Altered Physiological Functions and Ion Currents in Atrial Fibroblasts From

Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Altered physiological functions and ion currents in atrial ﬁbroblasts from patients with chronic atrial ﬁbrillation Claire Poulet1, Stephan Kunzel€ 1, Edgar Buttner€ 1, Diana Lindner2, Dirk Westermann2 & Ursula Ravens1 1 Department of Pharmacology and Toxicology, Medical Faculty Carl-Gustav-Carus, TU Dresden, Dresden, Germany 2 Department of General and Interventional Cardiology, University Heart Center Hamburg Eppendorf, Hamburg, Germany

Keywords Abstract Atrial fibrillation, electrophysiology, fibroblasts. The contribution of human atrial fibroblasts to cardiac physiology and pathophysiology is poorly understood. Fibroblasts may contribute to arrhythmogen- Correspondence esis through fibrosis, or by directly altering electrical activity in Claire Poulet, Imperial College London, Imperial cardiomyocytes. The objective of our study was to uncover phenotypic differ- Centre for Translational and Experimental ences between cells from patients in sinus rhythm (SR) and chronic atrial fib- Medicine, Hammersmith Campus, Du Cane rillation (AF), with special emphasis on electrophysiological properties. We Road, London W12 0NN, UK isolated fibroblasts from human right atrial tissue for patch-clamp experi- Tel: +44 207 594 2738 Fax: +44 207 594 3653 ments, proliferation, migration, and differentiation assays, and gene expression E-mail: [email protected] profiling. In culture, proliferation and migration of AF fibroblasts were strongly impaired but differentiation into myofibroblasts was increased. This Present Addresses was associated with a higher number of AF fibroblasts expressing functional Claire Poulet, Imperial College London, Nav1.5 channels. Strikingly Na+ currents were considerably larger in AF cells. National Heart and Lung Institute, London, UK Blocking Na+ channels in culture with tetrodotoxin did not affect proliferation, migration, or differentiation in neither SR nor AF cells. While freshly Edgar Buttner,€ Biotechnology Centre Dresden, TU Dresden, Dresden, Germany isolated fibroblasts showed mostly weak rectifier currents, fibroblasts in culture developed outward rectifier K+ currents of similar amplitude between the SR Funding Information and AF groups. Adding the K+ channel blockers tetraethylammonium and 4- This work was financially supported by the aminopyridin in culture reduced current amplitude and inhibited proliferation European Union (FP7-Health-F2-2010- in the SR group only. Analysis of gene expression revealed significant differ- 261057; EUTRAF). Some parts of the ences between SR and AF in genes encoding for ion channels, collagen, manuscript are included in S.K.’s medical growth factors, connexins, and cadherins. In conclusion, this study shows that thesis which was supported by Deutsche Herzstiftung e.V. with a “Kaltenbach under AF conditions atrial fibroblasts undergo phenotypic changes that are Doktorandenstipendium”. revealed in culture. Future experiments should be performed in situ to understand the nature of those changes and whether they affect cardiac electrical Received: 25 August 2015; Revised: 30 activity. November 2015; Accepted: 16 December 2015 doi: 10.14814/phy2.12681

Physiol Rep, 4 (2), 2016, e12681, 10.14814/phy2.12681

Introduction thereby ensuring a proper cardiac function, for review see (Camelliti et al. 2005; Souders et al. 2009). They are pri- Cardiac ﬁbroblasts are an essential cell population of the marily responsible for the homeostasis of the extracellular heart. Their role is to maintain the integrity of the tissue, matrix (ECM) through the synthesis and degradation of

ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2016 | Vol. 4 | Iss. 2 | e12681 the American Physiological Society and The Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Fibroblasts from Patients with Atrial Fibrillation C. Poulet et al. connective tissue components. Involved in an extensive thereby providing a substrate for AF (Li et al. 1999). And communication with their environment – through direct second, fibroblasts can alter atrial electrical activity by cell–cell or cell-matrix connections, and via the autocrine directly coupling with cardiomyocytes through gap and paracrine actions of various cytokines and growth junctions. factors – fibroblasts sense changes in mechanical and Although the presence of such connections between chemical signals and react appropriately to stress or fibroblasts and cardiomyocytes remains to be demon- injury. strated in human tissue, functional gap junctions were Upon pathological stimuli, such as myocardial infarc- observed in animal models (Camelliti et al. 2004a,b) and tion, fibroblasts proliferate, and migrate to the site of their consequences on the electrical activity of cardiomy- injury where they differentiate into cells resembling ocytes were thoroughly studied in co-culture systems. smooth muscle, and aptly called “myofibroblasts” (Majno Because of the less negative resting membrane potential et al. 1971). Similarities to smooth muscle cells include (RMP) of fibroblasts, such electrotonic interactions pri- an increase in the number and extent of connections to marily provoke the depolarization of cardiomyocytes, the ECM and to other cells, and the development of a fib- resulting in (1) slowing of conduction, due to the reduced rillar sytem of contractile proteins (Gabbiani et al. 1972). number of available Na+ channels and which facilitates The main protein forming the myofilaments, alpha- reentry (Miragoli et al. 2006; Zlochiver et al. 2008) and smooth muscle actin (aSMA), has been widely used as a (2) ectopic activity when the threshold potential for acti- marker of fibroblast differentiation until today. vation is reached (Miragoli et al. 2007). During the healing process, myofibroblasts help to In this context, exploring the electrophysiological prop- rebuild damaged tissue by producing large amounts of erties of fibroblasts is of primordial importance as the fibrillar collagen, replacing lost cardiomyocytes by scar expression of ion channels, and the resulting changes in tissue (Cleutjens et al. 1995). Equipped with a de novo electrical conductance might further impair cardiomy- contractile apparatus, they provide mechanical strength to ocyte activity. Multiple ion currents have been described the remodeling tissue which might reduce scarring (Gab- in variable proportions of cultured human fibroblasts, biani et al. 1972; Hinz et al. 2001). This reparative proce- including Na+ currents, various K+ currents (delayed rec- dure is essential to prevent dilation and wall thinning, tifier, inward rectifier, and Ca2+-activated channels of big but can lead to pathological fibrosis if an excessive myofi- conductance), and chloride currents (Wang et al. 2006; Li broblast activity persists. Indeed, while in most tissues et al. 2009; Chatelier et al. 2012). Interestingly, recent myofibroblasts usually undergo apoptosis, once the heal- studies using a canine model of atrial fibrillation reported ing process is over (Desmouliere et al. 1995), they remain the remodeling of K+ currents under disease conditions in infarct scars for years, where they continue to promote (Wu et al. 2014; Qi et al. 2015). The objective of this fibrosis (Willems et al. 1994). study was to compare atrial fibroblasts from patients with In other organs, including lung, kidney and liver, the a normal cardiac rhythm (sinus rhythm, SR) and with abnormal persistence of myofibroblasts and resulting pro- chronic AF to uncover any phenotypic differences, hence gressive fibrosis were shown to be associated with organ providing insight into the relationship between atrial failure, reviewed in (Hinz et al. 2012). Furthermore, after fibroblasts and atrial fibrillation. myocardial infarction, collagen deposition is not only found in the infarct scar, but also in noninfarcted areas of Material and Methods the heart (Volders 1993; Cleutjens et al. 1995), forming reactive fibrosis around cardiomyocytes that contributes to Ethical approval ventricular stiffness and dysfunction (Litwin et al. 1991). Fibrosis notably plays an important role in the patho- The study was approved by the ethics committee of Dres- physiology of atrial fibrillation (AF); reviewed in (Bur- den University of Technology (No. EK790799). Each stein and Nattel 2008). While atrial fibrosis might patient gave written, informed consent. Right atrial originate from other underlying cardiac diseases, rapid appendages were obtained from patients with SR and atrial pacing alone results in ECM remodeling in animal with chronic AF (AF >6 months) undergoing open-heart models (Li et al. 1999; Pan et al. 2007; Avitall et al. surgery for coronary artery and/or valve disease. More 2008), and interstitial fibrosis was found in patients with details on patients are available in Table 1. lone AF (Frustaci et al. 1997; Boldt et al. 2004). Structural remodeling indeed correlates with the development of Cell culture sustained AF (Xu et al. 2004) and might participate in the maintenance of the disease, reviewed in (Yue et al. Cells were isolated using the outgrowth technique and 2011). First, interstitial fibrosis impairs local conduction enzymatic digestion. For the outgrowth method, tissue

