Final Report Summary - HYPOXIA & CELL FATE (Oxygen sensing in cardiovascular progenitors cell fate decisions)
Cardiogenesis is a complex process that requires the maturation of different cardiovascular progenitors to compose the final functional compartments of the heart. Cardiac development takes place under low oxygen conditions but the direct function of hypoxia during cardiogenesis is mostly unknown. The main goal of this project is to characterize the role of embryonic hypoxia in cardiovascular development and homeostasis, specially at the level of early mesodermal and epicardial progenitors.
By means of embryonic stem cells and characterization of several mouse models of loss (LOF) and gain of function (GOF) we have determined that low oxygen tensions repress second heart field (SHF) mesodermal progenitors proliferation and at the time promotes differentiation towards cardiac muscle lineages co-expressing Nkx2.5 marker. Furthermore, we have defined for the first time a detailed oxygen map during cardiac development finding that at early stages (E9.5) the whole primitive cardiac tube is hypoxic while from E12.5 to E14.5 low oxygen regions reside in the epicardium and endocardium with the intermediate myocardium remaining well oxygenated. HIF1 alpha expression within this time window follows a completely different pattern, suggesting that HIF1 alpha subunit stabilization during cardiogenesis occurs in an oxygen-independent manner. Indeed HIF1 alpha is spatial and temporaly regulated in a way that higher HIF1 alpha protein levels could be detected in the compct myocardium layer compared to the trabecules, composed by more mature cardiomyocytes in terms of structure and contractility. GOF and LOF models in Nkx2.5 mesodermal progenitors show different defects in chamber formation, beeing the deletion of VHL embryonic lethal in this pool of precursors. Transcriptional analysys from E12.5 hearts of GOF/LOF models suggests that HIF1 regulates bioenergetics of embryonic cardiomyocytes mediating a metabolic compartmentalization that programs glycolytic metabolism in the high HIF1 alpha -expressing compact myocardium and allows oxidative metabolism in the trabecules with lower HIF1 alpha levels. This hypothesis is further supported by the observation of differential mithocondrial netwrok and functional activity between both myocardial layer and the loss of this heterogeneous pattern in GOF/LOF models.
In addition we have determine the role of hypoxia and HIF/VHL pathway in the biology of epicardial progenitors. We have generated a GOF model by deleting vhl in the Wt1 lineage that develops cardiac hypertrophy and present difused fibrosis, pericardial hemorrhages, miocarditis and severe dilatation of the coronaries. These mice show high lethality in the postnatal period and suddenly die pressumably by coronary rupture. Furthermore, recent studies indicate that these mice present systemic inflammation that could probably contribute to exacerbate the coronary vasculitis developed by these mice. In parallel we have generated a LOF model that demonstrate that the lack of HIF2 during heart formation is deleterious and induces myocardial dilatation and ventricular disfunction. These results show that a proper balance in HIF/VHL pathway is required for correct coronary development and cardiovascular homeostasis.
In summary our data demonstrate that hypoxia and HIF/VHL axis participates in esential processes during mammalian cardiogenesis like ventricular chamber formation or coronary development. We are currently characterizing the molecular mechanisms underlying the alterations observed in the different phenotypes described above. We consider that the mouse model generated could have biomedical interest as they might recapitulates congenital heart disease and defects not fully understood, thus providing a perfect experimental scenario to understand the molecular origin of human pathologies and offering the opportunity to test novel diagnostic/therapeutic opportunities.
By means of embryonic stem cells and characterization of several mouse models of loss (LOF) and gain of function (GOF) we have determined that low oxygen tensions repress second heart field (SHF) mesodermal progenitors proliferation and at the time promotes differentiation towards cardiac muscle lineages co-expressing Nkx2.5 marker. Furthermore, we have defined for the first time a detailed oxygen map during cardiac development finding that at early stages (E9.5) the whole primitive cardiac tube is hypoxic while from E12.5 to E14.5 low oxygen regions reside in the epicardium and endocardium with the intermediate myocardium remaining well oxygenated. HIF1 alpha expression within this time window follows a completely different pattern, suggesting that HIF1 alpha subunit stabilization during cardiogenesis occurs in an oxygen-independent manner. Indeed HIF1 alpha is spatial and temporaly regulated in a way that higher HIF1 alpha protein levels could be detected in the compct myocardium layer compared to the trabecules, composed by more mature cardiomyocytes in terms of structure and contractility. GOF and LOF models in Nkx2.5 mesodermal progenitors show different defects in chamber formation, beeing the deletion of VHL embryonic lethal in this pool of precursors. Transcriptional analysys from E12.5 hearts of GOF/LOF models suggests that HIF1 regulates bioenergetics of embryonic cardiomyocytes mediating a metabolic compartmentalization that programs glycolytic metabolism in the high HIF1 alpha -expressing compact myocardium and allows oxidative metabolism in the trabecules with lower HIF1 alpha levels. This hypothesis is further supported by the observation of differential mithocondrial netwrok and functional activity between both myocardial layer and the loss of this heterogeneous pattern in GOF/LOF models.
In addition we have determine the role of hypoxia and HIF/VHL pathway in the biology of epicardial progenitors. We have generated a GOF model by deleting vhl in the Wt1 lineage that develops cardiac hypertrophy and present difused fibrosis, pericardial hemorrhages, miocarditis and severe dilatation of the coronaries. These mice show high lethality in the postnatal period and suddenly die pressumably by coronary rupture. Furthermore, recent studies indicate that these mice present systemic inflammation that could probably contribute to exacerbate the coronary vasculitis developed by these mice. In parallel we have generated a LOF model that demonstrate that the lack of HIF2 during heart formation is deleterious and induces myocardial dilatation and ventricular disfunction. These results show that a proper balance in HIF/VHL pathway is required for correct coronary development and cardiovascular homeostasis.
In summary our data demonstrate that hypoxia and HIF/VHL axis participates in esential processes during mammalian cardiogenesis like ventricular chamber formation or coronary development. We are currently characterizing the molecular mechanisms underlying the alterations observed in the different phenotypes described above. We consider that the mouse model generated could have biomedical interest as they might recapitulates congenital heart disease and defects not fully understood, thus providing a perfect experimental scenario to understand the molecular origin of human pathologies and offering the opportunity to test novel diagnostic/therapeutic opportunities.