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Purpose of review: The cardiac conduction system, composed of pacemaker cells and conducting cardiomyocytes, orchestrates the propagation of electrical activity to synchronize heartbeats. The conduction system plays a crucial role in the development of cardiac arrhythmias. In the embryo, the cells of the conduction system derive from the same cardiac progenitors as the contractile cardiomyocytes and and the key question is how this choice is made during development. Recent Findings: This review focuses on recent advances in developmental biology using the mouse as animal model to better understand the cellular origin and molecular regulations that control morphogenesis of the cardiac conduction system, including the latest findings in single-cell transcriptomics. Summary: The conducting cell fate is acquired during development starting with pacemaking activity and last with the formation of a complex fast-conducting network. Cardiac conduction system morphogenesis is controlled by complex transcriptional and gene regulatory networks that differ in the components of the cardiac conduction system. [ABSTRACT FROM AUTHOR]

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. [ABSTRACT FROM AUTHOR]

To determine the function of VEGF-A in nervous system development, we have utilized the Nestin promoter-driven Cre recombinase transgene, in conjunction with a conditional and hypomorphic VEGF-A allele, to lower VEGF-A activity in neural progenitor cells. Mice with intermediate levels of VEGF-A activity showed decreased blood vessel branching and density in the cortex and retina, resulting in a thinner retina and aberrant structural organization of the cortex. Severe reductions in VEGF-A led to decreases in vascularity and subsequent hypoxia, resulting in the specific degeneration of the cerebral cortex and neonatal lethality. Decreased neuronal proliferation and hypoxia was evident at E11.5, leading to increased neuronal apoptosis in the cortex by E15.5. In order to address whether the observed changes in the structural organization of the nervous system were due to a direct and autocrine role of VEGF-A on the neural population, we conditionally inactivated the main VEGF-A receptor, Flk1, specifically in neuronal lineages, by using the Nestin Cre transgene. The normality of these mice ruled out the possibility that VEGF-A/Flk1 signaling has a significant autocrine role in CNS development. VEGF-A dosage is therefore a critical parameter regulating the density of the vascular plexus in the developing CNS that is in turn a key determinant in the development and architectural organization of the nervous system. Keywords: VEGF-A; Cre recombinase; Conditional gene inactivation; Blood vessel; Endothelium; Nervous system; Cortex; Retina

The ventricular conduction system (VCS) and the working myocardium (WM) derive from the same myogenic progenitors during the embryonic development. The VCS development is regulated by numerous genes including the T-box transcription factor 5 (Tbx5). In human, Tbx5 mutations cause Holt-Oram syndrome, an autosomal dominant disease associated with heart abnormalities such as arrhythmias. In adult mice, the deletion of the Tbx5 gene provoked a slowing of ventricular conduction due to a loss of the gap junction Connexin 40 and dysfunction of cardiac channels. However, it is still unknown how Tbx5 deletion affects the development and the function of the VCS. The aim is to study the consequences of the Tbx5 deletion in embryonic progenitors of the ventricular conduction system on cardiac function in adult mice. Conditional deletion of Tbx5 alleles in mice was induced by expressing the Cre recombinase under the control of the Connexin 40 (Cx40) gene. We studied two groups of mutants: Tbx5 deletion at embryonic days E13-14 targeting both the ventricular conduction system and the working myocardium ("VCS + WM" group; n = 40) and mice deleted at E18-P2 targeting only the ventricular conduction system ("VCS" group; n = 17). Surface ECGs were achieved monthly until 3 months of age, when immunostaining was performed on the left VCS. Immunostaining showed a reduced expression of the two major connexines in the ventricle (Cx40 and Cx43) and a reduced number of conductive cells in both mutants, suggesting a defective maturation of the VCS (Fig. 1). Furthermore, the loss of Cx43 was correlated with that of Tbx5, reflecting a direct transcriptional effect. Surface ECG revealed a significant increase of the ventricular activation duration in both mutants (P < 0.01). Moreover, premature ventricular contractions (PVCs) and a left QRS axis deviation were observed in both mutants. Finally, these PVCs disappeared with age in both mutants under resting conditions, and are unmasked when the heart is challenged with a β-1 adrenergic agonist. Our results provide new insights into Tbx5 targets in the VCS. Tbx5 deletion impacts the maturation and the anatomy of the VCS as proved by the deviation of the ventricular electrical axis, which even occurs when the VCS is already formed. Tbx5 appears to have a direct impact on the Gja1 gene, encoding the gap junction Cx43, which is reduced at the junction between the VCS and the WM in both mutants and may be responsible for PVCs. [ABSTRACT FROM AUTHOR]

