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Contractile and hemodynamic forces coordinate Notch1b-mediated outflow tract valve formation
Jeffrey J. Hsu, … , Alison L. Marsden, Tzung K. Hsiai
Jeffrey J. Hsu, … , Alison L. Marsden, Tzung K. Hsiai
Published April 11, 2019
Citation Information: JCI Insight. 2019;4(10):e124460. https://doi.org/10.1172/jci.insight.124460.
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Research Article Cardiology Development

Contractile and hemodynamic forces coordinate Notch1b-mediated outflow tract valve formation

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Abstract

Biomechanical forces and endothelial-mesenchymal transition (EndoMT) are known to mediate valvulogenesis. However, the relative contributions of myocardial contractile and hemodynamic shear forces remain poorly understood. We integrated 4D light-sheet imaging of transgenic zebrafish models with moving-domain computational fluid dynamics to determine effects of changes in contractile forces and fluid wall shear stress (WSS) on ventriculobulbar (VB) valve development. Augmentation of myocardial contractility with isoproterenol increased both WSS and Notch1b activity in the developing outflow tract (OFT) and resulted in VB valve hyperplasia. Increasing WSS in the OFT, achieved by increasing blood viscosity through EPO mRNA injection, also resulted in VB valve hyperplasia. Conversely, decreasing myocardial contractility by Tnnt2a morpholino oligonucleotide (MO) administration, 2,3-butanedione monoxime treatment, or Plcγ1 inhibition completely blocked VB valve formation, which could not be rescued by increasing WSS or activating Notch. Decreasing WSS in the OFT, achieved by slowing heart rate with metoprolol or reducing viscosity with Gata1a MO, did not affect VB valve formation. Immunofluorescent staining with the mesenchymal marker, DM-GRASP, revealed that biomechanical force–mediated Notch1b activity is implicated in EndoMT to modulate valve morphology. Altogether, increases in WSS result in Notch1b- and EndoMT-mediated VB valve hyperplasia, whereas decreases in contractility result in reduced Notch1b activity, absence of EndoMT, and VB valve underdevelopment. Thus, we provide developmental mechanotransduction mechanisms underlying Notch1b-mediated EndoMT in the OFT.

Authors

Jeffrey J. Hsu, Vijay Vedula, Kyung In Baek, Cynthia Chen, Junjie Chen, Man In Chou, Jeffrey Lam, Shivani Subhedar, Jennifer Wang, Yichen Ding, Chih-Chiang Chang, Juhyun Lee, Linda L. Demer, Yin Tintut, Alison L. Marsden, Tzung K. Hsiai

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Figure 6

Effects of hemodynamic modulation on endocardial cell differentiation during VB valve formation in the OFT.

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Effects of hemodynamic modulation on endocardial cell differentiation du...
Immunostaining was performed against DM-GRASP, a cell surface adhesion protein expressed by differentiated valve-forming endocardial cells, in Tg(cmlc:mCherry) zebrafish embryos at 4 dpf. (A–C) Confocal imaging (original magnification, ×20) demonstrates sections through the (A) ventricle, (B) AVC, and (C) OFT of a control embryo. Green fluorescent protein (GFP) signal is observed between the cmlc+ myocardial cells, as well as in the AV and VB valve leaflets, which lack a cmlc signal, suggesting these are differentiated endocardial/endothelial cells. Scale bar: 50 μm. (D) Confocal images through the OFT of embryos subjected to the indicated treatments. Arrows denote DM-GRASP+, cmlc– valve leaflets. Scale bar: 25 μm. The outline denotes the DM-GRASP+, cmlc– valve leaflets in green and the cmlc+ myocardium in red. (E) Quantification of the volumes of DM-GRASP+, cmlc– valve leaflets in the indicated treatment groups (n = 4 per group). (F) Quantification of the cell counts in the DM-GRASP+, cmlc– valve leaflets in the indicated treatment groups (n = 6 per group, except n = 5 for the NICD mRNA group). Data are presented as mean ± SD; *P < 0.05; †P < 0.01; ‡P < 0.001; §P < 0.0001; 1-way ANOVA with Dunnett’s multiple-comparisons test.

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