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

Abstract

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 PPARd 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. METHODSThe effects of digoxin on PPARd expression, intracellular calcium and TnI phosphorylation were investigated in cultured H9c2 cells, maintained in a HG medium, by using Western blot analysis. RESULTSDigoxin increased PPARd 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 PPARd was silenced by the addition of RNAi to the cells. Similar changes were observed on the contraction of H9c2 cells. CONCLUSIONThe results suggest that digoxin appears, through calcium-triggered signals, to reverse the reduced expression of PPARd in H9c2 cells caused by HG treatment.

Increase of PPARδ by dopamine mediated via DA-1 receptor-linked phospholipase C pathway in neonatal rat cardiomyocytes

“…Similar to previous report (Fan et al, 2010;Chen et al, 2011Chen et al, , 2012aChou et al, 2012), the increased PPARδ and calcium ions may induce cTnI phosphorylation which is responsible for cardiac contraction. This finding suggests a new mechanism of dopamine for cardiac tonic action in cardiomyocytes.…”

“…Ca 2+ is known to involve in muscle contraction (Bootman and Berridge, 1995;Metzger and Westfall, 2004;Layland et al, 2005;Ohtsuki and Morimoto, 2008). Thus, as the indicator of cardiac contraction similar to previous report (Chen et al, 2011(Chen et al, , 2012a, cTnI phosphorylation is elevated in neonatal rat cardiomyocytes by dopamine. Also, BAPTA-AM decreased the level of cTnI phosphorylation in dopamine-treated neonatal rat cardiomyocytes.…”

“…Phosphorylation of TnI may result in an increase of cross-bridge cycling rate leading to an increase in power output of heart (Metzger and Westfall, 2004;Layland et al, 2005). Thus, similar to previous report (Fan et al, 2010;Chen et al, 2011Chen et al, , 2012aChou et al, 2012), we used TnI phosphorylation as the indicator of cardiac contraction in this study. Otherwise, Ca 2+ is also involved in muscle contraction (Bootman and Berridge, 1995;Metzger and Westfall, 2004;Layland et al, 2005;Ohtsuki and Morimoto, 2008) and we compared the changes of Ca 2+ by dopamine in addition.…”

“…It has been established that PPARδ plays an important role in the regulation of cardiac performance and the phosphorylation of TnI in cardiomyocytes (Chen et al, 2011(Chen et al, , 2012bChou et al, 2012). Our previous study also demonstrated that treatment with the selective agonist GW0742 for the activation of PPARδ restored the cardiac performance in diabetic rats.…”

“…The medium was changed every 48 h. After 3 to 4 days in culture, the cells were exposed to hyperglycemic conditions. The high glucose-treated cardiomyocytes were generated by applying 30 mmol/L glucose to the cells for 24 h (Chen et al, 2011). This animal experiment was approved and conducted in accordance with local institutional guidelines for the care and use of laboratory animals in the Chi-Mei Medical Center (No.…”