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Extracellular mechanical forces drive endocardial cell volume decrease during cardiac valve morphogenesis

Hélène Vignes, Christina Vagena-Pantoula, Mangal Prakash, Caren Norden, Florian Jug, Julien Vermot

Preprint posted on July 27, 2021 https://www.biorxiv.org/content/10.1101/2021.07.27.453460v1

Physical environment matters! - How mechanical forces play an important role in heart tube remodeling.

Selected by Julia Grzymkowski

Background

During the complex process of cardiac morphogenesis, the primitive heart tube, consisting of an inner endocardium layer and outer myocardium layer, undergoes extensive remodeling to form the functioning heart (1). This requires endocardial cells (EdC) to coordinate responses to both genetic information and mechanical forces originating from different sources such as the extracellular matrix (i.e cardiac jelly), neighboring cells, or tissues, or within the heart tissue itself (2-3). During the remodeling process, endocardial cells are capable of sensing and transducing mechanical signals from their environment via mechano-sensitive membrane channels, resulting in changes to their cellular properties (3). However, how mechanical forces lead to the modulation of endocardial cell properties and how these changes result in tissue-level remodeling of the heart are not well understood. In this preprint, Vignes et al aim to address the role of mechanical forces in heart development during the formation of the atrioventricular canal (AVC) in zebrafish.

Key Findings

Taking advantage of zebrafish’s amenability for live imaging, the authors first utilized a transgenic line that fluorescently labels the nucleus and Golgi apparatus of EdC’s to study EdC polarity (i.e., nucleus-to-Golgi orientation) during AVC formation as a readout for collective cell movements. Before the establishment of a heartbeat (22 hours post fertilization (hpf)), EdC’s in all areas of the heart displayed a nucleus-to-Golgi orientation towards the outflow, which was sustained after blood flow initiation (28 hpf). Interestingly, between 28 and 48 hpf, EdC’s within the atrium remained oriented towards the outflow, while EdC’s in the ventricle reversed their nucleus-to-Golgi orientation towards the inflow. This suggests that a global orientation of the nucleus-to-Golgi axis of EdC’s towards the AVC coincides with tissue convergence at the AVC.

The authors posited that cells within the area of tissue convergence must experience a change in shape, so they analyzed changes in cell volume within the AVC over time. Cell volume was assessed through three different methods. First, using manual 3D segmentation, the mean cell volume was calculated based on cell height and area measurements, quantitative deep learning was then used to determine nuclei volume, which directly correlates with cell volume, and finally the distance between a nucleus and its three neighbors was measured. With these robust quantification methods, the authors found that mean cell volume within the AVC decreases over time between 36 and 48 hpf. This corresponded with an enrichment in actin filaments and phospho-myosin light chain within the AVC, suggesting that both cell volume decrease, and contractility are involved in heart tube remodeling. Clustering of EdC’s is observed during tissue convergence at the AVC, which could be caused by a difference in cell proliferation. Therefore, the authors inhibited cell proliferation in zebrafish embryos during heart development to see if this affected the cell volume decrease within the AVC. While the number of EdC’s within the hearts of treated embryos did not double over time as in control hearts, the hearts in treated embryos were able to loop properly, and the cell size ratio in the different regions of the heart was not significantly different between controls and treated embryos. In addition, cells were still smaller in the AVC of treated embryos, suggesting that a global scaling effect of EdC’s based on cell number and size can occur. Finally, global EdC nucleus-to-Golgi orientation towards the AVC was still observed in treated embryos. Altogether, these results suggest that tissue convergence during heart tube remodeling occurs independently of cell proliferation.

Cardiac remodeling occurs while the heart is beating, and blood is flowing. Blood flow is known to create mechanical forces on EdC’s, such as shear stress and circumferential stretch (3). To assess the role of mechanical forces on cardiac remodeling, the authors studied the cell size changes at the AVC in silent heart mutants (sih-/-), which carry a mutation in the gene troponin T2a (tnnt2a) and therefore have no heart contraction or blood flow. Interestingly, mutant embryos did not display smaller mean cell volumes within the AVC and actin enrichment at the AVC was absent. EdC’s within the atrium had a random nucleus-to-Golgi orientation and EdC’s within the ventricle did not reverse their nucleus-to-Golgi orientation towards the AVC. Results were confirmed in embryos injected with a diluted tnnt2a morpholino, suggesting that mechanical forces are required for cell volume decrease and tissue convergence at the AVC.

