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Optical mapping of electrical activation in the developing heart.

Specialized conduction tissues mediate coordinated propagation of electrical activity through the adult vertebrate heart. Following activation of the atria, the activation wave is slowed down in the atrioventricular canal or node, after which it spreads rapidly into the left and right ventricles via the His-Purkinje system (HPS). This results in the ventricles being activated from the apex toward the base, which is a hallmark of HPS function. The development of mature HPS function follows significant phases of cardiac morphogenesis. Initially, the cardiac impulse propagates in a slow, linear, and isotropic fashion from the sinus venosus at the most caudal portion of the tubular heart. Although the speed of impulse propagation gradually increases as it travels toward the anterior regions of the heart tube, the actual sequence of ventricular activation in the looped heart proceeds in the same direction as blood flow. Eventually, the immature base-to-apex sequence of ventricular activation undergoes an apparent reversal, changing to the mature apex-to-base pattern. Using an optical mapping approach, we demonstrate that the timing of this last transition shows striking dependence on hemodynamic loading of the ventricle, being accelerated by pressure overload and delayed in left ventricular hypoplasia. Comparison of chick and mammalian hearts revealed some striking similarities as well as key differences in the timing of such events during cardiac organogenesis.

Action Potentials↗

A new human homeobox gene OGI2X is a member of the most conserved homeobox gene family and is expressed during heart development in mouse.

Homeodomain (HD) proteins are transcription regulators controlling a variety of cell fates. The HD region characterizing this protein family is a domain of 60 amino acid residues that recognizes and binds a site in the regulatory region of the target gene. It has been suggested that regions outside the HD may determine the specific functions of the various HD proteins by forming additional contacts with DNA sequences or by interactions with other proteins. We have identified a 14 amino acid motif within the C-terminal region of the protein encoded by the RIEG1 gene that is conserved among several HD proteins. Overlapping expression of the genes encoding these proteins during craniofacial development suggested that they might interact with a common factor. In order to identify additional genes possessing this motif we screened a human craniofacial cDNA library with oligoprobes. A novel gene was identified, exhibiting the most homology to murine Og12x (formerly OG12) and the recently reported human SHOX gene. Human OG12X and murine Og12x are highly homologous and the OG12X and Og12x proteins are 100% identical. In situ hybridization on mouse embryos ranging from 9 to 16 days post-coitum localized murine Og12x mRNA in the heart, otic region, maxillary and mandibular components of the first branchial arch, nasal processes, eyelid, midbrain, medulla oblongata, limbs, dorsal root ganglia and genital tubercle. OG12X was mapped to human chromosome 3q22-26 and murine Og12x to the syntenic region on mouse chromosome 3. Based upon the expression pattern of its mouse cognate, OG12X represents a candidate for the blepharophimosis (BPES) and Cornelia de Lange syndromes previously mapped to this region.

Amino Acid Sequence↗

Expression of bone morphogenetic protein-10 mRNA during chicken heart development.

In this communication we describe the expression pattern of BMP10 mRNA during cardiac development in chickens. BMP10 is considered an important factor in the regulation of cardiac growth and trabeculation in the murine embryo. We identified chicken Ests, which are similar to mouse and human BMP10 in the UMIST database. The cDNA clone that contained most sequences was obtained, verified by sequence analysis, and used to determine the spatiotemporal pattern of gene expression. BMP10 mRNA is initially expressed at HH10 in the myocardium of the arterial pole of the heart tube, anterior to the interventricular groove. Between HH14 and HH22, BMP10 mRNA becomes broadly expressed in the outflow tract, the distal part of the inflow tract, and the trabeculated part of the developing ventricles and atria. From HH31 onward, BMP10 mRNA expression decreases in the ventricular myocardium by first disappearing from the compact myocardium and then from the tips of the trabecules. At HH44, BMP10 mRNA is expressed only in the trabeculated myocardium of the atria and the endocardium of the ventricles. The observed expression pattern of BMP10 mRNA suggests that it may play a role in regulating the formation of the ventricular wall and trabecules.

Animals↗

Selected views of early heart development by scanning electron microscopy.

This tutorial on cardiac development is designed to acquaint the novice student of embryology with the key events that occur during cardiogenesis. Each of the following events is depicted through a series of scanning electron micrographs which convey the spatial relationships between minute, yet essential structures: fusion of paired heart tubes; looping; partitioning of the common atrium, the atrioventricular canal, the primitive ventricle and the outflow tract. The cellular and biochemical mechanisms responsible for these events cannot be adequately determined nor illustrated by SEM and therefore will not be considered in detail herein.

