International Journal of Molecular Sciences Article Atrial Fibrillation in Heart Failure Is Associated with High Levels of Circulating microRNA-199a-5p and 22–5p and a Defective Regulation of Intracellular Calcium and Cell-to-Cell Communication Anna Garcia-Elias 1,2, *, Marta Tajes 2,3 , Laia Yañez-Bisbe 1,2 , Cristina Enjuanes 3,4 , Josep Comín-Colet 3,4,5 , Selma A. Serra 6 , José M. Fernández-Fernández 6 , Kathryn W. Aguilar-Agon 7 , Svetlana Reilly 7 , Julio Martí-Almor 2,8,9 and Begoña Benito 1,2,9,10,11, * 1 Vascular Biology and Metabolism Program, Vall d’Hebron Research Institute (VHIR), Passeig de la Vall d’Hebron 119-129, 08035 Barcelona, Spain; laiayb19@gmail.com 2 Group of Biomedical Research in Heart Diseases, Hospital del Mar Medical Research Institute (IMIM), Doctor Aiguader 88, 08003 Barcelona, Spain; cicelyflyn@gmail.com (M.T.); jmartialmor@gmail.com (J.M.-A.) 3  Research Group in Cardiovascular Disorders (BIOHEART), Bellvitge Biomedical Research  Institute (IDIBELL), Avinguda de la Granvia de l’Hospitalet, 199, 08908 L’Hospitalet de Llobregat, Spain; cristinaenjuanes@gmail.com (C.E.); josepcomin@gmail.com (J.C.-C.) Citation: Garcia-Elias, A.; Tajes, M.; 4 Cardiology Department, Hospital de Bellvitge, Carrer de la Feixa Llarga s/n, Yañez-Bisbe, L.; Enjuanes, C.; 08907 L’Hospitalet de Llobregat, Spain Comín-Colet, J.; Serra, S.A.; 5 Department of Clinical Sciences, University of Barcelona School of Medicine, 08036 Barcelona, Spain Fernández-Fernández, J.M.; 6 Laboratory of Molecular Physiology, Department of Experimental and Health Sciences, Aguilar-Agon, K.W.; Reilly, S.; Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain; selmaserra@gmail.com (S.A.S.); Martí-Almor, J.; et al. Atrial jmanuel.fernandez@upf.edu (J.M.F.-F.) 7 Radcliffe Department of Medicine, Division of Cardiovascular Medicine, John Radcliffe Hospital, Fibrillation in Heart Failure Is Associated with High Levels of University of Oxford, Oxford OX3 9DU, UK; kathryn.aguilar-agon@cardiov.ox.ac.uk (K.W.A.-A.); Circulating microRNA-199a-5p and svetlanareilly@aol.com (S.R.) 8 Cardiology Department, Hospital del Mar. Passeig de la Barceloneta 25, 08003 Barcelona, Spain 22–5p and a Defective Regulation of 9 Department of Medicine, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain Intracellular Calcium and Cell-to-Cell 10 Cardiology Department, Hospital Universitari Vall d’Hebron, Universitat Autonoma Barcelona, Communication. Int. J. Mol. Sci. 2021, Passeig de la Vall d’Hebron 119-129, 08035 Barcelona, Spain 22, 10377. https://doi.org/10.3390/ 11 Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBER-CV), ijms221910377 Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: anna.garcia-elias@upf.edu (A.G.-E.); begona.benito@vhir.org (B.B.); Academic Editor: Anastasios Tel.: +34-93-4894038 (A.G.-E.); +34-93-3160717 (B.B.) Lymperopoulos Abstract: MicroRNAs (miRNAs) participate in atrial remodeling and atrial fibrillation (AF) promo- Received: 31 August 2021 tion. We determined the circulating miRNA profile in patients with AF and heart failure with reduced Accepted: 21 September 2021 ejection fraction (HFrEF), and its potential role in promoting the arrhythmia. In plasma of 98 patients Published: 26 September 2021 with HFrEF (49 with AF and 49 in sinus rhythm, SR), differential miRNA expression was determined by high-throughput microarray analysis followed by replication of selected candidates. Validated Publisher’s Note: MDPI stays neutral miRNAs were determined in human atrial samples, and potential arrhythmogenic mechanisms with regard to jurisdictional claims in studied in HL-1 cells. Circulating miR-199a-5p and miR-22-5p were significantly increased in HFrEF published maps and institutional affil- iations. patients with AF versus those with HFrEF in SR. Both miRNAs, but particularly miR-199a-5p, were increased in atrial samples of patients with AF. Overexpression of both miRNAs in HL-1 cells resulted in decreased protein levels of L-type Ca2+ channel, NCX and connexin-40, leading to lower basal intracellular Ca2+ levels, fewer inward currents, a moderate reduction in Ca2+ buffering post-caffeine exposure, and a deficient cell-to-cell communication. In conclusion, circulating miR-199a-5p and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. miR-22-5p are higher in HFrEF patients with AF, with similar findings in human atrial samples of AF This article is an open access article patients. Cells exposed to both miRNAs exhibited altered Ca2+ handling and defective cell-to-cell distributed under the terms and communication, both findings being potential arrhythmogenic mechanisms. conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 