Changes in Cardiomyocyte Cell Cycle and Hypertrophic Growth During Fetal to Adult in Mammals.

The failure of adult cardiomyocytes to reproduce themselves to repair an injury results in the development of severe cardiac disability leading to death in many cases. The quest for an understanding of the inability of cardiac myocytes to repair an injury has been ongoing for decades with the identification of various factors which have a temporary effect on cell-cycle activity. Fetal cardiac myocytes are continuously replicating until the time that the developing fetus reaches a stage of maturity sufficient for postnatal life around the time of birth. Recent reports of the ability for early neonatal mice and pigs to completely repair after the severe injury has stimulated further study of the regulators of the cardiomyocyte cell cycle to promote replication for the remuscularization of injured heart. In all mammals just before or after birth, single-nucleated hyperplastically growing cardiomyocytes, 1X2N, undergo ≥1 additional DNA replications not followed by cytokinesis, resulting in cells with ≥2 nuclei or as in primates, multiple DNA replications (polyploidy) of 1 nucleus, 2X2(+)N or 1X4(+)N. All further growth of the heart is attributable to hypertrophy of cardiomyocytes. Animal studies ranging from zebrafish with 100% 1X2N cells in the adult to some strains of mice with up to 98% 2X2N cells in the adult and other species with variable ratios of 1X2N and 2X2N cells are reviewed relative to the time of conversion. Various structural, physiologic, metabolic, genetic, hormonal, oxygenation, and other factors that play a key role in the inability of post-neonatal and adult myocytes to undergo additional cytokinesis are also reviewed.

G-protein receptor kinases 2, 5 and 6 redundantly modulate Smoothened-GATA transcriptional crosstalk in fetal mouse hearts.

G-protein receptor kinases (GRKs) regulate adult hearts by modulating inotropic, chronotropic and hypertrophic signaling of 7-transmembrane spanning neurohormone receptors. GRK-mediated desensitization and downregulation of ß-adrenergic receptors has been implicated in adult heart failure; GRKs are therefore a promising therapeutic target. However, germ-line (but not cardiomyocyte-specific) GRK2 deletion provoked lethal fetal heart defects, suggesting an unexplained role for GRKs in heart development. Here we undertook to better understand the consequences of GRK deficiency on fetal heart development by creating mice and cultured murine embryonic fibroblasts (MEFs) having floxed GRK2 and GRK5 alleles on the GRK6 null background; simultaneous conditional deletion of these 3 GRK genes was achieved using Nkx2-5 Cre or adenoviral Cre, respectively. Phenotypes were related to GRK-modulated gene expression using whole-transcriptome RNA sequencing, RT-qPCR, and luciferase reporter assays. In cultured MEFs the atypical 7-transmembrane spanning protein and GRK2 substrate Smoothened (Smo) stimulated Gli-mediated transcriptional activity, which was interrupted by deleting GRK2/5/6. Mice with Nkx2-5 Cre mediated GRK2/5/6 ablation died between E15.5 and E16.5, whereas mice expressing any one of these 3 GRKs (i.e. GRK2/5, GRK2/6 or GRK5/6 deleted) were developmentally normal. GRK2/5/6 triple null mice at E14.5 exhibited left and right heart blood intermixing through single atrioventricular valves or large membranous ventricular septal defects. Hedgehog and GATA pathway gene expression promoted by Smo/Gli was suppressed in GRK2/5/6 deficient fetal hearts and MEFs. These data indicate that GRK2, GRK5 and GRK6 redundantly modulate Smo-GATA crosstalk in fetal mouse hearts, orchestrating transcriptional pathways previously linked to clinical and experimental atrioventricular canal defects. GRK modulation of Smo reflects convergence of conventional neurohormonal signaling and transcriptional regulation pathways, comprising an unanticipated mechanism for spatiotemporal orchestration of developmental gene expression in the heart.

