Abstract 12438: Novel 4-Dimensional Optical Technique to Elucidate Hemodynamic Shear Forces and Initiation of Trabeculation During Cardiac Morphogenesis

Abstract

Hemodynamic shear forces are intimately linked with cardiac development. However, the mechano-transduction mechanisms underlying this morphological event remain elusive. Trabeculation develops after cardiac looping, which is visualized in zebrafish embryos starting at 70 hours post-fertilization (hpf). We hypothesized that endocardial wall shear stress (WSS) up-regulates Notch signaling to initiate trabeculation. We developed a novel fast z-axis imaging acquisition system to reconstruct time-dependent 3-dimensional WSS in live zebrafish using single plane illuminating microscopy (SPIM). To assess the link between shear stress and trabeculation, we lowered the WSS by micro-injection of (1) gata1a-morpholino oligonucleotides (MO) at 2-cell stage to reduce erythropoiesis and viscosity by 70%, (2) tnnt2a-MO injection to inhibit cardiac contraction, and (3) use of weak atrium(wea) mutant to inhibit atrial contraction. To determine mechano-signal transduction underlying WSS and trabeculation, we assessed Notch signaling by in situ hybridization of NRG1 and notch1b. At 70 hpf, the control fish showed formation of trabeculation, whereas gata1 MO injected fish revealed a decrease in trabeculation in response to a reduction in shear stress by 90 %. Both wea mutant and tnnt2a MO injected fish exhibited smooth endocardial surfaces without trabecular networking. In situ hybridization of NRG1 and Notch 1b further showed a decrease in fluorescent intensity in the gata1 MO, wea mutant, and tnnt2a MO groups by 2.33±0.87-fold, 4.21±0.25-fold, and 4.96±0.17-fold, respectively (p < 0.05 vs. control fish, n=10). Thus, our findings indicate that hemodynamic shear forces are implicated in the initiation of cardiac trabecualtion via Notch signaling pathway, with a translational implication to understanding congenital noncompacted cardiomyopathy.