扩张型心肌病患者新型致室性心律失常和猝死的抗钙通道自身抗体的研究
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摘要
第一部分扩张型心肌病患者新型抗钙通道自身抗体的发现及致心律失常和猝死的机制
背景：室性心律失常和猝死是扩张型心肌病(dilated cardiomyopathy，DCM)严重的并发症，近年来研究发现自身免疫在DCM室性心律失常和猝死发生中起重要作用，而钙离子通道是DCM的免疫反应靶点之一。
目的：DCM患者体内是否存在抗钙通道自身抗体，抗钙通道自身抗体与DCM患者室性心律失常和猝死发生之间的关系及其机制。
方法：收集在我院住院治疗的80例DCM患者，以健康献血者80例作为对照，通过ELISA的方法检测抗钙通道自身抗体，免疫印迹和免疫荧光的方法研究抗钙通道自身抗体的特异性。Logistic回归等统计学方法分析抗钙通道自身抗体与DCM患者室性心律失常和猝死之间的关系，离体大鼠心脏灌流的方法进一步验证抗钙通道自身抗体致心律失常的作用。电生理实验探讨抗钙通道自身抗体致心律失常的机制。
结果：80例DCM患者中39例检测到抗钙通道自身抗体，对照组仅5例检测到抗钙通道自身抗体(48．8％vs 6．3％，P＜0．01)。24小时动态心电图检查，抗钙通道自身抗体阳性的DCM患者室性心律失常发生率高于抗钙通道自身抗体阴性患者(92．3%vs 70．7％，P=0．013)。经过32±8个月的随访，抗钙通道自身抗体阳性的DCM患者中8例死于猝死，高于抗钙通道自身抗体阴性的患者(20．5％vs 4．9％，P=0．045)。Logistic回归分析显示抗钙通道自身抗体阳性是DCM患者室性心律失常和猝死发生的独立危险因子。通过亲和层析的方法提纯抗体，免疫印迹和免疫荧光证实抗钙通道自身抗体能够与心肌细胞膜的钙通道特异性的结合。离体大鼠心脏灌流抗钙通道自身抗体可引起室性心动过速等室性心律失常。电生理实验证实抗钙通道自身抗体引起了大鼠心室肌细胞动作电位时程的延长和早期后除极的发生。将抗钙通道自身抗体作用于急性分离的人心房肌细胞，大鼠心室肌细胞和表达人Ca_v1．2通道的非洲爪蟾卵母细胞，结果均一致显示抗钙通道自身抗体可引起钙通道电流的增加。通过激光共聚焦显微镜检测到抗钙通道自身抗体还可引起胞内钙浓度的升高。
结论：本研究在DCM患者体内首次发现了一种新型的抗钙通道自身抗体，该抗体具有致室性心律失常的作用，可能是DCM患者发生室性心律失常乃至猝死的重要原因，其机制为抗钙通道自身抗体能够引起钙通道电流的增加和胞内钙浓度的升高，延长动作电位，导致早期后除极，触发可能引起猝死的室性心律失常。
第二部分钙通道抗原免疫大鼠致室性心律失常和猝死的研究
背景：DCM患者体内首次发现的新型抗钙通道自身抗体具有致室性心律失常的作用，可能是DCM患者发生室性心律失常乃至猝死的重要原因。
目的：通过钙通道抗原免疫大鼠的方法，建立抗钙通道抗体阳性的免疫大鼠模型，验证抗钙通道抗体致室性心律失常和猝死的作用及其机制。
方法：通过人工免疫钙通道抗原的方法建立抗钙通道抗体阳性的免疫大鼠模型。通过ELISA的方法检测免疫大鼠体内抗钙通道抗体的产生情况，免疫印迹和免疫荧光的方法研究抗钙通道抗体的特异性。心电图、心脏超声及病理学检查等检测免疫大鼠心脏形态与功能改变。离体大鼠心脏灌流的方法进一步验证抗钙通道抗体致心律失常的作用。电生理实验探讨抗钙通道抗体致心律失常的机制。
结果：ELISA方法检测到通过人工免疫钙通道抗原的方法成功的使大鼠体内产生了高滴度的抗钙通道抗体。通过亲和层析的方法纯化免疫大鼠体内产生的抗钙通道抗体，免疫印迹和免疫荧光方法证实抗钙通道抗体能够与心肌细胞膜的钙通道特异性的结合。抗钙通道抗体阳性的免疫大鼠免疫过程共死亡3只，均为猝死(3／20，15．0％)，而对照组大鼠均正常。10只抗钙通道抗体阳性的免疫大鼠(10／17，58．8％)检测到室性心律失常的发生，离体大鼠心脏灌流进一步证实抗钙通道抗体可引起室性早搏、室性心动过速等室性心律失常。电生理实验研究发现抗钙通道抗体引起了大鼠心室肌细胞动作电位时程的延长和早期后除极的发生。将抗钙通道抗体作用于大鼠心室肌细胞，可引起钙通道电流的增加。通过激光共聚焦显微镜检测到抗钙通道抗体引起了心肌细胞胞内钙浓度的升高，胞内钙超载甚至细胞死亡。台盼蓝细胞毒性试验证实了抗钙通道抗体的细胞毒性作用。
