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 patho- physiology 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 prolifera- tion, 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 under- stand 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