Right ventricular (RV) dysfunction can lead to complications after acute inferior myocardial infarction (MI). However, it is unclear how RV failure after MI contributes to left-sided dysfunction. The aim of the present study was to investigate the consequences of right coronary artery (RCA) ligation in mice. RCA ligation was performed in C57BL/6JRj mice (n = 38). The cardiac phenotypes were characterized using high-resolution echocardiography performed up to 4 wk post-RCA ligation. Infarct size was measured using 2,3,5-triphenyltetrazolium chloride staining 24 h post-RCA ligation, and the extent of the fibrotic area was determined 4 wk after MI. RV dysfunction was confirmed 24 h post-RCA ligation by a decrease in the tricuspid annular plane systolic excursion (P < 0.001) and RV longitudinal strain analysis (P < 0.001). Infarct size measured ex vivo represented 45.1 ± 9.1% of the RV free wall. RCA permanent ligation increased the RV-to-left ventricular (LV) area ratio (P < 0.01). Septum hypertrophy (P < 0.01) was associated with diastolic septal flattening. During the 4-wk post-RCA ligation, LV ejection fraction was preserved, yet it was associated with impaired LV diastolic parameters (E/E=, global strain rate during early diastole). Histological staining after 4 wk confirmed the remodeling process with a thin and fibrotic RV. This study validates that RCA ligation in mice is feasible and induces RV heart failure associated with the development of LV diastolic dysfunction. Our model offers a new opportunity to study mechanisms and treatments of RV/LV dysfunction after MI. [ABSTRACT FROM AUTHOR]

Left ventricular non-compaction (LVNC) is a rare cardiomyopathy associated with a hypertrabeculated phenotype and a large spectrum of symptoms. It is still unclear whether LVNC results from a defect of ventricular trabeculae development and the mechanistic basis that underlies the varying severity of this pathology is unknown. To investigate these issues, we inactivated the cardiac transcription factor Nkx2-5 in trabecular myocardium at different stages of trabecular morphogenesis using an inducible Cx40-creERT2 allele. Conditional deletion of Nkx2-5 at embryonic stages, during trabecular formation, provokes a severe hypertrabeculated phenotype associated with subendocardial fibrosis and Purkinje fiber hypoplasia. A milder phenotype was observed after Nkx2-5 deletion at fetal stages, during trabecular compaction. A longitudinal study of cardiac function in adult Nkx2-5 conditional mutant mice demonstrates that excessive trabeculation is associated with complex ventricular conduction defects, progressively leading to strain defects, and, in 50% of mutant mice, to heart failure. Progressive impaired cardiac function correlates with conduction and strain defects independently of the degree of hypertrabeculation. Transcriptomic analysis of molecular pathways reflects myocardial remodeling with a larger number of differentially expressed genes in the severe versus mild phenotype and identifies Six1 as being upregulated in hypertrabeculated hearts. Our results provide insights into the etiology of LVNC and link its pathogenicity with compromised trabecular development including compaction defects and ventricular conduction system hypoplasia. [ABSTRACT FROM AUTHOR]

