| [Background]Atrial fibrillation (AF), characterized by atrial rhythm disturbance, is the most common cardiac supraventricular tachyarrhythmias. The prevalence of AF is 0.4%-1%, and increases with age. Stroke and heart failure are two major hazards of AF, which are caused by embolus detachment and rapid ventricular rates, respectively. The overall mortality rate of AF patients is twice as high as that of healthy people. Although methods for treatment of AF, such as drugs, surgery and radiofrequency catheter ablation, have undergone continuous development, the therapeutic effects are less than satisfactory. The reasons are that the exact mechanisms of AF are still unknown. After atrial electrical and structure remodeling, autonomic neural remodeling (ANR) has been demonstrated to be involved in the pathogenesis of AF. ANR, defined as nerve regeneration and uneven distribution, acts as an important factor in the initiation and perpetuation of AF. Meanwhile, the occurrence of AF exacerbates the remodeling of the autonomic nerves. Therefore, ANR and AF are closely linked as cause-and-effect, which eventually develops into a vicious cycle. Stopping or even reversing the ANR may become a novel therapeutic approach for the treatment of AF. However, molecular mechanisms of cardiac ANR during AF remain elusive, which should be addressed by further studies.Long non-coding RNA (lncRNA) is a class of non-protein coding transcripts with length longer than 200 nucleotides. Albeit many lncRNAs are similar to mRNAs, such as 5’capped, spliced and polyadenylated, they lack open reading frames. Besides, lncRNAs usually exhibit a spatially and temporally restricted expression pattern. It has been found that lncRNAs can control gene expression through multiple mechanisms, including chromatin remodeling, transcriptional regulation, post-transcriptional assembly. In addition, lncRNAs act as master regulators in multiple biological processes by directly binding with proteins, DNAs and RNAs. Because of their strong specificity and diverse functions, lncRNAs, currently, have been emerging as a new research hotspot. lncRNAs have been implicated in the development of central and peripheral nervous systems and are associated with the pathogenesis of cardiovascular disease. Specifically, lncRNAs are involved in not only neuronal differentiation, brain development and synaptic plasticity, but also the occurrence and development of neurodegenerative disorders. Simultaneously, lncRNAs can modulate the pathogenic process of cardiovascular diseases, such as myocardial infarction, heart failure, dilated cardiomyopathy, hypertension, et al. These results support that lncRNAs may play important roles in the process of cardiac ANR during AF. But as to whether the expressions of lncRNAs are dysregulated and their roles in cardiac ANR during AF, no systematic study has been carried out.[Aims]The aim of our study is to establish canine AF models by right atrial tachypacing; characterize the lncRNA expression profiles in AF and non-AF canine anterior right fat pads (ARFPs) through high throughput sequencing technique; identify AF-related lncRNAs using a series of bioinformatics methods; investigate the influence of identified lncRNAs on canine AF and cardiac ANR by both in vitro and in vivo experiments; further illustrate the potential mechanisms of lncRNA-032546 and lncRNA-026102 in AF neural remodeling, thereby better elucidating the mechanisms of cardiac ANR of AF from the perspective of lncRNAs and providing novel targets for treatment of AF.[Methods]1. Establishment canine AF models6 adult beagles of either sex were randomly divided into two groups, the AF group (n= 3) and the control group (n=3). The endocardial electrode leads were inserted into the right atrial appendage in the two groups. The AF group underwent continuous atrial tachypacing (400 beats/min) for four weeks, while the control group was in the same way as the AF group but without pacing. Surface ECGs were recorded at least once a week to examine the occurrence of AF.2. Validation the occurrence of ANRARFPs, including the underlying muscles, were collected from all animals. Immunocytochemical staining was performed for protein gene product 9.5 (PGP 9.5), and the nerve density was quantified to valid the occurrence of ANR mediated by AF.3. High-throughput