Effects Of GCH1 On Atrial Autonomic Nerve Remodeling Through Regulating Tetrahydrobiopterin In Atrial-tachypacing Canines

Background and objectivesAtrial fibrillation(AF)is a common arrhythmia in clinical practice with high incidence,high morbidity and poor efficacy,which seriously affects patients' health and quality of life.Currently,treatments of AF include drug therapy,surgery and catheter ablation.But the mechanisms of AF has not yet been fully elucidated,which leads to poor therapeutic effects in clinical.Atrial remodeling is a key mechanism to induce AF,including structural remodeling,electrical remodeling and nerve remodeling.Cardiac autonomic nerve remodeling(ANR)is an important factor in triggering and maintaining AF.Autonomic nervous system(ANS)includes extrinsic ANS and intrinsic ANS.Intrinsic ANS is composed of ganglionated plexi(GPs)and a network of interconnecting neurons.ANR contributed to the development of AF and AF development exacerbated ANR.However,the mechanism of ANR in the initiation and maintenance of AF has not been fully illustrated yet.MicroRNAs(miRNAs)are a group of small noncoding RNAs which affects gene expression by post transcriptional regulation.In recent years,there has been a new understanding of the role of miRNAs in the remodeling of AF.Previous study has shown that miR-206 can promote ANR by regulating the expression of superoxide dismutase,which illustrates the important role of miR-206 in ANR of AF.However,whether miR-206 can affect ANR through other mechanisms has not been systematically investigated yet.Guanosine triphosphate cyclohydrolase I(GTPCH I),encoded by guanosine cyclohydrolase 1(GCH1)is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin(BH4).BH4 is an essential co-factor for nitric oxide(NO)generation.Previous studies have found that in animal models of AF,myocardial BH4 content significantly decreased,while exogenous BH4 supplement can reduce the incidence of AF,which proved that BH4 was involved in the pathogenesis of AF.However,there is no study about the role of BH4 pathway in ANR of AF.In this study,canine models of AF was established to investigate GCH1 expression changes in atrial tissues and the effect of miR-206 on GCH1/BH4 pathway.This study aims to provide new ideas for the study of ANR by clarifying whether miR-206 influences ANR by regulating the GCH1/BH4 pathway.Methods and Methods1.canine models of AF24 mongrel dogs weighing 10-20 kg,of either sex,were allocated into four groups:the atrial-tachypacing group(A-TP,n = 6),the A-TP+ miR-206 over-expression group(n = 6),the A-TP+ lenti-anti-miR-206 group(n = 6)and the control group(n = 6).The first three groups underwent continuous right A-TP(400 beats/min)for 4 weeks.In the control group,the dogs were sham-operated in the same way but without pacing.After 4 weeks of A-TP,dogs of the first three groups were injected with corresponding lentivirus into right upper fat pad(RSFP)while the control group canines were killed and atrial tissues were harvested from<0.5cm around RSFP.2.Construction of LentivirusesThe miR-206 over-expression lentiviruses(lenti-miR-206),miR-206 silencing lentiviruses(lenti-anti-miR-206),GCH1 over-expression lentiviruses(lenti-GCH1),and GCH1 silencing lentiviruses(lenti-anti-GCH1)were synthesized.3.Infection of LentivirusesAnother 24 dogs were randomly divided into four groups:the miR-206 over-expression group,the lenti-anti-miR-206 group,the GCH1 over-expression group and the lenti-anti-GCH1 group while each group contains 6 canines.Open chest and lentiviruses were directly injected into the RSFP.After 2 weeks of infection,atrial samples were collected.Cells from<0.5cm around RSFP were cultured and transfected with different lentivirus,including lenti-miR-206,lenti-anti-miR-206,lenti-GCH1,lenti-anti-GCH1,lenti-control and lenti-RNAi-NC.4.Detection of atrial effective refractory period(AERP)AERP in the miR-206 over-expression group,lenti-anti-miR-206 group,GCH1 over-expression group and lenti-anti-GCH1 group were determined before infection and 2 weeks after infection.The S1-S2 intervals started at 150ms,with a decrements of 5 ms(S1:S2 = 8:1).AERP was defined as the longest SI-S2 interval which can't produce a propagated atrial response.5.Quantitative real-time polymerase chain reaction(qRT-PCR)qRT-PCR was conducted to detect mRNA expressions of GCH1 and protein gene product 9.5(PGP9.5)in myocardial tissues and cells after A-TP and lentivirus infection.6.Immunohistochemical AnalysisThe expression of PGP9.5 protein in the GCH1 over-expression group,the control group and the GCH1 group were measured by immunohistochemical staining.7.Western blot analysisDetection of GCH1 and PGP9.5 protein expression changes in myocardial tissues and cells after A-TP and lentivirus infection by Western blot.8.Luciferase AssaysGCH1 was a direct target of miR-206 validated by Luciferase Assays.9.Measurement of NO production in RSFPNO content was measured using nitric reductase method.The optical density values of the tissues were measured at 550 nm on a spectrophotometer.10.Measurement of BH4 content in RSFPMyocardial BH4 content was measured with the ELISA kit instructions.The optical density values of the samples were measured at 450 nm on a spectrophotometer.Results1.The expression of PGP9.5 mRNA and protein content were significantly up-regulated in the A-TP group and the miR-206 over-expression group.While the increase was most significant in the ATP+lenti-miR-206 group.But in miR-206 silence group,PGP9.5 content decreased significantly.2.AERP in the miR-206 over-expression in group was significantly shortened,while AERP in the lenti-anti-miR-206 group was prolonged.AERP was obviously longer in the lenti-GCH1 group than the control group.In contrast,AERP in lenti-anti-GCH1 group were found decreased.3.Luciferase Assays verified that GCH1 is the target gene of miR-206.The GCH1mRNA and protein levels in miR-206 over-expression group were decreased,while the expression of GCH1 increased in the lenti-anti-miR-206 group.4.The content of BH4 and NO in the miR-206 over-expression group were decreased,while the BH4 and NO levels in the lenti-anti-miR-206 group were significantly higher.The contents of BH4 and NO were significantly increased in GCH1 over-expression group,while lenti-anti-GCH1 reduced the content of BH4 and NO.5.The mRNA expression,protein levels and nerve density of PGP9.5 were significantly decreased in the GCH1 over-expression group and decreased in the lenti-anti-GCH1 group.ConclusionsMiR-206 promotes ANR of AF by regulating GCH1/BH4 pathway.Over-expression of GCH1 can inhibit ANR development and the initiation of atrial fibrillation.