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Developmental etiologies causing complex congenital aortic root abnormalities are unknown. Here we show that deletion of Sox17 in aortic root endothelium in mice causes underdeveloped aortic root leading to a bicuspid aortic valve due to the absence of non-coronary leaflet and mispositioned left coronary ostium. The respective defects are associated with reduced proliferation of non-coronary leaflet mesenchyme and aortic root smooth muscle derived from the second heart field cardiomyocytes. Mechanistically, SOX17 occupies a Pdgfb transcriptional enhancer to promote its transcription and Sox17 deletion inhibits the endothelial Pdgfb transcription and PDGFB growth signaling to the non-coronary leaflet mesenchyme. Restoration of PDGFB in aortic root endothelium rescues the non-coronary leaflet and left coronary ostium defects in Sox17 nulls. These data support a SOX17-PDGFB axis underlying aortic root development that is critical for aortic valve and coronary ostium patterning, thereby informing a potential shared disease mechanism for concurrent anomalous aortic valve and coronary arteries.

Chromatin structure is determined by nucleosome positioning, histone modifications, and DNA methylation. How chromatin modifications are coordinately altered under pathological conditions remains elusive. Here we describe a stress-activated mechanism of concerted chromatin modification in the heart. In mice, pathological stress activates cardiomyocytes to express Brg1 (nucleosome-remodeling factor), G9a/Glp (histone methyltransferase), and Dnmt3 (DNA methyltransferase). Once activated, Brg1 recruits G9a and then Dnmt3 to sequentially assemble repressive chromatin—marked by H3K9 and CpG methylation—on a key molecular motor gene (Myh6), thereby silencing Myh6 and impairing cardiac contraction. Disruption of Brg1, G9a or Dnmt3 erases repressive chromatin marks and de-represses Myh6, reducing stress-induced cardiac dysfunction. In human hypertrophic hearts, BRG1–G9a/GLP–DNMT3 complex is also activated; its level correlates with H3K9/CpG methylation, Myh6 repression, and cardiomyopathy. Our studies demonstrate a new mechanism of chromatin assembly in stressed hearts and novel therapeutic targets for restoring Myh6 and ventricular function. The stress-induced Brg1–G9a–Dnmt3 interactions and sequence of repressive chromatin assembly on Myh6 illustrates a molecular mechanism by which the heart epigenetically responds to environmental signals. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Our developmental studies provide an insight into the pathogenesis of heart failure in adults. These studies reveal a mechanistic link between fetal cardiomyocytes and pathologically stressed adult cardiomyocytes at the level of chromatin regulation. In embryos, chromatin-regulating factors within the cardiomyocytes respond to developmental signals to program cardiac gene expression to promote cell proliferation and inhibit premature cell differentiation. In the neonatal period, the activity of these developmental chromatin regulators is quickly turned off in cardiomyocytes, coinciding with the cessation of cell proliferation and advance in cell differentiation toward adult maturity. When the mature hearts are pathologically stressed, those chromatin regulators essential for cardiomyocyte development in embryos are reactivated, triggering gene reprogramming to a fetal-like state and pathological cardiac hypertrophy. Furthermore, in the study of chromatin regulation and cardiac gene expression, we identified a long noncoding RNA that interacts with chromatin remodeling factor to regulate the cardiac response to environmental changes. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Cardiac organogenesis requires the control of gene expression at distinct developmental windows in order to organize morphogenetic steps in the correct sequence for heart development. This is facilitated by concerted regulation at three levels: chromatin, transcription, and post-transcriptional modifications. Epigenetic regulation at the chromatin level changes the chromatin scaffold of DNA to regulate accessibility of the DNA sequence to transcription factors for genetic activation or repression. At the genome, long non-coding RNAs work with epigenetic factors to alter the chromatin scaffold or form DNA-RNA complexes at specific genomic loci to control the transcription of genetic information. After RNA transcription, the expression of genetic information can be further modified by microRNAs. Each layer of gene regulation requires the participation of many factors, with their combinatorial interactions providing variations of genetic expression at distinct pathophysiological phases of the heart. The major functions of chromatin remodellers and non-coding RNAs are discussed.

Long noncoding RNAs (lncRNAs) are pivotal regulators of genome structure and gene expression. LncRNAs can directly interact with chromatin-modifying enzymes and nucleosome-remodeling factors to control chromatin structure and accessibility of genetic information. Moreover, lncRNA expression can be controlled by chromatin-remodeling factors, suggesting a feedback circuit of regulation. Here, we discuss the recent advances of lncRNA studies, focusing on the function and mechanism of lncRNA-chromatin interactions.

Despite the importance of cardiomyocyte proliferation in cardiac development and regeneration, the mechanisms that promote cardiomyocyte cell cycle remain incompletely understood. RE1 silencing transcription factor (REST) is a transcriptional repressor of neuronal genes. Here we show that REST also regulates the cardiomyocyte cell cycle. REST binds and represses the cell cycle inhibitor gene p21 and is required for mouse cardiac development and regeneration. Rest deletion de-represses p21 and inhibits the cardiomyocyte cell cycle and proliferation in embryonic or regenerating hearts. By contrast, REST overexpression in cultured cardiomyocytes represses p21 and increases proliferation. We further show that p21 knockout rescues cardiomyocyte cell cycle and proliferation defects resulting from Rest deletion. Our study reveals a REST-p21 regulatory axis as a mechanism for cell cycle progression in cardiomyocytes, which might be exploited therapeutically to enhance cardiac regeneration.

