Time-dependence of cardiomyocyte differentiation disturbed by peroxisome proliferator-activated receptor ? inhibitor GW6471 in murine embryonic stem cells in vitro

Abstract

To investigate the possible roles of peroxisome proliferator-activated receptor alpha(PPAR alpha) and the signal pathway regulating the transcription of PPAR alpha in the cardiomyocyte differentiation course of murine embryonic stem (ES) cells in vitro. The expression of PPAR alpha during cardiomyocyte differentiation was analyzed using both Western blotting and immunofluorescence. Cardiac specific genes and sarcomeric proteins were evaluated when embryoid bodies were challenged with PPAR alpha specific inhibitor GW6471 at different time courses. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) was studied in the differentiation process, and its specific inhibitor SB203580 was employed to study the function of p38 MAPK on cardiac differentiation and the expression of PPAR alpha. The expression of PPAR alpha was observed to be at a low level in undifferentiated ES cells and markedly induced with the appearance of beating clusters. The inhibition of PPAR alpha by its specific inhibitor GW6471 (1X10(-5) mol/L) significantly prevented cardiomyocyte differentiation and resulted in the reduced expression of cardiac sarcomeric proteins (ie alpha-actinin, troponin-T) and specific genes (ie alpha-MHC, MLC2v) in a time-dependent manner. In the differentiation course, p-p38 MAPK was maintained at a high level from d 3 followed by a decrease from d 10. The inhibition of the p38 MAPK pathway by SB203580 between d 3 and d 7 efficiently prevented cardiomyocyte differentiation and resulted in the capture of the upregulation of PPAR alpha. Taken together, these results showed a close association between PPAR alpha and cardiomyocyte differentiation in vitro, and p38 MAPK was partly responsible for the regulation of PPAR alpha.