Cardiac Progenitor Gene Research Articles

The Tbx20-TLE interaction is essential for the maintenance of the second heart field.

T-box transcription factor 20 (Tbx20) plays a multifaceted role in cardiac morphogenesis and controls a broad gene regulatory network. However, the mechanism by which Tbx20 activates and represses target genes in a tissue-specific and temporal manner remains unclear. Studies show that Tbx20 directly interacts with the Transducin-like Enhancer of Split (TLE) family of proteins to mediate transcriptional repression. However, a function for the Tbx20-TLE transcriptional repression complex during heart development has yet to be established. We created a mouse model with a two amino acid substitution in the Tbx20 EH1 domain, thereby disrupting the Tbx20-TLE interaction. Disruption of this interaction impaired crucial morphogenic events, including cardiac looping and chamber formation. Transcriptional profiling of Tbx20EH1Mut hearts and analysis of putative direct targets revealed misexpression of the retinoic acid pathway and cardiac progenitor genes. Further, we show that altered cardiac progenitor development and function contribute to the severe cardiac defects in our model. Our studies indicate that TLE-mediated repression is a primary mechanism by which Tbx20 controls gene expression.

Mutations in genes related to myocyte contraction and ventricular septum development in non-syndromic tetralogy of Fallot.

Eighty percent of patients with a diagnosis of tetralogy of Fallot (TOF) do not have a known genetic etiology or syndrome. We sought to identify key molecular pathways and biological processes that are enriched in non-syndromic TOF, the most common form of cyanotic congenital heart disease, rather than single driver genes to elucidate the pathogenesis of this disease. We undertook exome sequencing of 362 probands with non-syndromic TOF and their parents within the Pediatric Cardiac Genomics Consortium (PCGC). We identified rare (minor allele frequency <1 × 10-4), de novo variants to ascertain pathways and processes affected in this population to better understand TOF pathogenesis. Pathways and biological processes enriched in the PCGC TOF cohort were compared to 317 controls without heart defects (and their parents) from the Simons Foundation Autism Research Initiative (SFARI). A total of 120 variants in 117 genes were identified as most likely to be deleterious, with CHD7, CLUH, UNC13C, and WASHC5 identified in two probands each. Gene ontology analyses of these variants using multiple bioinformatic tools demonstrated significant enrichment in processes including cell cycle progression, chromatin remodeling, myocyte contraction and calcium transport, and development of the ventricular septum and ventricle. There was also a significant enrichment of target genes of SOX9, which is critical in second heart field development and whose loss results in membranous ventricular septal defects related to disruption of the proximal outlet septum. None of these processes was significantly enriched in the SFARI control cohort. Innate molecular defects in cardiac progenitor cells and genes related to their viability and contractile function appear central to non-syndromic TOF pathogenesis. Future research utilizing our results is likely to have significant implications in stratification of TOF patients and delivery of personalized clinical care.

Maternal obesity persistently alters cardiac progenitor gene expression and programs adult-onset heart disease susceptibility

ObjectiveHeart disease risk can be programmed by intrauterine exposure to obesity. Dysregulating key transcription factors in cardiac progenitors can cause subsequent adult-onset heart disease. In this study, we investigated the transcriptional pathways that are altered in the embryonic heart and linked to heart disease risk in offspring exposed to obesity during pregnancy. MethodsFemale mice were fed an obesogenic diet and mated with males fed a control diet. Heart function and genome-wide gene expression were analyzed in adult offspring born to obese and lean mice at baseline and in response to stress. Cross-referencing with genes dysregulated genome-wide in cardiac progenitors from embryos of obese mice and human fetal hearts revealed the transcriptional events associated with adult-onset heart disease susceptibility. ResultsWe found that adult mice born to obese mothers develop mild heart dysfunction consistent with early stages of disease. Accordingly, hearts of these mice dysregulated genes controlling extracellular matrix remodeling, metabolism, and TGF-β signaling, known to control heart disease progression. These pathways were already dysregulated in cardiac progenitors in embryos of obese mice. Moreover, in response to cardiovascular stress, the heart of adults born to obese dams developed exacerbated myocardial remodeling and excessively activated regulators of cell-extracellular matrix interactions but failed to activate metabolic regulators. Expression of developmentally regulated genes was altered in cardiac progenitors of embryos of obese mice and human hearts of fetuses of obese donors. Accordingly, the levels of Nkx2-5, a key regulator of heart development, inversely correlated with maternal body weight in mice. Furthermore, Nkx2-5 target genes were dysregulated in cardiac progenitors and persistently in adult hearts born to obese mice and human hearts from pregnancies affected by obesity. ConclusionsObesity during pregnancy alters Nkx2-5-controlled transcription in differentiating cardiac progenitors and persistently in the adult heart, making the adult heart vulnerable to dysregulated stress responses.