Insufficiency of surfactants is a core factor in respiratory distress syndrome, which causes apnea and neonatal death, particularly in preterm infants. Surfactant proteins are secreted by alveolar type II cells in the lung epithelium, the differentiation of which is regulated by Fgf10 elaborated by the adjacent mesenchyme. However, the molecular regulation of mesenchymal Fgf10 during lung development has not been fully understood. Here, we show that Pbx1, a homeodomain transcription factor, is required in the lung mesenchyme for the expression of Fgf10. Mouse embryos lacking Pbx1 in the lung mesenchyme show compact terminal saccules and perinatal lethality with failure of postnatal alveolar expansion. Mutant embryos had severely reduced expression of Fgf10 and surfactant genes (Spa, Spb, Spc, and Spd) that are essential for alveolar expansion for gas exchange at birth. Molecularly, Pbx1 directly binds to the Fgf10 promoter and cooperates with Meis and Hox proteins to transcriptionally activate Fgf10. Our results thus show how Pbx1 controls Fgf10 in the developing lung.

Ventricular trabeculation and compaction are two of the many essential steps for generating a functionally competent ventricular wall. A significant reduction in trabeculation is usually associated with ventricular compact zone deficiencies (hypoplastic wall), which commonly leads to embryonic heart failure and early embryonic lethality. In contrast, hypertrabeculation and lack of ventricular wall compaction (noncompaction) are closely related defects in cardiac embryogenesis associated with left ventricular noncompaction (LVNC), a genetically heterogenous disorder. Here we review recent findings through summarizing several genetically engineered mouse models that have defects in cardiac trabeculation and compaction.

SummaryHair follicle stem cells (bulge cells) are essential for hair regeneration and early epidermal repair after wounding. Here we show that Brg1, a key enzyme in the chromatin-remodeling machinery, is dynamically expressed in bulge cells to control tissue regeneration and repair. In mice, sonic hedgehog (Shh) signals Gli to activate Brg1 in bulge cells to begin hair regeneration, whereas Brg1 recruits NF-κB to activate Shh in matrix cells to sustain hair growth. Such reciprocal Brg1-Shh interaction is essential for hair regeneration. Moreover, Brg1 is indispensable for maintaining the bulge cell reservoir. Without Brg1, bulge cells are depleted over time, partly through the ectopic expression of the cell-cycle inhibitor p27Kip1. Also, bulge Brg1 is activated by skin injury to facilitate early epidermal repair. Our studies demonstrate a molecular circuit that integrates chromatin remodeling (Brg1), transcriptional regulation (NF-κB, Gli), and intercellular signaling (Shh) to control bulge stem cells during tissue regeneration.

Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1) , and a cell cycle inhibitor, p21 cip1 , to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.

Genes encoding angiotensin-converting enzymes (Ace and Ace2) are essential for heart function regulation. Cardiac stress enhances Ace, but suppresses Ace2, expression in the heart, leading to a net production of angiotensin II that promotes cardiac hypertrophy and fibrosis. The regulatory mechanism that underlies the Ace2-to-Ace pathological switch, however, is unknown. Here we report that the Brahma-related gene-1 (Brg1) chromatin remodeler and forkhead box M1 (FoxM1) transcription factor cooperate within cardiac (coronary) endothelial cells of pathologically stressed hearts to trigger the Ace2-to-Ace enzyme switch, angiotensin I-to-II conversion, and cardiac hypertrophy. In mice, cardiac stress activates the expression of Brg1 and FoxM1 in endothelial cells. Once activated, Brg1 and FoxM1 form a protein complex on Ace and Ace2 promoters to concurrently activate Ace and repress Ace2, tipping the balance to Ace2 expression with enhanced angiotensin II production, leading to cardiac hypertrophy and fibrosis. Disruption of endothelial Brg1 or FoxM1 or chemical inhibition of FoxM1 abolishes the stress-induced Ace2-to-Ace switch and protects the heart from pathological hypertrophy. In human hypertrophic hearts, BRG1 and FOXM1 expression is also activated in endothelial cells; their expression levels correlate strongly with the ACE/ACE2 ratio, suggesting a conserved mechanism. Our studies demonstrate a molecular interaction of Brg1 and FoxM1 and an endothelial mechanism of modulating Ace/Ace2 ratio for heart failure therapy.

