child Health Hospital, Lishui, 323000, China. 3. Department of Pharmacology of College of Medicine and Health, Lishui University, Lishui, 323000, China. 4. The Department of Pharmacology of Materia Medica (the State Key Laboratory of Functions and Applications of Medicinal Plants, The High Efficacy Application of Natural Medicinal Resources Engineering Center of Guizhou Province, The Key Laboratory of Optimal Utilizaiton of Natural Medicine Resources), School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guian New District, Guizhou, 550025, China. 5. The Department of Pharmacology of Materia Medica (the State Key Laboratory of Functions and Applications of Medicinal Plants, The High Efficacy Application of Natural Medicinal Resources Engineering Center of Guizhou Province, The Key Laboratory of Optimal Utilizaiton of Natural Medicine Resources), School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guian New District, Guizhou, 550025, China. Electronic address: sxc@gmc.edu.cn.
Abstract
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.
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.