Tripterygium And YK1101 Affect Myocardial Repolarization By Inhibiting The Potassium Current Of I _Kr

Human ether a-go-go related gene (HERG) encodes the a subunit of the rapid component of delayed rectifier current (IKr), which plays an important role at the terminalphase of repolarization in cardiac action potential.Rat ERG1is homologous to full-length HERG1.It is well known that the dysfunction in IKr channel either by application of drugs or by rare mutations in some familiesis associated witha potentially fatal disorder called long QT syndrome, a life-threatening arrhythmia,creating a concomitant risk of cardiac sudden death. A diverse group of drugs have been reported to be linked to the prolongation of QT intervals, including antiarrhythmics (especially Class1A and Class III), anti-psychotic agents, certain antibiotics (including quinolones and macrolides), anti-histamineagent(terfenadine), and anti-tumor compounds (Amsacrine) as well. Due to the awareness of the potential danger of such QT drugs, the risk assessment of HERG inhibition has become a major pharmacological safety concern.Celastrol is the first isolated compound of the Thunder God Vine(Tripterygium wilfordii Hook F.), a perennial celastraceae vine. An increasing number of studies have shown thatCelastrol plays a potent antioxidant and anti-inflammatory rolethrough multiple targets in the cell, such as the NF-κB, HSP90and its co-chaperone, proteasome, Akt/mTOR signal pathway and other cellular targets,raising interesting possibility thatcelastrol may serve as a novel drug fortherapies ofcancer and inflammatory diseases. However, in our previous study we observed an obvious apoptosis in NRVMs upon application of celastrol inmicromolar rangewhichis equivalent to the dose used for anti-tumor or anti-inflammitory treatment reported by others. More than that, we also observed a significant prolongation of cardiac action potential duration caused in response totreatment of celastrol in nonomolar range, implying that celastrol is of the potentialto induce arrhythmia by disturbance of cardiac replolarozation. YK1101is a novel compond ofhistone deacetylase inhibition, which has great potential to therapy ofcutaneous T-cell Lymphoma in early development stage. In order to further investigate the mechanism regarding to celastrol-induced the prolongation of action potential duration, and analyze the effect of YK1101on IKr, primary cultured rat nenatal ventricular myocytes (NRVMs) and HEK293cell line expressing HERG K+channels were used in the present study forelectrophysiological recording, molecular biology analysis.The main results were as follows: 1. Using whole-cell patch clamp recording, it was evident that celastrol induced inhibition ofIKr in NRWMs in a concentration dependent manner.2. Western blotting analysis indicated that under the chronic treatment of different concentration, celastrol significantly reduced the expression of mature and immature form of ERG1a in a time and concentration dependent manner. The data analysis showed that celastrol inhibited expression of mature ERG1aat the concentration lower than150nM, andinhibited expression both mature and immature ERG1aat the concentration over150nM, giving the ICsovalues of265.3nM and422.1nM for celastrol induced inhibition ofmature and immature ERG1a respectively. These result suggested that celastrol not only disrupted the trafficking of ERG1a protein but also affected the ERG1a protein expression.3. RT-qPCR analysis showed that celastrol induced reduction of ERG mRNA expression in RNVMs in a time and concentration dependent manner, but had no effect on that in HEK293cell linesheterologously expressing HERG potassium channel driven by a different promoter, suggestinga regulatory effect of celastrol on KCNH2at transcriptional level.4. Bioinformatics analysis combined withLuciferase and ChIP assays demonstrated that celastrol do has regulatoryeffect onKCNH2at transcriptional level, which were evidenced by the fact thatcelastrol caused a distinct suppression ofNF-KB itself and ability of NF-κB binding to promoter area in KCNH2, revealing a novel molecular mechanisms that contribute tocelastrol-induced inhibition of IKrpreviously observed in NRVMs.5. In HEK293cells heterologously expressing HERG channels,YK1101inhibited the IHERGincluding the step and tail current in a concentration-dependent manner, but had no effect on the voltage-dependence of IHERG.Moreover, YK1101accelerated inactivation of HERG at the voltage range from-10mV to+10mV and inhibited recovery from inactivation of HERG at the voltages range from-40to-20mV. Afitting of dose-response curve using Hill’s equation gave anICsovalue of97.40μMforIHERG-tailinhibition,which was32.5fold more than the Cmax of other analog compoundsin clinic use, illustratinga lesspotencyof YK1101to induce QT prolongation.In summary, celastrol and YK1101both exhibited inhibition on IKr current but in the different inhibition degree. Our results for the first time reveal a novel mechanism that celastrol exerts its arrhythmogenic cardiotoxcity. As a potential anti-tumor drug, the potential toxic effectsof celastrol on repolarization and QT prolongation can not be ignored.