Abstract
目的
糖尿病患者发生心律失常的风险增加，但高血糖及胰岛素治疗对人心肌细胞电生理特性的影响尚未完全明确。本研究探讨高糖与胰岛素对人诱导多潜能干细胞来源的心肌细胞(human induced pluripotent stem cells derived cardiomyocytes，hiPSC-CMs)电生理特性及诱发心律失常现象的影响。
方法
采用流式细胞术分析健康个体来源的hiPSC-CMs纯度。将hiPSC-CMs分为3组：对照组(NM组，培养基含5 mmol/L葡萄糖)、高糖组(HG组，培养基含15 mmol/L葡萄糖)和高糖+胰岛素组(HG+INS组，培养基含15 mmol/L葡萄糖+100 mg/L胰岛素)，各组干预时间均为4 d。采用微电极阵列技术检测3组hiPSC-CMs在干预前后的电生理改变，包括跳动频率(beating rate，BR)、阈电位时程[(field potential duration，FPD)，类似于心电图QT间期]、FPDc(经BR校正的FPD)、锋电位与传导速率(conduction velocity，CV)；此外，采用索他洛尔干预各组hiPSC-CMs，观察其诱发FPDc延长及心律失常现象发生的情况。
结果
hiPSC-CMs中心肌标志物肌钙蛋白T呈高表达，其纯度为99.06%。与NM组相比，HG组hiPSC-CMs的BR增快(9.14±0.8)%(P<0.01)；高糖处理后，HG组hiPSC-CMs的FPD从处理前的(460.4±9.0) ms延长至处理后的(587.6±23.7) ms，NM组FPD从(462.5±14.5) ms延长至(512.6±17.6) ms，与NM组相比，HG组FPD延长了(16.8±1.4)%(P<0.01)；高糖处理后，HG组hiPSC-CMs的FPDc从处理前的(389.1±13.7) ms延长至处理后的(478.3±31.5) ms，NM组从(387.7±21.6) ms延长至(422.6±32.9) ms，与NM组相比，HG组hiPSC-CMs的FPDc延长了(13.9±1.3)%(P<0.05)；但高糖处理后，NM组和HG组锋电位与CV均无明显变化(均P>0.05)。药物诱发实验发现10 µmol/L索他洛尔可使HG组各培养孔hiPSC-CMs诱发出心律失常现象。高糖+胰岛素处理后，HG+INS组BR相对HG组增快(8.3±0.5)%(P<0.05)；HG+INS组 FPD从处理前的(463.4±9.7) ms延长至处理后的(532.6±12.8) ms，HG组从处理前的(460.4±9.0) ms延长至(587.6±23.7) ms，与HG组比较，HG+INS组 FPD缩短了(12.7±1.9)%(P<0.01)；HG+INS组FPDc从处理前的(387.4±4.1) ms延长至处理后的(422.4±10.0) ms，HG组从处理前的(384.8±4.0) ms延长至(476.3±11.5) ms，与HG组相比，HG+INS组FPDc缩短了(14.7±1.1)%(P<0.01)；胰岛素处理后，hiPSC-CMs锋电位显著增加，HG+INS组锋电位从处理前的(3.12±0.46) mV增加至处理后的(4.35±0.64) mV，HG组从(3.06±0.35) mV增加至(3.33±0.41) mV，与HG组相比，HG+INS组锋电位增加了(30.8±3.7)%(P<0.05)；胰岛素处理后，HG+INS组CV从处理前的(0.23±0.08) mm/ms增加至处理后的(0.32±0.08) mm/ms，HG组CV从处理前的(0.21±0.04) mm/ms增加至处理后的(0.30±0.07) mm/ms，与HG组相比，HG+INS组CV无明显变化(P>0.05)；药物诱导实验发现10 µmol/L索他洛尔可使HG+INS组的FPDc延长(78.9±11.6)%，但各孔均未诱发出心律失常现象。
结论
高糖可诱导hiPSC-CMs的FPD/FPDc延长，并增加其药物诱发心律失常现象的风险；胰岛素可缩短高糖诱导的FPD/FPDc延长，并能降低高糖作用下药物诱发的心律失常现象。这为糖尿病患者心肌的电生理改变及胰岛素治疗对其电生理的影响提供了实验依据；进一步的机制研究将可能为获得性甚至遗传性长QT综合征的治疗提供新的思路与方法。
Keywords: 高糖, 胰岛素, 人诱导多潜能干细胞, 心肌细胞, 电生理学
Abstract
Objective
The risk of arrhythmia increases in diabetic patients. However, the effects of hyperglycemia and insulin therapy on the electrophysiological properties of human cardiomyocytes remain unclear. This study is to explore the effects of high glucose and insulin on the electrophysiological properties and arrhythmias of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs).
