Retinoic acid signaling in heart development: Application in the differentiation of cardiovascular lineages from human pluripotent stem cells.

Retinoic acid (RA) signaling plays an important role during heart development in establishing anteroposterior polarity, formation of inflow and outflow tract progenitors, and growth of the ventricular compact wall. RA is also utilized as a key ingredient in protocols designed for generating cardiac cell types from pluripotent stem cells (PSCs). This review discusses the role of RA in cardiogenesis, currently available protocols that employ RA for differentiation of various cardiovascular lineages, and plausible transcriptional mechanisms underlying this fate specification. These insights will inform further development of desired cardiac cell types from human PSCs and their application in preclinical and clinical research.

Hedgehog signalling controls sinoatrial node development and atrioventricular cushion formation.

Smoothened is a key receptor of the hedgehog pathway, but the roles of Smoothened in cardiac development remain incompletely understood. In this study, we found that the conditional knockout of Smoothened from the mesoderm impaired the development of the venous pole of the heart and resulted in hypoplasia of the atrium/inflow tract (IFT) and a low heart rate. The blockage of Smoothened led to reduced expression of genes critical for sinoatrial node (SAN) development in the IFT. In a cardiac cell culture model, we identified a Gli2-Tbx5-Hcn4 pathway that controls SAN development. In the mutant embryos, the endocardial-to-mesenchymal transition (EndMT) in the atrioventricular cushion failed, and Bmp signalling was downregulated. The addition of Bmp2 rescued the EndMT in mutant explant cultures. Furthermore, we analysed Gli2+ scRNAseq and Tbx5-/- RNAseq data and explored the potential genes downstream of hedgehog signalling in posterior second heart field derivatives. In conclusion, our study reveals that Smoothened-mediated hedgehog signalling controls posterior cardiac progenitor commitment, which suggests that the mutation of Smoothened might be involved in the aetiology of congenital heart diseases related to the cardiac conduction system and heart valves.

Network-driven discovery yields new insight into Shox2-dependent cardiac rhythm control.

The homeodomain transcription factor SHOX2 is involved in the development and function of the heart's primary pacemaker, the sinoatrial node (SAN), and has been associated with cardiac conduction-related diseases such as atrial fibrillation and sinus node dysfunction. To shed light on Shox2-dependent genetic processes involved in these diseases, we established a murine embryonic stem cell (ESC) cardiac differentiation model to investigate Shox2 pathways in SAN-like cardiomyocytes. Differential RNA-seq-based expression profiling of Shox2+/+ and Shox2-/- ESCs revealed 94 dysregulated transcripts in Shox2-/- ESC-derived SAN-like cells. Of these, 15 putative Shox2 target genes were selected for further validation based on comparative expression analysis with SAN- and right atria-enriched genes. Network-based analyses, integrating data from the Mouse Organogenesis Cell Atlas and the Ingenuity pathways, as well as validation in mouse and zebrafish models confirmed a regulatory role for the novel identified Shox2 target genes including Cav1, Fkbp10, Igfbp5, Mcf2l and Nr2f2. Our results indicate that genetic networks involving SHOX2 may contribute to conduction traits through the regulation of these genes.

GATA6 is a regulator of sinus node development and heart rhythm.

The sinus node (SAN) is the primary pacemaker of the human heart, and abnormalities in its structure or function cause sick sinus syndrome, the most common reason for electronic pacemaker implantation. Here we report that transcription factor GATA6, whose mutations in humans are linked to arrhythmia, is highly expressed in the SAN and its haploinsufficiency in mice results in hypoplastic SANs and rhythm abnormalities. Cell-specific deletion reveals a requirement for GATA6 in various SAN lineages. Mechanistically, GATA6 directly activates key regulators of the SAN genetic program in conduction and nonconduction cells, such as TBX3 and EDN1, respectively. The data identify GATA6 as an important regulator of the SAN and provide a molecular basis for understanding the conduction abnormalities associated with GATA6 mutations in humans. They also suggest that GATA6 may be a potential modifier of the cardiac pacemaker.