2016 | Vol. 4 | Iss. 2 | e12681 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 2 the American Physiological Society and The Physiological Society. C. Poulet et al. Fibroblasts from Patients with Atrial Fibrillation

consistent gap of 0.9 mm between the cells. After 2 weeks Table 1. Clinical parameters of the patients involved in the study. of culture following replating, cells were trypsinized and SR AF replated in 24-well plates containing the wound healing inserts at a density of 5 9 10 4 cells per well. After let- Patients, n 58 33 Gender, m/f 50/8 28/5 ting the cells attach overnight, the inserts were removed Age, year 65.3 1.5 70.8 1.4 to monitor the closure of the gap. Cells were stained with Body mass index, kg/m2 27.7 0.5 29.5 0.7 DAPI or the staining solution provided with the assay CAD, n (%) 45 (78%) 7 (21%) 24 h after the inserts were removed. The number of MVD/AVD, n (%) 7 (12%) 16 (48%) migrating cells was evaluated by counting the number of + CAD MVD/AVD, n (%) 6 (10%) 10 (30%) nuclei within the gap. Hypertension, n 54 (93%) 32 (97%) Diabetes, n 23 (40%) 17 (52%) Hyperlipidemia, n 50 (86%) 26 (79%) Gene expression analysis LVEF, % 51.4 1.9 48.1 2.3 Digitalis, n (%) 3 (5%) 13 (39%) Total RNA from cells was isolated using the peqGOLD ACE inhibitors, n (%) 37 (64%) 18 (54%) Total RNA kit (PeqLab, Erlangen, Germany) according to AT1 blockers, n (%) 10 (17%) 7 (21%) the manufacturer’s instructions. RNA was cleared off b -blockers, n (%) 45 (78%) 28 (85%) genomic DNA by DNase digestion (DNase I digest kit, Ca2+-antagonists, n (%) 13 (22%) 11 (33%) PeqLab). Diuretics, n (%) 20 (34%) 26 (79%) Nitrates, n (%) 5 (9%) 2 (6%) The GeneChip Human Gene 1.0 ST Array (Affyme- Lipid-lowering drugs, n (%) 47 (81%) 20 (61%) trix, Santa Clara, CA, USA) comprises 36,079 transcripts. Gene ST stands for sense target – the probes on the array SR, sinus rhythm; AF, chronic atrial fibrillation; CAD, coronary are selected to be distributed along the entire sequence of artery disease; MVD, mitral valve disease requiring valve replace- the transcript. First RNA integrity was checked by analyz- ment; AVD, aortic valve disease requiring valve replacement; LVEF, ing on the 2100 Bioanalyzer (Agilent technologies, PA, left ventricular ejection fraction; ACE, angiotensin-converting enzyme; AT, angiotensin receptor. USA). For the first cycle cDNA synthesis 300 ng of total RNA was used. During the following in vitro transcrip- tion reaction cRNA was obtained and used as starting was cut in small pieces (~ 1mm3) and placed in a material for the second cycle cDNA synthesis. The second 25 mm-diameter petri dish with 1 mL medium consisting cycle random-primed single-strand DNA synthesis of DMEM (Sigma-Aldrich, Steinheim, Germany), 10% resulted in a product containing incorporated deoxyuri- FCS and 1% penicillin/streptomycin (Biochrom, Berlin, nine at predefined ratios relative to thymidine (Ambion Germany). The medium was changed every second day, WT Expression Kit, Ambion, Austin, Texas, USA). Subse- and after 3 weeks of culture, cells were trypsinized, quently, 5.5 lg of this generated single-strand DNA was replated at a density of 2.5 9 10 3 cells per cm2 and fur- fragmented with a combination of uracil DNA glucosylase ther cultivated for 10–15 days before analysis. In some (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) experiments, the culture medium was supplemented with and then labeled using the WT terminal labeling kit 10 lmol/L tetrodotoxin (TTX) or 1 mM tetraethylammo- (Affymetrix Inc., Santa Clara, CA, USA). The biotinylated nium plus 1 mmol/L 4-aminopyridine (TEA + 4AP). DNA was added to a hybridization cocktail for hybridiza- Drugs were added freshly every time the medium was tion to the Human Gene 1.0ST Array, also containing 50 changed for 2 weeks. For the enzymatic isolation, small pM control oligonucleotide B2, eukaryotic hybridization pieces of tissue (~ 1mm3) were digested with collagenase controls (bioB, bioC, bioD, 1.5, 5, 25 pM, respectively), A (Sigma-Aldrich, Steinheim, Germany), 2 mg per according to the manufacturer’s protocol (Hybridization

100 mg of tissue in medium, for 5 h at 37°C, 5% CO2. Wash and Stain Kit, Affymetrix). The hybridization in the Following filtration and centrifugation, the pellet was Affymetrix hybridization oven 640 at 45°C and 60 rpm resuspended in 1 mL medium, and the cell suspension for 16 h, the staining and washing, processed in the Flu- was then plated and incubated for 12 h at 37°C, 5% CO2 idics Station 450 and the scanning on the GeneChip before patch-clamp experiments. Scanner 3000 G7 system were carried out as recom- mended by Affymetrix (Affymetrix Inc., Santa Clara, CA, USA). The CEL files were imported to the Partek Geno- Migration assay mics Suite 6.6 software. The data processing steps The migration capacity of fibroblasts was assessed with involved the background correction on the PM values, the Cytoselect wound healing assay (Biocat GmbH, the quantile probe normalization across all arrays of the Heildeberg, Germany) using inserts that generate a experiment as well as the log2 transformation. A mean

