Abstract
Tissue integrity is critical for organ formation and function. During heart development, cardiomyocytes differentiate and integrate to form a coherent tissue that contracts synchronously. However, the molecular mechanisms regulating cardiac tissue integrity are poorly understood. Here we show that proteolysis, via the E3 ubiquitin ligase ASB2, regulates cardiomyocyte maturation and tissue integrity. Cardiomyocytes in asb2b zebrafish mutants fail to terminally differentiate, resulting in reduced cardiac contractility and output. Mosaic analyses reveal a cell-autonomous requirement for Asb2b in cardiomyocytes for their integration as asb2b mutant cardiomyocytes are unable to meld into wild-type myocardial tissue. In vitro and in vivo data indicate that ASB2 negatively regulates TCF3, a bHLH transcription factor. TCF3 must be degraded for cardiomyocyte maturation, as TCF3 gain-of-function causes a number of phenotypes associated with cardiomyocyte dedifferentiation. Overall, our results show that proteolysis has an important role in cardiomyocyte maturation and the formation of a coherent myocardial tissue.
Highlights
Tissue integrity is critical for organ formation and function
We found that forced overexpression of asb2b in cardiomyocytes resulted in disrupted myofilaments (Supplementary Fig. 2c), indicating that tight regulation of asb2b expression levels is crucial for cardiac development
We found that asb2b mutant cardiomyocytes in wild type (WT) hearts failed to develop organized myofilaments and exhibited thin myofilaments that appeared discontinuous with the myofilaments of adjacent WT host cardiomyocytes (Fig. 3b and Supplementary Fig. 4b)
Summary
Observed that EGFP-Podocalyxin strongly accumulates in protruding membranes in asb2b mutant cardiomyocytes (Supplementary Fig. 3e). We found upregulation in the expression of several genes involved in actin rearrangement and cell migration in asb2b mutant hearts (Supplementary Fig. 3g and Supplementary Table 1)[19] These data suggest that asb2b mutant cardiomyocytes exhibit changes in cell morphology and cell–cell junctions, as well as loss of apicobasal. We observed that asb2b mutant cardiomyocytes in WT hearts exhibited irregular borders and membrane protrusions (Fig. 3d). Cardiomyocytes overexpressing Tcf3b exhibited severe myofilament disassembly or disorganized myofilaments, similar to the phenotype observed in asb2b mutant cardiomyocytes (Fig. 4e), as well as irregular cell shape and membrane protrusions (Fig. 4f). (a) Three-dimensional images of 50 hpf Tg(myl7:LIFEACT-GFP) WT and asb2b mutant hearts reveal differences in myofilament thickness and organization.
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