MicroRNA‐146b‐5p promotes atrial fibrosis in atrial fibrillation by repressing TIMP4

Abstract Alteration of tissue inhibitors of matrix metalloproteinases (TIMP)/matrix metalloproteinases (MMP) associated with collagen upregulation has an important role in sustained atrial fibrillation (AF). The expression of miR‐146b‐5p, whose the targeted gene is TIMPs, is upregulated in atrial cardiomyocytes during AF. This study was to determine whether miR‐146b‐5p could regulate the gene expression of TIMP4 and the contribution of miRNA to atrial fibrosis in AF. Collagen synthesis was observed after miR‐146b‐5p transfection in human induced pluripotent stem cell‐derived atrial cardiomyocytes (hiPSC‐aCMs)‐fibroblast co‐culture cellular model in vitro. Furthermore, a myocardial infarction (MI) mouse model was used to confirm the protective effect of miR‐146b‐5p downregulation on atrial fibrosis. The expression level of miR‐146b‐5p was upregulated, while the expression level of TIMP4 was downregulated in the fibrotic atrium of canine with AF. miR‐146b‐5p transfection in hiPSC‐aCMs‐fibroblast co‐culture cellular model increased collagen synthesis by regulating TIMP4/MMP9 mediated extracellular matrix proteins synthesis. The inhibition of miR‐146b‐5p expression reduced the phenotypes of cardiac fibrosis in the MI mouse model. Fibrotic marker MMP9, TGFB1 and COL1A1 were significantly downregulated, while TIMP4 was significantly upregulated (at both mRNA and protein levels) by miR‐146b‐5p inhibition in cardiomyocytes of MI heart. We concluded that collagen fibres were accumulated in extracellular space on miR‐146b‐5p overexpressed co‐culture cellular model. Moreover, the cardiac fibrosis induced by MI was attenuated in antagomiR‐146 treated mice by increasing the expression of TIMP4, which indicated that the inhibition of miR‐146b‐5p might become an effective therapeutic approach for preventing atrial fibrosis.


| INTRODUC TI ON
Atrial fibrillation (AF) is the most common arrhythmia with increasing prevalence and indidence over the last decade. 1,2 Electrical and structural remodelling have been implicated in the pathogenesis of AF, and atrial fibrosis is the hallmark of structural remodelling. 3 The fibrotic process is a complex process that involves increased production and activity of a variety of extracellular matrix (ECM) proteins secreted by fibroblasts, including enzymes that modify the ECM such as matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Numerous studies have suggested that an alteration of TIMP/MMP associated with the collagen upregulation plays an important role in progressing sustained AF. [4][5][6][7] MicroRNAs (miRNAs) are endogenous ~23-nt RNAs that can complementarily bind to messenger RNAs (mRNAs) of protein-coding genes and lead to their regulation posttranscriptionally. 8,9 The human miR-146 family has two genes: miRNA-146a and miRNA-146b. These two miRNAs, which located on different chromosomes, have different regulatory functions. 10 Most published studies have focussed on their effect on the immune system or in cancer, while only a few reported their role in cardiovascular disease. [11][12][13][14] In our previous study, 10 differentially expressed miRNAs and 624 differentially expressed mRNAs are identified and 1 miRNA-target gene pair, that is miR-146b-5p and TIMP4 from atrial cardiomyocytes (CMs), was constructed in the left atrial appendage (LAA) from patients with nonvalvular paroxysmal AF (NVPAF). Furthermore, we found that the 3-UTR region of TIMP-4 region has only 1 highly conserved miR-146b-5p binding site, and the ability of miR-146b-5p to repress the expression of TIMP-4 was further verified by Western blotting analysis in mouse cardiac fibroblasts transfected with miR-146b5p. However, the role of miR-146b-5p on atrial fibrosis remains mysterious. 15 This study hypothesized that miR-146b-5p could target the gene and contribute to the balance between TIMP4 and MMP9, which is associated with atrial fibrosis. We verified whether miR-146b-5p upregulation participates in the induction of collagen expression in human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) fibroblast contact co-culture system. Furthermore, a myocardial infarction (MI) mouse model was used to verify the effect of miR-146b-5p downregulation on cardiac fibrosis.

| MATERIAL S AND ME THODS
The expansion of the method section can be found in the Supplemental Materials.

| HiPSC derived atria-like cardiomyocytes
An early conducted research has documented a highly homogenous atrial-like cardiomyocytes (aCMs) obtained from human pluripotent stem cells. 16,17 Details are shown in the Supplemental Materials.

