【作者】 陈卉；

【导师】 黄从新；

【作者基本信息】 武汉大学 ， 内科学， 2014， 博士

【摘要】 第一部分：超极化激活环核苷酸门控通道HCN4在窦房结亚细胞的定位目的：研究HCN4通道在窦房结的亚细胞定位和这样的定位对窦房结起搏通道特性的影响。材料与方法：使用酶解法分离出兔窦房结细胞,然后按照非去污剂法分离窦房结富含脂筏的膜部分,通过不连续蔗糖梯度离心和western blot方法检测在脂筏区域和非脂筏区域HCN4蛋白的表达差异,采用膜片钳技术检测窦房结If电流的变化。结果：HCN4通道在窦房结定位在脂筏中,且与caveolin-3一起定位于膜的低密度区域。使用MβCD破坏脂筏会改变HCN4在脂筏的定位。MβCD处理使窦房结细胞的If电流的激活中点朝正向移动。MβCD处理后电流密度无明显变化,窦房结细胞显示出舒张期去极化斜率和速率增加。在通道动力学方面,f-通道在MβCD处理后去激活变慢。结论：起搏通道定位在脂筏,破坏脂筏会导致通道在膜内重新分配并改变其动力学特性。第二部分：心衰对兔窦房结功能和电生理特性的影响及心衰后窦房结小凹改变及caveolin-3、HCN4表达变化目的：旨在研究兔心衰后兔窦房结功能及电生理特性的变化及caveolin-3、 HCN4的mRNA及蛋白表达的变化。材料与方法：采用主动脉瓣返流联合腹主动脉缩窄的方法制作慢性心衰模型,造模前及造模后8周行超声心动图检查造模是否成功。然后使用32导生理记录仪测窦房结传导时间(SACT)和窦房结恢复时间(SNRT)的变化。采用标准玻璃微电极技术检测造模前后兔心脏窦房结组织的跨膜动作电位改变。通过透射电镜观察心衰前后兔窦房结组织小凹的变化,通过RT-PCR, western plot和免疫组织化学技术研究心衰后兔窦房结caveolin-3、HCN4的mRNA及蛋白表达变化。结果：与对照组相比,心衰组LVIDs、LVIDd、ESV、EDV值均降低(p<0.05),而EF和FS值升高(p<0.05)。心衰组SACT、SNRT、cSNRT均显著延长(p<0.05),心衰后窦房结小凹丰度下降,跨膜动作电位MDP、APA、DDR、RPF值均降低,而APD50、APD90则延长(p<0.05)。心衰显著降低窦房结caveolin-3、HCN4的mRNA及蛋白表达水平,且caveolin-3表达下降与HCN4表达降低有相关性。结论：心衰后窦房结小凹数量减少导致定位在小凹上HCN4通道表达改变,窦房结HCN4表达下调有助于CHF引起的窦房结功能障碍,窦房结HCN4表达下调可能与小凹caveolin-3蛋白的降低相关。这些发现为心脏冲动形成的正常和疾病相关损伤的分子基础和调控提供了新的理论依据。第三部分：caveolin-3对HCN4电生理特性的影响目的：旨在研究caveolin-3对在非洲爪蟾卵母细胞中异源表达的人HCN4通道电生理特性的影响材料与方法：HCN4与caveolin-3一起定位于脂筏的的低密度区域,体外转录人HCN4、Cav-3和p104L基因,酶解分离非洲爪蟾卵母细胞,显微注射人HCN4、 Cav-3和p104L的mRNA。实验分为HCN4、HCN4+Cav-3、HCN4+p104L、Cav-3和p104L共5组,其中Cav-3和p104L为阴性对照组。双电极电压钳技术记录各组中表达的通道电流。结果：在爪蟾卵母细胞中成功表达并记录到了特征性的HCN4电流：在超极化电位下呈现缓慢激活的内向电流,CsCl (5mM)处理可完全不可逆性抑制这种电流,而扎替雷定(10μM)可显著抑制人HCN4电流。Cav-3与HCN4基因共表达可以增加后者的电流幅值、加速HCN4通道的激活和去激活动力学、而不影响HCN4通道的电压依赖性和翻转电位。p104L与HCN4基因共表达可以降低后者的电流幅值,增加HCN4通道的激活时间常数,使稳态去激活电压向更负电位方向移动。结论：Cav-3与HCN4基因在非洲爪蟾卵母细胞中共表达,不仅可以调节HCN4通道的电生理学特性,而且可能具有生理相关性。更多还原