It is known that the nanoscale morphological organization of donors or acceptors in bulk heterojunction (BHJ) solar cells is critical to device performance and strongly affects carrier generation, transporting, and collection. This work demonstrates the dependence of nanocrystal dimensionality and organization on the polymer nanomorphology in P3HT:TiO2 hybrid bulk heterojunctions, which were revealed using grazing-incidence X-ray diffraction (GIXRD) using a synchrotron X-ray beam and electron tomography. We further performed a multiscale molecular dynamic simulation to understand the morphological orientation of a polymer blended with TiO2 nanoparticles (NPs) or nanorods (NRs). The correlation between polymer nanoscale morphology and the dimensionality and anisotropy of nanocrystals in P3HT:TiO2 hybrids clearly explains the observation of different optical absorption and carrier transport behaviors in directions perpendicular or parallel to the film substrate. Our results provide crucial information towar...

SummaryThe origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

Heart malformations are common congenital defects in humans. Many congenital heart defects involve anomalies in cardiac septation or valve development, and understanding the developmental mechanisms that underlie the formation of cardiac septal and valvular tissues thus has important implications for the diagnosis, prevention and treatment of congenital heart disease. The development of heart septa and valves involves multiple types of progenitor cells that arise either within or outside the heart. Here, we review the morphogenetic events and genetic networks that regulate spatiotemporal interactions between the cells that give rise to septal and valvular tissues and hence partition the heart.

SummaryThe molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.

Chromatin regulation provides an important means for controlling cardiac gene expression under different physiological and pathological conditions. Processes that direct the development of normal embryonic hearts and pathology of stressed adult hearts may share general mechanisms that govern cardiac gene expression by chromatin-regulating factors. These common mechanisms may provide a framework for us to investigate the interactions among diverse chromatin remodelers/modifiers and various transcription factors in the fine regulation of gene expression, essential for all aspects of cardiovascular biology. Aberrant cardiac gene expression, triggered by a variety of pathological insults, can cause heart diseases in both animals and humans. The severity of cardiomyopathy and heart failure correlates strongly with abnormal cardiac gene expression. Therefore, controlling cardiac gene expression presents a promising approach to the treatment of human cardiomyopathy. This review focuses on the roles of ATP-dependent chromatin-remodeling factors and chromatin-modifying enzymes in the control of gene expression during cardiovascular development and disease.

Heart valve malformations are one of the most common types of birth defects, illustrating the complex nature of valve development. Vascular endothelial growth factor (VEGF) signaling is one pathway implicated in valve formation, however its specific spatial and temporal roles remain poorly defined. To decipher these contributions, we use two inducible dominant negative approaches in mice to disrupt VEGF signaling at different stages of embryogenesis. At an early step in valve development, VEGF signals are required for the full transformation of endocardial cells to mesenchymal cells (EMT) at the outflow tract (OFT) but not atrioventricular canal (AVC) endocardial cushions. This role likely involves signaling mediated by VEGF receptor 1 (VEGFR1), which is highly expressed in early cushion endocardium before becoming downregulated after EMT. In contrast, VEGFR2 does not exhibit robust cushion endocardium expression until after EMT is complete. At this point, VEGF signaling acts through VEGFR2 to direct the morphogenesis of the AVC cushions into mature, elongated valve leaflets. This latter role of VEGF requires the VEGF-modulating microRNA, miR-126. Thus, VEGF roles in the developing valves are dynamic, transitioning from a differentiation role directed by VEGFR1 in the OFT to a morphogenetic role through VEGFR2 primarily in the AVC-derived valves.

This work presents polymer photovoltaic devices based on poly(3-hexylthiophene) (P3HT) and TiO2 nanorod hybrid bulk heterojunctions. Interface modification of a TiO2 nanorod surface is conducted to yield a very promising device performance of 2.20% with a short circuit current density (J(sc)) of 4.33 mA/cm2, an open circuit voltage (V(oc)) of 0.78 V, and a fill factor (FF) of 0.65 under simulated A.M. 1.5 illumination (100 mW/cm2). The suppression of recombination at P3HT/TiO2 nanorod interfaces by the attachment of effective ligand molecules substantially improves device performance. The correlation between surface photovoltage and hybrid morphology is revealed by scanning Kelvin probe microscopy. The proposed method provides a new route for fabricating low-cost, environmentally friendly polymer/inorganic hybrid bulk heterojunction photovoltaic devices.

Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMCs) by breeding mice possessing a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22alpha-Cre mice. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMCs that was also related to reduced proliferation. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of matrix metalloproteinase (MMP) 2 and 9 were reduced in E9.5 and E10.5 SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of BMPR1A by RNA interference in human pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP2 or MMP9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of BMPR1A reduced MMP2 activity and knock-down of either BMPR1A or MMP2 caused resistance to apoptosis. Thus, loss of Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for vascular dilatation and persistence of brain microvessels, leading to the impaired organogenesis documented in the brain.

SummaryDeveloping myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. We show that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer.

