Mechanisms Of Ribociclib-induced Cardiotoxicity By Targeting The HERG Channel

Objective(s):To investigate the cardiotoxic side effects caused by the CDK4/6 inhibitor ribociclib in breast cancer treatment,specifically studying the mechanism behind ribociclib-induced Long QT Syndrome(LQTS)and exploring potential solutions to the issue.This research aimed to provide a scientific solution for the safe use of ribociclib in breast cancer treatment.Methods:1.Guinea pigs were given an intraperitoneal injection of ribociclib,and electrocardiograms were recorded.QT intervals were measured and statistically analyzed to detect whether ribociclib can cause LQT.2.Using the patch clamp recording technique,the effects of ribociclib on cardiac action potentials in isolated guinea pig myocardial cells and human-induced pluripotent stem cell-derived cardiomyocytes(hi PSC-CMs)were examined.3.A long-term ribociclib-LQT model was established in C57BL/6 mice,and quantitative Real-Time PCR(q RT-PCR)was used to detect the effects of ribociclib on the expression levels of LQT-related genes in heart tissue,in order to facilitate identification of the target.4.The effects of ribociclib on the activity of LQT-related ion channels were detected by patch clamp recording technique to further clarify the critical target of ribociclib in causing LQT.Western blot was used to investigate the effects of ribociclib on the expression levels of key proteins.5.Patch clamp recording technique was performed to explore the effects of ribociclib on the kinetics of key ion channels in HEK293 T cells transfected with these channels.Point mutation experiments were conducted to identify the binding site of ribociclib with the target channel.6.Using the patch clamp recording technique,drugs that can reverse ribociclib-induced LQT were searched for.These drugs must activate the hERG current that is inhibited by ribociclib in HEK293 T cells and shorten the prolonged action potential of the myocardial cells that are extended by ribociclib.The effectiveness of these drugs was confirmed at the animal level by verifying whether they can shorten the prolonged QT interval caused by ribociclib.Results:1.Ribociclib prolonged QT interval in a concentration-dependent manner in guinea pigs.2.Ribociclib prolonged action potential duration(APD)in a concentration-dependent manner in cardiomyocytes.3.Ribociclib decreased the expression of LQT-related genes KCNQ1,KCNH2,SCN5 A,KCNJ2,SNTA1,and KCNJ5.4.Ribociclib reduced the expression of hERG channel protein on cell membranes.5.Ribociclib concentration-dependently inhibited hERG ion channels in the open state,exerting inhibitory function by binding to the Y652 and F656 sites to block the pore region.6.NS1643 could activate the hERG current inhibited by ribociclib in HEK293 T cells,shorten the prolongation of the cardiac action potential caused by ribociclib in cardiomyocytes,and shorten the QT interval prolongation caused by ribociclib at the animal level.Conclusion(s):Ribociclib downregulates the expression of LQT-related ion channel receptor genes such as KCNQ1,KCNH2,SCN5 A,KCNJ2,SNTA1,and KCNJ5.By screening heterologously expressed receptors that may be involved in regulating LQT in HEK293 T cells using the patch clamp recording technique,ribociclib was found to selectively inhibit hERG channels.The inhibition of hERG channels by ribociclib is concentration-dependent,and western blot analysis showed that ribociclib can reduce the expression of hERG channel proteins on the cell membrane.Therefore,ribociclib selectively inhibits hERG channels through a dual mechanism of direct blockage and reduction of hERG channel protein expression on the cell membrane.Ribociclib acts on the open state of the channel and exerts its inhibitory function by binding to the pore region at the Y652 and F656 sites.In addition,we found that the hERG channel activator NS1643 can activate the hERG channel inhibited by ribociclib.In cardiac cells,NS1643 can shorten the prolonged action potential caused by ribociclib,and at the animal level,it can also shorten the prolonged QT interval caused by ribociclib,reversing the LQT induced by ribociclib.