Anormalidades del tamaño del corazón fetal / Anomalies in the fetal heart disease

Introducción: el corazón fetal es la víscera más difícil de estudiar, debido a que es un órgano móvil con una anatomía compleja y presenta un número importante de anomalías posibles. Objetivos: exponer al alcance de los especialistas dedicados al diagnóstico prenatal una revisión de las posibles causas que modifican el tamaño del corazón fetal. Métodos: la evaluación de su tamaño se realiza a partir de la vista ecocardiográfica de las cuatro cámaras la cual es obtenida realizando un barrido ultrasonográfico desde el abdomen hasta el tórax. Cuando el tamaño es normal, ocupa un tercio del tórax fetal y la circunferencia cardíaca equivale aproximadamente a la mitad de la circunferencia torácica durante todo el curso del embarazo. Resultados: las anomalías del tamaño del corazón pueden ser ocasionadas por disminución o por incremento del radio cardiotorácico. En relación con su incremento es muy importante definir si este es debido a un crecimiento global del corazón o si está afectada una cavidad auricular, ventricular o ambas. En cuanto a la disminución del tamaño este siempre será debido a compresiones intratoráxicas. Conclusiones: la evaluación ecocardiográfica del corazón fetal resulta posible en casi la totalidad de los casos y aporta un elemento de valor al examen prenatal del feto(AU)

Introduction: Fetus heart is the most difficult viscera to be studied, due to the fact that it is a moving organ with a complex anatomy and an important amount of possible anomalies. Objective: To provide the specialists devoted to the prenatal diagnose with a review of the possible causes that modify the fetus heart size. Methods: The heart size is evaluated from an echocardiographic image of its four chambers, which is obtained in an ultrasonic scanning¨from the abdomen to the thorax. When the size is normal, it occupies one third of the fetal thorax and the cardiac circumference is roughly equal to half the thorax circumference throughout the pregnancy. Results: Heart size anomalies could be caused by a drop or an increase of the cardiothoracic radius. It is very important to define whether the increase is due to a global growth of the heart or to an affected atrial or ventricular cavity, or both. Size decreases will always be connected to an intrathoracic compression. Conclusions: Echocardiographic assessment of the fetal heart is possible in almost all cases and provides a valuable element to the fetus prenatal testing(AU)

The roadmap of WT1 protein expression in the human fetal heart.

The transcription factor Wilms' Tumor-1 (WT1) is essential for cardiac development. Deletion of Wt1 in mice results in disturbed epicardial and myocardial formation and lack of cardiac vasculature, causing embryonic lethality. Little is known about the role of WT1 in the human fetal heart. Therefore, as a first step, we analyzed the expression pattern of WT1 protein during human cardiac development from week 4 till week 20. WT1 expression was apparent in epicardial, endothelial and endocardial cells in a spatiotemporal manner. The expression of WT1 follows a pattern starting at the epicardium and extending towards the lumen of the heart, with differences in timing and expression levels between the atria and ventricles. The expression of WT1 in cardiac arterial endothelial cells reduces in time, whereas WT1 expression in the endothelial cells of cardiac veins and capillaries remains present at all stages studied. This study provides for the first time a detailed description of the expression of WT1 protein during human cardiac development, which indicates an important role for WT1 also in human cardiogenesis.

Cardiac structure/function, protein expression, and DNA methylation are changed in adult female mice exposed to diethylstilbestrol in utero.

The detrimental effects of in utero exposure to the non-steroidal estrogen diethylstilbestrol (DES) are particularly marked in women. Fetal hearts express estrogen receptors, making them potentially responsive to DES. To examine whether gestational exposure to DES would impact the heart, we exposed pregnant C57bl/6n dams to DES (0.1, 1.0, and 10.0 µg·(kg body mass)(-1)·day(-1)) on gestation days 11.5-14.5, and examined the measured cardiac structure/function and calcium homeostasis protein expression in adult females. At baseline, echocardiography revealed eccentric hypertrophy in mice treated with 10.0 µg·(kg body mass)(-1)·day(-1) DES, and immunoblots showed increased SERCA2a in all DES-treated mice. Mice were swim-trained to assess cardiac remodeling. Swim-trained vehicle-treated mice developed eccentric hypertrophy without changing SERCA2 or calsequestrin 2 expression. In contrast, no DES-treated mice hypertrophied, and all increased in SERCA2a and calsequestrin 2 expression after training. To determine whether DES-induced changes in DNA methylation is part of the mechanism for its long-term effects, we measured DNA methyltransferase expression and DNA methylation. Global DNA methylation and DNA methyltransferase 3a expression were unchanged. However, DES-treated mice had increased DNA methylation in the calsequestrin 2 promoter. Thus, gestational exposure to DES altered female ventricular DNA, cardiac structure/function, and calcium homeostasis protein expression. We conclude that gestational exposure to estrogenizing compounds may impact cardiac structure/function in adult females.

