Fibroblast growth factor 10.

Fibroblast growth factor 10 (FGF10) is a major morphoregulatory factor that plays essential signaling roles during vertebrate multiorgan development and homeostasis. FGF10 is predominantly expressed in mesenchymal cells and signals though FGFR2b in adjacent epithelia to regulate branching morphogenesis, stem cell fate, tissue differentiation and proliferation, in addition to autocrine roles. Genetic loss of function analyses have revealed critical requirements for FGF10 signaling during limb, lung, digestive system, ectodermal, nervous system, craniofacial and cardiac development. Heterozygous FGF10 mutations have been identified in human genetic syndromes associated with craniofacial anomalies, including lacrimal and salivary gland aplasia. Elevated Fgf10 expression is associated with poor prognosis in a range of cancers. In addition to developmental and disease roles, FGF10 regulates homeostasis and repair of diverse adult tissues and has been identified as a target for regenerative medicine.

A transposable element into the human long noncoding RNA CARMEN is a switch for cardiac precursor cell specification.

AIMS: The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The human lncRNA CARMEN, which is known to dictate specification toward the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced isoforms. METHODS AND RESULTS: The CARMEN locus can be manipulated to direct human primary cardiac precursor cells (CPCs) into specific cardiovascular fates. Investigating CARMEN isoform usage in differentiating CPCs represents therefore a unique opportunity to uncover isoform-specific functions in lncRNAs. Here, we identify one CARMEN isoform, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced. CONCLUSIONS: These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA. They further demonstrated the crucial importance of the lncRNA isoform CARMEN-201 in SMC specification during heart development.

PPARγ and NOTCH Regulate Regional Identity in the Murine Cardiac Outflow Tract.

BACKGROUND: The arterial pole of the heart is a hotspot for life-threatening forms of congenital heart defects (CHDs). Development of this cardiac region occurs by addition of Second Heart Field (SHF) progenitor cells to the embryonic outflow tract (OFT) and subsequently the base of the ascending aorta and pulmonary trunk. Understanding the cellular and genetic mechanisms driving arterial pole morphogenesis is essential to provide further insights into the cause of CHDs. METHODS: A synergistic combination of bioinformatic analysis and mouse genetics as well as embryo and explant culture experiments were used to dissect the cross-regulatory transcriptional circuitry operating in future subaortic and subpulmonary OFT myocardium. RESULTS: Here, we show that the lipid sensor PPARγ (peroxisome proliferator-activated receptor gamma) is expressed in future subpulmonary myocardium in the inferior wall of the OFT and that PPARγ signaling-related genes display regionalized OFT expression regulated by the transcription factor TBX1 (T-box transcription factor 1). Modulating PPARγ activity in ex vivo cultured embryos treated with a PPARγ agonist or antagonist or deleting Pparγ in cardiac progenitor cells using Mesp1-Cre reveals that Pparγ is required for addition of future subpulmonary myocardium and normal arterial pole development. Additionally, the non-canonical DLK1 (delta-like noncanonical Notch ligand 1)/NOTCH (Notch receptor 1)/HES1 (Hes family bHLH transcription factor 1) pathway negatively regulates Pparγ in future subaortic myocardium in the superior OFT wall. CONCLUSIONS: Together these results identify Pparγ as a regulator of regional transcriptional identity in the developing heart, providing new insights into gene interactions involved in congenital heart defects.

FGF10 promotes cardiac repair through a dual cellular mechanism increasing cardiomyocyte renewal and inhibiting fibrosis.

AIMS: Promoting cardiomyocyte renewal represents a major therapeutic approach for heart regeneration and repair. Our study aims to investigate the relevance of FGF10 as a potential target for heart regeneration. METHODS AND RESULTS: Our results first reveal that Fgf10 levels are up-regulated in the injured ventricle after MI. Adult mice with reduced Fgf10 expression subjected to MI display impaired cardiomyocyte proliferation and enhanced cardiac fibrosis, leading to a worsened cardiac function and remodelling post-MI. In contrast, conditional Fgf10 overexpression post-MI revealed that, by enhancing cardiomyocyte proliferation and preventing scar-promoting myofibroblast activation, FGF10 preserves cardiac remodelling and function. Moreover, FGF10 activates major regenerative pathways including the regulation of Meis1 expression levels, the Hippo signalling pathway and a pro-glycolytic metabolic switch. Finally, we demonstrate that elevated FGF10 levels in failing human hearts correlate with reduced fibrosis and enhanced cardiomyocyte proliferation. CONCLUSIONS: Altogether, our study shows that FGF10 promotes cardiac regeneration and repair through two cellular mechanisms: elevating cardiomyocyte renewal and limiting fibrosis. This study thus identifies FGF10 as a clinically relevant target for heart regeneration and repair in man.