value of 5 was set to be a detectable expression in the ments: y = 1/(1 + exp[(x–V0.5)/k]), where V0.5 is the investigated sample. To find differentially expressed genes, potential for half maximum activation or inactivation in analysis of variance (ANOVA) in a multifactor experi- mV, and k the slope factor. Nonselective ion currents ment was used (Fisher 1925). A step-up false discovery were measured with a voltage-ramp protocol and a higher rate (FDR) was applied to p values from the linear con- concentration of 20 mmol/L KCL in the bath solution to trasts to determine a cutoff for significantly differentially enhance the amplitude of inward rectifier K+ currents. expressed genes (Benjamini and Hochberg 1995). The rectifier factor (RF) was defined as the ratio of outward current to inward current measured 300 ms after and 300 ms before the reversal potential, respectively. The Immunostaining following drugs were diluted in bath solution and applied For immunostaining experiments, fibroblasts were culti- using a rapid solution exchanger (ALA Scientific Instru- vated on glass coverslips. Cells were fixed with 4% ments, NY, USA): TTX (100 nmol/L or 10 lmol/L), Bar- formaldehyde (Thermo scientific, Waltham, USA) for ium (1 mmol/L), TEA (1 mmol/L), 4-AP (1 mmol/L), 10 min, permeabilized using 0.5% Triton X-100 for Paxilline (1 lmol/L), and DIDS (200 lmol/L). The sys- 15 min and blocked with 10% FCS (Biochrom, Berlin, tem supplied only bath solution during drug-free periods Germany) in PBS for 30 min at room temperature. Incu- and for time-matched controls (TMC). bation with primary antibodies (all from Sigma-Aldrich, Steinheim, Germany) against vimentin (1:200), human Drugs and chemicals fibroblast surface protein (hFSP, 1:200), and a-smooth muscle actin (1:200) were done in blocking buffer for 2 h Tetrodotoxin was purchased from Carl Roth (Karlsruhe, at room temperature. Subsequent incubation with sec- Germany). All other compounds were from Sigma- ondary antibody anti-mouse Cy3 (1/1000) and counter- Aldrich (Steinheim, Germany). staining of nuclei with 40,6-diamidin-2-phenylindol (DAPI; 1/1000) were performed simultaneously in block- Statistical analysis ing buffer for 1 h at room temperature. Data are expressed as means SEM. In most cases, the number of experiments n represents the number of Electrophysiological recordings patients. For electrophysiological measurement, n = num- Electrophysiological recordings were performed on freshly ber of cells/number of patients. Statistical comparison isolated fibroblasts and cultured fibroblasts, 10–15 days between means was made using two-tailed paired or after replating. Membrane potentials and ion currents unpaired Student’s t-test. P < 0.05 was considered statisti- were measured 37°C with standard whole cell current- cally significant. Analysis of variance (ANOVA) was and voltage-clamp techniques. ISO-2 software (MFK, applied for analysis of gene expression. Niedernhausen, Germany) was used for data acquisition and analysis. Borosilicate glass microelectrodes had tip Results resistances of 2–5MΩ when filled with pipette solution. After gigaseal formation and breaking into the cell, cell Characterization of SR and AF cultured capacitance was measured using fast depolarizing ramp fibroblasts pulses (55 to 50 mV, 5 ms). Series resistance and cell capacitance were electronically compensated up to 75%. Fibroblasts growing out of explants could be observed All chemicals and drugs were purchased from Sigma- after 3–4 days of culture in both groups. The cells had Aldrich. All current–voltage relations and steady-state the stellar shape typical of fibroblasts and no morphologi- inactivation curves were measured within 5 min after cal difference between SR and AF cells was noted membrane rupture. Outward K+ currents and Na+ cur- (Fig. 1A). After 2– 3 weeks of culture, most cells rents were measured using voltage-step protocols with the expressed vimentin (SR: 161/177, 92.4 3.8%, n = 3; following solutions (mmol/L): NaCl 150, KCl 5.4, CaCl2 AF: 234/249, 89.4 7.7%, n = 4) and human fibroblast 2, MgCl2 2, glucose 11, Hepes 10 (pH 7.4, adjusted with surface protein (hFSP; SR: 136/143, 95.5 2.4%, n = 3; NaOH). The pipette solution consisted of (mmol/L): AF: 174/185, 94.2 1.8%, n = 4), confirming their NaCl 8, KCl 40, potassium aspartate 80, Tris-GTP 0.1, fibroblast phenotype (Fig. 1B). A fraction of cells

Mg-ATP 5, CaCl2 2, EGTA 5, Hepes 10 (pH 7.4, adjusted expressed aSMA-positive myofilaments, (SR: 810/1414, with KOH). Mean parameters for steady-state activation 56.0 4.4%, n = 6; AF: 744/1211, 59.9 5.0%, n = 7) and inactivation curves were calculated by fitting a Boltz- suggesting their differentiation into myofibroblasts. We mann function to the data points of individual experi- found a few isolated cells positive for the endothelial mar-

2016 | Vol. 4 | Iss. 2 | e12681 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 4 the American Physiological Society and The Physiological Society. C. Poulet et al. Fibroblasts from Patients with Atrial Fibrillation

Figure 1. Characterization of cultured fibroblasts. (A) Fibroblasts growing out of explants from SR and AF samples after 1 week of culture. Scale bar: 100 lm. (B) Staining for the expression of vimentin, human fibroblast surface protein (hFSP), alpha-smooth muscle actin (aSMA) and CD31 in AF fibroblasts before replating. Nuclei are stained in blue with DAPI. Cells expressing aSMA-positive myofilaments are easily distinguished from negative cells (arrows). Scale bar: 50 lm. (C) Cell count after 3 weeks of culture. The number of cells was normalized to the number of tissue pieces. *P < 0.05, unpaired Student’s t-test. (D) Staining for Vimentin and hFSP in AF fibroblasts after replating. Nuclei are stained in blue with DAPI. Scale bar: 50 lm. ker CD31 (SR: 24/585, 3.8 0.8%, n = 3; AF: 18/714, was much lower than that of SR cells (number of cells 1.8 1.6%, n = 4) which implies that the rare endothe- after 14 days, 9 104, SR: 4.4 0.5, n = 34; AF: lial cells that migrated out of the tissue did not prolifer- 2.2 0.2, n = 20; P < 0.01). Second, AF fibroblasts also ate, and further confirms that the cell population showed an impaired capacity for migration (number of consisted mostly of fibroblasts. A striking difference migrating cells after 24 h, SR: 102.9 10.0, n = 9; AF: between SR and AF samples was the number of cells 68.1 11.8, n = 7; P < 0.05). And finally, a larger num- obtained after 3 weeks of culture (Fig. 1C). AF biopsies ber of AF fibroblasts differentiated into myofibroblasts indeed yielded significantly less cells than SR samples (SR: 542/1065, 52.8 5.2; AF: 500/680, 75.0 3.9; (Number of cells/number of tissue pieces, 9 104, SR: P < 0.01). 0.33 0.04, n = 34; AF: 0.20 0.02, n = 18; P < 0.05). SR and AF fibroblasts showed overall similar expres- After fibroblasts were replated, 99–100% of SR and AF sion levels of extracellular matrix constituents and growth cells expressed vimentin (SR: 292/292: AF: 253/255, n = 4 factors (Fig. 3 and Table S1). They expressed high levels in both groups) and hFSP (SR: 282/282; AF: 234/234, of fibronectin and various members of the collagen fam- n = 4 in both groups, Fig. 1D). At this stage of culture, ily, mostly of type I, III, IV, V, and VI. Differences we observed important differences between the two between the two groups are noteworthy, such as a signifi- groups (Fig. 2). First the proliferation of AF fibroblasts cant lower expression of COL4A6 and COL10A1 in AF

Figure 2. Proliferation, migration, and differentiation of cultured fibroblasts. (A) Cells were counted after 7 and 14 days of culture following replating (SR, n = 34; AF, n = 20). (B) Migration was assessed using a wound healing assay (see Material and Methods). Left panel: example of the gap closure after 24 h. The graph summarizes the number of cells closing the gap. (C) Differentiation of fibroblasts into myofibroblasts was evaluated by staining the cells for a-smooth muscle actin (aSMA). Left panel: cells expressing aSMA-positive filaments are easily distinguished from negative cells (arrows). Right panel: The graph summarized the fraction of cells positive for aSMA. *P < 0.05, **P < 0.01, unpaired Student’s t-test.