【Abstract】 Part I Subcellular localization of HCN4pacemaker channel in the sinus nodeObjective:toinvestigate the sub-cellular localization of hyperpolarization-activated cyclic nucleotide-gated channel4(HCN4) expressed in rabbit sinoatrial (SAN) cells and the effects of such localization on the characteristics of HCN4channel in SAN cells.Methods:The preparations of sinoatrial node cells were isolated from rabbit heartusing enzymatic method, membrane cholesterol depletion was achieved by methyl-β-cyclodextrin (MβCD) treatment. Then we used a discontinuous sucrose gradient and Western blot analysis to detect HCN4proteins in SAN lipid raft-enriched fractions and nonlipid raft membranes, a whole-cell patch clamp technique was employed to record If in isolated rabbit SAN pacemaker cells.Results:HCN4proteins localized to low-density membrane together with caveolin-3, which was the scaffolding protein of caveolae. Lipid raft disruption with MβCD changedthe specific HCN4localization. It also shifted the midpoint of activation of If in SAN cells to the positive direction. In accordance with an increased If, MβCD-treated SAN cells showed large increases of diastolic depolarization slope and rate. We also found that the kinetics of native f-channel deactivation were slower after lipid raft disorganization.Conclusions:These results indicate thatHCN4channel localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties.Part II The changes of sinus node function and electrophysiological characteristics and caveolin-3, HCN4expression in rabbit heart failureObjective:The objective of this study was to investigate the change of function and electrophysiological properties in sinus node, and the change of mRNA levels and protein expressions of caveolin-3and HCN4in control group and heart failure group.Methods:HF was produced by combined aortic insufficiency and abdominal aortic constriction. Before modeling and after8weeks with echocardiography to detect whether the model was produced successfully. Then we use to test the changes of sinus node conduction time (SACT) and sinus node recovery time (SNRT). We detected the change of action potential with standard glass microelectrode technique. The change of caveolae in sinoatrial node tissue was observed by transmission electron microscopy. RT-PCR, western plot and immunohistochemistry were used to observe the mRNA levels and protein expression of caveolin-3and HCN4.Results:Compared with the control group, LVIDs, LVIDd, ESV, EDV values were lower in heart failure group (p<0.05), while the EF and FS values increased (p<0.05). HF group SACT, SNRT, cSNRT were significantly prolonged (p<0.05). MDP, APA, DDR, RPF values were reduced, while APD50, APD90were prolonged (p<0.05). mRNA and protein expression levels of caveolin-3and HCN4significantly reduced after heart failure, and HCN4abundance positively correlated with the reduction of caveolin-3.Conclusions:Reduction of caveolae number lead to the expression change of HCN4channel which location in caveolae, downregulation of HCN4expression might contribute to CHF induced sinus sinus node dysfunction,the reduction of HCN4may be associated with the change of caveolin-3proteins. These findings provide novel information about the molecular basis of normal and disease-related impairments of cardiac impulse formationPart Ⅲ The effects of caveolin-3on the hyperpolarization-activated cyclic nucleotide-gated channel heterologously expressed in Xenopus oocytesObjective:To study the effects of caveolin-3on human HCN4channel heterolog-ously expressed in Xenopus oocytes.Methods:We transcribed human HCN4, Cav-3and p104L DNA into complementary using mMESSAGE mMACHINE(?) T7Kit, then the cRNAs were injected in enzymatically isolated Xenopus oocytes.The experiment was divided into five groups:HCN4, HCN4+Cav-3, Cav-3, HCN4+p104L and p104L (as negative control) group. Human HCN4currents were recorded using two-electrode voltage clamp technique.Results:The human HCN4currents were successfully expressed and recorded, with characteristics as follows:1) the channel open in a time-dependent manner after a series of hyperpolarization voltages;2) the more hyperpolarizated, the faster the channel activated;3) zatebradine and CsCl significantly inhibited the channel.4) Cav-3increased the hHCN4current amplitude, accelerated the activation and deactivation kinetics without affecting the voltage dependence and reversal potential. P104L caused a two-fold increase in the activation time constant for IHCN4and shifted the voltage of the steady-state inactivation to a more negative potential.Conclusions:Cav-3and HCN4gene, co-expressed in Xenopus oocytes, can regulate the electrophysilological properties of HCN4channel, and may have a physiological correlation.更多还原