The Effects And Related Mechanisms Of DPP6 On Ventricular Electrical Activity In Guinea Pig

Ventricular fibrillation(VF)is the major cause of cardiac arrest and sudden cardiac death.Through the patient's etiology,genetics research and animal model study found that VF occurrence is often due to cardiac ion channel abnormalities.Idiopathic ventricular fibrillation(IVF)is one type of inherited ventricular fibrillation.Due to lack of etiology and atypical clinical symptom,IVF has a very high mortality.Recent study suggests the occurrence of IVF is closely related to the abnormal expression of dipeptidyl peptidase-like protein type 6(DPP6).However,the detailed underlying mechanism is currently unknown.DPP6 is a putative auxiliary subunit of Kv4 channels that encod cardiac transient outward potassium current(Ito)and play an important role in the maintenance of normal rhythm.However,the functions of DPP6 in heart,especially it's effects on arrhythmia are rarely reported currently.The present study prove that DPP6 abnormalities led to heart(especially Purkinje fiber)Ito current increase is a major cause of ventricular fibrillation.Though DPP6 is a putative auxiliary subunit of Kv4 channels,the study found that DPP6 had an impact on both A-type potassium current and sodium channel in the nervous system.Supriseingly,DPP6 was present at the reigon where Kv4 negatively expressed.Therefor we hypothesize that DPP6 also modulate other cardiac ion channels and thus affects the occurrence of ventricular fibrillation.To answer this scientific question,we constructed adenovirus mediated DPP6 overexpression vector and infected cultured guinea pig cardiomyocytes successfully.We detect the effects and mechanisms of DPP6 overexpression on cardiac electrical activity of guinea pig.This study will provide important experimental basis for revealing the etiology,pathology,prevention and treatment of DPP6 in a certain idiopathic ventricular fibrillation,withimportant basic and clinical significance.Objectives:(1)To evaluate the overexpression efficiency of adenovirus mediated DPP6 overexpressing vector in guinea pig ventricular myocytes.(2)To study the effects of DPP6 on the action potential of guinea pig ventricular myocytes;(2)To study the effects and mechanisms of DPP6 on the ion channels(sodium channel,L-type calcium channel,IKr,IKs)in the guinea pig ventricular myocytes.Methods:(1)Identifing the overexpression efficiency of adenovirus mediated DPP6 overexpression vector by fluorescence,real-time PCR and western-blot.(2)Using whole cell current-clamp technique to record action potential in guinea pig ventricular myocytes and to observe the changes of AP after overexpress DPP6.(3)Using voltage-clamp technique to detect the changes of sodium channel,L-type calcium channel and IKr,IKs.Results:(1)Fluorescence microscopy revealed that the ventricular cardiomyocytes infected with adenovirus had green fluorescent protein(GFP)expression.Analyzed by qPCR technique,DPP6 mRNA level was significantly increased in guinea pig ventricular myocytes infected with adenovirus after cultured for 48 hours.DPP6 protein level was significantly increased in guinea pig ventricular myocytes infected with adenovirus after cultured for 48 hours analyzed by western blot technique.(2)Overexpression of DPP6 in guinea pig ventricular myocytes shortened the action potential durations significantly and accelerated the phase 0 of up-stroke compared to control group;(3)Overexpression of DPP6 in guinea pig ventricular myocyte increased sodium current density significantly,both the activation curve and the inactivation curve were shifted to the hyperpolarization direction,and the activation curve moved more significantly.There is no change in recovery curves.The L-type calcium current density is markedly reduced,the activation curve moves to the depolarization direction,but there is no change in inactivation curves.Both IKr and IKs are not changed.Conclusions:(1)We successfully constructed adenovirus mediated DPP6 overexpression vector which could be used in further functional studies ofDPP6 in cardiomyocytes.(2)Overexpression of DPP6 induces a significant shortening of action potential duration in guinea pig ventricular myocytes and the speed of phase 0 was accelerated,which might be related to the increasement in sodium currents and reduction in calcium currents in ventricular myocytes overexpression of DPP6.The cause of action potential changes is that DPP6 overexpression increased sodium current and reduced calcium current significantly.All above suggest that the effect of DPP6 on myocardial electrical activity may have some pathological significance.