Reversal of slow growth and heartbeat through the restoration of mitochondrial function in clk-1-deficient mouse embryos by exogenous administration of coenzyme Q10.

The longevity gene clk-1/coq7 encodes an enzyme that is essential for the biosynthesis of coenzyme Q (CoQ) in mitochondria and regulates the lifespan and behavioral timing in Caenorhabditis elegans and the chronological lifespan in fission yeast. However, whether the mammalian clk-1/coq7 ortholog (clk-1) regulates these phenotypes in mammals remains to be fully evaluated due to the embryonic lethality of clk-1-deficient (clk-1(-/-)) mice. To investigate whether clk-1 regulates biological functions, such as growth and heartbeat, through CoQ in mouse embryos, we cultivated the cells and hearts of clk-1(-/-) mouse embryos at embryonic day 10.5 (E10.5) for at least 10 days in the presence of fetal bovine serum. In embryonic cells, cardiomyocytes, and hearts, the growth and heart rates were significantly slowed in clk-1(-/-) compared with wild-type or heterozygous mouse tissues. Moreover, frequent apoptosis and a significant reduction in mitochondrial functions, including membrane potential and ATP production, were observed in the clk-1(-/-) cells and hearts. The slowed growth and heart rates and the reduced mitochondrial function of clk-1(-/-) embryonic cells and hearts in culture were almost completely rescued by the administration of exogenous CoQ(10). The results indicate that clk-1 regulates growth and heart rates through CoQ-mediated mitochondrial functions in mouse embryos.

Natural history of prenatal ventricular septal defects and their association with foetal echocardiographic features.

OBJECTIVE: To describe the evolution of ventricular septal defects in infants from intra-uterine diagnosis to the age of 3 years or until documented echocardiographic closure of the defect, as well as any relationship between closure rate, time and foetal echocardiographic features. METHODS: Between January, 2004 and December, 2006, 268 cases of congenital cardiac defect were detected in 14,993 pregnancies referred to our hospital for routine foetal echocardiography; of these cases, 125 had isolated ventricular septal defect. The mothers were scheduled for regular ultrasonography every 2 weeks from diagnosis until the ventricular septal defect closed or 3 years postnatally. RESULTS: Of the 125 cases of ventricular septal defects, the pregnancy was terminated in 25, four resulted in death, two defects closed spontaneously in utero, 55 closed at a mean age of 13.7 months postnatally, 17 were treated with surgery, nine remained unclosed, and 13 cases were lost to follow-up. Only 7.7% of muscular ventricular septal defects remained patent as compared with 35.7% of perimembranous ventricular septal defects (p is less than 0.01). Muscular ventricular septal defects closed earlier than perimembranous ventricular septal defects. All the ventricular septal defects less than or equal to 3 millimetres closed, whereas only 79.5% of the defects greater than 3 millimetres closed before the age of 3 years; 60.9% of the defects less than or equal to 3 millimetres closed before the age of 1 year as compared with 41.7% of the defects greater than 3 millimetres. The velocity of right-to-left flow was negatively correlated with closure rate but not related to closure period. CONCLUSION: Ventricular septal defects can close in utero or during the postnatal period, and both the size and site play a role in the natural history, with small and muscular ventricular septal defects having a high closure rate and early closure.

Histone deacetylase 3 regulates smooth muscle differentiation in neural crest cells and development of the cardiac outflow tract.

RATIONALE: The development of the cardiac outflow tract (OFT) and great vessels is a complex process that involves coordinated regulation of multiple progenitor cell populations. Among these populations, neural crest cells make important contributions to OFT formation and aortic arch remodeling. Although numerous signaling pathways, including Notch, have been implicated in this process, the role of epigenetics in OFT development remains largely unexplored. OBJECTIVE: Because histone deacetylases (Hdacs) play important roles in the epigenetic regulation of mammalian development, we have investigated the function of Hdac3, a class I Hdac, during cardiac neural crest development in mouse. METHODS AND RESULTS: Using 2 neural crest drivers, Wnt1-Cre and Pax3(Cre), we show that loss of Hdac3 in neural crest results in perinatal lethality and cardiovascular abnormalities, including interrupted aortic arch type B, aortic arch hypoplasia, double-outlet right ventricle, and ventricular septal defect. Affected embryos are deficient in aortic arch artery smooth muscle during midgestation, despite intact neural crest cell migration and preserved development of other cardiac and truncal neural crest derivatives. The Hdac3-dependent block in smooth muscle differentiation is cell autonomous and is associated with downregulation of the Notch ligand Jagged1, a key driver of smooth muscle differentiation in the aortic arch arteries. CONCLUSIONS: These results indicate that Hdac3 plays a critical and specific regulatory role in the neural crest-derived smooth muscle lineage and in formation of the OFT.