Conjugated activation of myocardial-specific transcription of Gja5 by a pair of Nkx2-5-Shox2 co-responsive elements.

The sinoatrial node (SAN) is the primary pacemaker in the heart. During cardiogenesis, Shox2 and Nkx2-5 are co-expressed in the junction domain of the SAN and regulate pacemaker cell fate through a Shox2-Nkx2-5 antagonism. Cx40 is a marker of working myocardium and an Nkx2-5 transcriptional output antagonized by Shox2, but the underlying regulatory mechanisms remain elusive. Here we characterized a bona fide myocardial-specific Gja5 (coding gene of Cx40) distal enhancer consisting of a pair of Nkx2-5 and Shox2 co-bound elements in the regulatory region of Gja5. Transgenic reporter assays revealed that neither element alone, but the conjugation of both elements together, drives myocardial-specific transcription. Genetic analyses confirmed that the activation of this enhancer depends on Nkx2-5 but is inhibited by Shox2 in vivo, and its presence is essential for Gja5 expression in the myocardium but not the endothelial cells of the heart. Furthermore, chromatin conformation analysis showed an Nkx2-5-dependent loop formation between these two elements and the Gja5 promoter in vivo, indicating that Nkx2-5 bridges the conjugated activation of this enhancer by pairing the two elements to the Gja5 promoter.

Control of sinus venous valve and sinoatrial node development by endocardial NOTCH1.

AIMS: Sinus venous valve (SVV) and sinoatrial node (SAN) develop together at the sinoatrial junction during embryogenesis. SVV ensures unidirectional cardiac input and SAN generates sinus rhythmic contraction, respectively; both functions are essential for embryonic survival. We aim to reveal the potential role of endocardial NOTCH signalling in SVV and SAN formation. METHODS AND RESULTS: We specifically deleted Notch1 in the endocardium using an Nfatc1Cre line. This deletion resulted in underdeveloped SVV and SAN, associated with reduced expression of T-box transcription factors, Tbx5 andTbx18, which are essential for the formation of SVV and SAN. The deletion also led to decreased expression of Wnt2 in myocardium of SVV and SAN. WNT2 treatment was able to rescue the growth defect of SVV and SAN resulted from the Notch1 deletion in whole embryo cultures. Furthermore, the Notch1 deletion reduced the expression of Nrg1 in the SVV myocardium and supplement of NRG1 restored the growth of SVV in cultured Notch1 knockout embryos. CONCLUSION: Our findings support that endocardial NOTCH1 controls the development of SVV and SAN by coordinating myocardial WNT and NRG1 signalling functions.

Flap valve of the heart foramen ovale revisited: macroscopic and histologic observations of human near-term fetuses.

We assessed the flap valve of the foramen ovale (FO valve) by examining 30 hearts from human fetuses of gestational age 30-40 weeks. We dissected the hearts, examined their macroscopic morphology, and then prepared semiserial sagittal sections across the valve. Although the primary septum is expected to extend along the left atrial face, eight hearts had a superior rim of the fossa ovalis on the left atrial face that was too thick and high, so there was no smooth continuation with the valve. Moreover, three of these eight hearts each had a flap valve that was fused with a long and narrow plate arising from the caval orifice. Histological analysis indicated that 21 specimens each had a candidate primary septum that contained myocardium, although the left sinuatrial valve (LSAV) contained fibrous tissue, but little or no myocardium. In each of 17 hearts, a candidate primary septum was attached to the left atrial face of the fossa, and parts of the LSAV extended to and approached the right atrial face. However, seven of these 17 hearts each had a folded small primary septum. Another four of these 17 hearts each had an LSAV that extended widely to the fossa, and a candidate primary septum (which might be a remnant) attached to the left atrial side of the LSAV. These variations suggest that the LSAV makes a major contribution to the FO valve in some fetal hearts. Consequently, the fetal FO valve appears to have heterogeneous morphology and origin.