fibroblasts but a tendency for higher COL15A1 levels. We Ion currents in fresh and cultured found high levels of matrix metalloproteinase (MMP) 1, fibroblasts 2, and 14 and tissue Inhibitor of metalloproteinase (TIMP) 1, 2, and 3 with a tendency for reduced MMP1 In order to understand the impact of culture conditions on expression in AF fibroblasts. We found, in both SR and the phenotype of our cells, we measured ion currents in AF cells high levels of connective tissue (CTGF), fibrob- freshly isolated fibroblasts, 12 h after isolation, and cul- last growth factors (FGF) 1, 2, 5, and 7, as well as FGF- tured fibroblasts, 10–15 days after the first passage (Fig. 4). receptor 1. Most members of the platelet-derived growth As shown by cell capacitance measurements, fresh factor (PDGF), transforming growth factor (TGF)-Beta, fibroblasts were much smaller than replated cells (Fig. 4A) and vascular endothelial growth factor (VEGF) families with a capacitance of 14.2 1.8 pF (n = 14) for fresh SR were also highly expressed in both groups. AF fibroblasts cells vs. 68.1 4.0 pF (n = 66) for replated SR cells showed a significant upregulation of FGF7 and PDGFA (P < 0.0001); and 14.6 1.4 pF (n = 7) for fresh AF and a tendency for higher levels of FGF1, TGFb1, and fibroblasts versus 63.5 3.3 pF (n = 77) for replated AF VEGFC. Finally while most integrins (ITG) and cadherins cells (P < 0.0001). (CDH) were similarly expressed in AF and SR cells, The amplitude of inward currents in fresh fibroblasts CDH13 and CDH19 were significantly upregulated in the was more variable and significantly larger than in replated AF group. We also observed a tendency for higher levels cells (Fig. 4C). At 120 mV, current amplitude was of ITGA7 and CDH1 and lower levels of ITGA10, CDH8, 13.4 3.5 pA/pF (n = 14/4) in fresh SR cells versus CDH10, and CDH17. The cardiac-specific gap junction 1.3 0.1 pA/pF (n = 23/11) in replated SR fibroblasts protein connexin 43 (Cx43) was highly expressed in both (P < 0.0001); and 25.7 8.2 pA/pF (n = 7/4) in fresh groups. Cx26 was significantly downregulated in AF AF cells versus 2.0 0.3 pA/pF (n = 15/6) in replated fibroblasts. AF fibroblasts (P < 0.001). Inward current amplitude in

2016 | Vol. 4 | Iss. 2 | e12681 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 6 the American Physiological Society and The Physiological Society. C. Poulet et al. Fibroblasts from Patients with Atrial Fibrillation

ECM Proteins Growth factors Ion channels SR AF SR AF SR AF ELN Elastin & CTGF KCNJ1 (Kir1.1) ENI EGF KCNJ2 (Kir2.1) COL1A1 Fibronectin EGFP EGF KCNJ12 (Kir2.2) COL1A2 FGF1 KCNJ4 (Kir2.3) COL2A1 FGF2 KCNJ14 (Kir2.4) COL3A1 FGF3 KCN43 (Kir3.1) COL4A1 FGF4 KCNJ6 (Kir3.2) COL4A2 FGF5 KCNJ9 (Kir3.3) K channels COL4A3 FGF6 KCNJ5 (Kir3.4) ir COL4A4 FGF7 KCNJ10 (Kir4.1) COL4A5 FGF8 KCNJ15 (Kir4.2) COL4A6 FGF9 KCNJ16 (Kir5.1) COL5A1 FGF10 KCNJ8 (Kir6.1) COL5A2 FGF11 KCNJ11 (Kir6.2) COL5A3 FGF12 KCNJ13 (Kir7.1) COL6A1 FGF13 KCNMA1 (BK) COL6A2 FGF14 FGF KCNN1 (SK1) COL6A3 FGF16 KCNN2 (SK2) K channels COL6A6 FGF17 KCNN3 (SK3) Ca COL7A1 FGF18 KCNN4 (SK4) COL8A1 FGF19 KCNA1 (Kv1.1) COL8A2 FGF20 KCNA2 (Kv1.2) COL9A1 FGF21 KCNA3 (Kv1.3) COL9A2 FGF22 KCNA4 (Kv1.4) COL9A3 Collagens FGF23 KCNA5 (Kv1.5) COL10A1 FGFR1 KCNA6 (Kv1.6) COL11A1 FGFR2 KCNA7 (Kv1.7) COL11A2 FGFR3 KCNA10 (Kv1.8) COL12A1 FGFR4 KCNB1 (Kv2.1) COL13A1 IGF1 KCNB2 (Kv2.2) COL14A1 IGF1R KCNC1 (Kv3.1) COL15A1 IGF2 IGF KCNC2 (Kv3.2) COL16A1 IGF2R KCNC3 (Kv3.3) COL17A1 PDGFA KCNC4 (Kv3.4) COL18A1 PDGFB KCND1 (Kv4.1) COL19A1 PDGFC KCND2 (Kv4.2) COL20A1 PDGFD PDGF KCND3 (Kv4.3) COL21A1 PDGFRA KCNF1 (Kv5.1) COL22A1 PDGFRB KCNG1 (Kv6.1) COL23A1 TGFA KCNG2 (Kv6.2) Kv channels COL24A1 TGFB1 KCNG3 (Kv6.3) COL25A1 TGFB1I1 KCNG4 (Kv6.4) COL27A1 TGFB2 KCNQ1 (Kv7.1) COL28A1 TGFB3 TGF KCNQ2 (Kv7.2) COL29A1 TGFBI KCNQ3 (Kv7.3) LAMA1 TGFBR1 KCNQ4 (Kv7.4) LAMA2 TGFBR2 KCNQ5 (Kv7.5) LAMA3 TGFBR3 KCNS1 (Kv9.1) LAMA4 VEGFA KCNS2 (Kv9.2) LAMA5 VEGFB VEGF KCNS3 (Kv9.3) LAMB1 VEGFC KCNH1 (Kv10.1) LAMB2 KCNH5 (Kv10.2) LAMB3 KCNH2 (Kv11.1) LAMB4 Laminins KCNH6 (Kv11.2) LAMC1 KCNH7 (Kv11.3) LAMC2 Membrane Proteins KCNH8 (Kv12.1) LAMC3 KCNH3 (Kv12.2) MMP1 SR AF KCNH4 (Kv12.3) MMP2 SCN1A (Nav1.1) MMP3 GJA1 (Cx43) SCN2A (Nav1.2) MMP7 GJA3 (Cx46) SCN3A (Nav1.3) MMP8 GJA4 (Cx37) SCN4A (Nav1.4) MMP9 GJA5 (Cx40) SCN5A (Nav1.5) MMP10 GJA8 (Cx50) SCN7A (Nav2.1) MMP11 GJA10 (Cx58) SCN8A (Nav1.6) MMP12 GJBI (Cx32) SCN9A (Nav1.7) Nav channels MMP13 GJB2 (Cx36) SCN10A (Nav1.8) MMP14 GJB3 (Cx31) SCN11A (Nav1.9) MMP15 GJB4 (Cx30.3) Connexins SCN1B (NavB1) MMP16 GJB5 (Cx30.1) SCN2B (NavB2) MMP17 GJB6 (Cx30) SCN3B (NavB3) MMP19 MMPs GJB7 (Cx35) SCN4B (NavB4) MMP20 GJC1 (Cx45) CACNA1S (Cav1.1) MMP21 GJC2 (Cx47) CACNA1C (Cav1.2) MMP23B GJC3 (Cx30.2) CACNA1D (Cav1.3) MMP24 GJD2 (Cx36) CACNA1F (Cav1.4) MMP25 GJD3 (Cx31.9) CACNA1A (Cav2.1) MMP26 GJD4 (Cx39) CACNA1B (Cav2.2) MMP27 ITGA2 CACNA1E (Cav2.3) ITGA2B MMP28 CACNA1G (Cav3.1) Cav channels TIMP1 ITGA3 CACNA1H (Cav3.2) TIMP2 ITGA4 CACNA1I (Cav3.3) TIMP3 ITGA5 CACNB1 (CavB1) TIMP4 ITGA6 CACNB2 (CavB2) TIMPs ITGA7 CACNB3 (CavB3) ITGA8 CACNB4 (CavB4) ITGA9 HCN1 ITGA10 HCN2 ITGA11 HCN3 HCN channels ITGAD HCN4 ITGAE Integrins ANO1 ITGAL CLCN1 ITGAM CLCN2 ITGAV CLCN3 ITGAX CLCN4 Chloride ITGB1 CLCN5 channels <4 >13 ITGB2 CLCN6 ITGB3 CLCN7 ITGB4 CLCNKA ITGB5 CLCNKB ITGB6 TRPA1 ITGB7 TRPC1 ITGB8 TRPC2 CDH1 TRPC3 CDH2 TRPC4 CDH3 TRPC5 CDH4 TRPC6 CDH5 TRPC7 CDH6 TRPM1 CDH7 TRPM2 CDH8 TRPM3 CDH9 TRPM4 TRP channels CDH10 TRPM5 CDH11 Cadherins TRPM6 CDH12 TRPM7 CDH13 TRPM8 CDH15 TRPV1 CDH16 TRPV2 CDH17 TRPV3 CDH18 TRPV4 CDH19 TRPV5 CDH20 TRPV6 CDH29

Figure 3. Gene expression of major ECM components, growth factors, membrane proteins, and ion channels in cultured fibroblasts. Gene expression was quantified in SR (n = 8) and AF (n = 5) fibroblasts 2 weeks after replating using an Affymetrix GeneChip array (see Material and Methods). Colors represent log2-transformed expression levels from low (blue) to high (red) levels. Boxes denote significant (solid line, P < 0.05) and nonsignificant (dotted line, 0.05 < P < 0.1) differences between SR and AF. Absolute expression levels, fold change, and P values are given in Table S1.