In situ differentiation of human-induced pluripotent stem cells into functional cardiomyocytes on a coaxial PCL-gelatin nanofibrous scaffold.

Human-induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs) have been explored for cardiac regeneration and repair as well as for the development of in vitro 3D cardiac tissue models. Existing protocols for cardiac differentiation of hiPSCs utilize a 2D culture system. However, the efficiency of hiPSC differentiation to cardiomyocytes in 3D culture systems has not been extensively explored. In the present study, we investigated the efficiency of cardiac differentiation of hiPSCs to functional cardiomyocytes on 3D nanofibrous scaffolds. Coaxial polycaprolactone (PCL)-gelatin fibrous scaffolds were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. hiPSCs were cultured and differentiated into functional cardiomyocytes on the nanofibrous scaffold and compared with 2D cultures. To assess the relative efficiencies of both the systems, SEM, immunofluorescence staining and gene expression analyses were performed. Contractions of differentiated cardiomyocytes were observed in 2D cultures after 2weeks and in 3D cultures after 4weeks. SEM analysis showed no significant differences in the morphology of cells differentiated on 2D versus 3D cultures. However, gene expression data showed significantly increased expression of cardiac progenitor genes (ISL-1, SIRPA) in 3D cultures and cardiomyocytes markers (TNNT, MHC6) in 2D cultures. In contrast, immunofluorescence staining showed no substantial differences in the expression of NKX-2.5 and α-sarcomeric actinin. Furthermore, uniform migration and distribution of the in situ differentiated cardiomyocytes was observed in the 3D fibrous scaffold. Overall, our study demonstrates that coaxial PCL-gelatin nanofibrous scaffolds can be used as a 3D culture platform for efficient differentiation of hiPSCs to functional cardiomyocytes.

Induction of developmental toxicity and cardiotoxicity in zebrafish embryos/larvae by acetyl-11-keto-β-boswellic acid (AKBA) through oxidative stress

Acetyl-11-keto-β-boswellic acid (AKBA), a triterpenoid from Boswellia serrate, is regarded as an angiogenesis inhibitor. However, its toxicity is unknown. The aim of this study was to examine its developmental toxicity and cardiotoxicity. A developmental toxicity assay in zebrafish embryos/larvae from 4 to 96 hours post-fertilization (hpf) was performed and a cardiotoxicity assay was designed from 48 to 72 hpf. Markers of oxidative stress and related genes were selected to access the possible mechanisms. According to the results, AKBA induced pericardium edema, yolk-sac edema, abnormal melanin, spinal curvature, hatching inhibition and shortened body length. Further, increased SV-BA distance, reduced heart rate, increased pericardium area and decreased blood flow velocity were detected in AKBA treated groups. The inhibition of cardiac progenitor gene expression, such as Nkx2.5 and Gata4, may be related to cardiotoxicity. The activities of antioxidant enzymes were decreased and the content of MDA was increased. In addition, AKBA treatment decreased the expression levels of Mn-Sod, Cat, and Gpx. These results suggested that AKBA induced developmental toxicity and cardiotoxicity through oxidative stress. As far as we know, this is the first report on the toxicity of AKBA. It reminds us to pay attention to developmental toxicity and cardiotoxicity of AKBA.

Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential

Although resident cardiac stem cells have been reported, regeneration of functional cardiomyocytes (CMs) remains a challenge. The present study identifies an alternative progenitor source for CM regeneration without the need for genetic manipulation or invasive heart biopsy procedures. Unlike limb skeletal muscles, masseter muscles (MM) in the mouse head are developed from Nkx2–5 mesodermal progenitors. Adult masseter muscle satellite cells (MMSCs) display heterogeneity in developmental origin and cell phenotypes. The heterogeneous MMSCs that can be characterized by cell sorting based on stem cell antigen-1 (Sca1) show different lineage potential. While cardiogenic potential is preserved in Sca1+ MMSCs as shown by expression of cardiac progenitor genes (including Nkx2–5), skeletal myogenic capacity is maintained in Sca1- MMSCs with Pax7 expression. Sca1+ MMSC-derived beating cells express cardiac genes and exhibit CM-like morphology. Electrophysiological properties of MMSC-derived CMs are demonstrated by calcium transients and action potentials. These findings show that MMSCs could serve as a novel cell source for cardiomyocyte replacement.

Deletion of HP1\u03b3 in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth

BackgroundHeterochromatin, which is formed when tri-methyl lysine 9 of histone H3 (H3K9me3) is bound by heterochromatin 1 proteins (HP1s), plays an important role in differentiation and senescence by silencing cell cycle genes. Cardiac myocytes (CMs) accumulate heterochromatin during differentiation and demethylation of H3K9me3 inhibits cell cycle gene silencing and cell cycle exit in CMs; however, it is unclear if this process is mediated by HP1s. In this study, we created a conditional CM-specific HP1 gamma (HP1γ) knockout (KO) mouse model and tested whether HP1γ is required for cell cycle gene silencing and cardiac growth.ResultsHP1γ KO mice were generated by crossing HP1γ floxed mice (fl) with mice expressing Cre recombinase driven by the Nkx2.5 (cardiac progenitor gene) promoter (Cre). We confirmed that deletion of critical exons of HP1γ led to undetectable levels of HP1γ protein in HP1γ KO (Cre;fl/fl) CMs. Analysis of cardiac size and function by echo revealed no significant differences between HP1γ KO and control (WT, Cre, fl/fl) mice. No significant difference in expression of cell cycle genes or cardiac-specific genes was observed. Global transcriptome analysis demonstrated a very moderate effect of HP1γ deletion on global gene expression, with only 51 genes differentially expressed in HP1γ KO CMs. We found that HP1β protein, but not HP1α, was significantly upregulated and that subnuclear localization of HP1β to perinuclear heterochromatin was increased in HP1γ KO CMs. Although HP1γ KO had no effect on H3K9me3 levels, we found a significant reduction in another major heterochromatin mark, tri-methylated lysine 20 of histone H4 (H4K20me3).ConclusionsThese data indicate that HP1γ is dispensable for cell cycle exit and normal cardiac growth but has a significant role in maintaining H4K20me3 and regulating a limited number of genes in CMs.

Exposure to acrylamide induces cardiac developmental toxicity in zebrafish during cardiogenesis.

