β-受体阻滞剂恶化的室性心律失常信号转导机制研究

Ventricular arrhythmia is the common cause of cardiac sudden death. A great deal of data demonstrate that catecholamine secretion secondary to the activation of sympathetic system apparently promote ventricular arrhythmia. Guidelines recommend isoprenaline can be used for induction of idiopathic ventricular arrhythmia, which may facilitate mapping and ablation. The application of beta adrenoceptor blocker markedly reduce the incidence of ventricular arrhythmia and mortality..However, we accidentally found ß1 adrenoceptor blocker aggravated ventricular arrhythmia (BAVA) during our ablation procedure. In those patients, isoprenaline apparently inhibited the ventricular arrhythmia, whereas intravenous esmolol marked aggravated the arrhythmia.The phenomenon had good repeatability and was not associated with heart rate. Thus, those results suggest that signal transduction mechanism of catecholamine promoting ventricular arrhythmia might have other pathways.Our study by exome sequencing and further verification showed that missense mutation of PKA existed in some patients. .Hence, we suppose that the signal transduction of BAVA was via the activation of ERK pathway through β2 adrenoceptor. To test our hypothesis, we plan to study on the following aspects: (1) in vivo: The heart of rats (adriamycin-induced heart failure model) are selectively transfected with antisense PKA or ERK carried by rAAV9;β2 adrenoceptor stimulator and (or) β2 adrenoceptor blocker and electrophysiology stimulation are given; the incidence of ventricular arrhythmia, the change of threshold of ventricular fibrillation, ERP, and QTc are evaluated; the expression and activity of of ERK/PKA are determined;(2) organ or tissue: in Langendorff-perfused rat heart transfected antisense PKA or ERK, APD characteristic, repolarization dispersion, EAD, DAD, and the changes of ERP and QTc are evaluated by micro-electrode and ECG recording technique; (3) myocardial cell from transfected rat: myocardial cells are separated, then are treated with ERK/PKA inhibitor or β2 adrenoceptor stimulator and (or) β2 adrenoceptor blocker ; Ito/Ikr/Iks are assessed via whole cell patch clamp technique; (4) myocardial cell transfected with antisense ERK/PKA by rAAV9: cells are treated with ERK/PKA inhibitor or β2 adrenoceptor stimulator and (or) β2 adrenoceptor blocker, then the above parameters, and the expression/activity of ERK/PKA are determined.

室性心律失常是心源性猝死的主要原因，是世界公共卫生难题。已证实儿茶酚胺分泌显著促进室性心律失常。1）近期，我们意外发现，部分患者β1受体阻滞剂显著促进室性心律失常（BAVA)；2）全外显子测序和验证发现PKA错义突；3）我们推想BAVA的机制可能为儿茶酚胺经β2受体的ERK显著激活。为此拟研究：a）阿霉素诱导的心衰大鼠：rAAV9病毒心脏选择性转染，反义抑制PKA或ERK，再给予药物刺激β2受体兴奋剂/β1受体阻滞剂，电生理刺激，观察室性心律失常及、室颤阈值、ERP和QTc变化、PKA/ERK的表达及磷酸化；b）离体心脏 给予上述干预，记录APD特征、复极离散度、EAD、DAD、ERP和QTc；c)分离转染动物的心肌细胞/病毒直接转染心肌细胞 药物刺激或信号转导抑制剂，膜片钳记录Ito/Ikr/Iks等。上述研究中检测信号转导蛋白表达及活性。本研究可能为心源性猝死防治提供重要参考。

室性心律失常尤其是在合并严重器质性心脏病的患者，通常可导致室颤，猝死等严重后果，是各种心脏病死亡的常见原因，室性心律失常的防治是世界性公共卫生的重点和难题。目前国际指南主张采用β受体阻滞剂治疗室性心律失常。然而，我们在国内首次报告了β受体阻滞剂诱发的心律失常，我们目前称这类室性心律失常为β受体阻滞剂恶化的室性心律失常（BAVA）。其具体机制尚不太清楚。我们通过不断探究发现心脏特异性高表达Caveolin-3可降低心梗后交感神经异常激活引起的室性心律失常。在动物实验部分，我们通过重组腺相关病毒技术构建心脏特异性高表达Caveolin-3小鼠模型，研究心脏特异性高表达Caveolin-3对心梗后交感神经异常激活引起的室性心律失常的作用及其机制。结果证实心脏特异性高表达Caveolin-3可降低心梗后交感神经异常激活引起的室性心律失常发生率并改善心梗后心功能。在细胞实验中，我们进一步研究高表达Caveolin-3这一作用的机制，发现Caveolin-3可调控心律失常相关分子pkp2表达，并且钙成像显示高表达Caveolin-3可降低心梗后心肌细胞自发性钙释放发生的频率。进一步的机制研究证实这一抑制作用是由抑制RyR2Ser2814位点的过磷酸化而介导的。另外，我们还证实了pkp2是Caveolin-3对RyR2Ser2814位点的磷酸化抑制所需要的。通过本项目研究我们阐明了Caveolin-3抗心梗后交感神经异常激活引起室性心律失常的作用及机制，同时为预防和治疗心梗后室性心律失常和心源性猝死的发生提供新的靶点。