Methods
Immunofluorescent staining and flow cytometry were used to analyze the purity of hiPSC-CMs generated from human skin fibroblasts of a healthy donor. The hiPSC-CMs were divided into 3 group (treated with normal medium, high glucose and insulin for 4 days): a control group (NM group, containing 5 mmol/L glucose), a high glucose group (HG group, containing 15 mmol/L glucose), and a high glucose combined with insulin (HG+INS group, containing 15 mmol/L glucose+100 mg/L insulin). Electrophysiological changes of hiPSC-CMs were detected by microelectrode array (MEA) before or after treatment with glucose and insulin, including beating rate (BR), field potential duration (FPD) (similar to QT interval in ECG), FPDc (FPD corrected by BR), spike amplitude and conduction velocity (CV). Effects of sotalol on electrophysiological properties and arrhythmias of hiPSC-CMs were also evaluated.
Results
The expression of cardiac-specific marker of cardiac troponin T was high in the hiPSC-CMs. The purity of hiPSC-CMs was 99.06%. Compared with the NM group, BR was increased by (9.14±0.8)% in the HG group (P<0.01). After treatment with high glucose, FPD was prolonged from (460.4±9.0) ms to (587.6±23.7) ms in the HG group, while it was prolonged from (462.5±14.5) ms to (512.6±17.6) ms in the NM group. Compared with the NM group, FPD of hiPSC-CMs was prolonged by (16.8±1.4)% in the HG group (P<0.01). The FPDc of hiPSC-CMs was prolonged from (389.1±13.7) ms to (478.3±31.5) ms in the HG group, and that was prolonged from (387.7±21.6) ms to (422.6±32.9) ms in the NM group. Compared with the NM group, the FPDc of hiPSC-CMs was prolonged by (13.9±1.3)% in HG group (P<0.01). The spike amplitude and CV remained unchanged between the HG group and the NM group (P>0.05). Ten µmol/L of sotalol can induce significant arrhythmias from all wells in the HG group. After treatment with insulin and high glucose, compared with the HG group, BR was increased by (8.3±0.5)% in the HG+INS group (P<0.05). The FPD was prolonged from (463.4±9.7) ms to (532.6±12.8) ms in the HG+INS group, while it was prolonged from (460.4±9.0) ms to (587.6±23.7) ms in the HG group. Compared with the HG group, the FPD of hiPSC-CMs was shortened by (12.7±1.9)% in the HG+INS group (P<0.01). The FPDc of hiPSC-CMs was prolonged from (387.4±4.1) ms to (422.4±10.0) ms in the HG+INS group, and that was prolonged from (384.8±4.0) ms to (476.3±11.5) ms in HG group. Compared with the HG group, the FPDc of hiPSC-CMs was shortened by (14.7±1.1)% in HG group (P<0.01). After the insulin treatment, the spike amplitude of hiPSC-CMs was increased from (3.12±0.46) mV to (4.35±0.64) mV in the HG+INS group, while it was enhanced from (3.06±0.35) mV to (3.33±0.41) mV in the HG group. The spike amplitude of hiPSC-CMs was increased by (30.8±3.7)% in the HG+INS group compared with that in the HG group (P<0.05). The CV in the HG+INS group was increased from (0.23±0.08) mm/ms to (0.32±0.08) mm/ms after insulin treatment, which was increased from (0.21±0.04) mm/ms to (0.30±0.07) mm/ms in the HG group, but there was no significant difference in CV between the HG+INS group and the HG group (P>0.05). The induction experiment showed that 10 μmol/L of sotalol could prolong the FPDc of hiPSC-CMs by (78.9±11.6)% in the HG+INS group, but no arrhythmia was induced in each well.
