【作者】 余良主；

【导师】 唐明；

【作者基本信息】 华中科技大学 ， 生理学， 2010， 博士

【摘要】 背景：心律失常是临床上常见的疾病。其机制较为复杂。从根本上说,心肌细胞各种离子通道表达或功能的异常是形成各种心律失常最重要的病理生理基础。钠通道在心肌细胞动作电位起始与传播中起着重要的作用。近年来研究证实,钠通道门控特征改变是形成某些长QT综合征、Brugada’s syndrome和一些遗传性心脏传导疾病的根本原因。并且,在一些获得性心脏疾病如心肌缺血和心衰中,人们也发现钠通道门控特征发生了变化。另外,在患病心脏中,一些细胞内调节的信号途径如cAMP依赖的蛋白激酶A途径也发生了变化。改善衰竭心脏功能的一个实验性策略即是进行细胞移植。已有许多研究者正尝试着应用胚胎心肌、胚胎心脏前体细胞和骨髓干细胞进行移植研究。这些创新方法由于缺乏对正常心肌分化尤其是胚胎发育阶段的了解而受到阻碍。因此,研究胚胎心脏中Na+通道的门控特征变化及其调控,从病理生理和治疗学上来说都是具有十分重要的意义。目的：应用胚胎小鼠心室肌细胞,观察不同胚胎发育时期钠通道门控特征的变化；检测胚胎心脏中钠通道各亚单位的表达性改变,以探讨与功能变化相关的分子机制。方法：急性分离早期(胎龄10.5 d)和晚期(胎龄17.5 d)胚胎小鼠心脏,采用胶原酶Ⅱ,分离获得单个的心室肌细胞,并置于CO2培养箱中培养24-48h后进行电生理记录。用标准的全细胞膜片钳技术,记录胚胎小鼠心室肌细胞的钠电流；利用RT-PCR技术检测了胚胎心脏中钠通道六种α亚单位(Nav1.1-Nav1.6)和三种β亚单位(Navβ1-Navβ3)的表达量。结果：通过酶解法得到的胚胎小鼠心室肌细胞能够记录到相应的INa电流,表明其具有良好的细胞电生理特性。1.不同发育时期INa电流密度的变化：晚期心室肌细胞与早期心室肌细胞相比,峰INa电流密度在从-60至+20 mV的刺激电压范围内明显增大。在-30 mV处,INa电流密度从早期的-88.1±8.0 pA/pF (n=8)增大为晚期的-287.9±9.7pA/pF(n=11,P<0.01)。2.不同发育时期INa电流激活特征的变化：早期心室肌细胞与晚期细胞的激活特征相似。两种心肌细胞的半数激活电压(Va:-46.1±3.4 mV EDS vs.-47.0±3.4 mV LDS, P>0.05)和斜率(k:5.5±0.4 mV EDS vs.5.8±0.7 mV LDS, P>0.05)较为接近。另外,两种细胞在-60至0 mV范围内,其INa电流达峰时间相似。3.不同发育时期INa电流失活特征的变化：与早期心肌细胞(Vi:-71.6±2.4mV EDS, P<0.01)相比,晚期心室肌细胞(Vi:-82.7±4.4 mV LDS P<0.01)的半数失活电压明显向负电位方向偏移。而斜率并没有显著性差异(k:7.2±0.7 mV EDS vs.6.5±0.9 mV LDS, P>0.05).另外,在-30 mV电压处,钠电流的失活相均符合双指数拟和,都含有一个快失活成分和慢失活成分。两种细胞的快、慢失活时间相近。而就两种失活成分的相对幅度来说,晚期心肌细胞较之早期细胞具有更大幅度的快失活成分和更小幅度的慢失活成分。4.不同发育时期INa电流复活特征的变化：与失活特征相似,INa电流复活过程也包含有快、慢两种成分。与早期心室肌细胞相比,晚期心肌细胞的快慢时间常数均表现为明显减小。早期心室肌细胞INa电流的两种时间常数分别为τf=7.5±0.5ms和τs=198.6±12.3 ms；晚期心室肌细胞INa电流的两种时间常数分别为τf=5.5±0.4 ms和τs=122.5±4.5 ms。5.不同发育时期钠通道α亚单位和p亚单位表达量的改变：在钠通道α亚单位中,Nav1.1、Nav1.2和Nav1.3的表达量在胚胎心脏中始终是缺乏或处于低表达水平。Nav1.4、Nav1.5和Nav1.6的表达量随着小鼠胚胎发育而明显增大。另外,三种钠通道p亚单位(Navβ1、Navβ2、Navβ3)的表达量也是随着发育明显上调。6.不同发育时期总INa电流对河豚毒(TTX)的敏感性：为了比较早期、晚期心肌细胞总钠电流对TTX敏感性的不同,我们分析了TTX抑制早期心肌细胞和晚期心肌细胞总INa电流的剂量-浓度反应关系。结果发现,在这两种心肌细胞中,TTX均以浓度依赖性方式抑制了总INa电流。采用Hill公式拟和,得到相应半数抑制浓度IC50值为5.2μM(早期心肌细胞)和6.6μM(晚期心肌细胞)。结论：在小鼠胚胎发育时期,心脏钠通道存在着显著性功能特征改变；与功能变化相对应,钠通道各亚单位的表达也受到了胚胎发育过程的紧密调节；这些发现证实了电压门控性钠通道在心脏发育过程中起着一定的生理作用。更多还原