Sinus venosus incorporation: contentious issues and operational criteria for developmental and evolutionary studies.

The sinus venosus is a cardiac chamber upstream of the right atrium that harbours the dominant cardiac pacemaker. During human heart development, the sinus venosus becomes incorporated into the right atrium. However, from the literature it is not possible to deduce the characteristics and importance of this process of incorporation, due to inconsistent terminology and definitions in the description of multiple lines of evidence. We reviewed the literature regarding the incorporation of the sinus venosus and included novel electrophysiological data. Most mammals that have an incorporated sinus venosus show a loss of a functional valve guard of the superior caval vein together with a loss of the electrical sinuatrial delay between the sinus venosus and the right atrium. However, these processes are not necessarily intertwined and in a few species only the sinuatrial delay may be lost. Sinus venosus incorporation can be characterised as the loss of the sinuatrial delay of which the anatomical and molecular underpinnings are not yet understood.

Shox2: The Role in Differentiation and Development of Cardiac Conduction System.

The formation and conduction of electrocardiosignals and the synchronous contraction of atria and ventricles with rhythmicity are both triggered and regulated by the cardiac conduction system (CCS). Defect of this system will lead to various types of cardiac arrhythmias. In recent years, the research progress of molecular genetics and developmental biology revealed a clearer understanding of differentiation and development of the cardiac conduction system and their regulatory mechanisms. Short stature homeobox 2 (Shox2) transcription factor, encoded by Shox2 gene in the mouse, is crucial in the formation and differentiation of the sinoatrial node (SAN). Shox2 drives embryonic development processes and is widely expressed in the appendicular skeleton, palate, temporomandibular joints, and heart. Mutations of Shox2 can lead to dysembryoplasia and abnormal phenotypes, including bradycardiac arrhythmia. In this review, we provide a summary of the latest research progress on the regulatory effects of the Shox2 gene in differentiation and development processes of the cardiac conduction system, hoping to deepen the knowledge and understanding of this systematic process based on the cardiac conduction system. Overall, the Shox2 gene is intimately involved in the differentiation and development of cardiac conduction system, especially sinoatrial node. We also summarize the current information about human SHOX2. This review article provides a new direction in biological pacemaker therapies.

Comparative developmental biology of the cardiac inflow tract.

The vertebrate heart receives the blood through the cardiac inflow tract. This area has experienced profound changes along the evolution of vertebrates; changes that have a reflection in the cardiac ontogeny. The development of the inflow tract involves dynamic changes due to the progressive addition of tissue derived from the secondary heart field. The inflow tract is the site where oxygenated blood coming from lungs is received separately from the systemic return, where the cardiac pacemaker is established and where the proepicardium develops. Differential cell migration towards the inflow tract breaks the symmetry of the primary heart tube and determines the direction of the cardiac looping. In air-breathing vertebrates, an inflow tract reorganization is essential to keep separate blood flows from systemic and pulmonary returns. Finally, the sinus venosus endocardium has recently been recognized as playing a role in the constitution of the coronary vasculature. Due to this developmental complexity, congenital anomalies of the inflow tract can cause severe cardiac diseases. We aimed to review the recent literature on the cellular and molecular mechanisms that regulate the morphogenesis of the cardiac inflow tract, together with comparative and evolutionary details, thus providing a basis for a better understanding of these mechanisms.

RHOA-ROCK signalling is necessary for lateralization and differentiation of the developing sinoatrial node.