Animals↗

Regulation of voltage-dependent Ca2+ channels in the early developing heart: role of beta1 integrins.

In contrast to adult ventricular cardiomyocytes the developmentally early stage cardiomyocytes show a suppression of the basal voltage-dependent calcium channels (DCC L-type Ca2+ channels, I(Ca)) by carbachol (CCh). This effect is mediated by the endothelial NO-synthase (NOS III). In contrast late stage and adult cardiomyocytes a direct coupling of the muscarinic receptor to the adenylyl cyclase. Thus, NO may function as an early signal transduction molecule during development. This review elucidates the role of beta1-integrins in mediating signal transduction between muscarinic receptors and coupled downstream target proteins such as ion channels. The key finding is that in embryonic stem cell-derived cardiomyocytes deficient of beta1-integrins, the modulation of L-type Ca2+ channels via the M2 receptor is absent. Experiments indicate that this selective signaling defect occurs at the G-protein level. This suggests a novel critical role for integrins in membrane delimited signal transduction processes.

Animals↗

Biophysics of the developing heart. II. The interaction of the force-interval relationship with inotropic state and muscle length (preload).

The interaction of the force-interval relationship, muscle length (ventricular volume), and inotropy was evaluated in isolated fetal myocardial preparations and in the in utero chronically instrumented fetal lamb. In the isolated muscle, 93 to 141 days' gestation, a change in muscle length strengthened all contractions equally (multiplicatively) but produced no other changes in either the quantitative or the qualitative characteristics of the force-interval relationship; it was significantly altered by isoproterenol. Similarly, in the intact fetal lamb the qualitative and quantitative features of the relationship did not depend on ventricular volume, e.g., postextrasystolic potentiation was constant over a wide range of volumes. But isoproterenol produced a decrease in postextrasystolic potentiation. These results demonstrate that the force-interval relationship satisfies two major criteria for an index of cardiac contractility and suggest that the relationship may provide a basis for the evaluation of changes in contractility with development and birth.

Animals↗

Cloning and expression of fibroblast growth factor receptor-1 isoforms in the mouse heart: evidence for isoform switching during heart development.

Basic (b) fibroblast growth factor (FGF) mediates various biological responses including mitogenesis and angiogenesis by binding to specific cell surface receptors of the tyrosine kinase family. The bFGF receptor-1 FGFR1) exists in short and long isoforms due to alternate RNA splicing. Minor alterations in the amino acid sequence have also led to reports of different FGFR1 isoforms in different tissues even in the same species. In the absence of any sequence for heart FGFR1 and accumulating evidence for a role of bFGF in heart growth and differentiation, we cloned FGFR1 from embryonic mouse hearts. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to generate full-length short (2259 base pairs) and long (2526 base pairs) forms of FGFR1 cDNAs which generated 86 and 102 kDa proteins, respectively, following in vitro translation. Embryonic mouse heart FGFR1 differed by seven amino acids from the reported sequence for mouse neuroepithelial FGFR1 and appeared more similar to human placental FGFR1. A single FGFR1 transcript of approximately 4.3 kb was seen in RNA isolated from embryonic as well as adult mouse hearts. There was a decrease (approximately 8.5-fold) in FGFR1 RNA levels in the adult. The majority of FGFR1 transcripts in the adult as well as embryonic heart contained exon IIIc (FGFR1-IIIc) which is associated with isoforms that display the highest affinity for bFGF. However, the relative ratio of short versus long FGFR1 RNA expression was 0.5 in the embryonic heart compared to 5.9 in the adult heart. These results indicate that: (i) structurally distinct short and long FGFR1 isoform RNAs are expressed in the embryonic and adult heart; (ii) FGFR1-IIIc is the major form of receptor expressed in the embryonic as well as adult heart; (iii) the transition from the embryo to the adult stage is associated with a decrease but not absence of FGFR1 RNA expression; and (iv) long FGFR1-isoforms are more abundant in the embryo while short FGFR1 isoforms predominate in the adult.

Amino Acid Sequence↗

Baf60c is essential for function of BAF chromatin remodelling complexes in heart development.

