ES cell-derived cardiomyocytes: functional expression of all essential cardiac ion currents

Introduction

To date, in vitro preclinical electrophysiological studies rely mainly on recombinant cell systems like injected oocytes or mammalian cell lines expressing only one specific ion channel.

These recombinant cell lines are designed to analyze the properties of a single ion channel but lack the normal physiological cellular environment and functional humoral regulation compared to primary cardiomyocytes or primary tissues (eg, papillary muscle or Langendorff-perfused explanted hearts). On the other hand, the isolation of the primary cardiac cells is costly, time consuming and difficult to standardize. [1]

To provide scientists in basic or applied cardiology and toxicology a standardized and pure cardiac myocyte model with functional expression of all three essential cardiac ion currents, Axiogenesis has developed Cor.At cardiomyocytes. These mouse embryonic stem cell-derived cardiomyocytes are ready-to-use and 99.9 % pure without contamination by other cell types. Patch clamp recordings revealed that Cor.At cardiomyocytes exhibit typical cardiac action potentials and the three essential cardiac currents INa, ICa, and IK (Figure 1 and 2 below).

Cor.At cardiomyocytes enable scientists to adequately address the efficacy and safety pharmacology of potential lead compounds in drug discovery and development.

Results I

Cor.At cardiomyocytes are frozen and ready-to-use, primary-like cardiac cells. They recover completely after thawing and start to contract autonomously after a few hours in culture. When applied to current clamp experiments, Cor.At cardiomyocytes display typical cardiac action potentials as depicted in Figure 1 (data kindly provided by Dr. Davide Pau, Scottish Biomedical Ltd, Glasgow, UK). Cells were patched using whole cell configuration and stimulated at a frequency of 3 Hz.

The functionality of the three ion currents INa, ICa,L and IK in Cor.At cardiomyocytes was demonstrated by voltage clamp experiments (see Figure 2).

INa was elicited by a family of 100 ms depolarizations from a -90 mV holding potential (HP) to voltages ranging from -80 to +55 mV in 5 mV steps.

ICa,L was recorded during the application of depolarization steps of 100 ms from a -90 mV HP, and a following 67 ms pre-pulse to -40 mV, to voltages ranging from -60 to +50 mV in 10 mV steps.

IK was elicited by a family of 500 ms depolarizations from a -80 mV HP to voltages ranging from -40 mV to +60 mV in 10 mV steps.

Results II

The predictivity of Cor.At cardiomyocytes for assessment of drug efficacy or cardiac side effects was scrutinized in current clamp experiments. Figures 3 and 4 show representative recording sequences from perforated patch clamp experiments with autonomously contracting Cor.At cardiomyocytes. After control recording (vehicle only), cumulative dosing of drugs was performed, followed by a final washout period.

The ICa,L blocker nifedipine (Fig. 3A) caused beating arrest at 200 nM. The hERG blocker E-4031 induced decreased beating frequency, a reduction in maximum re- and depolarization potentials, beating arrest (Fig. 3B) and prolongation of APD90 (Fig. 5). The INa blocker lidocaine reduced the maximum depolarization and induced beating arrest at highest concentration (Fig. 3C). As shown in figure 4A, the β-adrenergic agonist epinephrine increased beating frequency, whereas the muscarinic agonist carbachol reduced the spontaneous beating frequency (Fig. 4B).

Summary

Electrophysiological characterization of the Cor.At cardiomyocytes demonstrated their primary-like phenotype. In current clamp recordings, the membrane potential follows the characteristics of a typical cardiac action potential. Voltage clamp recordings revealed the presence of all three essential cardiac ion currents.

Regular cardiac physiology of Cor.At cardiomyocytes was confirmed by their response to the ion channel blockers nifedipine, E-4031 and lidocaine. Additionally, Cor.At cardiomyocytes exhibit a functional and reversible humoral modulation of their electrophysiological properties. This is a benefit over recombinant cell models, which do not provide the cellular environment of a normal cardiomyocyte.

Our results clearly indicate that Cor.At cardiomyocytes exhibit primary cell-like qualities. The cells are therefore suitable for the preclinical assessment of drugs in regard to efficacy and safety pharmacology.

References:

[1] Kettenhofen R, Bohlen H. Preclinical assessment of cardiac toxicity. Drug Discov Today. 2008 Aug;13(15-16):702-7. Epub 2008 Jul 24.