| hiPSC-aCMs-fibroblast contact co-culture and rapid electrical field stimulation
After 24 h, the hiPSC-aCMs and fibroblasts were in the confluent phase and grew in monolayers, while the hiPSC-aCMs were still in spontaneous synchronized contraction. After 6 h of serum-free culture, the cells were moved into an incubator containing an atmosphere of air enriched with 5% CO2 at 37°C ( Figure S1) and a rapid electrical field stimulation (RES) was conducted. Details are available in the Supplemental Materials.

| Animals
All animal studies were done in compliance with the regulations and guidelines of Capital Medical University institutional animal care and conducted according to the AAALAC and the IACUC guidelines. All the C57BL/6 mice (6-8 weeks old, weighing 20-25 g) were housed in an environment with a temperature of 24 ± 1ºC, a relative humidity of 50 ± 1% and a light/dark cycle of 12/12 h and fed with food and water at libitum. Twelve healthy male Beagles, weighing between 8 and 12 kg, were individually housed in stainless steel cages from the time of arrival until euthanasia. Beagle dogs were separately housed in cages at a temperature of 13-16˚C, a relative humidity of 40-70% and a light/dark cycle of 12/12 h and fed with water and high-quality adult dog food (3-5% of its weight) twice a day. fibres were accumulated in extracellular space on miR-146b-5p overexpressed coculture cellular model. Moreover, the cardiac fibrosis induced by MI was attenuated in antagomiR-146 treated mice by increasing the expression of TIMP4, which indicated that the inhibition of miR-146b-5p might become an effective therapeutic approach for preventing atrial fibrosis.

| Canine model of AF
Male Beagles were randomly divided into two groups: sham control (n = 6) and atrial tachypacing (ATP, n = 6) group ( Figure S2). Details are available in the Supplemental Materials.

| Myocardial infarction (MI) wild-type mice
MI was induced in 10-week-old wild-type (WT) C57BL/6 mice by permanent ligation of the left anterior descending coronary artery (LAD). Details are available in the Supplemental Materials.

| Induction of AF in wild-type mice
Intracardiac pacing was performed in wild-type C57BL/6 mice by inserting an eight-electrode catheter (1.1F, octapolar EP catheter, Scisense) through the jugular vein and advancing it into the right atrium and ventricle. The inducibility of atrial arrhythmias was tested by applying 6-s bursts through the catheter electrodes using an automated stimulator that was part of the data acquisition system. The first 6-s burst had a cycle length of 40 ms, decreasing in each successive burst with a 2-ms decrement down to a cycle length of 20 ms. Then, 50 ng/kg carbachol was injected through the inner canthus veniplex. Two minutes later, the same combination of bursts was applied to the animals. Successful induction of AF was defined as a period of rapid, irregular atrial rhythm lasting at least 1 s.

| Statistical analysis
All results were stated as mean ±standard deviation of the mean for continuous variables, and categorical variables were stated as the number of patients and percentage. Differences between the groups were analysed using the Student t test or one-way analysis of variance for continuous variables and the chi-square test for categorical variables. All tests were two-tailed, and a P-value of 0.05 was statistically significant. The data were analysed in GraphPad Prism 8.0 (GraphPad Software Inc.).

| Increased miR-146 expression in a canine AF model induced by atrial tachypacing
Based on the previous results, we decided to examine ATP in AF canine recapitulated atrial fibrosis phenotypes and the increased expression of miR-146b-5p. No spontaneous AF was observed in the sham group. However, a typically induced atrial tachycardia by programmed electrical atrial pacing was inducible with average 3345 ± 146 s AF duration in all ATP canines (p < 0.0001 versus sham, Figure 1A-C).
Next, miRNA and mRNA expression profiles of the left atrium from canines in the AF (n = 5) and sham groups (n = 5) were compared. The miRNA expression profiles revealed that 20 miRNAs, including previously described miRNA (such as miR-146b-5p, miR-19, miR-142 and miR-155), were differentially expressed between two groups (>twofold change; p < 0.05) ( Table 1). A mRNA microarray was used to assay the gene expression profiles. We observed a upregulation of expression of 1174 genes and a downregulation of the expression of 218 genes in canines with AF (p < 0.05; Table S1-2). In particular, miR-146b-5p and fibrosis-related genes (TIMP4, MMP9, TGFB1 and COL1A1) indicated the significance in change among the expressed miRNAs and mRNAs respectively. In Figure 2A,B, the volcano plot has illustrated an elevation that is more than four times of the expression in canines with AF compared with shams.
The expression was elevated more than 4 times in canines with AF, comparing with shams that is clearly stated in the volcano plot shown in Figure 2A

