微管解聚对大鼠心肌细胞自主搏动和动作电位的影响及其机制

Effects of microtubule depolymerization on spontaneous beating and action potential of cardiac myo- cytes in rats and its mechanism

目的观察微管解聚对大鼠心肌细胞自主搏动频率、动作电位（AP）、耗氧量的影响并探究其机制。方法将180只SD大鼠乳鼠分12批处理进行实验，每批取15只大鼠乳鼠处死，剪取心脏组织分离培养心肌细胞，分别接种至1个铺有6块圆形盖玻片的12孔板、1个铺有6块方形盖玻片的12孔板、2个细胞培养瓶、2个细胞培养皿。将培养3d的各容器中细胞按随机数字表法分为正常对照组（加入3mL37℃复温的DMEM／F12培养液，常规培养3h）和微管解聚组（加入3mL37℃复温的含终浓度为8μmoL／L秋水仙碱的DMEM／F12培养液，常规培养3h），每组分别3孔、1瓶、1皿。免疫荧光染色后激光扫描共聚焦显微镜下观察细胞微管形态变化，蛋白质印迹法检测细胞游离态及聚合态α微管蛋白的含量变化。倒置显微镜下观察并计算细胞自主搏动频率。氧微电极监测系统测定含有心肌细胞的DMEM／F12培养液加入秋水仙碱前后的溶解氧浓度，另测定单纯培养液和秋水仙碱＋培养液溶解氧浓度。采用全细胞膜片钳记录模式记录细胞AP、延迟整流型钾离子通道电流（Ik）和L型钙离子通道电流（Lca-L）变化，绘制电流密度-电压（I-V）曲线。对数据行独立或配对样本t检验。结果（1）正常对照组细胞微管结构完整，围绕核周呈放射状分布，线性管状结构清晰。微管解聚组细胞微管结构破坏，呈现弥散性分布，线性管状结构粗糙而不光滑。（2）微管解聚组细胞游离态d微管蛋白含量为0．61±0．03，明显高于正常对照组的0．46±0．03，t=6．99，P〈0．05；聚合态a微管蛋白含量为0．57±0．04，明显低于正常对照组的0．88±0．04，t=9．09，P〈0．05。（3）微管解聚组细胞自主搏动频率为（59±8）次／min，较正常对照组的（41±7）次／min明显增加（t=5．62，P〈0．01）。（4）含心肌细胞的培养液溶解氧浓度为（138．4±2．5）μLmol／L，秋水仙碱处理后下降为（121．7±3．6）μmol／L，差异明显（t=26．31，P〈0．05）。单纯培养液和秋水仙碱＋培养液溶解氧浓度无明显差异（t=0．72，P〉0．05）。（5）与正常对照组比较，微管解聚组细胞AP形态发生明显变化，复极化平台期不明显，动作电位时程（APD）明显缩短。微管解聚组细胞的APD20、APD50、APD90分别为（36．2±3．8）、（73．7±5．7）、（115．1±8．0）ms，较正常对照组的（40．2±2．3）、（121．4±7．0）、（169．4±5．6）mS明显缩短（t值分别为2．61、15．88、16．75，P值均小于0．05）。（6）微管解聚组细胞I。的I．V曲线较正常对照组上移，激活后各测试电压（0—40mV）下微管解聚组I。电流密度均高于正常对照组（t值为2．70-3．76，P值均小于0．05）。（7）2组细胞IC-a-L的I-V曲线基本重叠，激活后各测试电压（-30-50mV）下ICa-L。电流密度相近（t值为-1．57—1．66，P值均大于0．05）。结论微管解聚后Ik增强，IC-L变化不明显，使得AP复极化增快，进而缩短APD，加快大鼠心肌细胞自主搏动频率，增加其耗氧量。

Objective To explore the effects of mierotubule depolymerization （MD） on the sponta- neous beating rate, action potential （ AP）, and oxygen consumption of cardiac myocytes in rats and its mechanism. Methods One-hundred and eighty neonatal SD rats divided into 12 batches were used in the experiment, and 15 rats in each batch were sacrificed for the isolation and culture of cardiac myocytes after the heart tissues were harvested. The cardiac myocytes were respectively inoculated in one 12-well plate filled with 6 round cover slips, one 12-well plate filled with 6 square cover slips, two cell culture flasks, and two cell culture dishes. After routine culture for three days, the cardiac myocytes from all the containers were divided into normal control group （ NC, routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 ℃for 3 h） and group MD （routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 ℃ and containing 8 μmol/L eolchicine for 3 h） according to the random number table, with 3 holes, 1 flask, or 1 dish in each group. The morphological changes in microtubules were observed with confocal laser scanning microscope af- ter immunofluorescent staining. The content of polymerized or dissociative α-tubulin was determined by Western blotting. Spontaneous beating rate of the cells was observed and calculated under inverted micro- scope. Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was determined by oxygen mieroelectrode system before and after the addition of colchieine. Additionally, dissolved oxygen concentration of DMEM/F12 solution and colchicine ＋ DMEM/F12 solution was determined. The whole-cell patch-clamp technique was used to record AP, delayed rectifier K ＋ current （IK） , and L-type Ca2＋ current （Ica-L） in cardiac myocytes; current density-voltage （I-V） curves were drawn based on the traces. Data were processed with independent or paired samples t -test. Results （ 1 ） In group NC, microtubules of cardiac myocytes were around the nucleus in radial distribution with intact and clear linear tubiform struc- ture. The microtubules in group MD were observed in dispersive distribution with damaged structure and rough linear tubiform structure. （2） In group MD, the content of dissociative a-tubulin of cells （0.61 ± 0. 03 ） was obviously higher than that in group NC （0.46 ± 0.03, t = - 6.99, P 〈 0.05）, while the content of polymerized ct-tubulin （0.57 ± 0.04） was significantly lower than that in group NC （0.88± 0.04, t = 9.09, P 〈0.05）. （3） Spontaneous beating rate of cells was （59 ± 8） times per min in group MD, which was distinctly higher than that in group NC [（41 ±7） times per min, t =5.62,P 〈0.01]. （4） Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was （ 138.4 ± 2.5） μmol/L, and it was reduced to （121.7±3.6） μmol/L after the addition ofcolchieine （ t =26.31, P 〈0.05）. There was no obvious difference in dissolved oxygen concentration between DMEM/F12 solution and colchicine ＋ DMEM/FI2 solution （ t = 0.72, P 〉 0.05）. （5） Compared with that of group NC, AP morphology of cells in group MD changed significantly, with unobvious repolarization plateau phase and shorter action potential duration （APD）. The APD2o, APDs0, and APDg0 were respectively （36.2 ±3.8）, （73.7 ±5.7）, and （115.1 ± 8. 0） Ins in group MD, which were significantly shorter than those of group NC [ （40.2 ± 2.3） , （121.4 ±7.0） , and （169.4 ±5.6） ms, with t values respectively 2.61,15.88, and 16.75, P values below 0. 05 ]. （6） Compared with that of group NC, the I-V curve of IK of cells in group MD moved up with higher current density under each test voltage （0 to 40 mV） after activation （ with t values from 2.70 to 3.76, P values below 0.05）. （7） There was not much alteration in current density of Ica-L under each test voltage （ -30 to 50 mV） between2 groups （withtvalues from -1.57 to 1.66, Pvalues above0.05）, and their I-V curves were nearly overlapped. Conclusions After MD, the IK is enhanced without obvious change in ICa-L, making AP repolarization faster and APD shortened. Then the rapid spontaneous beating rate increases oxygen consumption of cardiac myocytes of rats.