Objective: To investigate the effect and related mechanism of homocysteine (Hcy) on calcium overload in neonatal rat atrial cells (NRICs). Methods: NRICs were assigned to 9 groups after culture for 3 days: (1) control group; (2) Hcy group (0, 50, 100, 200, 500 μmol/L for 48 hours); (3) antioxidant group (NAC, 10 μmol/L for 24 hours); (4) Hcy+NAC group (500 μmol/L Hcy for 48 hours, then treated with 10 μmol/L NAC for 24 hours); (5) calcium/calmodulin dependent protein kinase Ⅱδ (CaMKⅡδ) inhibitor group (KN-93, 3 μmol/L KN-93 for 5 hours); (6) specific sodium current inhibitor group (ELE, 1 μmol/L ELE for 5 hours); (7) Hcy+KN-93 group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 for 5 hours); (8) Hcy+ELE group (500 μmol/L Hcy for 48 hours, then treated with 1 μmol/L ELE for 5 hours; (9) Hcy+KN-93+ELE group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 and 1 μmol/L ELE for 5 hours). Moreover, NRICs were also treated with CaMKⅡδ-siRNA lentivirus, and Nav1.5-siRNA lentivirus, negative lentivirus carrier containing green fluorescent protein (GFP) for 24 hours. The MOI values of the three groups were 10. Infection efficiency of lentivirus was determined by observing the percentage of GFP fluorescence under inverted fluorescence microscope after transfection for 24 hours, and cultured regularly with simultaneous Puro screening, then cells were grouped as Hcy+CaMKⅡδ-siRNA group, Hcy+Nav1.5-siRNA group and Hcy+negative group. The concentration of Ca(2+) in NRICs ([Ca(2+)]i) of various groups was detected through Fluo-4/AM fluorescence probe, then 2', 7'- two chlorofluorescein diacetate (DCFH-DA) was used as a probe to detect reactive oxygen species (ROS) in NRICs by flow cytometry. The malondialdehyde (MDA) was detected by the activity of superoxide dismutase (SOD) and xanthine oxidase was detected by thiobarbituric acid colorimetry. The protein and mRNA expression level of CaMKⅡδ and Nav1.5 in NRICs were detected by Western blot and quantitative real-time PCR. Results: (1) ROS, MDA and SOD were similar between NAC group and control group, ROS and MDA were significantly increased, while SOD was significantly reduced in Hcy group in a concentration-dependent manner. (2) [Ca(2+)]i: The level of [Ca(2+)]i was (155.57+7.25), (187.43+13.07), (248.98+27.22) and (307.36+15.09) nmol/L in 50, 100, 200 and 500 μmol/L Hcy groups, which was significantly higher than that in the control group ((123.18+7.24) nmol/L, P<0.01). In addition, the level of [Ca(2+)]i in Hcy+NAC group ((222.87+23.71)nmol/L) was significantly lower than that in Hcy 500 μmol/L group ((305.15+39.45) nmol/L, P<0.05), while [Ca(2+)]i level was similar between NAC group and the control group. (3) The protein expression of CaMKⅡδ and Nav1.5 was significantly upregulated in Hcy groups than in the control group. The protein expression level of CaMKⅡδ-Thr287 was significantly lower in NAC group than in Hcy 500 μmol/L group (P<0.01), however, there was no significant difference on the protein expression levels of CaMKⅡδ-Thr287 and Nav1.5 between NAC group and control group (all P>0.05). (4) The protein expression levels of CaMKⅡδ-Thr287 and the concentration of [Ca(2+)]i were significantly lower in Hcy+KN-93 group and Hcy+KN-93+ELE group than in Hcy 500 μmol/L group (P<0.05). [Ca(2+)]i concentration was significantly lower in Hcy+KN-93 group, Hcy+ELE group and KN-93+ELE+Hcy group than in Hcy 500 μmol/L group (P<0.05). (5) The mRNA and protein expression levels of CaMKⅡδ and Nav1.5 in each group infected with lentivirus: the GFP expression was ideal post lentivirus transfection for 24 hours (up to 90%), which was significantly lower in the CaMKⅡδ-siRNA group and Nav1.5-siRNA group than in the negative infection group (all P<0.05), which was similar between negative infection group and control group (P>0.05). Moreover, the mRNA and protein expression levels of CaMKⅡδ and CaMKⅡδ-Thr287 was significantly lower in Hcy+Nav1.5-siRNA group than in Hcy+negative infection group (P<0.05). The protein and mRNA levels of Nav1.5 were similar between Hcy+CaMKⅡδ-siRNA group and Hcy+negative infection group (P>0.05). Conclusions: Hcy can induce calcium overload in NRICs by increasing oxidative stress, upregulating the sodium channel protein, and activating the late sodium current and phosphorylating CaMKⅡδ.