A B

Figure 4. Ion currents in freshly isolated and cultured fibroblasts. (A) Capacitance was measured in freshly isolated fibroblasts from four SR patients and three AF patients; and in replated fibroblasts from 11 SR patients and 10 AF patients. (B) Left panel: voltage-ramp protocol for current activation. Right panel: Example of different types of currents recorded in AF (red) and SR (black) fresh or replated fibroblasts. + [K ]o = 20 mmol/L. (C) Current–voltage relationship of currents measured in freshly isolated (left) and replated fibroblasts (right). (D) Fraction of freshly isolated (left) and replated fibroblasts (right) cells exhibiting inward rectifier, weak rectifier and outward rectifier current [defined by rectifier factors (see Material and Methods) of <0.8, 0.8 to <1.6, and >1.6, respectively]. fresh AF cells was larger than in fresh SR cells but this current amplitude was 7.8 2.3 pA/pF (n = 14/4) in difference was not significant. In replated fibroblasts, fresh SR cells versus 2.5 0.3 pA/pF (n = 23/11) in however, inward currents in AF cells were significantly replated SR fibroblasts (P < 0.01); and 12.5 3.9 pA/pF larger than in SR cells at voltages from 120 to 60 mV. (n = 7/4) in fresh AF cells versus 2.4 0.4 pA/pF Barium, a nonselective blocker of inward rectifier K+ (n = 15/6) in replated AF fibroblasts (P < 0.01). (Kir) channels did not affect inward current amplitude in To further characterize ion currents measured in fresh replated fibroblasts (Table 2). We could detect transcripts and replated fibroblasts, we defined the rectifier factor for the IK1 subunits, Kir2.2 and Kir2.3 in both replated (RF) as the ratio of outward current to inward current SR and AF cells (Fig. 3 and Table S1), however, as these (see Material and Methods) and distinguished three cate- channels are sensitive to Ba2+, they are unlikely to be gories: inward rectifier (RF < 0.8), weak rectifier (0.8 < involved in the currents observed. RF < 1.6), and outward rectifier (1.6 < RF). We found Under these experimental conditions, small outward that while ion currents in most fresh cells were weak rec- currents could also be observed, activating at 0 mV in tifiers, outward rectification was observed in a large frac- fresh cells and -20 mV in replated cells. At +40 mV, tion of replated SR and AF fibroblasts (Fig. 4D).

2016 | Vol. 4 | Iss. 2 | e12681 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 8 the American Physiological Society and The Physiological Society. C. Poulet et al. Fibroblasts from Patients with Atrial Fibrillation

levels of Kv6.2 and Kv11.3 that we observed in AF ﬁbrob- Table 2. Effect of various drugs on ion currents. lasts (Fig. 3 and Table S1). SR AF Interestingly, blocking Kv channels in culture with

+ 1 mmol/L TEA plus 1 mmol/L 4-AP reduced the prolifer- Inward K currents TMC 99.9 3.7 (7/4) 91.9 4.8 (6/2) ation of SR fibroblasts to the level of AF cells while it did Barium, 1 mmol/L 104.7 1.7 (6/3) 92.2 10.4 (4/3) not affect the proliferation of AF fibroblasts (Fig. 6A). Outward currents The number of cells after 14 days (*104) was in SR con- TMC 100.1 4.7 (7/3) 97.2 3.1 (6/3) trol: 5.3 1.3; and in SR TEA + 4-AP: 1.7 0.2; TEA, 1 mmol/L 83.2 2.5 (7/4)** 86.6 4.7 (11/3) n = 10; (P < 0.05); in AF control: 2.3 0.2; and in AF * 4-AP, 1 mmol/L 86.3 2.9 (5/3) 94.8 2.1 (11/3) TEA + 4-AP: 2.0 0.4; n = 6. Neither migration, nor Paxilline, 1 lmol/L 97.8 6.3 (6/3) 97.2 5.2 (4/2) differentiation of SR and AF fibroblasts were affected by DIDS, 200 lmol/L 91.5 5.2 (4/3) 96.5 8.6 (5/2) the addition of TEA and 4-AP in the culture medium Inward currents were measured at 120 mV using a voltage-ramp (Fig. 6B and C). protocol and high external K+ (see Fig. 4). Outward currents were Although the chloride channel blocker 4,4’-diisothio- measured at +60 mV using a voltage-step protocol and low exter- cyano-2,2’-stilbenedisulfonic acid (DIDS) had no effect + nal K (See Fig. 5). Data are given as percentage of predrug con- on current amplitude (Table 2) and the currents recorded trol. were hence unlikely to be Cl- currents, we found very *P < 0.05; **P < 0.001, compared to TMC, unpaired Student’s t test. high levels of transcripts for various voltage sensitive Cl channels (Fig. 3 and Table S1).

Sodium currents in cultured fibroblasts Outward potassium currents in cultured fibroblasts SR and AF fibroblasts showed a highly variable resting membrane potential (RMP) ranging from almost 0 to 80 mV Outward rectifying currents were then further character- in both groups (Fig. 7A) and averaging at 35.1 4.2 mV ized in replated SR and AF fibroblasts using a voltage-step (n = 31/7) in SR cells, and 36.7 3.4 mV (n = 34/6) in protocol and a low external K+ concentration (Fig. 5). AF fibroblasts. We measured fast activating, slowly inactivating currents We found Na+ currents in fibroblasts from six of 10 in both SR and AF fibroblasts (Fig. 5B). These currents SR patients and seven of nine AF patients (Fig. 7C). The activated around 20 mV (Fig. 5C) and mean peak cur- presence of Na+ currents was not patient-dependent since rent density was, at +80 mV, 6.6 0.7 pA/pF (n = 31/ cells with and without current were measured within the 12) for SR and 7.0 1.1 (n = 40/9) for AF (n.s.). Late same isolation. Interestingly a larger proportion of AF current density at +80 mV was 4.3 0.5 pA/pF (n = 31/ fibroblasts developed these currents: 57% in the AF group 12) for SR and 4.0 0.6 (n = 40/9) for AF (n.s.). (28 of 49 cells) for only 32% in the SR group (13 of 41 Steady-state inactivation curve is shown in Fig. 5D. cells). Furthermore, current amplitude was much larger in The parameters from curve fitting are summarized in AF cells: 3.7 2.1 pA/pF (n = 11) in SR vs. Table 3. The steady-state inactivation curve of AF cells 16.4 3.3 pA/pF (n = 20) in AF at 15 mV, P = 0.01, was slightly shifted toward more negative potentials; how- (Fig. 7D). The specific Na+ channel blocker tetrodotoxin ever, this difference failed to reach the level of statistical (TTX) was used to confirm the nature of the currents significance. In both groups a comparable fraction of cur- and help identify the channel isoform involved in AF rent did not inactivate. fibroblasts (Fig. 7E). A low concentration of 100 nmol/L Although we found high level of transcript for TTX did not affect the currents, however, almost 80% of KCNMA1 (Fig. 3 and Table S1), Ca2+-activated potas- current amplitude was blocked in the presence of sium channels of big conductance (BKCa) did not seem 10 lmol/L TTX (At 15 mV: AF control, 18.7 2.0 to be involved, as currents were not affected by paxilline pA/pF; AF 10 lmol/L TTX, 3.9 0.4 pA/pF; P < 0.05). (Table 2). The nonspecific K+ channel blockers tetraethy- Similar results were obtained with TTX on SR cells (data lammonium (TEA) and 4-aminopyridine (4-AP) reduced not shown). These results strongly suggest that cardiac current amplitude in SR fibroblasts but did but affect cur- fibroblasts expressed the cardiac-specific Na+ channel iso- rents in AF cells. This confirms the presence of voltage- form Nav1.5 for which the IC50 of TTX is ~2 lmol/L gated K+ (Kv) channels in SR fibroblasts and implies that (Satin et al. 1992). On the mRNA level, the main sodium those channels are absent in the AF group. These results channel a-subunits expressed by both SR and AF fibrob- could be explained by the significant downregulation of lasts were Nav1.4, Nav1.5, and Nav1.7 (Fig. 3 and