Patients with Holt-Oram syndrome suffer from cardiac conduction disorders and are often associated with mutations in the gene encoding the cardiac transcription factor Tbx5. In mice, it has been shown that Tbx5 is essential for maintaining the structure and function of the atrioventricular node and His bundle. Conditional deletion of Tbx5 in the adult mouse heart is associated with conduction defects, slowing of the ventricular conduction system (VCS), QRS prolongation, bundle branch blocks and atrial fibrillation. However, there were no data on the morphological and functional consequences of a cardiac deletion of Tbx5 during embryonic development. The aim is to analyze the morphological and functional consequences in the adult heart of the deletion of the Tbx5 gene in the progenitor cells of the cardiac VCS at the fetal stage. Tbx5 gene was inactivated in VCS progenitors using Cx40-CreERT2:Tbx5 floxed/floxed transgenic mice. Cre-mediated recombination was induced by oral gavage of pregnant females at E13-14 during trabecular compaction. Surface ECGs were achieved monthly until 3 months of age, when immunofluorescence staining was performed on mouse hearts. In 3-week-old Tbx5Fl/Fl mice (n = 16), ECG analysis showed a decrease in P- (P < 0.01) and T-wave amplitudes (P < 0.01) and an increased QRS interval duration (P < 0.05) compared to controls (n = 12). At 2 months, the mice did not show any worsening of the phenotype. In addition, severe functional defects in Tbx5Fl/Fl mice, including atrial susceptibility, extrasystoles, and arrhythmias were observed by 3 weeks of age. Moreover, left bundle branches block and slowing of ventricular depolarization were also detected in Tbx5Fl/Fl mice. A β-adrenergic stimulation revealed a large tendency for atrial fibrillation and extrasystoles in 88% of Tbx5Fl/Fl mice against 50% of Tbx5Fl/+ mice and 43% of control mice. In 2-month-old Tbx5Fl/Fl mice, Purkinje Fiber cells composing the fast-VCS are still present, however, they lose the expression of Contactin-2 and Connexin-40. Furthermore, we observed a drastic loss of Connexin-43 in Tbx5-deleted contractile cardiomyocytes that are close to the VCS, which may have resulted in decreased signal propagation. In this study, we demonstrated that Tbx5 deletion in VCS progenitors prevents their maturation and hence the expression of rapid conduction markers causing disturbances in the propagation of ventricular electrical activity. [ABSTRACT FROM AUTHOR]

The transcription factor Nkx2-5 is known to be a key regulator of cardiac development and NKX2-5 mutations have been found in numerous cardiac diseases associated with conduction defects. This factor is required for the differentiation of Purkinje fibers (PF), which have been implicated in triggering ventricular arrhythmias. A better understanding of the development of the Purkinje system in normal and Nkx2-5 mutant hearts could provide insight into their involvement in conduction defects in cardiac diseases. However, analysis of the Purkinje network development is hampered by the lack of specific lineage tracers. Recently, the voltage-gated sodium channel Nav1-8 short transcript has been shown to be expressed in the ventricular conduction system (VCS). This study aims to characterize the expression domain of Nav1-8 in the murine VCS to determine whether it is a suitable tool for tracing conductive cells. This would also help to identify defects in conductive recruitment in congenital disease. Nav1-8-Flp mice were crossed with a GFP reporter line for genetic tracing analyses in WT and Nkx2-5 heterozygous mice. The specificity and efficiency of Nav1-8 labelling was measured from P1 to P21 based on the number of GFP-positive (Nav1-8 derivatives), Cntn2-positive (VCS) and double positive cells (Nav1-8 derivatives in the VCS). GFP reporter expression starts at birth and is restricted to the PF at the distal part of the VCS. The specificity of Nav1-8 as a marker of PF increased over time between P1 and P21, suggesting that Nav1.8 is expressed in PF progenitor cells prior to their recruitment to the conduction system. However, its efficiency is limited to 20% of PF being marked at a mature stage of development (P21). In Nkx2-5 mutant hearts, GFP labelling within the left ventricle delimits the PF scaffold and is almost absent in the complex ramifications of the PF network. This anatomical distribution is consistent with the hypoplastic Purkinje system of these mice. However, unlike in control hearts, additional GFP+ cells are found in the bundle branches at the proximal part of the VCS in Nkx2-5 mutants. Our study shows that the sodium channel Nav1-8 represents a unique and highly specific lineage tracer of the peripheral PF in mature hearts. In addition, we show an enlargement of the Nav1-8 expression domain to the bundle branches in Nkx2-5 mutants. This indicates that Nkx2-5 may influence cell fate choice within the VCS. [ABSTRACT FROM AUTHOR]

