The Study Of The Mechanism Of Ibrutinib,A Targeted Treatment Of Lymphoma,Induced Ventricular Arrhythmias

Bruton's tyrosine kinase(BTK)inhibitor Ibrutinib has been used more and more widely because of its remarkable efficacy in B-cell lymphoma treatment.The subsequent cardiac complications such as atrial fibrillation(AF)and more life-threating ventricular arrhythmias(VA)have resulted in a common concern and high attention in the field of Cardio-oncology and hematology,for which the usage has also been limited.Ibrutinib-related arrhythmias and its related mechanisms have become a hotspot in the field of Cardio-oncology,but few studies have been conducted.The purpose of this study is to try to answer the following questions:1)Ibrutinib-induced VA has only been reported in a small number of cases and in a retrospective clinical study.It is inconclusive whether Ibrutinib increases susceptibility to ventricular arrhythmias;2)Age and previous cardiac disease are independent risk factors for Ibrutinib-related atrial fibrillation.Whether Ibrutinib-related VA is related to age and previous heart disease needs to be clarified;3)Cytoplasmic calcium abnormalities and membrane electrophysiological abnormalities are common related mechanism of ventricular arrhythmias,which can be analyzed by optical mapping(calcium mapping and voltage mapping).Whether the Ibrutinib treatment will cause related electrophysiological abnormalities needs to be clarified;4)Chronic lymphocytic leukemia(CLL)can cause metabolic stress,and metabolic stress marker AMPactivated protein kinase(AMPK)is activated.The activation of AMPK level of Ibrutinib-sensitive CLL cells are reduced compared to Ibrutinibresistant CLL cells.Inhibition of AMPK activation has been shown to be associated with increased arrhythmia susceptibility in a few studies.In the study of the molecular mechanism of Ibrutinib related VA,it should be made clear whether VA is related to its related metabolic stress(AMPK regulation).The experiment was designed into two parts: 1)Acute study of Ibrutinib effect: The hearts of young(age: 10-14 weeks)and old(age: 10-14 months)spontaneously hypertensive rats(SHR)were harvested under anaesthesia and retrogradely perfused in a Langendorff perfusion system through the aorta.Ibrutinib(0.1 ?M)or vehicle(DMSO)was added to the perfusion system and the heart was exposed to the drug for 30 minutes.During this period,calcium and voltage-sensitive fluorescent dyes were added to the perfusion system for staining.Through a pacing device connected to the rat's heart "seat",the SHR heart was paced incrementally(9.0hz-12.5hz,every 0.5hz)for 30 s.Signals of the 4 seconds including the last two seconds of pacing and first two seconds after the pacing stopped were collected.Calcium dynamics and action potentials changes the LV epicardial of SHR hearts were analyzed with a custom program made by Matlab(Math Works),calcium transient parameters and action potential duration(APD)parameters were selected and processed.VA susceptibility was assessed by burst pacing induced ventricular fibrillation(VF).A ventricular fibrillation episode with a duration of ?10seconds after a burst pacing is considered to be VF successful induction.The following pathways and proteins were analyzed with Western blot method:(1)Calcium handling protein: Ry R2 receptor phosphorylation at Ser2814 and Ser2808 sites;PLB phosphorylation at Thr17 and Ser16sites;(2)PI3K-Akt pathway;(3)AMPK phosphorylation.2)In-vivo study of Ibrutinib effect: Old Sprague-Dawley(SD)rats were selected as the research animals,and Ibrutinib 10 mg/kg/d(equivalent to the clinical treatment dose of 420 mg / d)was chosen as the treatment dose.After two weeks and four weeks of oral gavage(control group(DMSO)and Ibrutinib group),the hearts of the SD rats were harvested,Langendorff-perfused and subjected to incremental stimulation(9.0hz-15.0hz,every 0.5hz)for 30 s.Then calcium mapping(same as above)were done.The following parameters were compared in the control group and the ibtinib group to determine the Ibrutinib induced VA animal model;(1)Frequency and threshold of Post incremental pacing VA;(2)Time duration of VF after pacing,the ratio of successful VF induction(VF ? 10 seconds)and sustained VF induction(VF ? 