Effects Of Diabetes On JPH-2 And Related Atrial Selective Ion Channels In Rat Atrial Remodeling

Background:Diabetes and atrial fibrillation are common chronic diseases that affect the world today.There is a correlation between the two diseases.Diabetes can cause atrial remodeling and form the substrate for atrial fibrillation.Atrial remodeling in diabetes includes structure,electrical remodeling,electromechanical coupling remodeling,autonomic nerve remodeling,and metabolic remodeling.The specific molecular mechanism has not yet been fully clarified.Atrial selective ion channels are expected to become targets for the treatment of atrial fibrillation in the future,and are also involved in the process of atrial remodeling.However,the specific changes and gating characteristics in the course of diabetes are not completely clear or controversial.Junctophilin-2（JPH-2）is a member of the protein family that connects the membrane complex in cardiomyocytes,is responsible for maintaining the subcellular structure of the T tube,and is also involved in intracellular calcium regulation and related ion channel regulation.It has the potential to bridge structural remodeling and electrical remodeling,but in diabetic atrial remodeling,the specific mechanism of action has not yet been fully determined.Objective:1.To establish diabetic rats model and observe the characteristics of atrial remodeling and electrical remodeling at the level of living animals and tissues;2.To observe the effects of diabetes on atrial remodeling at the microscopic level,that is,the instantaneous sodium channel current(I_Na),delayed sodium channel current(I_{Na L})and instantaneous outward potassium channel current(I_to)and their gating characteristics,as well as the effect on the expression of JPH-2;3.To observe the functional gene transcription of atrial JPH-2 and related atrial selective ion channels in diabetic conditions,screening and analyzing the possible interaction mechanism between JPH-2 and related atrial selective ion channels.Methods:Nithty SD rats,male,about 220 g,8 weeks old,were randomly divided into a control group and a diabetes group.After 2 weeks of overfeeding,diabetic models were established by injecting STZ into the tail vein（the control group was injected with saline）.After 6 weeks of feeding,the tail vein fasting blood glucose≥16.7 mmol/L was regarded as diabetes.The rats modeling were successful.All surviving rats underwent surface multi-lead electrocardiogram examination under chloral hydrate anesthesia,and recorded and analyzed the P wave graphic data.Experiments were conducted on rats in the control group and the diabetic group of the same feeding batch.After the first part of the rats received the ECG test,they continued to receive echocardiography under anesthesia to collect data;then the chests were opened and the hearts were taken out,and retrograde perfused through the aorta on the Langendorff system,and atrial electrophysiological examination was taken under the state of isolated perfusion.Focus on recording and analyzing the effective refractory period（ERP）of atrium,interatrial conduction time（IACT）and the induction of atrial arrhythmia.In the second part of the experiment,a part of the rats were directly heparinized and then opened the chest under anesthesia to take out the heart.The Langendorff system was used for retrograde perfusion to separate atrial myocytes.The whole cell patch clamp technique was used to measure the two groups.Draw I-V curve,activation curve,inactivation curve and recovery curve for I_Na,I_{Na L} and I_to of rat atrial myocytes,and compare and analyze current density and related gating parameters.Another part of the rats was opened chest and taken out the heart after the ECG test,separated the atria and ventricles,and then cryopreserved in liquid nitrogen,and then homogenized the tissues and extracted the protein.The expression of JPH-2 and ion channel protein in atrial muscle of the two groups were observed by Western Blot technology.In the third part of the experiment,the two groups of rats in the same batch were taken out of the heart and separated atrial and ventricular specimens after the ECG test,and frozen in liquid nitrogen;afterwards,the atrium of the control group and the diabetic group were screened by Illumina high-throughput sequencing technology.Differentially expressed genes,and perform gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）function annotation and enrichment analysis for differentially expressed genes.Results:1.After 6 weeks of modeling,compared with the control group,the fasting blood glucose of the rats in the diabetes group were significantly increased（5.93±0.49mmol/L vs.22.93±2.92 mmol/L,P<0.01）;and in diabetes group P wave widening（40.71±0.74 ms vs.42.46±0.61 ms,P<0.01）,PR interval prolongation（48.03±0.75ms vs.52.77±1.03 ms,P<0.01）,P-wave termina force in lead V₁ increased（1.84±0.81 m V·ms vs.2.49±1.22 m V·ms,P<0.01）,and the amplitude decreased in lead II,a VF,and V₁（P<0.01）.Compared with the control group,the inner diameter of the left atrium of the rats in the diabetes group increased（3.78±0.31 mm vs.4.73±0.27 mm,P<0.01）;the conduction time of the atrial chamber was prolonged（27.00±2.73 mm vs.31.43±2.51 mm,P<0.01）,the effective refractory period of the atrium was shortened（45.81±3.56 ms vs.30.83±3.29 ms in the right atrium;46.23±3.44 ms vs.30.97±3.10 ms in the left atrium,P<0.01,respectively）,and the diabetes group was more easily induce persistent atrial arrhythmia.2.