Myocardial smad4 is essential for cardiogenesis in mouse embryos.

Congenital heart diseases are the most commonly observed human birth defects and are the leading cause of infant morbidity and mortality. Accumulating evidence indicates that transforming growth factor-beta/bone morphogenetic protein signaling pathways play critical roles during cardiogenesis. Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of transforming growth factor-beta/bone morphogenetic protein signaling. The aim of this work was to investigate the roles of Smad4 during heart development. To overcome the early embryonic lethality of Smad4(-/-) mice, we specifically disrupted Smad4 in the myocardium using a Cre/loxP system. We show that myocardial-specific inactivation of Smad4 caused heart failure and embryonic lethality at midgestation. Histological analysis revealed that mutant mice displayed a hypocellular myocardial wall defect, which is likely the primary cause for heart failure. Both decreased cell proliferation and increased apoptosis contributed to the myocardial wall defect in mutant mice. Data presented in this article contradict a previous report showing that Smad4 is dispensable for heart development. Our further molecular characterization showed that expression of Nmyc and its downstream targets, including cyclin D1, cyclin D2, and Id2, were downregulated in mutant embryos. Reporter analysis indicated that the transcriptional activity of the 351-bp Nmyc promoter can be positively regulated by bone morphogenetic protein stimulation and negatively regulated by transforming growth factor-beta stimulation. Chromatin immunoprecipitation analysis revealed that the Nmyc promoter can form a complex with Smad4, suggesting that Nmyc is a direct downstream target of Smad4. In conclusion, this study provides the first mouse model showing that Smad4 plays essential roles during cardiogenesis.

The effect of vascular endothelial growth factor on in vitro embryonic heart development in rats.

In vitro effects of vascular endothelial growth factor (VEGF) on heart development and total embryonic growth were investigated in 84 rat embryos (obtained from nine pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), in <30 kDa + >50 kDa serum fractions [retenate (R)], and in R + VEGF. After 24-h culture, the embryos from each group were harvested and divided into two groups. One group was analysed morphologically and biochemically to obtain embryo protein content, the second group was serially sectioned and examined by light microscopy. Morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in R had significant embryonic retardation, whereas the addition of VEGF to R increased embryonic growth and development. The morphological scores for WRS, R and R + VEGF were 57.7 +/- 0.87, 46.6 +/- 1.90 and 52.1 +/- 0.97, somite numbers were 26.5 +/- 0.47, 20.1 +/- 0.63 and 24.4 +/- 0.46, crown-rump lengths were 3 +/- 0.07, 2.4 +/- 0.06 and 2.7 +/- 0.06 mm, and embryo protein contents were 160.5 +/- 7.41, 98.2 +/- 4.81 and 141.1 +/- 10.96 mug per embryo, respectively. The results of histological examination of heart development were similar. The hearts of embryos grown in R were unseptated and tubular. The atrioventricular endocardial cushions were incompletely developed. The addition of VEGF to R improved heart development. There were no gross morphological differences in the cardiac development between embryos grown in WRS and R + VEGF. In both groups, development of the muscular interventricular septum had begun. Development of the atrioventricular cushions was also similar in both groups and had caused narrowing of the atrioventricular canals, but the atrial septation was not observed.

Neural and mammary gland defects in ErbB4 knockout mice genetically rescued from embryonic lethality.

Mice lacking the epidermal growth factor receptor family member ErbB4 exhibit defects in cranial neural crest cell migration but die by embryonic day 11 because of defective heart development. To examine later phenotypes, we rescued the heart defects in ErbB4 mutant mice by expressing ErbB4 under a cardiac-specific myosin promoter. Rescued ErbB4 mutant mice reach adulthood and are fertile. However, during pregnancy, mammary lobuloalveoli fail to differentiate correctly and lactation is defective. Rescued mice also display aberrant cranial nerve architecture and increased numbers of large interneurons within the cerebellum.

