ß1 integrins regulate cellular behavior and cardiomyocyte organization during ventricular wall formation.

AIMS: The mechanisms regulating the cellular behavior and cardiomyocyte organization during ventricular wall morphogenesis are poorly understood. Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. This study aims to determine whether and how ß1 integrins regulate cardiomyocyte behavior and organization during ventricular wall morphogenesis in the mouse. METHODS AND RESULTS: We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/ß integrins and their ligands in the embryonic heart. Integrin ß1 subunit (ß1) and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, and fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1, which encodes the ß1, was deleted via Nkx2.5Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts display an absence of a trabecular zone but a thicker compact zone. The levels of hyaluronic acid and versican, essential for trabecular initiation, were not significantly different between control and B1KO. Instead, fibronectin, a ligand of ß1, was absent in the myocardium of B1KO hearts. Furthermore, B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. Mosaic clonal lineage tracing showed that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. CONCLUSIONS: ß1 is asymmetrically localized in the cardiomyocytes, and some of its ECM ligands are enriched along the luminal side of the myocardium, and fibronectin surrounds cardiomyocytes. ß1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1 leads to loss of ß1 and fibronectin and prevents cardiomyocytes from engaging the ECM network, resulting in failure to establish tissue architecture to form trabeculae.

Left Ventricular Noncompaction Is Associated with Valvular Regurgitation and a Variety of Arrhythmias.

Left ventricular noncompaction (LVNC) is a type of cardiomyopathy characterized anatomically by prominent ventricular trabeculation and deep intertrabecular recesses. The mortality associated with LVNC ranges from 5% to 47%. The etiology of LVNC is yet to be fully understood, although decades have passed since its recognition as a clinical entity globally. Furthermore, critical questions, i.e., whether LVNC represents an acquired pathology or has a congenital origin and whether the reduced contractile function in LVNC patients is a cause or consequence of noncompaction, remain to be addressed. In this study, to answer some of these questions, we analyzed the clinical features of LVNC patients. Out of 9582 subjects screened for abnormal cardiac functions, 45 exhibit the characteristics of LVNC, and 1 presents right ventricular noncompaction (RVNC). We found that 40 patients show valvular regurgitation, 39 manifest reduced systolic contractions, and 46 out of the 46 present different forms of arrhythmias that are not restricted to be caused by the noncompact myocardium. This retrospective examination of LVNC patients reveals some novel findings: LVNC is associated with regurgitation in most patients and arrhythmias in all patients. The thickness ratio of the trabecular layer to compact layer negatively correlates with fractional shortening, and reduced contractility might result from LVNC. This study adds evidence to support a congenital origin of LVNC that might benefit the diagnosis and subsequent characterization of LVNC patients.

The Spatiotemporal Expression of Notch1 and Numb and Their Functional Interaction during Cardiac Morphogenesis.

Numb family proteins (NFPs), including Numb and Numblike (Numbl), are commonly known for their role as cell fate determinants for multiple types of progenitor cells, mainly due to their function as Notch inhibitors. Previous studies have shown that myocardial NFP double knockout (MDKO) hearts display an up-regulated Notch activation and various defects in cardiac progenitor cell differentiation and cardiac morphogenesis. Whether enhanced Notch activation causes these defects in MDKO is not fully clear. To answer the question, we examined the spatiotemporal patterns of Notch1 expression, Notch activation, and Numb expression in the murine embryonic hearts using multiple approaches including RNAScope, and Numb and Notch reporter mouse lines. To further interrogate the interaction between NFPs and Notch signaling activation, we deleted both Notch1 or RBPJk alleles in the MDKO. We examined and compared the phenotypes of Notch1 knockout, NFPs double knockout, Notch1; Numb; Numbl and RBPJk; Numb; Numbl triple knockouts. Our study showed that Notch1 is expressed and activated in the myocardium at several stages, and Numb is enriched in the epicardium and did not show the asymmetric distribution in the myocardium. Cardiac-specific Notch1 deletion causes multiple structural defects and embryonic lethality. Notch1 or RBPJk deletion in MDKO did not rescue the structural defects in the MDKO but partially rescued the defects of cardiac progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis. Our study concludes that NFPs regulate progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis partially through Notch1 and play more roles than inhibiting Notch1 signaling during cardiac morphogenesis.