| Objective(s): Atrial fibrillation(AF)is one of the more common types of cardiovascular disease.Among the many risk factors for AF,hyperthyroidism(referred to as hyperthyroidism)is the most common.Atrial sympathetic remodeling plays a vital role throughout the disease progression of AF.However,the exact mechanism of action by which AF-related atrial sympathetic remodeling occurs has yet to be fully understood.Relevant evidence suggests that atrial myocyte apoptosis may be essential in this process.In this study,we investigated the effect of sympathetic nerves on atrial myocyte apoptosis caused by hyperthyroidism in a rabbit model of atrial fibrillation.We also looked into possible molecular mechanisms that could play a role in the in vitro development of left atrial myocytes and the pharmacological treatment of lactating rabbits.Methods: 1.Animal experiments:(1)Establishing a rabbit model of atrial fibrillation caused by hyperthyroidism: fifty mature,healthy Japanese large-eared rabbits were randomly split into three groups: model,model+metoprolol,and control.Among them,twenty rabbits were in the model group,twenty were in the model+metoprolol group,and ten were in the control group.Each group’s body weight and heart rate were recorded before the start of the experiment.For the model group,50 μg/kg of thyroxine solution was injected intraperitoneally daily according to the body weight of the rabbits;for the model+metoprolol group,9.5 mg/kg of metoprolol tartrate tablets were administered daily to the rabbits by gavage in addition to the intraperitoneal injection of thyroxine solution.An identical dosage of saline was given each day intraperitoneally to the control group.The experiment was repeated daily for four months.After four months,each group of rabbits’ body weight and heart rate were re-recorded.(2)Left atrial electrophysiological assay and collection of blood specimens and left atrial myocardial tissue from rabbits: The atrial effective refractory period(AERP)was measured in three groups of rabbits at two different stimulation perimeters(i.e.,200 ms and 150 ms).Atrial fibrillation stimulation lasting more than 10 minutes was considered successful,and each group’s evoked rate of atrial fibrillation was recorded separately.Afterward,blood was collected via a vein at the ear margin of the rabbit,and the left atrial muscle tissue was taken by opening the chest and tested centrally.(3)Molecular biology experiments:enzyme-linked immunosorbent assay(ELISA)was used to detect norepinephrine(NE)levels in the serum of three groups of rabbits.Real-time quantitative polymerase chain reaction(RT-q PCR)and immunohistochemistry(IHC)were used to detect the m RNA and protein expression levels of sympathetic remodeling markers such as growth-associated protein 43(GAP-43)and tyrosine hydroxylase(TH)in the left atrial muscle tissue of three groups of rabbits,respectively.Protein expression of Bax,B-cell lymphoma-2 gene(Bcl-2),Cleaved caspase-3(activated form of caspase-3),p38 mitogen-activated protein kinase(p38 MAPK),and p-p38 MAPK(phosphorylated p38 MAPK)was measured by western blot(WB)in three groups of rabbits.2.Cell experiments:(1)Culture and identification of left atrial myocytes in suckling rabbits: 10 Japanese large-eared suckling rabbits were executed within 48 hours of birth,left atrial myocardial tissue was taken for culture,then the cultured myocytes were identified using immunofluorescence staining.(2)Left atrial myocyte experimental grouping and drug intervention: after thriving culture and identification of left atrial myocytes,the cells were randomly divided into three groups: control group: left atrial myocytes+saline;NE group: left atrial myocytes+50μmol/L norepinephrine;NE+LY2228820(p38 MAPK inhibitor)group: left atrial myocytes+50μmol/L norepinephrine +50μmol/L LY2228820.Three groups intervened for 48 hours.(3)Following a 48-hour pharmacological intervention,the protein expression of Bax,Bcl-2,Cleaved-caspase 3,p38 MAPK,and p-p38 MAPK was found in three groups of cells using western blot analysis.Results: 1.Animal experiments:(1)Changes in body weight and heart rate of three groups of rabbits before and after the experiment: before the start of the experiment: comparison of body weight and heart rate of the three groups of rabbits,with no statistically significant differences.After four months of the experiment:Compared with the control group and the model+metoprolol group,the rabbits showed decreased body weight and accelerated heart rate(P<0.001);compared with the model+metoprolol group and the control group,P>0.05,the difference was not statistically significant.