4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation
Juhyun Lee, … , Rongsong Li, Tzung K. Hsiai
Juhyun Lee, … , Rongsong Li, Tzung K. Hsiai
Published May 2, 2016; First published March 28, 2016
Citation Information: J Clin Invest. 2016;126(5):1679-1690. https://doi.org/10.1172/JCI83496.
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Technical Advance Cardiology Development

4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

Abstract

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress–mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress–activated Notch signaling to modulate trabeculation.

Authors

Juhyun Lee, Peng Fei, René R. Sevag Packard, Hanul Kang, Hao Xu, Kyung In Baek, Nelson Jen, Junjie Chen, Hilary Yen, C.-C. Jay Kuo, Neil C. Chi, Chih-Ming Ho, Rongsong Li, Tzung K. Hsiai

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Figure 1

Fluorescent light sheets to image cardiac morphogenesis.

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Fluorescent light sheets to image cardiac morphogenesis. (A) The sample ...
(A) The sample is placed at the orthogonal intersection between the illumination lens (IL) and the detection lens (DL) in the SPIM system. (B) A cylindrical lens generates a thin laser light sheet, and the illumination lens excites a thin slice of the sample in a 2D plane. The fluorescence from the illuminated planes is orthogonally detected by the detection lens. (C) A schematic diagram illustrates the laser light-sheet sectioning across a zebrafish embryo. (D) The entire embryo can be imaged within 30 seconds at a single cellular resolution. Inset reveals the trabecular endocardium. (E) Magnification of the heart reveals the contracting cmlc2-gfp–labeled myocardium and flowing DsRed-labeled red blood cells across the atriventricular valve. (F) The integration of SPIM image with 4D synchronization algorithm reveals a beating 4D heart. A, atrium; V, ventricle. (G) Trabecular ridges were absent in the myocardium at approximately 50 hpf. (H) Ridges protruding into the ventricular cavity occurred at approximately 60 hpf near the myocardial lining experienced high ventricular inflow. (I) Distinct trabecular ridges occurred at approximately 70 hpf.