| BackgroundAtrial fibrillation（AF）is one of the most common arrhythmias in clinical practice.The morbidity rate of AF is high and increases with age.The total morbidity rate of AF in the world was about 1%-2%,and the number in our country was about 0.77%.AF is associated with stroke and heart failure,resulting in an enormous burden worldwide which is closely related to the high disability and mortality.Treatments for AF mainly include drug therapy,interventional therapy,and Maze procedure.But the therapeutic effect and prognosis of AF is still not satisfactory because of the mechanisms of AF has not been fully elucidated.Previous studies had confirmed that atrial energy metabolic remodeling plays important roles in the initiation and maintenance of AF.Atrial energy metabolic remodeling mainly includes myocardial cell energy metabolism disorder and metabolic pathway changes,for example alterations in adenine nucleotides,mitochondrial function,and a change in substrate.These alterations cause abnormal changes in myocardial physiological function and tissue structure,resulting in the occurrence of AF.LncRNAs are non-protein coding RNAs that longer than 200 nt in length.Previous studies had confirmed that IncRNAs are involved in gene transcription and protein translation.Recent studies revealed that lncRNAs play important roles in cardiac diseases,including heart failure,ventricular septal defect,myocardial infarction,electrical remodeling in AF and autonomic neural remodeling in AF.However,the roles and mechanisms of IncRNAs in atrial energy metabolic remodeling in AF have not been fully illustrated yet.ObjectivesIn this study,right atrial tachypacing was used to establish AF rabbit model;high-throughput sequencing technique（next generation high-throughput sequencing technique）was conducted to detect the IncRNA expression profiles of right atria in non-AF and AF rabbit models;a series of bioinformatics methods were used to to identify the lncRNAs related to atrial energy metabolic remodeling in AF.To investigate the roles of target IncRNA on atrial energy metabolic remodeling and inducibility of AF,we knockdown the target IncRNA.We aimed to clarify its molecular biological mechanism and provide new explanation for the occurrence of AF.Materials and Methods1.Establishment of AF rabbit modelsTwelve healthy adult New Zealand white rabbits,weighing 2.0-2.5 kg,of either sex,were randomly divided into control group（n=6）and AF group（n=6）.Both of the two groups were performed with pacemaker implantation,and the endocardial electrode leads were inserted into the right atria.The AF group underwent continuous right atrial tachypacing（600 beats/min）for one week to establish AF rabbit models,while the control group without pacing.Cardiac electrophysiological tests,including AF inducibility and atrial effective refractory period（AERP）were measured before and one week after.he pacemaker implantation.2.High-throughput sequencing of IncRNAs and bioinformatics analysisTotal RNAs of the right atria from control（n=3）and AF groups（n=3）were extracted with TRIzol reagent.High-throughput sequencing technique was conducted to detect the aberrantly expression profiles of IncRNAs in right atria from control and AF groups.The differentially expressed transcripts（DETs）were analyzed by a series of bioinformatics methods,including Gene Ontology（GO）enrichment analysis,Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis,co-expression genes analysis,tissue specificity analysis,and so on.TCONS00016478 and the target gene PGC-1α were identified to play important roles in atrial energy metabolic remodeling during AF.3.Mechanism prediction of the target lncRNA（1）cis-predictionAccording to cis-mechanisms,the nearby mRNAs reside within 10 kb of the target IncRNA were searched.The quantitative real-time polymerase chain reaction（qRT-PCR）was used to show the expression levels of the target IncRNA and nearby mRNAs,and statistical analysis was used to assesse the gene expression correlation.