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

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

Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1(-/-) mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1(-/-); Ccm2(lacZ/+) and Ccm2(lacZ/lacZ) mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity

Mutations in the gene for cardiac myosin-binding protein C and late- onset familial hypertrophic cardiomyopathy

Background: Mutations in the gene for cardiac myosin-binding protein C account for approximately 15 percent of cases of familial hypertrophic cardiomyopathy. The spectrum of disease-causing mutations and the associated clinical features of these gene defects are unknown. Methods: DNA sequences encoding cardiac myosin-binding protein C were determined in unrelated patients with familial hypertrophic cardiomyopathy. Mutations were found in 16 probands, who had 574 family members at risk of inheriting these defects. The genotypes of these family members were determined, and the clinical status of 212 family members with mutations in the gene for cardiac myosin- binding protein C was assessed. Results: Twelve novel mutations were identified in probands from 16 families. Four were missense mutations; eight defects (insertions, deletions, and splice mutations) were predicted to truncate cardiac myosin-binding protein C. The clinical expression of either missense or truncation mutations was similar to that observed for other genetic causes of hypertrophic cardiomyopathy, but the age at onset of the disease differed markedly. Only 58 percent of adults under the age of 50 years who had a mutation in the cardiac myosin-binding protein C gene (68 of 117 patients) had cardiac hypertrophy disease penetrance remained incomplete through the age of 60 years. Survival was generally better than that observed among patients with hypertrophic cardiomyopathy caused by other mutations in the genes for sarcomere proteins. Most deaths due to cardiac causes in these families occurred suddenly. Conclusions: The clinical expression of mutations in the gene for cardiac myosin-binding protein C is often delayed until middle age or old age. Delayed expression of cardiac hypertrophy and a favorable clinical course may hinder recognition of the heritable nature of mutations in the cardiac myosin-binding protein C gene. Clinical screening in adult life may be warranted for members of families characterized by hypertrophic cardiomyopathy

pak2a mutations cause cerebral hemorrhage in redhead zebrafish

The zebrafish is a powerful model for studying vascular development, demonstrating remarkable conservation of this process with mammals. Here, we identify a zebrafish mutant, redhead (rhdmi149), that exhibits embryonic CNS hemorrhage with intact gross development of the vasculature and normal hemostatic function. We show that the rhd phenotype is caused by a hypomorphic mutation in p21-activated kinase 2a (pak2a). PAK2 is a kinase that acts downstream of the Rho-family GTPases CDC42 and RAC and has been implicated in angiogenesis, regulation of cytoskeletal structure, and endothelial cell migration and contractility among other functions. Correction of the Pak2a-deficient phenotype by Pak2a overexpression depends on kinase activity, implicating Pak2 signaling in the maintenance of vascular integrity. Rescue by an endothelial-specific transgene further suggests that the hemorrhage seen in Pak2a deficiency is the result of an autonomous endothelial cell defect. Reduced expression of another PAK2 ortholog, pak2b, in Pak2a-deficient embryos results in a more severe hemorrhagic phenotype, consistent with partially overlapping functions for these two orthologs. These data provide in vivo evidence for a critical function of Pak2 in vascular integrity and demonstrate a severe disease phenotype resulting from loss of Pak2 function