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 David Milan |
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Science
Cardiac Repolarization
One of the most challenging problems in medicine today is variation in drug response. Whether it takes the form of variable efficacy of chemotherapy, or differences in toxic side effects, the unpredictability of this phenomenon increases the difficulty of therapeutic intervention. We are seeking to understand the molecular and genetic basis of variation in drug response using drug-induced QT prolongation as a model. For reasons that are not yet completely understood, the zebrafish has proven to be a remarkably good model for drug induced QT prolongation. We have developed this model and adapted it to high throughput analysis, enabling chemical and genetic screens. With this work we hope to better understand the molecular mechanisms that underlie cardiac repolarization and ultimately determine which patients are at risk for the potentially lethal side effect of QT prolongation.
Cardiac Valvulogenesis
Defects in cardiac valve formation are one of the most common congenital heart defects observed in humans, and the study of this process promises to provide mechanistic insights and lead to novel therapeutics for treatment of cardiac disease. During development, the boundary between the atrium and ventricle undergoes a highly orchestrated series of changes that ultimately result in the formation of the cardiac valves and decremental conduction tissue in the atrioventricular ring. These separate processes occur in two different tissue planes – the endothelium for the valve and the myocardium for the conduction system. The coordinate regulation of these processes is critical to the proper formation of the functioning heart. Furthermore, understanding the formation of the endocardial cushions, which involves a classical epithelial to mesenchymal transition, will have far reaching implications. We have are using the zebrafish as an experimental system for the investigation of the early events in this process. The traits of external fertilization coupled with their transparency during embryogenesis allow observation of early events in cardiac development without the need for invasive techniques.
Cardiac Physiology
After initial genetic screens were performed in zebrafish, it became clear that further progress would depend on the ability to discern more subtle phenotypes. We have developed novel high and medium throughput assays of cardiac physiology that will allow exploration of 1) determinants of cardiac rhythm, 2) development of specialized conduction tissue 3) modifiers of drug-responses and 4) the molecular mechanisms of cardiac repolarization. We are continuing to explore novel methods for the investigation of cardiac physiology in this zebrafish.
DEVELOPMENT OF NOVEL PHYSIOLOGIC ASSAYS
Heart Rate
One of the simplest quantitative cardiac traits is the rate at which the heart beats. In humans, the normal resting heart rate is between 60 and 90 beats per minute. In many of the more traditional models the heart rate is markedly different - for instance the heart rate of a mouse is 300-600 beats per minute. In zebrafish, the normal embryonic heart rate is much closer to humans at 120-180 beats per minute. We have developed a method for recording heart rate in embryonic zebrafish at high throughput. The initial assay required a fluorescent reporter to be expressed in the heart, but more recently we have improved on this assay to allow the determination of heart rate at high throughput in any zebrafish. This assay enables the exploration of the determinants of heart rate, sinus node function, and even drug responses, and is the basis for an ongoing pharmacogenetic screen to understand the variation in response to QT prolonging drugs.
Calcium Imaging
We have developed the use of calcium fluorescent imaging to study the patterns of myocardial activation during cardiac development. The technique allows real-time evaluation of calcium wavefronts non-invasively, allowing longitudinal measurements to be made.
Electrophysiology
We have applied the technique of optical voltage mapping to study the electrophysiology of the embryonic zebrafish heart. This technique enables detailed quantitative evaluation of cardiac conduction velocities and action potential duration and has allowed the confirmation of several key electrophysiologic parallels between humans and zebrafish. We are using this technique to further explore the molecular details of cardiac repolarization, the development of specialized conduction tissue, and to validate findings from zebrafish and human genetics as they relate to repolarization.
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