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 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 2

The dynamics of 3D cardiac architecture in response to genetic manipulations of the Tg(cmlc:gfp) zebrafish system.

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The dynamics of 3D cardiac architecture in response to genetic manipulat...
(A) A 3D Cartesian coordinate system provides the orientation of the ventricle. (B) The ventricular inflow tract from the atrium relates to the outflow tract from the 3D ventricle. (C) Trabecular network developed in the ventricle at 75 hpf in the WT (control). (D) Trabeculae formed a prominent sponge-like structure at 100 hpf in the WT. (E) gata1a MO microinjection at the 1- to 4-cell stage attenuated trabeculation at 75 hpf. (F) gata1a MO–treated fish delayed trabecular formation at 100 hpf. (G) Coinjection of Nrg1 mRNA promoted trabeculation at 75 hpf. (H) Coinjection of Nrg1 mRNA nearly restored trabecular network at 100 hpf. (I) Trabeculation was absent in the wea mutants with a small ventricle. (J) Injection of Nrg1 mRNA into the wea mutants promoted trabecular formation. (K) The tnnt2a MO microinjection inhibited trabeculation at both 75 hpf (not shown) and 100 hpf. (L) The clo mutants developed nontrabeculated endocardium at 100 hpf. Red arrows indicate trabecular ridges. Scale bars: 50 μm. Original magnification, ×10.