Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation
Graphical abstract
Introduction
Atrial Fibrillation (AF) is a supraventricular arrhythmia with increasing prevalence in countries with an aging population. Although AF is one of the most common cardiovascular causes of hospitalization [[1], [2], [3]], its pathophysiology is not fully elucidated, and it represents an unmet need for effective prevention and treatment. One hallmark of AF is its progressive nature. As AF becomes increasingly resistant over time to pharmacological or electrical attempts at conversion back to sinus rhythm (SR) [2], atrial tissue undergoes pronounced remodelling [2,4]. Structural and functional changes involve cell electrophysiological and tissue morphological alterations. Whilst electrical remodelling of atrial cardiomyocytes is characterised by a shortening of action potential duration and of effective refractory period, as well as by impaired adaptation of these parameters to changes in heart rate [5], fibrosis – a prominent feature of AF-related structural remodelling – may in parallel contribute to slowing of conduction. The combination of short effective refractory period and slow conduction favours maintenance of AF via re-entry mechanisms [6].
Many of the risk factors for AF, e.g. heart failure, hypertension, or valvulopathies, are accompanied by mechanical overload of the atria [7]. Since stretch enhances the susceptibility to AF induction [8,9], it has been suggested, that mechanical overload may contribute to initiation and perpetuation of AF in vivo [[10], [11], [12], [13]]. In addition, acute stretch of control atrial tissue induces complex and regionally varying changes in action potential shape [10] and diastolic depolarizations that may trigger extrasystoles [9,14,15]. This ‘mechano-electric feedback’ [16,17] requires cells to be able to sense their mechanical environment, and to translate this into an electrophysiologically relevant signal.
Ample evidence points to an essential role of stretch-activated ion channels (SAC) as mechano-sensors in cardiomyocytes (for reviews see [18,19]). SAC are also present and functional in human atrial fibroblasts [20,21], but it is currently not known whether SAC function is altered in AF in the various human heart cells, especially in fibroblasts, which are key players in fibrosis. Therefore, the aim of this study was to compare SAC function in atrial fibroblasts from patients in SR or in sustained AF. The cation-nonselective SAC Piezo1 [[22], [23], [24]] forms a plausible candidate, in line with recently reported Piezo1 effects on remodelling of non-cardiac tissues [25]. A second candidate, the potassium-selective Ca2+-activated channel of large conductance (BKCa), has been reported to respond to stretch, local Ca2+ concentration changes, transforming growth factor beta (TGF-β), and angiotensin II in several cardiac cell types [[26], [27], [28], [29], [30]]. BKCa is further known to modulate fibroblast proliferation [31], a critical event during pathological tissue remodelling in AF. Both Piezo1 and BKCa have previously been detected in human atrial fibroblasts [[31], [32], [33]].
In this study we report AF-related changes in Piezo1 and BKCa channel activity in human atrial fibroblasts, and establish functional interactions between the two channel types.
Section snippets
Tissue collection
Tissue samples were obtained from the right atrial appendage of patients undergoing open-heart surgery. Patients were either in SR, or in sustained AF (which includes patients with persistent, long-standing persistent and permanent AF, defined according to ESC Guidelines [34]). Tissue samples were processed by the Cardiovascular Biobank of the University Heart Center Freiburg Bad Krozingen (approved by the ethics committee of Freiburg University, No 393/16; 214/18) or the Clinical Center of the
Fibroblasts and myofibroblasts are the main constituents of right atrial outgrowth cell cultures
Cells obtained by the outgrowth technique from human right atrial appendage did not contain any cardiomyocytes. The majority (98%) of cells stained positive for vimentin, and 1% of the cells were positive for the endothelial cell marker CD31. Antibody functionality was verified in positive controls using human umbilical vein endothelial cell (HUVEC; Fig. S1A and B). Overall, results confirmed that the outgrowth technique yields predominantly fibroblasts-like cells with negligible contamination
Discussion
We confirm the presence, in human right atrial fibroblasts from patients in SR and AF, of at least two different types of ion currents that are activated during stretch: the cation-nonselective Piezo1 and the potassium-selective BKCa. Our main findings are: (i) activity and expression of Piezo1 are larger in non-passaged right atrial fibroblasts from AF patients than in cells from SR patients; (ii) activity, but not expression, of BKCa channels is lower in AF than in SR cells; (iii)
Author contributions
DJ, AK, SNH, PK, UR and RP contributed to conception, design and interpretation of the study. DJ, AK and ED performed and analysed electrophysiological experiments. DA, EARZ, SP and CS performed and analysed quantitative RT-PCR experiments. DJ and TG performed and analysed immunocytochemical experiments. DA and ASC isolated cells. BA and HG performed and analysed the co-immunoprecipitation and localisation experiments. RE performed the PCR to assess the presence of STREX. SRK provided HAF
Funding
This work was supported by the ERC Advanced Grant CardioNECT (project ID: #323099, PK), a research grant from the Ministry of Science, Research and Arts Baden-Württemberg (MWK-BW Sonderlinie Medizin, #3091311631, PK), and a DFG Emmy Noether Fellowship (to EARZ, 285 #396913060). This research was funded by the German Research Foundation (DFG) under the Excellence Strategy (CIBSS-EXC-2189-Project ID 390939984). ED, PK, UR and RP acknowledge support by Amgen Inc. ED, RE, ASC, EARZ, FB, FAK, CS,
Declaration of Competing Interest
None.
Acknowledgments
The authors thank all colleagues at the Department for Cardiovascular Surgery of the University Heart Centre Freiburg - Bad Krozingen, and at the CardioVascular BioBank Freiburg, for providing access to human atrial tissue. Special thanks for technical support go to Cinthia Walz, Anne Hetkamp, Kristina Kollmar and Gabriele Lechner. We would like to also thank Dr. Simone Nübling and Dr. Hannah Fürniss for their help concerning patient demographics. We thank Dr. Bo Bentzen (University of
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These authors contributed equally to the work.