Tissue-specific transcription factors regulate several important aspects of embryonic development. They must function in the context of DNA assembled into the higher-order structure of chromatin. Enzymatic complexes such as the Swi/Snf-like BAF complexes remodel chromatin to allow the transcriptional machinery access to gene regulatory elements. Here we show that Smarcd3, encoding Baf60c, a subunit of the BAF complexes, is expressed specifically in the heart and somites in the early mouse embryo. Smarcd3 silencing by RNA interference in mouse embryos derived from embryonic stem cells causes defects in heart morphogenesis that reflect impaired expansion of the anterior/secondary heart field, and also results in abnormal cardiac and skeletal muscle differentiation. An intermediate reduction in Smarcd3 expression leads to defects in outflow tract remodelling reminiscent of human congenital heart defects. Baf60c overexpressed in cell culture can mediate interactions between cardiac transcription factors and the BAF complex ATPase Brg1, thereby potentiating the activation of target genes. These results reveal tissue-specific and dose-dependent roles for Baf60c in recruiting BAF chromatin remodelling complexes to heart-specific enhancers, providing a novel mechanism to ensure transcriptional regulation during organogenesis.

Animals↗

Cardia bifida, defective heart development and abnormal neural crest migration in embryos lacking hypoxia-inducible factor-1alpha.

OBJECTIVES: Previous studies have revealed the essential role of hypoxia-inducible factor-1alpha (HIF-1alpha), a basic helix-loop-helix transcription factor, in cardiovascular development. We attempted to further characterize the underlying mechanisms resulting in abnormal cardiogenesis and defective angiogenesis in mice deficient for HIF-1alpha (HIF-1alpha(-/-)). METHODS: We analyzed cardiovascular development in HIF-1alpha(-/-) embryos at both the macroscopic and microscopic level. Gene expression was determined by RT-PCR, in situ hybridization and immunohistochemistry. Embryonic survival was studied using whole embryo culture. RESULTS: HIF-1alpha deficiency caused cardia bifida in some embryos, while cardiac looping was disturbed in others. These defects did not result from abnormal cardiomyocyte commitment or differentiation, but may relate to defective ventricle formation caused by reduced expression of myocyte enhancer factor 2C (MEF2C) and eHAND. In addition, remodeling of the aortic outflow tract and cephalic blood vessels was abnormal in HIF-1alpha(-/-) embryos. These malformations, together with the hypoplastic pharyngeal arches, are presumably induced by defective neural crest cell (NCC) migration. Impaired migration might be related to insufficient levels of semaphorin-3A (Sema3A). Hyperoxia prolonged survival but only partially rescued the developmental program of cultured HIF-1alpha(-/-) embryos. CONCLUSION: HIF-1alpha is essential for proper cardiac development by modulating both neural crest migration and ventricle formation.

Abnormalities, Multiple↗

Dynamic patterns of expression of BMP isoforms 2, 4, 5, 6, and 7 during chicken heart development.

Bone morphogentic proteins (BMPs) play an important role in cardiac development. Using an in vitro explant analysis, we show that BMPs are crucial for myocardium formation. As a first approach to identify which BMP may be involved in myocardium formation in intra- and extracardiac mesenchyme in vivo, a survey of the expression patterns of BMP2, -4, -5, -6, and -7 mRNA is prepared by in situ hybridization in chicken embryonic hearts from HH5 to 44. During recruitment of mesodermal cells to the outflow tract myocardium (HH10-23), BMP2, -4, -5, and -7 mRNA are expressed in the distal myocardial border and the flanking mesenchyme. After completion, BMP2 and -4 mRNA become restricted to the mesenchyme and BMP5 and -7 mRNA to the myocardium. At the venous pole, BMP2, -5, and -7 mRNA are expressed in the distal myocardial border of the caval vein, while BMP2, -5, -6, and -7 mRNA are expressed in the distal myocardium around the pulmonary vein. BMP4 mRNA is expressed in the adjacent mesenchyme at both sides. During muscularization of the atrioventricular cushions and the tricuspid valve, the cardiomyocytes that protrude into the mesenchyme express BMP2, -4, -5, and -7 mRNA, whereas BMP6 mRNA is expressed in the cushion mesenchyme. The myocardial protrusions formed in the mesenchymal proximal outlet septum express BMP4, -5, and -7 mRNA, while BMP2 and -6 mRNA are expressed in the mesenchyme. The spatiotemporal expression patterns of these BMPs in relation to myocardium formation at the distal ends and within the heart suggest a role for BMPs in myocardium formation. During delamination of the valves, BMP4 and -6 mRNA are expressed at the ventricular side of the forming mitral valve, BMP4 mRNA at the ventricular side of the forming tricuspid valve, and BMP2, -4, and -6 mRNA at the vascular side of the forming semilunar valves.

