Piezo1 and BK_Ca channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation

Highlights

• Piezo1 and BK_Ca are present and mechano-modulated in human atrial fibroblasts.

• Fibroblast Piezo1 activity is larger in atrial fibrillation compared to sinus rhythm.

• Fibroblast BK_Ca activity is smaller in atrial fibrillation compared to sinus rhythm.

• BK_Ca mechanosensitivity depends in part on stretch-activated Ca²⁺ influx via Piezo1.

• Piezo1 and BK_Ca channels are linked functionally, but not structurally.

Abstract

Aims

Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF.

Methods and results

Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent ‘big potassium channels’ (BK_Ca). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BK_Ca activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BK_Ca current during stretch. No co-immunoprecipitation of Piezo1 and BK_Ca was detected.

Conclusions

Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BK_Ca. While Piezo1 is directly stretch-activated, the increase in BK_Ca activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BK_Ca activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BK_Ca in human atrial fibroblasts in the absence of physical links between the two channel proteins.

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 (BK_Ca), 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]]. BK_Ca is further known to modulate fibroblast proliferation [31], a critical event during pathological tissue remodelling in AF. Both Piezo1 and BK_Ca have previously been detected in human atrial fibroblasts [[31], [32], [33]].

In this study we report AF-related changes in Piezo1 and BK_Ca channel activity in human atrial fibroblasts, and establish functional interactions between the two channel types.