Mechanical fracturing of the extracellular matrix patterns the vertebrate heart

‘Mechanical fracturing of the extracellular matrix patterns the vertebrate heart’ Study Summary [1] [2].

‘These cracks, They let the Developing Cells in: A Chronicle of Trabeculation in the developing Zebrafish Heart’ — The authors of this study looked at zebrafish embryos and a quantitative biophysical model. In these, interactions between the cardiac ECM and the contracting myocardium were observed and analyzed, to examine the initiating events that trigger trabecula formation in the zebrafish ventricle.

Background/ A brief overview of zebrafish heart development: As with most vertebrates, the zebrafish heart begins its journey as a linear tube, which eventually loops and acquires an S shape comprising a posteriorly placed atrium and an anterior ventricle. The ventricle expands anisotropically (for Definition see Glossary included at the end of this preLight) and eventually acquires a bean shape – the outer ‘bulge’ in this configuration is called the outer curvature (OC) and has been defined as the location in which trabeculation of the myocardial tissue commences. Trabeculation is a specialised process during which a monolayer of cardiac tissue in the ventricle transforms and assumes the intricacies of a 3D meshwork composed of an inner trabecular layer (TL) surrounded by an outer compact layer (CL). The process of trabeculation involves delamination, a name attributed to developmental processes that involve cells from an epithelial layer ‘breaking-away’ and populating adjacent areas – in the case of trabeculation, this culminates in the creation of a 3D trabecular meshwork.

What triggers this phenomenon?  Delamination occurs around the 55 hours post-fertilization (hpf) mark, triggered by local discrepancies in the tension of the actomyosin network, in the OC of the bean-shaped ventricle. The authors of this preprint started their study by finding out what doesn’t trigger delamination in the ventricle OC – it is not dependent on Nrg2a/Erbb2 signaling nor basement membrane remodeling (evaluated via fibronectin, laminin localization/ concentration). It is, however, associated with the appearance of ‘fractures’ in the cardiac jelly of the cardiac extracellular matrix (cECM), preceded by irregularities in cECM thickness throughout the OC, occurring around the 24-34 hpf mark. cECM fracturing is independent of Nrg/Nrg2a and Notch signaling, at least in the zebrafish, as well as enzymatic degradation of the matrix. Furthermore, while cECM fracturing events precede delamination, delamination itself is not the cause of these fractures – instead it can be described as a mechanical event, occurring due to the contracting movement of the developing ventricle.

What are the authors doing in this study? To evaluate the hypothesis that mechanical strain from a contracting ventricle leads to cECM fractures, in turn triggering delamination events throughout the OC, the authors turned to zebrafish embryos and a quantitative biophysical model. In this model, the cECM behaves as an isotropic viscoelastic sheet (for Definition see Glossary included at the end of this preLight), interacting frictionally with a beating myocardium which also undergoes cyclic loading during each cycle.

Key results of the study:

1. Areas where delamination will occur are associated with cECM thinning, evident in cECM thickness heatmaps generated by the authors – before delamination events, cECM thickness is non-uniform across regions with thinner cECM regions mainly in the OC of the ventricle.
2. Cyclic myocardial deformation triggers areas of slight cECM damage, which eventually appear as ‘fractures’ – these fractures become more pronounced with time, as it also normally occurs during in vivo zebrafish development.
3. Cells delaminate into the cECM fractures – eventually these cells transform into multicellular ridges.
4. Pharmacologic agents like IBMX and isoprenaline, which enhance ventricular contraction, increase cECM fracture occurrence – Conversely, agents such as nifedipine and lidocaine that reduce ventricular contraction, also reduce the rate of cECM fracture formation.
5. Genetic abrogation of heart function via morpholino knockdown (KD) of the gene tnnt2a, which encodes Troponin T, abolishes cECM fracture formation in the ventricle.
6. Genetic abrogation of ventricular function via generation of half-hearted mutants, where the atrium contracts and the ventricle does not, abolishes cECM fracture formation in the ventricle – cECM fracture formation is thus dependent on ventricular contraction.
7. Genetic abrogation of the stereotypical geometric S heart shape via morpholino KD of the gene tbx5a and via inhibition of BMP signaling during development with K02288 leads to linear cECM fractures which are also more evenly distributed across the length of the heart tube.