结论：钙通道抗原免疫大鼠可导致室性心律失常乃至猝死的发生，其机制为抗钙通道抗体能够引起钙通道电流的增加和胞内钙浓度的升高，延长动作电位，产生早期后除极，触发室性心律失常而导致猝死。
Part one
High sudden death incidence of patients with arrhythmogenicautoantibodies against calcium channel in dilated cardiomyopathy
Background: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(39/80,48.8％)and 5HBD(5/80,6.3％,P＜0.01).Ventricular arrhythmia was more common in CC-AAbspositive than in negative patients with DCM(92.3％vs 70.7％,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{[Ca~(2+)]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-L)and[Ca~(2+)]i.
Part two
Ventricular arrhythmias and sudden death in rats immunizedby calcium channel antigen
Background: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-L)was significantly increased in thepresence of CC-Abs in rat ventricular myocytes.Intracellular calcium concentration([Ca~(2+)]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-L)and [Ca~(2+)]i.
背景：室性心律失常和猝死是扩张型心肌病(dilated cardiomyopathy，DCM)严重的并发症，近年来研究发现自身免疫在DCM室性心律失常和猝死发生中起重要作用，而钙离子通道是DCM的免疫反应靶点之一。
目的：DCM患者体内是否存在抗钙通道自身抗体，抗钙通道自身抗体与DCM患者室性心律失常和猝死发生之间的关系及其机制。
方法：收集在我院住院治疗的80例DCM患者，以健康献血者80例作为对照，通过ELISA的方法检测抗钙通道自身抗体，免疫印迹和免疫荧光的方法研究抗钙通道自身抗体的特异性。Logistic回归等统计学方法分析抗钙通道自身抗体与DCM患者室性心律失常和猝死之间的关系，离体大鼠心脏灌流的方法进一步验证抗钙通道自身抗体致心律失常的作用。电生理实验探讨抗钙通道自身抗体致心律失常的机制。
结果：80例DCM患者中39例检测到抗钙通道自身抗体，对照组仅5例检测到抗钙通道自身抗体(48．8％vs 6．3％，P＜0．01)。24小时动态心电图检查，抗钙通道自身抗体阳性的DCM患者室性心律失常发生率高于抗钙通道自身抗体阴性患者(92．3%vs 70．7％，P=0．013)。经过32±8个月的随访，抗钙通道自身抗体阳性的DCM患者中8例死于猝死，高于抗钙通道自身抗体阴性的患者(20．5％vs 4．9％，P=0．045)。Logistic回归分析显示抗钙通道自身抗体阳性是DCM患者室性心律失常和猝死发生的独立危险因子。通过亲和层析的方法提纯抗体，免疫印迹和免疫荧光证实抗钙通道自身抗体能够与心肌细胞膜的钙通道特异性的结合。离体大鼠心脏灌流抗钙通道自身抗体可引起室性心动过速等室性心律失常。电生理实验证实抗钙通道自身抗体引起了大鼠心室肌细胞动作电位时程的延长和早期后除极的发生。将抗钙通道自身抗体作用于急性分离的人心房肌细胞，大鼠心室肌细胞和表达人Ca_v1．2通道的非洲爪蟾卵母细胞，结果均一致显示抗钙通道自身抗体可引起钙通道电流的增加。通过激光共聚焦显微镜检测到抗钙通道自身抗体还可引起胞内钙浓度的升高。
