| Objective:Type 2 diabetes mellitus(T2DM)is a complex,heterogeneous polygenic disease characterized by hyperglycemia and insulin resistance.Currently,T2 DM is prevalent worldwide and causes a variety of metabolic disorders and cardiovascular complications such as arrhythmias,cardiomyopathy,coronary atherosclerosis and heart failure and increases the risk of atrial arrhythmias such as atrial fibrillation(AF).Prevention and treatment of T2DM-related cardiovascular complications are urgent clinical issues that must be addressed.AF is a complex functional disorder associated with electrophysiological changes.It is one of the primary T2DM-related complications that increases mortality.Currently,it is believed that atrial ion channel remodeling is the primary cause of AF.However,the mechanism of remodeling is complex,and how diabetes induces AF remains unknown.As a result,treating AF is difficult.Therefore,a complete understanding of the mechanisms underlying the remodeling of various types of ion channels in diabetic atria is of great significance to the treatment of diabetes-induced arrhythmias.Several studies show that metformin,a first-line drug for diabetes management,reduces the risk of AF in patients with diabetes by improving glucose and lipid metabolism and reducing insulin resistance.In addition,metformin reduces oxidative stress and inflammatory responses independent of its hypoglycemic effects,but the molecular mechanisms are not fully understood.Studies have shown that atrial ion channel remodeling is the basis of AF.However,few studies have investigated the effect of metformin on the remodeling of atrial ion channels in DM,and the molecular mechanism remain unclear.Recent studies have shown that the remodeling of small conductance calcium-activated potassium channels(SK channels)is closely associated with AF.SK channels are widely distributed in the myocardial cell membrane and are involved in the initiation of action potential.They play an important role in the regulation of myocardial depolarization and the maintenance of normal cardiac electrical activity.We previously found that metformin regulates the ion current conducted by SK channels in the atria of rats with T2 DM as well as the mRNA and protein expression of the SK2 and SK3 subtypes of SK channels;however,the underlying signaling mechanisms remain unclear.The protein kinase C(PKC)/extracellular signal-regulated kinase(ERK)signaling pathway and the nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)/ p38 mitogen-activated protein kinase(p38MAPK)signaling pathway,which are extensively expressed throughout the cardiovascular system,plays an important role in the regulation of cardiac electrophysiology and function.Diabetes-induced hyperglycemia activates the PKC/ERK signaling pathway and the NOX4/p38 MAPK signaling pathway in the myocardium and promotes the release of various inflammatory factors,and eventually leading to bodily injury.Studies have shown that metformin inhibits the activation of both the signaling pathways and blocks the release of inflammatory mediators in vivo,thereby exerting a protective effect.However,whether the two signaling pathways participate in the regulation of SK2 and SK3 channels in the atria of T2 DM rats and whether metformin regulates the expression of SK2 and SK3 channel proteins through the two signaling pathways must be further investigated.The purpose of this study was to determine whether metformin regulates atrial SK2 and SK3 proteins in T2 DM rats via the the PKC/ERK signaling pathway and the NOX4/p38 MAPK signaling pathway to provide a theoretical basis for a mechanism of atrial ion channel remodeling in DM and the prevention and treatment of diabetes-induced AF.Methods: A rat model of T2 DM was established through a combination of high-fat,high-sugar diet administration and intraperitoneal injections of small doses of streptozotocin(STZ)using healthy male Wistar rats.Four weeks later(after overnight fasting),blood samples were collected by tail snipping.Fasting blood glucose(FBG)and fasting insulin(FINS)were measured,and the insulin sensitivity index(ISI)was calculated.In addition,an intraperitoneal glucose tolerance test was conducted.Rats with an FBG ? 11.1 mmol/L and a decreased ISI were selected(the model group)and used in the subsequent assays.Meanwhile,the control group(n = 8)was intraperitoneally injected with an equal volume of saline.Blood samples were then collected,and FBG and FINS were measured.All rat models were randomly divided into two groups: the untreated T2 DM group(DM,n = 8)and the metformin group(Met).The Met group was given 300 mg/kg/d metformin(via gavage)for 8 weeks,while the Con and the DM groups were given an equal volume of saline(via gavage)for 8 weeks.During the last two weeks,the Met group was further randomly divided into the following groups: the metformin-treated group(Met,n = 8),the group receiving intraperitoneal injections of the PKC agonist phorbol 12-myristate 13-acetate group(PMA,n = 8),the group receiving tail vein injections of the ERK agonist recombinant human epidermal growth factor(rh-EGF,n = 8),the group receiving subcutaneous injections of the nicotinamide adenine dinucleotide phosphate oxidase(NOX)inhibitor diphenyleneiodonium(DPI,n = 8),and the group receiving tail vein injections of the p38 MAPK agonist anisomycin(AN,n = 8).The PMA group rats were