Glossary

• Viscoelastic: Viscoelastic materials are described as such because they exhibit both elastic and viscous deformation when stress is applied, i.e. they behave as both solid and liquid [3] – Examples of materials with viscoelastic characteristics include rubber and memory foam. Various biological tissues, including the hyaluronan cardiac extracellular matrix (cECM) of the developing zebrafish heart, are also considered viscoelastic [1].
• Isotropic: Isotropic materials are so described because they exhibit the same properties despite the direction in which they are tested – examples include glass, plastic and metal.
• Anisotropic: Anisotropic materials are so described because they exhibit different properties depending on the direction in which they are tested – examples include materials containing fibers or natural materials. In these cases, the strength of the material can depend on whether force is applied in the same direction as the fibers present. In general, composite materials are anisotropic [4] — The myocardium has been described as macroscopically anisotropic due to the transmural variation in cardiomyocyte orientation (± 60 ° in humans), and the configuration of perimysial extracellular matrix surrounding the cardiomyocyte bundles in sheets [5].

 

Why this work is interesting: While the process of developmental patterning has been attributed to genetic prepatterning, organs subject to mechanical deformation such as the heart are also subject to patterning driven by mechanical deformation. While mechanical stresses are a normal physiological part of everyday heart function, this study uncovers processes with which these stresses also contribute to its embryonic development.

 

Why is the use of zebrafish in animal research ethical? Several characteristics of the species render it compliant with the 3R (Reduce, Refine, Replace) of Ethics for Animal Research. Regarding the first R ‘Reduce’ [Number of animals used], multiple embryos can be generated per week, allowing the evaluation of multiple parameters and reducing the need for higher mammal numbers in later stages. Regarding the second R ‘Refine’ [Experimental quality and precision], the unique characteristics of the larvae, including its transparent skin, allow for in vivo imaging in real-time, minimizing the risk of invasive procedures to the integrity of the organism and allowing the development of standardized experimental conditions for easier reproducibility. Regarding the third R ‘Replace’ [Use of a non-animal model], zebrafish allow for the testing of physiologically complex conditions without the ethical constraints of mammal-based animal modeling [6].

Questions I would like to ask the authors about their work:

1. Does excessive myocardial contraction provide continuously positive feedback on the formation of cECM fractures?
   1. Alternatively, will excessive myocardial contraction, after a particular point in the value of the force applied to the hyaluronan cECM matrix, negatively affect the architecture of the tissue and trabecula formation?
   2. Essentially, is there a contractile force limit that will lead to complete dissociation in ventricular architecture?

References:

[1] Chan Jin Jie C, Santos-Oliván D, Ramel M-C, Sánchez-Posada J, Andrews TGR, Paizakis P, et al. Mechanical fracturing of the extracellular matrix patterns the vertebrate heart 2025:2025.03.07.641942. https://doi.org/10.1101/2025.03.07.641942.

[2] Battista NA, Douglas DR, Lane AN, Samsa LA, Liu J, Miller LA. Vortex Dynamics in Trabeculated Embryonic Ventricles. Journal of Cardiovascular Development and Disease 2019;6:6. https://doi.org/10.3390/jcdd6010006.

[3] Arjona MI, Najafi J, Minc N. Cytoplasm mechanics and cellular organization. Current Opinion in Cell Biology 2023;85:102278. https://doi.org/10.1016/j.ceb.2023.102278.

[4] Huang Z-M. Chapter One – Mechanics theories for anisotropic or composite materials. In: Bordas SPA, editor. Advances in Applied Mechanics, vol. 56, Elsevier; 2023, p. 1–137. https://doi.org/10.1016/bs.aams.2022.09.004.

[5] Tueni N, Allain J-M, Genet M. On the structural origin of the anisotropy in the myocardium: Multiscale modeling and analysis. Journal of the Mechanical Behavior of Biomedical Materials 2023;138:105600. https://doi.org/10.1016/j.jmbbm.2022.105600.

[6] Canedo A, Saiki P, Santos AL, Carneiro K da S, Souza AM de, Qualhato G, et al. Zebrafish (Danio rerio) meets bioethics: the 10Rs ethical principles in research. Ciênc Anim Bras 2022;23:e. https://doi.org/10.1590/1809-6891v22e-70884.

Tags: biology, cardiac, cardiovascular, development, zebrafish

doi: https://doi.org/10.1242/prelights.40632

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