MiR-182 regulates IL-6 in the cardiac context: implications for human atrial electrical remodelling

MiR-182 regulates IL-6 in the cardiac context: implications for human atrial electrical remodelling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MiR-182 regulates IL-6 in the cardiac context: implications for human atrial electrical remodelling Elena Guzzolino, Valentina Balducci, Giada Allegro, Valentina Spinelli, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8808389/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background The progression of cardiac electrical remodelling and the onset of proarrhythmic events are multifactorial processes, with many factors contributing to the development of atrial fibrillation (AF). Recently, proinflammatory mediators have emerged as new key players: in particular, independent preclinical evidence has identified interleukin-6 (IL-6) and miR-182 as causative factors of arrhythmogenesis in animal models. MiR-182 regulates a wide range of pathways, including the expression of inflammatory mediators; however, in the cardiac context, the potential relationship between these two factors remains unknown. Methods Human induced pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (CMs) to study the role of miR-182-overexpression (OE) on IL-6 expression/secretion by RT-PCR/Elisa assays. Functional consequences were assessed by measuring spontaneous electrical activity by using MULTIPLE-High-Throughput and Intracell systems, with/without autonomic stimulation. Hearts from the Tg(myl7:GAL4,EGFP) x Tg(Sce.4xUAS:miR-182,cry:EGFP) zebrafish line [ Tg(myl7 > miR-182) ] were used to measure the expression of dre-il6 . Human IL-6 protein (5.4pg/nL) was microinjected in the pericardial region of 2dpf Tg(myl7:EGFP) wt -like zebrafish embryos and heart rate was recorded. Expression analyses were performed on human left atrial samples of 49 patients (11 controls, CT; 18 left atrial dilation (LA-D); 20 chronic AF). Results MiR-182-OE in hiPS-CMs significantly incremented IL-6 expression and secretion, and was associated with a reduced and irregular spontaneous beating rate, as well as enhanced response to acetylcholine. Accordingly, MiR-182-OE downregulated the expression of HCN4, encoding for the pacemaker f-current , and dysregulated genes associated with atrial pathology. On the contrary, 24-hour incubation with IL-6 (50ng/mL) did not change miR-182-5p expression levels in CT hiPS-CMs. The IL-6 receptor antagonist tocilizumab (TOC, 10µg/mL) partially rescued HCN4 expression in miR-182-OE hiPS-CMs. Zebrafish heart samples from Tg(myl7 > miR-182) exhibited increased il6 expression levels. Pericardial injection of human IL-6 in wt zebrafish embryos decreased heart rate. Finally, miR-182-5p was found to be overexpressed in human biopsies from patients with LA-D, with the highest expression levels observed in patients with permanent AF; remarkably, miR-182 levels positively correlated with IL-6 expression. Conclusion The results support the hypothesis of a causative link between miR-182-OE and IL-6 production in the cardiac context. This molecular axis may represent a prognostic factor predisposing to arrhythmogenesis. Overall, our findings reveal novel pathophysiological mechanisms and suggest novel pharmacological targets within the complex AF setting. miR-182-5p IL-6 hiPS-CMs Arrhythmias zebrafish Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Disturbances of the heart’s electrical activity (‘arrhythmias’) remain a leading cause of morbidity and mortality worldwide, reinforcing the need to advance mechanistic understanding and identify new targets for therapeutic interventions ( 1 , 2 ). Arrhythmias, including atrial fibrillation (AF), are progressive diseases that are caused and aggravated by multiple factors, such as genetics, epigenetics, aging, and life style ( 3 ). Appearance and persistence of arrhythmias is directed by the progressive occurrence of structural and functional remodelling of the cardiac tissue. Among several molecular mechanisms involved, the synthesis of key cardiac proteins may be disrupted by modifications of gene transcription and post-transcription due to changes in the concentration and activity of specific microRNAs ( 4 – 6 ). Our previous studies in animal models showed that the cardiac specific overexpression of miR-182 acts as an important promoter of adverse cardiac phenotype, triggering arrhythmogenic mechanisms, such as bradycardia, early and delayed after depolarization, and heart block. These modifications are also accompanied by the reduced expression of cardiac calcium channel subunits ( 7 ), an alteration commonly observed in atrial arrhythmias ( 8 ). Basal expression of miR-182-5p was previously documented in vertebrate and mammalian hearts ( 7 ), found to be negatively regulated by the T-box transcription factor 5 (TBX5), whose mutations are associated with the human rare genetic condition Holt-Oram Syndrome (HOS) and with AF, as revealed by genome-wide association studies ( 9 , 10 ). Microarray profiling of plasma obtained from patients with chronic congestive heart failure led to propose miR-182-5p as a potential prognostic marker for cardiac outcomes ( 11 ), in line with the evidence that miR-182-5p promotes myocardial apoptosis in experimental myocardial infarction ( 12 ). Notably, miR-182 is also involved in inflammatory processes ( 13 – 17 ) known to be active in atrial remodelling and arrhythmias ( 18 , 19 ). In line with this, our recent study provided a basis to link IL-6 to human atrial dilation, a condition that predisposes atria to arrhythmic dysfunction ( 20 ). The same study showed that IL-6 deregulates electrogenesis of human cardiomyocytes hampering the expression and function of the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels ( 21 ). Despite extensive understanding of the factors driving IL-6 upregulation and secretion in extra-cardiac tissue ( 22 ), the specific signals triggering IL-6 transcription in the heart still remain unclear. In this study we aimed to assess the functional role of miR-182 in the human cardiac context, hypothesizing that it contributes to the activation of inflammatory responses in cardiac cells, and addressed this question using a wide range of models and experimental approaches. Using human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes (CMs) and overexpressing miR-182, we explored the causal mechanism linking miR-182 to IL-6 transcription and secretion in CMs, and to alterations in electrogenesis. Then, using zebrafish embryos we assessed in vivo the functionality of miR-182/IL-6 axis and studied the direct effects of elevate IL-6 levels in the beating heart. Finally, we analysed left atrial samples from healthy donors and patients with different severity of atrial disease to establish a translational benchmark. Methods 2.1 Culture and cardiac differentiation from human induced pluripotent stem cells Human induced pluripotent stem cells (hiPSCs; wt-c11 or UCSFi001-A, obtained from fibroblasts of a healthy 30–33 years old man and gifted by Dr. Pioner, Department of Clinical and Experimental Medicine, Division of Physiology, University of Florence, Italy) were cultured on a Corning®Matrigel matrix using mTeSR Plus medium (Stem Cell Technologies) and splitted every 4–5 days with 0.5 mM EDTA in phosphate-buffered saline (PBS) solution, supplementing the medium with ROCK inhibitor (Y27632, 5µM). The monolayer cardiac differentiation protocol used the cardiac PSC Cardiomyocyte Differentiation Kit (Life Technologies, Thermo Fisher scientific), following manufacturer’s instructions. Initially, hiPSC colonies were dissociated into single cells using TrypLE for 5 minutes at 37°C, which were seeded into mTeSR Plus with Y27632 onto Matrigel-coated wells of a 24-well plate at a cell density of 80.000 cells/well ( 21 , 23 ). When cells reached 70–90% confluency (2/3 days later), the culture medium was changed to Cardiomyocyte Differentiation Medium A (day 0) to induce mesodermal differentiation, followed by subsequent medium change to Medium B on day 2 and to Medium C on day 4, which facilitated final cardiomyocyte differentiation. Spontaneously beating monolayers appeared around day 8–10, and at day 12 the Medium C was replaced with RPMI plus B27 supplement (Life Technologies, Thermo Fisher scientific) to further CMs maturation ( 23 ). 2.2 Transgenic hiPSC lines production To obtain a stable overexpression of miR-182 ( hiPSC wt-c11 miR-182) , 70%-80% confluent hiPSCs were transfected with the pLV-[hsa-mir-182] plasmid biosettia, via Lipo3000 (Invitrogen), following manufacturer instructions. After 2 days, cells were selected with mTeSR Plus containing Puromycin (1µg/mL) for 1 week. The transgenic hiPSC line used as control ( hiPSC wt-c11 CT ) was obtained transfecting the same plasmid, previously excided from the pre-miR-182 sequence, using XhoI restriction enzyme. The bona fide of the transgenic lines was tested with Trilineage differentiation, RT-PCR, MTT and Proliferation assays (Figures Supplemental 4–5 and Supplementary Methods). 2.3 IL-6 treatment in hiPS-CMs 29-day differentiated hiPS-CMs were serum-starved overnight in RPMI (B27 minus) medium. On the 30th day of cardiac differentiation, hiPS-CMs were incubated with TOC (Tocilizumab 10 µg/mL, MedChemExpress) solubilized in RPMI + B27 medium for 30 minutes and then stimulated with IL-6 (50 ng/mL, Sigma Aldrich) or vehicle (PBS) for 24 hours. After treatment, the medium was changed for electrophysiological and/or molecular analyses. 2.4 Electrophysiological recordings and analyses with IntraCell Recordings of intracellular-like APs from hiPS-CMs were obtained on microelectrode arrays (MEA) with the IntraCell system from Foresee Biosystems S.R.L. in combination with a MEA2100-Lite system from Multi Channel Systems GmbH ( 24 ). On the 27th day of maturation, the differentiated hiPS-CMs cultured in 24-well plate were incubated for 1h with RPMI supplemented with B27 and Y27632 (10 µM). The cells were then dissociated into single cells using TrypLE (10 minutes at 37°C) and centrifuged at 300g for 3 minutes. Following the resuspension in RPMI supplemented with B27, 10% fetal bovine serum, and 10 µM Y27632, the hiPS-CMs were seeded at a density of 16.000 cells per well onto 60-6wellMEA200/30iR-Ti-rcr MEA plates with 54 electrodes and 6 independent wells (9 electrodes per well) pre-coated with 8µL human fibronectin (1 µg/mL; Gibco). The following day, half of the medium was replaced with RPMI supplemented with B27. On the second day after the dissociation, the medium was refreshed, and cells recovered the spontaneous beating. On the third day, corresponding to the 30th day of cardiac maturation, the IntraCell system was used to apply laser-mediated cell poration on CMs in contact with the MEA electrodes to obtain intracellular-like APs. With this technique it is not possible to determine the absolute membrane potential, therefore data are expressed in arbitrary units (A.U.). 2.5 Electrophysiological recordings and analyses with High-Throughput MULTIPLE system The spontaneous activity of hiPS-CMs was recorded using the High-Throughput MULTIPLE system, as previous described ( 25 ). HiPS-CMs were incubated with a near-infrared voltage-sensitive dye (VSD) di-4-ANBDQPQ ( 26 ) (2 µg/mL) for 9 minutes and then washed with dye-free Tyrode’s solution (D-(+)-glucose 10 mM, NaCl 140 mM, KCl 5.4 mM, MgCl 2 1.2 mM, CaCl 2 1.8 mM, Hepes 5.0 mM, adjusted to pH 7.3 with NaOH). Cultures were placed on the High-Throughput MULTIPLE microscope stage, where the temperature was constantly maintained at 37°C during recordings. A red LED (SOLIS-623C, Thorlabs) followed by a band-pass filter (625PB50 Omega optical) illuminated the monolayer of hiPS-CMs and the fluorescence signal was collected in forward direction using a camera lens (MVL12M43, Thorlabs) placed in front of a sCMOS camera (ORCA-Flash 4.0 V3, Hamamatsu) operating at a frame rate of 100Hz. A long-pass filter (LP700; Omega optical) was placed in front of the camera lens. Fluorescent signals obtained from the High-Throughput recordings were associated with regions of interest (ROI) and were photo-bleached, corrected, normalized, and temporally filtered using LabVIEW (National Instruments, Austin, TX, United States), Fiji-ImageJ (National Institutes of Health, Bethesda, MD) and OriginLab 2023b (Northampton, MA, United States) software. OriginLab 2023b software was used to analyse AP frequency. With this technique it is not possible to determine the absolute membrane potential, therefore data are expressed in arbitrary units (A.U.). 2.6 Zebrafish lines husbandry The zebrafish facility has held the authorization n°297/2012-A since 12/21/2012. Fish were bred in standard laboratory conditions (Westerfield M zebrafish book) in Zebrafish Housing Systems (Tecniplast, Varese, Italy), as previously reported ( 27 ). All animal practices comply with fundamental ethical standards and the Directive 2010/63/EU of the European Parliament, which addresses the protection and welfare of animals utilized for scientific aims. Natural spawning was used to produce embryos, which were then incubated in E3 medium at 28 ± 0.5°C. The Tg(myl7:EGFP) transgenic line, in AB genetic background was used in this study and biobank materials from Tg(myl7:GAL4,EGFP)xTg(Sce.4xUAS:miR-182,cry:EGFP) transgenic lines [to simplify the term “ Tg(myl7 > miR-182)” will be used in the text meanwhile “Myl7-m182-OE”, or “Myl7-GAL4” for controls, will be used in images to refer at siblings (Fig. 4 )]. 2.7 RNA extraction and RT-PCR Atrial human samples from cardiac surgery were quickly frozen in liquid nitrogen and stored at − 80°C or used for RNA isolation ( 28 ). Briefly, RNA was extracted using QIAzol (Qiagen), following the manufacturer's instructions, and subsequently quantified with SmartDrop Nano Spectrophotometers (Accuris Instruments-Bioclass). RT-PCR were performed using CFX Opus 96 system (Biorad). For gene expression analyses the RPL19 gene was used as housekeeping reference gene, while for miRNA expression analyses the U6 was used as standard internal control. RNA was retro-transcribed in cDNA using iScript™ cDNA Synthesis Kit (BIORAD) or SuperScript™ IV VILO™ Master Mix (Thermo Fisher Scientific) for genes and using Mir-X™ miRNA First-Strand Synthesis Kit (TAKARA) for miRNAs. Heart tissues RNA from 4dpf zebrafish, obtained from Tg(myl7:GAL4,EGFP)xTg(Sce.4xUAS:miR-182,cry:EGFP) transgenic lines, are derived from a tissue biobank stored in the laboratory as a result of previous research studies ( 7 ). Beating heart tissues were surgically extracted from embryos, previously anesthetized with 0,016% tricaine (standard 1X MS-222; Sigma-Aldrich) for 15 minutes, and washed in L-15 (Leibovit’s) + 10%FBS to eliminate residual blood cells in the tissue before being stored in QIAzol for RNA extraction. The residual embryos were soon transferred in tricaine overdose solution (500mg/L) for 30 minutes and then in 1–1,5% of sodium hypochlorite (bleach solution) for 5–10 minutes, as described in paragraph 2.10. No additional embryos of this line were raised for this work, observing the ethics of biologists in accordance with the 3R principles. For gene expression analyses the eef1a1l1 and rpl13 genes were used to normalize data. 2.8 Pericardial microinjection in zebrafish embryos and video recordings At the zygote stage, transgenic zebrafish embryos of the Tg(myl7:EGFP) line were collected and raised in E3 medium at 28°C. At 2dpf, prior of the microinjection, embryos were transferred in E3 medium at room temperature (26.5°C) and acclimated for 2h before heart rate recordings to prevent bias in physiological measurements. Room temperature was maintained constant during the entire timeline of the experiments with the supply of an air conditioner. Before video recordings and microinjection, embryos were anesthetized with 0.016% tricaine (standard 1X MS-222; Sigma-Aldrich) for 15 minutes; the solution was prepared starting from a 25X stock (400mg/100 mL of buffered DD water at pH ˜ 7) and diluted in E3 medium at room temperature. During the experiments the solution temperature was measured with a bath thermometer to ensure its stability. Embryos of the same population were divided into 2 groups (pre-sham and pre-hIL-6) and the heart rate was measured before microinjection to verify the similarity of heart rate variability between the two groups and avoid stochastic bias caused by selection. Heart rate was 133.4 ± 12.3 and 133.3 ± 11.3 bpm in pre-sham and pre-IL-6, respectively; coefficient of variation was 9.2% and 8.4% for pre-sham and pre-IL-6, respectively. After initial (T0) video recordings, anesthetized zebrafish embryos were positioned on a plate filled with 1.5% solidified agar low melting, slumped laterally exposing the pericardial portion in opposition with the direction of needle (see Fig. 4 B). Embryo insensitivity to touch was assessed before the microinjection. For each embryo, 1 nL (corresponding to total 5.4 picograms) of human recombinant IL-6 protein solution (10µM, Sigma Aldrich), or 1nL PBS 1X, used as control, was microinjected in the pericardial region. After microinjection the embryos were transferred in E3 medium for 1 hour to avoid possible side effects of anaesthetic and in compliance with the good practices of laboratory animal care suggested by the institutional Animal Welfare Body (OPBA). Before the second round of video recordings, embryos were again anesthetized with 0.016% tricaine for 15 minutes. The heart rate was calculated a posteriori analysing video recorded using a Leica MZ10F microscope supplied with Leica DFC 3000G camera and LASX software. The procedures caused a 5–10% mortality. At the end of the experiments, we performed the euthanasia procedure as described in paragraph 2.10. 2.9 Euthanasia procedure After the experiments, the zebrafish embryos (< 5dpf) were euthanized with overdose of tricaine 500mg/L (buffered solution at pH ˜ 7) in E3 medium, in accordance with the European Union Directive 2010/63/EU and the transposed Italian law, Legislative Decree 4 March 2014, n.26. All euthanasia protocols were approved by the institutional Animal Welfare Body (OPBA). The embryos were kept in the tricaine solution for 30–50 minutes, after the cessation of opercular movement and heartbeat to ensure a complete and irreversible cessation of vital signs. A second step of extra-precaution was adopted prior to disposal, transferring no vital embryos in 1-1.5% sodium hypochlorite (bleach solution) for 5–10 minutes. 2.10 Patients and Ethical statement For this study we used cardiac tissue samples excided from left atrial appendage, obtained through collaborations between the University of Florence and the Cardio-Surgery Units of AOU-Careggi (Florence, Italy) and the “Policlinico Le Scotte” (Siena, Italy). Samples from control donor hearts not suitable for transplantation were from the University of Szeged (Hungary). The investigation conforms with the principles outlined in the “Declaration of Helsinki” of the World Medical Association and was approved by the local ethical committee ( 29 ). Informed consent for the use of tissue samples was obtained from patients before cardiac surgery. Basic demographic (age and sex) information was available for all samples, while clinical information (echocardiography and associated comorbidities, therapy) was acquired only for samples from patients undergoing surgery (Table 1 and Supplemental S- Table 1 ). To overcome this inconvenient we stratified samples according with cardiac phenotype severity, creating 3 clinical groups: control (CT); LA-D (pathological cardiac condition and different grades of atrial dilation) and AF (chronic atrial fibrillation). A total of 49 samples were collected: 11 CT samples were from subjects with no history of atrial diseases, undergoing surgery after accidental event or patients undergoing mitral valve replacement without showing a pathological cardiac condition and atrial dilation; 18 LA-D samples were obtained from patients undergoing cardiac surgery, principally aortic or mitral valve replacement, showing a pathological cardiac condition and different grades of atrial dilation. Among them, 16 LA-D samples were from patients in sinus rhythm, and 2 were from patients presenting paroxysmal AF. Finally, 20 samples were from patients with chronic AF and atrial dilation. Table 1 Main characteristics of the patients. Patients Total Sex Age LA volume (F) LA volume (M) Sinus rhythm n° (F - M) (min-max) (min-max) (mL) (min -max)(mL) (%) CT 11 3–8 40–50 - - 100% LA-D 18 11 − 7 26–84 40–130 45–153 89% AF 20 6 − 4 65–85 65–140 80–208 0% 2.11 Statistics and graphs Statistical analysis was performed using GraphPAD prism software. The statistical test used is indicated in figure legend. Normal distribution of data was tested to choose between parametric or non-parametric inference. We adopted *for p < 0,05; **for p < 0,01; ***for p < 0,001; ****for p < 0,0001, with α = 0,05. Figures were also prepared by using the Biorender software (ID: university-of-florence—dept-of-neuroscience). Part of the cartoon in Fig. 4 was created with GIMP software. Results 3.1 MiR-182 overexpression in hiPS-CMs decreases spontaneous AP frequency As a first step, we performed a preliminary in silico investigation to unveil if direct miR-182-5p targeted genes might be described in key GO and KEEG terms related to human cardiac arrhythmogenic pathways [see Supplemental Methods for details]. In line with our previous integrated analyses on putative target genes of miR-182-5p shared by mouse and zebrafish (7), the analysis showed that miR-182-5p directly modulates human cardiac genes involved in electrical coupling, muscle cellular homeostasis and calcium handling, suggesting a similar regulatory framework operating in our experimental setting [ Figures Supplemental 1-3 ]. To assess the functional consequences of miR-182–mediated dysregulation of genes involved in cardiac electrogenesis, we investigated the spontaneous electrical activity in wt-c11 CT and miR-182 CMs, which stably overexpresses miR-182 in differentiated cells ( Figure 1A ). We first confirmed that main cell line properties were not altered, documenting that pluripotency and trilineage differentiation ability were similar in wt-c11 CT and miR-182 cell lines ( Figure Supplemental 4 ), as measured in different pools of cells. Moreover, pluripotent wt-c11 miR-182 hiPSCs showed similar viability and rate of growth compared to wt-c11 CT hiPSCs, as demonstrated by MTT and proliferation assays ( Figure Supplemental 5 ). We then used the IntraCell platform system to study spontaneous action potentials (APs) of cultured hiPSC-CMs at 30 days of differentiation. Recordings revealed marked differences between AP traces obtained from wt-c11 miR-182 and wt-c11 CT CMs ( Figure 1B ), with the former exhibiting lower absolute values of spontaneous frequency ( Figure 1C ) and higher coefficient of variation (CV) in spontaneous frequency compared to CT CMs ( Figure 1D ). Average values of AP frequency was 74.5±0.9 beat per minute (bpm) in wt-c11 CT CMs and 42.7±3.0 bpm in wt-c11 miR-182 CMs (n=22-27, mean±SEM, p<0.0001 unpaired t-test). CV in beating rate was calculated from a series of consecutive APs recorded during a period of 10 seconds, and repeated for 3 to 4 times during the same recording. It was 2.59±3.43% in wt-c11 CT CMs and 15.28±7.76% in wt-c11 miR-182 CMs (n=7-8, mean±SD p<0.01 unpaired t-test). Overall, these data demonstrate the occurrence of cellular electrophysiological abnormalities induced by miR-182 overexpression. 