Chromatin provides a dynamic DNA scaffold that reacts to physiological and pathological signals to control the accessibility of DNA sequence and the genomic responses to environmental stimuli. Chromatin can be regulated by nucleosome remodeling, histone modification, and DNA methylation. Histone and DNA modifications occur by covalent alterations of the side chains of histone or bases of DNA, catalyzed by specific histone- and DNA-modifying enzymes, whereas nucleosome or chromatin-remodeling controls noncovalent changes of nucleosomes, including their position and histone composition, effected by adenosine triphosphate (ATP)-dependent chromatin-remodeling complexes. Within the nucleosome, the chromatin remodelers can replace canonical histones with variant forms of histones, which are involved in cardiac stress response. In addition, chromatin remodelers can interact with histone- and DNA-modifying enzymes to control chromatin structure and reprogram gene expression in pathologically stressed hearts. More recently, a chromatin-remodeling factor was found to interact with a cardiac-specific long noncoding RNA to control gene expression and maintain cardiac homeostasis. These functional aspects of chromatin remodelers are critical for the pathogenesis of cardiomyopathy and heart failure. This chapter is focused on the recent progress in understanding the roles of chromatin-remodeling factors in heart failure, new chromatin-based mechanisms, and potential therapeutic strategies for heart failure.

Interstitial fibrosis plays a key role in the development and progression of heart failure. Here, we show that an enzyme that crosslinks collagen—Lysyl oxidase-like 2 (Loxl2)—is essential for interstitial fibrosis and mechanical dysfunction of pathologically stressed hearts. In mice, cardiac stress activates fibroblasts to express and secrete Loxl2 into the interstitium, triggering fibrosis, systolic and diastolic dysfunction of stressed hearts. Antibody-mediated inhibition or genetic disruption of Loxl2 greatly reduces stress-induced cardiac fibrosis and chamber dilatation, improving systolic and diastolic functions. Loxl2 stimulates cardiac fibroblasts through PI3K/AKT to produce TGF-β2, promoting fibroblast-to-myofibroblast transformation; Loxl2 also acts downstream of TGF-β2 to stimulate myofibroblast migration. In diseased human hearts, LOXL2 is upregulated in cardiac interstitium; its levels correlate with collagen crosslinking and cardiac dysfunction. LOXL2 is also elevated in the serum of heart failure (HF) patients, correlating with other HF biomarkers, suggesting a conserved LOXL2-mediated mechanism of human HF., Lysyl oxidase-like 2 (LOXL2) is an enzyme that promotes scaffolding of extracellular matrix proteins. Here the authors show that LOXL2 is crucial for pressure-overload induced cardiac fibrosis, and that antibody-mediated inhibition or genetic disruption of Loxl2 in mice shows therapeutic potential for treatment of cardiac fibrosis.

Trisomy 21 results in Down's syndrome, but little is known about how a 1.5-fold increase in gene dosage produces the pleiotropic phenotypes of Down's syndrome. Here we report that two genes, DSCR1 and DYRK1A , lie within the critical region of human chromosome 21 and act synergistically to prevent nuclear occupancy of NFATc transcription factors, which are regulators of vertebrate development. We use mathematical modelling to predict that autoregulation within the pathway accentuates the effects of trisomy of DSCR1 and DYRK1A, leading to failure to activate NFATc target genes under specific conditions. Our observations of calcineurin-and Nfatc-deficient mice, Dscr1- and Dyrk1a-overexpressing mice, mouse models of Down's syndrome and human trisomy 21 are consistent with these predictions. We suggest that the 1.5-fold increase in dosage of DSCR1 and DYRK1A cooperatively destabilizes a regulatory circuit, leading to reduced NFATc activity and many of the features of Down's syndrome. More generally, these observations suggest that the destabilization of regulatory circuits can underlie human disease.

Background: The optimal cell-matrix combination for robust and sustained myocardial restoration has not been identified. The present study utilizes embryonic stem cells as the substrate of bioartificial myocardial tissue and evaluates engraftment in, and functional recovery of, the recipient heart. Methods: Collagen type I was populated with undifferentiated green fluorescent protein (GFP)-positive mouse embryonic stem cells. An intramural left ventricular pouch was fashioned after ligation of the left anterior descending artery in an athymic nude rat heterotopic heart transplant model. The bioartificial mixture (0.125 ml) was implanted in the infarcted area within the pouch. Echocardiography was performed to assess fractional shortening in: Group I, infarcted rats that received cell-matrix implants; Group II, rats given matrix implant without cells; Group III, rats given no matrix or cells; and Group IV, rats receiving transplanted hearts without ligation (n = 5/group). Hearts were stained for GFP, cardiac markers (connexin-43, α-sarcomeric actin), hematoxylin-eosin (H&E) and trichrome. Results: Embryonic stem cells formed stable intramyocardial grafts that were incorporated into the surrounding area without distorting myocardial geometry, thereby preventing ventricular wall thinning (anterior wall thickness was: Group I, 1.4 ± 0.1 mm; Group II, 1.0 ± 0.1 mm, Group III, 0.9 ± 0.2 mm; and Group IV, 1.3 ± 0.2 mm). The inoculated cells expressed connexin-43 and α-sarcomeric actin in vivo. Fractional shortening was better in embryonic stem cell-treated animals (Group I, 21.5 ± 3.5%; Group II, 12.4 ± 2.8%; Group III, 8.2 ± 2.9%; Group IV, 23.2 ± 4.2%). Conclusions: Embryonic stem cells are an efficient alternative substrate for myocardial tissue engineering and can prevent myocardial wall thinning and improve contractility after implantation into injured myocardium in a 3-dimensional matrix. Copyright © 2005 by the International Society for Heart and Lung Transplantation.

