Cardiotoxicity Studies in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 57

that drug effects are mediated via I Kr inhibition. Of note, submaximal doses of two
I Kr blocking drugs have been shown to have additive properties.
Classic drug-drug interactions have also been reproduced in this model. For
example, it was possible to recapitulate the potentiating effect of erythromycin on
cisapride, with a 10-fold decrease in the ED 50 for heart rate for cisapride observed
with increasing doses of erythromycin. Several other drug-drug interactions were
reproduced including that reported between cimetidine and terfenadine (Fig. 5.2e).
Taken together, these data suggest that the embryonic zebrafish model of QT drug
toxicity may prove useful for studying individual drug toxicities, drug-drug inter-
actions, and the pharmacogenetics of this drug response.
Bradycardia has been previously reported as a result of I Kr blockade in several
experimental and clinical situations (Eckardt et al., 1998; De Clerck et al., 2002).
This effect has been attributed to action potential prolongation and slowing of phase
4 depolarization in pacemaker tissue (Verheijck et al., 1995). Of the 23 known I Kr
blockers tested in our initial screen, 22 caused bradycardia when injection of
hydrophilic compounds was performed. Erythromycin is the one false negative
result in this assay. We deduced that erythromycin was absorbed by observing its
interaction with cisapride (Fig. 5.2d). Erythromycin prolongs the human QT
interval and can cause TdP at least in injectable form (Antzelevitch et al., 1996).
It is conceivable that the rare events associated with erythromycin may represent the
effects of the carrier, though QT prolongation resulting from CYP3A4-mediated
effects of erythromycin on other drugs is well documented. The recapitulation of
such drug-drug interactions demonstrates a major advantage of the zebrafish model
(Roden, 2000). In humans, these interactions have been shown to be pharmaco-
kinetic: the inhibition of hepatic metabolism by one drug resulting in increased
levels of the other.
This simple assay is useful as a screen, presenting an opportunity to test not only
large numbers of molecules, but also their quantitative interactions with a throughput
superior to current methods. Some of the effects seen in the fish (as in other models)
may reflect interactions with targets other than KCNH2. Clearly, not all drugs that
result in bradycardia do so through I Kr blockade; for example, propranolol and
clonidine both reduce heart rate in this assay.
5.4 HIGH-RESOLUTION ASSAYS
OF REPOLARIZATION
Given the lack of specificity of the simple heart rate assay, more direct assessment of
cardiac electrical events, including action potential duration, at the larval stage is
critical if the zebrafish is to become a robust tool for the exploration of drug-induced
cardiotoxicity. We therefore developed optical voltage mapping for high-resolution
electrophysiological analysis of the zebrafish heart.
Using this technology it is possible to record cardiac action potentials in the
zebrafish mutant breakdance that carries a mutation in KCNH2 (Chen et al., 1996;
Langheinrich et al., 2003), the major subunit responsible for I Kr (Bezzina et al., 2003;