结论：本研究在DCM患者体内首次发现了一种新型的抗钙通道自身抗体，该抗体具有致室性心律失常的作用，可能是DCM患者发生室性心律失常乃至猝死的重要原因，其机制为抗钙通道自身抗体能够引起钙通道电流的增加和胞内钙浓度的升高，延长动作电位，导致早期后除极，触发可能引起猝死的室性心律失常。
第二部分钙通道抗原免疫大鼠致室性心律失常和猝死的研究
背景：DCM患者体内首次发现的新型抗钙通道自身抗体具有致室性心律失常的作用，可能是DCM患者发生室性心律失常乃至猝死的重要原因。
目的：通过钙通道抗原免疫大鼠的方法，建立抗钙通道抗体阳性的免疫大鼠模型，验证抗钙通道抗体致室性心律失常和猝死的作用及其机制。
方法：通过人工免疫钙通道抗原的方法建立抗钙通道抗体阳性的免疫大鼠模型。通过ELISA的方法检测免疫大鼠体内抗钙通道抗体的产生情况，免疫印迹和免疫荧光的方法研究抗钙通道抗体的特异性。心电图、心脏超声及病理学检查等检测免疫大鼠心脏形态与功能改变。离体大鼠心脏灌流的方法进一步验证抗钙通道抗体致心律失常的作用。电生理实验探讨抗钙通道抗体致心律失常的机制。
结果：ELISA方法检测到通过人工免疫钙通道抗原的方法成功的使大鼠体内产生了高滴度的抗钙通道抗体。通过亲和层析的方法纯化免疫大鼠体内产生的抗钙通道抗体，免疫印迹和免疫荧光方法证实抗钙通道抗体能够与心肌细胞膜的钙通道特异性的结合。抗钙通道抗体阳性的免疫大鼠免疫过程共死亡3只，均为猝死(3／20，15．0％)，而对照组大鼠均正常。10只抗钙通道抗体阳性的免疫大鼠(10／17，58．8％)检测到室性心律失常的发生，离体大鼠心脏灌流进一步证实抗钙通道抗体可引起室性早搏、室性心动过速等室性心律失常。电生理实验研究发现抗钙通道抗体引起了大鼠心室肌细胞动作电位时程的延长和早期后除极的发生。将抗钙通道抗体作用于大鼠心室肌细胞，可引起钙通道电流的增加。通过激光共聚焦显微镜检测到抗钙通道抗体引起了心肌细胞胞内钙浓度的升高，胞内钙超载甚至细胞死亡。台盼蓝细胞毒性试验证实了抗钙通道抗体的细胞毒性作用。
结论：钙通道抗原免疫大鼠可导致室性心律失常乃至猝死的发生，其机制为抗钙通道抗体能够引起钙通道电流的增加和胞内钙浓度的升高，延长动作电位，产生早期后除极，触发室性心律失常而导致猝死。
Part one
High sudden death incidence of patients with arrhythmogenicautoantibodies against calcium channel in dilated cardiomyopathy
Background: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(39/80,48.8％)and 5HBD(5/80,6.3％,P＜0.01).Ventricular arrhythmia was more common in CC-AAbspositive than in negative patients with DCM(92.3％vs 70.7％,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{[Ca~(2+)]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-L)and[Ca~(2+)]i.
Part two
Ventricular arrhythmias and sudden death in rats immunizedby calcium channel antigen
Background: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-L)was significantly increased in thepresence of CC-Abs in rat ventricular myocytes.Intracellular calcium concentration([Ca~(2+)]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-L)and [Ca~(2+)]i.
引文
1 中华医学会心血管病分会、中华心血管病杂志编委会、中国心肌病诊断与治疗建议工作组．心肌病诊断与治疗建议．中华心血管病杂志．2007； 35：3-16．
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