| Rapid depolarization of hiPSC-aCMsfibroblast co-culture cellular model
The characterization of hiPSC-aCMs was described before. 17 Figure 3A shows the hiPSC-aCMs-fibroblast contact co-culture.
The beating of myocytes was crucial when distinguishing the hiPSC-aCMs from fibroblasts (Supplementary Video). Next, we tested whether co-culture could change electrophysiological properties in hiPSC-aCMs and fibrosis-related mRNA expression in the fibroblasts. We found that the action potential of hiPSC-aCMs and action potential duration (APD) had no changes after 24 h co-culture ( Figure S3A) The identical mRNA expression of miR-146b-5p, TIMP4, MMP9, TGFB1 and COL1A1 ( Figure S3B) was confirmed by the PCR analysis.
When using RES, we found that the APD of hiPSC-aCMs was markedly shortened, which was consistent with the atrial remodel-

| MiR-146b-5p transfection in hiPSC-aCMs induced increased interstitial collagen by decreasing the expression of TIMP4 in the cocultured cellular model
Seventy-two hours after miR-146b-5p transfection, a study of miR-146b-5p by qRT-PCR for mRNA levels is conducted on hiPSC-aCMs, which reflected that the miR-146b-5p inhibitor could critically decrease the expressionism of miR-146b-5p in CMs ( Figure S5).
Next, we analysed whether miR-146b-5p transfection (30 nmol/L, Figure S6 We also observed the ultrastructure of hiPSC-aCMs-fibroblast by transmission electron microscopy (TEM). The experimental results revealed that collagen fibres were accumulated in extracellular space after rapid depolarization of miR-146b-5p overexpressed hiPSC-aCMs-fibroblast co-culture cellular model by RES ( Figure 4C).
To sum up, phenotype change in miR-146b-5p transfected hiPSC-aCMs-fibroblast co-culture cellular model by RES suggested that the interaction mechanism of electrical and structural remodelling has a crucial role in AF.

| Inhibiting miR-146b-5p reduces cardiac fibrosis after MI in mice
Based on the previous discovery, our goal became to examine whether inhibiting the expression of miR-146b-5p reduces the phenotypes of cardiac fibrosis in the MI mouse model. The mice were subjected to experimental MI and pretreatment with injecting antagomiR-146b-5p by tail vein to knock down the endogenous origin of miR-146b-5p for 3 days. We found that antagomiR-146b-5p significantly improved post-MI survival during 8 weeks after MI ( Figure 5A). Echocardiography was performed at 4 and 8 weeks after MI to assess cardiac function ( Figure 5B). Within expectation, ejection fraction (EF) decreased in all groups of mice after MI (p < 0.001).
However, antagomiR-146b-5p mice demonstrated a significantly preserved EF than those two group mice at 4 and 8 weeks after There was a clear biatrial enlargement in mice after MI 8 weeks compared with WT mice (Figure 6A,B). Quantification of the atrial weight normalized to tibia length revealed that the atrial mass was significantly increased in all mice after MI compared with WT mice.
In order to determine the fibrotic response, we utilized Masson's trichrome staining to stain fibrotic areas within the myocardium ( Figure 6D-E). All mice after MI showcased a fibrosis increase in the LA and LV, while antagomiR-146b-5p treated MI mice only had a significant increase in LV fibrosis. Consistent with these results, the collagen volume fraction was significantly reduced in antago-miR-146 treated mice after MI in both regions. Immunofluorescence also showed that antagomiR-146 decreased in the expression of collagen-1 protein compared with the other 2 groups of mice after MI ( Figure 6F). Fibrotic marker MMP9, TGFB1 and COL1A1 were significantly downregulated, while TIMP4 was significantly upregulated at both mRNA and protein levels by miR-146b-5p inhibition in CMs of MI heart ( Figure 6G-H). These results suggest that the loss of miR-146b-5p can protect against MI-mediated cardiac fibrosis.