Kv4.1 expression and the tendency for lower transcript Table S1). The IC50 of TTX for Nav1.4 and Nav1.7 is in

Figure 5. Outward rectifier currents in cultured fibroblasts. (A) Voltage-step protocols for current activation (top) and steady-state inactivation + (bottom). (B) Examples of currents recorded at different potentials in a SR (black) and AF (red) fibroblast. [K ]o = 5.4 mmol/L. (C) Current– voltage relationship of outward peak and late currents. (D) Steady-state inactivation of outward peak currents. A Boltzmann function was fitted to the data points (see Material and Methods). Fitting parameters are given in Table 3. the nanomolar range (reviewed in (Catterall et al. 2005)) measured in AF cells were similar to those measured in confirming that the currents observed were most likely SR fibroblasts (Table 3). The overlap of the activation conducted through the cardiac-specific isoform Nav1.5. and inactivation curves defines a voltage range (“win- + The main Na channel b-subunits expressed was b1. dow”) where the channels are partially activated but not Steady-state activation and inactivation curves are fully inactivated. The probability of Na+ channel opening shown in Figure 7F. The parameters from curve fitting within that window was calculated as the product of the

activation and inactivation functions (1/{1 + exp[(V0.5act- V)/kact]} 9 1/{1 + exp[(VV0.5inact)/kinact]}) as described previously (Huang et al. 2011). In AF fibroblasts, we Table 3. Potassium and sodium current parameters. found a non-negligible probability to have persistent SR AF P opened channels between 60 mV and +20 mV with a peak value of 0.33% at 30 mV (Fig. 7G). In SR cells, IK parameters peak probability reached 0.56% at 25 mV. ss inactivation V0.5,mV 23.0 1.9 28.6 1.8 0.053 + l K 11.3 1.9 8.4 0.7 0.104 Blocking Na channels in culture with 10 mol/L TTX Noninactivating fraction % 51.9 3.7 43.6 4.5 0.213 did not affect proliferation, migration or differentiation n 9/5 15/5 – of SR and AF fibroblasts (Fig. 8A, B, and C). + + INa parameters Under the conditions used for Na and K currents Activation V ,mV 27.8 2.2 29.9 1.2 0.363 2+ 0.5 measurement ([Ca ]o = 2 mmol/L), we did not observe K 3.2 0.3 3.1 0.2 0.806 any inward currents with properties similar to voltage- n 11/6 20/7 – 2+ gated Ca currents. Additional experiments were per- ss inactivation V0.5,mV 63.2 3.6 64.7 1.7 0.372 + K 7.8 1.3 6.9 0.4 0.338 formed without Na in the bath solution and with 2+ 2+ n 5/3 16/6 – 10 mmol/L Ba as charge carrier to bypass Ca /calmodulin-dependent inactivation of Ca2+ channels (Levitan Voltage for half maximum activation and/or steady-state (ss) inac- 1999) We investigated the presence of functional voltage- 2+ tivation (V0.5) and the respective slope factors (k) were obtained gated Ca channels in 28 cells of five patients and never from fitting a Boltzmann function (see Material and Methods) to observed any inward currents (data not shown). It is, the data points shown in Figure 5D and Figure 6F. Data were however, interesting to note that the expression of several compared using a two-tailed unpaired Student’s t test. calcium channel subunits, including the cardiac-specific

2016 | Vol. 4 | Iss. 2 | e12681 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 10 the American Physiological Society and The Physiological Society. C. Poulet et al. Fibroblasts from Patients with Atrial Fibrillation

Figure 6. Effect of K+ current blockers on cellular functions of cultured fibroblasts. Fibroblasts were cultivated with normal culture medium (control) or medium supplemented with 1 mmol/L tetraethylammonium and 1 mmol/L 4-aminopyridine (TEA + 4-AP). Proliferation (A; SR, n = 10; AF, n = 6), migration (B; SR, n = 4; AF, n = 3), and differentiation (C; SR, n = 5; AF, n = 5) were assessed as described in Figure 2. *P < 0.05, two-tailed unpaired Student’s t-test. #P < 0.05, ##P < 0.01, paired Student’s t-test. channel Cav1.2, was significantly upregulated in AF from rapidly paced cardiomyocytes considerably reduced fibroblasts. proliferation of cultured right atrial fibroblasts and pro- moted their differentiation in myofibroblasts as shown by Discussion increased expression of aSMA and ECM proteins (Bur- stein et al. 2007). These results were corroborated by In this study, we have shown that AF fibroblasts have a in vivo data that showed increased levels of aSMA and reduced capacity for proliferation and migration in cul- ECM proteins in atrial tissue of atrial-tachypaced dogs. ture compared to SR cells. We found, however, that they Taken together, these results suggest that under rapid differentiate more readily and develop larger Na+ and Kir atrial activity such as AF, cardiomyocytes produce factors currents than the SR group. While the involvement of that alter fibroblast function, promoting their differentia- Na+ and Kir currents in fibroblast function remains to be tion into myofibroblasts. demonstrated, our results suggest that Kv currents regu- An intriguing result was the development of large Na+ lated the proliferation of SR fibroblasts. currents in more than half the population of AF fibroblasts, whereas much smaller currents were recorded in a few SR cells. TTX experiments showed that the major Fibroblast differentiation and sodium channel subunit responsible for these currents was the currents cardiac-specific isoform Nav1.5. Activation and inactiva- While recent studies have isolated fibroblasts by enzy- tion properties were, however, different from those matic dissociation of cardiac tissue, here we used the out- reported for human ventricular and atrial myocytes as growth technique as it enables the isolation of a larger both half maximum potential for activation and inactiva- number of cells and, in addition, is a more gentle proce- tion were shifted toward more positive voltages (Sakak- dure for ion channels which can be destroyed with stan- ibara et al. 1993; Feng et al. 1996). This difference is dard enzymatic digestion (Rajamani et al. 2006). We most likely due to the participation of a different panel of found that significantly fewer cells migrated out of AF tis- accessory subunits. Interestingly two other groups have sue. This could result from a reduced number of fibrob- recently reported the expression of functional Nav1.5 lasts in the fibrillating environment; an impaired potential channels in atrial fibroblasts (Chatelier et al. 2012; Koi- to migrate out of the tissue, and/or a reduced prolifera- vum€aki et al. 2014). In contrast to our study, Chatelier tive capacity once the cells are on the culture plate. Upon et al. observed Na+ currents in cells from SR patients replating, AF fibroblasts indeed showed reduced prolifera- after only 8 days of culture, and current density after tion and migration, but differentiated into myofibroblasts 15 days (~13 pA/pF) was almost as big as what we more readily than SR cells. A similar increase in differen- observed in replated AF fibroblasts (~16 pA/pF) (Chate- tiation has been previously reported for freshly isolated lier et al. 2012). Like in our experiments, the authors right atrial fibroblasts from patients with AF (Du et al. used right atrial appendage tissue obtained from 2010). In addition, Burstein et al. showed that medium patients undergoing cardiac bypass surgery and/or valve