Conclusion
High glucose can induce FPD/FPDc of hiPSC-CMs prolongation and increase the risk of arrhythmia induced by drugs. Insulin can reduce the FPD/FPDc prolongation and the risk of induced arrhythmia by high glucose.These results are important to understand the electrophysiological changes of the myocardium in diabetic patients and the impact of insulin therapy on its electrophysiology. Further study on the mechanism may provide new ideas and methods for the treatment of acquired and even inherited long QT syndrome.
Keywords: high glucose, insulin, human induced pluripotent stem cell, cardiomyocyte, electrophysiology
糖尿病已成为严重危害人类健康且发病仍呈快速增长的重大疾病之一[1]。糖尿病不仅可导致严重的大血管(如冠状动脉性疾病等)、微血管(如糖尿病视网膜病变等)病变，并且可导致相应的心肌发生纤维化重构[2]与收缩舒张功能障碍[3-4]。同时有研究[5-7]发现糖尿病患者发生心律失常的风险明显增加，提示长期高血糖可显著影响心肌的电生理特性，但高糖及胰岛素治疗对人心肌细胞电生理特性及诱发心律失常的影响却仍不完全清楚，本研究以人诱导多潜能干细胞来源的心肌细胞(human induced pluripotent stem cells derived cardiomyocytes，hiPSC-CMs)为模型，深入探讨高糖对hiPSC-CMs电生理特性及诱发心律失常现象的影响，以及胰岛素治疗对高糖所致的hiPSC-CMs电生理改变，为阐明糖尿病患者心肌的电生理改变及胰岛素治疗对其电生理的影响提供实验依据。
1. 材料与方法
1.1. 仪器与试剂
Maestro微电极矩阵(microelectrode array，MEA)记录系统、MEA细胞培养板购自美国Axion Biosystems公司；CytoFLEX型流式细胞仪购自美国Beckman Coulter公司；IXplore型荧光倒置显微镜购自日本Olympus公司。
纤连蛋白购自美国Roche Life Science公司；胰岛素、索他洛尔和二甲基亚砜(dimethylsulfoxide，DMSO)购自美国Millipore Sigma公司。hiPSC-CMs、接种细胞用培养基和培养细胞用培养基均购自美国Cellular Dynamics International(CDI)公司；心脏肌钙蛋白T(cardiac troponin T，cTnT)抗体购自英国Abcam公司。RNA提取miRNeasy Mini试剂盒、cDNA反转录试剂盒、SYBR green real-time PCR试剂盒和基因特异性引物购自美国Qiagen公司。
1.2. hiPSC-CMs的接种、培养及分组
hiPSC-CMs来自于美国CDI公司的iCell心肌细胞株，为健康个体来源，细胞培养同以往方法[8]：接种前用纤连蛋白(50 mg/L)预处理MEA培养板，进行细胞复苏、计数、离心(1 280 r/min，5 min)，调整细胞密度至1×107个/mL，培养板中每孔滴种5 μL细胞悬液，于37 ℃下孵育3 h，加入培养细胞用培养基，每48 h换液，1周后行纯度分析、药物干预及MEA记录。干预时hiPSC-CMs分为如下3组(干预4 d)：对照组(NM组，培养基中含5 mmol/L葡萄糖)、高糖组(HG组，培养基中含15 mmol/L葡萄糖)、高糖+胰岛素组(HG+INS组，培养基中含15 mmol/L葡萄糖+100 mg/L胰岛素)。
1.3. hiPSC-CMs细胞纯度检测