Comparison of a New 68Ga-Radiolabelled PET Imaging Agent sCD146 and RGD Peptide for In Vivo Evaluation of Angiogenesis in Mouse Model of Myocardial Infarction.

Ischemic vascular diseases are associated with elevated tissue expression of angiomotin (AMOT), a promising molecular target for PET imaging. On that basis, we developed an AMOT-targeting radiotracer, 68Ga-sCD146 and performed the first in vivo evaluation on a myocardial infarction mice model and then, compared AMOT expression and αvß3-integrin expression with 68Ga-sCD146 and 68Ga-RGD2 imaging. After myocardial infarction (MI) induced by permanent ligation of the left anterior descending coronary artery, myocardial perfusion was evaluated by Doppler ultrasound and by 18F-FDG PET imaging. 68Ga-sCD146 and 68Ga-RGD2 PET imaging were performed. In myocardial infarction model, heart-to-muscle ratio of 68Ga-sCD146 imaging showed a significantly higher radiotracer uptake in the infarcted area of MI animals than in sham (* p = 0.04). Interestingly, we also observed significant correlations between 68Ga-sCD146 imaging and delayed residual perfusion assessed by 18F-FDG (* p = 0.04), with lowest tissue fibrosis assessed by histological staining (* p = 0.04) and with functional recovery assessed by ultrasound imaging (** p = 0.01). 68Ga-sCD146 demonstrated an increase in AMOT expression after MI. Altogether, significant correlations of early post-ischemic 68Ga-sCD146 uptake with late heart perfusion, lower tissue fibrosis and better functional recovery, make 68Ga-sCD146 a promising radiotracer for tissue angiogenesis assessment after MI.

Cardiomyocyte proliferation, a target for cardiac regeneration.

Cardiac diseases, characterized by cardiomyocyte loss, lead to dramatic impairment of cardiac function and ultimately to congestive heart failure. Despite significant advances, conventional treatments do not correct the defects in cardiac muscle cell numbers and the prognosis of congestive heart failure remains poor. The existence, in adult mammalian heart, of low but detectable cardiomyocyte proliferative capacities has shifted the target of regenerative therapy toward new therapeutical strategy. Indeed, the stimulation of terminally differentiated cardiomyocyte proliferation represents the main therapeutic approach for heart regeneration. Increasing evidence demonstrating that the loss of mammalian cardiomyocyte renewal potential shortly after birth causes the loss of regenerative capacities, strongly support the hypothesis that a detailed understanding of the molecular mechanisms controlling fetal and postnatal cardiomyocyte proliferation is essential to identify targets for cardiac regeneration. Here, we will review major developmental mechanisms regulating fetal cardiomyocyte proliferation and will describe the impact of the developmental switch, operating at birth and driving postnatal heart maturation, on the regulation of adult cardiomyocyte proliferation, all these mechanisms representing potential targets for cardiac repair and regeneration.

FGF10 Signaling in Heart Development, Homeostasis, Disease and Repair.

Essential muscular organ that provides the whole body with oxygen and nutrients, the heart is the first organ to function during embryonic development. Cardiovascular diseases, including acquired and congenital heart defects, are the leading cause of mortality in industrialized countries. Fibroblast Growth Factors (FGFs) are involved in a variety of cellular responses including proliferation, differentiation, and migration. Among the 22 human/mouse FGFs, the secreted FGF10 ligand through the binding of its specific receptors (FGFR1b and FGFR2b) and subsequent activation of downstream signaling is known to play essential role in cardiac development, homeostasis and disease. FGF10 is one of the major marker of the early cardiac progenitor cells and a crucial regulator of differentiated cardiomyocyte proliferation in the developing embryo. Increasing evidence support the hypothesis that a detailed understanding of developmental processes is essential to identify targets for cardiac repair and regeneration. Indeed the activation of resident cardiomyocyte proliferation together with the injection of cardiac progenitors represent the most promising therapeutical strategies for cardiac regenerative medicine. The recent findings showing that FGF10 promotes adult cardiomyocyte cell cycle reentry and directs stem cell differentiation and cell reprogramming toward the cardiogenic lineage provide new insights into therapeutical strategies for cardiac regeneration and repair.

Interhelical interaction and receptor phosphorylation regulate the activation kinetics of different human ß1-adrenoceptor variants.