ObjectivesMost tissue-engineering approaches to restore injured heart muscle result in distortion of left ventricular geometry. In the present study we suggest seeding embryonic stem cells in a liquid matrix for myocardial restoration.MethodsUndifferentiated green fluorescent protein–labeled mouse embryonic stem cells (2 × 106) were seeded in Matrigel (B&D, Bedford, Mass). In a Lewis rat heterotopic heart transplant model an intramural left ventricular pouch was fashioned after ligation of the left anterior descending coronary artery. The liquid mixture (0.125 mL) was injected in the resulting infarcted area within the pouch and solidified within a few minutes after transplantation (37°C). Five recipient groups were formed: transplanted healthy hearts (group I), infarcted control hearts (group II), matrix recipients alone (group III), the study group that received matrix plus cells (group IV), and a group that received embryonic stem cells alone (group V). After echocardiography 2 weeks later, the hearts were harvested and stained for green fluorescent protein and cardiac muscle markers (connexin 43 and α-sarcomeric actin).ResultsThe graft formed a sustained structure within the injured area and prevented ventricular wall thinning. The inoculated cells remained viable and expressed connexin 43 and α-sarcomeric actin. Fractional shortening and regional contractility were better in animals that received bioartificial tissue grafts compared with control animals (infarcted, matrix only, and embryonic stem cells only: group I, 17.0% ± 3.5%; group II, 6.6% ± 2.1%; group III, 10.3% ± 2.2%; group IV, 14.5% ± 2.5%; and group V, 7.8% ± 1.8%).ConclusionsLiquid bioartificial tissue containing embryonic stem cells constitutes a powerful new approach to restoring injured heart muscle without distorting its geometry and structure.

Congenital obstructive nephropathy is the principal cause of renal failure in infants and children. The underlying molecular and cellular mechanisms of this disease, however, remain largely undetermined. We generated a mouse model of congenital obstructive nephropathy that resembles ureteropelvic junction obstruction in humans. In these mice, calcineurin function is removed by the selective deletion of Cnb1 in the mesenchyme of the developing urinary tract using the Cre/lox system. This deletion results in reduced proliferation in the smooth muscle cells and other mesenchymal cells in the developing urinary tract. Compromised cell proliferation causes abnormal development of the renal pelvis and ureter, leading to defective pyeloureteral peristalsis, progressive renal obstruction, and, eventually, fatal renal failure. Our study demonstrates that calcineurin is an essential signaling molecule in urinary tract development and is required for normal proliferation of the urinary tract mesenchymal cells in a cell-autonomous manner. These studies also emphasize the importance of functional obstruction, resulting from developmental abnormality, in causing congenital obstructive nephropathy.

In mouse developmental studies it is frequently desirable to isolate embryos of a specific age. However, the traditional staging of embryonic development based on postcoital dates often erroneously predicts the embryonic age, resulting in unwarranted sacrifice of the pregnant mother. Here we report a noninvasive way of staging embryonic development in utero. A clinical 14 MHz ultrasound system was employed to assess the morphology and size of developing embryos from embryonic day 7.5 to 18.5. We demonstrate that the developmental age of the mouse embryos can be accurately determined based on the sonographic morphology and size of the embryos. This noninvasive ultrasound application requires no anesthesia of the mice and the entire process of staging can be completed within 5-10 min. Empirically, this approach is applicable to mice of various genetic backgrounds and significantly enhances the efficiency of studying murine embryogenesis.

Hox homeodomain proteins are developmental regulators that determine body plan in a variety of organisms. A majority of the vertebrate Hox proteins bind DNA as heterodimers with the Pbx1 homeodomain protein. We report here the 2.35 A structure of a ternary complex containing a human HoxB1–Pbx1 heterodimer bound to DNA. Heterodimer contacts are mediated by the hexapeptide of HoxB1, which binds in a pocket in the Pbx1 protein formed in part by a three–amino acid insertion in the Pbx1 homeodomain. The Pbx1 DNA-binding domain is larger than the canonical homeodomain, containing an additional α helix that appears to contribute to binding of the HoxB1 hexapeptide and to stable binding of Pbx1 to DNA. The structure suggests a model for modulation of Hox DNA binding activity by Pbx1 and related proteins.

The DNA is tightly packed by chromatin, which controls the expression of genetic information encoded by the DNA sequence. The chromatin scaffold responds dynamically to physiological and pathological signals and can be altered by histone modification, DNA methylation, and nucleosome remodeling.1 Histone and DNA modifications include covalent modifications of histone side chains and DNA bases, whereas nucleosome remodeling involves non-covalent changes of the composition or positioning of nucleosomes along the genomic DNA. Remodeling of nucleosomes is executed by ATP-dependent chromatin-remodeling complexes; however, it is largely unknown how these chromatin remodelers target specific genomic sites to control local nucleosome dynamics. Our recent work revealed that a long non-coding RNA sequesters the core subunit of a chromatin-remodeling complex from its genomic targets to inhibit gene reprogramming in pathologically stressed hearts.2 This sheds new light on the pathogenesis of heart failure and the targeting mechanism of chromatin-remodeling complexes.

