Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart

Abstract

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis. The growth and development of vertebrates are critically dependent on efficient cardiac output to drive blood circulation. An essential step of heart development is the formation of heart valves, whose leaflets are made through a complex set of cellular rearrangements of endothelial cells. Endothelial cells experience high flow forces as blood circulates. Moreover, heart valves and associated structures can be malformed when flow forces are abnormal, suggesting that these flow forces are in fact required for proper valve formation. Whether it is the force of the blood flow, its directionality (forward or reverse), or both that are important is not clear. We studied the interplay during valve development between key genes known to be involved in the process and epigenetic influences such as flow forces. Using zebrafish, whose optical clarity allows analyzing blood flow patterns at high resolution, we identified the presence of reversing flows specifically at the level of valve precursors. By manipulating blood flow patterns, we show that reversing flows are essential for valve morphogenesis. Specifically, we show that the expression of the gene klf2a depends on the presence of reversing flows and is required for valve development. We predict that by influencing levels of klf2a, reversing flows constitute an important stimulus controlling the appropriate biological responses of endothelial cells during valve formation.