lncRNA sequencing and verificationTotal RNAs were isolated from each sample by using TRIzol reagent. Aberrantly expressed lncRNAs and mRNAs were examined through the next generation sequencing. Some novel transcripts were randomly selected and their expression levels were analyzed by real-time quantitative PCR (qRT-PCR) to validate the sequencing results.4. Identification of candidate lncRNAsDifferentially expressed genes were categorized and annotated by Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Based on a series of filtering pipelines, such as fold change, tissue specificity, target gene prediction and document retrieval, ANR-related lncRNAs, namely, lncRNA-032546 and lncRNA-026102, were selected.5. Loss of function experiments1) Infection of lentiviruses in vivoIn vitro, a scrambled shRNA and specific shRNAs targeting lncRNA-032546 and lncRNA-026102 were designed and the lentiviruses against specific shRNAs were constructed with a titer of 1×109 TU/ml. Fifteen adult beagle dogs of either sex were randomly allocated into three groups (n=5) according to infection with different lentiviruses. All animals were subjected to right thoracotomy. Subsequently, mixed lentiviruses were injected into ARFPs in vivo. Then, the chest was closed. And all animals were sacrificed 10 days after infection. Efficacy of transfection was assessed by immunofluorescence assay.2) Electrophysiological measurementsProgrammed electrical stimulation was performed to analyze the atrial effective refractory periods (AERPs) and the AF inducibilities after thoracotomy (before infection), immediately after infection and 10 days after infection, respectively. The catheter was placed in the right atrium tissue located< 0.5 cm around the ARFPs.3) Molecular biological assayThe ARFPs in control and transfection groups were used for detection of multiple indexes. Briefly, the immunocytochemical staining for PGP 9.5 was performed and the nerve density was determined to analyze the protein level of PGP 9.5. Besides, the qRT-PCR against PGP 9.5 and growth-associated protein 43 (GAP 43) were implemented to determine the mRNA expression levels of PGP 9.5 and GAP 43.6. Potential mechanisms of IncRNAs1) Cis predictionIncRNAs were categorized. According to cis mechanism, we searched for the nearby genes of the two IncRNAs, which reside within 300 kb of their locus. The expression levels of the neighboring protein-coding genes were examined by qRT-PCR. Combined with literature analysis, the relationships between IncRNAs and mRNAs were clarified.2) Trans predictionAiming at the given lncRNA, its co-expressed mRNAs were calculated. And based on Pearson correlation and P value, we selected the top 100 ranked mRNAs. Then we carried out a functional enrichment analysis in these co-expressed mRNAs. The enriched functional terms were considered as the trans mechanisms of the specific lncRNA. The lncRNA-related signal pathways were investigated through literature analysis. The pathway was further validated by examining the expression of key molecules through qRT-PCR.[Results]1. Canine AF models were successfully built by right atrial tachypacing. Compared with the control group, the density of the PGP 9.5-positive neurons was significantly elevated in the AF group.2. In total,61616 putative lncRNAs were acquired through high-throughout lncRNA sequencing. Based on a series of filter criteria,1164 candidate lncRNAs were yielded. Among them,576 transcripts displayed more than two-fold change, in which 410 transcripts were up-regulated and 166 were down-regulated. Besides,45 transcripts were identified as novel lncRNAs. Subsequently,6 new lncRNAs were randomly selected. And the sequencing results were confirmed to be reliable by validating the expression trends of the 6 novel transcripts through qRT-PCR.3. GO functional and KEGG pathway analysis indicated that the dysregulated genes were mainly associated with nervous system development, cell migration, and neurodegenerative diseases. According to fold change, the functions of target genes, and tissue specificity, we selected two novel down-regulated lncRNAs, namely, lncRNA-032546 and lncRNA-026102, which were associated with AF neural remodeling.4. We validated the transfection efficacy by immunofluorescence assay and the knockdown efficacy was examined via qRT-PCR. The cardiac