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

The chimeric oncoprotein E2a-Pbx1 results from fusion of the E2A and PBX1 genes following t(1;19) chromosomal translocations in B cell precursor acute leukemias. Experimentally B cell progenitors do not tolerate constitutive expression of E2a-Pbx1 which contrasts with transformation of several other cell types following its stable expression both in vitro and in vivo. To further investigate the effects of E2a-Pbx1 on the B cell progenitors, we conditionally expressed E2a-Pbx1 under control of a metal response element in hematopoietic precursor cell lines in vitro. Inducible expression of E2a-Pbx1 resulted in cell death with the morphologic and molecular features of apoptosis. A structure-function analysis demonstrated that induction of apoptosis was not a dominant-negative effect of the E2a moiety but, rather, required the DNA-binding homeodomain of Pbx1. E2a-Pbx1-induced apoptosis proceeded through a BCL2-responsive checkpoint eventuating in PARP inactivation but did require p53. Constitutive expression of E2a-Pbx1 did not induce apoptosis or continued cycling of Rat-1 fibroblasts in low serum conditions. These studies demonstrate that E2a-Pbx1 initiates programmed cell death of hematopoietic precursers by a mechanism that requires its chimeric transcriptional properties, but, unlike other nuclear oncoproteins, is independent of p53.

The success of semiconductor technology is largely ascribed to controlled impacts of strains and defects on the two-dimensional interfacial charges. Interfacial charges also appear in oxide heterojunctions such as LaAlO3/SrTiO3 and (Nd0.35Sr0.65)MnO3/SrTiO3. How the localized strain field of one-dimensional misfit dislocations, defects resulting from the intrinsic misfit strains, would affect the extended oxide-interfacial charges is intriguing and remains unresolved. Here we show the atomic-scale observation of one-dimensional electron chains formed in (Nd0.35Sr0.65)MnO3/SrTiO3 by the condensation of characteristic two-dimensional interfacial charges into the strain field of periodically arrayed misfit dislocations, using chemical mapping and quantification by scanning transmission electron microscopy. The strain-relaxed inter-dislocation regions are readily charge depleted, otherwise decorated by the pristine charges, and the corresponding total-energy calculations unravel the undocumented charge-reservoir role played by the dislocation-strain field. This two-dimensional-to-one-dimensional electronic condensation represents a novel electronic-inhomogeneity mechanism at oxide interfaces and could stimulate further studies of one-dimensional electron density in oxide heterostructures.

Aims Congenital coronary artery anomalies produce serious events that include syncope, arrhythmias, myocardial infarction, or sudden death. Studying the mechanism of coronary development will contribute to the understanding of the disease and help design new diagnostic or therapeutic strategies. Here, we characterized a new calcineurin–NFAT signalling which specifically functions in the epicardium to regulate the development of smooth muscle wall of the coronary arteries. Methods and results Using tissue-specific gene deletion, we found that calcineurin–NFAT signals in the embryonic epicardium to direct coronary smooth muscle cell development. The smooth muscle wall of coronary arteries fails to mature in mice with epicardial deletion of calcineurin B1 ( Cnb1 ), and accordingly these mutant mice develop cardiac dysfunction with reduced exercise capacity. Inhibition of calcineurin at various developmental windows shows that calcineurin–NFAT signals within a narrow time window at embryonic Day 12.5–13.5 to regulate coronary smooth muscle cell development. Within the epicardium, NFAT transcriptionally activates the expression of Smad2 , whose gene product is critical for transducing transforming growth factor β (TGFβ)–Alk5 signalling to control coronary development. Conclusion Our findings demonstrate new spatiotemporal and molecular actions of calcineurin–NFAT that dictate coronary arterial wall development and a new mechanism by which calcineurin–NFAT integrates with TGFβ signalling during embryonic development.

Metabolic remodeling is an integral part of heart failure. Current studies are largely focusing on glucose and fatty acid metabolism, while little is known about the changes in amino acid homeostasis during heart failing process. Branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, serve as not only essential building blocks for protein synthesis, but also important energy source and signaling molecules that have significant effects on cell growth and function. In this study, we demonstrated that the BCAA catabolic intermediate branched-chain keto acid (BCKA) accumulated in both mouse and human failing heart. BCAA catabolic genes were selectively and significantly down-regulated at both mRNA and protein levels in failing heart in mice, mimicking a similar expression pattern observed in neonatal heart. Using both in vitro and in vivo models, we established that BCAA catabolic genes were regulated by Krüppel-like factor 15 (KLF15), a key transcriptional regulator for glucose, fat, and amino acid nutrient homeostasis, suggesting that the KLF15-mediated BCAA catabolic regulation is part of the metabolic remodeling during heart failure. Genetic ablation of PP2Cm, a key regulator of BCAA catabolism, led to a significant impairment of BCAA catabolic activities and accumulation of BCKA in cardiac tissue. Importantly, PP2Cm deficiency accelerated heart failure under pressure overload. PP2Cm deficiency or elevated BCKA induced oxidative stress in cardiomyocytes and impairment of oxygen consumption and ATP production of mitochondria. Antioxidant treatment ameliorated the heart failure progression in PP2Cm deficient animals. Taken together, our data established for the first time that BCAA catabolic reprogramming is an integral component of metabolic remodeling during heart failure, and this remodeling can significantly contribute to heart failure progression.