The growth promoting effects of bFGF, PD-ECGF and VEGF on cultured postimplantation rat embryos deprived of serum fractions.

Serum components in which embryos are cultured in vitro are very important for normal embryonic development. In this study, rat serum was fractionated using Macrosep filters to study the effect of a single growth factor. The fractionated serum, both that containing only material greater than 30 kDa molecular weight (> 30 kDa) and that from which material between 30 kDa and 50 kDa had been removed (< 30 kDa+ > 50 kDa), caused significant embryonic growth retardation. Addition of different concentrations of basic fibroblast growth factor (bFGF, 18 kDa), vascular endothelial growth factor (VEGF, 45 kDa) and platelet-derived endothelial growth factor (PD-ECGF, 45 kDa), to fractionated serum (bFGF to > 30 kDa serum and VEGF or PD-ECGF to < 30 kDa+ > 50 kDa serum) partially restored embryonic growth and development according to a morphological scoring system and protein assay. This restoration was clear by all criteria, as well as in yolk sac vascularisation and heart development. The growth promoting effects of all 3 factors were significant but did not reach the level seen in embryos grown in whole rat serum. The effect of these growth factors was also investigated on anembryonic yolk sac development using a concentration for which maximum whole embryonic growth was seen (128 ng/ml bFGF, 1.6 ng/ml VEGF and 4 ng/ml PD-ECGF), and significant anembryonic yolk sac development was found. These findings suggest that the angiogenic factors may have a growth promoting effect on total embryonic development and vascularisation.

Heart mitochondrial DNA and enzyme changes during early human development.

Previous studies in our laboratory demonstrated significant changes in bovine heart mitochondrial bioenergetics during fetal growth and development. To further understand mitochondrial biogenesis in early human development, the activity and subunit content levels of specific mitochondrial enzymes in fetal and neonatal heart were determined. Comparing early gestation (EG, 45-65 day) later gestation (LG, 85-110 day) and neonate (birth-1 month), specific activity of citrate synthase (CS), a Krebs cycle enzyme showed a 2 fold increase from EG to LG and a 2 fold increase from LG to neonate. Specific activities of complex IV and complex V increased similarly 1.8-2 fold from EG to LG. However during the later fetal period from LG to neonate, complex IV activity increased only 1.3 fold and complex V showed no significant increase. Peptide content of COX-II subunit increased 2 fold from EG to LG and by 3.5 fold from LG to neonate. Levels of COX-IV and ATP synthase alpha subunits were undetectable in EG hearts, clearly detectable in LG heart and 3 fold increased from LG to neonate. Unexpectedly, mitochondrial transcription factor A (mt-TFA) levels were not significantly different during these developmental stages. Mitochondrial DNA (mtDNA) levels increased 1.8 fold from EG to LG, and 3.8 fold increase from EG to neonate and correlated with CS activity levels. In conclusion, these data indicate coordinated regulation of some nuclear-encoded (COX-IV and CS activity) and mitochondrial components (COX-II and mtDNA), and strongly suggest that mitochondrial content increases particularly during the early fetal cardiac development and reveal a distinct pattern of regulation for mt-TFA.

Physiology of angiogenesis.

Angiogenesis is a key prerequisite for growth in all vertebrate embryos and in many tumors. Rapid growth requires efficient transport of oxygen and metabolites. Hence, for a better understanding of tissue growth, biophysical properties of vascular systems, in addition to their molecular mechanisms, need to be investigated. The purpose of this article is twofold: (1) to discuss the biophysics of growing and perfused vascular systems in general, emphasizing non-sprouting angiogenesis and remodeling of vascular plexuses; and (2) to report on cellular details of sprouting angiogenesis in the initially non-perfused embryonic brain and spinal cord. It is concluded that (1) evolutionary optimization of the circulatory system corresponds to highly conserved vascular patterns and angiogenetic mechanisms; (2) deterministic and random processes contribute to both extraembryonic and central nervous system vascularization; (3) endothelial cells interact with a variety of periendothelial cells during angiogenesis and remodeling; and that (4) mathematical models integrating molecular, morphological and biophysical expertise improve our understanding of normal and pathological angiogenesis and account for allometric relations.

RXR alpha deficiency confers genetic susceptibility for aortic sac, conotruncal, atrioventricular cushion, and ventricular muscle defects in mice.

