Fast Optical Investigation of Cardiac Electrophysiology by Parallel Detection in Multiwell Plates.

Caterina Credi,U Munagala,Elisabetta Cerbai,Antoine A F De Vries,C Cianca,Leonardo Sacconi,Leslie M Loew,Francesco S Pavone,S Bigiarini,Valentina Balducci,Laura Sartiani

doi:10.3389/fphys.2021.692496

Abstract

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells. MULTIPLE combines optical detection of action potentials using a red-shifted voltage-sensitive fluorescent dye (di-4-ANBDQPQ) with optical stimulation, employing optogenetic actuators, to ensure excitation of cardiomyocytes at constant rates. MULTIPLE was first characterized in terms of interwell uniformity of the illumination intensity and optical detection performance. Then, it was applied for probing action potential features in HL-1 cells (i.e., mouse atrial myocyte-like cells) stably expressing the blue light-activatable cation channel CheRiff. Under proper stimulation conditions, we were able to accurately measure action potential dynamics across a 24-well plate with variability across the whole plate of the order of 10%. The capability of MULTIPLE to detect action potential changes across a 24-well plate was demonstrated employing the selective Kv11.1 channel blocker (E-4031), in a dose titration experiment. Finally, action potential recordings were performed in spontaneous beating human induced pluripotent stem cell derived cardiomyocytes following pharmacological manipulation of their beating frequency. We believe that the simplicity of the presented optical scheme represents a valid complement to sophisticated and expensive state-of-the-art optical systems for high-throughput cardiac electrophysiological investigations.

Highlights

Fast investigation of cardiac electrophysiology is found convenient for the assessment of cardiac ion channel activity of new chemical entities (Dunlop et al, 2008)
We describe a simple and cost-effective optical approach (MULTIPLE) that exploits high-power light-emitting diode (LED) arrays to globally illuminate a multiwell plate and an sCMOS sensor coupled with a camera lens for parallel detection of the fluorescence coming from multiple sites
Standard optical technologies to simultaneously control and monitor action potentials (APs) features of cells in multiwell plates are integrated in a compact platform

Summary

Introduction

Fast investigation of cardiac electrophysiology is found convenient for the assessment of cardiac ion channel activity of new chemical entities (Dunlop et al, 2008) In this respect, optical techniques for actuating and sensing cardiac action potentials (APs) represent a reference method for preclinical drug screening and cardiotoxicity testing, especially in combination with human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) (Klimas et al, 2016). Conventional methods for multisite optical interrogation generally require movement of the sample (McGlynn et al, 2018) or of the imaging system (Hansen et al, 2010). These approaches intrinsically limit the acquisition time and require complex and expensive fully automated microscope platforms. The system was successfully applied for imaging the contraction waves of multiple cardiac monolayers, the possibility of performing high-speed voltage imaging using a fluorescent voltage-sensitive dye (VSD) is precluded due to the requirement of coherent light in the RAP image formation mechanism

Methods

Results

Conclusion