Connexin 43 regulates epicardial cell polarity and migration in coronary vascular development

Abstract

Connexin 43 knockout (Cx43 KO) mice exhibit conotruncal malformations and coronary artery defects. We observed epicardial blisters in the Cx43 KO hearts that suggest defects in epicardial epithelial-mesenchymal transformation (EMT), a process that generates coronary vascular progenitors. Analysis using a three-dimensional collagen gel invasion assay showed that Cx43 KO epicardial cells are less invasive and that, unlike wild-type epicardial cells, they fail to organize into thin vessel-like projections. Examination of Cx43 KO hearts using Wt1 as an epicardial marker revealed a disorganized pattern of epicardial cell infiltration. Time-lapse imaging and motion analysis using epicardial explants showed a defect in directional cell migration. This was associated with changes in the actin/tubulin cytoskeleton. A defect in cell polarity was indicated by a failure of the microtubule-organizing center to align with the direction of cell migration. Forced expression of Cx43 constructs in epicardial explants showed the Cx43 tubulin-binding domain is required for Cx43 modulation of cell polarity and cell motility. Pecam staining revealed early defects in remodeling of the primitive coronary vascular plexuses in the Cx43 KO heart. Together, these findings suggest an early defect in coronary vascular development arising from a global perturbation of the cytoarchitecture of the cell. Consistent with this, we found aberrant myocardialization of the outflow tract, a process also known to be EMT dependent. Together, these findings suggest cardiac defects in the Cx43 KO mice arise from the disruption of cell polarity, a process that may be dependent on Cx43-tubulin interactions.

FGF10/FGFR2b signaling is essential for cardiac fibroblast development and growth of the myocardium

“…We posit that decreased myocardial proliferation in hearts lacking epicardial FGF signaling could result indirectly from consequences of decreased numbers of interstitial cardiac fibroblasts. This is consistent with small heart size phenotypes that result from other mutations that disrupt pro-epicardial migration: defects in epicardial EMT and EPDC migration into the myocardium (Rhee et al, 2009;Martinez-Estrada et al, 2010;Wu et al, 2010).…”

Epithelial-mesenchymal transitions: insights from development

“…Perturbation of -catenin disrupts adherens junctions, causing an abnormal distribution of Numb and randomized spindle orientation (Wu et al, 2010). The gap junction protein connexin 43 (Cx43; Gja1 -Mouse Genome Informatics) regulates the microtubule-organizing center of epicardial cells through tubulin binding (Rhee et al, 2009). Indeed, Cx43-knockout mice show a coronary vascular developmental defect due to failed alignment between the microtubule-organizing center and the direction of epicardial cell migration.…”

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

“…Despite the presence of this binding site, microtubule blockers do not block trafficking or gap junctions assembly, which can be explained by redundancies that allow for alternative trafficking pathways to the cell membrane (Jordan et al, 1999;Majoul et al, 2009;del Castillo et al, 2010). Additionally, removal of the microtubule-binding domain or complete removal of the Cx43 C terminus does not stop plasma membrane trafficking or gap junction assembly (although gap junction plaques are smaller) (Jordan et al, 1999;Omori and Yamasaki, 1999;Rhee et al, 2009). However, under these conditions, there is a reduction in available hemichannels at the plasma membrane and subsequent transition to gap junctions (Johnson et al, 2002;Maass et al, 2007).…”