Cardiac repolarization abnormalities, often leading to life-threatening arrhythmias, represent one of the most important types of drug toxicity. Drugs that cause clinical arrhythmias are often associated with prolongation of the measured QT interval on the electrocardiogram (EKG) but this can vary from beat to beat. The majority of the drugs that result in clinical problems exhibit IKr blocking activity, but in vitro IKr inhibition is of limited predictive value. There is accumulating evidence that the lack of predictive utility of current assays reflects a failure to capture the true complexity both of drug effects (which may involve multiple targets) and the underlying substrate. We have developed a model of drug-induced repolarization abnormalities in the larval fish in order to characterize the fundamental biology of these toxic drug responses. Using heart rate (HR) response, we have established an excellent correlation with known adult human repolarization cardiac toxicity and recapitulated clinically relevant, drug-drug interactions. We now propose to extend this preliminary work, exploiting tools for genetic manipulation of the zebrafish to create a panel of 'reporter' fish modeling repolarization reserve and several proarrhythmic human disease states. We will use these reporter fish to generate a profile of repolarization responses for cardiotoxic and non-cardiotoxic drugs in order to identify features predictive of human proarrhythmia.
Specific aims are as follows; I. The generation of sensitized reporter zebrafish modeling multiple aspects of repolarization and human disease states. i) Initial cloning and characterization of zebrafish orthologs of the target genes ii) Morpholino knockdown in larval zebrafish iii) Assembly of existing mutants and generation of specific transgenic reporter lines II. Detailed characterization of the repolarization effects of a derivation set of cardiotoxic and noncardiotoxic drugs in a panel of reporter fish, i) Measurement of calcium transients in larval zebrafish hearts ii) Optical mapping of action potential duration in larval and adult zebrafish hearts III Validation of an essential predictive combination of reporter fish and repolarization parameters in a blinded, second drug set.
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