10377. https://doi.org/10.3390/ijms221910377 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 10377 2 of 17 Keywords: heart failure; atrial fibrillation; atrial remodeling; microRNA; biomarkers; HL-1 cells; calcium regulation; L-type calcium channels; connexin 40; NCX1 1. Introduction Heart failure (HF), affecting 1–2% of the adult population and up to > 10% of individ- uals over 70 years, is the cardiovascular disorder associated with the highest morbidity and mortality rates, particularly in the subgroup of patients with reduced ejection fraction [1]. Atrial fibrillation (AF) occurs in 25–40% of patients with HF and reduced ejection fraction (HFrEF) [2]. The coexistence of both conditions is facilitated by the common pathogenic mechanisms shared between HF and AF, which promote one another [3,4]. For example, impaired regulation of intracellular Ca2+ , electrical remodeling and the development of fibrosis are seen in the atria of AF patients and in the ventricles of failing hearts [3,4]. These mechanisms favor atrial arrhythmia susceptibility and impaired ventricular contraction [4]. The presence of AF in HF patients has serious prognostic implications. In patients with HFrEF, a preexisting AF increases the risk of cardiovascular death or hospital readmission by more than 20% at a three-year follow-up [5]. Moreover, the development of new-onset AF in patients with HF has been associated with doubled mortality in follow-up [5–7]. These observations emphasize the need to treat and prevent the occurrence of AF in patients with HF. Defining the mechanisms underlying AF occurrence and progression in HF patients is critical for the development of new treatment tools. Despite sharing similar cardiac substrates, AF requires the presence of an electrical trigger to generate the arrhythmia, an element that is not obligatory in HF [8,9]. It is plausible that the presence of AF in the setting of HF is accompanied by certain mechanistic features, related to electrical or structural remodeling, that are specific to this condition and are differential to those that are present in HF without concomitant AF. During the last decade, microRNAs (miRNAs), 21 to 25 nucleotides sequences that reg- ulate gene expression, have been implicated in the pathogenesis of numerous diseases [10]. In the setting of HF and AF, a number of miRNAs like miR-21, miR-30, miR-31, miR-133 and miR-29 have been shown to mediate structural remodeling, an important mechanism underlying both ventricular dysfunction and the occurrence of AF [11–14]. The presence of miRNAs in circulation makes them attractive as potential biomarkers of disease [15]. For example, use of multi-miRNA panels in conjunction with the levels of NT-proBNP has been found to improve diagnostic accuracy of HF [16]. Similarly, a particular miRNAs signature in blood, characterized by increased expression of miRNA-103a, -107, -320d, -486, andlet-7b, has also been related to the presence of persistent but not paroxysmal AF [17]. While previous reports focused majorly on the role of selected miRNAs [13,18,19], instances of unbiased profiling of circulating miRNA using high-throughput screenings in patients with combined HF and AF are scarce. Here, we tested whether the presence of AF in patients with HFrEF would be associ- ated with a distinct systemic miRNA profile in plasma, which could be reflective of the particular mechanistic pathways altered when both entities co-exist. We first characterized the circulating miRNA profiles of two very homogeneous populations of HFrEF patients with and without AF, following a two-phase approach of screening and replication. We observed increase of miR-199a-5p and miR-22-5p in plasma of patients with AF + HFrEF compared to those with HFrEF without the arrhythmia, and in the atrial tissue obtained from patients with AF. HL-1 cells transfected with both miRNAs had altered Ca2+ handling that correlated with decreased expression and functionality of L-type calcium channel (LTCC) and NCX1, and defective cell-to-cell communication due to a decrease in connexin 40 (Cx40). Our findings, therefore, identify significant upregulation of circulating levels of miR-22-5p and miR-199a-5p in patients with HFrEF and AF that may potentially facilitate the promotion of the arrhythmia in this setting. Int. J. Mol. Sci. 2021, 22, 10377 3 of 17 2. Results 2.1. HFrEF Patients with AF Have Higher Plasmatic Levels of miRNA-199a-5p and miRNA-22-5p The clinical characteristics of patients included in the discovery phase are shown in Supplementary File, Table S1. Briefly, 55% were men and the mean age was 75 in both groups. The prevalence of cardiovascular