Primary focus is on the study of the electrical properties of cardiomyocytes derived from pluripotent stem cells. These include mouse and human embryonic stem cells and induced pluripotency (iPS cells). Most recent additions to the research programme are stem cells bearing genetic mutations or modifications associated with cardiac disease: cells bearing ion channel or sarcomeric defects leading to channelopathies and hypertrophy respectively are of particular interest. Early and fully developed cardiomyocytes are characterized using:
- Whole cell current and voltage patch clamp
- Single ion channel recording
- External field potential recordings from MEAs (Multi Electrode Arrays). We investigate molecular and biophysical mechanisms underlying the excitability of cardiomyocytes. We characterize expression patterns of membrane proteins and ion channels which reflect the electrophysiological parameters of the individual conductances.
Research is of direct importance for understanding the differentiation state and maturity of cardiomyocytes and how this affected during development and by genetic disease. Primary human fetal cardiomyocytes are used as controls for comparison. In addition to basic cardiomyocyte characterization, we also investigate the effects of a variety of cardiac and non-cardiac drugs on electrophysiological behaviour, largely using MEA readouts but also patch clamp electrophysiology, which is most appropriate for determining action potential characteristics.
Braam SR, Tertoolen L, van de Stolpe A, Meyer T, Passier R, Mummery CL. Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes. Stem Cell Res. 2010 4(2):107-16.
Chuva de Sousa Lopes SM, Hassink RJ, Feijen A, van Rooijen MA, Doevendans PA, Tertoolen L, Brutel de la Rivière A, Mummery CL. Dev Dyn. 2006 Patterning the heart, a template for human cardiomyocyte development. Jul;235(7):1994-2002.
Mummery C, Ward-van Oostwaard D, Doevendans P, Spijker R, van den Brink S, Hassink R, van der Heyden M, Opthof T, Pera M, de la Riviere AB, Passier R, Tertoolen L. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation. 2003 Jun 3;107(21):2733-40. Epub 2003 May 12.
Patch clamp electrophysiology
What is patch clamp?
Patchclamp electrophysiology is a technique developed by Neher and Sakmann in the 1970’s, designed as a versatile technique to investigate the electrical properties of ion channels in the cell membrane. It is a modification and powerful extension of classical electrophysiology, originally developed in the 1950’s by Hodgekin and Katz. For developmental biology, it has been as particularly useful analytical tool to investigate the electrical properties of cells differentiating into specialized cell types like (cardiac) muscle cells and neural cells. For the heart, it has allowed distinction between atrial and ventricular myocytes in the developing heart.
How does it work?
The setup consists of a inverted microscope, a patchclamp amplifier, an oscilloscope to visualize the currents and a computer to digitize and store the derived data.
Schematic diagram of the technique
Each living cell is surrounded by a membrane which separates the world within the cell from its exterior. In this membrane there are channels, through which the cell communicates with its surroundings. These channels consist of single molecules and have the ability to allow passage of charged atoms, i,.e. ions. The regulation of ion channels influences the life of the cell and its functions under normal and pathological conditions. The Nobel prize for Physiology or Medicine in 1991 was awarded for research on the function of ion channels. The two German physiologists Erwin Neher and Bert Sackman together developed a technique that allows the registration of incredibly small electrical currents (as low as the picoampere 10 -12 A range) that passes though a single ion channel. The technique is unique in that it records how a single channel molecule alters its shape and in such a way that the flow of current within a time frame of a few millionths of a second is controlled. Neher and Sackman established conclusively that ion channels do exist and how they function. They demonstrated what happens during the opening and closing of an ion channel with a diameter corresponding to that of a single sodium ion or chloride ion. Several ion channels are regulated by a receptor localized on one part of the channel molecule which upon activation alters its shape. Neher and Sackman demonstrated which parts of the molecule constitute the "sensor" and the interior wall of the channel. They also showed how the channel regulates the passage of positively or negatively charged ions. This new knowledge and this new analytical tool has over the past ten years revolutionized modern biology, facilitated research, and contributed to the understanding of cellular mechanisms underlying several diseases, including diabetes and cystic fibrosis.
From the press release of the Nobel price for Neher and Sackmann of the Nobel Assembly in Stockholm 7 October 1991 (in part):
Single channel patchclamp
A glass pipette with a diameter of ~ 8 mm is gently pushed against the cell membrane, while a small flow of liquid is continuously passed through the pipette. When the liquid flow is stopped and suction is gently applied a giga seal is formed between the glass of the pipette and the cell membrane. A giga seal is a very high electrically insulated contact (> 10 giga Ohm) in order to measure the pico 10 -12 Ampere currents of one ion channel.
In this configuration single channel currents can be measured (diagram below a. ).
Whole cell patchclamp
When strong suction is applied directly after formation of the giga seal, the membrane inside the pipette is ruptured, and the sum of all channels present in the cell membrane can be measured (diagram above b.).
The multi electrode array (MEA)
Cells are seeded on a glass substrate containng 64 electrodes of TiN (Titanium Nitrate). The electrode area is +/- 2x2 mm.
The MEA amplifier Field potentials from one electrode modulated by the drug (Quinidine)
Recording from clusters of stem cell derived cardiomyocytes can be made as extracellular field potentials on two-dimensional microelectrode arrays (MEAs). The two-dimensional information allows analysis of the conduction velocity and to generation of excitation maps of the cardiac tissue (see movie below).
Stem cell derived human cardiomyocytes can potentially be used for pharmaceutical safety studies, testing drugs for their potential cardiac safety risks, specifically field potential prolongation.
Human embryonic stem cell derived cardiomyocytes. Contractions similar to those in the heart can be seen at regular intervals. The currents are here analysed using the MEA technique.