Grego-Bessa, J. | Search bioRxiv

bioRxiv2022

Neuregulin1 regulates cardiomyocyte dynamics, proliferation, and maturation during ventricular chamber morphogenesis

BACKGROUNDCardiac ventricles are essential for providing the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for fundamental biology and regenerative medicine. Trabeculation is dependent on the signaling protein Neuregulin-1 (Nrg1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood. METHODSIn this study we investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with conditional cardiac-specific inactivation or overexpression of Nrg1. RESULTSAnalysis of cardiac-specific-Nrg1 mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 signaling and pErk activation. Early endothelial loss of Nrg1 and below normal pErk activation diminished cardiomyocyte Pard3 and Crumbs2 protein, and altered cytoskeletal gene expression and organization. These changes were associated with aberrant expression of genes involved in mitotic spindle organization and a directional shift from perpendicular to parallel/obliquely-oriented cardiomyocyte division. Further analysis indicated that Nrg1 is required for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchyme transition (EMT)-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, maintained high EMT-like gene expression and prolonged trabeculation, blocking compact myocardium maturation. Likewise, alterations of myocardial trabecular patterning resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with disorganization of the sarcomere actin cytoskeleton. The Nrg1 loss- and gain-of-function transcriptomes were enriched for yes-associated protein-1 (Yap1) gene signatures, identifying Yap1 as a potential downstream effector. Biochemical and imaging data showed that pErk activation and nuclear-cytoplasmic distribution of Yap1 during trabeculation are dependent on Nrg1. CONCLUSIONSThese data establish the Nrg1-ErbB2/4-pErk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development. Moreover, our data identify a Nrg1-dependent signaling cascade that could be leveraged for future cardiac regenerative therapies. Novelty and Significance WHAT IS KNOWN?O_LIMyocardial trabeculae play important roles in ventricular chamber growth, development of the conduction system, and formation of the coronary arteries. C_LIO_LITrabeculae are formed through oriented cell division (OCD), and their growth is driven by directional migration. C_LIO_LIThe membrane glycoprotein Neuregulin-1 (Nrg1) mediates cell-cell signaling and is essential for trabecular development. C_LI WHAT NEW INFORMATION DOES THIS ARTICLE CONTRIBUTE?O_LINrg1 signaling is essential for the expression of cardiomyocyte polarity genes and the organization of the cytoskeleton during the oriented cell division process underlying trabeculation. C_LIO_LINrg1 is required for the formation of the inner ventricular wall but not the coronaries. C_LIO_LINrg1 regulates motility and cell-cycle progression during ventricular wall growth. C_LIO_LIEctopic expression of Nrg1 leads to excessive trabeculation of the myocardium and disrupts compaction. C_LIO_LINrg1 regulates ventricular patterning mediated by cytoskeletal dynamics and modulates pErk-dependent Yap1 S274 phosphorylation during trabeculation. C_LIO_LINrg1 is not required for ventricular compaction. C_LI

developmental biology↗

bioRxiv2022

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.

genetics↗

bioRxiv2019

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase MFng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

developmental biology↗

bioRxiv2018

Myocardial Notch-Rbpj deletion does not affect heart development or function

During vertebrate cardiac development NOTCH signaling activity in the endocardium is essential for the crosstalk between endocardium and myocardium that initiates ventricular trabeculation and valve primordium formation. This crosstalk leads later to the maturation and compaction of the ventricular chambers and the morphogenesis of the cardiac valves, and its alteration may lead to disease. Although endocardial NOTCH signaling has been shown to be crucial for heart development, its physiological role in the myocardium has not been clearly established. Here we have used a genetic strategy to evaluate the role of NOTCH in myocardial development. We have inactivated the unique and ubiquitous NOTCH effector RBPJ in the early cardiomyocytes progenitors, and examined its consequences in cardiac development and function. Our results demonstrate that mice with cTnT-Cre-mediated myocardial-specific deletion of Rbpj develop to term, with homozygous mutant animals showing normal expression of cardiac development markers, and normal adult heart function. Similar observations have been obtained after Notch1 deletion with cTnT-Cre. We have also deleted Rbpj in both myocardial and endocardial progenitor cells, using the Nkx2.5-Cre driver, resulting in ventricular septal defect (VSD), double outlet right ventricle (DORV), and bicuspid aortic valve (BAV), due to NOTCH signaling abrogation in the endocardium of cardiac valves territories. Our data demonstrate that NOTCH-RBPJ inactivation in the myocardium does not affect heart development or adult cardiac function.

developmental biology↗