60seconds).If VF persisted for more than 60 s,3J defibrillation is delivered.At the same time,the calcium kinetics was analyzed again(protocol same as acute study).The following pathways and proteins were analyzed with Western blot method:(1)Calcium handling proteins: Ry R2 receptor expression and phosphorylation at Ser2814 and Ser2808 sites;PLB expression and phosphorylation at Thr17 and Ser16 sites;SERCA2a expression;(2)PI3K-Akt pathway(3)Expression and activation of AMPK.In the acute part,we found that Ibrutinib significantly increased susceptibility to VA in old SHR(27.5% vs.5.71% [control group],p =0.015),but in young SHR,this increase was not statistically significant(8% vs.0% [control group],p = 0.49).In old SHR,after Ibrutinib treatment,the calcium transient duration 50(Ca TD50)was prolonged(p= 0.007),calcium transient amplitude alternans ratio was significantly reduced(p = 0.03),and the time to peak(TPP)of calcium transient was shortened(p <0.001).Changes in the above parameters suggested that thecalcium release rate of Ry R2 receptors was accelerated,and the ability of SERCA2 a to uptake Ca2+ from the cytoplasm was dysfunctioned.In young SHR,no differences in calcium dynamics parameters between the two groups were observed.Membrane electrophysiological parameters show that in old SHR rats,the left ventricular epicardial action potential duration 80(APD80)alternation(p <0.001)and APD alternation spatial discordance in the Ibrutinib treatment group(P <0.001)were more prominent,which meant that the SHR rats treated with Ibrutinib had increased temporal and spatial discordance of left ventricular epicardial depolarizations.However,western blot results related to aged SHR rats showed that there was no difference between the two groups in Ry R2 receptor phosphorylation,PLB phosphorylation,AMPK activation,PI3 K expression,and Akt activation.In considering of the experimental design,short time of contact with the drug is possibly the reason.Also,the changes in the function of Ry R2 receptor and SERCA2 a by Ibrutinib might not be regulated through phosphorylation.In summary,acute study showed that Ibrutinib can increase the inducibility of ventricular arrhythmias in SHR,especially in the old and those with previous existing cardiac disease;abnormal calcium handling and left ventricular epicardial repolarization discordance is the electrophysiological mechanism of increased susceptibility to VA caused by Ibrutinib.In the in vivo part,10 mg / kg / d Ibrutinib was oral gavaged to the SD rats for 4 weeks to establish an animal model of Ibrutinib induced VA.10 mg / kg / d Ibrutinib Oral gavage can significantly increase the rate of VA after incremental pacing(15.38% vs 5.77%,p = 0.04).At the same time,the threshold of post pacing VA in Ibrutinib group was significantly lower than that of control group(10.7 ± 0.9 hz vs 13.7 ± 0.5 hz,p = 0.02).After bursting pacing,in the four-week oral gavaged rats,the total duration of VF after burst pacing was significantly prolonged comparedwith the control group(190.3 ± 18.4s vs 82.5 ± 3.9s,p = 0.0017);Ibrutinib group increased the proportion of successful VF induction(70%vs 60%),but the increase was not statistically significant(p = 0.48).Meanwhile,for sustained VF inductions,compared with the control group,Ibrutinib group also significantly increased the induction success rate(40% vs 10%,p = 0.004).At the same time,the VF induction result compositions in the two groups after four weeks treatment was classified into without VF,VF self-terminated,and VF required defibrillation treatment.After burst pacing,the proportion of non-VF in the Ibrutinib treatment group and the control group was close(20% vs 15%),but VF needs defibrillation in the Ibrutinib group was significantly higher than the control group(40% vs 10%).),Self-terminating VF was significantly lower than the control group(40% vs 75%).For calcium dynamics analysis,in Ibrutinib-treated group,Ca TD80 was prolonged(p = 0.01),calcium transient amplitude alternans ratio was reduced(p = 0.008),and time to peak was shortened(p = 0.046),while no differences were observed in Ca TD50,Ca TD50 / Ca TD80 ratio,and SCa E(p = 0.76,0.45,and 0.38,respectively).The molecular mechanism showed that compared with the control group,the Ibrutinib group reduced PI3K110? expression(p = 0.01),decreased AMPK phosphorylation level(p = 0.04),and reduced Akt phosphorylation level(p = 0.05).Expression of AMPK,Akt were not statistically different between the two groups(p values were0.89,0.26 respectively).Similarly,there was no significant change in Ry R2 receptor expression and phosphorylation and SERCA2 a receptor expression and phosphorylation between the two groups.An animal model of ventricular arrhythmia caused by Ibrutinib was provided in the maintext;it was also verified that ibrutinib affected both Ry R2 sarcoplasmic reticulum calcium release function and SERCA2 a calciumuptake capacity;Ibrutinib related VA may be related to inhibition of AMPK activation and its associated calcium abnormalities.Conclusion:Ibrutinib can increase the inducibility of ventricular arrhythmias,especially in the elderly and those with existing cardiac diseases;abnormal calcium handling and dysregulated membrane electrophysiology repolarization are important electrophysiological mechanisms;Ibrutinib induced VA may be related to inhibition of AMPK activation and its associated calcium handling abnormalities.