hiPSC-CMs复苏后按4×106个/孔的密度接种于纤连蛋白(10 mg/L)预处理的6孔板中，培养1周使细胞达到90%融合后，采用流式细胞术分析其纯度[8]。用1.25%胰酶于37 ℃消化3~5 min，以1 280 r/min离心 5 min，加入4%甲醛于4 ℃固定30 min，再用0.2%的Triton X-100处理15 min，按照1꞉100加入cTnT一抗，于室温下孵育2 h；按照1꞉500加入FITC荧光二抗，于室温下孵育2 h，重悬细胞并将其置于流式细胞仪上分析。
1.4. 免疫荧光检测
hiPSC-CMs复苏后，种植在纤连蛋白(50 mg/L)预处理的1 cm×1 cm玻片上，培养1周后用PBS冲洗，加入4%多聚甲醛室温下固定30 min，再用0.2% Triton X-100穿膜15 min，采用5%BSA在室温下封闭30 min。按照1꞉100加入cTnT一抗，于4 ℃孵育过夜；用PBS冲洗，按照1꞉500加入荧光二抗和异硫氰酸荧光素(fluorescein isothiocyanate，FITC)，室温孵育1 h，再用4',6二脒基-2-苯吲哚(4',6-diamidino-2-phenylindole，DAPI)复染细胞核，封片后于荧光显微镜下观察。
1.5. Real-time PCR检测
采用real-time PCR分析3组hiPSC-CMs干预前后的心脏各离子通道基因mRNA的表达，方法同文献[8]，以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase，GAPDH)为内参，各离子通道基因的mRNA相对表达使用2-ΔΔCt公式计算[ΔΔCt=(Ct目的基因-CtGAPDH)-(Ct对照基因-CtGAPDH)]。
1.6. MEA检测
hiPSC-CMs培养1周后行MEA检测，方法同文献[9]，待温度稳定于37 ℃，CO2浓度稳定于5%时，将细胞培养板放入Maestro电极接触槽内，稳定平衡10 min，3 min后记录数据。将记录的文件导入到CiPA Analysis数据分析软件中，手动确定每孔记录到的T波顶点位置以确定阈电位时程(field potential duration，FPD)(图1A)，同时分析判定有无心律失常现象(图1B)以及心律失常现象的类型(包括A、B、C、D 4种类型[10])，CiPA Analysis软件根据T波顶点位置计算出FPD值，同时根据记录的电活动数据自动测定跳动间期(beat period，BP)(相当于心电图的R-R间期)、传导速率(conduction velocity，CV)(图1C)、锋电位(图1D)，根据BP(图1E)计算出跳动频率(beating rate，BR)(BR=60/BP)，根据FPD及BP计算出校正的FPD(FPD corrected，FPDc)(FPDc=FPD/BP1/3)。
图1.
微电极阵列记录人诱导多潜能干细胞来源的心肌细胞电活动代表图
Figure 1 Representative of electrophysiological activities of human induced pluripotent stem cells derived cardiomyocytes recorded by microelectrode array
A: Representative figure of field potential duration changes in human induced pluripotent stem cell derived cardiomyocytes after insulin intervention for 4 d; B:Representative figure of arrhythmia induced by sotalol intervention in human induced pluripotent stem cell derived cardiomyocytes (red arrow); C: Representative figure of electric conduction of human induced pluripotent stem cell derived cardiomyocytes; D: Representative figure of spike recording; E: Representative figure of beat period of human induced pluripotent stem cell derived cardiomyocytes (16 microelectrodes in the same well).
