Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

The transcription factor ISL1 is thought to be key for conveying the multipotent and proliferative properties of cardiac precursor cells. Here, we investigate its function upon cardiac induction of human embryonic stem cells. We find that ISL1 does not stabilize the transient cardiac precursor cell state but rather serves to accelerate cardiomyocyte differentiation. Conversely, ISL1 depletion delays cardiac differentiation and respecifies nascent cardiomyocytes from a ventricular to an atrial identity. Mechanistic analyses integrate this unrecognized anti-atrial function of ISL1 with known and newly identified atrial inducers. In this revised view, ISL1 is antagonized by retinoic acid signaling via a novel player, MEIS2. Conversely, ISL1 competes with the retinoic acid pathway for prospective cardiomyocyte fate, which converges on the atrial specifier NR2F1. This study reveals a core regulatory network putatively controlling human heart chamber formation and also bears implications for the subtype-specific production of human cardiomyocytes with enhanced functional properties.

Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.

While gene regulatory networks involved in cardiogenesis have been characterized, the role of bioenergetics remains less studied. Here we show that until midgestation, myocardial metabolism is compartmentalized, with a glycolytic signature restricted to compact myocardium contrasting with increased mitochondrial oxidative activity in the trabeculae. HIF1α regulation mirrors this pattern, with expression predominating in compact myocardium and scarce in trabeculae. By midgestation, the compact myocardium downregulates HIF1α and switches toward oxidative metabolism. Deletion of the E3 ubiquitin ligase Vhl results in HIF1α hyperactivation, blocking the midgestational metabolic shift and impairing cardiac maturation and function. Moreover, the altered glycolytic signature induced by HIF1 trabecular activation precludes regulation of genes essential for establishment of the cardiac conduction system. Our findings reveal VHL-HIF-mediated metabolic compartmentalization in the developing heart and the connection between metabolism and myocardial differentiation. These results highlight the importance of bioenergetics in ventricular myocardium specialization and its potential relevance to congenital heart disease.

Heart cells are the unitary elements that define cardiac function and disease. The recent identification of distinct families of cardiovascular progenitor cells begins to build a foundation for our understanding of the developmental logic of human cardiovascular disease, and also points to new approaches to arrest and/or reverse its progression, a major goal of regenerative medicine. In this review, we highlight recent clarifications, revisions, and advances in our understanding of the many lives of a heart cell, with a primary focus on the emerging links between cardiogenesis and heart stem cell biology.

Most of the genes induced by hypoxia are regulated by a family of transcription factors termed hypoxia-inducible factors (HIF). Under normoxic conditions, HIF␣proteins are very unstable due to hydroxylation by a recently described family of proline hydroxylases termed EGL-Nine homologs (EGLN). Upon hydroxylation, HIF␣ is recognized by the product of the tumor suppressor vhl and targeted for proteosomal degradation. Since EGLNs require oxygen to catalize HIF hydroxylation, this reaction does not efficiently occur under low oxygen tension. Thus, under hypoxia, HIF␣ escapes from degradation and transcribes target genes. The mRNA levels of two of the three EGLNs described to date are induced by hypoxia, suggesting that they might be novel HIF target genes; however, no proof for this hypothesis has been reported. Here we show that the induction of EGLN1 and -3 by hypoxia is found in a wide range of cell types. The basal levels of EGLN3 are always well below those of EGLN1 and EGLN2, and its induction by hypoxia is larger than that found for EGLN1. The inhibitor of transcription, actinomycin D, prevents the increase of EGLN3 mRNA induced by hypoxia, indicating that it is due to enhanced gene expression. Interestingly, EGLN1 and EGLN3 mRNAs were also triggered by EGLN inhibitors, suggesting the involvement of HIF␣ in the control of its transcription. In agreement with this possibility, pVHL-deficient cell lines, which present high HIF activity under normoxia, also showed dramatically increased normoxic levels of EGLN3. Moreover, the overexpression of an oxygen-insensitive mutant form of HIF␣ resulted in increased normoxic levels of EGLN3 mRNA. Finally, hypoxic induction of EGLNs was not observed in cells lacking functional HIF␣.Mammalian cells are critically dependent on oxygen for survival; thus, it is not surprising that an evolutionarily conserved machinery able to sense oxygen levels is present in almost every cell type studied. The activity of a variety of signaling molecules has been found to be altered in response to changes in oxygen tension, including ion channels (1), kinases (2-7), and transcription factors (2, 8 -13). Among them, the most common and best understood response is the activation of a group of basic helix-loop-helix-Per/Aryl hydrocarbon receptor translocator/Sim domain transcription factors, called hypoxiainducible factors (HIF) 1 (14). In fact, HIF is responsible for the induction of the vast majority of genes up-regulated in response to decreased oxygen tension. HIF transcription factors are heterodimers of a constitutively expressed ␤ subunit (HIF-␤; also known as the aryl hydrocarbon receptor translocator) and an oxygen-regulated ␣ subunit (HIF-␣). Although there are three types of ␣ subunits, HIF-1␣, HIF-2␣ (also known as the endothelial Per/Aryl hydrocarbon receptor translocator/Sim domain), and HIF-3␣, encoded by different genes, it is currently accepted that they are regulated in a similar fashion by oxygen availability. The expression of HIF␤ and ␣ subunits genes, their mRNA stability, ...

