T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway.

OBJECTIVE

To investigate the effect of T-2 toxin on murine embryonic stem cells (ESCs) cardiac differentiation and mitochondrial biogenesis in vitro.

METHODS

Cardiac differentiation of the mouse ESCs was initiated by embryoid bodies (EBs) formation in hanging drops. EBs were exposed to 0.5ng/ml T-2 toxin for 24, 72 and 120h. Cultures were observed daily for the appearance of contracting clusters, and cardiac-specific protein (α-actiniin) were measured by Western blot and immunocytochemistry. Mitochondrial ultrastructure was observed by confocal laser scanning microscopy and transmission EM photography. Reactive oxygen species (ROS) was monitored by H2-dichlorofluorescein-diacetate (H2DCF-DA). The phosphorylation of the p38 (p-p38) and p38 mitogen-activated protein kinase (MAPK) and the expression of mitochondrial biogenesis proteins, including peroxisome proliferator activated receptor coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), and mitochondrial respiratory chain complex IV (COXIV) were analyzed using Western blot. In some experiments, mESCs were pre-treated with the antioxidant Trolox (200μM) for 30min, then exposed to Trolox (200μM) and T-2 toxin (0.5ng/ml) for 72h.

RESULTS

Contracting clusters were observed under the microscope light and cardiac-specific protein (α-actinin) expressed positively indicated mESCs directly differentiated in cardiomyocytes. However, the cardiac differentiation was inhibited by T-2 toxin treatment 72 and 120h. ROS accumulated in murine ES cells in a time-dependent manner. The expression of p-p38 significantly increased in 24h group and decrease in 72 and 120h groups. The decrease of mitochondrial number and the mitochondrial biogenesis-related proteins expression, including PGC-1α, NRF-1, mtTFA, and COXIV decreased in a time-dependent manner with T-2 toxin treatment. However, the inhibition of mitochondrial biogenesis by T-2 toxin in differentiated mESCs was recovered significantly in the presence of the antioxidant Trolox.

CONCLUSION

Taken together, T-2 toxin decreased the expression of PGC-1α, NRF-1, and mtTFA, inhibited mitochondrial biogenesis, and then inhibited the cardiac differentiation of murine ES cells, and the effect was partly responsible for the p38 MAPK mediated by ROS.