药物干预hiPSC-CMs时，在干预前16 h左右完全更换新鲜培养基270 µL，记录基线的MEA数据，随后每孔加入30 μL药物(浓度为最终工作浓度的10倍)处理细胞，每10 min记录1次MEA数据，每次记录3 min，共记录1 h，分析加入药物后30 min的数据以评价药物的作用。
1.7. 统计学处理
采用GraphPad 8.0统计学软件进行分析，计量资料采用均数±标准差( ±s)表示，两组间比较采用t检验，多组间比较采用方差分析；计数资料组间比较采用χ2检验；心律失常现象经过CiPA Analysis软件自动分析与人工再次确认分析，如若处理组≥3/5的培养孔出现心律失常现象，则不再分析FPD及FPDc(心律失常现象影响FPD分析的准确性)，只标注、比较心律失常现象发生的孔数(计数资料)，组间比较采用Fisher确切概率法。检验水准α=0.05，P<0.05为差异有统计学意义。
2. 结 果
2.1. hiPSC-CMs的形态学特征及纯度
hiPSC-CMs接种培养72 h后，可观察到局部细胞的收缩活动；培养7 d后，可见hiPSC-CMs连接成片，并出现有规律的收缩活动，此时hiPSC-CMs多呈类圆形、椭圆形或类长杆状，单核或双核，核多居中，胞质内可见肌节样结构，细胞高表达心肌标志物cTnT(图2A)。流式细胞术结果显示99.06%的细胞表达cTnT(图2B)。
图2.
人诱导多潜能干细胞来源的心肌细胞的形态学特征及纯度
Figure 2 Morphological characteristics and purification of human induced pluripotent stem cells derived cardiomyocytes
A: Morphological characteristics of human induced pluripotent stem cells derived cardiomyocytes by immunofluorescence staining (×400) [cytoplasm (green), nucleus (blue)]; B: Analysis of purity of human induced pluripotent stem cells derived cardiomyocytes by flow cytometry. VIL: Volume of sample 1 in the left; VIR: Volume of sample 1 in the right; cTnT: Cardiac troponin T.
2.2. 高糖对hiPSC-CMs电生理特征的影响
hiPSC-CMs经15 mmol/L葡萄糖处理4 d后，HG组与NM组的BR较前均减慢，HG组BR从(31.9±0.5) 次/min降低至处理后的(27.9±1.4) 次/min，NM组BR从(32.2±0.6) 次/min降低至(25.1±0.9) 次/min，但HG组BR相对于NM组增加了(9.14±0.8)%(F=12.78，P<0.01；图3A)；经高糖处理后，HG组hiPSC-CMs的FPD显著延长，处理前为(460.4±9.0) ms，处理后为(587.6±23.7) ms，NM组FPD从(462.5±14.5) ms延长至(512.6±17.6) ms，HG组FPD相对于NM组延长(16.8±1.4)%(F=25.61，P<0.001；图3B)；高糖处理后，hiPSC-CMs的FPDc从处理前的(389.1±13.7) ms延长至处理后的(478.3±31.5) ms，NM组从(387.7±21.6) ms延长至(422.6±32.9) ms，HG组FPDc相对于NM组延长(13.9±1.3)%(F=5.399，P<0.01；图3C)；高糖处理后，HG组hiPSC-CMs的锋电位从(3.06±0.35) mV增加至(3.33±0.41) mV，NM组从(3.08±0.22) mV增加至(3.36±0.41) mV，两组锋电位变化差异无统计学意义(F=0.002，P>0.05；图3D)；高糖处理后，HG组hiPSC-CMs的CV无明显改变，处理前CV为(0.21±0.04) mm/ms，处理后为(0.27±0.07) mm/ms，NM组CV分别为(0.20±0.03) mm/ms与(0.21± 0.01) mm/ms，HG组CV相对于NM组虽然增加了(25.6±2.1)%，但差异无明显统计学意义(F=1.838，P>0.05；图3E)。Real-time PCR分析显示：与NM组相比，HG组编码快速激活延迟整流钾电流(rapidly activated delayed rectifier potassium current，IKr)的离子通道基因KCNH2 mRNA表达降低，为NM组的(0.86±0.02)倍 (t=11.12，P<0.01)，HG组心脏其他主要离子通道(SCN5A和SCN1B、CACNA1C和CACNB2、KCND3、KCNE2、KCNQ1和KCNE1、KCNJ2)基因的mRNA表达与NM组相比差异无统计学意义(均P>0.05，图3F)。
图3.
