| Background and objectiveAtrial fibrillation (AF for short) is one of the most common arrhythmia, in which the incidence in people over 70 years of age up to 10%, has become a threat to human health and quality of life one of the important diseases. Occurrence of atrial fibrillation can be induced by atrial effective refractory period (AERP) is characterized by reduced atrial electrical remodeling and the structure of adaptive and non adaptive changes caused by remodeling."Calcium overload" is the key to electrical remodeling, atrial fibrillation and may also be an important basis for continuing. Calcium / calmodulin-dependent protein kinaseⅡ(calcium / calmodulin-dependent protein kinaseⅡ, CaMKⅡ) as a Ca2 + / CaM-regulated protein family of one of the main members, the pathophysiological process in the cell play an important biological role. Research has shown that CaMKⅡatrial fibrillation significantly increased protein content, which increased more significantly in the left atrium, and with the AF time CaMKⅡprotein content also increased gradually. Therefore, the study of atrial fibrillation, calcium overload and the relationship between CaMKⅡmay help the prevention and treatment of atrial fibrillation. In this study, calcium ionophore ionomycin Neonatal rat atrial muscle cell calcium overload model, based on the observation of this CaMKⅡinhibitor KN93 on neonatal rat atrial myocytes of load, and cell Detection of changes in CaMKⅡfor CaMKⅡinhibitors on the prevention and treatment of atrial fibrillation feasibility study lays the foundation.Methods1. The atrial myocytes were disassociated from neonate rats with digestion method and cultured for 96 hours. Co-incubated with the ionomycin(0,0.5,1.0,2.0μmol/L, respectively), the cells were divided into control group and experiment group(E1, E2 and E3) and loaded with Ca2+ indicator Fluo-3 /AM. The level of intracellular Ca2+ ([Ca2+]i) were measured with laser confocal scanning microscope.2. The atrial muscle cells were primarily cultured for 96 h and a model of calcium overload for atrial muscle cell in neonate rat was established by using calcium ionophore(ionomycin, 1.0μmol/L). In the present of Fluo-3 /AM(an indicator of calcium), intracellular calcium and the expression of CaMKⅡwere detected under the intervention of KN93(0.25,0.5,1.0μmo/L).Results:1. More than 90% of cultured cells were positive toα-actin antibody. The activity ratio of cells in control group was 73.00士2.37%. Compared with the each subgroup of experiment, there was no significant difference(p>0.05). One hour after treated with different concentrations of ionomycin, the [Ca2+]i of atrial muscle cell was significantly increased in all the experimental groups(431.54±20.97, 705.87±28.34, 1305.05±53.69 vs 257.08±18.63, respectively, p<0.05). There was significant difference between each subset of the experimental group. [Ca2+]i level and the fluorescence intensity of the myocytes increased gradually in a dose-dependent manner with the concentration of ionomycin.2. Compared with the control group, the intracellular Ca2+ was increased significantly by ionomycin (660.16士108.47 vs 376.12士57.57,p<0.01) and KN93 has no effect on it(389.00士64.01 vs 376.12士57.57, p>0.05). Added KN93 (0.25,0.5,1.0μmo/L) in advance, the fluorescence intensity of intracellular Ca2+ induced by ionomycin was significantly decreased (vs 660.16士108.47, p<0.01). the expression of CaMKⅡwas increased in calcium overload group( vs control group, p<0.01) and KN93 has no effect on it. a Added KN93 (0.25,0.5,1.0μmo/L) in advance, the expression of CaMKⅡin cell of calcium overload was significantly reduced (vs calcium overload group,p<0.01).Conclusion1. calcium overload model with primary atrial myocytes of neonate rats can be built by ionomycin.2. KN93, an inhibitor of CaMKⅡ, can reduce the calcium load of atrial muscle cell induced by ionomycin and decrease the expression of CaMKⅡ.
|
PDF Full Text Request