The Purkinje network (PN) is the most distal part of the cardiac conduction system and ensures fast conduction of the electrical impulse through the ventricles. Despite its small volume in the heart, the PN has a disproportionally high pathogenic role. Indeed, defects in PN architecture or electrophysiology trigger conduction blocks and/or ventricular arrythmia associated with poor clinical outcomes. Purkinje fibers (PF) are specialized cardiomyocytes with a low proliferative capacity and are recruited from bipotent cardiomyocyte progenitors throughout development up to the neonatal period. Late PF recruitment is dependent on maximal levels of the transcription factor Nkx2-5. A decade ago, a neonatal regenerative window was identified in mice following myocardial infarction (MI). While damaged contractile myocardium regenerates within weeks, neither PN structure nor function has been investigated in this context. This study aims to determine whether the PN is capable of regeneration in the neonatal period, and whether PN maturation is compromised in the regenerating context. Neonatal MI was performed by permanent ligation of the left coronary artery on one-day-old mice. PN structure and function were characterized by histology and electrocardiogram. PF recruitment was investigated by clonal analysis in wildtype and Nkx2-5 haploinsufficent mice. Damage caused by the MI to the PN is spatially restricted and PFs are not replaced after regeneration, leaving a gap in the network. Surprisingly, we also observed PN hyperplasia (Fig. 1A) in regions that were not directly affected by the MI, attesting to abnormal PN maturation in the context of regeneration. At the cellular level, ectopic PFs have abnormal shapes and gap junction content. Consistently, ventricular conduction was slowed, shown by an increased QRS duration (Fig. 1B). Nkx2-5± mice, which are incompetent for late PF recruitment, do not develop MI-induced hyperplasia. Together with clonal analysis, this indicates that the hyperplastic PN in WT mice arises from ectopic PF recruitment during regeneration. Our study reveals the incapacity of the PN to regenerate after direct damage. We also uncover an extended window of PF recruitment following MI, which leads to permanent structural defects and impaired electrical conduction. [ABSTRACT FROM AUTHOR]