(2)Results of left atrial AERP in three groups of rabbits: at both stimulation perimeters,the AERP was shortened in the model group compared with the control group and the model+metoprolol group(P<0.001);and in the model+metoprolol group compared with the control group(200 ms: P<0.001;150 ms:P<0.01).(3)Comparison of atrial fibrillation induction rates in three groups of rabbits:the rate of atrial fibrillation induction was high in the model group compared with the control group and the model+metoprolol group(P<0.05);the difference was not statistically significant in the model+metoprolol group compared with the control group,P>0.05.(4)ELISA results of serum NE in three groups of rabbits: NE levels were significantly higher in the model group compared with the control group and the model+metoprolol group(P<0.01 vs.control group;P<0.05 vs.model+metoprolol group).(5)RT-q PCR results of GAP-43 and TH in the three groups of rabbits: the m RNA expression levels of GAP-43 and TH were higher in the model group compared with the control group and the model+metoprolol group(P<0.001 vs.control group;P<0.01 vs.model+metoprolol group).(6)The results of immunohistochemical detection of GAP-43 and TH in the three groups of rabbits: the protein expression levels of GAP-43 and TH were higher in the model group compared with the control group and the model+metoprolol group(P<0.001).(7)Western blot results of Bax,Bcl-2,Cleaved-caspase 3,p38 MAPK and p-p38 MAPK in three groups of rabbits: the model group had higher protein expression levels of Bax and Cleaved-caspase 3 than the control and model+metoprolol groups(P<0.001 vs.control;P<0.01 vs.model+metoprolol).The model group had lower protein expression levels of Bcl-2 compared with the control group and the model+metoprolol group(P<0.001 vs.control group;P<0.01 vs.model+metoprolol group).The protein expression levels of p-p38 MAPK/p38 MAPK were higher in the model group compared with the control group and the model+metoprolol group(P<0.001 vs.control group;P<0.01 vs.model+metoprolol group).2.Cellular experiments:(1)Lactating rabbit left atrial myocyte culture and identification: after three days of culture: the number of cells was small,and the cells were irregularly polygonal in shape;after five days of culture: the number of cells increased compared with the previous one,and most of the cells were interconnected and formed a cluster arrangement;after seven days of culture: the number of cells reached more than80%-90%,and pseudopods entangled the cells to create a network and fused into a concentric radial arrangement;immunofluorescence staining: α-transverse muscle actin was labeled by red fluorescence,and Microfilaments were visible and neatly arranged.In contrast,blue DAPI fluorescence labeled the nucleus,and the structure was visible.The two fluorescence channels were then superimposed together for merging.It suggests that the isolated,cultured cells are cardiomyocytes.(2)Results of the three cell groups’ western blot assays: The NE group had higher protein expression levels of Bax,Cleaved-caspase 3,and p-p38 MAPK/p38 MAPK than the control and NE+LY2228820 groups(P<0.001 vs.control;P<0.01 vs.NE+LY2228820group).The NE group had lower protein expression levels of Bcl-2 compared with the control and NE+LY2228820 groups(P<0.001 vs.control;P<0.01 vs.NE+LY2228820group).Conclusion(s): 1.Prolonged high thyroid hormone levels contribute to the onset,development,and maintenance of AF through activation of sympathetic nerves.2.Sympathetic nerve activation in AF may mediate atrial myocyte apoptosis through the p38 MAPK signaling pathway.3.The β-blocker class of drugs may prevent atrial myocytes from undergoing apoptosis by inhibiting sympathetic remodeling in rabbit hyperthyroid-derived AF and thereby inhibiting activation of the p38 MAPK signaling pathway. |
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