AIMS: RHOA-ROCK signalling regulates cell migration, proliferation, differentiation, and transcription. RHOA is expressed in the developing cardiac conduction system in chicken and mice. In early development, the entire sinus venosus myocardium, including both the transient left-sided and the definitive sinoatrial node (SAN), has pacemaker potential. Later, pacemaker potential is restricted to the right-sided SAN. Disruption of RHOA expression in adult mice causes arrhythmias including bradycardia and atrial fibrillation, the mechanism of which is unknown but presumed to affect the SAN. The aim of this study is to assess the role of RHOA-ROCK signalling in SAN development in the chicken heart. METHODS AND RESULTS: ROCK signalling was inhibited chemically in embryonic chicken hearts using Y-27632. This prolonged the immature state of the sinus venosus myocardium, evidenced by up-regulation of the transcription factor ISL1, wide distribution of pacemaker potential, and significantly reduced heart rate. Furthermore ROCK inhibition caused aberrant expression of typical SAN genes: ROCK1, ROCK2, SHOX2, TBX3, TBX5, ISL1, HCN4, CX40, CAV3.1, and NKX2.5 and left-right asymmetry genes: PITX2C and NODAL. Anatomical abnormalities in pulmonary vein development were also observed. Patch clamp electrophysiology confirmed the immature phenotype of the SAN cells and a residual left-sided sinus venosus myocardium pacemaker-like potential. CONCLUSIONS: RHOA-ROCK signalling is involved in establishing the right-sided SAN as the definitive pacemaker of the heart and restricts typical pacemaker gene expression to the right side of the sinus venosus myocardium.

Current concepts of anatomy and electrophysiology of the sinus node.

The sinoatrial node, or sinus node, of humans is the principal pacemaker of the heart. Over the last century, studies have unraveled the complex molecular architecture of the sinus node and the expression of unique ion channels within its specialized myocytes. Aim of this review is to describe the embriology, the anatomy, the histology and the electrophisiology of the sinus node.

New Approaches to Biological Pacemakers: Links to Sinoatrial Node Development.

Irreversible degeneration of the cardiac conduction system is a common disease that can cause activity intolerance, fainting, and death. While electronic pacemakers provide effective treatment, alternative approaches are needed when long-term indwelling hardware is undesirable. Biological pacemakers comprise electrically active cells that functionally integrate with the heart. Recent findings on cardiac pacemaker cells (PCs) within the sinoatrial node (SAN), along with developments in stem cell technology, have opened a new era in biological pacing. Recent experiments that have derived PC-like cells from non-PCs have brought the field closer than ever before to biological pacemakers that can faithfully recapitulate SAN activity. In this review, I discuss these approaches in the context of SAN biology and address the potential for clinical translation.

Transcription factor ISL1 is essential for pacemaker development and function.

The sinoatrial node (SAN) maintains a rhythmic heartbeat; therefore, a better understanding of factors that drive SAN development and function is crucial to generation of potential therapies, such as biological pacemakers, for sinus arrhythmias. Here, we determined that the LIM homeodomain transcription factor ISL1 plays a key role in survival, proliferation, and function of pacemaker cells throughout development. Analysis of several Isl1 mutant mouse lines, including animals harboring an SAN-specific Isl1 deletion, revealed that ISL1 within SAN is a requirement for early embryonic viability. RNA-sequencing (RNA-seq) analyses of FACS-purified cells from ISL1-deficient SANs revealed that a number of genes critical for SAN function, including those encoding transcription factors and ion channels, were downstream of ISL1. Chromatin immunoprecipitation assays performed with anti-ISL1 antibodies and chromatin extracts from FACS-purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes required for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct ISL1 targets. Together, our results demonstrate that ISL1 regulates approximately one-third of SAN-specific genes, indicate that a combination of ISL1 and other SAN transcription factors could be utilized to generate pacemaker cells, and suggest ISL1 mutations may underlie sick sinus syndrome.

Stem cell-derived nodal-like cardiomyocytes as a novel pharmacologic tool: insights from sinoatrial node development and function.

