The Influence And Its Mechanism Of MicroRNA-206 On Intrinsic Cardiac Autonomic Nerve Remodeling In Canines With Experimental Atrial Fibrillation

Atrial fibrillation (AF) is a highly prevalent disease associated with pronounced morbidity and mortality, which affects the health and life quality of patients seriously, but the mechanism is still not very clear. It is found that atrial structural remodeling and electrical remodeling play important roles in AF. Accumulating evidences have demonstrated that cardiac ANR is involved in the pathogenesis of AF. Cardiac nervous innervation involves the extrinsic and intrinsic cardiac autonomic nervous systems (ANS). The former collectively includes the ganglia in the brain and along the spinal cord and their axons. The latter consists of cardiac ganglionated plexuses (GPs), which are usually embedded in the homologous fat pads (FPs), including the superior left GP (SLGP), inferior left GP (ILGP), the anterior right GP (ARGP), and the inferior right GP (IRGP). Nerve regeneration in GPs is more intensive than other places in the atrium, but the biological mechanism of autonomic nerve remodeling (ANR) around the GPs has remained incompletely clarified.MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs. MiRNAs have been demonstrated to participate in many essential biological events and to be associated with the pathogenesis of various diseases, such as neurodegenerative diseases, metabolic diseases and cardiovascular diseases. Additionally, it is reported that miRNAs may be involved in the associated atrial structural and electrical remodeling in AF and in the pathogenesis of nervous systems.Although the studies referenced above have provided promising evidence in support of the role of miRNAs in extrinsic cardiac ANR, whether the expression of miRNAs is dysregulated during intrinsic cardiac ANR is unclear. In addition, the underlying molecular mechanisms in AF and ANR remain unknown. This study aimed to clarify the cardiac ANR in canine LSFP in AF models, and illustrate the underlying mechanism of ANR from miRNA angle, including two parts:Part Ⅰ:Expression profile of microRNAs associated with intrinsic cardiac autonomic nerve remodeling in canines with experimental atrial fibrillationObjectivesAccumulating evidence has demonstrated that cardiac autonomic nerve remodeling (ANR) is involved in the pathogenesis of atrial fibrillation (AF). However, the expressions and the roles of miRNAs on autonomic nerve remodeling in AF is still unclear. This study was designed to investigate whether the ANR existed at the SLGP in right atrial pacing canine model and to identify miRNAs in SLFPs by high-throughput sequencing assays.Materials and Methods1. Canine Models of AFAdult mongrel dogs of either sex were randomly divided into two groups:the control group (control, n= 6) and the atrial tachypacing group (A-TP, n= 6). The dogs were subjected to continuous A-TP at 400 bpm/min for 4 weeks in the A-TP group.2. Detection of AERPThe atrial effective refractory period (AERP) was measured in the posterior wall of left atrium with S1-S2 programmed electrical stimulation (PES). AF was induced by PES with burst stimulation.3. Immunohistochemical AnalysisThe nerve markers, including tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT), were stained. Nerve densities, were determined in control and A-TP groups.4. miRNA Expression ProfilingThe miRNAs were sequenced with high-throughput sequencing systems. To identify miRNAs in canine LSFPs, genome-wide comparison was conducted with all of the canine miRNAs in the miRBase database. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to quantify miRNA levels.ResultsCompared to the lenti-control group, AERP were significantly shortened, and immunohistochemical staining showed that the densities of TH-and ChAT-positive nerves in the LSFPs were all significantly increased in A-TP dogs. A total of 241 miRNAs were found to be expressed, of which 16 miRNAs were found significantly differentially expressed between the two groups. Among them,6 miRNAs were significantly upregulated, and 10 miRNAs were downregulated in the A-TP group. qRT-PCR analysis showed that the expression of miR-206, miR-224, miR-137 and miR-203 were all significantly dysregulated in A-TP tissues.ConclusionAutonomic neural remodeling existed at SLGP in rapid right atrial pacing canine models. Plenty of miRNAs were found to be dysregulated in LSFP of AF.Part Ⅱ:MicroRNA-206 accelerates cardiac autonomic nerve remodeling by regulating SOD1 in atrial fibrillationObjectivesPrior studies showed that miR-206 was expressed in the central nervous system and peripheral nervous system specifically, and accelerated nerve regeneration. The role of miR-206 on intrinsic cardiac autonomic nervous system is still unknown. We selected miR-206, which was differently expressed, to clarify its influence on the nerve remodeling and to reveal its molecular mechanism of miR-206 on target gene and downstream pathways.Materials and Methods1. Grouping and Infection of LentivirusesFor the in vivo studies,30 mongrel dogs of either sex were randomly