Fig 1: Enhanced binding activity of toll‐like receptor 4 (TLR4) in chronic heart failure (CHF) cardiomyocytes to lipopolysaccharide (LPS) and heat shock protein 60 (HSP60). Isolated cardiomyocytes were cultured in a CO2 incubator at 37°C for 24 hrs, then the binding assay was performed at 4°C for 30 min. To block TLR4, cultured cardiomyocytes were incubated with TLR4 neutralizing antibody (anti‐TLR4, 5 μg/ml) at 37°C for 15 min., and subsequently incubated with FITC‐LPS or OG‐HSP60 at 4°C for 30 min. (A) Representative fluorescent images of isolated cardiomyocytes after the incubation with FITC‐LPS (green) or OG‐HSP60 (green). (B) Binding curves of FITC‐LPS to cardiomyocytes. (C) Binding curves of OG‐HSP60 to cardiomyocytes.
Fig 2: Increased cytokine production mediated by toll‐like receptor 4 (TLR4) in chronic heart failure (CHF) cardiomyocytes. Cultured cardiomocytes from sham and CHF rats were treated with lipopolysaccharide (LPS; 1 μg/ml) or heat shock protein 60 (HSP60; 1 μg/ml) for 6 hrs. TLR4 neutralizing antibody (anti‐TLR4, 5 μg/ml) was added 15 min before LPS or HSP60 treatment. (A) Tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 mRNA levels (n = 6/group). (B) The amount of TNF‐α and IL‐6 released into culture supernatant (n = 6/group). (C) Representative Western blot images and quantification of p65 in the nuclei of cardiomyocytes from three independent experiments (data are means ± SD, a P < 0.05, A P < 0.01 versus respective sham; b P < 0.05, B P < 0.01 versus sham‐blank; c P < 0.05, C P < 0.01 versus CHF‐blank; d P < 0.05, D P < 0.01 versus respective LPS; e P < 0.05, E P < 0.01 versus respective HSP60).
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