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