Cyclic stretch enhances the expression of toll-like receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-kappaB pathway

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-alpha antibody, and p38alpha 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-kappaB 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-kappaB siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580, TNF-alpha antibody, and TLR4 siRNA attenuated the adherence of monocyte. TNF-alpha and Ang II significantly increased TLR4 protein expression. Addition of losartan, TNF-alpha antibody, or p38alpha siRNA 30 min before Ang II and TNF-alpha stimulation significantly blocked the increase of TLR4 protein by AngII and TNF-alpha.

Conclusions: Cyclic mechanical stretch enhances TLR4 expression in cultured rat neonatal cardiomyocytes. The stretch-induced TLR4 is mediated through activation of p38 MAP kinase and NF-kappaB pathways. TLR4 up-regulation by cyclic stretch increases monocyte adherence.

Figure 1

Cyclic stretch increases toll-like receptor 4 (TLR4) protein and mRNA expression in cardiomyocytes. (A) Representative Western blots for TLR4 in cardiomyocytes subjected to cyclic stretch by 20% or 10% for various periods of time. (B) Quantitative analysis of TLR4 protein levels. The values from stretched cardiomyocytes have been normalized to values in control cells and the data are from 4 independent experiments. *P < 0.001 vs. control. **P < 0.01 vs. control. (n = 4 per group) (C) Fold increases in TLR4 mRNA as a result of cyclic stretch by 20% for various periods of time. The values from stretched cardiomyocytes have been normalized to matched GAPDH measurement and then expressed as a ratio of normalized values to mRNA in control cells (n = 4 per group). *P < 0.01 vs. control.

Figure 2

p38 MAP kinase and tumor necrosis factor-α (TNF-α) are important regulators that mediate stretch-induced TLR4 expression in cardiomyocytes. (A) Representative Western blots for TLR4 protein levels in cardiomyocytes subjected to cyclic stretch for 6 h or control cells without stretch in the absence or presence of different inhibitors, and siRNA. CM indicates conditioned medium. (B) Quantitative analysis of TLR4 protein levels. The values from stretched cardiomyocytes have been normalized to values in control cells (n = 4 per group). *P < 0.001 vs. stretch 6 h. **P < 0.01 vs. stretch 6 h. ⁺P < 0.001 vs. control.

Figure 3

Effect of cyclic stretch on expression of p38 kinase in cardiomyocytes. (A) Representative Western blots for phosphorylated and total p38 kinases in cardiomyocytes after stretch by 20% for various periods of time and in the presence of SB203580 and p38 siRNA. (B) Quantitative analysis of phosphorylated p38 protein levels. The values from stretched cardiomyocytes have been normalized to matched GAPDH and corresponding total protein measurement and then expressed as a ratio of normalized values to each phosphorylated protein in control cells. Data are from 4 independent experiments. *P < 0.001 vs. control. ⁺P < 0.001 vs. stretch 6 h.

Figure 4

Angiotensin II mediates the increase of TLR4 by cyclic stretch through angiotensin II receptor. (A) Representative Western blots for TLR4 in cardiomyocytes subjected to cyclic stretch by 20% for 6 h or without stretch in the presence or absence of inhibitors. (B) Quantitative analysis of TLR4 protein levels. The values from stretched cardiomyocytes have been normalized to values in control cells and the data are from 4 independent experiments.

Figure 5

Cyclic stretch increases release of TNF-α and angiotensin II (AngII) from cardiomyocytes subjected to 20% of stretch for various periods of time. The cultured medium were collected for measurement of TNF-α (A) and Ang II (B) in cultured cardiomyocytes after stretch for various periods of time via immunoassay (n = 4). *P < 0.001 vs. control. **P < 0.05 vs. control. ⁺P < 0.001 vs. stretch 2 h.

Figure 6

Cyclic stretch increases NF-κB-binding activity and NF-κB protein phosphorylation. (A) Representative EMSA showing protein binding to the NF-κB oligonucleotide in nuclear extracts of cardiomyocytes after cyclic stretch for various periods of time and in the presence of inhibitors. Similar results were found in another two independent experiments. Cold oligo means unlabeled NF-κB oligonucleotides. A supershifted complex is observed after addition of p65 antibody. (B) Representative Western blots for phosphorylated and total NF-κB in cardiomyocytes after stretch by 20% for various periods of time and in the presence of SB203580, losartan, and TNF-α antibody. (C) Quantitative analysis of phosphorylated NF-κB protein levels. The values from stretched cardiomyocytes have been normalized to matched C23 and corresponding total protein measurement and then expressed as a ratio of normalized values to each phosphorylated protein in control cells. Data are from 4 independent experiments. *P < 0.001 vs. control. **P < 0.01 vs. control. ⁺P < 0.001 vs. stretch 2 h. ^‡ P < 0.001 vs. stretch 2 h.

Figure 7

Cyclic stretch increases TLR4 promoter activity. Upper panel, constructs of TLR4 promoter gene. Lower panel, quantitative analysis of TLR4 promoter activity. Cardiomyocytes were transiently transfected with pTLR4-Luc by gene gun. The luciferase activity in cell lysates was measured and was normalized with renilla activity (n = 3 per group). *P < 0.001 vs. control. **P < 0.001 vs. 2 h.

Figure 8

Cyclic stretch increases adhesion of monocyte to stretched cardiomyocytes. A, Representative microscopic image for monocyte adhesion assay with (left panel) or without green fluorescence (right panel) in cardiomyocytes subjected to cyclic stretch for 6 h or control cells without stretch in the absence or presence of inhibitors. B, Quantitative analysis of the positive fluorescent cells. (n = 4 per group). *P < 0.001 vs. 6 hr. **P < 0.01 vs. 6 hr