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Cyclic stretch enhances the expression of Toll-like Receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-B pathway

Toll-like receptor 4 (TLR4) plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes. Methods: Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected | Shyu et al. Journal of Biomedical Science 2010 17 15 http www.jbiomedsci.eom content 17 1 15 JOURNAL OF BIOMEDICAL SCIENCE RESEARCH Open Access Cyclic stretch enhances the expression of Toll-like Receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-d B pathway Kou-Gi Shyu1 2 Bao-Wei Wang3 Chiu-Mei Lin4 Hang Chang4 Abstract Background Toll-like receptor 4 TLR4 plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes. Methods Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20 of maximum elongation at 60 cycles min. Western blot real-time polymerase chain reaction and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected. Results Cyclic stretch significantly increased TLR4 protein and mRNA expression after 2 h to 24 h of stretch. Addition of SB203580 TNF-a antibody and p38a MAP kinase siRNA 30 min before stretch inhibited the induction of TLR4 protein. Cyclic stretch increased while SB203580 abolished the phosphorylated p38 protein. Gel shifting assay showed significant increase of DNA-protein binding activity of NF-kB after stretch and SB203580 abolished the DNA-protein binding activity induced by cyclic stretch. DNA-binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased TLR4 promoter activity while SB203580 and NF-kB siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580 TNF-a antibody and TLR4 siRNA attenuated the adherence of monocyte. TNF-a and Ang II significantly increased TLR4 protein expression. Addition of losartan TNF-a antibody or p38a siRNA 30 min .