Low oxygen tension areas are found in inflamed or diseased tissues where hypoxic cells induce survival pathways by regulating the hypoxia-inducible transcription factor (HIF). Macrophages are essential regulators of inflammation and, therefore, we have analyzed their response to hypoxia. Murine peritoneal elicited macrophages cultured under hypoxia produced higher levels of IFN-␥ and IL-12 mRNA and protein than those cultured under normoxia. A similar IFN-␥ increment was obtained with in vivo models using macrophages from mice exposed to atmospheric hypoxia. Our studies showed that IFN-␥ induction was mediated through HIF-1␣ binding to its promoter on a new functional hypoxia response element. The requirement of HIF-␣ in the IFN-␥ induction was confirmed in RAW264.7 cells, where HIF-1␣ was knocked down, as well as in resident HIF-1␣ null macrophages. Moreover, Ag presentation capacity was enhanced in hypoxia through the up-regulation of costimulatory and Ag-presenting receptor expression. Hypoxic macrophages generated productive immune synapses with CD8 T cells that were more efficient for activation of TCR/CD3, CD3 and linker for activation of T cell phosphorylation, and T cell cytokine production. In addition, hypoxic macrophages bound opsonized particles with a higher efficiency, increasing their phagocytic uptake, through the upregulated expression of phagocytic receptors. These hypoxia-increased immune responses were markedly reduced in HIF-1␣-and in IFN-␥-silenced macrophages, indicating a link between HIF-1␣ and IFN-␥ in the functional responses of macrophages to hypoxia. Our data underscore an important role of hypoxia in the activation of macrophage cytokine production, Ag-presenting activity, and phagocytic activity due to an HIF-1␣-mediated increase in IFN-␥ levels.

One drawback of chemotherapy is poor drug delivery to tumor cells, due in part to hyperpermeability of the tumor vasculature. Extracellular superoxide dismutase (SOD3) is an antioxidant enzyme usually repressed in the tumor milieu. Here we show that specific SOD3 re-expression in tumor-associated endothelial cells (ECs) increases doxorubicin (Doxo) delivery into and chemotherapeutic effect on tumors. Enhanced SOD3 activity fostered perivascular nitric oxide accumulation and reduced vessel leakage by inducing vascular endothelial cadherin (VEC) transcription. SOD3 reduced HIF prolyl hydroxylase domain protein activity, which increased hypoxia-inducible factor-2α (HIF-2α) stability and enhanced its binding to a specific VEC promoter region. EC-specific HIF-2α ablation prevented both the SOD3-mediated increase in VEC transcription and the enhanced Doxo effect. SOD3, VEC, and HIF-2α levels correlated positively in primary colorectal cancers, which suggests a similar interconnection of these proteins in human malignancy.