Role of NRG1 in Mouse Heart Development

Abstract

During embryonic development, cellular interactions are crucial to orchestrate the processes that give rise to the final body plan. The heart is the first organ to form and function during development. The formation of trabeculae, myocardial protrusions covered by endocardium that grow towards the lumen of the ventricles, is the first sign of chamber development, and occurs in response to signals from the endocardium that activate the overlying myocardium. Trabeculae are crucial for increasing the internal ventricular surface, favouring oxygen exchange and nourishment of the cardiomyocytes. Previous studies in mice have shown that NEUREGULIN1 (NRG1), a ligand belonging to the Epidermal Growth Factor family expressed in the endocardium, and its more widespread receptors -ERBB2,4- are crucial for ventricular trabeculation, but the cellular processes affected by Nrg1 loss in the heart are not well understood. To gain an insight into the role of NRG1 in heart development, we have used conditional loss- and gain-of-function mouse models. We have found that endothelial-specific, Tie2Cre-mediated, Nrg1 inactivation disrupts trabecular morphology and patterning, and reduces ventricular cardiomyocyte proliferation, while paradoxically, the compact myocardium appeared thickened. Global gene expression analysis in embryonic hearts by RNA-seq revealed a dysregulation of apico-basal polarity marker genes. We thus examined whether cellular polarity and oriented cell division were affected in chamber cardiomyocytes. We observed a loss of polarity (LAMININS-ITGα6, PKC, N-CADHERIN) in cardiomyocytes, and an increase in parallel divisions in chamber cardiomyocytes of Nrg1flox;Tie2Cre mutants. This finding would explain the thickened compact myocardium and the impaired trabeculation of these mice. In addition to trabeculation defects, Nrg1flox;Tie2Cre mutant hearts show hypoplastic valves, due to impaired epithelial-mesenchyme transition (EMT) of presumptive valve endocardial cells, presumably because binding and activation of the receptor ERBB2,3 by NRG1 in this region is impaired. To study the function of NRG1 signalling at later stages of ventricular chamber development, we have induced Nrg1 deletion during compaction, using the Cdh5CreERT2 driver. Late Nrg1 inactivation in cardiac endothelium leads to a thinner compact myocardium, and defective myocardial patterning and coronary vessel morphogenesis. We have also generated a conditional gain-of-function transgenic line for NRG1. Tie2Cre-mediated Nrg1 overexpression lead to thickened valves, and Nkx2.5Cre-mediated Nrg1 lead also to thickened valves, ventricular septal defect and ventricular chamber dilation. Our results indicate that NRG1 is essential from early to late stages of ventricular wall development. NRG1 is required for cardiomyocyte polarization and oriented cell division during trabeculation, for endocardial cushion formation during valve development, and for chamber maturation and coronary vessel formation during compaction.