The Effect Of Metformin On Atrial Structure And Small Conductance Calcium Activated Potassium Channel In GK Rats

Objective:With the number of people with diabetes is growing around the world,T2DM accounts for about 90 to 95%of all diagnosed cases of diabetes,and the prevalence and incidence of T2DM have also increased rapidly throughout the world,which makes the risk of related cardiovascular diseases increased accordingly.Although coronary artery disease and related cardiac events are the most documented diabetic cardiovascular complications,the cardiac electrical system is also an important target for diabetic damage.On the other hand,there is growing evidence in recent years about the relationship between hypoglycemic episodes and arrhythmic disorders.However,the relationship between DM and atrial electrophysiology is not yet fully understood,and there is a growing population of DM patients every day.Recently,GWAS identified the SK channel genes as a new locus for AF.SK channels have recently been shown to be expressed in cardiac tissue,and there is a significant atrioventricular difference of its distribution.That's,the expression level of atrium is much higher than that of ventricle.The SK channels is an extroverted current activated during the contraction,and then participates in the repolarization of the cardiac AP.It can shorten the APD during repolarization.As a consequence of their activation by increased intracellular Ca²⁺,SK channels may be especially important during rapid cardiac electrical activity such as AF.Furthermore,SK channels seem to be involved in atrial remodeling in several experimental AF models.However,how SK channels are involved in atrial arrhythemia during T2DM conditions is still unknown.Metformin is considered as the first-line pharmacological therapy in T2DM.In recent years,the antiarrhythmic effect of metformin has been gradually recognized.But whether metformin has a restoring effect on the dysfunction of SK channels in atrial myocytes impaired in T2DM remains unknown.The aim of our study was to assess whether the SK channels exhibit abnormalities in atrial myocytes isolated from GK rats at the chronic stage of T2DM and if so,whether treatment with metformin?for 12 weeks?could normalize such responses.Methods:1.Effect of Met on atrial histological structure:10-11 weeks old male GK and Wistar rats,during an 1-week adaption period?about 12 weeks old of the animals?,the FBG,FINS,IPGTT were detected.The identification of T2DM model is though evaluating the level of fasting blood glucose and insulin resistance of the rats.When the diabetic state was already established among the GK rats,they were randomly divided into 2 groups:GK group?GK?and GK+Met group?Met?.Wistar rats were used as control group in this experiment.Altogether there were 3 groups during this experiment:Control group?Con?,GK group?GK?,GK+Met group?Met?.Then the physiological parameters and biochemical indicators of each group were detected.The morphology and structure of atrial tissue were observed by HE staining and Masson staining,and the percentage of atrial fibrosis was counted.2.Effect of Met on atrial small conductance calcium activated potassium channels expression:The m RNA and protein expression of KCa2.1,KCa2.2 and KCa2.3 in T2DM atrial tissue were detected by Real-time PCR and Western blot.3.Effect of Met on atrial small conductance calcium activated potassium channel currents and action potential:Whole-cell patch clamp techniques was performed to record the atrial I_SKK currents and related action potential parameters in 3 groups.Results:1.The FBG and FINS levels of GK rats were significantly higher?P<0.01?,and there was a decrease IPGTT when compared to control?P<0.01?.GK rats atrial myocytes were disordered and distorted,and increasing interstitial collagen fibrils while 12 weeks of Met treatment partially restored these phenomenon.2.Compared with Con group,the m RNA and protein expression of KCa2.2 in GK group decreased by 62.98%and 52.10%?P<0.01?,respectively,while the mRNA and protein expression of KCa2.3 increased by 2.81±0.5 fold and 1.98±0.5 fold?P<0.01?.Compared with the GK group,the mRNA and protein expression of KCa2.2 increased by 2.38±0.5 times?P<0.05?and 1.75±0.5 times?P<0.01?,and KCa2.3 expression level decreased by 59.43%,P<0.01)54.55%?P<0.05?.The mRNA and protein expression levels of KCa2.1 were not significantly different between groups.3.Atrial I_SKK currents and related action potential parameters were recored by whole-cell patch clamp techniques.Total K⁺currents?I_K?was recorded directly under the setted stimulating protocol,and then recording the potassium current(I_K')once more after adding SK channels specific blocker apamin?100pM 15min?.Therefore,I_SK=I_K-I_K'.At-40m V,I_SKK in GK group was significantly decreased by 67.34%compared to the Con group?P<0.001?,while Met significantly alleviated the suppression by 1.98±0.5 fold compared with the GK group?P<0.01?.Compared with Con group,APD50and APD90 in group GK were significantly prolonged?3.44±0.5,1.78±0.5 fold,P<0.01?,APD50 and APD90 in Met group were shorter than those in GK group?67.07%and 57.03%,respectively,P<0.01?.No statistically significant differences of APA and RP were noted among the 3 groups?P>0.05?.However,T2DM will lead to the prolongation of APD.This is the combined action of various ion channels.We can simulate the independent role of SK channels in maintaining atrial action potential by adding SK channel specific blocker apamin?100pM,15min?.After the addition of Apamin,APD50 and APD90 in Con group were prolonged by 25%and 28%,respectively,and APD50 and APD90 were prolonged by 9%and 13%in Met group,but there was no significant change in GK group.It was confirmed that T2DM greatly weakened the function of the SK channels,and this phenomenon could be improved through the treatment of Met.Conclusion:1.The disease state of GK rats is similar to that of human T2DM.It is a stable and authoritative T2DM model.In addition,Met can improve the metabolic disorders,inflammatory changes and atrial structural damage in GK rats.2.Met can upregulate the expression of KCa2.2 in the atria of GK rats,inhibit the expression of KCa2.3 in atrial myocytes of GK rats,but have little effect on the expression of KCa2.1.3.Met significantly reduced the transmembrane I_SKK current density and inhibited the prolonged action potential duration?APD?in atrial myocytes of GK rats.In addition,the protein subtype of KCa2.2 plays a leading role in the transmembrane currents of the SK channels.