Action potentials in cardiac myocytes have durations in the order of magnitude of 100 milliseconds. In biomedical
investigations the documentation of the occurrence of action potentials is often not sufficient, but a recording of the
shape of an action potential allows a functional estimation of several molecular players. Therefore a temporal resolution
of around 500 images per second is compulsory. In the past such measurements have been performed with photometric
approaches limiting the measurement to one cell at a time. In contrast, imaging allows reading out several cells at a time
with additional spatial information. Recent developments in camera technologies allow the acquisition with the required
speed and sensitivity. We performed action potential imaging on isolated adult cardiomyocytes of guinea pigs utilizing
the fluorescent membrane potential sensor di-8-ANEPPS and latest electron-multiplication CCD as well as scientific
CMOS cameras of several manufacturers. Furthermore, we characterized the signal to noise ratio of action potential
signals of varying sets of cameras, dye concentrations and objective lenses. We ensured that di-8-ANEPPS itself did not
alter action potentials by avoiding concentrations above 5 μM. Based on these results we can conclude that imaging is a
reliable method to read out action potentials. Compared to conventional current-clamp experiments, this optical approach
allows a much higher throughput and due to its contact free concept leaving the cell to a much higher degree
undisturbed. Action potential imaging based on isolated adult cardiomyocytes can be utilized in pharmacological cardiac
safety screens bearing numerous advantages over approaches based on heterologous expression of hERG channels in cell
lines.
Identifying cardiac safety is becoming increasingly important for new drugs under development, since it is compulsory
for the approval of almost all pharmaceutical drugs. In contrast to conventional electrophysiological in vitro assays that
are based on a single entity, the hERG channel, primary cardiomyocytes based readouts seem to be more comprehensive.
Such an action potential readout for those cells can be performed with contact-free optical methods. Here we reveal the
details of both, the optical arrangement and the procedure to screen cardiac myocytes based on naïve action potentials.
Furthermore we evaluate the differences between neonatal and adult cardiac myocytes from rats based on a selection of
test substances (quinidine, 4-aminopyridine and E-4031) and relate it to the human situation. Finally the results are
discussed in the context of emerging genetically encoded potential sensors and latest development in optical detection
technologies.
Here we describe the cell- and molecular-biological concepts to utilise excitable primary isolated cells, namely
cardiomyocytes, for optical high content screens. This starts with an optimised culture of human adult cardiomyocytes,
allowing culture with diminished dedifferentiation for one week. To allow fluorescence based molecular imaging
genetically encoded biosensors need to be expressed in the cardiomyocytes. For transduction of end-differentiated
primary cells such as neurons or cardiomyocytes, a viral gene transfer is necessary. Several viral systems were balanced
against each other and an adenoviral system proofed to be efficient. This adenoviral transduction was used to express the
calcium sensors YC3.6 and TN-XL in cardiomyocytes. Example measurements of calcium transients were performed by
wide-field video imaging. We discuss the potential application of these cellular and molecular tools in basic research,
cardiac safety screens and personalised diagnostics.
Cardiac failure is still one of the mayor reasons for death in the Western population but the pathophysiology of the
molecular processes in the heart is far from being completely understood. Therefore further basic research is necessary.
With recent developments of optical technologies novel tools to investigate cardiac physiology and pathophysiology
became available. They comprise non-linear imaging techniques such as second harmonic generation imaging and fast
two-photon excitation imaging of cardiac tissue. In addition, high-speed multi-beam two-photon imaging as well as
ultra-high speed single beam single photon 2D-confocal imaging offer novel approaches to study cellular and
subcellular signalling events in cardiac tissue and/or single cardiac myocytes. Here we introduce and discuss these new
technologies and their practical application to study cardiac physiology and pathophysiology.
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