given intraperitoneal injections of 2 ?g/kg PMA in dimethyl sulfoxide(DMSO)every other day;the EGF group was given tail vein injections of 10 ?g/kg/day rh-EGF in distilled water;the DPI group was received subcutaneous injections of 1mg/kg/d DPI in DMSO;the AN group was received tail vein injections of 5 mg/kg anisomycin in sterile phosphate-buffered saline every other day;and the Con,DM,and Met groups were injected with an equal volume of liquid.Finally,the rats were sacrificed and atrial tissues were rapidly isolated.Adipose tissues and connective tissues were carefully removed.The activity of PKC in the atrial tissues of the related rat groups was detected by enzyme-linked immuno sorbent assay(ELISA)method using a PKC kinase activity assay kit.Real-time quantitative polymerase chain reaction(RT-PCR)was performed to examine the expression levels of SK2,SK3,NOX4,p38 MAPK and ERK1/2 mRNAs in the atria of relevant groups.Western blot was applied to examine the expression levels of SK2,SK3,NOX4,phospho-ERK(pERK),and phospho-p38MAPK(p-p38MAPK)proteins in the relevant groups.Immunohistochemistry(IHC)was applied to examine the expression levels of SK2 and SK3 proteins in the relevant groups.Results: 1.A stable rat model of T2 DM,characterized by typical metabolic disorders of T2 DM,was induced by a combination of 8-week high-fat,high-sugar diet administration and intraperitoneal injections of small doses of STZ(30mg/kg).We observed that the body weights(BW)significantly decreased(P < 0.01),the FBG and FINS significantly increased(P < 0.01),and the calculated ISI values significantly decreased(P < 0.01)in diabetic rats compared with those of the rats in the Con group.We also observed a significant elevation of blood glucose levels during the intraperitoneal glucose tolerance test in diabetic rats(P < 0.01).These results indicated that the diabetic rats had typical characteristics of T2 DM,such as blood glucose elevation,glucose tolerance impairment,and insulin resistance.2.The mRNA levels of SK2,SK3,NOX4,p38 MAPK and ERK1/2 in the atrial tissues of the Con,DM and Met groups were detected by RT-PCR method.Compared with the control group,the rats with T2 DM exhibited a decreased SK2 mRNA expression(P < 0.01)and an increased expression of SK3 and NOX4 mRNAs in the atria(P < 0.01)while showing no change in p38 MAPK and ERK1/2 mRNA expression.SK2 mRNA expression was significantly upregulated and SK3 and NOX4 mRNA expression was significantly downregulated after 8 weeks of metformin treatment(P < 0.01).3.The PKC activity in the atrial tissues of the Con,DM,Met and PMA groups were detected by ELISA.The expression of SK2,SK3 and pERK proteins in the atrial tissues of the Con,DM,Met,PMA and EGF groups were detected by Western blot and IHC.Compared with the Control group,atrial PKC activity and pERK and SK3 protein expression were significantly increased(P < 0.01),while SK2 protein expression was decreased in atrial tissues of T2 DM rats(P < 0.01).Eight weeks of metformin treatment inhibited the PKC activity(P < 0.01)and pERK and SK3 protein expression(P < 0.01),while elevated SK2 protein expression(P < 0.01)compared with the T2 DM group.Compared with the Met group,the injection of rh-EGF increased pERK and SK3 protein expression(P < 0.01),and decreased SK2 protein expression(P < 0.01);the injection of PMA increased PKC activity(P< 0.01)and SK3 protein expression(P< 0.01),and decreased SK2 protein expression(P < 0.01).In addition,the injection with PMA significantly elevated the expression of pERK(P < 0.01).4.The expression of SK2,SK3,NOX4 and p-p38 MAPK proteins in the atrial tissues of the Con,DM,Met,DPI and AN groups were detected by Western blot and IHC.Compared with the control group,the expression levels of NOX4 and p-p38 MAPK proteins were significantly elevated in the atria of rats with T2DM(P < 0.01).In addition,SK2 protein expression was reduced(P < 0.01),while SK3 protein expression was increased(P < 0.01)in the atria of T2 DM rats.The 8-week treatment with metformin markedly reduced the expression levels of NOX4 and p-p38 MAPK proteins(P < 0.01),upregulated the protein expression of SK2(P < 0.01)and downregulated the protein expression of SK3(P < 0.01).Compared with the Met group,tail vein injection with the p38 MAPK agonist anisomycin significantly increased the phosphorylation of p38MAPK(P < 0.01)while further inhibiting the expression of SK2 protein(P < 0.01)and enhancing the expression of SK3 protein(P < 0.01).Subcutaneous injection with the NOX inhibitor DPI considerably inhibited the expression of NOX4 protein(P < 0.01),further enhanced the expression of SK2 protein(P < 0.01)and suppressed the expression of SK3 protein(P< 0.01).In addition,subcutaneous injection with DPI markedly suppressed the phosphorylation of p38MAPK(P < 0.01).Conclusion: 1.Atrial SK2 channel protein is down-regulated and SK3 channel protein is up-regulated in T2 DM rats,and metformin reversely regulates the abnormal expression of SK2 and SK3 channel proteins.2.The PKC/ERK and the NOX4/p38 MAPK signaling pathways mediates the downregulation of SK2 protein and the upregulation of SK3 protein in the atria of rats with T2 DM.3.Long-term metformin treatment may partially restore the remodeling of SK2 and SK3 ion channels by inhibiting the PKC/ERK and the NOX4/p38 MAPK signaling pathways. |
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