Background: 14-3-3ε plays an important role in the maturation of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via p27kip1. However, additional cardiac defects are possible given the ubiquitous expression pattern of this protein. Results: Germ line deletion of 14-3-3ε led to malalignment of both the outflow tract (OFT) and atrioventricular (AV) cushions, with resulting tricuspid stenosis and atresia, mitral valve abnormalities, and perimembranous ventricular septal defects (VSDs). We confirmed myocardial non-compaction and detected a spongy septum with muscular VSDs and blebbing of the epicardium. These defects were associated with abnormal patterning of p27kip1 expression in the subendocardial and possibly the epicardial cell populations. In addition to abnormal pharyngeal arch artery patterning, we found deep endocardial recesses and paucity of intramyocardial coronary vasculature as a result of defective coronary plexus remodeling. Conclusions: The malalignment of both endocardial cushions provides a new explanation for tricuspid and mitral valve defects, while myocardial non-compaction provides the basis for the abnormal coronary vasculature patterning. These abnormalities might arise from p27kip1 dysregulation and a resulting defect in epithelial-to-mesenchymal transformation. These data suggest that 14-3-3ε, in addition to left ventricular non-compaction (LVNC), might be linked to different forms of congenital heart disease (CHD). Developmental Dynamics 245:1107-1123, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Purpose: To propose, assess, and validate a semiautomatic method allowing rapid and reproducible measurement of trabeculated and compacted left ventricular (LV) masses from cardiac magnetic resonance imaging (MRI).Materials and Methods: We developed a method to automatically detect noncompacted, endocardial, and epicardial contours. Papillary muscles were segmented using semiautomatic thresholding and were included in the compacted mass. Blood was removed from trabeculae using the same threshold tool. Trabeculated, compacted masses and ratio of noncompacted to compacted (NC:C) masses were computed. Preclinical validation was performed on four transgenic mice with hypertrabeculation of the LV (high-resolution cine imaging, 11.75T). Then analysis was performed on normal cine-MRI examinations (steady-state free precession [SSFP] sequences, 1.5T or 3T) obtained from 60 healthy participants (mean age 49 ± 16 years) with 10 men and 10 women for each of the following age groups: [20,39], [40,59], and [60,79]. Interobserver and interexamination segmentation reproducibility was assessed by using Bland-Altman analysis and by computing the correlation coefficient.Results: In normal participants, noncompacted and compacted masses were 6.29 ± 2.03 g/m(2) and 62.17 ± 11.32 g/m(2) , respectively. The NC:C mass ratio was 10.26 ± 3.27%. Correlation between the two observers was from 0.85 for NC:C ratio to 0.99 for end-diastolic volume (P < 10(-5) ). The bias between the two observers was -1.06 ± 1.02 g/m(2) for trabeculated mass, -1.41 ± 2.78 g/m(2) for compacted mass, and -1.51 ± 1.77% for NC:C ratio.Conclusion: We propose a semiautomatic method based on region growing, active contours, and thresholding to calculate the NC:C mass ratio. This method is highly reproducible and might help in the diagnosis of LV noncompaction cardiomyopathy. J. Magn. Reson. Imaging 2016;43:1398-1406. [ABSTRACT FROM AUTHOR]

Background: Coronary artery (CA) stems connect the ventricular coronary tree with the aorta. Defects in proximal CA patterning are a cause of sudden cardiac death. In mice lacking Tbx1, common arterial trunk is associated with an abnormal trajectory of the proximal left CA. Here we investigate CA stem development in wild-type and Tbx1 null embryos. Results: Genetic lineage tracing reveals that limited outgrowth of aortic endothelium contributes to proximal CA stems. Immunohistochemistry and fluorescent tracer injections identify a periarterial vascular plexus present at the onset of CA stem development. Transplantation experiments in avian embryos indicate that the periarterial plexus originates in mesenchyme distal to the outflow tract. Tbx1 is required for the patterning but not timing of CA stem development and a Tbx1 reporter allele is expressed in myocardium adjacent to the left but not right CA stem. This expression domain is maintained in Sema3c−/− hearts with a common arterial trunk and leftward positioned CA. Ectopic myocardial differentiation is observed on the left side of the Tbx1−/− common arterial trunk. Conclusions: A periarterial plexus bridges limited outgrowth of the aortic endothelium with the ventricular plexus during CA stem development. Molecular differences associated with left and right CA stems provide new insights into the etiology of CA patterning defects. Developmental Dynamics 245:445-459, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Aims Capillary and arterial endothelial cells share many common molecular markers in both the neonatal and adult hearts. Herein, we aim to establish a genetic tool that distinguishes these two types of vessels in order to determine the cellular mechanism underlying collateral artery formation. Methods and results Using Apln-GFP and Apln-LacZ reporter mice, we demonstrate that APLN expression is enriched in coronary vascular and results endothelial cells. However, APLN expression is reduced in coronary arterial endothelial cells. Genetic lineage tracing, using an Apln-CreER mouse line, robustly labelled capillary endothelial cells, but not arterial endothelial cells. We leveraged this differential activity of Apln-CreER to study collateral artery formation following myocardial infarction (MI). In a neonatal heart MI model, we found that Apln-CreER-labelled capillary endothelial cells do not contribute to the large collateral arteries. Instead, these large collateral arteries mainly arise from pre-existing, infrequently labelled coronary arteries, indicative of arteriogenesis. Furthermore, in an adult heart MI model, Apln-CreER activity also distinguishes large and small diameter arteries from capillaries. Lineage tracing in this setting demonstrated that most large and small coronary arteries in the infarcted myocardium and border region are derived not from capillaries, but from pre-existing arteries. Conclusion Apln-CreER-mediated lineage tracing distinguishes capillaries from large arteries, in both the neonatal and adult hearts. Through genetic fate mapping, we demonstrate that pre-existing arteries, but not capillaries, extensively contribute to collateral artery formation following myocardial injury. These results suggest that arteriogenesis is the major mechanism underlying collateral vessel formation. [ABSTRACT FROM AUTHOR]