Acrylamide (AA), an environmental pollutant, has been linked to neurotoxicity, genotoxicity and carcinogenicity. AA is widely used to synthesize polymers for industrial applications, is widely found in Western-style carbohydrate-rich foods and cigarette smoke, and can also be detected in human umbilical cord blood and breast milk. This is the first study that demonstrated the cardiac developmental toxicity of AA in zebrafish embryos. Post-fertilization exposure to AA caused a clearly deficient cardiovascular system with a shrunken heart and abortive morphogenesis and function. Disordered expression of the cardiac genes, myl7, vmhc, myh6, bmp4, tbx2b and notch1b, as well as reduced number of myocardial cells and endocardial cells, indicated the collapsed development of ventricle and atrium and failed differentiation of atrioventricular canal (AVC). Although cell apoptosis was not affected, the capacity of cardiomyocyte proliferation was significantly reduced by AA exposure after fertilization. Further investigation showed that treatment with AA specifically reduced the expressions of nkx2.5, myl7 and vmhc in the anterior lateral plate mesoderm (ALPM) during the early cardiogenesis. In addition, AA exposure disturbed the restricted expressions of bmp4, tbx2b and notch1b during atrioventricular (AV) valve development and cardiac chambers maturation. Our results showed that AA-induced cardiotoxicity was related to decreased cardiac progenitor genes expression, reduced myocardium growth, abnormal cardiac chambers morphogenesis and disordered AVC differentiation. Our study demonstrates that AA exposure during a time point analogous to the first trimester in humans has a detrimental effect on early heart development in zebrafish. A high ingestion rate of AA-containing products may be an underlying risk factor for cardiogenesis in fetuses.

Abstract 441: Role of Nitric Oxide and S-nitrosylation in Cardiomyogenesis by iPSCs

Introduction: The mechanism by which signaling pathways, such as Wnt and BMP interact and modulate each other’s function is crucial to our understanding of cardiomyogenesis and cardiomyocyte proliferation. Nitric oxide (NO) is a signaling molecule that can trigger cardiac differentiation of stem cells, suggesting a cardiogenic function of NO synthase(s) (NOS). Hypothesis: NO modulates transcription factor function during pluripotency and differentiation toward a cardiac phenotype. Methods: Induced pluripotent stem cells (iPSCs) were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR -/- ), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into functional cardiomyocytes from embryoid bodies (EBs) via the hanging-drop method. Results: During differentiation into cardiomyocytes, GSNOR -/- iPSC-derived cardiomyocytes exhibited reduced expression of mesoderm induction-related ( Brachyury ), cardiac mesoderm (Kdr , Isl-1 ) and cardiac progenitor genes ( Nkx2.5 , GATA4 ). Axin-1, an inducer of apoptosis and negative regulator of the Wnt signaling pathway and MAPK pathways, specifically p38, were increased on EB-Day (D)4. In contrast, SMAD1/5/8, members of the BMP canonical signaling pathway, were reduced beginning on EB-D8. Increased p38 is associated with reduced GATA4 expression and differentiation of human ES cells into cardiomyocytes. Decreased SMAD1/5/8 is likely at least in part responsible for the reduced expression of Nkx2.5. Conclusions: Our findings support that the absence of GSNOR modulates Wnt/β-catenin and BMP signaling pathways during cardiogenesis, resulting in reduced expression of mesoderm, cardiac mesoderm and cardiac progenitor genes. These findings are expected to have important implications for regenerative medicine and can provide new targets for iPS cell-based therapy.

Finding Expandable Induced Cardiovascular Progenitor Cells.

Expandable Cardiovascular Progenitor Cells Reprogrammed From Fibroblasts Zhang et al Cell Stem Cell . 2016;18:368–381. Lineage Reprogramming of Fibroblasts Into Proliferative Induced Cardiac Progenitor Cells by Defined Factors Lalit et al Cell Stem Cell . 2016;18:354–367. Cardiovascular progenitor cells (CPCs) are a promising cell source for cardiac regenerative therapy because of their ability for self-renewal and differentiation into various cardiovascular cell types beneficial for myocardial repair: cardiomyocytes, smooth muscle cells, and endothelial cells. Previous evaluations of exogenously derived CPCs have focused mainly on their capacity for trilineage differentiation rather than self-renewal because of the lack of an effective protocol to maintain and expand CPCs long-term in culture. In a recent issue of Cell Stem Cell , 2 groups of investigators independently reported their success in isolating, maintaining, and expanding mouse CPCs in culture for >18 to 20 passages (1010- to 1015-fold expansion), with faithful preservation of progenitor phenotype and ability for trilineage-restricted differentiation in both cell culture and mouse models of myocardial infarction (MI). This commentary will discuss the unique findings of these 2 studies, highlight the strengths and weaknesses of each CPC derivation/expansion technique, and propose additional steps necessary to accelerate their clinical translation. The past 2 decades have witnessed some of the most dramatic developments in the field of cardiac regeneration. Central to numerous controversies is the debate on whether certain stem cell types can differentiate into functional cardiomyocytes at a clinically relevant efficiency after implantation for repair of MI. Bone marrow–derived hematopoietic stem cells1,2 and c-kit+ resident cardiac stem cells3 are 2 notable cell types at the center of contention. Although many adult stem cells can still confer modest therapeutic benefits via paracrine effects, the search for a bona fide progenitor cell type that can generate a significant …

