| Objective:At present,diabetes is a disease with high incidence in the world.A large number of studies have found that there is a high incidence of cardiovascular disease and mortality in patients with diabetes,and the proportion of arrhythmias in patients with diabetes is also increasing year by year.Previous studies mainly focused on ventricular arrhythmias,but there are few studies on atrial arrhythmias,while more and more diabetes-related atrial fibrillation has attracted the attention of many researchers.Calcium channel protein is an important protein in the body,which participates in many pathophysiological processes such as growth and development,neuroregulation and so on.It has been found that the level of intracellular free calcium increases significantly in animal atrial myocytes during atrial fibrillation,indicating that intracellular calcium overload may be related to the occurrence and development of atrial fibrillation.On the surface of cardiomyocytes,it has been confirmed that L-type and T-type calcium channels are involved in the regulation of calcium homeostasis.Intracellular mitochondria are considered to be the most important regulatory organs and targets for calcium signal transduction.Studies have shown that mitochondria can rapidly absorb Ca2+,through mitochondrial calcium uniporter?MCU?and then regulate the changes of calcium concentration in cytoplasm and mitochondria and participate in many basic biological processes.At present,the traditional antiarrhythmic drugs used in clinic can cause arrhythmia,so the prevention and treatment of atrial fibrillation need safe and effective drugs.There is evidence that statins may have additional effects independent of their reduction of low density lipoprotein cholesterol.These are called"multiple effects".It includes the function of prevention and treatment of atrial fibrillation.At present,it has been found that the main mechanisms of statins include:?1?enhancing vascular endothelial function;?2?inhibiting platelet aggregation;?3?anti-inflammation and antioxidation;?4?improving cardiomyocyte autonomy and mitochondrial function;?5?directly affecting myocardial electrical remodeling,structural remodeling and autonomic nerve remodeling.A large number of scientific studies and meta-analyses have shown that the use of statins can significantly reduce the risk of atrial fibrillation.However,the molecular role of statins in atrial myocyte-associated calcium channels in patients with type 2 diabetes mellitus is not clear.In this study,GK rats were used as a model,combined with basic experimental methods,whole-cell patch clamp technique and bioinformatics methods to comprehensively analyze the protein expression of associated calcium channel protein and mitochondrial calcium unidirectional transporter?MCU?in atrial myocytes of GK rats,the changes of transmembrane ICa-L and ICa-T in atrial myocytes,the three-dimensional structure of some calcium channels and the possible targets of statins.To provide ideas for exploring new action targets of statins.Methods:Healthy male GK and Wistar rats,aged 11 weeks and weighing 250ml/300g,were fed with SPF adaptively for one week.After the diabetic model of GK rats was identified,30 GK rats were randomly divided into two groups:untreated diabetic group?GK group?and treated diabetic group?GK group?with 15 rats in each group.Wistar rats were randomly divided into two groups:control group?Con group?and drug control group?Con group?with 15 rats in each group.Under the condition of normal feeding and diet,the rats in GK group and Con group were given RSV?10mg/kg?every day for 12weeks,while GK group and Con group were given the same dose of normal saline at the same time.After 12 weeks of intragastric administration,the electrocardiogram of rats in the first four groups was examined to observe the effect of rosuvastatin on P wave of electrocardiogram in GK rats.The parameters related to L-type and T-type calcium currents in four groups of atrial myocytes were recorded by whole-cell patch clamp,and the effect of rosuvastatin on atrial calcium channel currents in GK rats was observed.The expression of tissue-related calcium channel proteins?Cav1.2,Cav3.1,Cav3.2 and MCU?in atrial myocytes of each group was detected by Westernblot method,and the effect of rosuvastatin on atrial tissue-related calcium channel protein in GK rats was observed.Bioinformatics methods were used to analyze the structure of related calcium channels and the possible targets of statins.Firstly,after screening the related templates,the structures of Cav3.1 and MCU are constructed by using SWISS-MODEL homologous modeling server,then the molecular dynamics optimization of the modeled structure is carried out by using the Desmond module in Schr?dinger software package,and the