The recent discovery of the capacity of the mammalian heart to completely regenerate during a short period of time after birth opened a new area of research in the cardiovascular field. The revascularization of the ischemic area is an important process to favor cardiac regeneration and we have previously shown that preexisting coronary arteries undergo major remodeling events following adult MI. This mechanism of arteriogenesis is known as collateral growth but it has not been well described during cardiac regeneration. We hypothesize that collateral growth occurs more frequently in regenerative hearts compared to non-regenerative ones. To investigate the coronary arteries remodeling and collateral growth during cardiac regeneration after neonatal myocardial infarction (MI). Neonatal MI was performed by the permanent ligation of the left coronary artery on one day-old (P1) and seven day-old (P7) mice. MI surgery was performed on P1 or P7 pups from the Cx40-GFP mouse line in which the fluorescent protein GFP is specifically expressed in coronary arteries. Hearts were collected at 1, 2, 4, 7 and 14 dpi and processed for clearing using CUBIC solution. 3D reconstruction was acquired from images of cleared hearts. Our data show the dynamic remodeling of the ligated coronary artery, which completely regenerates in two weeks, starting one day-post-injury (dpi) by the migration of isolated arterial endothelial cells migrating from the injured artery to form a new vessel at 4dpi. We observed a larger number of collaterals branching from the injured left artery to healthy right or septal arteries in regenerating hearts (P1) in comparison with non-regenerating hearts (P7). These results highlight the rapid phase of arterial remodeling that would play an important role in inducing cardiac regeneration. [ABSTRACT FROM AUTHOR]

The mammalian heart can regenerate only during the first week of life. This window coincides with maturation of the myocardial wall through compaction of ventricular trabeculae, transient invaginations at the inner surface of the developing heart. Trabeculae give rise both to contractile cardiomyocytes (CM) and to Purkinje fibers composing the ventricular conductive system (VCS). Given the regenerative capacities of the uncompacted neonatal mammalian heart, we hypothesized that ventricular trabeculae possess superior regenerative capacities than compact myocardium. We also questioned the capacity of the VCS to undergo repair during the regenerative window. Investigate the contribution of ventricular trabeculae to the regeneration of contractile and conductive cardiac tissue after neonatal myocardial infarction (MI). Neonatal MI was performed by permanent ligation of the left coronary artery on one-day-old mice. Trabecular derivatives were genetically traced using Connexin40 driven inducible Cre recombinase together with a fluorescent conditional reporter gene (Cx40-CreERT2, Rosa26-YFP). VCS structure and function were respectively assessed by histology or electrocardiogram (ECG) analysis in a longitudinal study. Trabecular derivatives were found in an extended number in the regenerated myocardium after neonatal MI, compared to the same area in sham or remote regions of MI-operated hearts. Moreover, trabecular derivatives in the border zone show reactivation of Cx40 indicating a switch of these CM towards an immature phenotype. In contrast to regeneration of contractile myocardium, damage to the VCS after MI was not repaired, despite being spatially restricted. Moreover, maturation defects, characterized by a global hyperplasia of the VCS, was observed after regeneration and persists over time. The abnormal structure of the Purkinje fiber network in regenerated hearts is accompanied by a slowed ventricular conduction as shown by a prolonged QRS on ECG recording (Fig. 1). Trabeculae show an increased participation in the regeneration of the contractile compartment relative to that of the compact layer. Contrary to contractile myocardium, VCS is not repaired and its maturation is perturbed in the regenerative context, which permanently perturbed conduction. Together our data identify cellular mechanisms involved in cardiac repair and reveal limits to cardiac regeneration during the postnatal window. [ABSTRACT FROM AUTHOR]

