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Integrating Light-Sheet Imaging and Cardiac Hemodynamic Shear Forces to Study Trabeculation


Hemodynamic forces such are intimately linked with cardiac morphogenesis. Defects in genetic programming during cardiac development result in functional and functional anomalies. During heart development, the trabeculae form a network of branching outgrowths from the ventricular wall, and both trabeculation and compaction are essential for normal cardiac contractile function. A significant reduction in trabeculation is usually associated with ventricular compact zone deficiencies (hypoplastic wall), whereas hypertrabeculation (non-compaction) is closely associated with left ventricular non-compaction (LVNC). The former condition predisposes patients to embryonic heart failure and lethality, and the latter to arrhythmia and heart failure with preserved ejection fraction. Left ventricular non-compaction (LVNC) is the third most common cardiomyopathy after dilated and hypertrophic cardiomyopathy in the pediatric population. Its prevalence was estimated from 4.5 to 26 per 10,000 adult patients referred for echocardiographic diagnosis. 1, 2 Despite these important clinical observations over the past 3 decades, the etiology of LVNC is largely unknown. While the majority of clinical studies is based on the anatomical feature of LVNC in the adult patients, there remains a poor genotype-phenotype association in LVNC patients, and a paucity of knowledge on the mechano-signal transduction of trabeculation and compaction during cardiac development. Notch signaling, and Notch pathway mediates differentiation and proliferation of trabecular myocytes. However, the mechanotransduction mechanisms underlying endocardial shear stress and the initiation of trabeculation remain elusive.

The goal of my work was to study how endocardial WSS activates Notch signaling to promote the initiation of trabeculation. Zebrafish was utilized as our model organism to study the changes in Notch signaling during trabeculation since they provide many advantages in studying these developmental phenomena, such as rapid organ development, high fecundity, and are optically transparent during early embryonic stages. Furthermore, the genes related to congenital heart disease genes in humans are conserved in zebrafish. This study will provide a deeper understanding of the mechanisms that drive of cardiac morphogenesis in zebrafish model and contribute to developing therapies for humans with CHD.

Zebrafish cardiac development can be visually assessed in transgenic zebrafish with fluorescently labeled proteins to visualize the heart or endocardium. Through a series of acquired image, we computationally reconstructed 4-dimensional (x,y,z,t) in vivo cardiac images using Selective Plane Illumination Microscopy (SPIM) using a custom synchronization algorithm. These 4-D SPIM images provided the visual assessment of zebrafish cardiac mechanics and cardiac flow, which allowed for the quantification of endocardial WSS by Computational Fluid Dynamics (CFD). Zebrafish blood viscosity, a key parameter in calculating endocardial WSS, has not been previously measured due to technical challenges with small sample size and extremely limited blood volumes. To address this issue, we have developed a capillary pressure-driven microfluidic channel to measure viscosity across a range of shear rates. This allowed us to also determine the relationship between zebrafish hematocrit and blood viscosity in our studies. We then modulated zebrafish endocardial WSS by genetically reducing blood viscosity and hematopoeisis via gata1a-MO injection to examine changes in trabeculation. We observed changes in Notch signaling related genes expressions which was supported by Tg(tp1:gfp) which provided spatial Notch signaling expression levels. Furthermore, co-injection of Nrg1 mRNA with gata1a-MO was applied to rescue Notch activation by promoting cardiac contractility induced by trabeculation. This multidisciplinary approach to study ventricular morphogenesis via Notch signaling clarifies the mechanistic understanding of NC.

Furthermore, we utilized adult zebrafish to study cardiac regeneration in addition to the embryonic model. Unlike the human heart, zebrafish hearts naturally regenerate and undergo rapid scar degradation. To study the electrical and mechanical changes in the zebrafish heart, we developed an ECG gated pulse-wave Doppler ultrasound device to monitor changes in cardiac electrical-mechanical properties during regeneration. Although zebrafish hearts become fully regenerated 90 days after injury, their cardiac electrical conduction remains damaged, as indicated by longer QT intervals in injured fish. This synchronous ECG gated pulse-wave Doppler provides synergistic information that would otherwise be undetected. This integrated approach can be further applied to assess changes in electrophysiological cardiac diseases, including arrhythmias and atrial fibrillation.

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