目的： 探讨同型半胱氨酸（Hcy）对乳鼠心房肌细胞（NRIC）钙超载的作用及其机制。 方法： 分离出生1~3 d的SD大鼠NRIC。NRIC接种后第3天分成以下几组：（1）对照组（不做任何处理）；（2）Hcy不同浓度组：NRIC中分别加入终浓度为50、100、200、500 µmol/L的Hcy培养48 h；（3）抗氧化剂（NAC）组：NRIC中加入10 µmol/L NAC培养24 h；（4）Hcy+NAC组：NRIC中加入终浓度为500 µmol/L的Hcy培养48 h，然后加入10 µmol/L NAC培养24 h；（5）钙/钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡδ）抑制剂（KN-93）组：NRIC加入3 µmol/L的KN-93孵育5 h；（6）特异性钠电流抑制剂（ELE）：NRIC中加入1 µmol/L的ELE孵育5 h；（7）Hcy+KN-93组：NRIC加入终浓度为500 µmol/L的Hcy培养48 h，然后加入3 µmol/L的KN-93孵育5 h；（8）Hcy+ELE组：NRIC中加入终浓度为500 µmol/L的Hcy培养48 h，后加入1 µmol/L的ELE孵育5 h；（9）Hcy+KN-93+ELE组：NRIC中加入终浓度为500 µmol/L的Hcy培养48 h，然后加入3 µmol/L的KN-93和1 µmol/L的ELE孵育5 h。慢病毒感染NRIC的分组及其干预：将NRIC以2×10(6)个细胞/ml的密度接种于6孔板，培养48 h至细胞融合度达70%~80%。将包装的慢病毒载体原液加入完全培养基中，分别组成CaMKⅡδ-siRNA组和Nav1.5-siRNA组。阴性感染组仅加入含绿色荧光蛋白（GFP）的阴性慢病毒载体原液。3组MOI值均为10，感染24 h。在倒置荧光显微镜下观察感染后24 h各组细胞GFP荧光百分比，判断慢病毒感染效率，定期传代并进行Puro筛选，培养扩增稳转细胞，在构建成功的慢病毒干扰组的基础上加入Hcy处理，并分为Hcy+CaMKⅡδ-siRNA组、Hcy+Nav1.5-siRNA组和Hcy+阴性感染组3组。采用Fluo-4/AM荧光探针检测NRIC内Ca(2)+浓度（[Ca(2+)]i）。采用以2'，7'-二氯荧光黄双乙酸盐(DCFH-DA)作为探针,用流式细胞仪检测NRIC内活性氧（ROS），硫代巴比妥酸比色法检测丙二醛（MDA）和黄嘌呤氧化酶法检测超氧化物歧化酶（SOD）活力。Western blot法和实时荧光定量PCR法分别检测NRIC中CaMKⅡδ和Nav1.5蛋白和mRNA的表达水平。 结果： （1）各组NRIC内ROS水平和MDA、SOD活力：Hcy 50、100、200和500 μmol/L组NRIC内ROS水平和MDA活力均明显高于对照组（P均<0.05），SOD活力则均明显低于对照组（P均<0.05），且均呈现一定的浓度依赖性。另外，NAC组NRIC内ROS水平与对照组比较差异无统计学意义。（2）各组NRIC[Ca(2+)]i：Hcy 50、100、200和500 µmol/L组NRIC [Ca(2+)]i分别为（155.57±7.25）、（187.43±13.07）、（248.98±27.22）和（307.36±15.09）nmol/L，其中Hcy 500 µmol/L组明显高于对照组[123.18±7.24）nmol/L，P<0.01]。另外，Hcy+NAC组NRIC[Ca(2+)]i明显低于Hcy 500 µmol/L组[（222.87±23.71）nmol/L比（305.15±39.45）nmol/L，P<0.05],NAC组与对照组比较差异则无统计学意义。（3）各组NRIC内CaMKⅡδ和Nav1.5蛋白表达：Hcy 50、100、200和500 µmol/L组NRIC内CaMKⅡδ和Nav1.5蛋白表达水平均高于对照组，且后3组差异均有统计学意义（P均<0.01），Hcy对NRIC内CaMKⅡδ和Nav1.5蛋白表达水平的影响呈现一定的浓度依赖性。另外，NAC组NRIC内CaMKⅡδ-Thr287蛋白表达水平明显低于Hcy 500 µmol/L组（P<0.01），NAC组与对照组NRIC内CaMKⅡδ-Thr287和Nav1.5蛋白表达水平差异均无统计学意义。（4）各组NRIC内CaMKⅡδ-Thr287蛋白表达水平和[Ca(2+)]i：Hcy+KN-93组和Hcy+KN-93+ELE组NRIC内CaMKⅡδ-Thr287蛋白表达水平均明显低于Hcy 500 µmol/L组（P均<0.05），且以Hcy+KN-93+ELE组为甚，Hcy+ELE组亦低于Hcy 500 µmol/L组，但差异无统计学意义。Hcy+KN-93组、Hcy+ELE组和KN-93+ELE+Hcy组NRIC [Ca(2+)]i均明显低于Hcy 500 µmol/L（P均<0.05），且以KN-93+ELE+Hcy组为甚。（5）慢病毒感染各组NRIC内CaMKⅡδ、Nav1.5蛋白和mRNA表达：慢病毒感染NRIC 24 h时GFP表达最理想（可达90%），CaMKⅡδ-siRNA组和Nav1.5-siRNA组NRIC内CaMKⅡδ、Nav1.5蛋白和mRNA表达水平均明显低于阴性感染组（P均<0.05），而阴性感染组与对照组比较差异则均无统计学意义。另外，Hcy+Nav1.5-siRNA组NRIC内CaMKⅡδ-Thr287和CaMKⅡδ蛋白以及mRNA表达水平均明显低于Hcy+阴性感染组（P均<0.05），Hcy+CaMKⅡδ-siRNA组与Hcy+阴性感染组比较Nav1.5蛋白和mRNA水平差异则均无统计学意义。 结论： 高Hcy可诱导NRIC内钙超载，其机制可能为高Hcy使机体处于高氧化应激状态，通过上调钠通道蛋白，激活晚钠电流，并磷酸化CaMKⅡδ，从而共同引起钙超载。.
Keywords: Ca(2+) overload; Heart atria; Homocysteine; Muscle cells.