divided into 5 groups:1) the lenti-control group (n= 6):control lentiviruses were injected into LSFPs without A-TP for 2 weeks; 2) A-TP group (n=6):atrial tachypacing for 4 weeks; 3) the A-TP+lenti-miR-206 group (n= 6):miR-206 lentiviruses were injected into LSFPs after A-TP for 2 weeks; 4) the A-TP+lenti-anti-miR-206 group (n= 6): anti-miR-206 lentiviruses were injected into LSFPs after A-TP for 2 weeks; 5) the lenti-miR-206 group (n= 6):miR-206 lentiviruses were injected into LSFPs for 2 weeks without A-TP.In vitro, primary canine myocardial cells from< 0.5 cm around the LSFPs were isolated from the control canine myocardium. Canine myocardial cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. Lentivirus of miR-206 overexpression (lenti-miR-206), miR-206 silencing (lenti-anti-miR-206) and superoxide dismutase 1 (SOD1) silencing were used to infect cells for 48 h in different groups.2. Detection of AERP and Inducibility of AFThe AERP was measured in the posterior wall of left atrium with S1-S2 programmed electrical stimulation (PES). AF was induced by PES with burst stimulation. Successful induction of AF was defined as a period of rapid irregular atrial rhythm lasting at least 30 seconds.3. Immunohistochemical Analysis and Western BlotImmunohistochemical Analysis was used to determine the nerve markers (PGP9.5, TH and ChAT). And Western blot analysis were used to determine the protein expression of SOD 1.4. Luciferase AssaysLuciferase vectors with wild-type (WT) SOD1 full-length 3’UTR (pGL3-SOD1-UTR-WT) and SOD1 full-length 3’UTR with a miR-206 binding site deletion (pGL3-SOD1-UTR-Del) were constructed. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System.5. ROS Generation AssayThe levels of reactive oxygen species (ROS) were determined using the ROS-sensitive dye 2’,7’-dichlorofluorescein diacetate (DCF-DA). The level of DCF fluorescence, which reflects the concentration of ROS, was measured.6. Microarray Analysis of miR-206-affected GenesMicroarray-based mRNA expression profiling was performed in the miR-206-overexpressed and lenti-control groups. The validation was performed by qRT-PCR in all canines.Results1. Forced Expression of miR-206 Promoted the Occurrence of Atrial ANR and the Inducibility of AF(1) miR-206 Influenced AERP and AF InducibilityAfter 4 weeks of A-TP, the AERP in LSFPs of the atrium was obviously shorter in the A-TP group, and it was even shorter in the A-TP plus lenti-miR-206 group. In contrast, dogs treated with A-TP plus lenti-anti-miR-206 showed a significantly longer AERP than that in the A-TP group. After 4 weeks, AF was induced in 3 of 6 animals in the A-TP group, while AF did not occur in the lenti-control group. Furthermore, AF inducibility was increased to 5 of 6 in the A-TP plus lenti-miR-206 group and decreased to 1 of 6 in the A-TP plus lenti-anti-miR-206 group.(2) miR-206 Modulated ANRImmunohistochemical staining showed that the densities of PGP9.5-, TH- and ChAT-positive nerves in the SLGPs were all significantly increased in A-TP dogs and lenti-miR-206 infected group. However, the opposite results were obtained in the A-TP in combination with lenti-anti-miR-206 group.(3) SOD1 Was a Direct Target of miR-206Overexpression of miR-206 caused a substantial reduction of luciferase activity in pGL3-SOD1-UTR-WT-transfected cells. This decrease was prevented by the deletion of the predicted miR-206 binding site in the SOD1 3’UTR.(4) miR-206 downregulated SOD1In vivo results from Western Blot analysis showed that forced expression of miR-206 repressed the expression of SOD1. The protein level of SOD1 in A-TP dogs was downregulated to approximately 39% of that in the lenti-control group, and the level of SOD1 in the A-TP plus lenti-miR-206 group decreased to approximately 34% of that in the A-TP tissues.(5) miR-206 regulated ROS via SOD1In vitro, infection of lenti-miR-206 increased the baseline level of cellular ROS by-2-fold. The infection of lenti-anti-miR-206 significantly reduced the level of radiation-induced ROS to 50%. In vivo, the ROS level of lenti-miR-206-infected dogs was-1.6-fold of the lenti-control dogs. Additionally, the level of ROS was significantly increased in the A-TP in combination with lenti-miR-206 group compared to the A-TP dogs, and it was efficiently reversed by infection with lenti-anti-miR-206. SOD1 knockdown in myocardial cells resulted in upregulation of ROS levels. Lenti-anti-miR-206-mediated ROS reduction was reversed by SOD1 silencing, which verified that miR-206 regulated ROS levels by target-SOD1.2. miR-206 modulated mRNA expressionThe microarray profiling analysis demonstrated that the expression levels of 1254 mRNAs were differentially expressed by 2-fold or more in the lenti-miR-206 group. The GO and KEGG pathway analysis indicated that the genes affected by forced expression of miR-206 were enriched in many pathways involved the nervous system and cardiovascular system.ConclusionOur results showed that miR-206 overexpression induced cardiac ANR. The underlying mechanism was that miR-206 increased the production of ROS by targeting SOD1 and modulated multiple downstream pathways.