[Homocysteine induces calcium overload in neonatal rat atrial cells through activation of sodium current and CaMKⅡδ].

To investigate the effect and related mechanism of homocysteine (Hcy) on calcium overload in neonatal rat atrial cells (NRICs). 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. (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, 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, 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 (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 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 (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 (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 0.05), which was similar between negative infection group and control group (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 (0.05). The protein and mRNA levels of Nav1.5 were similar between Hcy+CaMKⅡδ-siRNA group and Hcy+negative infection group (0.05). 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.