3.2 MiR-182 overexpression in hiPSC-CMs regulates HCN4 expression via upregulation and secretion of IL-6 Considering the reduction of spontaneous AP frequency, its increase of variation in CMs overexpressing miR-182, and the role of f- channels in the spontaneous beating rate, we hypothesized that these electrical disturbances might be related to a reduced functional expression of the major cardiac f- channel isoform, HCN4. RT-PCR analyses confirmed our hypothesis of a downregulation of HCN4 ( Figure 2A ). Then, based on the recognized regulation of inflammatory pathways (30,31) by miR-182, and our previous report on HCN downregulation induced by IL-6 on hiPSC-CMs (21), we wondered whether in our setting overexpression of miR-182 regulates HCN4 channel transcript through the increased expression and activity of IL-6 levels. Indeed, differentiated CMs overexpressing miR-182 displayed significantly higher levels of IL-6 transcription compared to wt-c11 CT CMs ( Figure 2B-E) , suggesting that miR-182 acts as upstream regulator of this cytokine in human CMs. To determine whether the enhanced levels of IL-6 mRNA translate into increased secretion of protein, IL-6 concentrations in cell culture media were measured by ELISA assay. IL-6 protein levels were significantly higher in the media of wt-c11 miR-182 CMs compared with wt-c11 CT CMs, demonstrating that enhanced IL-6 secretion by CMs parallels the increased transcription of IL-6 ( Figure 2C-E ). In order to evaluate a possible opposite cause-effect mechanism between IL-6 and miR-182 levels in human CMs, hiPSC-CMs at 30 days of maturation were treated with 50 ng/mL recombinant IL-6 for 24h, with or without 10 µg/mL tocilizumab (TOC), a monoclonal antibody that inhibits IL-6 signalling activation by binding to IL-6 receptors ( Figure Supplemental 5-A) . IL-6 treatment did not increase miR-182-5p expression levels in hiPSC-CMs ( Figure Supplemental 5-B ), as well as that of its pri-miRNA ( Figure Supplemental 5-C ), which also contains miR-183 and miR-96 (7,32), thus excluding IL-6 is an upstream regulator of miR-182-5p in the cardiac context, at least in short term response. To further support the evidence that miR-182 indirectly affects HCN4 transcript levels through IL-6 overproduction, we treated wt-c11 miR-182 CMs with 10 µg/mL TOC for 24h to block IL-6 receptors. Results demonstrated that TOC significantly rescued HCN4 expression ( Figure 2E ), suggesting that at least part of the reduction of HCN4 transcript passes through the activation of IL-6 expression and the stimulation of IL-6 receptors. 3.3 MiR-182 overexpression in hiPS-CMs alters muscarinic response Being HCN channels key players regulated by the cardiac autonomic system, we investigated the functional impact of miR-182 overexpression on the responses to acetylcholine (ACh) and Isoprenaline (Iso) to mimic the muscarinic and β-adrenergic effect, respectively. ACh, at a concentration ranging from 0.1 to 10 μM, dose-dependently reduced spontaneous AP rate ( Figure 3 ), as expected from activation of muscarinic receptors in cardiac pacemaker-like cells. The effect was significantly greater in wt-c11 miR-182 compared to the wt-c11 CT CMs, suggesting an increased muscarinic signalling induced by miR-182 overexpression. Hence, the spontaneous beating even ceased in some cells exposed to the highest ACh concentrations. Conversely, Iso (1 μM) significantly increased the beating rate in wt-c11 CT CMs treated with 0.1 and 1 μM ACh, as well as in wt-c11 miR-182 CMs exposed to ACh 0.1 μM, while it failed to modify the rate when ACh was applied at 1 μM concentration. Overall, these data suggest that the response to autonomic signals is also altered by miR-182 overexpression in hiPSC-CMs. 3.4 Cardiac selective overexpression of miR-182 upregulates il6 in zebrafish heart To reveal if cardiac miR-182 overexpression upregulates cardiac IL-6 in vivo , we studied 4dpf zebrafish Tg(myl7>m182) embryos characterized by a cardiac selective overexpression of miR-182. We compared them with age-matched control Tg(Myl7:GAL4,EGFP) zebrafish embryos ( Myl7-GAL4 ) (7). In zebrafish, miR-182 overexpression induces heart morphological defects in a dose dependent manner, recapitulating the main features of cardiac HOS pathology and triggering arrhythmias even in embryos without cardiac morphological defects (7). As previously described, heterozygous Myl7-m182-OE zebrafish embryos did not show any structural and morphological cardiac defects, while they developed cardiac arrhythmias as early as 3dpf (7). Expression analysis on isolated hearts demonstrated a significant overexpression of cardiac il6 in Myl7-m182-OE compared to Myl7-GAL4 zebrafish embryos ( Figure 4-A ), suggesting that cardiac miR-182/IL-6 axis represents a molecular circuit that is conserved across vertebrates. 3.5 Human IL-6 protein decreases heart rate in zebrafish Little is known about the role of il6 in non-immune cells during zebrafish development and literature lack of information about its role in heart organogenesis and function. Analyses of IL-6 gene among species reveals a good grade of structural conservation through phylogenetic evolution and the zebrafish il6 protein shares a significant grade of similarity with human IL-6 (hIL-6) protein tertiary structure, as modelled by Valera et al., 2012 (33). On this basis and given that zebrafish is increasingly used as a powerful model to investigate the molecular circuits involved in human pathology (34–36), we sought to use zebrafish embryos to validate in vivo the hypothesis that human IL-6 hampers cardiac pacemaking. We assessed the heart rate of two groups of 2dpf Tg(myl7:EGFP) zebrafish embryos, selected to have values of cardiac frequency equally distributed within a physiological range ( Figure 4-B ). After 1h from microinjection into the pericardial space of 1nL of 10 mM hIL-6 solution or vehicle (control solution), heart rate of hIL-6 group displayed a significant reduction compared to the sham group, while the coefficients of variation were 21.2% and 9.8% in the hIL-6 and sham group, respectively ( Figure 4-C ). 3.6 MiR-182 overexpression deregulates the expression of genes involved in human CMs function and remodelling Given that the molecular machinery involved in the regulation of cardiac rhythm employs a complex interplay of multiple targets (37) and that the deregulation of single miRNAs inside cells can unbalance extensive molecular pathways (38), we thought to obtain a wider view of the impact of miR-182 overexpression on several genes involved in cardiac electrogenesis. We compared wt-c11 miR-182 and wt-c11 CT CMs at 30 days of maturation by transcription analysis of key genes involved in cardiac function and other genes known to be associated to cardiac remodelling and arrhythmogenesis. Results ( Figure 5; Figure Supplemental 7 ) evidenced several modifications of genes directly involved in intracellular calcium handling, as expected by previous in silico investigations and in vivo characterization(7), which showed a significant downregulation of RYR and a significant upregulation of CACNB4 in wt-c11 miR-182 compared to wt-c11 CT CMs. NCX, CACNA1C and CACNB2 transcripts showed a clear, albeit not significant, trend toward reduction in wt-c11 miR-182 compared to wt-c11 CT CMs, while CACNA2D1 showed a not significant trend toward increase. Expression levels of other ion channels were also modified: beyond HCN4 that was significantly downregulated in wt-c11 miR-182 compared to wt-c11 CT CMs, HCN2, HCN3 and SCN5A were all significantly upregulated. We also observed a significant downregulation of HCN1, CHRMR2 and ADRB3 in wt-c11 miR-182 compared to wt-c11 CT CMs, while ADRB1 was up-regulated. Finally, CAMKIIγ was significantly upregulated in wt-c11 miR-182 compared to wt-c11 CT CMs. In line with the increased expression of IL-6 levels, the expression of its well-known regulator NF-κB was also upregulated in wt-c11 miR-182 CMs ( Figure 6 ), unveiling the parallel activation of an inflammatory signalling (39). We tested the expression of IL-1β, TNFα and TGFβ ( Figure 6 ), also regulated by NF-κB in inflammation (40) and in heart disease (41), but interestingly only IL-6 resulted upregulated by miR-182 overexpression in the hiPSC-CMs. We also tested the expression of IL-10, but it resulted not detectable by RT-PCR in wt-c11 hiPSC-CMs. Finally, to gain deeper insights into the regulatory crosstalk within the miR-182/IL-6 axis, we investigated the transcriptional response of endoplasmic reticulum (ER) stress-related genes, such as EDEM1, GADD34, CHOP and ATF4, as EDEM1 and other genes belonging to the “Path:hsa04141-Protein processing in endoplasmic reticulum” resulted direct targets of miR-182-5p in our in silico analyses (not shown). The ER-stress signalling is known to be associated with cardiac adverse remodelling and enhanced risk for AF (42,43). As evident in Figure 6 , all genes EDEM1, GADD34, CHOP and ATF4 resulted upregulated in wt-c11 miR-182 compared to CT CMs, enforcing the hypothesis of an indirect activation of IL-6 through an intracellular cell stress-sensing signalling regulating NF-κB overexpression, a known transcription factor for IL-6 (44). Additional RT-PCR are reported in Figure Supplemental 7 . 3.7 MiR-182-5p is expressed in left human atria in accordance with disease severity and positively correlates with IL-6 expression levels We reasoned that a conserved cardiac miR-182/IL-6 axis could represent a relevant biomarker in the clinical setting. Thus, we performed RT-PCR analysis of left atrial (LA) samples obtained from 49 patients undergoing surgery to analyse miR-182-5p expression levels. Samples were divided into 3 groups according to disease severity: 11 control patients (CT), with no history of atrial pathology, 18 patients with LA dilation and elevate risk to develop AF (LA-D) (45), and 20 patients with chronic AF (AF) ( Figure 7A ). The main features are summarized in Table 1 . Compared to CT, miR-182-5p expression levels resulted significantly higher in LA-D and AF patients ( Figure 7B ). Despite a clear trend toward increased values of miR-182-5p in AF patients, the difference with LA-D patients was not significant, likely due to the large range of variation in data distribution. In the same atrial samples, IL-6 expression levels were significantly increased in AF compared to CT patients, while the difference did not reach the statistical significance in LA-D ( Figure 7C ). When plotted against miR-182-5p expression levels, IL-6 levels and miR-182-5p expression levels showed a similar trend with significant correlation, suggesting that these two markers of left atrial myopathy go hand in-hand. ( Figure 7D ). As previously mentioned, the pri-miR-182 is transcribed as a single transcript including the sequences of pre-miR-183 and pre-miR-96, which are both part of the highly conserved polycistronic miR ˜ 183 cluster (32). To uncover differences of expression among mature miRNAs belonging to miR ˜ 183 cluster, RT-PCR analyses of the same atrial samples evidenced that miR-183-5p and miR-96-5p were both significantly up-regulated in LA-D samples compared to CT, while no difference was present for AF patients ( Figure Supplemental 8 ). MiR-183-5p and miR-96-5p levels did not correlate with IL-6 expression ( Figure Supplemental 8 ), suggesting the unique major interplay of miR-182-5p with IL-6 in left human atria. Discussion MiR-182-5p was previously identified as negative modulator of TBX5 action, resulting overexpressed in Holt-Oram syndrome (HOS) murine and zebrafish animal models, a rare genetic autosomal conserved disease associated to TBX5 mutations (7). TBX5 is also involved in pacemaker formation (46), and cardiac electromechanical dysfunction in congenital diseases (47). In the hierarchical panel of cardiac gene expression, TBX5 is upstream of the transcription factor ISL-1, a gene identified as direct target of miR-182-5p by DIANA (48) and a key regulator of pacemaker formation and HCN4 expression (49). Even in the absence of gross cardiac malformations, electrical anomalies are the most striking events in some families and HOS patients often require pacemaker implantation (50). Early onset AF may appear in the childhood (51), with dramatic consequences on patients’ lifespan. The role of IL-6 in the development and progression of cardiac disease has never been inferred in this setting, and literature lacks information about the relationship between TBX5 and IL-6. Our observation linking miR-182 to IL-6 expression and secretion in cardiac myocytes provides important insight and warrants further investigation into the role of inflammatory signals within the cardiac context, rather than solely at systemic level, where it may exacerbate the pathogenesis of pre-existing or acquired molecular imbalances. These factors deserve further evaluation in view of their value in forecasting the progression of the arrhythmic phenotypes in paediatric cohorts, particularly in patients coping with complex scenarios. Indeed, systemic inflammation is associated with electric atrial remodelling and IL-6 is able to downregulate atrial connexins (52). We have previously shown that exposure of cardiomyocytes to IL-6 directly decreases the expression and function of HCN channels (21), which contribute to spontaneous activity in immature cardiomyocytes and to pacemaker rhythm in sinoatrial node cells. Similarly, wt-c11 miR-182 CMs exhibited a decreased expression of HCN4 along with a reduction of spontaneous rhythm compared to control CMs. The role of IL-6 was further reinforced by a partial rescue of HCN4 expression in wt-c11 miR-182 CMs exposed to tocilizumab (TOC, Figure 2E ). TOC represents the first humanized monoclonal antibody blocking IL-6 signalling by targeting the IL-6 receptor (53) and recent clinical works reported its safety and efficacy in several settings of cardiovascular diseases (54) . This finding adds consistent translational value to current knowledge and support future investigation. The biological regulation of miR ˜ 183 cluster expression has been extensively studied in the immune system, showing that IL-6 upregulates the expression of miR-183 in T helper 17 cells, via STAT3 signalling (55). This finding prompted us to infer that, also in hiPSC-CMs, IL-6 could regulate miR-182 expression: instead, our results demonstrated the opposite path, i.e. that miR-182 overexpression causes the up-regulation of IL-6 in hiPSC-CMs. Vice versa , exposing control hiPSC-CMs to IL-6 did not result in increased miR-182 overexpression, thus establishing a downstream cascade between the two markers detected in the bioptic samples, which has never been reported. Another interesting, original observation of our study refers to the altered response to acetylcholine of wt-c11 miR-182 CMs. When exposed to ACh, the slow intrinsic rate of CMs overexpressing miR-182 was further reduced, an effect significantly more pronounced than in control CMs. Since these data were obtained by measuring frequency of spontaneous APs, we cannot infer whether the hypersensitivity to ACh depends on a prominent response of ACh-sensitive potassium channels (and consequent hyperpolarization) or to a more marked negative shift of the f-current , due to inhibition of adenylyl cyclase and intracellular cAMP reduction, or a combination of these mechanisms (56–59). A more detailed electrophysiological study is warranted. However, the observation that adding Iso could partially restore spontaneous rhythm, likely through recovery of intracellular cAMP levels, suggests that the second mechanism plays a role at least at the lowest ACh concentrations (60). As previously described, fish with myocardial-specific miR-182 overexpression show irregular heart rate, passing through slow rate to bursts of activity (7). This behaviour resembles somehow that observed in the in vitro model of hiPS-CMs, as shown in Figure 4 , further suggesting a conserved role of miR-182 in cell-autonomous functions of CMs through evolution. More directly related to our hypothesis, here we report original observations obtained in vivo in zebrafish lines, supporting the conserved pathway linking miR-182 and IL-6 ( Figure 4A ) and the role of IL-6 as a direct modulator of cardiac rhythm ( Figure 4C ). The prognostic stratification of patients at risk of developing AF remains challenging, due to multifactorial pathogenesis of this arrhythmia (61). Among biomarkers, miRNAs have long been proposed as promising candidates in cardiovascular diseases (CVDs) (62,63). Our work showed a clear-cut overexpression of miR-182 in human samples from diseased atria and suggests, for the first time, a direct correlation between this miRNA and IL-6 expression ex vivo . IL-6 is a pro-inflammatory cytokine expressed by cells during stress conditions. Recent studies suggest IL-6 levels in blood serum as a prognostic marker for the propensity to AF (64,65), as well as for an increased risk of recurrence following electrical cardioversion and catheter ablation (66). In the context of atrial remodelling, increased circulating levels of IL-6 were found to be significantly associated with a greater risk of stroke and all-cause mortality in AF patients, leading to propose IL-6 as prognostic marker of AF (64). The evidence on the possible causal link between miRNA-182 and IL-6 could not be inferred based on the correlation measured in human atrial samples and, for obvious reasons, is not possible to provide longitudinal clinical data to directly link the level of miR-182-5p and the consequent development of AF, as well as performing multivariate adjustment or retrospective assessment with our clinical information. However, the two factors resulted concordantly present in the groups and principally higher in both patients with atrial dilation, a clinical condition recognized as risk factor for AF, and with chronic AF (67). Notwithstanding the complex and multifactorial AF pathogenesis, the novel evidence of the miRNA-182/IL-6 axis in atrial myopathy in patients in sinus rhythm, claims the attention to early markers, with the aim to detect and prevent the progression toward chronic arrhythmias. Besides the effect on IL-6 transcription and release, the overall modified gene expression profile in wt-c11 miR-182 CMs at 30 days of maturation supports the hypothesis that miR-182 overexpression may contribute to an increased risk for the development of AF. In our experimental samples ( Figure 5 ) we observed upregulation of CAMKIIγ, whose overexpression is reported to contribute to atrial pathogenesis (68). As expected, our molecular analyses confirmed a completely deregulated arrangement in gene expression of the genes involved in calcium handling, being the calcium signalling a pivotal actor in both the development and progression of AF (69–71) . We also reported an increased expression of SCN5A, encoding for the alpha subunit of the Nav1.5 cardiac sodium channel; although it is difficult to speculate further, it is worth to mention a recent clinical report of a gain-of-function variant that has been associated with AF (72) . Finally, we observed that the increment of miR-182 levels in hiPS-CMs caused ER stress, as confirmed by the upregulation of ER-stress activated genes ( Figure 6 ), closing the molecular circuit between miR-182 and IL-6 axis, through the expression of NF-κB regulator similarly to other cellular contexts (73–76). However, at the moment, we cannot exclude a feed-forward molecular loop between IL-6 and NF-κB in the cardiac context (44). As a limitation of our work, we did not directly measure the increase in IL-6 specifically in CMs. However, the increased release of the cytokine into the medium and the recovery HCN4 expression following IL-6R blockade both suggest an upregulation of IL-6 synthesis and release by CMs. The characterization of CMs differentiated from wt-c11 miR-182 hiPSCs allowed us to demonstrate the overexpression of IL-6 gene and – in parallel – the increased release of this cytokine in the culture medium. Also, we did not attempt to carry out a thorough electrophysiological characterization of wt-c11 m182 CMs in basal conditions and after stimulation with Ach. Since altered ion channel function is a major contributor to arrhythmia susceptibility, such a study should be conducted in hiPSC derived CMs, possibly differentiated into the atrial phenotype. In this study we used the Tg(myl7>miR-182) zebrafish line, in which the deregulation of miR-182 is confined in the myocardium; nevertheless, we cannot exclude cell-cell communication signalling among different cardiac cell phenotypes, including potential involvement of the endocardium. However, the use of the zebrafish model was essential for its “simplicity” in manipulation to demonstrate a short-term response in heart rate variability, exploring the direct possibility to microinject hIL-6 directly in the pericardial space. The expression of il6 and its receptors in zebrafish was detectable early during the first week of development, also in heart tissue, with an increment after 5dpf (33). Of note, the timeline used to test hIL-6 microinjected directly in the pericardial space of 2dpf zebrafish embryos was strategic because the innate immune system is rudimental and the terminal maturation phase occurs later, despite the signals for primitive haematopoiesis already started (77,78). A compelling question for future research is whether a stable myocardial miR-182 overexpression is effective to trigger the activation of resident immune cells in juvenile and adult fish. Exploring this path could offer a unique opportunity to decode the consequences on heart function and tissue remodelling and better refine the ability to predict cardiac outcomes in the in vivo model. While the two-chambered heart of zebrafish precludes the modelling of AF, this organism remains a valuable platform for assessing drug responses and validating key molecular circuitries at the transcriptional level. Future investigations in large mammal models may provide the necessary framework to overcome these biological constraints. Exploiting the in silico miRDB tool (79), both IL-6 and HCN4 are reported as putative targets of miR-182, albeit with a low score, being supported only by 3 and 5 predictive algorithms, respectively (not shown). We cannot exclude the possibility that miR-182-5p directly recognize these target genes in the cardiac context, nor the possibility it may act as small activating RNA to enhance IL-6 expression. In perturbed states, miRNA overexpression can drive the occupancy of weak binding sites that are otherwise neglected under physiological conditions, thereby expanding the miRNA's regulatory landscape (80). Future analyses may help resolve these possibilities. A relevant and original new result from our study is the evidence of an increased miR-182 level hand to hand with the overexpression of IL-6 in non-genetic AF. The miR-182 upregulation in myocardial tissue emerged from a recent clinical study of AF and it resulted more negatively correlated among its predicted targets (81), however no experimental data are reported in literature about its role in electrogenesis and arrhythmias. At the moment we are not able to dissect the multifactorial mechanisms leading to the up-regulation of miR-182-5p, likely different from the mechanisms mediated by genetic factors, as in the HOS. A recent experimental study in the rat animal model of pressure overload-induced systolic heart failure, a known risk factor predisposing AF (82,83), focused the attention on miRNAs roles and, even not directed to miR-182, this miRNA resulted in the cluster analyses (84). Compelling future perspective on possible epigenetic/multifactorial factors (like aging, enduring exercise, environmental contaminants) (85–87) might unearth what are the triggers activating miR-182 and its subsequent contribution to pathological cardiac remodelling. In conclusion, our findings propose miR-182 overexpression as a marker for the propensity to AF, possibly linked to a burst of IL-6 production. Further investigation should assess miR-182-5p expression not only in left atrial samples of patients undergoing surgery, but also in blood serum and in co-morbidity conditions. Abbreviations ACh= acetylcholine AF= atrial fibrillation CMs= cardiomyocytes CT= control/controls CV= coefficient of variation dpf= days post fertilization GO= Gene Ontology Enrichment HCN= Hyperpolarization-activated Cyclic Nucleotide-gated channels hiPSCs= Human induced pluripotent stem cells HOS= Holt-Oram syndrome IL-6= interleukin 6 Iso= isoprenaline KEEG= Kyoto Encyclopedia of Genes and Genomes LA-D= left atrial dilation Myl7-GAL4= Tg(Myl7:GAL4,EGFP) OPBA= Institutional Animal Welfare Body TBX5= transcription factor 5 Tg(myl7>miR-182)/ Myl7-m182-OE = Tg(myl7:GAL4,EGFP) x Tg(Sce.4xUAS:miR-182,cry:EGFP) TOC= Tocilizumab Declarations Ethics approval and consent to participate The Manuscript report studies involving biologic materials from human tissue. For this study cardiac tissue samples excided from left atrial appendage were used, obtained through collaborations between the University of Florence and the Cardio-Surgery Units of AOU-Careggi (Florence, Italy) and the “Policlinico Le Scotte” (Siena, Italy). Samples from control donor hearts not suitable for transplantation were from the University of Szeged (Hungary). The investigation conforms with the principles outlined in the “Declaration of Helsinki” of the World Medical Association and was approved by the local ethical committee. Informed consent for the use of tissue samples was obtained from patients before cardiac surgery. The Manuscript report studies involving biologic materials from animals ( Danio rerio ). All animal practices comply with fundamental ethical standards and the Directive 2010/63/EU of the European Parliament, which addresses the protection and welfare of animals utilized for scientific aims. All euthanasia protocols were approved by the institutional Animal Welfare Body (OPBA). Consent for publication The authors declare to accept the Editorial Policies of the Journal, they approve the Ethics and they consent to participate in this publication. Availability of data and material Additional detailed Methods descriptions and additional Data are provided in Supplementary Material file. Competing Interests The authors declare no competing interests. Funding This work was supported by the Italian Ministry of Health [SG-2019-12369183] to EG; was supported by the Italian Ministry of University and Research [M.I.U.R PRIN2017, M.I.U.R PRIN2022] to EC; was supported by National Recovery and Resilience Plan–NextGeneration EU [MNESYS–SPOKE1 code:PE000006] to LeS, [MNESYS–PE12_SPOKE3] to LaS [Tuscany Health Ecosystem] to EC; was supported by Fondazione Cassa di Risparmio di Firenze ( project human brain optical mapping ) to EC; was supported by ISPRO-institutional funding to LP; was supported by Fondazione Cassa di Risparmio di Pistoia e Pescia (project INCITE) to LaS. Author Contribution Statement Elena Guzzolino (EG) , Valentina Balducci (VB) , Giada Allegro (GA) , Valentina Spinelli (VS) , Cesare Sala (CS) , Andrea Ninu (AN) , Leonardo Sacconi (LeS) , Francesca Lo Presti (FL) , Camilla Volpicini ( CV ),Matteo Cameli (MC) , Giulia Elena Mandoli ( GEM ), Perluigi Stefano (PS) , Matteo Lulli ( ML ), Martina Lucia Boccitto (MLB) ,Laura Poliseno (LP) , Raffaella De Paolo (RD) , Laura Sartiani (LaS) , Elisabetta Cerbai (EC) . Conceptualization and Project administration : EG , LaS and EC . Supervision : LaS and EC . Investigation and Visualization : EG , VB , GA , CV , VS , CS , AN , FL , CV , MLB , ML and RD . EG and VB equally experimentally contributed to this work. CS and EG performed the bioinformatics in silico analyses. EG, VB and GA performed the hiPS-CMs culture and differentiation, hiPSCs transfection, Intracell biosystem and MULTIPLE recordings. VB, GA and CV performed the electrophysiological analyses. EG , VS , AG, FL, CV and MLB performed RNA isolation and RT-PCR. EG, CV and RD supported zebrafish experiments. EG , VB , GA , CS and AN participated in creating the stable transgenic hiPS cell lines, immunofluorescence assays and cell line validation. GA performed the ELISA experiments. VB and VS performed the IL6+TOC experiments. ML performed confocal imaging. EG created the illustrations. Data curation : EG , VB , MC , GEM , ML , LaS and EC . Formal analysis : EG and VB . Resources : MC , GEM , PS , LeS , ML , LP , LaS and EC . Funding Acquisition : EG , LaS , LeS, LP and EC . Writing-original draft : EG , VB , LaS and EC . Writing-review & editing : All authors Acknowledgments We are truly grateful to Prof. Andras Varro of the University of Szeged (Hungary) for the collaboration. We thank Dr. Josè Manuel Pioner of University of Florence (Italy) who bestowed the wt-c11 hiPSC line. We very thank Dr. Letizia Pitto, the doctoral co-tutor of Dr. Guzzolino, for the kind bequest of the biobank in the laboratory after her retirement. We acknowledge the support and the professionality of Dr. Michele Dipalo and all Foresee biosystems group. We thank Fondazione Veronesi that supported the fellowship of Dr. Raffaella De Paolo. References Geng EH, Powell BJ, Goss CW, Lewis CC, Sales AE, Kim B. When the parts are greater than the whole: how understanding mechanisms can advance implementation research. Implement Sci. 2025;20(1):22. Thomas D, Christ T, Fabritz L, Goette A, Hammwöhner M, Heijman J, et al. German Cardiac Society Working Group on Cellular Electrophysiology state-of-the-art paper: impact of molecular mechanisms on clinical arrhythmia management. Clin Res Cardiol. 2019;108(6):577–99. Wakili R, Voigt N, Kääb S, Dobrev D, Nattel S. Recent advances in the molecular pathophysiology of atrial fibrillation. J Clin Invest. 2011;121(8):2955–68. Balan AI, Scridon A. MicroRNAs in atrial fibrillation - have we discovered the Holy Grail or opened a Pandora’s box? Front Pharmacol. 2025;16:1535621. Vardas EP, Theofilis P, Oikonomou E, Vardas PE, Tousoulis D. MicroRNAs in Atrial Fibrillation: Mechanisms, Vascular Implications, and Therapeutic Potential. Biomedicines. 2024;12(4):811. Popat A, Jnaneswaran G, Yerukala Sathipati S, Sharma PP. MicroRNAs in cardiac arrhythmias: Mechanisms, biomarkers, and therapeutic frontiers. Heart Rhythm. 2025;22(11):2971–82. Guzzolino E, Pellegrino M, Ahuja N, Garrity D, D’Aurizio R, Groth M, et al. miR-182-5p is an evolutionarily conserved Tbx5 effector that impacts cardiac development and electrical activity in zebrafish. Cell Mol Life Sci. 2020;77(16):3215–29. Brundel BJ, Van Gelder IC, Henning RH, Tieleman RG, Tuinenburg AE, Wietses M, et al. Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation. Circulation. 2001;103(5):684–90. Grunert M, Dorn C, Rickert-Sperling S. Cardiac Transcription Factors and Regulatory Networks. Adv Exp Med Biol. 2024;1441:295–311. van Ouwerkerk AF, Hall AW, Kadow ZA, Lazarevic S, Reyat JS, Tucker NR, et al. Epigenetic and Transcriptional Networks Underlying Atrial Fibrillation. Circ Res. 2020;127(1):34–50. Cakmak HA, Coskunpinar E, Ikitimur B, Barman HA, Karadag B, Tiryakioglu NO, et al. The prognostic value of circulating microRNAs in heart failure: preliminary results from a genome-wide expression study. J Cardiovasc Med (Hagerstown). 2015;16(6):431–7. Niu N, Miao H, Ren H. Effect of miR-182-5p on apoptosis in myocardial infarction. Heliyon. 2023;9(11):e21524. Liang Q, Chen H, Xu X, Jiang W. miR-182-5p Attenuates High-Fat -Diet-Induced Nonalcoholic Steatohepatitis in Mice. Ann Hepatol. 2019;18(1):116–25. Zhang A, Jin Y. MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway. Open Med (Wars). 2020;15(1):1202–12. Alhadidi QM, Xu L, Sun X, Althobaiti YS, Almalki A, Alsaab HO, et al. MiR-182 Inhibition Protects Against Experimental Stroke in vivo and Mitigates Astrocyte Injury and Inflammation in vitro via Modulation of Cortactin Activity. Neurochem Res. 2022;47(12):3682–96. Wang Z, Dai R, Ahmed SA. MicroRNA-183/96/182 cluster in immunity and autoimmunity. Front Immunol. 2023;14:1134634. Sameti P, Tohidast M, Amini M, Bahojb Mahdavi SZ, Najafi S, Mokhtarzadeh A. The emerging role of MicroRNA-182 in tumorigenesis; a promising therapeutic target. Cancer Cell Int. 2023;23(1):134. Ihara K, Sasano T. Role of Inflammation in the Pathogenesis of Atrial Fibrillation. Front Physiol. 2022;13:862164. Scott L, Li N, Dobrev D. Role of inflammatory signaling in atrial fibrillation. Int J Cardiol. 2019;287:195–200. Katkenov N, Mukhatayev Z, Kozhakhmetov S, Sailybayeva A, Bekbossynova M, Kushugulova A. Systematic Review on the Role of IL-6 and IL-1β in Cardiovascular Diseases. J Cardiovasc Dev Dis. 2024;11(7):206. Spinelli V, Laurino A, Balducci V, Gencarelli M, Ruzzolini J, Nediani C, et al. Interleukin-6 Modulates the Expression and Function of HCN Channels: A Link Between Inflammation and Atrial Electrogenesis. Int J Mol Sci. 2024;25(22):12212. Kishimoto T. The biology of interleukin-6. Blood. 1989;74(1):1–10. Balducci V, Credi C, Sacconi L, Romanelli MN, Sartiani L, Cerbai E. The HCN channel as a pharmacological target: Why, where, and how to block it. Prog Biophys Mol Biol. 2021;166:173–81. Iachetta G, Melle G, Colistra N, Tantussi F, De Angelis F, Dipalo M. Long-term in vitro recording of cardiac action potentials on microelectrode arrays for chronic cardiotoxicity assessment. Arch Toxicol. 2023;97(2):509–22. Credi C, Balducci V, Munagala U, Cianca C, Bigiarini S, de Vries AAF, et al. Fast Optical Investigation of Cardiac Electrophysiology by Parallel Detection in Multiwell Plates. Front Physiol. 2021;12:692496. Matiukas A, Mitrea BG, Qin M, Pertsov AM, Shvedko AG, Warren MD, et al. Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium. Heart Rhythm. 2007;4(11):1441–51. Guzzolino E, Milella MS, Forini F, Borsò M, Rutigliano G, Gorini F, et al. Thyroid disrupting effects of low-dose dibenzothiophene and cadmium in single or concurrent exposure: New evidence from a translational zebrafish model. Sci Total Environ. 2021;769:144703. Stillitano F, Lonardo G, Giunti G, Del Lungo M, Coppini R, Spinelli V, et al. Chronic Atrial Fibrillation Alters the Functional Properties of I f in the Human Atrium. Cardiovasc electrophysiol. 2013;24(12):1391–400. World Medical Association Declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects. Cardiovasc Res. 1997;35(1):2–3. Pei G, Chen L, Wang Y, He C, Fu C, Wei Q. Role of miR-182 in cardiovascular and cerebrovascular diseases. Front Cell Dev Biol. 2023;11:1181515. Wang Z, Dai R, Ahmed SA. MicroRNA-183/96/182 cluster in immunity and autoimmunity. Front Immunol. 2023;14:1134634. Dambal S, Shah M, Mihelich B, Nonn L. The microRNA-183 cluster: the family that plays together stays together. Nucleic Acids Res. 2015;43(15):7173–88. Varela M, Dios S, Novoa B, Figueras A. Characterisation, expression and ontogeny of interleukin-6 and its receptors in zebrafish (Danio rerio). Dev Comp Immunol. 2012;37(1):97–106. Zang L, Torraca V, Shimada Y, Nishimura N. Editorial: Zebrafish Models for Human Disease Studies. Front Cell Dev Biol. 2022;10:861941. Briggs JP. The zebrafish: a new model organism for integrative physiology. Am J Physiology-Regulatory Integr Comp Physiol. 2002;282(1):R3–9. Choi TY, Choi TI, Lee YR, Choe SK, Kim CH. Zebrafish as an animal model for biomedical research. Exp Mol Med. 2021;53(3):310–7. Nattel S, Heijman J, Zhou L, Dobrev D. Molecular Basis of Atrial Fibrillation Pathophysiology and Therapy: A Translational Perspective. Circ Res. 2020;127(1):51–72. O’Brien J, Hayder H, Zayed Y, Peng C. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front Endocrinol (Lausanne). 2018;9:402. Libermann TA, Baltimore D. Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol. 1990;10(5):2327–34. Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Sig Transduct Target Ther. 2017;2(1):17023. Gordon JW, Shaw JA, Kirshenbaum LA. Multiple Facets of NF-κB in the Heart: To Be or Not to NF-κB. Circul Res. 2011;108(9):1122–32. Wiersma M, Meijering RAM, Qi X, Zhang D, Liu T, Hoogstra-Berends F, et al. Endoplasmic Reticulum Stress Is Associated With Autophagy and Cardiomyocyte Remodeling in Experimental and Human Atrial Fibrillation. JAHA. 2017;6(10):e006458. Sirish P, Diloretto DA, Thai PN, Chiamvimonvat N. The Critical Roles of Proteostasis and Endoplasmic Reticulum Stress in Atrial Fibrillation. Front Physiol. 2022;12:793171. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, et al. Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA. 1993;90(21):10193–7. Beltrami M, Palazzuoli A, Padeletti L, Cerbai E, Coiro S, Emdin M, et al. The importance of integrated left atrial evaluation: From hypertension to heart failure with preserved ejection fraction. Int J Clin Pract. 2018;72(2):e13050. Puskaric S, Schmitteckert S, Mori AD, Glaser A, Schneider KU, Bruneau BG, et al. Shox2 mediates Tbx5 activity by regulating Bmp4 in the pacemaker region of the developing heart. Hum Mol Genet. 2010;19(23):4625–33. Zhu Y, Gramolini AO, Walsh MA, Zhou YQ, Slorach C, Friedberg MK, et al. Tbx5-dependent pathway regulating diastolic function in congenital heart disease. Proc Natl Acad Sci USA. 2008;105(14):5519–24. Kameswaran V, Bramswig NC, McKenna LB, Penn M, Schug J, Hand NJ, et al. Epigenetic Regulation of the DLK1-MEG3 MicroRNA Cluster in Human Type 2 Diabetic Islets. Cell Metabol. 2014;19(1):135–45. Liang X, Zhang Q, Cattaneo P, Zhuang S, Gong X, Spann NJ, et al. Transcription factor ISL1 is essential for pacemaker development and function. J Clin Invest. 2015;125(8):3256–68. Skwarek-Dziekanowska A, Wójtowicz-Ściślak A, Sobieszek G. Holt-Oram syndrome. Eur Rev Med Pharmacol Sci. 2024;28(1):336–41. Ma JF, Yang F, Mahida SN, Zhao L, Chen X, Zhang ML, et al. TBX5 mutations contribute to early-onset atrial fibrillation in Chinese and Caucasians. Cardiovasc Res. 2016;109(3):442–50. Lazzerini PE, Laghi-Pasini F, Acampa M, Srivastava U, Bertolozzi I, Giabbani B et al. Systemic Inflammation Rapidly Induces Reversible Atrial Electrical Remodeling: The Role of Interleukin‐6–Mediated Changes in Connexin Expression. JAHA [Internet]. 2019 Aug 20 [cited 2025 May 23];8(16). Available from: https://www.ahajournals.org/doi/ 10.1161/JAHA.118.011006 Nishimoto N, Kishimoto T, Humanized Antihuman. IL-6 Receptor Antibody, Tocilizumab. In: Chernajovsky Y, Nissim A, editors. Therapeutic Antibodies [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited 2025 Jun 8]. pp. 151–60. (Starke K, editor. Handbook of Experimental Pharmacology; vol. 181). Available from: http://link.springer.com/ 10.1007/978-3-540-73259-4_7 Xie F, Yun H, Levitan EB, Muntner P, Curtis JR. Tocilizumab and the Risk of Cardiovascular Disease: Direct Comparison Among Biologic Disease-Modifying Antirheumatic Drugs for Rheumatoid Arthritis Patients. Arthritis Care Res. 2019;71(8):1004–18. Ichiyama K, Gonzalez-Martin A, Kim BS, Jin HY, Jin W, Xu W, et al. The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression. Immunity. 2016;44(6):1284–98. DiFrancesco D, Borer JS. The Funny Current: Cellular Basis for the Control of Heart Rate. Drugs. 2007;67(Supplement 2):15–24. DiFrancesco D. The Role of the Funny Current in Pacemaker Activity. Circul Res. 2010;106(3):434–46. Difrancesco D. The pacemaker current in the sinus node. Eur Heart J. 1987;8(suppl L):19–23. Saponaro A, DiFrancesco D. Structure mirroring function: What’s the ‘matter’ with the funny current? J Physiol. 2025;JP287209. DiFrancesco D, Tromba C. Muscarinic control of the hyperpolarization-activated current (if) in rabbit sino‐atrial node myocytes. J Physiol. 1988;405(1):493–510. Ardhianto P, Yuniadi Y. Biomarkers of Atrial Fibrillation: Which One Is a True Marker? Cardiol Res Pract. 2019;2019:1–8. Vardas EP, Theofilis P, Oikonomou E, Vardas PE, Tousoulis D. MicroRNAs in Atrial Fibrillation: Mechanisms, Vascular Implications, and Therapeutic Potential. Biomedicines. 2024;12(4):811. Popat A, Jnaneswaran G, Sathipati SY, Sharma PP. MicroRNAs in cardiac arrhythmias: mechanisms, biomarkers and, therapeutic frontiers. Heart Rhythm. 2025;S1547-5271(25)02512-3. Jia X, Cheng X, Wu N, Xiang Y, Wu L, Xu B, et al. Prognostic value of interleukin-6 in atrial fibrillation: A cohort study and meta-analysis. Anatol J Cardiol. 2021;25(12):872–9. Zhou P, Waresi M, Zhao Y, Lin HC, Wu B, Xiong N, et al. Increased serum interleukin-6 level as a predictive biomarker for atrial fibrillation: A systematic review and meta-analysis. Rev Port Cardiol. 2020;39(12):723–8. Wu N, Xu B, Xiang Y, Wu L, Zhang Y, Ma X, et al. Association of inflammatory factors with occurrence and recurrence of atrial fibrillation: A meta-analysis. Int J Cardiol. 2013;169(1):62–72. Scardigli M, Cannazzaro S, Coppini R, Crocini C, Yan P, Loew LM, et al. Arrhythmia susceptibility in a rat model of acute atrial dilation. Prog Biophys Mol Biol. 2020;154:21–9. Heijman J, Voigt N, Wehrens XHT, Dobrev D. Calcium dysregulation in atrial fibrillation: the role of CaMKII. Front Pharmacol. 