高糖对人诱导多潜能干细胞来源的心肌细胞电生理特性的影响
Figure 3 Impact of high glucose on electrophysiological properties of human induced pluripotent stem cells derived cardiomyocytes
A: Beating rate (BR) changes after high glucose intervention; B: Field potential duration (FPD) changes after high glucose intervention; C: Field potential duration corrected by beating rate (FPDc) changes after high glucose intervention; D: Spike changes after high glucose intervention; E: Conduction velocity (CV) changes after high glucose intervention; F: Changes of expression of KCNH2 mRNA after high glucose intervention. **P<0.01, ***P<0.001.
2.3. 胰岛素对高糖作用下hiPSC-CMs电生理特性的影响
hiPSC-CMs经高糖(15 mmol/L葡萄糖)+胰岛素(100 mg/L胰岛素)处理4 d，HG+INS组与HG组的BR较前均减慢，HG+INS组BR从(32.0±0.6) 次/min降低至(30.6±1.4) 次/min，HG组从(31.9±0.5) 次/min降低至(27.9±1.4) 次/min，但HG+INS组BR与HG组相比增加(8.3±0.5)%(F=7.875，P<0.01；图4A)；HG+INS组处理前hiPSC-CMs的FPD为(463.4±9.7) ms，处理后为(532.6±12.8) ms，HG组从(460.4±9.0) ms延长至(587.6±23.7) ms，HG+INS组FPD与HG组相比缩短(12.7±1.9)%(F=18.67，P<0.01；图4B)；HG+INS组hiPSC-CMs的FPDc从处理前的(387.4±4.1) ms延长至处理后的(422.4±10.0) ms，HG组从(384.8±4.0) ms延长至(476.3±11.5) ms，HG+INS组FPDc与HG组相比缩短(14.7±1.1)%(F=59.97，P<0.001；图4C)；胰岛素处理后，hiPSC-CMs锋电位显著增加，HG+INS组锋电位从(3.12±0.46) mV增加至(4.35±0.64) mV，HG组从(3.06±0.35) mV增加至(3.33± 0.41) mV，HG+INS组锋电位与HG组相比增加了(30.8±3.7)%(F=5.112，P<0.05；图4D)；胰岛素处理后，hiPSC-CMs传导速率无明显变化(F=0.198，P>0.05)，HG+INS组CV处理前为(0.23±0.08) mm/ms，处理后为(0.32±0.08) mm/ms，HG组处理前后的CV分别为(0.21±0.04) mm/ms与(0.30±0.07) mm/ms(图4E)。心脏离子通道基因表达分析显示：与HG组相比，HG+INS组高表达钠通道基因SCN5A，为HG组的(1.85±0.15)倍(t=13.69，P<0.001；图4F)，高表达编码IKr钾通道基因KCNH2，为HG组的(1.63±0.14)倍 (t=11.41，P<0.001；图4G)，而心脏其他各主要离子通道基因(SCN1B、CACNA1C、CACNB2、KCND3、KCNE2、KCNQ1、KCNE1、KCNJ2)的mRNA表达差异均无统计学意义(均P>0.05)。
图4.
胰岛素与高糖对人诱导多潜能干细胞来源的心肌细胞电生理特性的影响
Figure 4 Effect of insulin and high glucose on electrophysiology of human induced pluripotent stem cells derived cardiomyocytes
A: Beating rate (BR) changes after insulin and high glucose intervention; B: Field protential duration (FPD) changes after insulin and high glucose intervention; C: Field protential duration corrected by beating rate (FPDc) changes after insulin and high glucose intervention; D: Spike changes after insulin and high glucose intervention; E: Conduction velocity (CV) changes after insulin and high glucose intervention; F: Changes of relative expression of SCN5A mRNA after insulin and high glucose intervention; G: Changes of relative expression of KCNH2 mRNA after insulin and high glucose intervention. *P<0.05, **P<0.01, ***P<0.001.