risk factors and comorbidities was high (Table S1). Mean left ventricular ejection fraction (LVEF) was around 30%, and New York Heart Association (NYHA) class was similar between patients in permanent AF (permAF, including long-standing persistent and permanent AF) and those in sinus rhythm (SR). In this population, the microarray study revealed 76 miRNAs that were differentially ex- pressed between both groups. Based on their higher statistical significance, plasma stability or clinical relevance, 18 were selected for replication (Supplementary File, Table S2). Characteristics of patients included in the replication cohort are shown in Table 1. Mean age was 73 for both groups, and 77% were men. BMI was higher in patients with permAF. Prevalence of hypertension, diabetes and CKD was high in both groups. HF status was similar in patients with permAF and SR, with a mean LVEF 32% and no significant differences in treatment, although patients with permAF tended to present more advanced NYHA class (Table 1). Table 1. Characteristics of the replication cohort. permAF (n = 40) SR (n = 40) p-Value Patient Characteristics Sex, men (%) 31 (77.5) 31 (77.5) NS Age, y (SD) 73.2 (10.1) 73.5 (10.3) NS BMI, kg/m2 (SD) 30.5 (6.8) 27.5 (3.7) 0.02 Medical history Tobacco history (%) 22 (55) 22 (55) NS Hypertension (%) 30 (75) 31 (77.5) NS Diabetes (%) 14 (35) 18 (45) NS Hypercholesterolemia (%) 20 (50) 21 (52.5) NS COPD (%) 6 (15) 11 (27.5) NS CKD (%) 11 (27.5) 8 (20.0) NS Previous stroke (%) 8 (20.0) 4 (10.0) NS HF parameters Ischemic etiology (%) 16 (40) 24 (60) NS Heart rate, bpm (SD) 76.5 (16.6) 70.0 (14.6) NS NYHA class (%): NS I-II 16 (51.6) 24 (75) III-IV 15 (48.4) 8 (25) LVEF, % (SD) 32.5 (6.7) 32.8 (6.5) NS Log Pro-BNP, mean (SD) 3.4 (0.4) 3.3 (0.6) NS HF treatment ACEI/ARB (%) 34 (85.0) 33 (82.5) NS Betablockers (%) 37 (92.5) 37 (92.5) NS Diuretics (%) 40 (100) 37 (92.5) NS Heart rhythm <0.001 Permanent SR 0 40 History of parox/pers AF * 0 0 Permanent AF # 40 0 BMI: body mass index; COPD: chronic obstructive pulmonary disease; CKD: chronic kidney disease; NYHA: New York Heart Association; LVEF: left ventricular ejection fraction; ACEI: angiotensin-converting-enzyme inhibitors; ARB: angiotensin-receptor blockers; SR: sinus rhythm; AF: atrial fibrillation. * Includes episodes of paroxysmal or short-standing persistent AF; # includes long-standing (>1 year) persistent or permanent AF. NS: non-significant. The results of the replication study are summarized in Table 2. Two microRNAs (miR-199a-5p and miR-22-5p) remained differentially expressed between groups, showing 1.92-fold and 1.55-increased expression in permAF versus SR, respectively. miR-125a-5p 1.395 miR-126-5p 1.244 miR-133a-3p 1.554 miR-16-5p 0.955 Int. J. Mol. Sci. 2021, 22, 10377 miR-17-5p 1.230 4 of 17 miR-199a-5p 1.921 miR-19a-3p 1.526 Table 2. Results ofmiR-20a-5p the replication study. 1.158 miR-22-5p 1.549 2−44Ct (permAF vs. SR) p-Value miR-23a-3p 1.278 miR-106a-5p 1.292 0.378 miR-26a-5p miR-106b-5p 1.311 1.322 0.275 miR-27b-3p miR-125a-5p 1.395 1.402 0.110 miR-126-5p miR-301a-3p 1.244 1.115 0.258 miR-133a-3p 1.554 0.073 miR-324-5p 1.531 miR-16-5p 0.955 0.783 miR-374a-5p miR-17-5p 1.230 1.239 0.283 miR-199a-5p miR-425-3p 1.921 1.450 0.028 * miR-19a-3p 1.526 0.117 * Significant p-value <0.05. miR-20a-5p 1.158 0.452 miR-22-5p 1.549 0.033 * miR-23a-3p 1.278 0.219 2.2. MiR-199a-5p miR-26a-5p and 22–5p Expression 1.322 Levels Are Higher0.187 in Atrial Samples o miR-27b-3p 1.402 0.153 TomiR-301a-3p assess whether circulating 1.115 levels of miR-199a-5p 0.765 and miR-22-5p the atrial levels of both miRNAs,1.531 miR-324-5p we assessed miRNA0.088 expression in hum miR-374a-5p 1.239 0.433 atrial appendages miR-425-3p obtained from 22 patients with AF0.119 1.450 or SR (no AF) refe cardiac surgery * Significant p-value < 0.05. (Supplementary File, Table S3 for clinical characteristic Figure 1, miR-199-5p 2.2. MiR-199a-5p expression and 22–5p Expression Levels levels were Are Higher increased in Atrial in Patients Samples of AF both RAA (p = (p = To 0.014) in AF circulating assess whether versus no AF levels patients, whereas of miR-199a-5p and miR-22-5p miR-22-5p showed correlated with the a significant atrial levels of trend. both miRNAs, we assessed miRNA expression in human right and left atrial appendages obtained from 22 patients with AF or SR (no AF) referred for elective cardiac These observations indicate that local overexpression of miR-199a-5p surgery (Supplementary File, Table S3 for clinical characteristics). As shown in Figure 1, ismiR-199-5p associated withlevels expression AF, were andincreased suggestin that the (p both RAA increase in LAA = 0.034) and