3Rs-Centre Utrecht Life Sciences

Faculteit Dierengeneeskunde
July 2016
Cardiac safety screening with human cardiomyocytes potentially reduces animal use 

During drug development, screening of compounds is a prerequisite to assess the safety and efficacy of potentially dangerous compounds. A commonly observed and deleterious side effect of new drugs is that they are able to trigger cardiac rhythm disorders. To study these effects, existing animal models do not suffice. Therefore, cardiomyocytes derived from human stem cells are being studied as a potential alternative tool by Dr. Toon van Veen of the Department of Medical Physiology, Division Heart & Lungs, University Medical Center Utrecht. This new approach could potentially reduce and replace animal use. However, the immature phenotype of the cardiomyocytes are a challenging problem to tackle.

Many drugs (cardiac and non-cardiac) can induce cardiac arrhythmia by modulating ion channels in the heart. A potentially lethal cardiac disease is called “Torsade de Pointes”, characterized by abnormal heart rhythm. The clinical incidence of antiarrhythmic drugs causing Torsade de Pointes arrhythmias is 1-5%1.

Different electrophysiological properties

To test whether new drugs cause these cardiac arrhythmias, Dr. Toon van Veen makes use of human stem cells in vitro. The need for better alternative models is high, addresses Dr. van Veen. Dogs and rabbits are used the most, to obtain the Purkinje fibers (Roden and Hoffman, 1985). The applicability of these current animal models, used by the pharmaceutical industry in preclinical cardiac safety screening, has some important limitations. The electrophysiological properties of the heart of, especially, small mammals are very different from humans. More specifically, the expression of several types of ion channels (conducting different currents) in the heart is not consistent between humans and animals and therefore animal experiments are not providing the whole picture. Also, testing on isolated animal cardiomyocytes implies the lack of a multicellular compartment. In conclusion, the currently used models are not sensitive enough to detect all parameters of interest. No more than 1 out of 9 drugs that enters a clinical trial results in a registration of the drug2.
Human stem cell derived cardiomyocytes could potentially bridge the gap between preclinical and clinical testing. Above all, this could reduce and replace animal use for tissue extraction in cardiac safety testing.

Foto: stichting Proefdiervrij

Human stem cells

Instead of using animal cardiac tissue, human stem cells can be used. Human stem cells are, when triggered with the right stimuli and experimental conditions, able to differentiate into ventricular cardiomyocytes3. The research group of Dr. van Veen proved to be able to generate this type of cardiomyocytes. The next step was to identify selection criteria for 3D clusters having the ventricular phenotype4. After 42-56 days of differentiation, experiments were performed. The selected human embryonic stem cell-derived cardiomyocytes were compared in their response to arrhythmic stimuli with contemporary used models, like for example the rabbit Purkinje Fibers. Results showed that cells were capable of responding in a similar manner, qualitatively and quantitatively.

Tackle the problem: immature phenotype

Despite the promising first results, some problems had to be tackled. The electrophysiological properties of human adult cardiomyocytes and human stem cell- derived cardiomyocytes are quite similar, but there is one important difference. A potassium ion channel is lacking in the human embryonal stem cell phenotype, and as a consequence, this results in a relatively positive (less negative) resting membrane potential. Several ion channels differ in their activity compared to the real-life situation, which results in aberrant current densities, limiting the potential use of the model5. The comparison with the adult human situation is incomplete, therefore Toon van Veen speaks of an immature electrical phenotype.

However, a potential solution has been found to add the missing ion channel. With a technique called “dynamic clamp”, a computer helps by simulating dynamic electrophysiological processes and couples these to living cells. By simulating the missing potassium ion channel, a current is injected into the living cell, which receives the same current as if it would have contained in the adult situation. In this way, the lack of that particular potassium ion current can be ‘repaired’ and they can obtain a mature phenotype.

Reducing animal use in cardiac safety screening

The maturation of the electrical phenotype is a prerequisite for future implementation of the model in arrhythmogenic safety testing. After this has been accomplished, the in vitro model is ready to be used as a drug screening method. This is acknowledged by the pharmaceutical industry, which is involved as a funding partner in the project (read more below).

Dr. Toon van Veen is also eager to find out why the lacking potassium ion channel is not being expressed endogenously in the embryonal stem cell phenotype. Not earlier than until this is elucidated, can this promising innovative method be used as alternative to animal testing.

Funding: More Knowledge with Fewer Animals

The research project of Toon van Veen has been funded within the module “Innovative Testing” (2011-2017) of the Funding Program More Knowledge with Fewer Animals, of the Netherlands Organization for Health Research and Development6 (ZonmW). As an industry partner, Roche Innovation Center is also involved and is interested in using van Veen’s new approach in drug screening assays. Co-funding in this program is provided by the Dutch Society for the Replacement of Animal Testing (Stichting Proefdiervrij) and the Dutch Heart Foundation (Hartstichting).

1: Pedersen et al., 2007. Risk factors and predictors of Torsade de pointes ventricular tachycardia in patients with left ventricular systolic dysfunction receiving Dofetilide. American Journal of Cardiology, Vol. 100, No. 5, 2007, p. 876-80.
2: Kola & Landis, 2004. Can the pharmaceutical industry reduce attrition rates? Nature Reviews Drug Discovery 3, 711-716.
3: Synnergren J. et al., 2008. Molecular signature of cardiomyocyte clusters derived from human embryonic stem cells. Stem Cells 26, 1831–1840.
4: Jonsson M.K.B. et al., 2010. Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes, Stem Cell Research (2010) 4, 189–200. 
5: Jonsson M.K.B. et al., 2012. Application of human stem cell-derived cardiomyocytes in safety pharmacology requires caution beyond hERG, Journal of Molecular and Cellular Cardiology 52 (2012) 998–1008.
6: Read more here (in Dutch only).