乙醇及其代谢产物对新生大鼠原代培养心房肌细胞乙酰胆碱敏感型钾通道Kir3．1蛋白表达的影响

Effect of ethanol and its metabolites on acetylcholine-sensitive K ＋ channel Kir3. 1 protein expression of neonatal rat primary atrial cardiomyocytes

目的明确乙醇及其代谢产物乙醛对新生SD大鼠原代心房肌细胞乙酰胆碱敏感型钾通道Kir3．1蛋白表达的影响，并探讨该通道及乙醛在急性乙醇中毒引发心律失常的过程中所发挥的效应。方法采用胰蛋白酶及Ⅱ型胶原酶提取并培养150只新生SD大鼠原代心房肌细胞，并采用免疫荧光法进行肌钙蛋白I鉴定。采用细胞计数套件-8（CCK-8）法分别测定200-800mmol／L乙醇及50—500μmol／L乙醛处理24h后细胞的生存率，以确定导致新生sD大鼠原代心房肌细胞凋亡所需的乙醛及乙醇浓度。建立以最大非凋亡浓度（200mmol／L）的乙醇处理24h及相应剂量（100Ixmol／L）乙醛处理24h的新生SD大鼠原代心房肌细胞模型。采用Westernblot法检测乙醇处理组、乙醛处理组及对照组心房肌细胞Kir3．1蛋白的表达情况，并对结果进行统计学分析。结果（1）成功培养新生SD大鼠原代心房肌细胞，细胞免疫荧光鉴定所培养细胞为心肌细胞，且90％以上细胞肌钙蛋白I染色阳性。（2）CCK-8法测定显示400mmol／L以上的乙醇处理组的心肌细胞生存率明显低于对照组（P〈0．05），而200mmol／L及以下的乙醇处理组心肌细胞生存率与对照组相比差异无统计学意义（P〉0．05）；400μmol／L以上的乙醛处理组的心肌细胞生存率明显低于对照组（P〈0．05），而350μmol／L及以下的乙醛处理组心肌细胞生存率与对照组相比差异无统计学意义（P〉0．05）。（3）Westernblot检测显示200mmol／L乙醇及100vmol／L的乙醛处理24h的新生SD大鼠原代房心肌细胞Kit3．1蛋白表达分别高于对照组（44．52±23．07）％及（45．04±22．01）％（P〈0．01），而乙醇组与乙醛组之间Kir3．1蛋白表达差异无统计学意义（P〉0．05）。结论急性乙醇及乙醛处理均能够明显上调乙酰胆碱敏感型钾通道Kir3．1蛋白的表达。

Objective To identify the effect of ethanol and its metabolite acetaldehyde on acetylcholine-sensitive K ＋ channel Kir3. 1 protein expression, and explore the potential role of this channel and acetaldehyde in arrhythmia caused by acute alcoholic intoxication. Methods Primary atrial cardiomyocytes were isolated from 150 newborn SD rats by typsin and type Ⅱ collagenase, cultured and troponin I was determined by immunofluorescence. Cell survival in 200 -800 mmoL/L ethanol or 50 -500 μmol/L acetaldehyde treated cells for 24 hours was measured by CCK-8 assay to determine the concentration of ethanol and acetaldehyde for inducing apoptosis in cardiomyocytes. The highest non-apoptotic concentration（200 mmol/L） of ethanol and acetaldehyde（ 100 μmol/L） was used in the main study. Kir3.1 protein expression was detected by Western blot. Results （ 1 ） Cellular immunofluorescence results showed that cultured cells are cardiomyocytes, and more than 90% of these cells are troponin I positive. （2）CCK-8 assay demonstrated that the survival rate of cardiomyocytes in the groups treated by ethanol over 400 mmol/L for 24 hours or acetaldehyde over 400 μmol/L was significantly lower than that of the control group （ P 〈0.05） ,while the survival rate was similar in cardiomyocytes treated by ethanol less than 200 mmol/L or acetaldehyde less than 350 ~mol/L for 24 hours and the control group （ P 〉 0. 05 ）. （ 3 ） Western-bolt assay revealed that ethanol and acetaldehyde treatment for 24 hours upregulated Kir3.1 protein expression in primary atrial cardiomyocytes of newborn SD rats by （ 44. 52 ± 23.07 ） % and （ 45.04 ± 22.01 ） % respectively compared with the control group（ all P 〈 0.01）. Conclusions Acute ethanol and acetaldehyde treatment could significantly upregulate the protein expression of acetylcholine-sensitive K ＋ channel Kir3.1, this might serve as a potential mechanism for arrhythmia caused by acute alcoholic intoxication.