2.4. 高糖与胰岛素对hiPSC-CMs诱发心律失常现象的影响
经高糖与胰岛素处理后的hiPSC-CMs给予IKr阻滞剂索他洛尔干预(图5)，结果发现：1 μmol/L的索他洛尔使NM组hiPSC-CMs的FPDc延长(17.9±2.0)%，使HG组的FPDc延长(32.1±5.1)%，两组间差异存在统计学意义(t=3.597，P<0.05)；而HG+INS组hiPSC-CMs的FPDc延长(19.6±2.3)%，与HG组相比，HG+INS组FPDc显著缩短(t=3.169，P<0.05)，但与NM组相比，HG+INS组FPDc差异无统计学意义 (t=0.427，P>0.05)。3 μmol/L的索他洛尔使NM组hiPSC-CMs的FPDc延长(41.0±6.4)%，使HG组FPDc延长(58.6±7.5)%，两组间差异存在统计学意义(t=4.474，P<0.01)；而HG+INS组hiPSC-CMs的FPDc延长(43.5±7.6)%，与HG组相比，FPDc显著缩短(t=3.848，P<0.01)，但与NM组相比，HG+INS组FPDc差异无统计学意义(t=0.626，P>0.05)。10 μmol/L的索他洛尔使NM组hiPSC-CMs的FPDc延长(86.7±13.6)%，且1个培养孔诱发出了心律失常现象，而HG组5个孔均诱发出了心律失常现象，两组间差异存在统计学意义(t=19.69，P<0.001)；而HG+INS组hiPSC-CMs的FPDc延长78.9%±11.6%，各孔均未诱发出心律失常现象，与HG组相比，心律失常现象风险显著降低(t=16.57，P<0.01)。同时，hiPSC-CMs对IKr阻滞剂索他洛尔的反应在各组中均呈剂量效应依赖性。
图5.
高糖与胰岛素对索他洛尔诱发人诱导多潜能干细胞来源的心肌细胞FPDc的影响
Figure 5 Effect of high glucose and insulin on induced FPDc of human induced pluripotent stem cells derived cardiomyocytes by sotalol
FPDc: Field protential duration corrected by beating rate. *P<0.05, **P<0.01, ***P<0.001.
3. 讨 论
糖尿病不但可以导致心脏冠状动脉病变和直接心肌损伤，也可引起心肌电传导异常[11]，进而可引起心肌QT间期延长，可能引发危及生命的尖端扭转型室性心动过速而增加心脏性猝死的风险[11]。有研究[12]发现12%的1型糖尿病患者出现校正的QT间期(corrected QT interval，QTc)延长，1型糖尿病患者发生QTc延长的风险是正常对照者的4倍。相关横断面研究[13]也发现：在501名2型糖尿病患者中有44.1%的患者QTc>440 ms，2%的患者QTc>500 ms。在老年2型糖尿病患者中，27.27%的患者QTc>440 ms[14]。这些提示糖尿病可增加QT间期延长的风险，但高糖对人心肌细胞电生理特性、心律失常现象发生风险的影响以及胰岛素治疗后对其电生理特性的进一步影响尚不完全清楚。随着hiPSC-CMs研究[10, 15-16]的进展，hiPSC-CMs已成为体外研究药物对人心肌作用的重要新平台。本研究以hiPSC-CMs为模型，发现相对于正常对照组，高糖可显著延长hiPSC-CMs的FPD及FPDc，同时也增加了索他洛尔诱发心律失常的风险，但对代表钠电流的锋电位及心肌传导速率无明显影响，同时还发现高糖处理后可降低编码IKr电流的HERG(human ether-a-Go-Go)通道基因KCNH2的表达，这提示高糖可能通过抑制HERG通道基因KCNH2的表达而减少心肌复极电流IKr，从而延长人心肌QT/QTc间期，同时也增加了HERG通道阻滞剂索他洛尔诱发的心律失常风险。