【Abstract】 Background:Cardiac arrhythmia is common in the clinical practice, and its mechanism is complex. Fundamentally, the abnormalities of expression and function of the membrane ion channels can contribute to the development of cardiac arrhythmias.Na+ channel plays a key role in the generation and conduction of action potential in the heart. It has been showed that altered Na+ channel gating might underlie multiple cardiac diseases, such as long QT syndrome (LQTS), the Brugada syndrome and some inheritable cardiac conduction disorders. Na+ channel gating is altered in acquired diseases such as cardiac ischemia and heart failure. In addition, some signaling pathways that regulate Na+ channel function are altered in the diseased heart, such as cAMP-dependent protein kinase A (PKA).An experimental strategy to improve cardiac function in failing hearts is cell transplant. Some investigator has attempted to engraft fetal cardiomyocytes, and bone marrow stem cell. However, progress in this approach is hampered by lack of knowledge about normal differentiation of cardiomyocytes especially at embryonic stage. Thus, it would be of great interest to determine the developmental changes in gating properties of Na+ channel from a pathophysiological and therapeutic standpoint.Objective:To study the developmental changes in gating properties of Na+ channel during embryogenesis and to determine the changes in Na+ channel subunits expression that might be associated with functional changes.Methods:Single ventricular myocytes from embryos of early developmental stage (10.5 days postcoitum) and late developmental stage (17.5 days postcoitum) were obtained by enzymatic dissociation method and kept in the incubator for 24-48 hours until use. Whole-cell voltage-clamp technique was used to record Na+ currents in ventricular myocytes of early (EDS) and late (LDS) developmental stages in embryonic mice。Additionally, RT-PCR was performed to determine the transcripts of six Na+ channel a subunits (Nav1.1-Nav1.6) and threeβsubunits (Navβ1-Navβ3)。Results:Na+ current could be recorded in the isolated ventricular myocytes of both developmental stages, which indicated these myocytes had satisfactory electrophysiological properties.1. Developmental changes in peak INa current density:Peak Na+ current density was significantly larger in LDS cells (-60 to +20 mV) than in EDS cells (P<0.01). Na+ current density at -30 mV increased significantly from -88.1±8.0 pA/pF (n=8) in EDS to -287.9±9.7 pA/pF (n=11,P<0.01) in LDS.2. Developmental changes in activation properties of Na+ channels:The voltage dependence of activation in both cell types were similar. There was no significant difference in the voltage of half activation (Va) and slope factor (k) between EDS and LDS myocytes (Va:-46.1±3.4 mV EDS vs.-47.0±3.4 mV LDS, P>0.05; k:5.5±0.4 mV EDS vs.5.8±0.7 mV LDS, P>0.05). In addition, the time-to-peaks in both cell types were similar at potentials over -60 to 0 mV.3. Developmental changes in inactivation properties of Na+ channels:The voltage of half inactivation (V;) was shifted to more negative potentials in LDS than EDS cells (Vi:-82.7±4.4 mV LDS vs.-71.6±2.4 mV EDS, P<0.01). K values for EDS and LDS cells were not significantly different (k:7.2±0.7 mV EDS vs.6.5±0.9 mV LDS, P>0.05).In addition, the time course of inactivation at a test potential of-30 mV was well described by a bi-exponential fit in both cell types, containing a large fast component and a small slow component. Both the time constants of Na+ channel inactivation in both cell types were similar. But LDS myocytes had significantly larger amplitude of fast (Af) inactivation component and smaller amplitude of slow (As) inactivation component than EDS myocytes.4. Developmental changes in recovery properties of Na+ channels:Similarly, the time course of recovery from inactivation in both cell types also included a large fast component and a small slow component. The fast (if) and slow (τs) time constants for Na+ channel recovery were significantly smaller in LDS (τf:5.5±0.4 ms LDS vs. 7.5±0.5 ms EDS, P<0.01;τs:122.5±4.5 ms LDS vs.198.6±12.3 ms EDS, P<0.01) than in EDS cells.5. Expression of Na+ channel a subunits andβsubunits in embryonic cardiomyocytes:Transcripts of Navl.1, Nav1.2 and Nav1.3 were absent or present at very low levels in embryonic hearts. The amount of Nav1.4, Nav1.5 and Nav1.6 mRNA were increased with age during embryogenesis. Additionally, three Na+ channel 3 subunits (Navβ1-Navβ3) were upregulated during embryogenesis.6. Sensitivity of total Na+ currents to TTX in EDS and LDS cardiocytes:To determine the sensitivity of total Na+ current in both embryonic cardiomyocytes types, we analyzed the relationship between the dose of TTX and the blocking effect of TTX on total INa·Total Na+ currents were decreased in a dose-dependent manner by TTX in both myocyte types. Fitting the dose-response relationship with Hill equation yielded an IC50 of 5.2μM (EDS) and 6.6μM (LDS).Conclusions:These results suggest significantly functional changes in Na+ channels occur in cardiomyocytes of mouse embryo and that different Na+ channel subunits genes are strongly regulated during embryogenesis, and further support a physiological role for voltage-gated Na+ channels during heart development.更多还原