2014;5:30. Denham NC, Pearman CM, Caldwell JL, Madders GWP, Eisner DA, Trafford AW et al. Calcium in the Pathophysiology of Atrial Fibrillation and Heart Failure. Front Physiol [Internet]. 2018 Oct 4 [cited 2025 May 23];9. Available from: https://www.frontiersin.org/article/ 10.3389/fphys.2018.01380/full Dobrev D, Voigt N, Wehrens XHT. The ryanodine receptor channel as a molecular motif in atrial fibrillation: pathophysiological and therapeutic implications. Cardiovascular Res. 2011;89(4):734–43. Denham NC, Pearman CM, Caldwell JL, Madders GWP, Eisner DA, Trafford AW, et al. Calcium in the Pathophysiology of Atrial Fibrillation and Heart Failure. Front Physiol. 2018;9:1380. Darbar D, Kannankeril PJ, Donahue BS, Kucera G, Stubblefield T, Haines JL, et al. Cardiac Sodium Channel ( SCN5A ) Variants Associated with Atrial Fibrillation. Circulation. 2008;117(15):1927–35. Gao G, Dudley SC, Redox Regulation NF-κB, Fibrillation A. Antioxid Redox Signal. 2009;11(9):2265–77. Tam AB, Mercado EL, Hoffmann A, Niwa M. ER Stress Activates NF-κB by Integrating Functions of Basal IKK Activity, IRE1 and PERK. Koritzinsky M, editor. PLoS ONE. 2012;7(10):e45078. Prell T, Lautenschläger J, Weidemann L, Ruhmer J, Witte OW, Grosskreutz J. Endoplasmic reticulum stress is accompanied by activation of NF-κB in amyotrophic lateral sclerosis. J Neuroimmunol. 2014;270(1–2):29–36. Zhu X, Huang L, Gong J, Shi C, Wang Z, Ye B, et al. NF-κB pathway link with ER stress-induced autophagy and apoptosis in cervical tumor cells. Cell Death Discov. 2017;3(1):17059. Miao KZ, Kim GY, Meara GK, Qin X, Feng H. Tipping the Scales With Zebrafish to Understand Adaptive Tumor Immunity. Front Cell Dev Biol. 2021;9:660969. Franza M, Varricchio R, Alloisio G, De Simone G, Di Bella S, Ascenzi P, et al. Zebrafish (Danio rerio) as a Model System to Investigate the Role of the Innate Immune Response in Human Infectious Diseases. IJMS. 2024;25(22):12008. Tokar T, Pastrello C, Rossos AEM, Abovsky M, Hauschild AC, Tsay M, et al. mirDIP 4.1—integrative database of human microRNA target predictions. Nucleic Acids Res. 2018;46(D1):D360–70. Mukherji S, Ebert MS, Zheng GXY, Tsang JS, Sharp PA, Van Oudenaarden A. MicroRNAs can generate thresholds in target gene expression. Nat Genet. 2011;43(9):854–9. Van Den Berg NWE, Kawasaki M, Nariswari FA, Fabrizi B, Neefs J, Van Der Made I, et al. MicroRNAs in atrial fibrillation target genes in structural remodelling. Cell Tissue Res. 2023;394(3):497–514. Chen Y, Wakili R, Xiao J, Wu CT, Luo X, Clauss S, et al. Detailed characterization of microRNA changes in a canine heart failure model: Relationship to arrhythmogenic structural remodeling. J Mol Cell Cardiol. 2014;77:113–24. Chen YC, Voskoboinik A, Gerche AL, Marwick TH, McMullen JR. Prevention of Pathological Atrial Remodeling and Atrial Fibrillation. J Am Coll Cardiol. 2021;77(22):2846–64. Ruppert M, Korkmaz-Icöz S, Benczik B, Ágg B, Nagy D, Bálint T, et al. Pressure overload-induced systolic heart failure is associated with characteristic myocardial microRNA expression signature and post-transcriptional gene regulation in male rats. Sci Rep. 2023;13(1):16122. Al-Othman S, Boyett MR, Morris GM, Malhotra A, Mesirca P, Mangoni ME, et al. Symptomatic bradyarrhythmias in the athlete—Underlying mechanisms and treatments. Heart Rhythm. 2024;21(8):1415–27. Li P, Zhu X, Liu M, Wang Y, Huang C, Sun J, et al. Impact of gene-environment interactions on atrial fibrillation and cardiac structure. Sci Rep. 2025;15(1):16893. Wass SY, Hahad O, Asad Z, Li S, Chung MK, Benjamin EJ, et al. Environmental Exposome and Atrial Fibrillation: Emerging Evidence and Future Directions. Circul Res. 2024;134(8):1029–45. 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Firenze","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Sartiani","suffix":""},{"id":612030006,"identity":"7b210759-3478-4209-a4fb-5fd5c5c20bfe","order_by":17,"name":"Elisabetta Cerbai","email":"","orcid":"","institution":"University of Florence: Universita degli Studi di Firenze","correspondingAuthor":false,"prefix":"","firstName":"Elisabetta","middleName":"","lastName":"Cerbai","suffix":""}],"badges":[],"createdAt":"2026-02-06 14:50:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8808389/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8808389/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105641701,"identity":"77d9b955-041a-4cb2-b353-a1456e2e7be8","added_by":"auto","created_at":"2026-03-28 16:34:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38310111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-182 overexpression reduces spontaneous AP frequency of hiPSC-CMs.\u003c/strong\u003e A) Cartoon showing the experimental plan used to obtain and test \u003cem\u003ewt-c11 CT\u003c/em\u003e and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e hiPSC transgenic lines. B) Cartoon showing the experimental plan used to differentiate \u003cem\u003ewt-c11 CT\u003c/em\u003e and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e hiPSC lines into CMs and the maturation timeline used for functional analyses. C) Representative traces of the spontaneous APs recorded in \u003cem\u003ewt-c11 CT\u003c/em\u003e CMs and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e CMs by the IntraCell platform. D) Plots showing the spontaneous AP frequency (bpm) in \u003cem\u003ewt-c11CT\u003c/em\u003e (n=22 TR of 7 BR) CMs and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e (n=27 TR of 8 BR) CMs at 30 days of maturation, recorded using the IntraCell platform. Statistics: Student's t-test with α=0,05, ***\u003cem\u003ep\u003c/em\u003e=0,0002. E) Plots showing the coefficient of variation (%) of spontaneous\u003c/p\u003e\n\u003cp\u003eAP frequency in \u003cem\u003ewt-c11 CT\u003c/em\u003e (n=7 BR) CMs and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e (n=8 BR) CMs at 30 days of maturation. Statistics: Student's t-test with α=0,05, **\u003cem\u003ep\u003c/em\u003e=0,0016. BR: \u003cu\u003eBiological Replicates\u003c/u\u003e, TR: \u003cu\u003eTechnical Replicates\u003c/u\u003e.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/c61b3fb30180effd61dee1b9.png"},{"id":105641700,"identity":"f80e4c1a-0e9f-4590-a06e-2ef92d4dd378","added_by":"auto","created_at":"2026-03-28 16:34:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43215821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-182 overexpression in hiPS-CMs downregulates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHCN4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression and regulates IL-6. \u003c/strong\u003eA) Cartoon showing the experimental plan used to test if miR-182 expression regulates IL-6 in hiPS-CMs.B) Plots representing \u003cem\u003eHCN4\u003c/em\u003e expression levels measured by RT-PCR assay in \u003cem\u003ewt-c11 CT\u003c/em\u003e (n=10 BR) CMs and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e (n=9 BR) CMs at 30 days of maturation. Statistics: Student’ t-test with α= 0.05, *\u003cem\u003ep\u003c/em\u003e=0,038. C) Plot representing \u003cem\u003eIL-6\u003c/em\u003e expression levels measured by RT-PCR in \u003cem\u003ewt-c11\u003c/em\u003e \u003cem\u003eCT\u003c/em\u003e (n=10 BR) and \u003cem\u003ewt-c11\u003c/em\u003e \u003cem\u003emiR-182\u003c/em\u003eCMs (n=9 BR) at 30 days of maturation. D) Plot representing IL-6 protein (pg/mL) analysed by ELISA assay on growth media of \u003cem\u003ewt-c11 miR-182 \u003c/em\u003e(n=12 BR) and \u003cem\u003ewt-c11 CT\u003c/em\u003e (n=12 BR) CMs at 30 days of maturation. Statistics: Student's t-test with α=0,05; ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0,0001. E) Plot representing \u003cem\u003eHCN4\u003c/em\u003e expression levels measured by RT-PCR assay in \u003cem\u003ewt-c11 miR-182 \u003c/em\u003eCMs, with/without TOC (n=3/3 BR, n=6/6 TR). Statistics: Student’ t-test with α= 0.05, *\u003cem\u003ep\u003c/em\u003e=0,0247. BR: \u003cu\u003ebiological Replicates\u003c/u\u003e, TR: \u003cu\u003eTechnical Replicates\u003c/u\u003e. F) Cartoon summarizing the relationship between miR-182 and IL-6 in hiPS-CMs.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/9f95ef6af8cd0beb199abd9b.png"},{"id":105728949,"identity":"f4cdb027-7d48-4acc-af1f-3297c987516b","added_by":"auto","created_at":"2026-03-30 11:13:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24944372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-182 overexpression in hiPS-CMs impairs muscarinic response.\u003c/strong\u003e A) Plot shows the variation of spontaneous AP rate recorded by MULTIPLE system on \u003cem\u003ewt-c11 CT\u003c/em\u003e(black circles) and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e(red circles) CMs in the presence of0.1-1-10µM ACh or 0.1-1µM ACh plus 1µM Iso. Only statistically different values are marked, while not significant are omitted. Statistics: Student's t-test with α=0.05; ****p\u0026lt;0.0001 0.1µM ACh \u003cem\u003eCT\u003c/em\u003e (n=12 BR) vs 0.1µM ACh \u003cem\u003emiR-182\u003c/em\u003e(n=14 BR); ****p\u0026lt;0.0001 1µM ACh \u003cem\u003eCT\u003c/em\u003e (n=20 BR) vs 1µM ACh \u003cem\u003emiR-182\u003c/em\u003e(n=11 BR); *** \u003cem\u003ep\u003c/em\u003e=0,000310µM ACh \u003cem\u003eCT\u003c/em\u003e (n=8 BR) vs 10µM ACh \u003cem\u003emiR-182\u003c/em\u003e(n=8 BR); **\u003cem\u003ep\u003c/em\u003e=0.0017 0.1µM ACh + 1µM Iso \u003cem\u003eCT\u003c/em\u003e (n=12 BR) vs 0.1µM ACh + 1µM Iso \u003cem\u003emiR-182\u003c/em\u003e (n=11 BR); *\u003cem\u003ep\u003c/em\u003e=0.0212 1µM ACh + 1µM Iso \u003cem\u003eCT\u003c/em\u003e(n=18 BR) vs 1µM ACh + 1µM Iso \u003cem\u003emiR-182\u003c/em\u003e (n=8 BR). B) Representative traces of spontaneous APs recorded from \u003cem\u003ewt-c11 CT\u003c/em\u003e (solid lines) and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e(dashed lines) CMs in control conditions (grey), after exposure to ACh (black) and to ACh+Iso (red). BR: \u003cu\u003eBiological replicates\u003c/u\u003e, TR: \u003cu\u003eTechnical replicates\u003c/u\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/8eb66f742b4e41e8df4af529.png"},{"id":105641702,"identity":"c61275f9-00a8-4f0a-aa11-306010cf478c","added_by":"auto","created_at":"2026-03-28 16:34:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60900345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cardiac selective overexpression of miR182 on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIl6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression in zebrafish heart and direct effect of hIL-6 on heart rate. \u003c/strong\u003eA) Histogram representing mean values±SEM of \u003cem\u003eil6 \u003c/em\u003eexpression measured by RT-PCR (3 TR) in hearts dissected from 4dpf \u003cem\u003eMyl-7-m182-OE \u003c/em\u003eor \u003cem\u003eMyl7-GAL4 \u003c/em\u003ezebrafish embryos (3-pools of 20 dissected hearts each line were collected and gathered for a total of 60 heart each group). Statistics: Student’ t-test with α=0.05; ***\u003cem\u003ep\u003c/em\u003e=0,0002. B) Cartoon representing the experimental plan used to test the effect of hIL-6 protein on zebrafish heart rate [microinjection in the pericardial space in zebrafish embryos A (red)=atrium; V (yellow)=ventricle]. The arrow indicates the pericardium(P). The needle is inserted in the pericardial space of the embryo (Image created with GIMP software by Dr. Guzzolino). C)Plot representing the heart rate of \u003cem\u003eTg(myl7:EGFP)\u003c/em\u003eembryos at 2dpf microinjected with hIL-6 (n=55 BR), or \u003cem\u003esham\u003c/em\u003e solution (n=59 BR), into the pericardial space. Statistics: Student’ t-test with α=0.05; **p=0,0017. BR: \u003cu\u003eBiological Replicates\u003c/u\u003e, TR: \u003cu\u003eTechnical Replicates.\u003c/u\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/15f4120f883d9f7057a8a568.png"},{"id":105728684,"identity":"4047095e-44fa-421f-87db-55a2fad03720","added_by":"auto","created_at":"2026-03-30 11:12:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30668790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-182 overexpression alters the expression of genes involved in human CMs function and remodelling. \u003c/strong\u003ePlots showing expression levels of genes involved in CMs intracellular calcium handling (RYR, NCX, CASQ2, ATP2A2), membrane associated voltage-dependent ion channels (CACNB4, CACNA2D1, CACNB2, CACNA1C, SCN5A, HCN1, HCN2, HCN3), regulatory kinase (CAMKII), peptides (NPPA), muscarinic receptors (CHRM2) and adrenergic receptors (ADRB1, ADRB2, ADRB3), measured by RT-PCR in \u003cem\u003ewt-c11 CT \u003c/em\u003e(n=10 BR)\u003cem\u003e and wt-c11 miR-182 \u003c/em\u003e(n=9 BR) CMs at 30 days of maturation.\u003c/p\u003e\n\u003cp\u003eStatistics: Student’ t-test with α=0.05; RYR *\u003cem\u003ep\u003c/em\u003e=0,0106; NCX \u003cem\u003ep\u003c/em\u003e=0,104; ATP2A2 \u003cem\u003ep\u003c/em\u003e=0,66; CACNA1C \u003cem\u003ep\u003c/em\u003e=0,16; CACNB2 \u003cem\u003ep\u003c/em\u003e=0,105; CACNA2D1 ***\u003cem\u003ep\u003c/em\u003e=0,0002; CACNB4 *\u003cem\u003ep\u003c/em\u003e=0,0103; HCN1 *\u003cem\u003ep\u003c/em\u003e=0,022; HCN2 **\u003cem\u003ep\u003c/em\u003e=0,0039; HCN3 ***\u003cem\u003ep\u003c/em\u003e=0,0001; SCN5A **\u003cem\u003ep\u003c/em\u003e=0,0039; CASQ2 \u003cem\u003ep\u003c/em\u003e=0,13; CHRM2 *\u003cem\u003ep\u003c/em\u003e=0,019; NPPA *\u003cem\u003ep\u003c/em\u003e=0,0329; CAMKIIγ ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0,0001; CAMKIIδ \u003cem\u003ep\u003c/em\u003e=0,108; CHRNG *\u003cem\u003ep\u003c/em\u003e=0,04; TNFα \u003cem\u003ep\u003c/em\u003e=0,22; ADRB1 ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0,0001; ADRB2 \u003cem\u003ep\u003c/em\u003e=0,23; ADRB3 *\u003cem\u003ep\u003c/em\u003e=0,016. BR: \u003cu\u003eBiological Replicates\u003c/u\u003e.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/c40911f9c7c215a86ac1462a.png"},{"id":105641699,"identity":"be039fde-592c-43ee-b8bf-6c320dad6502","added_by":"auto","created_at":"2026-03-28 16:34:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":37374997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-182 overexpression upregulates NF-κB and ER-stress related genes in human CMs\u003c/strong\u003e. Plots showing expression levels of genes involved in inflammatory response and ER-stress measured by RT-PCR in \u003cem\u003ewt-c11 CT\u003c/em\u003e (n=10 BR) and \u003cem\u003ewt-c11 miR-182\u003c/em\u003e (n=9 BR) CMs at 30 days of maturation. Statistics: Student’ t-test with α=0.05. NF-κB **\u003cem\u003ep\u003c/em\u003e=0,0057; IL-1β **\u003cem\u003ep\u003c/em\u003e=0,0079; TNFα \u003cem\u003ep\u003c/em\u003e=0,26; TGFβ \u003cem\u003ep\u003c/em\u003e=0,067; GADD34 ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0,0001; ATF *\u003cem\u003ep\u003c/em\u003e=0,016; EDEM1 ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0,0001; CHOP ***\u003cem\u003ep\u003c/em\u003e=0,0007. BR: \u003cu\u003eBiological Replicates\u003c/u\u003e.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/097965f2ad710a5ae0c40b9a.png"},{"id":105641695,"identity":"eb2cf4c9-25b3-4754-aa5d-ff32af027b4c","added_by":"auto","created_at":"2026-03-28 16:34:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":25681913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMiR-182-5p is differently expressed in left atrial samples accordance with atrial disease severity and correlates with IL-6 expression. \u003c/strong\u003eA) Cartoon describing the main steps conducted for measuring the gene expression analyses in human left atrial samples, categorized by atrial disease severity. B-C) Plots representing miR-182-5p (B)/\u003cem\u003eIL-6\u003c/em\u003e (C) expression levels in CT (n=11), LA-D (n=18) and AF (n=20) patients, measured by RT-PCR analysis. Statistics: Kruskal-Wallis test with α=0,05 (B) **\u003cem\u003ep\u003c/em\u003e=0,0025/(C) *\u003cem\u003ep\u003c/em\u003e=0,0241 [Dunn’s comparison: (B) LA-D vs CT *MRD=-15.85; AF vs CT **MRD=-17,70; LA-D vs AF MRD=-1,844; (C) LA-D vs CT MRD=-11,17; AF vs CT *MRD=-14,45; LA-D vs AF MRD=-3.283]. D) Graph representing the correlation between miR-182-5p vs IL-6 mRNA expression in human left atrial samples, in semi-logarithmic scale [x=Log(x)]. Equation γ=4,505χ; *\u003cem\u003ep\u003c/em\u003e=0,0134. (Note: the semi-logarithmic scale better represented the relationship among data, however data expressed in linear scale followed the equation γ=0,1099χ+3,275 with *\u003cem\u003ep\u003c/em\u003e=0,0212).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/e2d5fd9da23cba66d3dbfa3e.png"},{"id":105641696,"identity":"39ee2525-b445-495c-aaf3-d483ad4c2328","added_by":"auto","created_at":"2026-03-28 16:34:58","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4940981,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/45e6d02a0a25445478a8a9b7.png"},{"id":105641694,"identity":"c4c2223e-d401-4026-8f65-e0b1ed42ec13","added_by":"auto","created_at":"2026-03-28 16:34:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2760195,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialJTM.docx","url":"https://assets-eu.researchsquare.com/files/rs-8808389/v1/a8c0df2df795a7aa93965a93.docx"}],"financialInterests":"","formattedTitle":"MiR-182 regulates IL-6 in the cardiac context: implications for human atrial electrical remodelling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDisturbances of the heart\u0026rsquo;s electrical activity (\u0026lsquo;arrhythmias\u0026rsquo;) remain a leading cause of morbidity and mortality worldwide, reinforcing the need to advance mechanistic understanding and identify new targets for therapeutic interventions (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Arrhythmias, including atrial fibrillation (AF), are progressive diseases that are caused and aggravated by multiple factors, such as genetics, epigenetics, aging, and life style (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Appearance and persistence of arrhythmias is directed by the progressive occurrence of structural and functional remodelling of the cardiac tissue. Among several molecular mechanisms involved, the synthesis of key cardiac proteins may be disrupted by modifications of gene transcription and post-transcription due to changes in the concentration and activity of specific microRNAs (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Our previous studies in animal models showed that the cardiac specific overexpression of miR-182 acts as an important promoter of adverse cardiac phenotype, triggering arrhythmogenic mechanisms, such as bradycardia, early and delayed after depolarization, and heart block. These modifications are also accompanied by the reduced expression of cardiac calcium channel subunits (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), an alteration commonly observed in atrial arrhythmias (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Basal expression of miR-182-5p was previously documented in vertebrate and mammalian hearts (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), found to be negatively regulated by the T-box transcription factor 5 (TBX5), whose mutations are associated with the human rare genetic condition Holt-Oram Syndrome (HOS) and with AF, as revealed by genome-wide association studies (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Microarray profiling of plasma obtained from patients with chronic congestive heart failure led to propose miR-182-5p as a potential prognostic marker for cardiac outcomes (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), in line with the evidence that miR-182-5p promotes myocardial apoptosis in experimental myocardial infarction (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, miR-182 is also involved in inflammatory processes (\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) known to be active in atrial remodelling and arrhythmias (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In line with this, our recent study provided a basis to link IL-6 to human atrial dilation, a condition that predisposes atria to arrhythmic dysfunction (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The same study showed that IL-6 deregulates electrogenesis of human cardiomyocytes hampering the expression and function of the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite extensive understanding of the factors driving IL-6 upregulation and secretion in extra-cardiac tissue (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), the specific signals triggering IL-6 transcription in the heart still remain unclear.\u003c/p\u003e \u003cp\u003eIn this study we aimed to assess the functional role of miR-182 in the human cardiac context, hypothesizing that it contributes to the activation of inflammatory responses in cardiac cells, and addressed this question using a wide range of models and experimental approaches. Using human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes (CMs) and overexpressing miR-182, we explored the causal mechanism linking miR-182 to IL-6 transcription and secretion in CMs, and to alterations in electrogenesis. Then, using zebrafish embryos we assessed \u003cem\u003ein vivo\u003c/em\u003e the functionality of miR-182/IL-6 axis and studied the direct effects of elevate IL-6 levels in the beating heart. Finally, we analysed left atrial samples from healthy donors and patients with different severity of atrial disease to establish a translational benchmark.