atrial (p =miRNA 0.014) lev in AF versus no AF patients, whereas miR-22-5p showed contribute to the elevated systemic levels observed in AF. a similar but non-significant trend. Figure 1. MicroRNA determination in human left and right atrial appendages. Expression levels Figure 1. MicroRNA of miR-199a-5p determination and miR-22-5p in human of AF patients (white columns)left and right compared atrial to those appendages. in sinus rhythm Ex (no-AF, black columns). Scatter dot blots represent each individual patient, miR-199a-5p and miR-22-5p of AF patients (white columns) compared to those in and the number within the column represents the total of independent experiments performed. * p < 0.05, RAA: right atrial AF, black columns). Scatter dot blots represent each individual patient, and the n appendage, LAA: left atrial appendage. column represents the total of independent experiments performed. * p < 0.05, appendage, LAA: left atrial appendage. Int. J. Mol. Sci. 2021, 22, 10377 5 of 17 These observations indicate that local overexpression of miR-199a-5p and miR-22-5p is associated with AF, and suggest that the increase in atrial miRNA levels may, in part, contribute to the elevated systemic levels observed in AF. 2.3. Overexpression of miR-199a-5p and miR-22-5p Is Associated with Reduced Expression of Proteins Involved in Ca2+ Homeostasis and Cell-to-Cell Communication Since circulating expression of both miRNAs was confirmed in atrial samples of AF patients, we sought to evaluate the potential role of miR-199a-5p and miR-22-5p in AF promotion by studying the effects of simultaneous overexpression of both miRNAs (miR-199a+miR-22) in atria-derived HL-1 cardiomyocytes versus cells transfected with miR-negative control (miR-NC). The efficiency of miRNA transfection was assessed by qPCR (Supplementary File, Figure S1). Among all predicted targets for both miRNAs (TargetScan), we focused on those molecules involved in Ca2+ homeostasis and cell-to-cell communication, key elements in AF pathophysiology [20,21]. We first evaluated the expression of the main target genes involved in both pathways in miR-199a+miR-22 and miR-NC cells. Results are summarized in Figure 2 and Figure S3 (Supplementary File). Gene expression of CACNA1C (encoding the alpha-subunit of the LTCC, Figure 2A) showed a trend, though it did not reach statistical significance, towards a reduction in miR-199a+miR-22 HL-1 cells compared to miR-NC. Western blot (WB) revealed a significant 30% reduction in Cav1.2 expression (Figure 2D,G, p < 0.0001). Gene expression of SLC8A1 (encoding NCX) was significantly reduced in miR-199a+miR-22 compared to miR-NC cells (Figure 2B), which translated into a 20% reduction in the protein expression (Figure 2E,G). Gene expression of RYR2, encoding the ryanodine receptor, was reduced in miR-199a+miR-22 cells, with no change in ATP2A2, PLN, or CAMK2A (encoding SERCA2, phospholamban and the alpha-subunit of the calcium-calmodulin-dependent kinase II, respectively (Supplementary File, Figure S3). The expression studies of the three main connexins in the heart revealed that only GJA5, encoding Cx40, the main connexin at the atrial level, was significantly reduced in miR-199a+miR-22 compared to miR-NC cells (p = 0.029, Figure 2C), with no significant changes in the genes encoding Cx43 and Cx45 (GJA1 and GJC1, respectively). Similarly, protein expression of Cx40 was decreased by 25% in cells transfected with miR-199a+miR- 22 (p = 0.0036, Figure 2F,G). 2.4. Overexpression of miR-199a-5p and miR-22-5p Is Associated with Reduced Intracellular Ca2+ Levels and a Decrease in Barium Currents (IBa ) To investigate the functional consequences of the decrease of Cav1.2, we monitored the intracellular calcium measurements under resting conditions, which revealed a prominent reduction in cytosolic Ca2+ levels of miR-199a+miR-22 HL-1 cells compared to miR-NC (p < 0.0001, Figure 3A). Patch-clamp experiments showed a marked reduction in IBa , the surrogate for the Ca2+ currents, in miR-199a+miR-22 HL-1 cells compared to miR-NC (p = 0.026, Figure 3B). Calcium influx through Cav1.2 was also impaired in miR-199a+miR- 22 HL-1 cells under depolarizing conditions induced by high extracellular potassium levels (Figure 3C, p = 0.004). These findings, together with the fact that no differences were observed in the fast-inactivation kinetics nor in cell capacitance between miR-199a+miR-22 and miR-NC HL-1 cells (Supplementary File, Figure S2), suggest that the reduction of inward currents present in miR-199a+miR-22 HL-1 cells is due to a decrease in the total protein content of channel rather than a functional defect. Int.Int. J. Mol.Sci. J. Mol. Sci.2021, 