Human umbilical cord blood-derived mononuclear cells improve murine ventricular function upon intramyocardial delivery in right ventricular chronic pressure overload

IntroductionStem cell therapy has emerged as potential therapeutic strategy for damaged heart muscles. Umbilical cord blood (UCB) cells are the most prevalent stem cell source available, yet have not been fully tested in cardiac regeneration. Herein, studies were performed to evaluate the cardiovascular safety and beneficial effect of mononuclear cells (MNCs) isolated from human umbilical cord blood upon intramyocardial delivery in a murine model of right ventricle (RV) heart failure due to pressure overload.MethodsUCB-derived MNCs were delivered into the myocardium of a diseased RV cardiac model. Pulmonary artery banding (PAB) was used to produce pressure overload in athymic nude mice that were then injected intramyocardially with UCB-MNCs (0.4 × 10^6 cells/heart). Cardiac functions were then monitored by telemetry, echocardiography, magnetic resonance imaging (MRI) and pathologic analysis of heart samples to determine the ability for cell-based repair.ResultsThe cardio-toxicity studies provided evidence that UCB cell transplantation has a safe therapeutic window between 0.4 to 0.8 million cells/heart without altering QT or ST-segments or the morphology of electrocardiograph waves. The PAB cohort demonstrated significant changes in RV chamber dilation and functional defects consistent with severe pressure overload. Using cardiac MRI analysis, UCB-MNC transplantation in the setting of PAB demonstrated an improvement in RV structure and function in this surgical mouse model. The RV volume load in PAB-only mice was 24.09 ± 3.9 compared to 11.05 ± 2.09 in the cell group (mm3, P-value <0.005). The analysis of pathogenic gene expression (BNP, ANP, Acta1, Myh7) in the cell-transplanted group showed a significant reversal with respect to the diseased PAB mice with a robust increase in cardiac progenitor gene expression such as GATA4, Kdr, Mef2c and Nkx2.5. Histological analysis indicated significant fibrosis in the RV in response to PAB that was reduced following UCB-MNC’s transplantation along with concomitant increased Ki-67 expression and CD31 positive vessels as a marker of angiogenesis within the myocardium.ConclusionsThese findings indicate that human UCB-derived MNCs promote an adaptive regenerative response in the right ventricle upon intramyocardial transplantation in the setting of chronic pressure overload heart failure.

Induced expression of Fndc5 significantly increased cardiomyocyte differentiation rate of mouse embryonic stem cells

Fibronectin type III domain-containing 5 protein (Fndc5) is an exercise hormone and its transcript profile in mouse showed high degree of expression in heart, skeletal muscle and brain. Our previous studies indicated a significant increase (approximately 10 fold) in mRNA level of Fndc5 when embryonic stem cells were differentiated into beating bodies. As a step closer to identify the involvement of Fndc5 in the process of cardiomyocyte differentiation, we generated a stably inducible transduced mouse embryonic stem cell (mESC) line that overexpressed Fndc5 following Doxycycline induction. Our results indicated that the overexpression of Fndc5 during spontaneous cardiac differentiation significantly increased not only at RNA levels for mesodermal markers but also at the transcriptional levels for cardiac progenitor and cardiac genes. These data suggest that Fndc5 may be involved in cardiomyocyte differentiation. Therefore, a new hope will be arisen for potential application of this myokine for regeneration of damaged cardiac tissues especially in cardiac failure.