Ventricular trabeculation and compaction are two of the many essential steps for generating a functionally competent ventricular wall. A significant reduction in trabeculation is usually associated with ventricular compact zone deficiencies (hypoplastic wall), which commonly leads to embryonic heart failure and early embryonic lethality. In contrast, hypertrabeculation and lack of ventricular wall compaction (noncompaction) are closely related defects in cardiac embryogenesis associated with left ventricular noncompaction (LVNC), a genetically heterogenous disorder. Here we review recent findings through summarizing several genetically engineered mouse models that have defects in cardiac trabeculation and compaction.

The role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodelling. An estimated 70% of mouse genes undergo antisense transcription, including myosin heavy chain 7 (Myh7), which encodes molecular motor proteins for heart contraction. Here we identify a cluster of lncRNA transcripts from Myh7 loci and demonstrate a new lncRNA-chromatin mechanism for heart failure. In mice, these transcripts, which we named myosin heavy-chain-associated RNA transcripts (Myheart, or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1-Hdac-Parp chromatin repressor complex to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodelling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. It does so by binding to the helicase domain of Brg1, a domain that is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic-acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized--but not naked--DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodelling. A Mhrt-Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify a cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodelling factors, and establish a new paradigm for lncRNA-chromatin interaction.

Pbx cofactors are implicated to play important roles in modulating the DNA-binding properties of heterologous homeodomain proteins, including class I Hox proteins. To assess how Pbx proteins influence Hox DNA-binding specificity, we used a binding-site selection approach to determine high-affinity target sites recognized by various Pbx-Hox homeoprotein complexes. Pbx-Hox heterodimers preferred to bind a bipartite sequence 5'-ATGATTNATNN-3' consisting of two adjacent half sites in which the Pbx component of the heterodimer contacted the 5' half (ATGAT) and the Hox component contacted the more variable 3' half (TNATNN). Binding sites matching the consensus were also obtained for Pbx1 complexed with HoxA10, which lacks a hexapeptide but requires a conserved tryptophan-containing motif for cooperativity with Pbx. Interactions with Pbx were found to play an essential role in modulating Hox homeodomain amino-terminal arm contact with DNA in the core of the Hox half site such that heterodimers of different compositions could distinguish single nucleotide alterations in the Hox half site both in vitro and in cellular assays measuring transactivation. When complexed with Pbx, Hox proteins B1 through B9 and A10 showed stepwise differences in their preferences for nucleotides in the Hox half site core (TTAT to TGAT, 5' to 3') that correlated with the locations of their respective genes in the Hox cluster. These observations demonstrate previously undetected DNA-binding specificity for the amino-terminal arm of the Hox homeodomain and suggest that different binding activities of Pbx-Hox complexes are at least part of the position-specific activities of the Hox genes.

Heart Failure (HF) is a major cause of morbidity and mortality in US. Fibrosis is an important feature in structural remodeling of the left ventricle (LV) and causes arrhythmia, cardiac dysfunction in HF. The lysyl oxidase (LOX) family crosslinks collagen and contributes to extracellular matrix

Apelin is an endogenous peptide hormone recently implicated in glucose homeostasis. However, whether apelin affects glucose uptake in myocardial tissue remains undetermined. In this study, we utilized in vivo, ex vivo and in vitro methods to study apelin's effect on myocardial glucose uptake. Pyroglutamated apelin-13 (2mg/kg/day) was administered to C57BL6/J mice for 7 days. In vivo myocardial glucose uptake was measured by FDG-PET scanning, and GLUT4 translocation was assessed by immunofluorescence imaging. For in vitro studies, differentiated H9C2 cardiomyoblasts were exposed to pyroglutamated apelin-13 (100 nM) for 2h. To test their involvement in apelin-stimulated myocardial glucose uptake, the energy sensing protein kinase AMPK were inhibited by pharmacologic inhibition (compound C) and RNA interference. IRS-1 phosphorylation was assessed by western blotting using an antibody directed against IRS-1 Ser-789-phosphorylated form. We found that apelin increased myocardial glucose uptake and GLUT4 membrane translocation in C57BL6/J mice. Apelin was also sufficient to increase glucose uptake in H9C2 cells. Apelin-mediated glucose uptake was significantly decreased by AMPK inhibition. Finally, apelin increased IRS-1 Ser-789 phosphorylation in an AMPK-dependent manner. The results of our study demonstrated that apelin increases myocardial glucose uptake through a pathway involving AMPK. Apelin also facilitates IRS-1 Ser-789 phosphorylation, suggesting a novel mechanism for its effects on glucose uptake.