相关动物实验[17]发现：与正常大鼠相比，2型糖尿病大鼠心室肌细胞动作电位时程延长，可引起大鼠心室肌L型钙电流(L-type calcium current，ICa-L)和瞬时外向钾电流(transient outward potassium currents，Ito)的电流密度减低，并能使其相关蛋白Cav1.2和Kv4.3表达水平的降低。也有动物实验[18]发现：1型和2型糖尿病模型小鼠的心室肌动作电位时程也明显延长，而这种电生理改变与动作电位中的晚钠电位的增加有关。上述动物模型研究发现高血糖导致心肌细胞动作电位时程延长的具体离子通道机制不同，与本研究发现的高糖抑制HERG通道基因表达的机制也不同，这可能与不同种属来源的心肌细胞存在的电生理差别有关，已有研究[19]指出动物心肌细胞不能完全代表存在明显差别而又具有复杂电生理特征的人心肌细胞。
本研究在进一步用胰岛素干预后发现：与HG组相比，HG+INS组hiPSC-CMs的BR增快，FPD及FPDc显著缩短，锋电位增加，而心肌传导速率无明显变化；索他洛尔诱发hiPSC-CMs的FPDc延长及心律失常现象风险也较HG组明显降低。这提示胰岛素可改善高糖诱导的人心肌细胞电生理重构，可缩短高糖诱导的QT间期，并能降低高糖作用下药物诱发心律失常现象的风险。相关临床研究[20]发现：胰岛素敏感指数降低与2型糖尿病患者的QTc间期延长相关。最新的研究[21]也发现2型糖尿病患者接受每日3~4次的胰岛素注射治疗，可使其QTc从(439±24) ms缩短至(427±26) ms。这些研究提示胰岛素可缩短高糖导致的人心肌QTc的延长，本研究结果与此相符；本研究进一步通过药物诱发心律失常现象证实了胰岛素可降低高糖所增加的心律失常风险，提示胰岛素治疗对2型糖尿病患者预防心脏性猝死可能有重要作用；本研究进一步发现胰岛素缩短高糖所致的QTc延长及降低心律失常风险可能与胰岛素导致人心肌编码Ikr电流的HERG通道基因表达增加有关，但胰岛素导致人心肌HERG通道基因表达增加的具体机制尚需进一步研究。
另外，本研究发现NM组hiPSC-CMs从7 d到11 d的培养过程中，BR逐渐减慢，FPD逐渐延长，FPDc、锋电位、CV无显著变化，这提示hiPSC-CMs培养7 d后电生理特性基本趋于稳定，BR逐渐减慢可能是细胞更加趋于成熟的一种电生理改变；相关研究[22]发现：随着培养时间的延长，hiPSC-CMs跳动频率逐渐减慢，当培养至80~120 d时，跳动频率减慢至(9.3±3.1) 次/min，同时细胞体积、肌纤维大小与排列、肌节等结构明显趋于成熟。
综上所述，本研究发现胰岛素可显著缩短高糖引起的人心肌细胞的QT间期延长，并能降低高糖作用下药物诱发心律失常现象的风险，此作用可能与胰岛素促进心肌细胞HERG通道基因的表达相关，这为人糖尿病心肌的电生理改变及胰岛素治疗对其电生理的影响提供了实验依据；后续进一步的机制研究将可能为获得性、甚至遗传性长QT综合征的治疗提供新的思路与方法。
基金资助
国家自然科学基金(81000063)；陕西省自然科学基础研究项目(2020JM-375)。
This work was supported by the National Natural Science Foundation (81000063) and the Natural Science Basic Research Program of Shaanxi (2020JM-375), China.
利益冲突声明
作者声称无任何利益冲突。
作者贡献
魏峰 课题设计，实验实施，结果分析，论文构想、撰写与修订；张玉顺，王星烨 课题设计指导，参与统计分析；霍建华 课题设计、论文修改与统计分析。所有作者阅读并同意最终的文本。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202205610.pdf
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