electrophysiological results showed that the AERPs in ARFPs had no statistical significance before and immediately after infection. However,10 days after transfection, repressing the expression of lncRNA-032546 significantly shortened the AERP, correspondingly, paroxysmal atrial tachycardia and AF could be easily induced. While down-regulated lncRNA-026102 could significantly prolong the AERP and no paroxysmal atrial tachycardia or AF induction was observed.5. Immunohistochemistry results of PGP 9.5 demonstrated that the nerve density in the down-regulated lncRNA-032546 group was significantly higher than that in the control group. However, the result was opposite to that in the lenti-RNAi-lncRNA-026102 group. The qRT-PCR results indicated that the expression trends of GAP 43 were similar to those of immunohistochemical staining results. With regard to PGP 9.5, silencing of lncRNA-032546 could significantly increase its mRNA level, while there was no significant difference between the lenti-RNAi-lncRNA-026102 and control groups.6. Both lncRNA-032546 and lncRNA-026102 belong to intergenic lncRNAs, which could control the expression of the nearby genes through cis regulation. Thus, based on cis prediction, the mRNAs residing within 300 kb of the two lncRNAs were identified. And qRT-PCR results indicated that the expression levels of CCND1-FGF19-FGF4-FGF3 gene cluster and SLC25A4 were significantly elevated in the lenti-RNAi-lncRNA-032546 and lenti-RNAi-lncRNA-026102 groups, respectively. Then, CCND1-FGF19-FGF4-FGF3 gene cluster and SLC25A4 were confirmed to be associated with neural remodeling by document retrieval. Furthermore, based on bioinformatics, the co-expression coefficients were calculated between lncRNAs and their corresponding neighboring genes. On the basis of the above results, we speculated that CCND1-FGF19-FGF4-FGF3/SLC25A4 was the potential target gene of lncRNA-032546/lncRNA-026102, respectively.7. According to trans prediction, the top 100 ranked co-expressed mRNAs of the two lncRNAs were found out. Then, GO functional analysis and KEGG pathway analysis were performed in this set of mRNAs. The studies suggested that mitogen-activated protein kinase (MAPK) pathway is tightly linked with CCND1-FGF19-FGF4-FGF3 gene cluster in neurogenesis. And the qRT-PCR results confirmed that the expression levels of ERK1, ERK2, JNK, and p38 were higher in the lenti-RNAi-lncRNA-032546 group than control group, which suggested that lncRNA-032546-induced neural remodeling may be ascribed to the activation of MAPK pathway. Meanwhile, NF-kappa B pathway plays crucial roles in nervous growth and development. Apoptosis could be induced by overexpression of SLC25A4 through recruiting of NF-kappa B. lncRNA-026102 knockdown lead to both the up-regulation of SLC25A4 and the down-regulation of nerve density. Therefore, we predicted that the biological effect of lncRNA-026102 might be attributed to an increased expression of SLC25 A4 through the NF-kappa B pathway.[Conclusions]1. We identified a series of abberantly expressed lncRNAs in ARFPs of AF and non-AF canines. The differentially expressed lncRNAs were involved in the development of AF neural remodeling.2. Two novel lncRNAs, namely, lncRNA-032546 and lncRNA-026102, were identified to be related to neural remodeling. Based on loss of function experiments, we found that lncRNA-032546 promoted the neurogenesis, which in turn facilitated the occurrence of AF, while lncRNA-026102 exhibited an opposite result.3. We predicted the mechanisms of the two lncRNAs. It was suggested that the biological effects of lncRNA-032546 were mediated by controlling the expression of CCND1-FGF19-FGF4-FGF3 gene cluster through cis regulation and modulating MAPK pathway through trans regulation. While, lncRNA-026102 might act in cis to regulate the expression of SLC25 A4 and act in trans to regulate NF-kappa B pathway.[Novelty]1. From the perspective of lncRNAs, we investigated the effects of their different levels on cardiac ANR of AF.2. The molecular mechanisms of cardiac ANR of AF were explored by illustrating lncRNA-regulated nearby genes as well as signal pathways via cis and trans prediction.3. The influence of lncRNAs on canine AF and cardiac ANR and their potential mechanisms were investigated by using lentivirus transfection in vivo. |
PDF Full Text Request