G protein-coupled receptors represent the largest class of drug targets, but genetic variation within G protein-coupled receptors leads to variable drug responses and, thereby, compromises their therapeutic application. One of the most intensely studied examples is a hyperfunctional variant of the human ß1-adrenoceptor that carries an arginine at position 389 in helix 8 (Arg-389-ADRB1). However, the mechanism underlying the higher efficacy of the Arg-389 variant remained unclear to date. Despite its hyperfunctionality, we found the Arg-389 variant of ADRB1 to be hyperphosphorylated upon continuous stimulation with norepinephrine compared with the Gly-389 variant. Using ADRB1 sensors to monitor activation kinetics by fluorescence resonance energy transfer, Arg-389-ADRB1 exerted faster activation speed and arrestin recruitment than the Gly-389 variant. Both activation speed and arrestin recruitment depended on phosphorylation of the receptor, as shown by knockdown of G protein-coupled receptor kinases and phosphorylation-deficient ADRB1 mutants. Structural modeling of the human ß1-adrenoceptor suggested interaction of the side chain of Arg-389 with opposing amino acid residues in helix 1. Site-directed mutagenesis of Lys-85 and Thr-86 in helix 1 revealed that this interaction indeed determined ADRB1 activation kinetics. Taken together, these findings indicate that differences in interhelical interaction regulate the different activation speed and efficacy of ADRB1 variants.

FGF10 promotes regional foetal cardiomyocyte proliferation and adult cardiomyocyte cell-cycle re-entry.

AIMS: Cardiomyocyte proliferation gradually declines during embryogenesis resulting in severely limited regenerative capacities in the adult heart. Understanding the developmental processes controlling cardiomyocyte proliferation may thus identify new therapeutic targets to modulate the cell-cycle activity of cardiomyocytes in the adult heart. This study aims to determine the mechanism by which fibroblast growth factor 10 (FGF10) controls foetal cardiomyocyte proliferation and to test the hypothesis that FGF10 promotes the proliferative capacity of adult cardiomyocytes. METHODS AND RESULTS: Analysis of Fgf10(-/-) hearts and primary cardiomyocyte cultures reveals that altered ventricular morphology is associated with impaired proliferation of right but not left-ventricular myocytes. Decreased FOXO3 phosphorylation associated with up-regulated p27(kip) (1) levels was observed specifically in the right ventricle of Fgf10(-/-) hearts. In addition, cell-type-specific expression analysis revealed that Fgf10 and its receptor, Fgfr2b, are expressed in cardiomyocytes and not cardiac fibroblasts, consistent with a cell-type autonomous role of FGF10 in regulating regional specific myocyte proliferation in the foetal heart. Furthermore, we demonstrate that in vivo overexpression of Fgf10 in adult mice promotes cardiomyocyte but not cardiac fibroblast cell-cycle re-entry. CONCLUSION: FGF10 regulates regional cardiomyocyte proliferation in the foetal heart through a FOXO3/p27(kip1) pathway. In addition, FGF10 triggers cell-cycle re-entry of adult cardiomyocytes and is thus a potential target for cardiac repair.

A polymorphism-specific "memory" mechanism in the ß(2)-adrenergic receptor.

Signaling through G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptors is affected by polymorphisms in receptor-encoding genes. Using fluorescence resonance energy transfer, we found that the ß(2)-adrenergic receptor (ß(2)AR) responded to repeated activation with altered activation kinetics. Polymorphic variants of the ß(2)AR displayed divergent changes of ß(2)AR activation kinetics that closely mimicked their different efficacies to generate cyclic adenosine 3',5'-monophosphate. More efficacious variants became faster in their activation kinetics, whereas less efficacious variants became slower, compared to their initial activation. These differences depended on phosphorylation of the receptor by G protein-coupled receptor kinases. Our findings suggest an intrinsic, polymorphism-specific property of the ß(2)AR that alters activation kinetics upon continued stimulation and that may account for individual drug responses.

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome.

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of beta-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1(lacZ) (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1(-)(/)(-) mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures.

Hes1 is expressed in the second heart field and is required for outflow tract development.

BACKGROUND: Rapid growth of the embryonic heart occurs by addition of progenitor cells of the second heart field to the poles of the elongating heart tube. Failure or perturbation of this process leads to congenital heart defects. In order to provide further insight into second heart field development we characterized the insertion site of a transgene expressed in the second heart field and outflow tract as the result of an integration site position effect. RESULTS: Here we show that the integration site of the A17-Myf5-nlacZ-T55 transgene lies upstream of Hes1, encoding a basic helix-loop-helix containing transcriptional repressor required for the maintenance of diverse progenitor cell populations during embryonic development. Transgene expression in a subset of Hes1 expression sites, including the CNS, pharyngeal epithelia, pericardium, limb bud and lung endoderm suggests that Hes1 is the endogenous target of regulatory elements trapped by the transgene. Hes1 is expressed in pharyngeal endoderm and mesoderm including the second heart field. Analysis of Hes1 mutant hearts at embryonic day 15.5 reveals outflow tract alignment defects including ventricular septal defects and overriding aorta. At earlier developmental stages, Hes1 mutant embryos display defects in second heart field proliferation, a reduction in cardiac neural crest cells and failure to completely extend the outflow tract. CONCLUSIONS: Hes1 is expressed in cardiac progenitor cells in the early embryo and is required for development of the arterial pole of the heart.