Brief report: SRF-dependent MiR-210 silences the sonic hedgehog signaling during cardiopoesis

Serum response factor (SRF) wields potent gene silencing activity through its regulation over numerous microRNAs (miRs). Here, SRF directs embryonic stem cell (ESC) progenitor cell lineage specification in part by silencing genes through miR-210. Viral expression of miR-210 in murine ESCs-derived embryoid bodies (EBs) inhibited cell growth and inhibited the appearance of cardiac progenitor markers Nkx2.5 and Gata4 and terminal differentiated contractile proteins Mlc2v and βMHC. Knockdown of miR-210 expression via antisense RNA activated cardiac progenitor gene activity. miR-210 inhibitory activity was attributed to silencing of the Sonic hedgehog (Shh) signaling pathway, which fosters the cardiac progenitor program. miR-210 directly silenced Shh via targeting of the Shh 3'UTR, comparable to the chemical Shh inhibitor, cyclopamine. miR-210 silencing of Shh/Gli1 signaling also blocked expression of the cell cycle regulators Cyclin D1 and Cyclin D2, and EB cell expansion. Absence of SRF expression in SRF null EBs blocked miR-210 expression, coincident with enhanced Shh, and Gli1 gene activity. Thus, SRF-dependent miR-210 expression may operate as a novel silencer of the Shh signaling pathway.

Yin-Yang 1, a New Player in Early Heart Development

In this issue of Circulation Research , Gregoire et al1 report their discovery that Yin-Yang 1 (YY1) is a critical factor for the transition from cardiac progenitor to differentiated cardiomyocyte. The authors made clever use of currently available transcription factor (TF)-binding databases to perform a bioinformatic-based screen for factors regulating cardiac progenitor genes. They compared their data set of genes enriched in mouse embryonic stem (ES) cell–derived cardiac progenitor cells (CPCs) with freely available TF–binding databases. They initially screened 32 candidate TFs and, surprisingly, found that YY1, a known repressor of sarcomeric gene expression, is resident on many CPC genes. Article, see p 900 The heart is the first organ to develop in the vertebrate embryo. Precardiogenic mesoderm is initially derived from anterior lateral plate mesoderm. Signals from surrounding tissues promote lateral plate to form cardiogenic mesoderm by inducing a cardiogenic transcriptional network. The cardiogenic mesoderm contains CPCs that differentiate into primitive cardiomyocytes. The transition from CPCs to cardiomyocytes is a critical developmental transition point that needs further study and is the focus of the study by Gregoire. Importantly, the cardiogenic developmental process can be mimicked …

Mature Cardiomyocytes Recall Their Progenitor Experience Via Polycomb Repressive Complex 2