Mutations of the cardiac transcription factor NKX2-5 cause hypoplastic development of the AV node and conduction block. How the anatomy of the mutant AV node relates to its function is unknown. We thus studied conduction through the AV nodal region in ex vivo preparations of wild-type and Nkx2-5+/− mouse hearts in which the central conduction system was highlighted by a transgenic Cx40- eGFP reporter. Fluorescence imaging guided electrode placement and pacing of the inferior and superior approaches to the AV node. Nkx2-5+/− hearts had a prolonged atrio-His interval compared to the wild type, consistent with previous in vivo observations. The conduction time to the His bundle from the Cx40− AV nodal region that is superior to and immediately adjacent to the Cx40+ lower node is slightly, but not significantly greater in Nkx2-5+/− than wild-type hearts. A novel phenotype was also observed. Pacing the Cx40− inferior approach to the AV node with increasing stimulus strength led to progressive shortening of the stimulus-to-His conduction interval in wild-type but not Nkx2-5+/− hearts. The strength of pacing at the Cx40− superior approach had no effect on the conduction interval in either group. The prolonged AV delay in the Nkx2-5+/− heart appears to arise before the Cx40+ lower node. Whether the pacing phenotype explains the mutant's conduction defect is uncertain, but the observation adds to a number of unique properties of the inferior approach to the AV node. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is an angiogenic factor and a potent stimulator of microvascular permeability. It is a mitogen specific for endothelial cells. The expression of VEGF and its two receptors, Flk-1 and Flt-1, is pivotal for proper formation of blood vessels in embryogenesis as shown by gene-targeting experiments. Interestingly, the loss of even a single allele of VEGF led to embryonic lethality between day E9.5 and day E10.5 in the mouse. To assess the role of VEGF during embryonic development we decided to tag VEGF expression with LacZ, by inserting an IRES (internal ribosome entry site)-LacZ reporter cassette into the 3[prime] untranslated region of the gene. This alteration enabled us to monitor VEGF expression throughout embryonic development at single-cell resolution. [Beta]-Galactosidase expression from the altered VEGF locus was first observed prior to gastrulation and was detectable at all stages of vascular development in the embryo. Later, the specific cellular distribution and the level of VEGF expression indicated its pleiotropic role in development. High expression levels seemed to be associated with vasculogenesis and permeability, whereas lower levels were associated with angiogenesis and cell migration. In addition, we found VEGF expression in a subtype of endothelial cells present in the endocardium. We believe that the LacZ-tagged allele we have generated offers a precise means of detecting VEGF expression under a variety of physiological and pathological conditions. Key Words: vascular endothelial growth factor; endothelial cells; gene targeting; IRES-lacZ knock-in; embryonic development; vasculogenesis; angiogenesis.

Background: The ventricular conduction system (VCS) coordinates the heartbeat and is composed of central components (the atrioventricular node, bundle, and right and left bundle branches) and a peripheral Purkinje fiber network. Conductive myocytes develop from common progenitor cells with working myocytes in a bimodal process of lineage restriction followed by limited outgrowth. The lineage relationship between progenitor cells giving rise to different components of the VCS is unclear. Results: Cell lineage contributions to different components of the VCS were analysed by a combination of retrospective clonal analysis, regionalized transgene expression studies, and genetic tracing experiments using Connexin40-GFP mice that precisely delineate the VCS. Analysis of a library of hearts containing rare large clusters of clonally related myocytes identifies two VCS lineages encompassing either the right Purkinje fiber network or left bundle branch. Both lineages contribute to the atrioventricular bundle and right bundle branch that segregate early from working myocytes. Right and left VCS lineages share the transcriptional program of the respective ventricular working myocytes and genetic tracing experiments discount alternate progenitor cell contributions to the VCS. Conclusions: The mammalian VCS is comprised of cells derived from two lineages, supporting a dual contribution of first and second heart field progenitor cells. Developmental Dynamics 242:665-677, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS. [ABSTRACT FROM AUTHOR]

The article offers information on a study which defined the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. A review of the related literature on the ventricular conduction system (VCS) is given. It describes the research methods. The classes of clusters obtained are those composed of conductive and working myocytes, and smaller unmixed clusters that are composed exclusively of conductive cells.