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Culture and cardiac differentiation from human induced pluripotent stem cells\u003c/h2\u003e \u003cp\u003eHuman induced pluripotent stem cells (hiPSCs; \u003cem\u003ewt-c11\u003c/em\u003e or UCSFi001-A, obtained from fibroblasts of a healthy 30\u0026ndash;33 years old man and gifted by Dr. Pioner, Department of Clinical and Experimental Medicine, Division of Physiology, University of Florence, Italy) were cultured on a Corning\u0026reg;Matrigel matrix using mTeSR Plus medium (Stem Cell Technologies) and splitted every 4\u0026ndash;5 days with 0.5 mM EDTA in phosphate-buffered saline (PBS) solution, supplementing the medium with ROCK inhibitor (Y27632, 5\u0026micro;M). The monolayer cardiac differentiation protocol used the cardiac PSC Cardiomyocyte Differentiation Kit (Life Technologies, Thermo Fisher scientific), following manufacturer\u0026rsquo;s instructions. Initially, hiPSC colonies were dissociated into single cells using TrypLE for 5 minutes at 37\u0026deg;C, which were seeded into mTeSR Plus with Y27632 onto Matrigel-coated wells of a 24-well plate at a cell density of 80.000 cells/well (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). When cells reached 70\u0026ndash;90% confluency (2/3 days later), the culture medium was changed to Cardiomyocyte Differentiation Medium A (day 0) to induce mesodermal differentiation, followed by subsequent medium change to Medium B on day 2 and to Medium C on day 4, which facilitated final cardiomyocyte differentiation. Spontaneously beating monolayers appeared around day 8\u0026ndash;10, and at day 12 the Medium C was replaced with RPMI plus B27 supplement (Life Technologies, Thermo Fisher scientific) to further CMs maturation (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Transgenic hiPSC lines production\u003c/h2\u003e \u003cp\u003eTo obtain a stable overexpression of miR-182 (\u003cem\u003ehiPSC wt-c11 miR-182)\u003c/em\u003e, 70%-80% confluent hiPSCs were transfected with the pLV-[hsa-mir-182] plasmid biosettia, via Lipo3000 (Invitrogen), following manufacturer instructions. After 2 days, cells were selected with mTeSR Plus containing Puromycin (1\u0026micro;g/mL) for 1 week. The transgenic hiPSC line used as control (\u003cem\u003ehiPSC wt-c11 CT\u003c/em\u003e) was obtained transfecting the same plasmid, previously excided from the pre-miR-182 sequence, using \u003cem\u003eXhoI\u003c/em\u003e restriction enzyme. The \u003cem\u003ebona fide\u003c/em\u003e of the transgenic lines was tested with Trilineage differentiation, RT-PCR, MTT and Proliferation assays (Figures Supplemental 4\u0026ndash;5 and Supplementary Methods).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 IL-6 treatment in hiPS-CMs\u003c/h2\u003e \u003cp\u003e29-day differentiated hiPS-CMs were serum-starved overnight in RPMI (B27 minus) medium. On the 30th day of cardiac differentiation, hiPS-CMs were incubated with TOC (Tocilizumab 10 \u0026micro;g/mL, MedChemExpress) solubilized in RPMI\u0026thinsp;+\u0026thinsp;B27 medium for 30 minutes and then stimulated with IL-6 (50 ng/mL, Sigma Aldrich) or vehicle (PBS) for 24 hours. After treatment, the medium was changed for electrophysiological and/or molecular analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Electrophysiological recordings and analyses with IntraCell\u003c/h2\u003e \u003cp\u003eRecordings of intracellular-like APs from hiPS-CMs were obtained on microelectrode arrays (MEA) with the IntraCell system from Foresee Biosystems S.R.L. in combination with a MEA2100-Lite system from Multi Channel Systems GmbH (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). On the 27th day of maturation, the differentiated hiPS-CMs cultured in 24-well plate were incubated for 1h with RPMI supplemented with B27 and Y27632 (10 \u0026micro;M). The cells were then dissociated into single cells using TrypLE (10 minutes at 37\u0026deg;C) and centrifuged at 300g for 3 minutes. Following the resuspension in RPMI supplemented with B27, 10% fetal bovine serum, and 10 \u0026micro;M Y27632, the hiPS-CMs were seeded at a density of 16.000 cells per well onto 60-6wellMEA200/30iR-Ti-rcr MEA plates with 54 electrodes and 6 independent wells (9 electrodes per well) pre-coated with 8\u0026micro;L human fibronectin (1 \u0026micro;g/mL; Gibco). The following day, half of the medium was replaced with RPMI supplemented with B27. On the second day after the dissociation, the medium was refreshed, and cells recovered the spontaneous beating. On the third day, corresponding to the 30th day of cardiac maturation, the IntraCell system was used to apply laser-mediated cell poration on CMs in contact with the MEA electrodes to obtain intracellular-like APs. With this technique it is not possible to determine the absolute membrane potential, therefore data are expressed in arbitrary units (A.U.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Electrophysiological recordings and analyses with High-Throughput MULTIPLE system\u003c/h2\u003e \u003cp\u003eThe spontaneous activity of hiPS-CMs was recorded using the High-Throughput MULTIPLE system, as previous described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). HiPS-CMs were incubated with a near-infrared voltage-sensitive dye (VSD) di-4-ANBDQPQ (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) (2 \u0026micro;g/mL) for 9 minutes and then washed with dye-free Tyrode\u0026rsquo;s solution (D-(+)-glucose 10 mM, NaCl 140 mM, KCl 5.4 mM, MgCl\u003csub\u003e2\u003c/sub\u003e 1.2 mM, CaCl\u003csub\u003e2\u003c/sub\u003e 1.8 mM, Hepes 5.0 mM, adjusted to pH 7.3 with NaOH). Cultures were placed on the High-Throughput MULTIPLE microscope stage, where the temperature was constantly maintained at 37\u0026deg;C during recordings. A red LED (SOLIS-623C, Thorlabs) followed by a band-pass filter (625PB50 Omega optical) illuminated the monolayer of hiPS-CMs and the fluorescence signal was collected in forward direction using a camera lens (MVL12M43, Thorlabs) placed in front of a sCMOS camera (ORCA-Flash 4.0 V3, Hamamatsu) operating at a frame rate of 100Hz. A long-pass filter (LP700; Omega optical) was placed in front of the camera lens. Fluorescent signals obtained from the High-Throughput recordings were associated with regions of interest (ROI) and were photo-bleached, corrected, normalized, and temporally filtered using LabVIEW (National Instruments, Austin, TX, United States), Fiji-ImageJ (National Institutes of Health, Bethesda, MD) and OriginLab 2023b (Northampton, MA, United States) software. OriginLab 2023b software was used to analyse AP frequency. With this technique it is not possible to determine the absolute membrane potential, therefore data are expressed in arbitrary units (A.U.).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Zebrafish lines husbandry\u003c/h2\u003e \u003cp\u003eThe zebrafish facility has held the authorization n\u0026deg;297/2012-A since 12/21/2012. Fish were bred in standard laboratory conditions (Westerfield M zebrafish book) in Zebrafish Housing Systems (Tecniplast, Varese, Italy), as previously reported (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). All animal practices comply with fundamental ethical standards and the Directive 2010/63/EU of the European Parliament, which addresses the protection and welfare of animals utilized for scientific aims. Natural spawning was used to produce embryos, which were then incubated in E3 medium at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C. The \u003cem\u003eTg(myl7:EGFP)\u003c/em\u003e transgenic line, in AB genetic background was used in this study and biobank materials from \u003cem\u003eTg(myl7:GAL4,EGFP)xTg(Sce.4xUAS:miR-182,cry:EGFP)\u003c/em\u003e transgenic lines [to simplify the term \u0026ldquo;\u003cem\u003eTg(myl7\u0026thinsp;\u0026gt;\u0026thinsp;miR-182)\u0026rdquo;\u003c/em\u003e will be used in the text meanwhile \u0026ldquo;Myl7-m182-OE\u0026rdquo;, or \u0026ldquo;Myl7-GAL4\u0026rdquo; for controls, will be used in images to refer at siblings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA extraction and RT-PCR\u003c/h2\u003e \u003cp\u003eAtrial human samples from cardiac surgery were quickly frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C or used for RNA isolation (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Briefly, RNA was extracted using QIAzol (Qiagen), following the manufacturer's instructions, and subsequently quantified with SmartDrop Nano Spectrophotometers (Accuris Instruments-Bioclass). RT-PCR were performed using CFX Opus 96 system (Biorad).\u003c/p\u003e \u003cp\u003eFor gene expression analyses the RPL19 gene was used as housekeeping reference gene, while for miRNA expression analyses the U6 was used as standard internal control. RNA was retro-transcribed in cDNA using iScript\u0026trade; cDNA Synthesis Kit (BIORAD) or SuperScript\u0026trade; IV VILO\u0026trade; Master Mix (Thermo Fisher Scientific) for genes and using Mir-X\u0026trade; miRNA First-Strand Synthesis Kit (TAKARA) for miRNAs.\u003c/p\u003e \u003cp\u003eHeart tissues RNA from 4dpf zebrafish, obtained from \u003cem\u003eTg(myl7:GAL4,EGFP)xTg(Sce.4xUAS:miR-182,cry:EGFP)\u003c/em\u003e transgenic lines, are derived from a tissue biobank stored in the laboratory as a result of previous research studies (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Beating heart tissues were surgically extracted from embryos, previously anesthetized with 0,016% tricaine (standard 1X MS-222; Sigma-Aldrich) for 15 minutes, and washed in L-15 (Leibovit\u0026rsquo;s)\u0026thinsp;+\u0026thinsp;10%FBS to eliminate residual blood cells in the tissue before being stored in QIAzol for RNA extraction. The residual embryos were soon transferred in tricaine overdose solution (500mg/L) for 30 minutes and then in 1\u0026ndash;1,5% of sodium hypochlorite (bleach solution) for 5\u0026ndash;10 minutes, as described in paragraph 2.10. No additional embryos of this line were raised for this work, observing the ethics of biologists in accordance with the 3R principles. For gene expression analyses the \u003cem\u003eeef1a1l1\u003c/em\u003e and \u003cem\u003erpl13\u003c/em\u003e genes were used to normalize data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Pericardial microinjection in zebrafish embryos and video recordings\u003c/h2\u003e \u003cp\u003eAt the zygote stage, transgenic zebrafish embryos of the \u003cem\u003eTg(myl7:EGFP)\u003c/em\u003e line were collected and raised in E3 medium at 28\u0026deg;C. At 2dpf, prior of the microinjection, embryos were transferred in E3 medium at room temperature (26.5\u0026deg;C) and acclimated for 2h before heart rate recordings to prevent bias in physiological measurements. Room temperature was maintained constant during the entire timeline of the experiments with the supply of an air conditioner. Before video recordings and microinjection, embryos were anesthetized with 0.016% tricaine (standard 1X MS-222; Sigma-Aldrich) for 15 minutes; the solution was prepared starting from a 25X stock (400mg/100 mL of buffered DD water at pH\u003csub\u003e˜\u003c/sub\u003e7) and diluted in E3 medium at room temperature. During the experiments the solution temperature was measured with a bath thermometer to ensure its stability. Embryos of the same population were divided into 2 groups (pre-sham and pre-hIL-6) and the heart rate was measured before microinjection to verify the similarity of heart rate variability between the two groups and avoid stochastic bias caused by selection. Heart rate was 133.4\u0026thinsp;\u0026plusmn;\u0026thinsp;12.3 and 133.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3 bpm in pre-sham and pre-IL-6, respectively; coefficient of variation was 9.2% and 8.4% for pre-sham and pre-IL-6, respectively. After initial (T0) video recordings, anesthetized zebrafish embryos were positioned on a plate filled with 1.5% solidified agar low melting, slumped laterally exposing the pericardial portion in opposition with the direction of needle (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Embryo insensitivity to touch was assessed before the microinjection. For each embryo, 1 nL (corresponding to total 5.4 picograms) of human recombinant IL-6 protein solution (10\u0026micro;M, Sigma Aldrich), or 1nL PBS 1X, used as control, was microinjected in the pericardial region. After microinjection the embryos were transferred in E3 medium for 1 hour to avoid possible side effects of anaesthetic and in compliance with the good practices of laboratory animal care suggested by the institutional Animal Welfare Body (OPBA). Before the second round of video recordings, embryos were again anesthetized with 0.016% tricaine for 15 minutes. The heart rate was calculated a posteriori analysing video recorded using a Leica MZ10F microscope supplied with Leica DFC 3000G camera and LASX software. The procedures caused a 5\u0026ndash;10% mortality. At the end of the experiments, we performed the euthanasia procedure as described in paragraph 2.10.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Euthanasia procedure\u003c/h2\u003e \u003cp\u003eAfter the experiments, the zebrafish embryos (\u0026lt;\u0026thinsp;5dpf) were euthanized with overdose of tricaine 500mg/L (buffered solution at pH\u003csub\u003e˜\u003c/sub\u003e7) in E3 medium, in accordance with the European Union Directive 2010/63/EU and the transposed Italian law, Legislative Decree 4 March 2014, n.26. All euthanasia protocols were approved by the institutional Animal Welfare Body (OPBA). The embryos were kept in the tricaine solution for 30\u0026ndash;50 minutes, after the cessation of opercular movement and heartbeat to ensure a complete and irreversible cessation of vital signs. A second step of extra-precaution was adopted prior to disposal, transferring no vital embryos in 1-1.5% sodium hypochlorite (bleach solution) for 5\u0026ndash;10 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Patients and Ethical statement\u003c/h2\u003e \u003cp\u003e For this study we used cardiac tissue samples excided from left atrial appendage, obtained through collaborations between the University of Florence and the Cardio-Surgery Units of AOU-Careggi (Florence, Italy) and the \u0026ldquo;Policlinico Le Scotte\u0026rdquo; (Siena, Italy). Samples from control donor hearts not suitable for transplantation were from the University of Szeged (Hungary). The investigation conforms with the principles outlined in the \u0026ldquo;Declaration of Helsinki\u0026rdquo; of the World Medical Association and was approved by the local ethical committee (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Informed consent for the use of tissue samples was obtained from patients before cardiac surgery.\u003c/p\u003e \u003cp\u003eBasic demographic (age and sex) information was available for all samples, while clinical information (echocardiography and associated comorbidities, therapy) was acquired only for samples from patients undergoing surgery (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplemental \u003cb\u003eS-\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To overcome this inconvenient we stratified samples according with cardiac phenotype severity, creating 3 clinical groups: control (CT); LA-D (pathological cardiac condition and different grades of atrial dilation) and AF (chronic atrial fibrillation). A total of 49 samples were collected: 11 CT samples were from subjects with no history of atrial diseases, undergoing surgery after accidental event or patients undergoing mitral valve replacement without showing a pathological cardiac condition and atrial dilation; 18 LA-D samples were obtained from patients undergoing cardiac surgery, principally aortic or mitral valve replacement, showing a pathological cardiac condition and different grades of atrial dilation. Among them, 16 LA-D samples were from patients in sinus rhythm, and 2 were from patients presenting paroxysmal AF. Finally, 20 samples were from patients with chronic AF and atrial dilation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMain characteristics of the patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePatients\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eLA volume (F)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eLA volume (M)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eSinus rhythm\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u0026deg;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003e(F - M)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e(min-max)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003e(min-max) (mL)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e(min -max)(mL)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003e(%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026ndash;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40\u0026ndash;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLA-D\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u0026thinsp;\u0026minus;\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u0026ndash;84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u0026ndash;130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45\u0026ndash;153\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAF\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u0026thinsp;\u0026minus;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65\u0026ndash;85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65\u0026ndash;140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e80\u0026ndash;208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistics and graphs\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPAD prism software. The statistical test used is indicated in figure legend. Normal distribution of data was tested to choose between parametric or non-parametric inference. We adopted *for p\u0026thinsp;\u0026lt;\u0026thinsp;0,05; **for p\u0026thinsp;\u0026lt;\u0026thinsp;0,01; ***for p\u0026thinsp;\u0026lt;\u0026thinsp;0,001; ****for p\u0026thinsp;\u0026lt;\u0026thinsp;0,0001, with α\u0026thinsp;=\u0026thinsp;0,05. Figures were also prepared by using the \u003cem\u003eBiorender\u003c/em\u003e software (ID: university-of-florence\u0026mdash;dept-of-neuroscience). Part of the cartoon in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e was created with GIMP software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 MiR-182 overexpression in hiPS-CMs decreases spontaneous AP frequency\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a first step, we performed a preliminary \u003cem\u003ein silico\u003c/em\u003e investigation to unveil if direct miR-182-5p targeted genes might be described in key GO and KEEG terms related to human cardiac arrhythmogenic pathways [see \u003cstrong\u003eSupplemental Methods\u003c/strong\u003e for details]. In line with our previous integrated analyses on putative target genes of miR-182-5p shared by mouse and zebrafish\u0026nbsp;(7), the analysis showed that miR-182-5p directly modulates human cardiac genes involved in electrical coupling, muscle cellular homeostasis and calcium handling, suggesting a similar regulatory framework operating in our experimental setting [\u003cstrong\u003eFigures Supplemental 1-3\u003c/strong\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo assess the functional consequences of\u0026nbsp;miR-182\u0026ndash;mediated dysregulation of genes involved in cardiac electrogenesis, we investigated the spontaneous electrical activity in \u003cem\u003ewt-c11\u003c/em\u003e CT and \u003cem\u003emiR-182\u003c/em\u003e CMs, which stably overexpresses miR-182 in differentiated cells (\u003cstrong\u003eFigure 1A\u003c/strong\u003e). We first confirmed that main cell line properties were not altered, documenting that pluripotency and trilineage differentiation ability were similar in \u003cem\u003ewt-c11\u003c/em\u003e CT and \u003cem\u003emiR-182\u0026nbsp;\u003c/em\u003ecell lines (\u003cstrong\u003eFigure Supplemental 4\u003c/strong\u003e), as measured in different pools of cells. Moreover, pluripotent \u003cem\u003ewt-c11\u003c/em\u003e \u003cem\u003emiR-182\u003c/em\u003e hiPSCs showed similar viability and rate of growth compared to \u003cem\u003ewt-c11\u003c/em\u003e CT hiPSCs, as demonstrated by MTT and proliferation assays (\u003cstrong\u003eFigure Supplemental 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe then used the IntraCell platform system to study spontaneous action potentials (APs) of cultured hiPSC-CMs at 30 days of differentiation. Recordings revealed marked differences between AP traces obtained from \u003cem\u003ewt-c11 miR-182\u003c/em\u003e and \u003cem\u003ewt-c11 CT\u003c/em\u003e CMs (\u003cstrong\u003eFigure 1B\u003c/strong\u003e), with the former\u0026nbsp;exhibiting lower absolute values of spontaneous frequency (\u003cstrong\u003eFigure 1C\u003c/strong\u003e) and higher coefficient of variation (CV) in spontaneous frequency compared to CT CMs (\u003cstrong\u003eFigure 1D\u003c/strong\u003e). Average values of AP frequency was 74.5\u0026plusmn;0.9 beat per minute (bpm) in \u003cem\u003ewt-c11 CT\u0026nbsp;\u003c/em\u003eCMs and 42.7\u0026plusmn;3.0 bpm in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003eCMs (n=22-27, mean\u0026plusmn;SEM, p\u0026lt;0.0001 unpaired t-test). CV in beating rate was calculated from a series of consecutive APs recorded during a period of 10 seconds, and repeated for 3 to 4 times during the same recording. It was 2.59\u0026plusmn;3.43% in \u003cem\u003ewt-c11 CT\u0026nbsp;\u003c/em\u003eCMs and 15.28\u0026plusmn;7.76% \u003cem\u003ein wt-c11 miR-182\u003c/em\u003e CMs (n=7-8, mean\u0026plusmn;SD p\u0026lt;0.01 unpaired t-test). Overall, these data demonstrate the occurrence of cellular electrophysiological abnormalities induced by miR-182 overexpression.