Novel Arrhythmogenic Autoantibodies Against Calcium Channel Lead To Sudden Death In Dilated Cardiomyopathy

Part oneHigh sudden death incidence of patients with arrhythmogenicautoantibodies against calcium channel in dilated cardiomyopathyBackground:Ventricular arrhythmias and sudden death were severe complications ofdilated cardiomyopathy(DCM).Autoantibodies have emerged as a new upstream target ofboth ventricular arrhythmias and sudden death in DCM,while calcium channel plays animportant role in the autoimmune pathogenesis of DCM.Objective:We sought to validate the hypothesis that there are independentautoantibodies against L-type calcium channel(CC-AAbs)which have an arrhythmogeniceffect and lead to sudden death in patients with DCM.Methods:We surveyed sudden death in 80 patients with DCM and exploredrelationship between the CC-AAbs and ventricular arrhythmia by statistics analysis andLangendorff perfused isolated rat heart models.80 health blood donors(HBD)wereselected as a control group.Immunoblotting and immunofluorescence were used to confirmthe specificity of CC-AAbs.Electrophysiological experiments were performed to study thearrhythmogenic mechanism of CC-AAbs.Results:CC-AAbs were detected in 39 patients with DCM(39/80,48.8ï¼…)and 5HBD(5/80,6.3ï¼…,P＜0.01).Ventricular arrhythmia was more common in CC-AAbspositive than in negative patients with DCM(92.3ï¼…vs 70.7ï¼…,P=0.013).After a mean follow-up period of 32±8 months,the incidence of sudden death was higher in theCC-AAbs positive patients than the negative ones(20.5% vs 4.9%,P=0.045).Bymultivariate logistic regression analysis,the presence of CC-AAbs was the independentpredictorof both ventricular tachycardia(VT)and sudden death.The CC-AAbs werepurified from sera of patients with DCM by affinity chromatography.The CC-AAbsrecognized L-type calcium channel specifically by immunoblotting andimmunofluorescence.VT generated by CC-AAbs perfusions in isolated perfused rat heartmodel.CC-AAbs not only prolonged the action potential duration(APD),but also inducedtheearly afterdepolarization(EAD).L type calcium currents(I_Ca-L)was significantlyincreased in the presence of CC-AAbs on human atrial myocytes,rat ventricular myocytesand Xenopus oocytes expressing Cav1.2 channels,while intracellular calcium concentration{[Ca²⁺]i} was enhanced by CC-AAbs.Conclusions:For the first time,we have found high sudden death incidence ofpatients with novel CC-AAbs in DCM.The CC-AAbs could induce sudden death and VTby prolongation of APD and triggered activity of EAD which resulted from enhancement ofI_Ca-Land[Ca²⁺]i.Part twoVentricular arrhythmias and sudden death in rats immunizedby calcium channel antigenBackground:We have found novel arrhythmogenic autoantibodies against L-typecalcium channel(CC-AAbs)which induce ventricular arrhythmias and sudden death inpatients with DCM. Objective:We sought to immunize rats by calcium channel antigen and make theimmunized-rat model with the antibodies against L-type calcium channel(CC-Abs).Thearrhythmogenic mechanisms of CC-Abs were further studied.Methods:The immunized-rat model with the CC-Abs was made by artificialimmunity of calcium channel antigen.The CC-Abs were detected by ELISA.Immunoblotting and immunofluorescence were used to confirm the specificity of CC-Abs.The morphology and function of immunized rats' hearts were detected byelectrocardiogram,cardiac ultrasound and pathology.Langendorff perfused isolated ratheart models were used to verify the arrhythmogenic effects of CC-Abs.Electrophysiological experiments were performed to study the arrhythmogenic mechanismof CC-Abs.Results:The immunized-rat model with high titers of CC-Abs was madesuccessfully by artificial immunity.The CC-Abs were purified from immunized rats byaffinity chromatography and specific CC-Abs were found in immunized rats byimmunoblotting and immunofluorescence.Ventricular arrhythmias were found inimmunized rats(10/17,58.8%),while 3(3/20,15%)immunized rats died from sudden death.VT generated by CC-Abs perfusions in isolated perfused rat heart model.CC-Abs not onlyprolonged the APD,but also induced the EAD.I_Ca-Lwas significantly increased in thepresence of CC-Abs in rat ventricular myocytes.Intracellular calcium concentration([Ca²⁺]i)was enhanced by CC-Abs and lead to calcium overload.The toxic effect ofCC-Abs was confirmed by typan blue toxicity test.Conclusions:We made the immunized-rat model with the CC-Abs successfully.The CC-Abs were arrhythmogenic and could induce VT by prolongation of APD andtriggered activity of EAD which resulted from enhancement of I_Ca-Land [Ca²⁺]i.