Up-regulated TLR4 in cardiomyocytes exacerbates heart failure after long-term myocardial infarction

Abstract

It remains unclear whether and how cardiomyocytes contribute to the inflammation in chronic heart failure (CHF). We recently reviewed the capacity of cardiomyocytes to initiate inflammation, by means of expressing certain immune receptors such as toll-like receptors (TLRs) that respond to pathogen- and damage-associated molecular patterns (PAMP and DAMP). Previous studies observed TLR4-mediated inflammation within days of myocardial infarction (MI). This study examined TLR4 expression and function in cardiomyocytes of failing hearts after 4 weeks of MI in rats. The increases of TLR4 mRNA and proteins, as well as inflammatory cytokine production, were observed in both the infarct and remote myocardium. Enhanced immunostaining for TLR4 was observed in cardiomyocytes but not infiltrating leucocytes. The injection of lentivirus shRNA against TLR4 into the infarcted heart decreased inflammatory cytokine production and improved heart function in vivo. Accordingly, in cardiomyocytes isolated from CHF hearts, increases of TLR4 mRNA and proteins were detected. More robust binding of TLR4 with lipopolysaccharide (LPS), a PAMP ligand for TLR4, and heat shock protein 60 (HSP60), a DAMP ligand for TLR4, was observed in CHF cardiomyocytes under a confocal microscope. The maximum binding capacity (Bmax ) of TLR4 was increased for LPS and HSP60, whereas the binding affinity (Kd) was not significantly changed. Furthermore, both LPS and HSP60 induced more robust production of inflammatory cytokines in CHF cardiomyocytes, which was reduced by TLR4-blocking antibodies. We conclude that the expression, ligand-binding capacity and pro-inflammatory function of cardiomyocyte TLR4 are up-regulated after long-term MI, which promote inflammation and exacerbate heart failure.

Keywords: cardiomyocyte; heart failure; inflammation; toll-like receptor.

Figure 1

Myocardial infarction resulted in chronic heart failure (CHF) in rats after 4 weeks of coronary ligation. (A) Gross view and microscopic photos of heart sections stained with haematoxylin and eosin. Cross‐sections were cut at the midhorizontal plane of the fixed paraffin‐embedded heart, and stained with haematoxylin and eosin reagents. (B) Heart‐to‐body weight ratio. (C) Representative M‐mode ultrasound images of sham and CHF rats taken at the midpapillary level. (D) LV end‐systolic diameter (LVESD). (E) LV end‐diastolic diameter (LVEDD). (F) Fractional shortening (%) of the left ventricle. (G) Serum brain natriuretic peptide (BNP) level (data are means ± SD, n = 6–9/group, **P < 0.01 versus sham).

Figure 2

Inflammation in chronic heart failure (CHF) rats. (A) Tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 mRNA levels in infarct and remote myocardium. (B) TNF‐α and IL‐6 protein levels in infarct and remote myocardium. (C) Serum content of TNF‐α and IL‐6 in sham and CHF rats. (D) TNF‐α and IL‐6 mRNA levels in cardiomyocytes isolated from sham and CHF rats. (E) TNF‐α and IL‐6 protein contents in cardiomyocytes isolated from sham and CHF rats (data are means ± SD, n = 4–6/group, *P < 0.05, **P < 0.01 versus respective sham).

Figure 3

Increased toll‐like receptor 4 (TLR4) expression in the myocardium of chronic heart failure (CHF) rats. (A) TLR4 mRNA levels in infarct and remote myocardium of sham and CHF rats (n = 6/group). (B) Representative Western blot images and (C) quantification of TLR4 proteins in infarct and remote myocardium of sham and CHF rats (n = 4/group). (D) Representative immunohistochemistry images of heart sections stained with TLR4 (green) and CD45 (red). The yellow box indicates the enlarged area shown on the right (data are means ± SD, *P < 0.05, **P < 0.01 versus respective sham).

Figure 4

Increased toll‐like receptor 4 (TLR4) expression in the surviving cardiomyocytes of chronic heart failure (CHF) rats. (A) Representative immunofluorescent images of TLR4 in cardiomyocytes isolated from sham and CHF rats. (B) TLR4 mRNA levels in cardiomyocytes isolated from sham and CHF rats. (C) Representative Western blot images and (D) quantification of TLR4 proteins in cardiomyocytes isolated from sham and CHF rats (data are means ± SD, n = 6/group, **P < 0.01 versus sham).

Figure 5

Toll‐like receptor 4 (TLR4)‐shRNA lentivirus reduced myocardial inflammation and improved heart function after myocardial infarction (MI). The rats received intra‐myocardial injection of normal saline (NS), control‐shRNA lentivirus or TLR4‐shRNA lentivirus (1 × 10⁹ TU/ml, 100 μl/heart) just after left anterior descending coronary artery (LAD) ligation or sham operation. All examinations were performed after 4 weeks of MI. (A) Expression of green fluorescent protein (GFP; green), the marker gene carried by TLR4‐shRNA lentivirus, in the myocardium. The nuclei were counter‐stained with Hoechst 33258 (blue). (B) Representative Western blot images and quantification of TLR4 proteins in sham and chronic heart failure (CHF) myocardium. (C) tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 protein contents in infarct and remote myocardium. (D) Representative images of Masson's trichrome staining (upper panel) and quantification (lower panel) of post‐infarct failing hearts, showing that TLR4‐shRNA lentivirus reduced cardiac fibrosis. Cross‐sections were cut at the midhorizontal plane of the fixed paraffin‐embedded heart, and stained with Masson's trichrome reagents. (E) Infarct size of post‐infarct failing hearts. (F) Fractional shortening (%) of the left ventricle (data are means ± SD, n = 4/group, ^a P < 0.05, ^A P < 0.01 versus respective sham‐NS; ^B P < 0.01 versus respective CHF‐NS).

Figure 6

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.

Figure 7

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).