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.2 MiR-182 overexpression in hiPSC-CMs regulates HCN4 expression via upregulation and secretion of IL-6\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the reduction of spontaneous AP frequency, its increase of variation in CMs overexpressing miR-182, and the role of \u003cem\u003ef-\u003c/em\u003echannels in the spontaneous beating rate, we hypothesized that these electrical disturbances might be related to a reduced functional expression of the major cardiac \u003cem\u003ef-\u003c/em\u003echannel isoform, HCN4. RT-PCR analyses confirmed our hypothesis of a downregulation of HCN4 (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). Then, based on the recognized regulation of inflammatory pathways (30,31) by miR-182, and our previous report on HCN downregulation induced by IL-6 on hiPSC-CMs (21), we wondered whether in our setting overexpression of miR-182 regulates HCN4 channel transcript through the increased expression and activity of IL-6 levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIndeed, differentiated CMs overexpressing miR-182 displayed significantly higher levels of IL-6 transcription compared to \u003cem\u003ewt-c11 CT\u0026nbsp;\u003c/em\u003eCMs\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u003cstrong\u003eFigure 2B-E)\u003c/strong\u003e, suggesting that miR-182 acts as upstream regulator of this cytokine in human CMs. To determine whether the enhanced levels of IL-6 mRNA translate into increased secretion of protein,\u0026nbsp;IL-6 concentrations in cell culture media were measured by ELISA assay. IL-6 protein levels were significantly higher in the media of wt-c11 miR-182 CMs compared with wt-c11 CT CMs, demonstrating that enhanced IL-6 secretion by CMs parallels the increased transcription of IL-6 (\u003cstrong\u003eFigure 2C-E\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to evaluate a possible opposite cause-effect mechanism between IL-6 and miR-182 levels in human CMs, hiPSC-CMs at 30 days of maturation were treated with 50 ng/mL recombinant IL-6 for 24h, with or without 10 \u0026micro;g/mL tocilizumab (TOC), a monoclonal antibody that inhibits IL-6 signalling activation by binding to IL-6 receptors (\u003cstrong\u003eFigure Supplemental 5-A)\u003c/strong\u003e. IL-6 treatment did not increase miR-182-5p expression levels in hiPSC-CMs (\u003cstrong\u003eFigure Supplemental 5-B\u003c/strong\u003e), as well as that of its pri-miRNA (\u003cstrong\u003eFigure Supplemental 5-C\u003c/strong\u003e), which also contains miR-183 and miR-96\u0026nbsp;(7,32), thus excluding IL-6 is an upstream regulator of miR-182-5p in the cardiac context, at least in short term response.\u003c/p\u003e\n\u003cp\u003eTo further support the evidence that miR-182 indirectly affects HCN4 transcript levels through IL-6 overproduction, we treated \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003eCMs with 10 \u0026micro;g/mL TOC for 24h to block IL-6 receptors. Results demonstrated that TOC significantly rescued HCN4 expression (\u003cstrong\u003eFigure 2E\u003c/strong\u003e), suggesting that at least part of the reduction of HCN4 transcript passes through the activation of IL-6 expression and the stimulation of IL-6 receptors.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.3 MiR-182 overexpression in hiPS-CMs alters muscarinic response\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeing HCN channels key players regulated by the cardiac autonomic system, we investigated the functional impact of miR-182 overexpression on the responses to acetylcholine (ACh) and Isoprenaline (Iso) to mimic the muscarinic and \u0026beta;-adrenergic effect, respectively. ACh, at a concentration ranging from 0.1 to 10 \u0026mu;M, dose-dependently reduced spontaneous AP rate (\u003cstrong\u003eFigure 3\u003c/strong\u003e), as expected from activation of muscarinic receptors in cardiac pacemaker-like cells. The effect was significantly greater in \u003cem\u003ewt-c11 miR-182\u003c/em\u003e compared to the \u003cem\u003ewt-c11 CT\u0026nbsp;\u003c/em\u003eCMs, suggesting an increased muscarinic signalling induced by miR-182 overexpression. Hence, the spontaneous beating even ceased in some cells exposed to the highest ACh concentrations. Conversely, Iso (1 \u0026mu;M) significantly increased the beating rate in \u003cem\u003ewt-c11 CT\u003c/em\u003e CMs treated with 0.1 and 1 \u0026mu;M ACh, as well as in \u003cem\u003ewt-c11 miR-182\u003c/em\u003e CMs exposed to ACh 0.1 \u0026mu;M, while it failed to modify the rate when ACh was applied at 1 \u0026mu;M concentration. Overall, these data suggest that the response to autonomic signals is also altered by miR-182 overexpression in hiPSC-CMs.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e3.4 Cardiac selective overexpression of miR-182 upregulates \u003cem\u003eil6\u003c/em\u003e in zebrafish heart\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo reveal if cardiac miR-182 overexpression upregulates cardiac IL-6 \u003cem\u003ein vivo\u003c/em\u003e, we studied 4dpf zebrafish \u003cem\u003eTg(myl7\u0026gt;m182)\u0026nbsp;\u003c/em\u003eembryos characterized by a cardiac selective overexpression of miR-182. We compared them with age-matched control \u003cem\u003eTg(Myl7:GAL4,EGFP)\u003c/em\u003e zebrafish embryos (\u003cem\u003eMyl7-GAL4\u003c/em\u003e) (7). In zebrafish, miR-182 overexpression induces heart morphological defects in a dose dependent manner, recapitulating the main features of cardiac HOS pathology and triggering arrhythmias even in embryos without cardiac morphological defects (7). As previously described, heterozygous \u003cem\u003eMyl7-m182-OE\u003c/em\u003e zebrafish embryos did not show any structural and morphological cardiac defects, while they developed cardiac arrhythmias as early as 3dpf (7). Expression analysis on isolated hearts demonstrated a significant overexpression of cardiac \u003cem\u003eil6\u003c/em\u003e in \u003cem\u003eMyl7-m182-OE\u003c/em\u003e compared to \u003cem\u003eMyl7-GAL4\u0026nbsp;\u003c/em\u003ezebrafish embryos (\u003cstrong\u003eFigure 4-A\u003c/strong\u003e),\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003esuggesting that cardiac miR-182/IL-6 axis represents a molecular circuit that is conserved across vertebrates.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e3.5 Human IL-6 protein decreases heart rate in zebrafish\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLittle is known about the role of \u003cem\u003eil6\u0026nbsp;\u003c/em\u003ein non-immune cells during zebrafish development and literature lack of information about its role in heart organogenesis and function. Analyses of \u003cem\u003eIL-6\u003c/em\u003e gene among species reveals a good grade of structural conservation through phylogenetic evolution and the zebrafish il6 protein shares a significant grade of similarity with human IL-6 (hIL-6) protein tertiary structure, as modelled by Valera et al., 2012 (33). On this basis and given that zebrafish is increasingly used as a powerful model to investigate the molecular circuits involved in human pathology (34\u0026ndash;36), we sought to use zebrafish embryos to validate \u003cem\u003ein vivo\u003c/em\u003e the hypothesis that human IL-6 hampers cardiac pacemaking. We assessed the heart rate of two groups of 2dpf \u003cem\u003eTg(myl7:EGFP)\u003c/em\u003e zebrafish embryos, selected to have values of cardiac frequency equally distributed within a physiological range (\u003cstrong\u003eFigure 4-B\u003c/strong\u003e). After 1h from microinjection\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003einto the pericardial space of 1nL of 10\u0026nbsp;mM hIL-6 solution or vehicle (control solution), heart rate of hIL-6 group displayed a significant reduction compared to the sham group, while the coefficients of variation were 21.2% and 9.8% in the hIL-6 and sham group, respectively (\u003cstrong\u003eFigure 4-C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.6 MiR-182 overexpression deregulates the expression of genes involved in human CMs function and remodelling\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that the molecular machinery involved in the regulation of cardiac rhythm employs a complex interplay of multiple targets (37) and that the deregulation of single miRNAs inside cells can unbalance extensive molecular pathways (38), we thought to obtain a wider view of the impact of miR-182 overexpression on several genes involved in cardiac electrogenesis. We compared \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003eand \u003cem\u003ewt-c11 CT\u003c/em\u003e CMs at 30 days of maturation by transcription analysis of key genes involved in cardiac function and other genes known to be associated to cardiac remodelling and arrhythmogenesis. Results (\u003cstrong\u003eFigure 5; Figure Supplemental 7\u003c/strong\u003e) evidenced several modifications of genes directly involved in intracellular calcium handling, as expected by previous \u003cem\u003ein silico\u003c/em\u003e investigations and \u003cem\u003ein vivo\u003c/em\u003e characterization(7), which showed a significant downregulation of RYR and a significant upregulation of CACNB4 in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003ecompared to \u003cem\u003ewt-c11 CT\u003c/em\u003e CMs. NCX, CACNA1C and CACNB2 transcripts showed a clear, albeit not significant, trend toward reduction in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003ecompared\u003cem\u003e\u0026nbsp;to wt-c11 CT\u003c/em\u003e CMs, while CACNA2D1 showed a not significant trend toward increase. Expression levels of other ion channels were also modified: beyond HCN4 that was significantly downregulated in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003ecompared\u003cem\u003e\u0026nbsp;\u003c/em\u003eto\u003cem\u003e\u0026nbsp;wt-c11 CT\u003c/em\u003e CMs, HCN2, HCN3 and SCN5A were all significantly upregulated. We also observed a significant downregulation of HCN1, CHRMR2 and ADRB3 in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003ecompared\u003cem\u003e\u0026nbsp;\u003c/em\u003eto\u003cem\u003e\u0026nbsp;wt-c11 CT\u003c/em\u003e CMs, while ADRB1 was up-regulated. Finally, CAMKII\u0026gamma; was significantly upregulated in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003ecompared\u003cem\u003e\u0026nbsp;\u003c/em\u003eto\u003cem\u003e\u0026nbsp;wt-c11 CT\u003c/em\u003e CMs. In line with the increased expression of IL-6 levels, the expression of its well-known regulator NF-\u0026kappa;B was also upregulated in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003eCMs (\u003cstrong\u003eFigure 6\u003c/strong\u003e), unveiling the parallel activation of an inflammatory signalling\u0026nbsp;(39). We tested the expression of IL-1\u0026beta;, TNF\u0026alpha; and TGF\u0026beta; (\u003cstrong\u003eFigure 6\u003c/strong\u003e), also regulated by NF-\u0026kappa;B in inflammation (40) and in heart disease (41), but interestingly only IL-6 resulted upregulated by miR-182 overexpression in the hiPSC-CMs. We also tested the expression of IL-10, but it resulted not detectable by RT-PCR in \u003cem\u003ewt-c11\u0026nbsp;\u003c/em\u003ehiPSC-CMs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, to gain deeper insights into the regulatory crosstalk within the miR-182/IL-6 axis, we investigated the transcriptional response of endoplasmic reticulum (ER) stress-related genes, such as EDEM1, GADD34, CHOP and ATF4, as EDEM1 and other genes belonging to the \u0026ldquo;Path:hsa04141-Protein processing in endoplasmic reticulum\u0026rdquo; resulted direct targets of miR-182-5p in our \u003cem\u003ein silico\u003c/em\u003e analyses (not shown). The ER-stress signalling is known to be associated with cardiac adverse remodelling and enhanced risk for AF (42,43). As evident in \u003cstrong\u003eFigure 6\u003c/strong\u003e, all genes\u0026nbsp;EDEM1, GADD34, CHOP and ATF4 resulted upregulated in\u003cem\u003e\u0026nbsp;wt-c11 miR-182\u0026nbsp;\u003c/em\u003ecompared to\u003cem\u003e\u0026nbsp;CT\u0026nbsp;\u003c/em\u003eCMs, enforcing the hypothesis of an indirect activation of IL-6 through an intracellular cell stress-sensing signalling regulating NF-\u0026kappa;B overexpression, a known transcription factor for IL-6 (44). Additional RT-PCR are reported in \u003cstrong\u003eFigure Supplemental 7\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.7 MiR-182-5p is expressed in left human atria in accordance with disease severity and positively correlates with IL-6 expression levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe reasoned that a conserved cardiac miR-182/IL-6 axis could represent a relevant biomarker in the clinical setting. Thus, we performed RT-PCR analysis of left atrial (LA) samples obtained from 49 patients undergoing surgery to analyse miR-182-5p expression levels. Samples were divided into 3 groups according to disease severity: 11 control patients (CT), with no history of atrial pathology, 18 patients with LA dilation and elevate risk to develop AF (LA-D) (45), and 20 patients with chronic AF (AF) (\u003cstrong\u003eFigure 7A\u003c/strong\u003e). The main features are summarized in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompared to CT, miR-182-5p expression levels resulted significantly higher in LA-D\u003cem\u003e\u0026nbsp;\u003c/em\u003eand AF patients (\u003cstrong\u003eFigure 7B\u003c/strong\u003e). Despite a clear trend toward increased values of miR-182-5p in AF patients, the difference with LA-D\u003cem\u003e\u0026nbsp;\u003c/em\u003epatients was not significant, likely due to the large range of variation in data distribution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the same atrial samples, IL-6 expression levels were significantly increased in AF compared to CT patients, while the difference did not reach the statistical significance in LA-D (\u003cstrong\u003eFigure 7C\u003c/strong\u003e). When plotted against miR-182-5p expression levels, IL-6 levels and miR-182-5p expression levels showed a similar trend with significant correlation, suggesting that these two markers of left atrial myopathy go hand in-hand. (\u003cstrong\u003eFigure 7D\u003c/strong\u003e). As previously mentioned, the pri-miR-182 is transcribed as a single transcript including the sequences of pre-miR-183 and pre-miR-96, which are both part of the highly conserved polycistronic miR\u003csub\u003e\u0026tilde;\u003c/sub\u003e183 cluster\u0026nbsp;(32). To uncover differences of expression among mature miRNAs belonging to miR\u003csub\u003e\u0026tilde;\u003c/sub\u003e183 cluster, RT-PCR analyses of the same atrial samples evidenced that miR-183-5p and miR-96-5p were both significantly up-regulated in LA-D\u003cem\u003e\u0026nbsp;\u003c/em\u003esamples compared to CT, while no difference was present for AF patients (\u003cstrong\u003eFigure Supplemental 8\u003c/strong\u003e). MiR-183-5p and miR-96-5p levels did not correlate with IL-6 expression (\u003cstrong\u003eFigure Supplemental 8\u003c/strong\u003e), suggesting the unique major interplay of miR-182-5p with IL-6 in left human atria.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMiR-182-5p was previously identified as negative modulator of TBX5 action, resulting overexpressed in Holt-Oram syndrome (HOS) murine and zebrafish animal models, a rare genetic autosomal conserved disease associated to TBX5 mutations\u0026nbsp;(7). TBX5 is also involved in pacemaker formation\u0026nbsp;(46), and cardiac electromechanical dysfunction in congenital diseases\u0026nbsp;(47). In the hierarchical panel of cardiac gene expression, TBX5 is upstream of the transcription factor ISL-1, a gene identified as direct target of miR-182-5p by DIANA\u0026nbsp;(48)\u0026nbsp;and a key regulator of pacemaker formation and HCN4 expression\u0026nbsp;(49). Even in the absence of gross cardiac malformations, electrical anomalies are the most striking events in some families and HOS patients often require pacemaker implantation\u0026nbsp;(50). Early onset AF may appear in the childhood\u0026nbsp;(51), with dramatic consequences on patients\u0026rsquo; lifespan. The role of IL-6 in the development and progression of cardiac disease has never been inferred in this setting, and literature lacks information about the relationship between TBX5 and IL-6. Our observation linking miR-182 to IL-6 expression and secretion in cardiac myocytes provides important insight and warrants further investigation into the role of inflammatory signals within the cardiac context, rather than solely at systemic level, where it may exacerbate the pathogenesis of pre-existing or acquired molecular imbalances. These factors deserve further evaluation in view of their value in forecasting the progression of the arrhythmic phenotypes in paediatric cohorts, particularly in patients coping with complex scenarios.\u003c/p\u003e\n\u003cp\u003eIndeed, systemic inflammation is associated with electric atrial remodelling and IL-6 is able to downregulate atrial connexins\u0026nbsp;(52). We have previously shown that exposure of cardiomyocytes to IL-6 directly decreases the expression and function of HCN channels\u0026nbsp;(21), which contribute to spontaneous activity in immature cardiomyocytes and to pacemaker rhythm in sinoatrial node cells. Similarly, \u003cem\u003ewt-c11 miR-182\u003c/em\u003e CMs exhibited a decreased expression of HCN4 along with a reduction of spontaneous rhythm compared to control CMs. The role of IL-6 was further reinforced by a partial rescue of HCN4 expression in \u003cem\u003ewt-c11 miR-182\u003c/em\u003e CMs exposed to tocilizumab (TOC, \u003cstrong\u003eFigure 2E\u003c/strong\u003e). TOC represents the first humanized monoclonal antibody blocking IL-6 signalling by targeting the IL-6 receptor\u0026nbsp;(53)\u0026nbsp;and recent clinical works reported its safety and efficacy in several settings of cardiovascular diseases (54)\u003cem\u003e.\u003c/em\u003e This finding adds consistent translational value to current knowledge and support future investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe biological regulation of miR\u003csub\u003e\u0026tilde;\u003c/sub\u003e183 cluster expression has been extensively studied in the immune system, showing that IL-6 upregulates the expression of miR-183 in T helper 17 cells, via STAT3 signalling\u0026nbsp;(55). This finding prompted us to infer that, also in hiPSC-CMs, IL-6 could regulate miR-182 expression: instead, our results demonstrated the opposite path, i.e. that miR-182 overexpression causes the up-regulation of IL-6 in hiPSC-CMs. \u003cem\u003eVice versa\u003c/em\u003e, exposing control hiPSC-CMs to IL-6 did not result in increased miR-182 overexpression, thus establishing a downstream cascade between the two markers detected in the bioptic samples, which has never been reported.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother interesting, original observation of our study refers to the altered response to acetylcholine of \u003cem\u003ewt-c11 miR-182\u003c/em\u003e CMs. When exposed to ACh, the slow intrinsic rate of CMs overexpressing miR-182 was further reduced, an effect significantly more pronounced than in control CMs. Since these data were obtained by measuring frequency of spontaneous APs, we cannot infer whether the hypersensitivity to ACh depends on a prominent response of ACh-sensitive potassium channels (and consequent hyperpolarization) or to a more marked negative shift of the \u003cem\u003ef-current\u003c/em\u003e, due to inhibition of adenylyl cyclase and intracellular cAMP reduction, or a combination of these mechanisms\u0026nbsp;(56\u0026ndash;59). A more detailed electrophysiological study is warranted. However, the observation that adding Iso could partially restore spontaneous rhythm, likely through recovery of intracellular cAMP levels, suggests that the second mechanism plays a role at least at the lowest ACh concentrations\u0026nbsp;(60).\u003c/p\u003e\n\u003cp\u003eAs previously described, fish with myocardial-specific miR-182 overexpression show irregular heart rate, passing through slow rate to bursts of activity\u0026nbsp;(7). This behaviour resembles somehow that observed in the \u003cem\u003ein vitro\u003c/em\u003e model of hiPS-CMs, as shown in \u003cstrong\u003eFigure 4\u003c/strong\u003e, further suggesting a conserved role of miR-182 in cell-autonomous functions of CMs through evolution. More directly related to our hypothesis, here we report original observations obtained \u003cem\u003ein vivo\u003c/em\u003e in zebrafish lines, supporting the conserved pathway linking miR-182 and IL-6 (\u003cstrong\u003eFigure 4A\u003c/strong\u003e) and the role of IL-6 as a direct modulator of cardiac rhythm (\u003cstrong\u003eFigure 4C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe prognostic stratification of patients at risk of developing AF remains challenging, due to multifactorial pathogenesis of this arrhythmia\u0026nbsp;(61). Among biomarkers, miRNAs have long been proposed as promising candidates in cardiovascular diseases (CVDs)\u0026nbsp;(62,63). Our work showed a clear-cut overexpression of miR-182 in human samples from diseased atria and suggests, for the first time, a direct correlation between this miRNA and IL-6 expression \u003cem\u003eex vivo\u003c/em\u003e. IL-6 is a pro-inflammatory cytokine expressed by cells during stress conditions. Recent studies suggest IL-6 levels in blood serum as a prognostic marker for the propensity to AF\u0026nbsp;(64,65), as well as for an increased risk of recurrence following electrical cardioversion and catheter ablation\u0026nbsp;(66). In the context of atrial remodelling, increased circulating levels of IL-6 were found to be significantly associated with a greater risk of stroke and all-cause mortality in AF patients, leading to propose IL-6 as prognostic marker of AF\u0026nbsp;(64).