Animals↗

Acidic fibroblast growth factor and heart development. Role in myocyte proliferation and capillary angiogenesis.

Proliferative growth of the ventricular myocyte (cardiomyocyte) is primarily limited to fetal and early neonatal periods of development. In concert with the neonatal "transition" from proliferative to hypertrophic growth, ventricular remodeling of the nonmyocyte compartment is characterized by increased extracellular matrix synthesis/deposition and capillary angiogenesis. A role for locally generated and bioactive ventricular acidic fibroblast growth factor (aFGF) in these processes is proposed and substantiated by the following: 1) colocalization of aFGF peptide and fibroblast growth factor receptor (flg) transcripts to the developing fetal cardiomyocyte by immunohistochemistry, immunoelectron microscopy, and in situ hybridization, 2) continued localization of aFGF peptide and transcripts to the neonatal/mature cardiomyocyte, and 3) localization of flg immunoreactivity and transcripts to specific neonatal ventricular nonmuscle cell types. Specific ventricular cell types at distinct developmental stages appear to be responsive to ventricular myocyte-derived aFGF (myocytes in the fetal heart and nonmyocytes/endothelial cells in the neonatal heart). These data indicate that expression of aFGF and one of its receptors (flg) are most pronounced in the fetal to early neonatal ventricle, the presence of both suggesting an autocrine/paracrine growth regulatory function. As the animal matures, ventricular capillary angiogenesis may be facilitated by "release" of cardiomyocyte-derived fibroblast growth factors into the surrounding extracellular space/matrix functioning as a "paracrine" angiogenic stimuli. Therefore, the results of our study suggest that myocyte-derived aFGF may function to increase the fetal ventricular cardiomyocyte population in absolute number as well as to facilitate the subsequent increase in capillary angiogenesis that occurs during cardiomyocyte maturation and ventricular remodeling.

Animals↗

Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.

The homeobox gene tinman and the nuclear receptor gene seven-up are expressed in mutually exclusive dorsal vessel cells in Drosophila, however, the physiological reason for this distinction is not known. We demonstrate that tin and svp-lacZ expression persists through the larval stage to the adult stage in the same pattern of cells expressing these genes in the embryo. In the larva, six pairs of Svp-expressing cells form muscular ostia, which permit hemolymph to enter the heart for circulation, however, more anterior Svp-expressing cells form the wall of the dorsal vessel. During pupation, the adult heart forms from a chimera of larval and imaginal muscle fibers. The portion of the dorsal vessel containing the larval ostia is histolyzed and the anterior Svp-expressing cells metamorphose into imaginal ostia. This is the first demonstration that the significant molecular diversity of cardial cells identified in the embryonic heart correlates with the formation of physiologically and functionally distinct muscle cells in the animal. Furthermore, our experiments define the cellular changes that occur as the larval heart is remodeled into an imaginal structure in an important model organism.

Animals↗

Acute phase lipocalin Ex-FABP is involved in heart development and cell survival.

Ex-FABP is an extracellular fatty acid binding protein, expressed during chicken embryo development in cartilage, muscle fibers, and blood granulocytes. Transfection of chondrocytes and myoblasts with anti-sense Ex-FABP cDNA results in inhibition of cell proliferation and apoptosis induction. Ex-FABP expression is dramatically enhanced by inflammatory stimuli and in pathological conditions. In this paper, by in situ whole mount and immunohistochemistry analysis we show that, at early developmental stage, Ex-FABP is diffuse in all tissues of chick embryos. Particularly high level of transcript and protein are expressed in the heart. During acute phase response (APR) induced by endotoxin LPS injection, a marked increase of Ex-FABP mRNA was observed in embryos, highest Ex-FABP expression being in heart and liver. To investigate in vivo the biological role of Ex-FABP, we have directly microinjected chicken embryos with antibody against Ex-FABP. Almost 70% of chicken embryos died and the target tissue was the heart. We detected in heart of the treated embryos a significant increase of apoptotic cells and high level of fatty acids. We propose that the accumulation of fatty acid, specific ligand of Ex-FABP, in the cell microenvironment is responsible of heart cell death, and we suggest that Ex-FABP may act as a survival protein by playing a role as scavenger for fatty acids.