Retinoid-dependent pathways play a central role in regulating cardiac morphogenesis. Recently, we characterized gene-targeted RXR alpha -/- embryos, which display an atrial-like ventricular phenotype with the development of heart failure and lethality at embryonic day 14.5. To quantitate the frequency and complexity of cardiac morphogenic defects, we now use microdissection and scanning electron microscopy to examine 107 wild-type, heterozygous, and homozygous embryos at embryonic day 13.5, 14.5, and 15.5. RXR alpha -/- embryos display complex defects, including ventricular septal, atrioventricular cushion, and conotruncal ridge defects, with double outlet right ventricle, aorticopulmonary window, and persistent truncus arteriosus. In addition, heterozygous RXR alpha embryos display a predisposition for trabecular and papillary muscle defects, ventricular septal defects, conotruncal ridge defects, atrioventricular cushion defects, and pulmonic stenosis. Lastly, we show that the intermediate anatomic phenotype displayed by heterozygous embryos is mirrored in the molecular marker MLC-2a. The intermediate phenotype of RXR alpha heterozygous embryos documents a gene dosage effect for RXR alpha in maintaining normal cardiac morphogenesis. In addition, some defects in RXR alpha mutant mice are phenocopies of human congenital heart defects, thereby suggesting that a relative deficiency in RXR alpha or molecules downstream in its signaling pathway may represent congenital heart disease-susceptibility genes.

Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier.

Few markers specific for mouse endothelium exist. We describe here one such marker, MECA-32, a monoclonal antibody which shows high specificity for mouse endothelium in both embryonic and mature tissues. The MECA-32 antigen has a M(r) of 50-55 x 10(3) under reducing conditions and M(r) of 100-120 x 10(3) under nonreducing conditions. It is expressed on most endothelial cells in the embryonic and in the adult mouse, with the exception of the brain, skeletal, and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle only small arterioles and venules express the MECA-32 antigen, while in the brain its expression is negatively correlated with the differentiation of the vasculature to form the blood brain barrier. Interestingly, during embryonic development the antigen occurs on the brain vasculature up to day 16 of gestation (E16), whereupon it disappears. The embryonic brain is an avascular organ anlage which is vascularized by ingrowth of external blood vessels. Differentiation of the vasculature to form the blood brain barrier occurs at approximately E16 in the mouse. This differentiation correlates with the downregulation of MECA-32 antigen expression. Between E12 and E16 MECA-32 detects most endothelial cell surfaces of the blood vessels in the brain. No MECA-32 antigen is found in the brain at E17 or any later stage of development with the exception of the vasculature of the circumventricular organs. The results suggest that MECA-32 antigen expression is temporally and spatially correlated with the development of the blood brain barrier.

A molecular approach towards the understanding of early heart development: an emerging synthesis.

In the past decade we have made an inventory of the changing three-dimensional patterns of expression of a number of key proteins involved in contraction, energy metabolism and conduction in developing and adult chicken, rat, bovine and human hearts. These integrated morphological and immunohistochemical studies were complemented with electrophysiological studies in developing chicken hearts and have resulted in a preliminary model of heart development, that explains how the embryonic heart can function without valves and without an atrioventricular conduction system that is indispensable for the adult heart. Cardiomyocyte-specific proteins are first expressed in the cardiogenic plate when 6 somites have developed, while electrical activity becomes detectable only slightly later. Development proceeds as follows: 1. Upon its formation 'primary' myocardium is characterised by anteroposterior gradients in gene expression. Therefore cardiogenesis resembles many other developmental processes in the embryo. It serves as source for endocardial cells and cells specialized in mechanical contraction and in impulse generation/conduction supporting the view that a single population of cells (the 'primary' myocardium) serves as a precursor for these distinct cell types. 2. 'Primary' myocardium is characterized by the expression of alpha and beta myosin, acetylcholinesterase and the absence of fast sodium channels and of connexin 43. It has a peristaltoid contraction form due to a relatively slow propagation of the impulse. 3. In the looping stage, two cardiac segments appear due to the development of atrial and ventricular working myocardium, that is characterized by the expression of either alpha or beta myosin, connexin 43, fast sodium channels, the disappearance of acetylcholinesterase and by a relatively fast conduction.2+ sinuatrial and atrioventricular nodes.