Patch-Clamp Recordings of Action Potentials From Human Atrial Myocytes: Optimization Through Dynamic Clamp.

Arie O Verkerk,Antoine H G Driessen,Gerard A Marchal,Ronald Wilders,Carol Ann Remme,Jan G Zegers,Joris R De Groot,Makiri Kawasaki

doi:10.3389/fphar.2021.649414

Arie O Verkerk, Antoine H G Driessen + Show 6 more

Open Access

https://doi.org/10.3389/fphar.2021.649414

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Abstract

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Consequently, novel therapies are being developed. Ultimately, the impact of compounds on the action potential (AP) needs to be tested in freshly isolated human atrial myocytes. However, the frequent depolarized state of these cells upon isolation seriously hampers reliable AP recordings. Purpose: We assessed whether AP recordings from single human atrial myocytes could be improved by providing these cells with a proper inward rectifier K+ current (IK1), and consequently with a regular, non-depolarized resting membrane potential (RMP), through “dynamic clamp”. Methods: Single myocytes were enzymatically isolated from left atrial appendage tissue obtained from patients with paroxysmal AF undergoing minimally invasive surgical ablation. APs were elicited at 1 Hz and measured using perforated patch-clamp methodology, injecting a synthetic IK1 to generate a regular RMP. The injected IK1 had strong or moderate rectification. For comparison, a regular RMP was forced through injection of a constant outward current. A wide variety of ion channel blockers was tested to assess their modulatory effects on AP characteristics. Results: Without any current injection, RMPs ranged from −9.6 to −86.2 mV in 58 cells. In depolarized cells (RMP positive to −60 mV), RMP could be set at −80 mV using IK1 or constant current injection and APs could be evoked upon stimulation. AP duration differed significantly between current injection methods (p < 0.05) and was shortest with constant current injection and longest with injection of IK1 with strong rectification. With moderate rectification, AP duration at 90% repolarization (APD90) was similar to myocytes with regular non-depolarized RMP, suggesting that a synthetic IK1 with moderate rectification is the most appropriate for human atrial myocytes. Importantly, APs evoked using each injection method were still sensitive to all drugs tested (lidocaine, nifedipine, E-4031, low dose 4-aminopyridine, barium, and apamin), suggesting that the major ionic currents of the atrial cells remained functional. However, certain drug effects were quantitatively dependent on the current injection approach used. Conclusion: Injection of a synthetic IK1 with moderate rectification facilitates detailed AP measurements in human atrial myocytes. Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs.

Highlights

Atrial fibrillation (AF) is the most common cardiac arrhythmia
We patched 58 single human atrial myocytes enzymatically isolated from left atrial appendage (LAA) that were obtained from eight patients with paroxysmal AF (Table 1)
In the depolarized cells (RMP positive to −60 mV), resting membrane potential (RMP) could be set at −80 mV using IK1 or constant current injection, and action potential (AP) could be evoked upon stimulation

Summary

Introduction

Atrial fibrillation (AF) is the most common cardiac arrhythmia. novel therapies are being developed. Various in vitro (Papke and Smith-Maxwell, 2009; Ebert et al, 2012; Devalla et al, 2015; Hu et al, 2018) and in vivo animal models (Olgin and Verheule, 2002; Finet et al, 2009; Nishida et al, 2010), including AF models, are available for atrial drug testing, all with their individual strengths and limitations While these models provide an excellent starting point for atrial and AFrelated drug studies, assessments of action potentials (APs) of native human atrial cardiomyocytes remain essential due to distinct inter-species differences in ion channel expression profiles (Varró et al, 2021), and because drugs frequently act through AP shortening or prolonging effects, which may be frequency dependent (Peyronnet and Ravens, 2019). Generally only a fraction of cells, approximately 5–20%, have an RMP sufficiently negative to elicit APs (Amos et al, 1996; Schreieck et al, 2000), and even within this fraction highly depolarized myocytes are frequently excluded from analysis (Loose et al, 2014)

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