Animals↗

Opioid antagonist modulation of rat heart development.

Endogenous opioids are known to regulate morphogenesis in both neural and non-neural systems. This study examined whether endogenous opioids influence cardiac development. Naltrexone, a potent opioid antagonist that blocks the interaction of opioid peptides and opioid receptors, was administered acutely (50 mg/kg) to 1-day old rats. The numbers of myocardial and epicardial cells in the ventricles and atria that synthesized DNA, as determined by [3H]-thymidine incorporation and autoradiography, were markedly increased from control levels. Labeling indices were significantly elevated for at least 12 hr following a single injection of naltrexone. Examination of 10-day old rats exposed to naltrexone from birth revealed higher labeling indices, as well as increases in body and heart weights and in areal measurements of the entire heart and the ventricles. The effects of naltrexone were not mediated through the sympathetic nervous system or thyroid hormone. These results lead one to suggest that an opioid peptide is tonically acting as a negative regulatory factor in the formation of the heart. Alterations in the endogenous opioid system in early life may contribute to cardiac dysmorphogenesis. Moreover, these data indicate that opioid antagonists could act as an important therapeutic influence with regard to cardiac malformations.

Aging↗

Regulation of the epithelial-mesenchymal transformation through gap junction channels in heart development.

Analyses of mice lacking the gap junction protein, connexin45 (Cx45), have provided new insights into the essential roles of gap junction channels in early embryogenesis. Of great surprise is the function of Cx45 in the endothelium, where it is essential for synchronized activation of the transcription factor Nfatc1. This laterally synchronized regulation model extends the generally accepted vertical model, in which interactions between the endocardium and the myocardium induce endocardial cushion formation through the epithelial-mesenchymal transformation.

Animals↗

Trichloroethylene, trichloroacetic acid, and dichloroacetic acid: do they affect fetal rat heart development?

Trichloroethylene (TCE), trichloroacetic acid (TCA), and dichloroacetic acid (DCA) are commonly found as groundwater contaminants in many regions of the United States. Cardiac birth defects in children have been associated with TCE, and laboratory studies with rodents report an increased incidence of fetal cardiac malformations resulting from maternal exposures to TCE, TCA, and DCA. The objective of this study was to orally treat pregnant CDR(CD) Sprague-Dawley rats with large bolus doses of either TCE (500 mg/kg), TCA (300 mg/kg), or DCA (300 mg/kg) once per day on days 6 through 15 of gestation to determine the effectiveness of these materials to induce cardiac defects in the fetus. All-trans retinoic acid (RA) dissolved in soybean oil was used as a positive control. Soybean oil is commonly used as a dosing vehicle for RA teratology studies and was also used in this study as a dosing vehicle for TCE. Water was used as the dosing vehicle for TCA and DCA. Fetal hearts were examined on gestation day (GD) 21 by an initial in situ, cardiovascular stereomicroscope examination, and then followed by a microscopic dissection and examination of the formalin-fixed heart. The doses selected for TCA and DCA resulted in a modest decrease in maternal weight gain during gestation (3% to 8%). The fetal weights on GD 21 in the TCA and DCA treatment groups were decreased 8% and 9%, respectively, compared to the water control group and 21% in the RA treatment group compared to soybean oil control group. The heart malformation incidence for fetuses from the TCE-, TCA-, and DCA-treated dams did not differ from control values on a per fetus or per litter basis. The rate of heart malformations, on a per fetus basis, ranged from 3% to 5% for TCE, TCA, and DCA treatment groups compared to 6.5% and 2.9% for soybean oil and water control groups. The RA treatment group was significantly higher with 33% of the fetuses displaying heart defects. For TCE, TCA, and DCA treatment groups 42% to 60% of the litters contained at least one fetus with a heart malformation, compared to 52% and 37% of the litters in the soybean oil and water control groups. For the RA treatment group, 11 of 12 litters contained at least one fetus with a heart malformation. Further research is needed to quantify the spontaneous rates of heart defects for vehicle control rats and to explain the disparity between findings in the present study and other reported findings on the fetal cardiac teratogenicity of TCE, TCA, and DCA.

Animals↗