Molecular Mechanism Of Cardiac Toxicity In Tripterygium Wilfordii

Celastrol, a bioactive compound isolated from Tripterygium wilfordii Hook.f. extracts, has recently attracted much attention for its potential to relief inflammation and inhibit various cancer. Recent reports have shown that celastrol exerts inhibitory effects on surface expression of hERG and Kir2.1potassium channels heterologously expressed in HEK293cells, suggesting its potential to cause cardiac arrhythmia. However, there are disadvantages for studying drug induced cardiotoxicity in non-cardiomyocyte. Besides, whether celastrol has any adverse effects on native cardiomyocyte is still unknown. Thus, the present study was designed to investigate the cardiotoxicity of celastrol using C57BL/6J mice and neonatal rat ventricular myocytes.To evaluate the possible cardiotoxicity of celastrol in vivo, healthy mice were subjected to injection (i.p) of celastrol for4weeks with a dose of3mg/kg/2day. It was found that treatment of mice with celastrol caused a loss of body weight and a decrease in food intake and a reduction of left ventricular contractile function.At in vitro level, chronic incubation with more than1μM celastrol caused dramatic morphological changes (cell rounding and pronounced cytosolic vacuolization) as well as decreased cell viability of primary cardiomyocytes, while the following Hoechst33342staining demonstrated that celastrol induced apoptosis in primary cardiomyocytes, which was evidenced again by the detection of the dissipation of mitochondrial membrane potential and the increased Caspase-3activity. Meanwhile, celastrol induced necrosis was observed using propidium iodide (PI) and Hoechst33342co-staining. In addition, celastrol upregulated the ratio of LC3II/LC3I in a time and dose dependent manner, suggesting autophagy was induced by celastrol in primary cardiomyocytes. Interestingly, autophagy inhibitor3-MA aggravated celastrol induced primary cardiomyocytes viability decrease. The following results further showed that ER stress signaling targeted gene GRP78and CHOP were up-regulated by celastrol in early stage and celastrol also activated three sensors of ER stress, including IRE1, PERK and ATF6. Moreover, suppressing ER stress induced proapoptotic transcription factor CHOP could partially attenuate the apoptosis and cell viability decrease induced by celastrol. These results suggested that ER stress was an early event responsed to celastrol treatment and CHOP dependent pathway played important role in celastrol-induced cardiomyocyte death.Although low concentration celastrol did not impair the cardiomyocyte viability markedly, chronic incubation with400nM celastrol significantly caused a prolongation of the action potential duration (APD) in primary cardiomyocytes, which was associated with the reduction in IKr. The IC50of celastrol for IKr was220.3nM. Western blotting analysis for ERGla protein expression showed that celastrol inhibited expression of full glycosylated ERGla at a low concentration (<150nM) and reduced expression of core glycosylated ERGla at a high concentration. The IC50of celastrol for full glycosylatedand core glycosylated ERGla were265.3nM and422.1nM, respectively. These result suggested that celastrol not only disrupted the trafficking of ERG1a protein but also affected the ERG1a protein expression at a high concentration. Furthermore, celastrol induced reduction of ERG1a mRNA may be account for the reduction of ERG1a protein expression. In addition, celastrol inhibited several other currents in primary cardiomyocytes, including ICa, It0and IK1, but had no effect on INa.Taken together, our present studies showed that celastrol may exert cardiotoxicity through inducing cardiomyocyte death or causing cardiac repolarization disorder. Therefore, individuals who are on celastrol should be followed closely for symptoms of left ventricular dysfunction and/or long QT interval.