HMGB1 enhances mechanical stress-induced cardiomyocyte hypertrophy in vitro via the RAGE/ERK1/2 signaling pathway

Abstract

Pressure overload‑induced cardiac hypertrophy is associated with a complex spectrum of pathophysiological mechanisms, including the inflammation response. High mobility group box‑1 (HMGB1), a pro‑inflammatory cytokine, is not only increased in myocardium under pressure overload, but also exacerbates pressure overload‑induced cardiac hypertrophy and dysfunction; however, the underlying mechanisms have remained elusive. In the present study, cultured cardiomyocytes were stimulated by mechanical stress and/or HMGB1 for various durations to examine the role of HMGB1 in cardiomyocyte hypertrophy, and to detect the expression of receptor for advanced glycation end products (RAGE), toll‑like receptor 4 (TLR‑4) and the activation status of mitogen‑activated protein kinases (MAPKs) and Janus kinase 2 (JAK2)/STAT3. The results indicated that HMGB1 aggravated mechanical stress‑induced cardiomyocyte hypertrophy. Furthermore, mechanical stress and HMGB1 stimulation activated extracellular signal‑regulated kinase 1/2 (ERK1/2), P38 and JAK2/STAT3 signaling in cardiomyocytes, but an additive effect of the combined stimuli was only observed on the activation of ERK1/2. In addition, mechanical stress caused a prompt upregulation of the expression of RAGE and TLR‑4 in cardiomyocytes, while the activation of ERK1/2 by HMGB1 was inhibited by blockage of RAGE, but not by blockage of TLR‑4. In summary, the present results indicated that extracellular HMGB1 enhanced mechanical stress‑induced cardiomyocyte hypertrophy in vitro, at least partially via the RAGE/ERK1/2 signaling pathway.

Figure 1

HMGB1 enhances mechanical stress-induced cardiomyocyte hypertrophy. H9c2 cells were transfected with a GFP-expressing plasmid, and 48 h later, they were stimulated with 100 ng/ml HMGB1 and/or mechanical stretch for 24 h. (A) Western blot analysis confirmed successful GFP transfection in H9c2 cells. (B) Positive staining was observed under a fluorescence microscope (scale bar, 10 µm). (C) Quantification of cell surface area. Values are expressed as the mean ± standard error from three independent experiments. ^**P<0.01 vs. control; ^#P<0.05 vs. mechanical stretch. HMGB1, high mobility group box-1; GFP, green fluorescence protein.

Figure 2

Mechanical stress and HMGB1 stimulation activate mitogen-activated protein kinase signaling in cardiomyocytes. Neonatal rat cardiomyocytes were stimulated with 100 ng/ml HMGB1 and/or mechanical stretch for 5, 10 or 30 min, or 1 h. Protein expression levels were then analyzed by western blotting. (A) Representative images and (B) quantification of p-ERKs and t-ERKs levels. (C) Representative images and (D) quantification of p-P38 and t-P38 levels. Values are expressed as the mean ± standard error from three independent experiments. ^*P<0.05 vs. control; ^#P<0.05 vs. groups with the same stimuli at 5 min; ^&P<0.05 vs. groups with the same stimuli at 10 min; ^$P<0.05 vs. mechanical stretch treatment alone for 30 min. ^¥P <0.05 vs. HMGB1 alone for 30 min. HMGB1, high mobility group box-1; p-, phosphorylated; ERK, extracellular signal-regulated kinase; t-, total.

Figure 3

Mechanical stress and HMGB1 stimulation activate JAK2/STAT3 in cardiomyocytes. HMGB1 (100 ng/ml) and/or mechanical stress were applied to cultured cardiomyocytes for 5, 10, 30 or 60 min. Protein expression levels were then analyzed by western blotting. (A) Representative images and (B) quantification of p-JAK2 and t-JAK2 levels. (C) Representative images and (D) quantification of p-STAT3 and t-STAT3 levels. Values are expressed as the mean ± standard error from three independent experiments. ^*P<0.05 vs. control; ^#P<0.05 vs. groups with the same stimuli at 5 min. HMGB1, high mobility group box-1; JAK2, Janus kinase 2; p-, phosphorylated; t-, total.

Figure 4

Mechanical stress induces the expression of RAGE and TLR-4 in cardiomyocytes. Cultured cardiomyocytes were stimulated with mechanical stress for 2, 4, 8, 12 and 24 h, respectively. Protein expression levels were then analyzed by western blotting. (A) Representative images and (B) quantification of RAGE levels. (C) Representative images and (D) quantification of TLR-4 levels. Values are expressed as the mean ± standard error from three independent experiments. ^*P<0.05 vs. control. RAGE, receptor for advanced glycation end products; TLR-4, toll-like receptor 4.

Figure 5

Roles of RAGE and TLR-4 receptors in the activation of signaling pathways by HMGB1. Cardiomyocytes were pretreated either a RAGE-neutralizing antibody (20 µg/ml) or a TLR-4-neutralizing antibody (20 µg/ml) for 30 min, and stimulated with HMGB1 for a further 30 min. Cells were then analyzed by western blotting for the activation of (A and B) ERKs, (C and D) JAK2 and (E and F) STAT3. Values are expressed as the mean ± standard error from three independent experiments. ^*P<0.05 vs. control; ^#P<0.05 vs. HMGB1 group. RAGE, receptor for advanced glycation end products; TLR-4, toll-like receptor 4; HMGB1, high mobility group box-1; ERK, extracellular signal-regulated kinase; JAK2, Janus kinase 2; p-, phosphorylated; t-, total.