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe evidence on the possible causal link between miRNA-182 and IL-6 could not be inferred based on the correlation measured in human atrial samples and, for obvious reasons, is not possible to provide longitudinal clinical data to directly link the level of miR-182-5p and the consequent development of AF, as well as performing multivariate adjustment or retrospective assessment with our clinical information. However, the two factors resulted concordantly present in the groups and principally higher in both patients with atrial dilation, a clinical condition recognized as risk factor for AF, and with chronic AF\u0026nbsp;(67). Notwithstanding the complex and multifactorial AF pathogenesis, the novel evidence of the miRNA-182/IL-6 axis in atrial myopathy in patients in sinus rhythm, claims the attention to early markers, with the aim to detect and prevent the progression toward chronic arrhythmias.\u003c/p\u003e\n\u003cp\u003eBesides the effect on IL-6 transcription and release, the overall modified gene expression profile in \u003cem\u003ewt-c11 miR-182\u0026nbsp;\u003c/em\u003eCMs at 30 days of maturation supports the hypothesis that miR-182 overexpression may contribute to an increased risk for the development of AF. In our experimental samples (\u003cstrong\u003eFigure 5\u003c/strong\u003e) we observed upregulation of CAMKII\u0026gamma;, whose overexpression is reported to contribute to atrial pathogenesis\u0026nbsp;(68). As expected, our molecular analyses confirmed a completely deregulated arrangement in gene expression of the genes involved in calcium handling, being the calcium signalling a pivotal actor in both the development and progression of AF\u0026nbsp;(69\u0026ndash;71)\u003cem\u003e.\u003c/em\u003e We also reported an increased expression of SCN5A, encoding for the alpha subunit of the Nav1.5 cardiac sodium channel; although it is difficult to speculate further, it is worth to mention a recent clinical report of a gain-of-function variant that has been associated with AF\u0026nbsp;(72)\u003cem\u003e.\u003c/em\u003e Finally, we observed that the increment of miR-182 levels in hiPS-CMs caused ER stress, as confirmed by the upregulation of ER-stress activated genes (\u003cstrong\u003eFigure 6\u003c/strong\u003e), closing the molecular circuit between miR-182 and IL-6 axis, through the expression of NF-\u0026kappa;B regulator similarly to other cellular contexts\u0026nbsp;(73\u0026ndash;76). However, at the moment, we cannot exclude a feed-forward molecular loop \u0026nbsp;between IL-6 and NF-\u0026kappa;B in the cardiac context\u0026nbsp;(44).\u003c/p\u003e\n\u003cp\u003eAs a limitation of our work, we did not directly measure the increase in IL-6 specifically in CMs. However, the increased release of the cytokine into the medium and the recovery HCN4 expression following IL-6R blockade both suggest an upregulation of IL-6 synthesis and release by CMs. The characterization of CMs differentiated from \u003cem\u003ewt-c11 miR-182\u003c/em\u003e hiPSCs allowed us to demonstrate the overexpression of IL-6 gene and \u0026ndash; in parallel \u0026ndash; the increased release of this cytokine in the culture medium. Also, we did not attempt to carry out a thorough electrophysiological characterization of \u003cem\u003ewt-c11 m182\u003c/em\u003e CMs in basal conditions and after stimulation with Ach. Since altered ion channel function is a major contributor to arrhythmia susceptibility, such a study should be conducted in hiPSC derived CMs, possibly differentiated into the atrial phenotype.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study we used the \u003cem\u003eTg(myl7\u0026gt;miR-182)\u003c/em\u003e zebrafish line, in which the deregulation of miR-182 is confined in the myocardium; nevertheless, we cannot exclude cell-cell communication signalling among different \u0026nbsp;cardiac cell phenotypes, including potential involvement of the endocardium. However, the use of the zebrafish model was essential for its \u0026ldquo;simplicity\u0026rdquo; in manipulation to demonstrate a short-term response in heart rate variability, exploring the direct possibility to microinject hIL-6 directly in the pericardial space. The expression of \u003cem\u003eil6\u0026nbsp;\u003c/em\u003eand its receptors in zebrafish was detectable early during the first week of development, also in heart tissue, with an increment after 5dpf\u0026nbsp;(33). Of note, the timeline used to test hIL-6 microinjected directly in the pericardial space of 2dpf zebrafish embryos was strategic because the innate immune system is rudimental and the terminal maturation phase occurs later, despite the signals for primitive haematopoiesis already started\u0026nbsp;(77,78). A compelling question for future research is whether a stable myocardial miR-182 overexpression is effective to trigger the activation of resident immune cells in juvenile and adult fish. Exploring this path could offer a unique opportunity to decode the consequences on heart function and tissue remodelling and better refine the ability to predict cardiac outcomes in the \u003cem\u003ein vivo\u003c/em\u003e model. While the two-chambered heart of zebrafish precludes the modelling of AF, this organism remains a valuable platform for assessing drug responses and validating key molecular circuitries at the transcriptional level. Future investigations in large mammal models may provide the necessary framework to overcome these biological constraints.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExploiting the \u003cem\u003ein silico\u003c/em\u003e miRDB tool\u0026nbsp;(79), both IL-6 and HCN4 are reported as putative targets of miR-182, albeit with a low score, being supported only by 3 and 5 predictive algorithms, respectively (not shown). We cannot exclude the possibility that miR-182-5p directly recognize these target genes in the cardiac context, nor the possibility it may act as small activating RNA to enhance IL-6 expression. In perturbed states, miRNA overexpression can drive the occupancy of weak binding sites that are otherwise neglected under physiological conditions, thereby expanding the miRNA\u0026apos;s regulatory landscape\u0026nbsp;(80). Future analyses may help resolve these possibilities.\u003c/p\u003e\n\u003cp\u003eA relevant and original new result from our study is the evidence of an increased miR-182 level hand to hand with the overexpression of IL-6 in non-genetic AF. The miR-182 upregulation in myocardial tissue emerged from a recent clinical study of AF and it resulted more negatively correlated among its predicted targets\u0026nbsp;(81), however no experimental data are reported in literature about its role in electrogenesis and arrhythmias. At the moment we are not able to dissect the multifactorial mechanisms leading to the up-regulation of miR-182-5p, likely different from the mechanisms mediated by genetic factors, as in the HOS. A recent experimental study in the rat animal model of pressure overload-induced systolic heart failure, a known risk factor predisposing AF\u0026nbsp;(82,83), focused the attention on miRNAs roles and, even not directed to miR-182, this miRNA resulted in the cluster analyses\u0026nbsp;(84). Compelling future perspective on possible epigenetic/multifactorial factors (like aging, enduring exercise, environmental contaminants)\u0026nbsp;(85\u0026ndash;87)\u0026nbsp;might unearth what are the triggers activating miR-182 and its subsequent contribution to pathological cardiac remodelling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, our findings propose miR-182 overexpression as a marker for the propensity to AF, possibly linked to a burst of IL-6 production. Further investigation should assess miR-182-5p expression not only in left atrial samples of patients undergoing surgery, but also in blood serum and in co-morbidity conditions.\u003c/p\u003e\n"},{"header":"Abbreviations","content":"\u003cp\u003eACh= acetylcholine \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAF= atrial fibrillation\u003c/p\u003e\n\u003cp\u003eCMs= cardiomyocytes\u003c/p\u003e\n\u003cp\u003eCT= control/controls\u003c/p\u003e\n\u003cp\u003eCV= coefficient of variation\u003c/p\u003e\n\u003cp\u003edpf= days post fertilization\u003c/p\u003e\n\u003cp\u003eGO= Gene Ontology Enrichment\u003c/p\u003e\n\u003cp\u003eHCN= Hyperpolarization-activated Cyclic Nucleotide-gated channels\u003c/p\u003e\n\u003cp\u003ehiPSCs= Human induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003eHOS= Holt-Oram syndrome\u003c/p\u003e\n\u003cp\u003eIL-6= interleukin 6\u003c/p\u003e\n\u003cp\u003eIso= isoprenaline\u003c/p\u003e\n\u003cp\u003eKEEG= Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003eLA-D= left atrial dilation\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMyl7-GAL4= Tg(Myl7:GAL4,EGFP)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOPBA= Institutional Animal Welfare Body\u003c/p\u003e\n\u003cp\u003eTBX5= transcription factor 5\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTg(myl7\u0026gt;miR-182)/\u003c/em\u003e\u003cem\u003eMyl7-m182-OE\u003c/em\u003e =\u003cem\u003e\u0026nbsp;Tg(myl7:GAL4,EGFP)\u003c/em\u003ex\u003cem\u003eTg(Sce.4xUAS:miR-182,cry:EGFP)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTOC= Tocilizumab\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Manuscript report studies involving biologic materials from human tissue. For this study cardiac tissue samples excided from left atrial appendage were used, obtained through collaborations between the University of Florence and the Cardio-Surgery Units of AOU-Careggi (Florence, Italy) and the \u0026ldquo;Policlinico Le Scotte\u0026rdquo; (Siena, Italy). Samples from control donor hearts not suitable for transplantation were from the University of Szeged (Hungary). The investigation conforms with the principles outlined in the \u0026ldquo;Declaration of Helsinki\u0026rdquo; of the World Medical Association and was approved by the local ethical committee. Informed consent for the use of tissue samples was obtained from patients before cardiac surgery.\u003c/p\u003e\n\n\u003cp\u003eThe Manuscript report studies involving biologic materials from animals (\u003cem\u003eDanio rerio\u003c/em\u003e). All animal practices comply with fundamental ethical standards and the Directive 2010/63/EU of the European Parliament, which addresses the protection and welfare of animals utilized for scientific aims. All euthanasia protocols were approved by the institutional Animal Welfare Body (OPBA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare to accept the Editorial Policies of the Journal, they approve the Ethics and they consent to participate in this publication. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional detailed Methods descriptions and additional Data are provided in Supplementary Material file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Italian Ministry of Health [SG-2019-12369183] to EG; was supported by the Italian Ministry of University and Research [M.I.U.R PRIN2017, M.I.U.R PRIN2022] to EC; was supported by National Recovery and Resilience Plan\u0026ndash;NextGeneration EU [MNESYS\u0026ndash;SPOKE1 code:PE000006] to LeS, [MNESYS\u0026ndash;PE12_SPOKE3] to LaS [Tuscany Health Ecosystem] to EC; was supported by Fondazione Cassa di Risparmio di Firenze (\u003cem\u003eproject human brain optical mapping\u003c/em\u003e) to EC; was supported by ISPRO-institutional funding to LP; was supported by Fondazione Cassa di Risparmio di Pistoia e Pescia (project INCITE) to LaS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElena Guzzolino\u003csup\u003e \u003c/sup\u003e\u003cstrong\u003e(EG)\u003c/strong\u003e, Valentina Balducci \u003cstrong\u003e(VB)\u003c/strong\u003e, Giada Allegro \u003cstrong\u003e(GA)\u003c/strong\u003e, Valentina Spinelli \u003cstrong\u003e(VS)\u003c/strong\u003e, Cesare Sala \u003cstrong\u003e(CS)\u003c/strong\u003e, Andrea Ninu\u003cstrong\u003e (AN)\u003c/strong\u003e, Leonardo Sacconi \u003cstrong\u003e(LeS)\u003c/strong\u003e, Francesca Lo Presti \u003cstrong\u003e(FL)\u003c/strong\u003e, Camilla Volpicini (\u003cstrong\u003eCV\u003c/strong\u003e),Matteo Cameli \u003cstrong\u003e(MC)\u003c/strong\u003e, Giulia Elena Mandoli (\u003cstrong\u003eGEM\u003c/strong\u003e), Perluigi Stefano \u003cstrong\u003e(PS)\u003c/strong\u003e, Matteo Lulli (\u003cstrong\u003eML\u003c/strong\u003e), Martina Lucia Boccitto\u003cstrong\u003e (MLB)\u003c/strong\u003e,Laura Poliseno \u003cstrong\u003e(LP)\u003c/strong\u003e, Raffaella De Paolo \u003cstrong\u003e(RD)\u003c/strong\u003e, Laura Sartiani \u003cstrong\u003e(LaS)\u003c/strong\u003e, Elisabetta Cerbai \u003cstrong\u003e(EC)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eConceptualization\u003c/u\u003e\u003c/em\u003e and \u003cem\u003e\u003cu\u003eProject administration\u003c/u\u003e\u003c/em\u003e:\u003cstrong\u003e EG\u003c/strong\u003e, \u003cstrong\u003eLaS\u003c/strong\u003e and \u003cstrong\u003eEC\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eSupervision\u003c/u\u003e\u003c/em\u003e: \u003cstrong\u003eLaS\u003c/strong\u003e and \u003cstrong\u003eEC\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eInvestigation\u003c/u\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003e\u003cu\u003eVisualization\u003c/u\u003e\u003c/em\u003e:\u003cstrong\u003e EG\u003c/strong\u003e, \u003cstrong\u003eVB\u003c/strong\u003e, \u003cstrong\u003eGA\u003c/strong\u003e, \u003cstrong\u003eCV\u003c/strong\u003e, \u003cstrong\u003eVS\u003c/strong\u003e, \u003cstrong\u003eCS\u003c/strong\u003e,\u003cstrong\u003e AN\u003c/strong\u003e,\u003cstrong\u003e FL\u003c/strong\u003e,\u003cstrong\u003e CV\u003c/strong\u003e, \u003cstrong\u003eMLB\u003c/strong\u003e,\u003cstrong\u003e ML \u003c/strong\u003eand\u003cstrong\u003e RD\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEG\u003c/strong\u003e and \u003cstrong\u003eVB\u003c/strong\u003e equally experimentally contributed to this work. \u003cstrong\u003eCS\u003c/strong\u003e and \u003cstrong\u003eEG\u003c/strong\u003e performed the bioinformatics \u003cem\u003ein silico\u003c/em\u003e analyses. \u003cstrong\u003eEG, VB\u003c/strong\u003e and \u003cstrong\u003eGA\u003c/strong\u003e performed the hiPS-CMs culture and differentiation, hiPSCs transfection, Intracell biosystem and MULTIPLE recordings. \u003cstrong\u003eVB, GA and CV\u003c/strong\u003e performed the electrophysiological analyses. \u003cstrong\u003eEG\u003c/strong\u003e, \u003cstrong\u003eVS\u003c/strong\u003e, \u003cstrong\u003eAG, FL, CV\u003c/strong\u003e and \u003cstrong\u003eMLB\u003c/strong\u003e performed RNA isolation and RT-PCR. \u003cstrong\u003eEG, CV and RD \u003c/strong\u003esupported zebrafish experiments. \u003cstrong\u003eEG\u003c/strong\u003e, \u003cstrong\u003eVB\u003c/strong\u003e, \u003cstrong\u003eGA\u003c/strong\u003e, \u003cstrong\u003eCS\u003c/strong\u003e and \u003cstrong\u003eAN\u003c/strong\u003e participated in creating the stable transgenic hiPS cell lines, immunofluorescence assays and cell line validation. \u003cstrong\u003eGA\u003c/strong\u003e performed the ELISA experiments. \u003cstrong\u003eVB\u003c/strong\u003e and \u003cstrong\u003eVS\u003c/strong\u003e performed the IL6+TOC experiments. \u003cstrong\u003eML \u003c/strong\u003eperformed confocal imaging. \u003cstrong\u003eEG\u003c/strong\u003e created the illustrations. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eData curation\u003c/u\u003e\u003c/em\u003e\u003cem\u003e: \u003c/em\u003e\u003cstrong\u003eEG\u003c/strong\u003e, \u003cstrong\u003eVB\u003c/strong\u003e, \u003cstrong\u003eMC\u003c/strong\u003e, \u003cstrong\u003eGEM\u003c/strong\u003e, \u003cstrong\u003eML\u003c/strong\u003e, \u003cstrong\u003eLaS\u003c/strong\u003e and \u003cstrong\u003eEC\u003c/strong\u003e\u003cem\u003e. \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eFormal analysis\u003c/u\u003e\u003c/em\u003e: \u003cstrong\u003eEG\u003c/strong\u003e and \u003cstrong\u003eVB\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eResources\u003c/u\u003e\u003c/em\u003e:\u003cstrong\u003e MC\u003c/strong\u003e, \u003cstrong\u003eGEM\u003c/strong\u003e, \u003cstrong\u003ePS\u003c/strong\u003e,\u003cstrong\u003e LeS\u003c/strong\u003e,\u003cstrong\u003e ML\u003c/strong\u003e,\u003cstrong\u003e LP\u003c/strong\u003e,\u003cstrong\u003e LaS \u003c/strong\u003eand\u003cstrong\u003e EC\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eFunding Acquisition\u003c/u\u003e\u003c/em\u003e:\u003cstrong\u003e EG\u003c/strong\u003e, \u003cstrong\u003eLaS\u003c/strong\u003e,\u003cstrong\u003e LeS, LP\u003c/strong\u003e and \u003cstrong\u003eEC\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eWriting-original draft\u003c/u\u003e\u003c/em\u003e: \u003cstrong\u003eEG\u003c/strong\u003e, \u003cstrong\u003eVB\u003c/strong\u003e, \u003cstrong\u003eLaS\u003c/strong\u003e and \u003cstrong\u003eEC\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eWriting-review \u0026amp; editing\u003c/u\u003e\u003c/em\u003e: \u003cstrong\u003eAll authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are truly grateful to Prof. Andras Varro of the University of Szeged (Hungary) for the collaboration. We thank Dr. Jos\u0026egrave; Manuel Pioner of University of Florence (Italy) who bestowed the wt-c11 hiPSC line. We very thank Dr. Letizia Pitto, the doctoral co-tutor of Dr. Guzzolino, for the kind bequest of the biobank in the laboratory after her retirement. We acknowledge the support and the professionality of Dr. Michele Dipalo and all Foresee biosystems group. We thank Fondazione Veronesi that supported the fellowship of Dr. Raffaella De Paolo. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGeng EH, Powell BJ, Goss CW, Lewis CC, Sales AE, Kim B. When the parts are greater than the whole: how understanding mechanisms can advance implementation research. Implement Sci. 2025;20(1):22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas D, Christ T, Fabritz L, Goette A, Hammw\u0026ouml;hner M, Heijman J, et al. German Cardiac Society Working Group on Cellular Electrophysiology state-of-the-art paper: impact of molecular mechanisms on clinical arrhythmia management. Clin Res Cardiol. 2019;108(6):577\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWakili R, Voigt N, K\u0026auml;\u0026auml;b S, Dobrev D, Nattel S. Recent advances in the molecular pathophysiology of atrial fibrillation. J Clin Invest. 2011;121(8):2955\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalan AI, Scridon A. MicroRNAs in atrial fibrillation - have we discovered the Holy Grail or opened a Pandora\u0026rsquo;s box? Front Pharmacol. 2025;16:1535621.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVardas EP, Theofilis P, Oikonomou E, Vardas PE, Tousoulis D. MicroRNAs in Atrial Fibrillation: Mechanisms, Vascular Implications, and Therapeutic Potential. Biomedicines. 2024;12(4):811.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePopat A, Jnaneswaran G, Yerukala Sathipati S, Sharma PP. MicroRNAs in cardiac arrhythmias: Mechanisms, biomarkers, and therapeutic frontiers. Heart Rhythm. 2025;22(11):2971\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzzolino E, Pellegrino M, Ahuja N, Garrity D, D\u0026rsquo;Aurizio R, Groth M, et al. miR-182-5p is an evolutionarily conserved Tbx5 effector that impacts cardiac development and electrical activity in zebrafish. Cell Mol Life Sci. 2020;77(16):3215\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrundel BJ, Van Gelder IC, Henning RH, Tieleman RG, Tuinenburg AE, Wietses M, et al. Ion channel remodeling is related to intraoperative atrial effective refractory periods in patients with paroxysmal and persistent atrial fibrillation. Circulation. 2001;103(5):684\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrunert M, Dorn C, Rickert-Sperling S. Cardiac Transcription Factors and Regulatory Networks. Adv Exp Med Biol. 2024;1441:295\u0026ndash;311.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Ouwerkerk AF, Hall AW, Kadow ZA, Lazarevic S, Reyat JS, Tucker NR, et al. Epigenetic and Transcriptional Networks Underlying Atrial Fibrillation. Circ Res. 2020;127(1):34\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCakmak HA, Coskunpinar E, Ikitimur B, Barman HA, Karadag B, Tiryakioglu NO, et al. The prognostic value of circulating microRNAs in heart failure: preliminary results from a genome-wide expression study. J Cardiovasc Med (Hagerstown). 2015;16(6):431\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu N, Miao H, Ren H. Effect of miR-182-5p on apoptosis in myocardial infarction. Heliyon. 2023;9(11):e21524.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang Q, Chen H, Xu X, Jiang W. miR-182-5p Attenuates High-Fat -Diet-Induced Nonalcoholic Steatohepatitis in Mice. Ann Hepatol. 2019;18(1):116\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang A, Jin Y. MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway. Open Med (Wars). 