The Ion And Molecular Mechanism Of Age-Related Trigger Arrhythmia In Homozygous R33Q Calsequestrin2 Knock-in Mouse

Background:Inherited arrhythmogenic diseases(IADs)can cause sudden cardiac death(SCD),threating human life and health.Catecholaminergic polymorphic ventricular tachycardia(CPVT)is one of the IADs,which has a low morbidity but a high-risk of sudden death.With no organic structural changes,the clinical manifestation of CPVT shows the different symptoms such as torsades de points or polymorphic ventricular tachycardia,syncope,and even sudden death.The sudden death rate increases as high as 30%-50%of patients with age to 40 years old in the absence of treatment.Nowadays,some researches show that the main causative genes of CPVT consist of RYR2 mutation and CASQ2 mutation.CASQ2 mutation can affect sarcoplasmic reticulum calcium storage,by participating in the calcium release function of RYR2,lead to imbalance of intracellular calcium homeostasis and cause cardiomyocytes ion channel currents disorders,inducing malignant arrhythmia.It is found to have different incidences of DAD and TA in the CASQ2R33Q/R33Q knock-in mouse in different ages.Therefore,we studied the ion and molecular mechanism of malignant arrhythmia of CASQ2R33Q/R33Q knock-in mouse with aging.Objective:To study the ion and molecular mechanism of the increased incidence of DAD and TA with aging in the CASQ2R33Q/R33Q knock-in mouse,and then providing a theoretical basis to explore the mechanism of arrhythmia.Methods:It was studied the function and cellular electrophysiology of the age in 3-mo,9-mo,12-mo groups of CASQ2R33Q/R33Q knock-in mouse and wild type mouse,respectively.With HE and Masson staining technique,the myocardial structure and fibrosis were observed.Cardiac function was evaluated by ultrasound,and T tube structure was determinated by fluorescence staining.The structure of the j SR and t-tube coupling was mensurated by transmission electron microscope.Langendorff perfusion device was employed to achieve well-conditioned ventricular myocytes with the double-enzymatic separation method.Using patch clamp technique to record the action potential(AP)of single ventricular myocyte and ICa,L and Iti currents,and explore the occurrence of DAD and TA.The occurrence of calcium release,calcium spark and calcium wave in ventricular myocytes were mensurated by confocal microscopy and IonOptix microscopy.The expression of calcium transport proteins were analyzed by Western Blotting method.Results:(1)The incidence of DAD and TA in the CASQ2R33Q/R33Q knock-in mouse with the age of 9-mo group and 12-mo group increased significantly compared to 3-mo group and the control group(P<0.05).The densities of Iti current in the CASQ2R33Q/R33Q knock-in mouse of 9-mo group(-2.1 ±0.2 pA/pF)and 12-mo group(-2.2±0.1 pA/pF)was markedly higher than that of the 3-mo group(-0.9 + 0.1pa/pF)(P<0.05).(2)The cardiac size and function between CASQ2R33Q/R33Q knock-in mouse and control group were not altered,and no any disorder of myocardial fibers and fibrosis.(3)In the case of CASQ2R33Q/R33Q mutation,ultrastructure of jSR and t-tube coupling was abnormality.Compared with the 3-mo group and the control group with age-match wild type,jSR structure of the 12-mo group was swelling and fracture severity,and the density granule material decreased significantly.(4)In contrast with 3-mo knock-in mouse group,ICa,L currents were obviously increaed and the time course for recovery from inactivation were shortened,protein expression of Cav1.2 were clearly up-regulation in 9-mo group and 12-mo group(P<0.01).(5)Compared with 3-mo knock-in mouse group,the amplitude of calcium release of 9-mo and 12-mo knock-in mouse groups reduced sharply,while calcium release delay time prolonged significantly.With the stimulation of isoproterenol,the incidence of spontaneous calcium release,calcium spark and calcium wave increased significantly.(6)Along with the aging,protein expression of CASQ2 was down-regulation significantly,while protein expression CaMKII and P-RYR2 increased in CASQ2R33Q/R33Q knock-in mouse.The expression of protein RYR2,SERCA2a,and NCX1.1 related to calcium recycle were not remakrable difference.Conclusion:(1)The CASQ2R33Q/R33Q mutation resulted in a significant increase in the incidence of DAD and TA related to aging.(2)Along with the aging,the jSR and T tube coupling structural damage of CASQ2R33Q/R33Q mutation mouse was aggravating.(3)It were the down-regulation of CASQ2 protein expression and up-regulation of P-RYR2 protein expression of CASQ2 mutation mouse with aging,result in diastolic calcium leak.(4)ICa,L density of CASQ2R33Q/R33Q knock-in mouse was increased with the shorter recovery time from inactivation and up-regulation of Cav1.2 protein expression along with aging.(5)The incidence of spontaneous calcium release,calcium sparks and calcium wave of CASQ2R33Q/R33Q knock-in mouse were increased significantly.Intracellular calcium overload with diastolic calcium leakage and calcium reuptake delay leads to increasement of Iti current,which may be the current and molecular mechanism of malignant arrhythmia aggravation.