Figure 7. Sodium currents in cultured fibroblasts. (A) Resting membrane potential (RMP) was measured in cells from seven SR patients and six AF patients. (B) Voltage-step protocols for current activation (top) and steady-state inactivation (bottom). (C) Examples of currents recorded at the indicated potentials in a SR (black) and AF (red) fibroblast. (D) Current–voltage relationships. (E) Effect of 100 nmol/L and 10 lmol/L tetrodotoxin (TTX) on Na+ currents measured in AF fibroblasts (n = 4/2). (F) Activation (SR: black empty squares, n = 11/6; AF: red empty circles, n = 20/7) and steady-state inactivation (SR: black filled squares, n = 5/3; AF: red filled circles, n = 16/6) of Na+ currents measured in AF fibroblasts. Activation curves were calculated as normalized conductance values from the IV curves assuming a reversal potential of +78 mV. A Boltzmann function was fitted to the data points (see Material and Methods). Fitting parameters are given in Table 3. (G) Window current in SR and AF fibroblasts calculated as the product of the activation and inactivation functions (Huang et al. 2011). replacement, but a major difference, which could account in their differentiation. This is in fact in agreement with for this discrepancy, was the cell isolation protocol. While our results that showed, in the AF group, both a larger we used the outgrowth technique and let fibroblasts number fibroblasts with Na+ currents and a larger num- migrate out of the explants for 3 weeks before replating; ber of cells with aSMA fibers. Chatelier et al. isolated fibroblasts by enzymatic digestion The role of voltage-gated Na+ channel in nonexcitable of the tissue. The two isolation techniques might indeed cells remains unclear. Numerous studies have demon- provide different populations of fibroblasts regarding their strated their association with cancer progression, reviewed differentiation state: while the outgrowth technique selects in (Fraser et al. 2014; Patel and Brackenbury 2015). Most only for fibroblasts that are able to migrate out of the tis- Na+ channel a-subunits, including Nav1.5, were indeed sue, and which are possibly less committed, the enzymatic shown to enhance migration and invasion of various types digestion is likely to enable the isolation of fibroblasts at of cancer cells, thereby promoting metastasis. Na+ channels all stages of differentiation. Chatelier et al. actually corre- have also been linked to migration, endocytosis, and secre- lated the presence of Na+ currents to the expression of tion of human aortic smooth muscle cells (Meguro et al. aSMA and suggested that the development of such cur- 2009), as well as angiogenesis (Andrikopoulos et al. 2011). rents happens during the differentiation process. The fas- One of the mechanisms through which Na+ are likely ter appearance of Na+ currents in culture could therefore to modulate cell function is via the activation of the Na+/ be explained by the presence of fibroblasts more advanced Ca2+ exchanger (NCX) in reverse mode, resulting in the

2016 | Vol. 4 | Iss. 2 | e12681 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 12 the American Physiological Society and The Physiological Society. C. Poulet et al. Fibroblasts from Patients with Atrial Fibrillation

Figure 8. Effect of tetrodotoxin on cellular functions of cultured fibroblasts. Fibroblasts were cultivated with normal culture medium (control) or medium supplemented with 10 lmol/L tetrodotoxin (TTX). Proliferation (A; SR, n = 7; AF, n = 7), migration (B; SR, n = 7; AF, n = 4) and differentiation (C; SR, n = 5; AF, n = 3) were assessed as described in Figure 2. *P < 0.05, unpaired Student’s t-test. entry of Ca2+ and the subsequent activation of Ca2+- currents are involved in the proliferation of SR fibrob- dependent signaling cascades (Andrikopoulos et al. 2011). lasts. Such a role for Kv channels in cell cycle progression Ca2+ influx through NCX was indeed shown to regulate has been well documented in many other cell types, proliferation, migration, and contraction of rat ventricular reviewed in (Blackiston et al. 2009; Urrego et al. 2014). fibroblasts (Raizman et al. 2007). Similar to our results with proliferation, TEA and 4-AP In this study, we did not find any connection between blocked mitogenesis in T lymphocytes (DeCoursey et al. Na+ currents and proliferation, migration, or differentia- 1984). This differs from the more recent study by Wu tion of atrial fibroblasts, and their function remains elu- et al., which showed enhanced proliferation of dog car- sive. Nevertheless, a fraction of SR and AF fibroblasts had diac fibroblasts following incubation with TEA or 4-AP RMP below 50 mV, potentials at which Na+ channel (Wu et al. 2014). are available for activation. Furthermore, at less negative The mechanisms through which Kv currents affect cell potentials, Na+ channels are responsible for a small win- proliferation include modulation of RMP, which controls dow current, which may be involved in other functions of cell cycle progression (Blackiston et al. 2009); regulation fibroblasts, such as production of ECM components or of cell volume; and increase in driving force for Ca2+ secretion of cytokines. The persistent entry of Na+ in which can activate downstream Ca2+-dependent signaling fibroblasts potentially coupled to cardiomyocytes might pathways. In addition, Kv channels might act through also affect cardiac electrical activity. Koivum€aki et al. noncanonical mechanisms involving direct interactions recently investigated this possibility using a mathematical with members of signaling cascades (Hegle et al. 2006). model of fibroblast-myocyte coupling with the membrane Although Kv current density was similar in SR and AF potential of fibroblasts set at 35 mV or 65 mV to pre- fibroblasts, TEA and 4-AP reduced K+ currents in SR cells vent complete inactivation of Na+ channels (Koivum€aki only. This suggests a modulation of Kv channels expres- et al. 2014). Even at the more negative potential, intro- sion and/or regulation in AF fibroblasts, which might be ducing Na+ currents in the fibroblast population did not the cause for the altered physiological functions we result in any significant effect on the cardiomyocyte observed in AF cells, especially the reduced proliferation. action potential. Although further experiments are needed Our gene expression data identified three candidates, to confirm these observations, this work implies that if Kv4.1, Kv6.2, and Kv11.3, with reduced expression in AF Na+ channels are involved in the pathophysiology of AF, fibroblasts. Further experiments are needed to investigate it is more likely via the modulation of fibroblasts function the importance of these specific channel subunits in the rather than through the direct alteration of cardiomyocyte regulation of fibroblasts proliferation. electrical activity. Through their modulation of RMP, Kir channels have long been linked to physiological functions of cardiac fibroblasts, including proliferation and contractility (Chil- Fibroblasts proliferation and potassium ton et al. 2005). A recent study by Qi et al. showed that currents freshly isolated left atrial fibroblasts from a dog model of While we did not find any connection between Na+ cur- CHF display enhanced proliferation and differentiation, rents and fibroblasts function, our results suggest that Kv larger Ba2+-sensitive Kir currents, and a more negative