The article presents a study that shows that a subdomain of myocardium normally observed at the base of pulmonary trunk is minimized and malpositioned in Tbx1 mutant hearts, based on a range of genetic crosses using transgene markers of second heart field derived myocardium and coronary endothelial cells. Results of the study show second heart field development in coronary artery patterning, and offers new insights in the connection between conotruncal defects and coronary artery anomalies.

Objective: The aim of this work was to target a vital reporter gene in the mouse cardiac conduction system (CS) to distinguish this tissue from the surrounding myocardium in the adult heart. Methods: A transgenic mouse line has been created in which EGFP is expressed under the control of the Cx40 gene. Correlative investigations associating EGFP imaging and electrophysiological techniques were carried out on the adult heart and isolated cardiomyocytes. Results: In the heart of the Cx40EGFP/+ mice, EGFP signal was seen in the coronary arteries, the atria, the atrioventricular (AV) node and the His–Purkinje system. The latter was found to be structurally and functionally asymmetrical. The anatomical asymmetry was apparent in both the number of strands or fasciculi making up the His bundle branches (BBs) (1 strand on the right, 20 or so on the left), and the density (low on the right, high on the left) of the network of Purkinje fibers (PFs) that extends over the ventricular wall surfaces. The profiles of the electrical activation patterns recorded on the right and left flanks of the septum were also asymmetrical, mirroring the architecture of the branches. EGFP made it easy to identify the Purkinje cells in populations of dissociated cardiomyocytes and they were investigated using the patch-clamp technique. The hyperpolarization-activated current (If) was recorded in all spontaneously active Purkinje cells. Conclusions: This investigation provides positive evidence of the asymmetry of the His–Purkinje system of the adult mouse, and the first patch-clamp recording data on murine cardiac Purkinje cells. This mouse model opens up new perspectives for investigating the contribution of specific genes to the morphology and function of the His–Purkinje system. [Copyright &y& Elsevier]

It has long been known that gap junctions are required for the propagation of electrical impulse in the heart. A good deal later, the connexins (Cxs), which are probably exclusive components of the junctional channels that constitute the gap junctions, were identified. More recently, the in vivo functions of cardiac Cxs have been investigated by the analysis of genetically modified mice. These studies have confirmed that Cxs are involved in cardiac impulse conduction, and, unexpectedly, in heart morphogenesis. In addition, cardiac abnormalities described in mice genetically modified for Cx genes, and those observed in certain human cardiac diseases, have been proven to be similar. [Copyright &y& Elsevier]

During development, hematopoiesis initiates in the yolk sac through a process that depends on VEGF/Flk1 signaling and on the function of the SCL/Tall transcription factor. Here we show that VEGF modifies the developmental potential of primitive erythroid progenitors and prolongs their life span. Furthermore, the survival of yolk sac erythrocytes in vivo depends on the dose of VEGF. Interestingly, in Vegflo/lo embryos carrying a hypomorph allele, Flkl-positive cells reach the yolk sac at E8.5, but are severely compromised in their ability to generate primitive erythroid precursors. These observations indicate that during embryonic development, different thresholds of VEGF are required for the migration and clonal expansion of hematopoietic precursors. The near absence of primitive erythroid precursors in Vegflo/lo embryos correlates with low levels of Scl in the yolk sac. Strikingly, gain-of-function of SCL partially complements the hematopoietic defect caused by the hypomorph Veglo allele, and re-establishes the survival of erythroid cells and the expression of erythroid genes (Gata1 and βH1). This indicates that SCL functions downstream of VEGF to ensure an expansion of the hematopoietic compartment. [ABSTRACT FROM AUTHOR]