Characterization of the mechanisms of the increase in PPARδ expression induced by digoxin in the heart using the H9c2 cell line

Abstract

Background and purpose: Digoxin has been used as an inotropic agent in heart failure for a long time. Troponin I (TnI) phosphorylation is related to cardiac contractility, and the genes are regulated by peroxisome proliferator-activated receptors (PPARs). Our previous studies indicated that cardiac abnormality related to the depressed expression of PPARδ in the hearts of STZ rats is reversed by digoxin. However, the cellular mechanisms for this effect of digoxin have not been elucidated. The aim of the present study was to investigate possible mechanisms for this effect of digoxin using the H9c2 cell line cultured in high glucose (HG) conditions.

Methods: The effects of digoxin on PPARδ expression, intracellular calcium and TnI phosphorylation were investigated in cultured H9c2 cells, maintained in a HG medium, by using Western blot analysis.

Results: Digoxin increased PPARδ expression in H9c2 cells subjected to HG conditions, and increase the intracellular calcium concentration. This effect of digoxin was blocked by BAPTA-AM at concentrations sufficient to chelate calcium ions. In addition, the calcineurin inhibitor cyclosporine A and KN93, an inhibitor of calcium/calmodulin-dependent protein kinase, inhibited this action. Digoxin also increased TnI phosphorylation and this was inhibited when PPARδ was silenced by the addition of RNAi to the cells. Similar changes were observed on the contraction of H9c2 cells.

Conclusion: The results suggest that digoxin appears, through calcium-triggered signals, to reverse the reduced expression of PPARδ in H9c2 cells caused by HG treatment.

Figure 1

Effects of digoxin on PPARδ expression in H9c2 cells. (A) H9c2 cells were cultured with or without glucose at 30 mmol·L⁻¹ and treated with digoxin, which was also incubated with 5.5 mmol·L⁻¹ glucose. (B) Cells were also exposed to 24.5 mmol·L⁻¹ mannitol to produce the same osmolarity (317 mOsmol·L⁻¹) as that produced with the highest concentration of glucose (30 mmol·L⁻¹). The embryonic rat H9c2 cells (A), rat lung L2 cells and MDCK cells (C) were cultured with 30 mmol·L⁻¹ glucose for 24 h, and then treated with digoxin (0.01–1 µmol·L⁻¹) for 30 min. Also, the possible effects of digoxin (1 µmol·L⁻¹) on the expressions of PPARα and PPARγ in H9c2 cells were also investigated (D). These cells were harvested to determine the protein levels by Western blot analysis. All values are presented as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with the HG-treated cells.

Figure 2

Effects of BAPTA on digoxin- and caffeine-induced calcium release and increased PPARδ expression in HG-treated H9c2 cells. (A) The intracellular calcium ion concentrations in H9c2 cells exposed to digoxin at final concentrations of 0.001, 0.01, 0.1 and 1 µM compared that control cells not treated with digoxin and (B) the effects of the various treatments on the changes in intracellular calcium concentration, as detected by the fura-2 probe using a fluorescence spectrofluorometer. (C) Effect of BAPTA on the digoxin-induced PPARδ expression in the HG-treated cells. Cells were treated with 25 mmol·L⁻¹ BAPTA for 30 min before incubation with 1 µmol·L⁻¹ digoxin or 1 µmol·L⁻¹ caffeine for 30 min, and then harvested to measure PPARδ protein expression by Western blot analysis. The response to caffeine at a concentration sufficient to increase intracellular calcium was used as positive control. All values are expressed as mean ± SEM (n = 4 per group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with the HG-treated cells. ##P < 0.01 and ###P < 0.001 as compared with the HG-treated cells incubated with digoxin or caffeine.

Figure 3

Effects of calcium-mediated signalling inhibitors on digoxin-induced PPARδ expression in HG-treated H9c2 cells. The HG-treated cells were treated with 1 µmol·L⁻¹ cyclosporine A (CsA, calcineurin inhibitor) (A) or 1 µmol·L⁻¹ KN93 (CaMK inhibitor) (B) for 30 min before incubation with 1 µmol·L⁻¹ digoxin for 30 min. The cells were then harvested to measure PPARδ protein expression (A and B) by Western blot analysis. All values are expressed as mean ± SEM (n = 4 per group). **P < 0.01 and ***P < 0.001 as compared with the HG-treated H9c2 cells. ##P < 0.01 and ###P < 0.001 as compared with the HG-treated cells incubated with digoxin.

Figure 4

Effects of PPARδ-specific siRNA on digoxin-induced PPARδ expression and cardiac TnI phosphorylation in HG-treated H9c2 cells. The HG-treated H9c2 cells were incubated with siRNA-PPARδ or siRNA-control for 48 h before incubation with 1 µmol·L⁻¹ digoxin for 30 min, and were then harvested to measure PPARδ protein expression (A) and TnI phosphorylation (B) by Western blot analysis. All values are expressed as mean ± SEM (n = 4 per group). **P < 0.01 as compared with the HG-treated cells, and ##P < 0.01 as compared with the HG-treated cells incubated with siRNA-control.

Figure 5

Effects of PPARδ-specific siRNA on digoxin-induced cell contraction in HG-treated H9c2 cells. The HG-treated H9c2 cells were incubated with siRNA-PPARδ (Si) or siRNA-control cells (Sc) for 48 h before incubation with 1 µmol·L⁻¹ digoxin for 30 min and then applied for measurement of the planar surface area. The upper panel (A) shows the representative change of cells in each group. The lower panel (B) expresses the differences between four groups as mean ± SEM (n = 6 per group). Both digoxin-treated group and Sc treated with digoxin group were markedly different compared to the controls (*P < 0.05 and **P < 0.01).