Extended culture and imaging of normal and regenerating adult zebrafish hearts in a fluidic device.

Myocardial infarction and heart failure are leading causes of death worldwide, in large part because adult human myocardium has extremely limited regeneration capacity. Zebrafish are a powerful model for identifying new strategies for human cardiac repair because their hearts regenerate after relatively severe injuries. Zebrafish are also relatively scalable and compatible with many genetic tools. However, characterizing the regeneration process in live adult zebrafish hearts has proved challenging because adult fish are opaque, preventing live imaging in vivo. An alternative strategy is to explant and culture intact adult zebrafish hearts and investigate them ex vivo. However, explanted hearts maintained in conventional culture conditions experience rapid declines in morphology and physiology. To overcome these limitations, we designed and fabricated a fluidic device for culturing explanted adult zebrafish hearts with constant media perfusion that is also compatible with live imaging. We then compared the morphology and calcium activity of hearts cultured in the device, hearts cultured statically in dishes, and freshly explanted hearts. After one week of culture, hearts in the device experienced significantly less morphological degradation compared to hearts cultured in dishes. Hearts cultured in devices for one week also maintained capture rates similar to fresh hearts, unlike hearts cultured in dishes. We then cultured explanted injured hearts in the device and used live imaging techniques to continuously record the myocardial revascularization process over several days, demonstrating how our device is compatible with long-term live imaging and thereby enables unprecedented visual access to the multi-day process of adult zebrafish heart regeneration.

Late developing cardiac lymphatic vasculature supports adult zebrafish heart function and regeneration.

The cardiac lymphatic vascular system and its potentially critical functions in heart patients have been largely underappreciated, in part due to a lack of experimentally accessible systems. We here demonstrate that cardiac lymphatic vessels develop in young adult zebrafish, using coronary arteries to guide their expansion down the ventricle. Mechanistically, we show that in cxcr4a mutants with defective coronary artery development, cardiac lymphatic vessels fail to expand onto the ventricle. In regenerating adult zebrafish hearts the lymphatic vasculature undergoes extensive lymphangiogenesis in response to a cryoinjury. A significant defect in reducing the scar size after cryoinjury is observed in zebrafish with impaired Vegfc/Vegfr3 signaling that fail to develop intact cardiac lymphatic vessels. These results suggest that the cardiac lymphatic system can influence the regenerative potential of the myocardium.