Signaling pathways controlling second heart field development.

Insight into the mechanisms underlying congenital heart defects and the use of stem cells for cardiac repair are major research goals in cardiovascular biology. In the early embryo, progenitor cells in pharyngeal mesoderm contribute to the rapid growth of the heart tube during looping morphogenesis. These progenitor cells constitute the second heart field (SHF) and were first identified in 2001. Direct or indirect perturbation of SHF addition to the heart results in congenital heart defects, including arterial pole alignment defects. Over the last 3 years, a number of studies have identified key intercellular signaling pathways that control the proliferation and deployment of SHF progenitor cells. Here, we review data concerning Wnt, fibroblast growth factor, bone morphogenetic protein, Hedgehog, and retinoic acid signaling that have begun to identify the ligand sources and responding cell types controlling SHF development. These studies have revealed the importance of signals from pharyngeal mesoderm itself, as well as critical inputs from adjacent pharyngeal epithelia and neural crest cells. Proliferation is emerging as a central checkpoint in the regulation of SHF development. Together, these studies contribute to defining the niche of cardiac progenitor cells in the early embryo, and we discuss the implications of these findings for the regulation of resident stem cell populations in the fetal and postnatal heart. Characterization of signals that maintain, expand, and regulate the differentiation of cardiac progenitor cells is essential for understanding both the etiology of congenital heart defects and the biomedical application of stem cell populations for cardiac repair.

Real-time optical recording of beta1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol.

Antagonists of beta-adrenergic receptors (beta-ARs) have become a main therapeutic regimen for the treatment of heart failure even though the mechanisms of their beneficial effects are still poorly understood. Here, we used fluorescent resonance energy transfer-based (FRET-based) approaches to directly monitor activation of the beta(1)-AR and downstream signaling. While the commonly used beta-AR antagonists metoprolol, bisoprolol, and carvedilol displayed varying degrees of inverse agonism on the Gly389 variant of the receptor (i.e., actively switching off the beta(1)-AR), surprisingly, only carvedilol showed very specific and marked inverse agonist effects on the more frequent Arg389 variant. These specific effects of carvedilol on the Arg389 variant of the beta(1)-AR were also seen for control of beating frequency in rat cardiac myocytes expressing the 2 receptor variants. This FRET sensor permitted direct observation of activation of the beta(1)-AR in living cells in real time. It revealed that beta(1)-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use.

Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases.

A current challenge in cellular signaling is to decipher the complex intracellular spatiotemporal organization that any given cell type has developed to discriminate among different external stimuli acting via a common signaling pathway. This obviously applies to cAMP and cGMP signaling in the heart, where these cyclic nucleotides determine the regulation of cardiac function by many hormones and neuromediators. Recent studies have identified cyclic nucleotide phosphodiesterases as key actors in limiting the spread of cAMP and cGMP, and in shaping and organizing intracellular signaling microdomains. With this new role, phosphodiesterases have been promoted from the rank of a housekeeping attendant to that of an executive officer.

Gs activation is time-limiting in initiating receptor-mediated signaling.

To analyze individual steps of G(S)-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor G(S) activation in living cells. This was done by coexpressing a Galpha(s) mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gbetagamma subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the G(S) signaling cascade. When A(2A)-adenosine or beta(1)-adrenergic receptors are coexpressed with G(S) in HEK293T cells, the receptor-G(S) interaction was on the same time scale as A(2A) receptor activation with a time constant of <50 ms. G(S) activation was markedly slower and around 450 ms with similar kinetics following activation of A(2A)- or beta(1)-receptors. Taken together, our kinetic measurements demonstrate that the rate of G(S) activation limits initiation of G(S)-coupled receptor signaling.

A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes.

Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-AR regulation of I(Ca,L), whereas only PDE4 controlled Glu-R regulation of I(Ca,L) thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.

cAMP-binding protein Epac induces cardiomyocyte hypertrophy.

cAMP is one of the most important second messenger in the heart. The discovery of Epac as a guanine exchange factor (GEF), which is directly activated by cAMP, raises the question of the role of this protein in cardiac cells. Here we show that Epac activation leads to morphological changes and induces expression of cardiac hypertrophic markers. This process is associated with a Ca2+-dependent activation of the small GTPase, Rac. In addition, we found that Epac activates a prohypertrophic signaling pathway, which involves the Ca2+ sensitive phosphatase, calcineurin, and its primary downstream effector, NFAT. Rac is involved in Epac-induced NFAT dependent cardiomyocyte hypertrophy. Blockade of either calcineurin or Rac activity blunts the hypertrophic response elicited by Epac indicating these signaling molecules coordinately regulate cardiac gene expression and cellular growth. Our results thus open new insights into the signaling pathways by which cAMP may mediate its biological effects and identify Epac as a new positive regulator of cardiac growth.