Formation of the cardiac outflow tract and great arteries involves complex morphogenetic processes, whose abnormities result in several clinically important diseases. Studies of these developmental processes are therefore important for understanding congenital vascular defects. However, the three-dimensional structure of arteries makes it challenging to analyze the pattern of vasculature using conventional histological approaches. Here we describe a vascular casting method to visualize the branching and connections of great arteries in developing embryos as well as in adult mice. This technique can be used to study the development of cardiac outflow tract, semilunar valves, and great arteries as demonstrated previously (Circ Res, 2008; Development 135: 3577-3586, 2008).

Congenital heart defects occur in approximately 1% of newborns and are a major cause of morbidity and mortality in infants and children. Many adult cardiac diseases also have developmental basis, such as heart valve malformations, among others. Therefore, dissecting the developmental and molecular mechanisms underlying such defects in embryos is of great importance in prevention and developing therapeutics for heart diseases that manifest in infants or later in adults. Whole embryo culture is a valuable tool to study cardiac development in midgestation embryos, in which ventricular chambers are specified and expand, and the myocardium and endocardium interact to form various cardiac structures including heart valves and trabecular myocardium (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008). This technique is essentially growing a midgestation embryo ex utero in a test tube. One of the strengths of embryo culture is that it allows an investigator to easily manipulate or add drugs/chemicals directly to the embryos to test specific hypotheses in situations that are otherwise very difficult to perform for embryos in utero. For instance, embryo culture permits pharmacological rescue experiments to be performed in place of genetic rescue experiments which may require generation of specific mouse strains and crosses. Furthermore, because embryos are grown externally, drugs are directly acting on the cultured embryos rather than being degraded through maternal circulation or excluded from the embryos by the placenta. Drug dosage and kinetics are therefore easier to control with embryo culture. Conversely, drugs that compromise the placental function and are thus unusable for in utero experiments are applicable in cultured embryos since placental function is not required in whole embryo culture. The applications of whole embryo culture in the studies of molecular pathways involved in heart valve formation, myocardial growth, differentiation, and morphogenesis are demonstrated previously (Cell 118: 649-663, 2004; Dev Cell 14: 298-311, 2008; Nature 446: 62-67, 2010). Here we describe a method of embryo culture in a common laboratory setting without using special equipments.

Malformations of heart valves are one of the most common serious congenital defects. Heart valves are developed from endocardial cushions of the heart. The endocardial cushion in early heart development consists of two cell layers: an outer myocardial cell layer and an inner endocardial cell layer with abundant extracellular matrix (cardiac jelly) in between. Endocardial cells of the cushion, triggered by signals from myocardial cells, delaminate from the surface of the endocardial cushion and undergo transdifferentiation into mesenchymal cells. This process of endocardial-to-mesenchymal transformation (EMT) begins in the atrioventricular canal at embryonic day 9 (E9) and in the cardiac outflow tract at E10 of mouse development. Once formed by the EMT, the mesenchymal cells invade the cardiac jelly, proliferate, and populate the endocardial cushion. The cellularized endocardial cushion then undergoes morphological remodeling; it lengthens and matures into a thin elongated valve leaflet. Here we describe a method to culture endocardial cushions and measure EMT ex vivo. EMT can thus be analyzed independent of other concurrent developmental defects in mice. This culture method also enables ex vivo manipulations of signaling or gene function during EMT to delineate molecular pathways essential for heart valve development.

Site-specific recombination reactions in higher eukaryotes are uncommon, perhaps because of the potential genomic instability that they may create. We focus this review on the issues of site-specificity, reaction fidelity and immunologic diversity in the V(D)J recombination reaction.

Malformations of heart valves are one of the most common serious congenital defects. Heart valves are developed from endocardial cushions of the heart. The endocardial cushion in early heart development consists of two cell layers: an outer myocardial cell layer and an inner endocardial cell layer with abundant extracellular matrix (cardiac jelly) in between. Endocardial cells of the cushion, triggered by signals from myocardial cells, delaminate from the surface of the endocardial cushion and undergo transdifferentiation into mesenchymal cells. This process of endocardial-to-mesenchymal transformation (EMT) begins in the atrioventricular canal at embryonic day 9 (E9) and in the cardiac outflow tract at E10 of mouse development. Once formed by the EMT, the mesenchymal cells invade the cardiac jelly, proliferate, and populate the endocardial cushion. The cellularized endocardial cushion then undergoes morphological remodeling; it lengthens and matures into a thin elongated valve leaflet. Here we describe a method to culture endocardial cushions and measure EMT ex vivo. EMT can thus be analyzed independent of other concurrent developmental defects in mice. This culture method also enables ex vivo manipulations of signaling or gene function during EMT to delineate molecular pathways essential for heart valve development.

Formation of the cardiac outflow tract and great arteries involves complex morphogenetic processes, whose abnormities result in several clinically important diseases. Studies of these developmental processes are therefore important for understanding congenital vascular defects. However, the three-dimensional structure of arteries makes it challenging to analyze the pattern of vasculature using conventional histological approaches. Here we describe a vascular casting method to visualize the branching and connections of great arteries in developing embryos as well as in adult mice. This technique can be used to study the development of cardiac outflow tract, semilunar valves, and great arteries as demonstrated previously (Circ Res, 2008; Development 135: 3577-3586, 2008).