Poster Q2 - Snai1b Mediates Myocardial Trabeculation and Growth in Response to Mechanical Stimulation

Introduction: Snail family genes have long been recognized as inducers of epithelial-to-mesenchymal transition (EMT) in various cell types involved in cardiac development and injury, including endocardial cells, epicardial cells, and fibroblasts. The activation of Snail genes is crucial to processes such as valvulogenesis, fibrosis, and regeneration. However, the activation of Snail genes within cardiomyocytes (CMs) has yet to be characterized.
Although recent works reported that a zebrafish Snail gene, snai1b, plays a role in guiding the migration of myocardial progenitors and maintaining the architecture of the cardiac wall, the spatiotemporal dynamics of snai1b expression and the subsequent modulation of trabeculation are still poorly understood.

Materials and

Methods: To visualize the spatiotemporal expression of snai1b, we crossed the Tg(snai1b:EGFP) reporter line with different tissue-specific reporter lines, including Tg(myl7:mCherry) and Tg(myl7:mCherry-zCdt1). A tissue-specific Tol2-CRISPR interference/activation system was used to repress/overexpress snai1b in CMs. To alter the myocardial strain, we treated the embryos with isoproterenol hydrochloride (ISO), a -adrenergic agonist, and two different myosin inhibitors, 2,3-butanedione 2-monoxime (BDM) or para-amino-blebbistatin (pAB). The change in strain was confirmed by performing strain mapping in the 3D+time in vivo images of 5dpf hearts. The spatial patterning of snai1b and Notch signaling was visualized using a Tg(TP1:EGFP; myl7:mCherry) reporter line. We then performed single-cell RNA seq analysis to uncover the enriched gene pathways within snai1b+ CMs in embryonic hearts and validated the predictions via in situ hybridization.

Results, Conclusions, and Discussions: We discovered that snai1b is primarily expressed within a small population of CMs at the bulbus-ventricular annulus at 5 days post-fertilization (dpf). As the fish reaches juvenile and adult stages, snai1b begins to appear within epicardium and lymphatic vessels, but rarely in trabecular CMs.
Following ISO treatment, we can observe the activation of snai1b within the trabeculation of embryonic and juvenile hearts. The activation of snai1b is positively correlated with the duration of ISO treatment and can be attenuated by the myosin inhibitors, indicating its mechanosensitive nature. Additionally, the location of snai1b expression is spatially linked to the increase of myocardial strain, as demonstrated by 4D confocal imaging and strain mapping. A closer examination showed that ISO treatment inhibited Notch signaling in CMs, while activating snai1b in the delaminating CMs. This finding corroborates with the established Notch-ErbB2 signaling model of trabeculation, where Notch signaling in adjacent CMs reinforces the cell fate of trabecular CMs.
Via a CM-specific CRISPR interference/activation system, we showed that overexpression of snai1b led to extensive trabeculation and large myocardial clones at 14 dpf, while snai1b repression significantly restricted the trabecular formation and the number of cells per clone. As it has been shown that Notch signaling does not directly affect CM proliferation during trabeculation, we conclude that snai1b may also facilitate cardiac proliferation. Finally, the single-cell transcriptome of snai1b+ CMs revealed the enrichment of genes related to growth, cell proliferation, and ECM production.
In conclusion, we not only identified a novel mechanism of myocardial trabeculation and growth in response to mechanical forces but also for the first time, revealed that snai1b plays an EMT-independent role in trabeculation. As Snail family genes are often tissue-specific, our data may pave the way for future therapeutics by targeting myocardial-specific Snail programs.

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