2020;15(1):1202\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlhadidi QM, Xu L, Sun X, Althobaiti YS, Almalki A, Alsaab HO, et al. MiR-182 Inhibition Protects Against Experimental Stroke in vivo and Mitigates Astrocyte Injury and Inflammation in vitro via Modulation of Cortactin Activity. Neurochem Res. 2022;47(12):3682\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Dai R, Ahmed SA. MicroRNA-183/96/182 cluster in immunity and autoimmunity. Front Immunol. 2023;14:1134634.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSameti P, Tohidast M, Amini M, Bahojb Mahdavi SZ, Najafi S, Mokhtarzadeh A. The emerging role of MicroRNA-182 in tumorigenesis; a promising therapeutic target. Cancer Cell Int. 2023;23(1):134.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIhara K, Sasano T. Role of Inflammation in the Pathogenesis of Atrial Fibrillation. Front Physiol. 2022;13:862164.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott L, Li N, Dobrev D. Role of inflammatory signaling in atrial fibrillation. Int J Cardiol. 2019;287:195\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatkenov N, Mukhatayev Z, Kozhakhmetov S, Sailybayeva A, Bekbossynova M, Kushugulova A. Systematic Review on the Role of IL-6 and IL-1β in Cardiovascular Diseases. J Cardiovasc Dev Dis. 2024;11(7):206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpinelli V, Laurino A, Balducci V, Gencarelli M, Ruzzolini J, Nediani C, et al. Interleukin-6 Modulates the Expression and Function of HCN Channels: A Link Between Inflammation and Atrial Electrogenesis. Int J Mol Sci. 2024;25(22):12212.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKishimoto T. The biology of interleukin-6. Blood. 1989;74(1):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalducci V, Credi C, Sacconi L, Romanelli MN, Sartiani L, Cerbai E. The HCN channel as a pharmacological target: Why, where, and how to block it. Prog Biophys Mol Biol. 2021;166:173\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIachetta G, Melle G, Colistra N, Tantussi F, De Angelis F, Dipalo M. Long-term in vitro recording of cardiac action potentials on microelectrode arrays for chronic cardiotoxicity assessment. Arch Toxicol. 2023;97(2):509\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCredi C, Balducci V, Munagala U, Cianca C, Bigiarini S, de Vries AAF, et al. Fast Optical Investigation of Cardiac Electrophysiology by Parallel Detection in Multiwell Plates. Front Physiol. 2021;12:692496.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatiukas A, Mitrea BG, Qin M, Pertsov AM, Shvedko AG, Warren MD, et al. Near-infrared voltage-sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium. Heart Rhythm. 2007;4(11):1441\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzzolino E, Milella MS, Forini F, Bors\u0026ograve; M, Rutigliano G, Gorini F, et al. Thyroid disrupting effects of low-dose dibenzothiophene and cadmium in single or concurrent exposure: New evidence from a translational zebrafish model. Sci Total Environ. 2021;769:144703.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStillitano F, Lonardo G, Giunti G, Del Lungo M, Coppini R, Spinelli V, et al. Chronic Atrial Fibrillation Alters the Functional Properties of I\u003csub\u003ef\u003c/sub\u003e in the Human Atrium. Cardiovasc electrophysiol. 2013;24(12):1391\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Medical Association Declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects. Cardiovasc Res. 1997;35(1):2\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePei G, Chen L, Wang Y, He C, Fu C, Wei Q. Role of miR-182 in cardiovascular and cerebrovascular diseases. Front Cell Dev Biol. 2023;11:1181515.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Dai R, Ahmed SA. MicroRNA-183/96/182 cluster in immunity and autoimmunity. Front Immunol. 2023;14:1134634.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDambal S, Shah M, Mihelich B, Nonn L. The microRNA-183 cluster: the family that plays together stays together. Nucleic Acids Res. 2015;43(15):7173\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarela M, Dios S, Novoa B, Figueras A. Characterisation, expression and ontogeny of interleukin-6 and its receptors in zebrafish (Danio rerio). Dev Comp Immunol. 2012;37(1):97\u0026ndash;106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZang L, Torraca V, Shimada Y, Nishimura N. Editorial: Zebrafish Models for Human Disease Studies. Front Cell Dev Biol. 2022;10:861941.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBriggs JP. The zebrafish: a new model organism for integrative physiology. Am J Physiology-Regulatory Integr Comp Physiol. 2002;282(1):R3\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi TY, Choi TI, Lee YR, Choe SK, Kim CH. Zebrafish as an animal model for biomedical research. Exp Mol Med. 2021;53(3):310\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNattel S, Heijman J, Zhou L, Dobrev D. Molecular Basis of Atrial Fibrillation Pathophysiology and Therapy: A Translational Perspective. Circ Res. 2020;127(1):51\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Brien J, Hayder H, Zayed Y, Peng C. Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front Endocrinol (Lausanne). 2018;9:402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLibermann TA, Baltimore D. Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol. 1990;10(5):2327\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Sig Transduct Target Ther. 2017;2(1):17023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGordon JW, Shaw JA, Kirshenbaum LA. Multiple Facets of NF-κB in the Heart: To Be or Not to NF-κB. Circul Res. 2011;108(9):1122\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWiersma M, Meijering RAM, Qi X, Zhang D, Liu T, Hoogstra-Berends F, et al. Endoplasmic Reticulum Stress Is Associated With Autophagy and Cardiomyocyte Remodeling in Experimental and Human Atrial Fibrillation. JAHA. 2017;6(10):e006458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSirish P, Diloretto DA, Thai PN, Chiamvimonvat N. The Critical Roles of Proteostasis and Endoplasmic Reticulum Stress in Atrial Fibrillation. Front Physiol. 2022;12:793171.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, et al. Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA. 1993;90(21):10193\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeltrami M, Palazzuoli A, Padeletti L, Cerbai E, Coiro S, Emdin M, et al. The importance of integrated left atrial evaluation: From hypertension to heart failure with preserved ejection fraction. Int J Clin Pract. 2018;72(2):e13050.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuskaric S, Schmitteckert S, Mori AD, Glaser A, Schneider KU, Bruneau BG, et al. Shox2 mediates Tbx5 activity by regulating Bmp4 in the pacemaker region of the developing heart. Hum Mol Genet. 2010;19(23):4625\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Gramolini AO, Walsh MA, Zhou YQ, Slorach C, Friedberg MK, et al. Tbx5-dependent pathway regulating diastolic function in congenital heart disease. Proc Natl Acad Sci USA. 2008;105(14):5519\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKameswaran V, Bramswig NC, McKenna LB, Penn M, Schug J, Hand NJ, et al. Epigenetic Regulation of the DLK1-MEG3 MicroRNA Cluster in Human Type 2 Diabetic Islets. Cell Metabol. 2014;19(1):135\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang X, Zhang Q, Cattaneo P, Zhuang S, Gong X, Spann NJ, et al. Transcription factor ISL1 is essential for pacemaker development and function. J Clin Invest. 2015;125(8):3256\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkwarek-Dziekanowska A, W\u0026oacute;jtowicz-Ściślak A, Sobieszek G. Holt-Oram syndrome. Eur Rev Med Pharmacol Sci. 2024;28(1):336\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa JF, Yang F, Mahida SN, Zhao L, Chen X, Zhang ML, et al. \u003cem\u003eTBX5\u003c/em\u003e mutations contribute to early-onset atrial fibrillation in Chinese and Caucasians. Cardiovasc Res. 2016;109(3):442\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLazzerini PE, Laghi-Pasini F, Acampa M, Srivastava U, Bertolozzi I, Giabbani B et al. Systemic Inflammation Rapidly Induces Reversible Atrial Electrical Remodeling: The Role of Interleukin‐6\u0026ndash;Mediated Changes in Connexin Expression. JAHA [Internet]. 2019 Aug 20 [cited 2025 May 23];8(16). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ahajournals.org/doi/\u003c/span\u003e\u003cspan address=\"https://www.ahajournals.org/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1161/JAHA.118.011006\u003c/span\u003e\u003cspan address=\"10.1161/JAHA.118.011006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishimoto N, Kishimoto T, Humanized Antihuman. IL-6 Receptor Antibody, Tocilizumab. In: Chernajovsky Y, Nissim A, editors. Therapeutic Antibodies [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2008 [cited 2025 Jun 8]. pp. 151\u0026ndash;60. (Starke K, editor. Handbook of Experimental Pharmacology; vol. 181). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://link.springer.com/\u003c/span\u003e\u003cspan address=\"http://link.springer.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-540-73259-4_7\u003c/span\u003e\u003cspan address=\"10.1007/978-3-540-73259-4_7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie F, Yun H, Levitan EB, Muntner P, Curtis JR. Tocilizumab and the Risk of Cardiovascular Disease: Direct Comparison Among Biologic Disease-Modifying Antirheumatic Drugs for Rheumatoid Arthritis Patients. Arthritis Care Res. 2019;71(8):1004\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIchiyama K, Gonzalez-Martin A, Kim BS, Jin HY, Jin W, Xu W, et al. The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression. Immunity. 2016;44(6):1284\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiFrancesco D, Borer JS. The Funny Current: Cellular Basis for the Control of Heart Rate. Drugs. 2007;67(Supplement 2):15\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiFrancesco D. The Role of the Funny Current in Pacemaker Activity. Circul Res. 2010;106(3):434\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDifrancesco D. The pacemaker current in the sinus node. Eur Heart J. 1987;8(suppl L):19\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaponaro A, DiFrancesco D. Structure mirroring function: What\u0026rsquo;s the \u0026lsquo;matter\u0026rsquo; with the funny current? J Physiol. 2025;JP287209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiFrancesco D, Tromba C. Muscarinic control of the hyperpolarization-activated current (if) in rabbit sino‐atrial node myocytes. J Physiol. 1988;405(1):493\u0026ndash;510.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArdhianto P, Yuniadi Y. Biomarkers of Atrial Fibrillation: Which One Is a True Marker? Cardiol Res Pract. 2019;2019:1\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVardas EP, Theofilis P, Oikonomou E, Vardas PE, Tousoulis D. MicroRNAs in Atrial Fibrillation: Mechanisms, Vascular Implications, and Therapeutic Potential. Biomedicines. 2024;12(4):811.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePopat A, Jnaneswaran G, Sathipati SY, Sharma PP. MicroRNAs in cardiac arrhythmias: mechanisms, biomarkers and, therapeutic frontiers. Heart Rhythm. 2025;S1547-5271(25)02512-3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia X, Cheng X, Wu N, Xiang Y, Wu L, Xu B, et al. Prognostic value of interleukin-6 in atrial fibrillation: A cohort study and meta-analysis. Anatol J Cardiol. 2021;25(12):872\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou P, Waresi M, Zhao Y, Lin HC, Wu B, Xiong N, et al. Increased serum interleukin-6 level as a predictive biomarker for atrial fibrillation: A systematic review and meta-analysis. Rev Port Cardiol. 2020;39(12):723\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu N, Xu B, Xiang Y, Wu L, Zhang Y, Ma X, et al. Association of inflammatory factors with occurrence and recurrence of atrial fibrillation: A meta-analysis. Int J Cardiol. 2013;169(1):62\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScardigli M, Cannazzaro S, Coppini R, Crocini C, Yan P, Loew LM, et al. Arrhythmia susceptibility in a rat model of acute atrial dilation. Prog Biophys Mol Biol. 2020;154:21\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeijman J, Voigt N, Wehrens XHT, Dobrev D. Calcium dysregulation in atrial fibrillation: the role of CaMKII. Front Pharmacol. 2014;5:30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenham NC, Pearman CM, Caldwell JL, Madders GWP, Eisner DA, Trafford AW et al. Calcium in the Pathophysiology of Atrial Fibrillation and Heart Failure. Front Physiol [Internet]. 2018 Oct 4 [cited 2025 May 23];9. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.frontiersin.org/article/\u003c/span\u003e\u003cspan address=\"https://www.frontiersin.org/article/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fphys.2018.01380/full\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2018.01380/full\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDobrev D, Voigt N, Wehrens XHT. The ryanodine receptor channel as a molecular motif in atrial fibrillation: pathophysiological and therapeutic implications. Cardiovascular Res. 2011;89(4):734\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenham NC, Pearman CM, Caldwell JL, Madders GWP, Eisner DA, Trafford AW, et al. Calcium in the Pathophysiology of Atrial Fibrillation and Heart Failure. Front Physiol. 2018;9:1380.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarbar D, Kannankeril PJ, Donahue BS, Kucera G, Stubblefield T, Haines JL, et al. Cardiac Sodium Channel (\u003cem\u003eSCN5A\u003c/em\u003e) Variants Associated with Atrial Fibrillation. Circulation. 2008;117(15):1927\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao G, Dudley SC, Redox Regulation NF-κB, Fibrillation A. Antioxid Redox Signal. 2009;11(9):2265\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTam AB, Mercado EL, Hoffmann A, Niwa M. ER Stress Activates NF-κB by Integrating Functions of Basal IKK Activity, IRE1 and PERK. Koritzinsky M, editor. PLoS ONE. 2012;7(10):e45078.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrell T, Lautenschl\u0026auml;ger J, Weidemann L, Ruhmer J, Witte OW, Grosskreutz J. Endoplasmic reticulum stress is accompanied by activation of NF-κB in amyotrophic lateral sclerosis. J Neuroimmunol. 2014;270(1\u0026ndash;2):29\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu X, Huang L, Gong J, Shi C, Wang Z, Ye B, et al. NF-κB pathway link with ER stress-induced autophagy and apoptosis in cervical tumor cells. Cell Death Discov. 2017;3(1):17059.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao KZ, Kim GY, Meara GK, Qin X, Feng H. Tipping the Scales With Zebrafish to Understand Adaptive Tumor Immunity. Front Cell Dev Biol. 2021;9:660969.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranza M, Varricchio R, Alloisio G, De Simone G, Di Bella S, Ascenzi P, et al. Zebrafish (Danio rerio) as a Model System to Investigate the Role of the Innate Immune Response in Human Infectious Diseases. IJMS. 2024;25(22):12008.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTokar T, Pastrello C, Rossos AEM, Abovsky M, Hauschild AC, Tsay M, et al. mirDIP 4.1\u0026mdash;integrative database of human microRNA target predictions. Nucleic Acids Res. 2018;46(D1):D360\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMukherji S, Ebert MS, Zheng GXY, Tsang JS, Sharp PA, Van Oudenaarden A. MicroRNAs can generate thresholds in target gene expression. Nat Genet. 2011;43(9):854\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Den Berg NWE, Kawasaki M, Nariswari FA, Fabrizi B, Neefs J, Van Der Made I, et al. MicroRNAs in atrial fibrillation target genes in structural remodelling. Cell Tissue Res. 2023;394(3):497\u0026ndash;514.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Wakili R, Xiao J, Wu CT, Luo X, Clauss S, et al. Detailed characterization of microRNA changes in a canine heart failure model: Relationship to arrhythmogenic structural remodeling. J Mol Cell Cardiol. 2014;77:113\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen YC, Voskoboinik A, Gerche AL, Marwick TH, McMullen JR. Prevention of Pathological Atrial Remodeling and Atrial Fibrillation. J Am Coll Cardiol. 2021;77(22):2846\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuppert M, Korkmaz-Ic\u0026ouml;z S, Benczik B, \u0026Aacute;gg B, Nagy D, B\u0026aacute;lint T, et al. Pressure overload-induced systolic heart failure is associated with characteristic myocardial microRNA expression signature and post-transcriptional gene regulation in male rats. Sci Rep. 2023;13(1):16122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Othman S, Boyett MR, Morris GM, Malhotra A, Mesirca P, Mangoni ME, et al. Symptomatic bradyarrhythmias in the athlete\u0026mdash;Underlying mechanisms and treatments. Heart Rhythm. 2024;21(8):1415\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi P, Zhu X, Liu M, Wang Y, Huang C, Sun J, et al. Impact of gene-environment interactions on atrial fibrillation and cardiac structure. Sci Rep. 2025;15(1):16893.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWass SY, Hahad O, Asad Z, Li S, Chung MK, Benjamin EJ, et al. Environmental Exposome and Atrial Fibrillation: Emerging Evidence and Future Directions. Circul Res. 2024;134(8):1029\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"miR-182-5p, IL-6, hiPS-CMs, Arrhythmias, zebrafish","lastPublishedDoi":"10.21203/rs.3.rs-8808389/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8808389/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe progression of cardiac electrical remodelling and the onset of proarrhythmic events are multifactorial processes, with many factors contributing to the development of atrial fibrillation (AF). Recently, proinflammatory mediators have emerged as new key players: in particular, independent preclinical evidence has identified interleukin-6 (IL-6) and miR-182 as causative factors of arrhythmogenesis in animal models. MiR-182 regulates a wide range of pathways, including the expression of inflammatory mediators; however, in the cardiac context, the potential relationship between these two factors remains unknown.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman induced pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (CMs) to study the role of miR-182-overexpression (OE) on IL-6 expression/secretion by RT-PCR/Elisa assays. Functional consequences were assessed by measuring spontaneous electrical activity by using MULTIPLE-High-Throughput and Intracell systems, with/without autonomic stimulation. Hearts from the \u003cem\u003eTg(myl7:GAL4,EGFP)\u003c/em\u003ex\u003cem\u003eTg(Sce.4xUAS:miR-182,cry:EGFP)\u003c/em\u003e zebrafish line [\u003cem\u003eTg(myl7\u0026thinsp;\u0026gt;\u0026thinsp;miR-182)\u003c/em\u003e] were used to measure the expression of \u003cem\u003edre-il6\u003c/em\u003e. Human IL-6 protein (5.4pg/nL) was microinjected in the pericardial region of 2dpf \u003cem\u003eTg(myl7:EGFP) wt\u003c/em\u003e-like zebrafish embryos and heart rate was recorded. Expression analyses were performed on human left atrial samples of 49 patients (11 controls, CT; 18 left atrial dilation (LA-D); 20 chronic AF).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMiR-182-OE in hiPS-CMs significantly incremented IL-6 expression and secretion, and was associated with a reduced and irregular spontaneous beating rate, as well as enhanced response to acetylcholine. Accordingly, MiR-182-OE downregulated the expression of HCN4, encoding for the pacemaker \u003cem\u003ef-current\u003c/em\u003e, and dysregulated genes associated with atrial pathology. On the contrary, 24-hour incubation with IL-6 (50ng/mL) did not change miR-182-5p expression levels in CT hiPS-CMs. The IL-6 receptor antagonist tocilizumab (TOC, 10\u0026micro;g/mL) partially rescued HCN4 expression in miR-182-OE hiPS-CMs. Zebrafish heart samples from \u003cem\u003eTg(myl7\u0026thinsp;\u0026gt;\u0026thinsp;miR-182)\u003c/em\u003e exhibited increased \u003cem\u003eil6\u003c/em\u003e expression levels. Pericardial injection of human IL-6 in \u003cem\u003ewt\u003c/em\u003e zebrafish embryos decreased heart rate. Finally, miR-182-5p was found to be overexpressed in human biopsies from patients with LA-D, with the highest expression levels observed in patients with permanent AF; remarkably, miR-182 levels positively correlated with IL-6 expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe results support the hypothesis of a causative link between miR-182-OE and IL-6 production in the cardiac context. This molecular axis may represent a prognostic factor predisposing to arrhythmogenesis. Overall, our findings reveal novel pathophysiological mechanisms and suggest novel pharmacological targets within the complex AF setting.\u003c/p\u003e","manuscriptTitle":"MiR-182 regulates IL-6 in the cardiac context: implications for human atrial electrical remodelling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-28 16:34:48","doi":"10.21203/rs.3.rs-8808389/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-17T18:04:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-25T12:54:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-09T09:05:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2026-02-08T05:15:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6abb7df7-c098-4300-a7c4-71e3fd78873e","owner":[],"postedDate":"March 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-28T16:34:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-28 16:34:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8808389","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8808389","identity":"rs-8808389","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}