### Epigenetic Repression of Cardiac Progenitor Gene Expression by Ezh2 is Required for Postnatal Cardiac Homeostasis Delgado-Olguin et al Nat Genet . 2012;44:343–347. Polycomb repressive complex 2 (PRC2) deposits repressive epigenetic marks that silence inappropriate gene expression programs. Recent work from Delgado-Olguin et al1 in conjunction with studies from other laboratories2–4 show that PRC2 activity in cardiac progenitors influences growth and gene expression of mature cardiomyocytes. These studies illustrate the power of epigenetic mechanisms to retain information about the heart's developmental history and deploy it to influence mature heart function. Physiological and pathophysiological stresses experienced by the heart during gestation and postnatal life have longstanding effects on heart function. How does the heart remember its past experiences? Recent work by Delgado-Olguin et al1 and concurrently published work from our group2,3 and another laboratory4 highlight the key role of epigenetic mechanisms in shaping myocardial function and provide a molecular mechanism that likely underpins molecular implementation of cardiac memory (Figure). Figure. EZH2-mediated gene silencing in cardiac progenitors is required for normal regulation of adult cardiomyocyte growth and function. EZH2 deposits repressive H3K27me3 epigenetic marks in cardiac progenitor cells (CPCs), establishing a chromatin landscape that is required for silencing of Six1 . EZH2 ablation in CPCs blocks initial deposition of these marks, derepressing Six1 even in later stages when EZH2 activity is dispensible. SIX1 activates ectopic skeletal muscle gene expression, which contributes to adult cardiomyocyte hypertrophy and fibrosis. However, SIX1 upregulation alone is not sufficient to cause the phenotype seen in EZH2 mutants. For example, upregulation of the Ink4a/b cell cycle inhibitors likely contributes to myocardial hypoplasia. In current parlance, epigenetics refers to mechanisms active over rounds of cell division or between generations that lead to heritable gene expression changes without involving changes to the DNA sequence. Epigenetic regulatory mechanisms involve modifications to DNA (eg, DNA methylation) or histones, or higher-order packaging of DNA around nucleosomes. These …

Epigenetic repression of cardiac progenitor gene expression by Ezh2 is required for postnatal cardiac homeostasis

Adult-onset diseases can be associated with in utero events, but mechanisms for this remain unknown1,2. The polycomb histone methyltransferase, Ezh2, stabilizes transcription by depositing repressive marks during development that persist into adulthood3–9, but its function in postnatal organ homeostasis is unknown. We show that Ezh2 stabilizes cardiac gene expression and prevents cardiac pathology by repressing the homeodomain transcription factor Six1, which functions in cardiac progenitors but is stably silenced upon cardiac differentiation10. Ezh2 deletion in cardiac progenitors caused postnatal myocardial pathology and destabilized cardiac gene expression with activation of Six1-dependent skeletal muscle genes. Six1 induced cardiomyocyte hypertrophy and skeletal muscle gene expression. Furthermore, genetically reducing Six1 levels rescued the pathology of Ezh2-deficient hearts. Thus, Ezh2-mediated repression of Six1 in differentiating cardiac progenitors is essential for stable postnatal heart gene expression and homeostasis. Our results suggest that epigenetic dysregulation in embryonic progenitor cells predisposes to adult disease and dysregulated stress responses.

Bmp Signaling Regulates Myocardial Differentiation from Cardiac Progenitors Through a MicroRNA-Mediated Mechanism

MicroRNAs (miRNAs) are small, noncoding RNAs thatregulate gene expression posttranscriptionally. We investigated the hypothesis that bone morphogenetic protein (Bmp) signaling regulates miRNAs in cardiac progenitor cells. Bmp2 and Bmp4 regulate OFT myocardial differentiation via regulation of themiRNA-17-92 cluster. In Bmp mutant embryos, myocardial differentiation was delayed, and multiple miRNAs encoded by miRNA-17-92 were reduced. We uncovered functional miRNA-17-92 seed sequences within the 3' UTR of cardiac progenitor genes such as Isl1 and Tbx1. In both Bmp and miRNA-17-92 mutant embryos, Isl1 and Tbx1 expression failed to be correctly downregulated. Transfection experiments indicated that miRNA-17 and miRNA-20a directly repressed Isl1 and Tbx1. Genetic interaction studies uncovered a synergistic interaction between miRNA-17-92 cluster and Bmp4, providing direct invivo evidence for the Bmp-miRNA-17-92 regulatory pathway. Our findings indicate that Bmp signaling directly regulates a miRNA-mediated effector mechanism that downregulates cardiac progenitor genes and enhances myocardial differentiation.

An Nkx2-5/Bmp2/Smad1 Negative Feedback Loop Controls Heart Progenitor Specification and Proliferation

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.