Averaging in vitro cardiac field potential recordings obtained with microelectrode arrays

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Abstract

Extracellular field potential (FP) recordings with microelectrode arrays (MEAs) from cardiomyocyte cultures offer a non-invasive way of studying the electrophysiological properties of these cells at the population level. Several studies have examined the FP properties of cardiomyocytes of various origins, including stem cell-derived cardiomyocytes. This focus reflects growing importance and interest in the field of MEA. High-quality cardiac FP signals are often difficult to obtain, especially from stem cell-derived cardiomyocyte cultures, which represent an important new field in cardiac electrophysiology. One way to improve the quality of these recordings is to average the cardiac FP signals. To date, however, no studies have examined the effect of averaging on cardiac FP signals. We report here that cardiac FP averaging can yield higher-quality signals than original individual FPs, and therefore promise more accurate detection of different phases and analysis of the cardiac FP signal. Averaged signals improved the signal-to-noise ratio (SNR), and obtaining reliable averages required approximately 50 cardiac cycles. We therefore propose that routine cardiac FP averaging can serve as a tool to compare the effects of different experimental conditions or stimuli on the properties of cardiac FPs.

Introduction

Extracellular field potential (FP) recordings of cardiomyocytes with microelectrode arrays (MEA) enable the study of cardiac electrophysiological properties at the population level [1]. Previous studies have shown cardiac FPD on MEA to correspond with QT interval properties in the electrocardiogram [2]. Thus, FP recordings of beating cardiomyocyte aggregates offer insight into the electrical function of myocardial tissue in vitro [3]. The MEA platform has served extensively in the study of cardiomyocytes of various origins, such as the chick heart [4], mouse embryonic stem cell (ESC)-derived cardiomyocytes [5], mouse induced pluripotent stem (iPS) cell-derived cardiomyocytes [6], human ESC-derived cardiomyocytes [7], [8], [9] and human induced pluripotent stem (iPS) cell-derived cardiomyocytes [10].

Signals of good quality, especially from pluripotent stem cell-derived cardiomyocytes, are sometimes difficult to obtain. In the case of human embryonic stem cell-derived cardiomyocytes (hESC-CMs), the quality of the signal is often limited by low numbers of cardiomyocytes in the clusters. Irregular beating rhythms can also present a challenge in analysing the signals [11]. Obtaining a clear signal is crucial for determining different parameters, especially the end-point of the field potential duration (FPD), which indicates the end of repolarisation and thus the end of electrical activation in one cardiac cycle. Low signal-to-noise ratio (SNR) recordings can yield higher quality via averaging. To date, however, no studies have investigated the direct effects of averaging on cardiac FP signals.

We hypothesised that averaging several FP cycles from recordings with a stable beating rhythm would yield more accurate and reliable results than would measuring the parameter values from one or a few representative individual FP cycles. This would be especially important when poor quality due to a low signal-to-noise ration obscures identification of the end of the cardiac replolarisation phase in the FP signals. Indeed, our data suggest that averaging can serve to produce reliable results from the cardiac field potential recordings of neonatal rat cardiomyocytes (NRC) and hESC-CMs.

Section snippets

Human embryonic stem cell culture

H7 hESCs (WiCell) were cultured on mitomycin C inactivated mouse embryonic fibroblasts (MEF) in hES medium, which consisted of DMEM/F-12 (Invitrogen) supplemented with 20% KnockOut serum replacement (Invitrogen), 1% non-essential amino acids (Lonza), 2 mM Glutamax (Invitrogen), 50 U/ml penicillin/streptomycin (Lonza), 0.1 mM beta mercaptoethanol (Invitrogen), and 7.8 ng/ml basic fibroblast growth factor (R&D Systems). The medium was refreshed daily, and the hESC colonies were passaged onto a new

Peak detection validation

The automatic peak detection algorithms yielded an average comparable to that generated using manual peak detection. Fig. 2A illustrates the original signal along with automatically selected minimum amplitude values on the left hand panel. The right hand panel in Fig. 2A illustrates the average cardiac FP cycle generated from a 2-min recording of the NRC population utilising automatic peak detection from the Matlab-based program. Fig. 2B illustrates the same situation for the same NRC

Discussion

In vitro population-level cardiac electrophysiology will most likely become more significance in the coming years. The electrical properties of single cells have traditionally been studied with the patch clamp technique [15]. This method is time consuming, however, and requires a vast amount of training. Combining MEA technology with human embryonic stem cell-derived cardiomyocytes potentially allows for medium throughput drug screening for adverse cardiac side effects in vitro [2], [16].

Competing interests

The authors declare no competing financial interests.

Acknowledgements

We kindly thank Dr Christine Mummery for the END-2 cells. We also thank Henna Venäläinen for her technical help with the cell culturing. This research was funded by grants from the Academy of Finland (decision numbers: 126888, 122947 and 122959), the Finnish Heart Foundation, competitive funding from Pirkanmaa Hospital District and the Finnish Cultural Foundation. The MEA system was funded by BioneXt, Tampere, Finland.

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1

These authors contributed equally to this work.

2

Current address: Finnish Red Cross, Blood Service, Helsinki, Finland.

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