22, x10377 2021,22, 6 of 17 6 of 1 Figure2.2.Gene Figure Geneand andprotein proteinexpression expression (A–C). (A–C). Expression Expression levels levels of CACNA1C, of CACNA1C, SLC8A1 SLC8A1 and the con and the nexin members, connexin members,GJA5, GJA5, GJA1 GJA1 andandGJC1 GJC1ininHL-1 HL-1 cells cells transfected transfected withwith miR-NC miR-NC (black(black columns) o columns) miR-199a+miR-22 or miR-199a+miR-22 (white columns). Normalized (white columns). Normalized to to miR-NC miR-NC (D–F). (D–F). Protein Protein expression expression levels levels o CaCav1.2, of V1.2, NCX1 NCX1and and Cx40, Cx40, normalized normalized totothe theendogenous endogenous control, control, tubulin tubulin (G). (G). Representative W Representative experiments WB experimentsin in HL-1 HL-1 cells cellstransfected transfectedwith with miR-22+199a miR-22+199a and andmiR-NC. miR-NC.All Allproteins proteins (CaV1.2, NCX (Cav1.2, connexin NCX1, 40 and connexin 40tubulin) and tubulin)werewere blotted on the blotted same on the gel.gel. same This is aisrepresentative This a representativeimage imageofoffour inde pendent experiments. Numbers within columns represent the number four independent experiments. Numbers within columns represent the number of independent of independent experiment done. * p < done. experiments 0.05, *** * p p< <0.05, 0.0001, *** p n.s.: non-significant. < 0.0001, n.s.: non-significant. 2.5. Cells Transfected with miR-199a-5p and miR-22-5p Have Higher Ca2+ Levels in the 2.4. Overexpression of miR-199a-5p and miR-22-5p 2+ Is Associated with Reduced Intracellular Sarcoplasmic Reticulum and a Slower Caffeine-Induced Ca Decay Ca Levels and a Decrease in Barium Currents (IBa) 2+ Given the reduced expression of NCX1, we evaluated the rapid Ca2+ buffering after a To investigate caffeine-induced the functional response, driven by the consequences SERCA2 pumpofatthe the decrease of Ca sarcoplasmic V1.2, we monitore reticulum (SR) the NCX1 and intracellular calciummembrane. at the cytosolic measurementsFigureunder resting conditions, 4A represents the dynamicswhich of Ca2+ revealed release a prom inent in reduction response to 10 mMin cytosolic CamiR-199a+miR-22 caffeine in 2+ levels of miR-199a+miR-22 HL-1 cells compared to miR and miR-NC cells after normalizing to basal 2+ 2+ release was NC (p 1 year) or permanent AF (permAF), or otherwise those with no previous history of AF (sinus rhythm, SR), were included. SR patients were required to be in permanent SR if they had any intracardiac electronic device implanted or to be persistently found in SR in all on-site follow-up visits. Patients with other forms of AF were excluded from the study. Blood was collected from all patients in stable clinical status and immediately cen- trifuged at 1550× g for 10 min. Plasma was transferred to RNase/DNAse-free tubes and stored at −80 ◦ C until use. The study was conducted in accordance with the Declara- tion of Helsinki, and approved by the local Ethics Committee. All patients gave written informed consent. 4.1.2. Experimental Design This was a case-control study in which the miRNA expression of HFrEF patients with permAF (cases) was compared to that of HFrEF and SR (controls) following a two-step approach: (1) discovery phase using microarray data; (2) replication phase of those miRNAs showing the greatest association in the microarray study. For the discovery phase, a total of 18 patients (9 per group) were selected, matched for sex, age, body mass index and HF parameters in order to diminish confounding factors and guarantee that the populations of study were very similar except for the presence of AF. For the replication phase, we selected an independent cohort of 80 patients (40 per group) matched exclusively for age and sex, accepting the potential interference of confounding factors in an attempt to prove the association of biomarkers with AF irrespective to other baseline clinical conditions. The experimental design of the study is summarized in Figure 6. 