（1）Compared with the control group,the I_Na peak current density of rats in the diabetic group decreased（at-50 m V,-101.13±11.73 p A/p F vs.-55.70±23.16 p A/p F;at-40 m V,-94.75±9.68 p A/p F vs.-70.06±5.61 p A/p F,both P<0.01）;the half activation voltage(V_1/2,a)and the activation curve slope（K_a）both increased,the half deactivation voltage(V_1/2,i)decreased,the time constant of the recovery curve（τ）decreased,P<0.05,respectively;（2）Compared with the control group,the I_Na decay time constantτ’value of rats in the diabetic group decreased（at-45 m V,-40m V and-35m V,P<0.05,respectively）;the I_{Na L} peak current density of rats in the diabetic group decreased（at-50 m V,-0.53±0.23 p A/p F vs.-0.99±0.12 p A/p F,P<0.01）（3）Compared with the control group,the I_to peak current density of rats in the diabetic group decreased（at+60 m V,40.68±9.94 p A/p F vs.28.89±3.13 p A/p F,P<0.01）;V_1/2,a and K_a increased,V_1/2,i also increased,τvalue decreased,all P<0.05;（4）Compared with the control group,the atrial JPH-2 and SK2 protein expression in the diabetic group were down-regulated.3.According to Illumina transcriptome sequencing analysis,a total of 895 m RNAs were differentially expressed in the atrial muscle of the control group and the diabetic group,526 of the diabetic group had up-regulated expression,and a total of 369 had down-regulated expression;Among the 8 selected genes of interest in the experimental basis of the first part and the second part,7 were significantly down-regulated in the atria of the diabetes group,namely Jph2,Kcnd1,Kcnn2,Ryr2,Scn5a,Scn1b,and Camk2a.In the GO biological process group,the biological regulation process,cellular process,metabolic process,biological adhesion and rhythm control process that accompany diabetic remodeling are all significantly enriched types;in the cell component group,cell part,membrane Part and cell connection part are both significantly enriched types.In the molecular function grouping,the binding molecule component tops the list.At the same time,signal transduction activity,molecular function regulation and electroporter activity were all significantly enriched types.After KEGG significant enrichment analysis and literature review,the signal pathways that may be regulated by JPH-2 and related membrane ion channels are:PI3K-Akt signaling pathway,c GMP-PKG signaling pathway,AGE-RAGE signaling pathway,AMPK signaling pathway and Ca MKII signaling pathway.Conclusion:1.The T1DM model of diabetes group was successfully established;the left atrium of diabetic rats was enlarged,the P wave duration was widened and the amplitude was reduced,the interatrial conduction time was prolonged,and the effective refractory period was shortened,that is,structural remodeling and electrical remodeling occurred.2.The atrium current density of diabetic rats decreases,and the I_Na channel becomes difficult to activate,but it is easy to inactivate and recover after activation;at the same time,the attenuation of I_Na is accelerated,but the attenuated I_{Na L} increases;the I_to current density also decreases,and the I_to channel becomes difficult to activate,and it is not easy to inactivate after activation,but it is easy to recover.In addition,the expression of JPH-2 and SK2 proteins in the atria of diabetic rats was down-regulated.That is,the atria of diabetic rats are remodeled at the level of cell ultrastructure and ion channels.3.In the diabetic state,except for Cav1.2,the expression of JPH-2 and the detected ion channel function genes in the atrium of rats was significantly down-regulated;the signal pathways that may be regulated by JPH-2 and related membrane ion channels are screened:PI3K-Akt signaling pathway,c GMP-PKG signaling pathway,AGE-RAGE signaling pathway,AMPK signaling pathway and Ca MKII signaling pathway.4.The above results,literature review and analysis show that the Ca MKII signaling pathway may be linked to the regulation of JPH-2 and its related various ion channels and transporters in the diabetic state,including LTCC,SK channel,NCX1,I_to channel and I_Na channel.So as to realize the bridging of cell ultrastructure and cell electrophysiological reconstruction.