RMP than control cells (Qi et al. 2015). They identified fibroblasts (El Chemaly et al. 2014). While we did not KCNJ2 as the likely candidate for these changes and find any functional evidence for the presence of such cur- demonstrated that KCNJ2 expression modulates RMP rents in our fibroblasts, we found high levels of tran- and cell cycle progression. In this study, significantly lar- scripts for the corresponding channel subunits, ANO1 ger inward rectifier currents were observed in cultured AF being significantly upregulated in AF cells. fibroblasts compared to cultured SR cells. While current density was similar to that observed in dog atrial fibrob- Differences between fresh and cultured lasts (Qi et al. 2015), currents were not sensitive to Ba2+ fibroblasts suggesting that different channels are involved. Kir chan- 2+ nels insensitive to Ba include the KATP subunits Kir6.1 Culture time strongly influences fibroblasts properties. and Kir6.2, as well as Kir3.3 and Kir7.1 which are Using a dog model of CHF, Dawson et al. compared involved in setting the resting membrane potential of var- fresh and cultured atrial fibroblasts (Dawson et al. 2012). ious cell types other than cardiac cells (Kubo et al. 2005). They reported that while they observed significant differ- Interestingly, Kir6.1 activation was shown to be associated ences between fresh control and diseased cells regarding with increased proliferation of murine ventricular fibrob- expression of ECM proteins and amplitude of K+ cur- lasts and reduced secretion of interleukin 6 (Benamer rents, these differences disappeared after only 48 h of cul- et al. 2009). While we detected moderate levels of tran- ture. In this study, we compared ion currents in fresh scripts for Kir3.3, Kir6.1, and Kir6.2, the expression level and replated fibroblasts using a voltage-ramp from 120 for Kir7.1 was very low. Further experiments with other to 40 mV. We found that ion currents in fresh cells were specific inhibitors, such as the KATP channel blocker weak rectifiers, with a reversal potential at 0 mV, possibly glibenclamide, are needed to identify the currents conducted through leak or TRP channels. In replated observed in SR and AF fibroblasts. fibroblasts, current amplitude was largely diminished but The importance of fibroblast Kir currents on cardiac half of the cells showed outward rectifier currents that electrical activity has been highlighted in a recent study activated at 20 mV. Our results therefore confirm that (Aguilar et al. 2014). Using a mathematical model of car- ion currents are extensively remodeled when fibroblasts diomyocyte-fibroblast coupling, Aguilar et al. indeed are kept in culture and further highlight the caution showed that upregulation of Kir currents in fibroblasts needed when extrapolating data obtained from cells kept has profibrillatory consequences due mainly to shortening in such conditions. For instance, we and others have of action potential duration. observed the development of voltage-gated Na+ currents Another group of channels which might have been together with differentiation in myofibroblasts in vitro, responsible for the ion currents observed in fresh and but whether this phenomenon also occurs in vivo and is replated fibroblasts is the TRP channel family. These non- not only an artifact of culture conditions is a key point selective cation channels have recently aroused much that needs to be addressed. interest in the context of fibrogenesis. Four members, Nevertheless, we observed differences between cultured TRPC3, TRPC6, TRPM7, and TRPV4 were shown to be SR and AF fibroblasts up to 5 weeks after isolation, involved in (TGFB1-induced) activation of fibroblasts, implying that their phenotype had been affected not only most likely through calcium influx and the subsequent by culture time but also by disease conditions. The fibril- activation of calcium-dependent downstream signaling lating environment induced changes in atrial fibroblasts, pathways (Du et al. 2010; Davis et al. 2012; Harada et al. the consequences of which were revealed in our artificial 2012; Adapala et al. 2013). In addition, TRPC3 and system. As mentioned above, these changes might include TRPM7 were upregulated in AF fibroblasts, and might reduced proliferative capacity and/or increased differentia- thus contribute to the pathophysiology of the disease (Du tion into myofibroblasts, which could exhaust the pool of et al. 2010; Harada et al. 2012). In our study, however, fibroblasts available in the heart. Further experiments are the expression of TRPC3 and TRPM7 was similar needed to investigate these points, for instance character- between SR and AF samples, a discrepancy that might izing fibroblast/myofibroblast populations in SR and AF result from differences in culture conditions (see below). tissue sections would provide an insight into the impact Other ion channels that have been linked to prolifera- of atrial fibrillation on atrial fibroblasts. tion and differentiation of cardiac fibroblasts include BKCa and the H+/Cl- transporter CLCN3 which were Limitations shown to regulate cell cycle progression in human ventricular fibroblasts (He et al. 2011). It was also recently The use of pharmacological agents in culture medium to suggested that the calcium activated Cl- channel ANO1 investigate the role of ion currents in major cell functions contributes to angiotensin II-induced activation of is a limitation in this study. We cannot exclude that the

2016 | Vol. 4 | Iss. 2 | e12681 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 14 the American Physiological Society and The Physiological Society. C. Poulet et al. Fibroblasts from Patients with Atrial Fibrillation effects observed were due to off-target actions of the mediate cardiac fibroblast differentiation by integrating drugs. In addition, ion channels might act through non- mechanical and soluble signals. J. Mol. Cell. Cardiol. 54:45– canonical mechanisms involving direct interactions with 52. downstream signaling partners. Downregulating specifi- Aguilar, M., X. Y. Qi, H. Huang, P. Comtois, and S. Nattel. cally the expression of ion channels, using siRNA for 2014. Fibroblast electrical remodeling in heart failure and instance, would therefore provide valuable information potential effects on atrial fibrillation. Biophys. J . 107:2444– on their involvement in fibroblasts function as well as 2455. their mechanism of action. Andrikopoulos, P., S. P. Fraser, L. Patterson, Z. Ahmad, H. Microarrays are a great tool to assess the expression of Burcu, D. Ottaviani, et al. 2011. Angiogenic functions of a substantial number of genes but measurement of mRNA voltage-gated Na+ channels in human endothelial cells: levels is less accurate than with quantitative PCR which is Modulation of vascular endothelial growth factor (VEGF) – a more sensitive technique. The main purpose of our signaling. J. Biol. Chem. 286:16846 16860. gene expression profiling is to give directions for further Avitall, B., J. Bi, A. Mykytsey, and A. Chicos. 2008. Atrial and investigations and any difference, or suspicion of differ- ventricular fibrosis induced by atrial fibrillation: evidence to – ence, in mRNA levels between SR and AF should there- support early rhythm control. Heart Rhythm 5:839 845. fore be confirmed by quantitative PCR. Benamer, N., H. Moha Ou Maati, S. Demolombe, A. Cantereau, A. Delwail, P. Bois, et al. 2009. Molecular and functional characterization of a new potassium conductance Conclusions in mouse ventricular fibroblasts. J. Mol. Cell. Cardiol. 46:508–517. Cardiac fibroblasts are a primordial component of the Benjamini, Y., and Y. Hochberg. 1995. Controlling the false heart as they support cardiac function by ensuring the discovery rate - a practical and powerful approach to integrity of the tissue. But excessive fibroblast activity multiple testing. J R Stat Soc Ser B 57:289–300. under pathological conditions can lead to detrimental Blackiston, D. J., K. A. McLaughlin, and M. Levin. 2009. fibrosis and be involved in cardiac dysfunction. This Bioelectric controls of cell proliferation: ion channels, study shows that in the context of chronic atrial fibrilla- membrane voltage and the cell cycle. Cell Cycle 8:3519– tion, fibroblasts undergo phenotypic changes that are 3528. revealed in culture. While their capacity for proliferation Boldt, A., U. Wetzel, J. Lauschke, J. Weigl, J. Gummert, G. and migration is impaired, their differentiation in myofi- Hindricks, et al. 2004. Fibrosis in left atrial tissue of patients broblasts is increased. This is accompanied by changes in with atrial fibrillation with and without underlying mitral electrophysiological properties, which might modulate valve disease. Heart 90:400–405. physiological functions and alter cardiac electrical activity; Burstein, B., and S. Nattel. 2008. Atrial fibrosis: mechanisms and thereby contribute to the pathophysiology of the and clinical relevance in atrial fibrillation. J. Am. Coll. disease. Cardiol. 51:802–809. Burstein, B., X. Y. Qi, Y. H. Yeh, A. Calderone, and S. Nattel. Acknowledgments 2007. Atrial cardiomyocyte tachycardia alters cardiac fibroblast function: A novel consideration in atrial The authors wish to thank the cardiac surgeons Drs. remodeling. Cardiovasc. Res. 76:442–452. Klaus Matschke and Michael Knaut for obtaining Camelliti, P., G. P. Devlin, K. G. Matthews, P. Kohl, and C. 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