4.2. MicroRNA Extraction and Profiling MiRNAs were isolated from plasma using the miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany), and retrotranscribed into cDNA with the TaqMan Advanced miRNA cDNA Synthesis Kit. In the discovery phase, an OpenArray® technology was used to screen 754 of the most expressed human miRNAs. Expression levels of all miRNAs were analyzed with the Cloud software and the mean global normalization was used. Those miRNAs found to have a significant differential expression among groups (p < 0.05 and fold change >2 or <0.5) were selected for replication. MiRNA determination in the replication phase was performed by qPCR using the Fast Advanced master mix with specific TaqMan® miRNA probes (Life Technologies). MiR-660, miR-92a and miR-10b showed high stability between groups, and were therefore used as controls for normalization. Results were analyzed using the Expression Suite software. All reagents and sequencing technologies were from ThermoFisher, Waltham, MA, USA. Mol. Sci. 2021, 22, x 12 of 17 Int. J. Mol. Sci. 2021, 22, 10377 12 of 17 Figure 6. Study design. Of 495 patients with HFrEF, 18 (9 with permAF and 9 with SR) were selected for the discovery phase, and 80Figure (40 per6.group) Study design. Of 495 patients for the replication with phase. HFrEF, variables Matching 18 (9 withfor permAF and 9are each phase with SR) werewith presented, selected the accepted for the discovery phase, and 80 (40 per group) for the replication phase. Matching range in parenthesis. Abbreviations: HF-pEF, mrEF, and rEF: heart failure with preserved (pEF), mid-rangevariables for each (mrEF) and reducedphase are fraction ejection presented, withrespectively, (rEF), the accepted according range in parenthesis. to the ESCAbbreviations: HF-pEF, guidelines; (41) mrEF, permAF: and rEF: AF; SR: permanent heart failure with preserved (pEF), mid-range (mrEF) and reduced ejection fraction (rEF), respec- permanent sinus rhythm (no history of past or present AF); BMI: body mass index; CKD: chronic kidney disease; Hb: tively, according to the ESC guidelines;(41) permAF: permanent AF; SR: permanent sinus rhythm hemoglobin; LVEF: left ventricular ejection fraction; LVEDD: left ventricular end-diastolic diameter; Log(proBNP): pro-BNP (no history of past or present AF); BMI: body mass index; CKD: chronic kidney disease; Hb: hemo- values at baseline in Log globin; expression; LVEF: * included left ventricular matching ejection for treatment fraction; LVEDD:withleft betablockers, angiotensin-converting-enzyme ventricular end-diastolic diameter; inhibitors (ACEI), angiotensin-receptor blockers (ARB), and diuretics. Log(proBNP): pro-BNP values at baseline in Log expression; * included matching for treatment with betablockers, angiotensin-converting-enzyme inhibitors (ACEI), angiotensin-receptor blockers 5. Human Atrial Samples (ARB), and diuretics. 5.1. Cardiac Tissue Collection 4.1.3. MicroRNA Extraction and homogenates Whole-tissue Profiling from samples of the right and left atrial appendages MiRNAs(RAA, LAA) were were isolated fromobtained from the plasma using patients in AFSerum/Plasma miRNeasy or SR (no AF)kitwho were admitted for (Qiagen, the first-time elective on-pump cardiac surgery in the John Hilden, Germany), and retrotranscribed into cDNA with the TaqMan Advanced miRNA Radcliffe Hospital (Oxford, cDNA Synthesis Kit. In the discovery phase, an OpenArray technology was used to use. Experi- UK). Tissues were quickly rinsed of blood and snap-frozen ® until experimental screen 754 of mental the mostwork in human expressed specimens human miRNAs.wasExpression approved by the local levels of allResearch miRNAsEthics were Committees (REF #18/SC/0404) and all patients gave informed written consent. analyzed with the Cloud software and the mean global normalization was used. Those miRNAs found to have a significant differential expression among groups (p < 0.05 and 5.2. MicroRNA Profiling fold change >2 or <0.5) were selected for replication. MiRNA determination in the replica- Expression tion phase was performed bylevels qPCRofusing hsa-miR-199a-5p and hsa-miR-22-5p the Fast Advanced master mix transcripts in human RAA with specific TaqMan miRNA probes (Life Technologies). MiR-660, miR-92a and miR-10b showed mirVana Kit ® and LAA were measured by qPCR. Total RNA was extracted using the high stability(Applied Biosystems, between groups, Waltham, and were MA, used therefore USA)asaccording to the controls for manufacturer’s normalization. Re- instructions. Quantitative PCR was carried out using the TaqMan Advanced miRNA cDNA synthe- sults were analyzed using the Expression Suite software. All reagents and sequencing sis kit, TaqMan Advanced miRNA stem-loop primers (Supplementary File, Table S4), technologies were from ThermoFisher, Waltham, MA, USA. and TaqMan Universal Master Mix (all from Applied Biosystems, Waltham, MA, USA). Each reaction was performed in duplicate using the QuantStudio 7 Flex Real-Time PCR sys- 4.2. Human Atrial Samples tem (Applied Biosystems, Waltham, MA, USA) using miRNA stem-loop primers (Table S1), 4.2.1. Cardiacand Tissue Collection TaqMan Universal Master Mix. Relative quantification was calculated using the com- Whole-tissue homogenates parative threshold cycle samples(2−4 frommethod Ct ); the of the right and left of