stereochemical parameters of the model structure are evaluated by Procheck program.Molecular docking was carried out by modeling reasonable structure,and the binding mode of Cav3.1 and MCU with existing antagonists and rosuvastatin was analyzed,and the possible action mechanism of rosuvastatin on Cav3.1 and MCU was discussed.Results:1.The results of P wave analysis of electrocardiogram of rats in four groups showed that the P wave duration in GK group was significantly longer than that in control group?20.29±2.72 msvs.14.91±2.09 Ms;P<0.05?.However,the P wave duration of rats in GK group?n=15?was significantly shorter than that in GK group?16.57±3.82msvs.20.29±2.72 Ms;P>0.05?.2.The results of whole-cell patch clamp detection of L-type and T-type calcium currents in atrial myocytes of rats in the four groups showed that the ICa-L peak current density of+10mV GK group was-15.45±7.41 pA/pF,and the ICa-L peak current density of Con group was-7.49±2.11 pA/pF,which was 106.3%higher than that of Con group,and the difference was statistically significant.After 12 weeks of RSV treatment,the ICa-L peak current density of rats in the GK+RSV group was-7.71±2.68 pA/pF.Compared with the GK group,the ICa-L peak current density in the GK+RSV group decreased by 50.1%,and the difference was statistically significant?P<0.05?.The ICa-T peak current density of rats in the-30mV GK group was-0.71±0.42 pA/pF,and the ICa-T peak current density of Con group was-0.20±0.31 pA/pF,which was 2.55 times higher than that of Con group,and the difference was statistically significant.After 12 weeks of RSV treatment,the ICa-Ta-T peak current density of rats in the GK+RSV group wa-0.42±0.34 pA/pF.Compared with the GK group,the ICa-T peak current density in the GK+RSV group decreased by 40.8%,and the difference was statistically significant?P<0.05?.3.Western blot method was used to detect the expression of related proteins in atrial myocytes of rats in four groups:the protein expression of Cav1.2 in atrial muscle of rats in GK group was 86.3%higher than that in Con group?P<0.05.The protein expression of Cav1.2 in atrial muscle in?,GK group was 43.6%lower than that in GK group?P<0.05?.The protein expression of Cav3.1 in GK group was 68.0%higher than that in Con group?P<0.05?.The protein expression of Cav3.1 in atrial muscle of rats in),GK group was 28.1%lower than that in GK group?P<0.05?.The protein expression of MCU in GK group was 23.9%lower than that in Con group?P<0.05?.The protein expression of MCU in atrial muscle of rats in GK group was 25.1%higher than that in GK group?P<0.05?.There was no significant difference in the protein expression of Cav3.2 among the three groups.4.Using the existing three-dimensional structure of rabbit-derived Cav1.1 channel as template,the structure model of human-derived Cav3.1 channel was constructed by SWISS-MODEL homologous modeling server modeling,Procheck program evaluation,molecular dynamics optimization and molecular docking by Schr?dinger software.Through molecular docking with known Cav3.1 antagonists?including diltiazem,nifedipine,verapamil,mibefadil,NNC and TTA?and rosuvastatin,it was found that rosuvastatin may play a pharmacological role by binding to the amino acid Ser383,Gln922,Asn952 target of Cav3.1.5.In this study,the known N.fischeri MCU crystal structure is used as the template,and the homologous modeling server of SWISS-MODEL is used to construct the structure model of human MCU.The molecular dynamics is optimized by Desmond software and evaluated by Procheck program.Then,the binding mode of rosuvastatin with known antagonist DS16570511 and target drug rosuvastatin was discussed by molecular docking method.It was found that rosuvastatin had a good spatial match with MCU and interacted with the following amino acids:Leu67?Gly66?Met64?Lys62?Val61?Thr285?Met284?Tyr281?Ala280?Leu301?Gln298?Asn297?Ala294.Conclusion:1.The ECG P wave of GK rats was significantly prolonged,accompanied by a significant increase in the maximum current density of ICa-L and ICa-T,as well as an increase in the expression of Cav1.2 and Cav3.1 and a decrease in the expression of MCU in atrium.Rosuvastatin could improve the above pathological phenomena.2.The expression of calcium channel protein in atrial myocytes of GK rats:Cav1.2 and Cav3.1 were significantly increased,while MCU was significantly decreased.Rosuvastatin could improve this pathological phenomenon.3.Through molecular simulation,it is found that rosuvastatin may combine with Cav3.1to play a pharmacological effect,thus improving the occurrence and development of AF.4.Molecular simulation method is used to predict that rosuvastatin can combine with MCU and exert pharmacological effects,so as to improve the occurrence and development of AF. |
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