（2）trans-predictionThe co-expressed mRNAs of the target lncRNA were selected based on Pearson correlation coefficient（PCC）>0.85 and P<0.05,then GO enrichment analysis and KEGG pathway analysis were conducted to predict the trans-mechanisms.The target mRNAs,signal pathways and molecular mechanisms of lncRNA were based on qRT-PCR and literature retrieval.4.Loss-of-function of the target lncRNA（1）Infection of lentiviruses in vitroNegative control shRNAs and shRNAs specifically silencing TCONS 00016478 were constructed with a titer of 1×109 TU/mL.The primary atrial myocytes,extracted from the new born rabbits,were cultured and randomly divided into four groups:blank control group,with no infection;negative control group,infected with negative control lentiviruses;lenti-RNAi-TCONS 00016478 group,infected with lentiviruses for silencing TCONS 00016478;co-infected lentiviruses group,infected with lentiviruses for silencing TCONS00016478 and lentiviruses for over express PGC-1α.The cells were collected 96 h after infection,and would be used for further experiments.（2）Infection of lentiviruses in vivoEighteen adult New Zealand white rabbits of either sex were randomly divided into the sham group（n=6）,performed by sterile thoracotomy merely;negative control group（n=6）,right atria were infected with negative control lentiviruses;and lenti-RNAi-TCONS 00016478 group（n=6）,right atria were infected with lentiviruses for silencing TCONS 00016478.AF inducibility and AERP were measured after thoracotomy and one week after infection.After one week infection,atrial samples were collected for further experiments.5.Molecular biological assayThe gene expression levels of TCONS 00016478,PGC1-α and the downstream genes were determined by qRT-PCR;the protein expression levels of PGC1-α and the downstream genes were determined by Western Blotting.6.Adenine nucleotides determinationThe atrial tissues were cut into pieces and homogenized,then the adenine nucleotides（ATP,ADP and AMP）were extracted and assessed by high performance liquid chromatography（HPLC）.77.Tissue special stainingThe accumulation of glycogens were assessed by PAS staining.The accumulation of lipid droplets were assessed by Oil Red O staining.Results].The AF rabbit models were successfully established.Compared with the contral group,AERP was shortened in AF group,and the AF inducibility was increased.2.According to the high-throughput RNA-Seq,a total of 99843 new transcripts were detected in the right atria of AF group and control group.There are 1220 differentially expressed transcripts（DETs）,in which 237 DETs were up-regulated and 983 DETs were down-regulated.3.A new transcript TCONS00016478 was selected to explore its roles in atrial energy metabolic remodeling after a series of bioinformatics,such as GO enrichment analysis,KEGG pathway analysis,co-expressed genes analysis,target genes prediction,tissue-specific gene expression analysis,literature search,and so on.4.Sequence analysis revealed that TCONS,00016478 and PGC1-α were located on chromosome 2,TCONS00016478 was a downstream gene of PGC-1α,the distance was 1637 bp（<3 kb）.Compared with the control group,the gene expression of TCONS00016478 and PGC-la were both decreased in AF group,the expression of PGC-la had co-expression correlation with TCONS00016478.TCONS00016478 was a cis-regulator to regulate the taeget gene PGC-1α.5.Loss-of-function of TCONS00016478 verified that silencing TCONS00016478 in vitro could inhibit PGC-1α/PPARy pathway genes related to energy metabolism,include PGC1-α,PPARy,GLUT4,and CPT1.Silencing TCONS00016478 and overexpressing PGC1-α,the gene expression levels of PPARγ,GLUT4,and CPT1 were not statistically different from the control group.The results indicated that TCONS00016478 indirectly regulated the expression of PPARy,GLUT4 and CPT1 by regulating PGC1-α.6.Silencing TCONS00016478 in vivo could inhibit PGC-1α/PPARy pathway genes related to energy metabolism,resulting in the glycogens accumulation,lipid droplets accumulation and decrease in adenine nucleotides concentrations.After infection,the AERP was shortened and AF inducibility was increased in TCONS 00016478 silencing group,however,no significant difference in AERP or AF inducibility was found between the sham group and negative control group.ConclusionsTCONS00016478 plays important roles in modulating atrial energy metabolic remodeling in rabbits of AF by regulating PGC-1α/PPARγ pathway.Our study provided new explanation for the occurrence of AF and found new targets for prevention/treatment of AF. |