expression atrial appendages the target was normalized to (RAA, LAA) were obtained the mean of thefrom two patients in AF or housekeeping SR (no genes, AF) who wereand hsa-miR-191-5p admitted for the hsa-miR-26a-5p. Int. J. Mol. Sci. 2021, 22, 10377 13 of 17 6. Molecular Studies 6.1. Cell Cultures and Transfection Atrial HL-1 cardiomyocytes were cultured and maintained in Claycomb medium (Sigma Aldrich, Sain Louis, MO, USA) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% norepinephrine and 1% glutamine. They were transfected using lipofectamine RNAiMAX with 30 nM of mimic mmu-miR-199a-5p (assay MC10893) and 30 nM of miRNA mimic mmu-miR-22-5p (assay MC11752), in the text, miR-199a+miR-22, or with 60 nM of mir- Vana™ miRNA Mimic Negative Control #1, in the text, miR-NC. Experiments were per- formed 24 h after transfection. Transfection reagents were all from ThermoFisher, Waltham, MA, USA. 6.1.1. qPCR and Western Blot Analyses To evaluate gene expression, RNA was extracted using the Nucleospin® RNA extrac- tion kit (Macherey-Nagel, Duren, Germany). RNA was retro-transcribed into cDNA and the qPCR was performed using the specific TaqMan probes cycled in a 7500 ThermoCycler (Applied Biosystems, Waltham, MA, USA). HPRT was used as the endogenous control. Each transfection condition was repeated in 4 independent experiments. Expression level measurements are expressed as the mean ± standard error of the mean (SEM) of the 4 results. For protein determination, total protein was extracted from HL-1 cell cultures. In each lane, 25 µg of protein was loaded and separated on 4–12% SDS-PAGE gels before transferring to a nitrocellulose membrane. Membranes were blocked for 1 h at room tem- perature with TBST buffer containing 5% non-fat milk and incubated overnight at 4 ◦ C with the following primary antibodies, all at a 1/1000 dilution: anti-Cav1.2 (L-type calcium channel alpha subunit, ACC-003, Alomone Labs, Jerusalem, Israel), anti-NCX1 (ANX-011, Alomone Labs, Jerusalem, Israel), anti-Cx40 (36–4900, Thermo Fisher Scientific, Waltham, MA, USA) and anti-beta-tubulin antibody (ab6046, Abcam, Cambridge, UK). Membranes were washed with TBST and incubated for 1 h at room temperature with the secondary antibodies (Agilent-Dako, Santa Clara, CA, USA). After washing twice with TBST, bands were visualized with Super Signal (Pierce, Rockford, IL, USA) and developed with the Quantity One system. Optical density analysis of the bands was carried out with the Quantity One software. All proteins were normalized to the loading control beta-tubulin. 6.1.2. Recordings of Intracellular Ca2+ and Patch Clamp Studies The intracellular Ca2+ recordings were obtained from HL-1 cells transfected with miR-22-5p + miR-199a-5p or miR-NC and loaded with 5µM Fura-2AM (Molecular Probes, Eugene, OR, USA) during 45 min. Calcium measurements were obtained using an Olym- pus IX70 inverted microscope (Olympus, Hamburg, Germany). Cells were excited at 340 and 380 nm using a polychrome IV monochromator (Till Photonics, Munich, Germany) and fluorescence images were collected by a digital charge-coupled device camera (Hama- matsu Photonics, Japan) and viewed with the AquaCosmos software program (Hamamatsu Photonics, Japan). Throughout the experiments, cells were kept in an isotonic solution containing (in mM): 140 NaCl, 2.5 KCl, 1.2 CaCl2 , 0.5 MgCl2 , 5 glucose and 10 HEPES, pH 7.3 and 300–310 mOsmol/L. Intracellular calcium levels were recorded in the follow- ing conditions: basal resting conditions, following 10 mM of caffeine and after applying a hyperpolarizing solution containing (in mM): 60 NaCl, 75 KCl, 1.2 MgCl2 , 5 CaCl2 , 10 glucose and 10 HEPES, pH 7.3 and 300–310 mOsmol/L. Cytosolic Ca2+ levels are graphed as the ratio of emitted fluorescence (510 nm) after excitation at 340 and 380 nm relative to the baseline. 340/380 nm ratio images were obtained every 5 s. Calcium decay post-caffeine stimulation was inferred by calculating the time constant Tau with the use of a script written for this paper. Ba2+ currents through Cav1.2 channels were recorded miRNA-transfected HL-1 cells. Currents obtained by patch-clamp technique in a whole-cell configuration were recorded with a D-6100 Darmstadt amplifier (List Medical, Germany). Three independent transfec- Int. J. Mol. Sci. 2021, 22, 10377 14 of 17 tions were patched. Pipettes had a resistance of 2-3 MΩ when filled with a solution con- taining (in mM): 140 CsCl, 1 EGTA, 4 Na2 ATP, 0.1 Na3 GTP and 10 HEPES, pH 7.2–7.3 and 290–300 mOsmol/L. The external solution contained (in mM): 140 tetraethylammonium-Cl (TEA-Cl), 2.5 BaCl2 , 1.2 MgCl2 , 10 glucose and 10 HEPES, pH 7.4 and 300–310 mOsmol/L. pClamp8 software (Molecular Devices, San Jose, CA, USA) was used for pulse generation, data acquisition and subsequent analysis. Membrane potential was kept at −30 mV and maximal peak IBa were recorded at +20 mV for 500 ms. 6.1.3. Gap-Junction Monitoring Gap-junctions were monitored by the diffusion of a fluorescent dye between at- tached cell pairs, as previously described [42]. HL-1 cells transfected with miR-22-5p + miR-199a-5p or miR-NC were seeded at very low confluences in gelatin-coated 12 mm diameter glass coverslips and incubated at 37 ◦ C for 24 h in supplemented Claycomb medium. Afterwards, coverslips were mounted on a Zeiss Axiovert 10 microscope (Zeiss, Jena, Germany. Only attached cell pairs were selected, and one cell was injected with 0.1 pL of the molecule Alexa Fluor 488 (Alexa 570 MW, ThermoFisher, Waltham, MA, USA) using a FemtoJet 4i electric microinjector (Eppendorf, Hamburg, Germany). Images were acquired at 5 min intervals over a 90 min period to monitor the diffusion of the fluores- cent dye from the injected cell to the adjacent one. These images were collected using a digital camera ZEISS Axiocam MRc and operated by the imaging software Zen (ZEISS, Jena, Germany). 6.2. Statistical Analyses Selection of patients following the predefined matching criteria was performed with the statistical R software. Clinical data are reported as mean (SD) or frequency (%) as appropriate. MiRNA expression results are expressed with the 2− ∆∆Ct method [43]. Comparisons between groups were performed with Student’s t-test or chi-square analy- sis. For big-data analysis of OpenArray® determinations, the specific CLOUD Software from Life Technologies (ThermoFisher, Waltham, MA, USA) was used to detect miRNAs differentially expressed among groups, with the mean global normalization approach. Tar- getScanHuman7.2 software was used to predict genes that could be potential targets of the miRNAs of interest. Statistical analyses were performed using the Statistical Package for Social Sciences 23.1 software package (SPSS Inc.) For all analyses in the study, differences were considered significant when p < 0.05. 7. Conclusions This work demonstrates that 199a-5p and miR-22-5p are increased in plasma of HFrEF patients with AF compared to those in SR, and in atrial tissue obtained from patients with AF. Overexpression of both miRNAs in cultured atrial cells induces pro-arrhythmogenic responses. The potential role of high circulating levels of miR-199a-5p and miR-22-5p as biomarkers of AF in other contexts should be addressed in future studies. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms221910377/s1. Author Contributions: A.G.-E., J.C.-C., C.E., S.A.S., J.M.F.-F., J.M.-A. and B.B. designed the study. A.G.-E., M.T., L.Y.-B., K.W.A.-A., S.R. and S.A.S. performed the experiments and analyzed the results. J.C.-C., C.E. and B.B. recruited the patients and provided their clinical information. B.B.: obtained competitive funding for the present work. A.G.-E. and B.B. supervised the statistical analyses and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the following grants, awarded to B.B.: Sociedad Española de Cardiología, Sección de Arritmias y Electrofisiología 2012; Sociedad Española de Cardiología, Sección de Insuficiencia Cardíaca 2013; Fondo Investigación Sanitaria (FIS)—Instituto Carlos III 2013 (PI13/01830); and Societat Catalana de Cardiologia 2016. Awarded to K.W.A-A. and S.R.: British Heart Foundation (BHF) Intermediate Research Fellowship. Awarded to J.M.F.F.: grant Int. J. Mol. Sci. 2021, 22, 10377 15 of 17 from the Spanish Ministry of Science and Innovation (RTI2018-094809-B-I00). “María de Maeztu” Programme for Units of Excellence in R&D to the Departament de Ciències Experimentals i de la Salut (MDM-2014-0370) and FEDER (Fondo Europeo de Desarrollo Regional) also contributed to this work. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the local Research Ethics Committee of IMIM, Barcelona, Spain (protocol code 2004/1788) and the John Radcliffe Hospital, Oxford, UK (REF#18/SC/0404). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available in this paper and supplementary file. Acknowledgments: Authors would like to acknowledge the work of: Feng Xiong, from the Montreal Heart Institute (Canada) for writing the script used to calculate the Tau shown in Figure 4C; Sarah Bonnin from the CRG Bioinformatics Core Facilities (Centre for Genomic Regulation, Barcelona, Spain) for her help in the bioinformatic analysis of the OpenArrays. Authors would also like to thank the Alfonso Martín Escudero Foundation for the fellowship awarded to A.G.-E. in 2018. 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