| DISCUSS ION
This research discussed the function of miR-146b-5p in hiPSC-aCMsfibroblast co-culture cellular model in vitro and murine heart in vivo.
miR-146b-5p has a critical role in the regulation of atrial fibrosis, contributing to the structural remodelling in AF. It demonstrated that collagen fibres were accumulated in extracellular space after RES on miR-146b-5p overexpressed co-culture cellular model. Moreover, the cardiac fibrosis induced by MI was reduced in antagomiR-146 treated mice, indicating that the inhibition of miR-146b-5p might become an effective therapeutic approach to prevent atrial fibrosis.
To our understanding, this will be the initial study that investigated the unknown mechanism of miR-146b-5p on atrial fibrosis in AF. Our proposed working model of the miR-146b-5p mediated atrial fibrosis in AF is shown in Figure 7.
TIMP4 is endogenously one of the key modulators of MMP9, which is activated in atrial fibrosis. 18 The activation of MMP9 beyond a certain limit is deleterious to the heart, which worsens cardiac remodelling. 5,19,20 The hyperearly research discovered that when the expression of miR-146b-5p increases, TIMP4 is down-regulated due to repression in cardiomyocytes in patients with AF. 15 In the present study, we further evaluated miR-146b-5p and TIMP4 in ATP canine and observed a significant negative correlation between them. As a result, there is a shift in the TIMP4-MMP9 axis towards the MMP9, which has been reported to activate latent TGF-β1 to its active form and to induce TGF-β1 production, which interacts with neighbouring fibroblasts and promote collagen synthesis, as well as cardiac fibrosis. 21 In patients with AF, electrical and structural remodelling normally comes together; thus, their effects are difficult to separate.
ATP animal models typically develop some degree of structural remodelling. 22 The highly homogeneous aCMs with normal electrophysiological properties, generated from hiPSC, stand for a promising and powerful method for screening anti-arrhythmic drugs in vitro on a wide spectrum. Studies have shown that tachy-paced aCMs promotes protein synthesis by atrial fibroblasts. 23 Thus, we used a direct hiPSC-aCMs-fibroblast contact co-culture system to investigate the possible molecular mechanism of atrial fibrosis.
After using RES, we found that the APD of hiPSC-aCMs was markedly shortened with a marked reduction in I Ca,L , which was  and COL1A1 were found after RES in our study. 23 After transfection of miR-146b-5p in hiPSC-aCMs, TIMP4 mRNA expression level was significantly decreased, while MMP9, TGFB1 and F I G U R E 2 A, Hot map and volcano plot showed that miR-146b-5p is the most pronounced change among the 20 differentially expressed miRNAs in atrial cardiomyocytes from dogs with AF. B, The volcano plot showed fibrotic genes were significantly dysregulated in atrial cardiomyocytes from dogs with AF. C, Pathway enrichment analysis predicted the TGF-β1pathway was the most significant involved pathway based on the mRNA profiling results. D, PCR analysis showed a significant upregulation of miR-146b-5p, MMP9, TGFB1and COL1A1, with a significant downregulation of TIMP4 expression in dogs with AF. E, Western blot analysis showed protein expression of TIMP4, MMP9, TGF-β1 and COL-I in atrial cardiomyocytes from dogs with AF and sinus rhythm. F, Histopathological changes and the collagen volume fraction in left atrium after staining with Masson's trichrome stain from dogs with AF and sinus rhythm. ** p < 0.001 versus sham. AF, Atrial fibrillation COL1A1 expression were significantly increased without RES.
Moreover, after using RES, TGFB1 and COL1A1, mRNA expression was most significantly increased compared with those before RES.
These results showed that our co-culture cellular model maximum This study has certain limitations. First, although hiPSC-aCMsfibroblast co-culture was an AF cellular model, specific inherent restriction generating from the hiPSC-aCM model will be needing further study. Specifically, the problem with immaturity remains critical. Second, this research primarily studied the miR-146b-5p regulation of TIMP4 proteins, since the importance of the TIMP4/ MMP9 axis in the predominance of structural remodelling changes.
However, the differences in the miR-146b-5p level possibly lead to arrhythmogenesis by adjusting restructuring in cardiomyocytes. This is an area that merits further exploration.

| CON CLUS IONS
Our results demonstrated that the overexpression of miR-146b-5p activates collagen synthesis and deposition in an AF cellular model, while its inhibition could attenuate the atrial fibrosis and preserve heart function after MI in the mouse heart. This study was the first to demonstrate a mechanical link between miR-146b-5p and atrial fibrosis in AF. Our findings also indicated that the inhibition of miR-146b-5p hinders the TIMP4/MMP9 unbalance related restructuring in AF and might compose a new anti-AF method aiming atrial fibrosis.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.