EdC’s express the mechanosensitive membrane channels Transient Receptor Potential Polycystin 2 (TRPP2) and Transient Receptor Potential Vanilloid 4 (TRPV4). The authors utilized trpp2 and trpv4 mutants to determine their role in cell volume decrease during AVC formation. Double trpv4-/-; trpp2-/- mutants displayed similar phenotypes as sih-/-, with no observable cell volume decrease and reduced ventricular nucleus-to-Golgi orientation towards the inflow, indicating their importance for cell volume decrease and tissue convergence at the AVC. TRPV4 is known to interact with aquaporins during cell volume modulation, so the authors studied the expression of two aquaporins during heart development. Aqp8a.1 and aqp1a.1 are both expressed specifically in AVC cells starting at 30 hpf, with aqp1a.1 showing distinct AVC and outflow tract expression at 48 hpf. Intriguingly, aqp8a.1 and aqp1a.1 expression was absent in sih-/- embryos, suggesting their expression depends on heart function.

The cardiac jelly is the ECM separating the endocardium and myocardium and is largely composed of glycosaminoglycans (GAGs), the most common being hyaluronic acid (HA). GAG accumulation is known to apply osmotic pressure, therefore the authors wanted to assess AVC cell volume changes in the absence of HA by injecting hyaluronidase within the hearts of embryos. Removing HA from the cardiac jelly inhibited the AVC cell volume decrease and F-actin remodeling, while also reducing the ventricular nucleus-to-Golgi orientation towards the inflow.

Together, these data allowed the authors to propose a model of AVC formation, where HA accumulation creates an osmotic pressure between the EdCs and cardiac jelly, which promotes the cell volume decrease observed in the AVC, leading to tissue convergence and heart tube remodeling.

Why I like this preprint

I chose to highlight this preprint because I believe it is important to study the physical environment of a developing system and how physical forces play a role in normal development. In an embryo, organs form near other organs and tissues, so understanding how they sense their environment and respond accordingly during their development is an interesting topic. Using multiple mutant zebrafish lines and robust quantitative approaches, this preprint offers key insight into heart development that will help us better understand how different cardiac abnormalities could arise.

Questions for the authors

  1. In proliferation-inhibited embryos, do the hearts function normally? Could you say inhibition of proliferation would not cause heart defects?
  2. Do you think 1 mutant embryos would display reduced AVC cell volume decrease and tissue convergence?
  3. Do you think physical forces originating outside of the heart (pressure from the yolk sac or other developing organs) have any role in AVC formation?

References:

  1. Derrick, C. J., & Noël, E. S. (2021). The ECM as a driver of heart development and repair. Development (Vol. 148, Issue 5).
  2. Boselli, F., Steed, E., Freund, J. B., & Vermot, J. (2017). Anisotropic shear stress patterns predict the orientation of convergent tissue movements in the embryonic heart. Development, 144(23), 4322–4327.
  3. Campinho, P., Vilfan, A., & Vermot, J. (2020). Blood Flow Forces in Shaping the Vascular System: A Focus on Endothelial Cell Behavior. Frontiers in Physiology (Vol. 11).

Tags: biophysics, heart development, zebrafish

Posted on: 25th August 2021 , updated on: 30th August 2021

doi: https://doi.org/10.1242/prelights.30394

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Author's response

Julien Vermot shared

1. In proliferation-inhibited embryos, do the hearts function normally? Could you say inhibition of proliferation would not cause heart defects?

We treat embryos from 30hpf until 48hpf using a combination of two drugs that inhibit cell proliferation. After treatment, we did not see any defects in the heart volume, the beating of the heart and the heart also looped correctly. So, we could say that the heart is functioning normally when proliferation is inhibited for a short time-period. However, many embryos display morphological defects at later developmental stages. So, not surprisingly, long term cell proliferation inhibition affects heart development.

2. Do you think aqp1a.1 mutant embryos would display reduced AVC cell volume decrease and tissue convergence?

This is a very interesting point that we are currently addressing. Hopefully more to come soon.

3. Do you think physical forces originating outside of the heart (pressure from the yolk sac or other developing organs) have any role in AVC formation?

Overall, the wall shear stress generated by the blood flow, the hydrostatic pressure, the cyclic strain due to stretch and the forces originating from the ECM are most likely dominating in our system. However, we cannot exclude that the forces you mention are acting more generally on heart morphogenesis.

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