Since the first reports on the isolation and differentiation of stem cells, and in particular since the early success in driving these cells down a cardiac lineage, there has been interest in the potential of such preparations in cardiac regenerative therapy. Much of the focus of such research has been on improving mechanical function after myocardial infarction; however, electrophysiologic studies of these preparations have revealed a heterogeneous mix of action potential characteristics, including some described as "pacemaker" or "nodal-like," which in turn led to interest in the therapeutic potential of these preparations in the treatment of rhythm disorders; several proof-of-concept studies have used these cells to create a biologic alternative to electronic pacemakers. Further, there are additional potential applications of a preparation of pacemaker cells derived from stem cells, for example, in high-throughput screens of new chronotropic agents. All such applications require reasonably efficient methods for selecting or enriching the "nodal-like" cells, however, which in turn depends on first defining what constitutes a nodal-like cell since not all pacemaking cells are necessarily of nodal lineage. This review discusses the current state of the field in terms of characterizing sinoatrial-like cardiomyocytes derived from embryonic and induced pluripotent stem cells, markers that might be appropriate based on the current knowledge of the gene program leading to sinoatrial node development, what functional characteristics might be expected and desired based on studies of the sinoatrial node, and recent efforts at enrichment and selection of nodal-like cells.

Abnormal sinoatrial node development resulting from disturbed vascular endothelial growth factor signaling.

BACKGROUND: Sinus node dysfunction is frequently observed in patients with congenital heart disease (CHD). Variants in the Vascular Endothelial Growth Factor-A (VEGF) pathway are associated with CHD. In Vegf(120/120) mice, over-expressing VEGF120, a reduced sinoatrial node (SAN) volume was suggested. Aim of the study is to assess the effect of VEGF over-expression on SAN development and function. METHODS: Heart rate was measured in Vegf(120/120) and wildtype (WT) embryos during high frequency ultrasound studies at embryonic day (E)12.5, 14.5 and 17.5 and by optical mapping at E12.5. Morphology was studied with several antibodies. SAN volume estimations were performed, and qualitative-PCR was used to quantify expression of genes in SAN tissues of WT and Vegf(120/120) embryos. RESULTS: Heart rate was reduced in Vegf(120/120) compared with WT embryos during embryonic echocardiography (52 ± 17 versus 125 ± 31 beats per minute (bpm) at E12.5, p<0.001; 123 ± 37 vs 160 ± 29 bmp at E14.5, p=0.024; and 177 ± 30 vs 217 ± 34 bmp, at E17.5 p=0.017) and optical mapping (81 ± 5 vs 116 ± 8 bpm at E12.5; p=0.003). The SAN of mutant embryos was smaller and more vascularized, and showed increased expression of the fast conducting gap junction protein, Connexin43. CONCLUSIONS: Over-expression of VEGF120 results in reduced heart rate and a smaller, less compact and hypervascularized SAN with increased expression of Connexin43. This indicates that VEGF is necessary for normal SAN development and function.

RNA sequencing of mouse sinoatrial node reveals an upstream regulatory role for Islet-1 in cardiac pacemaker cells.

RATIONALE: Treatment of sinus node disease with regenerative or cell-based therapies will require a detailed understanding of gene regulatory networks in cardiac pacemaker cells (PCs). OBJECTIVE: To characterize the transcriptome of PCs using RNA sequencing and to identify transcriptional networks responsible for PC gene expression. METHODS AND RESULTS: We used laser capture microdissection on a sinus node reporter mouse line to isolate RNA from PCs for RNA sequencing. Differential expression and network analysis identified novel sinoatrial node-enriched genes and predicted that the transcription factor Islet-1 is active in developing PCs. RNA sequencing on sinoatrial node tissue lacking Islet-1 established that Islet-1 is an important transcriptional regulator within the developing sinoatrial node. CONCLUSIONS: (1) The PC transcriptome diverges sharply from other cardiomyocytes; (2) Islet-1 is a positive transcriptional regulator of the PC gene expression program.

Phosphorylation of Shox2 is required for its function to control sinoatrial node formation.

BACKGROUND: Inactivation of Shox2, a member of the short-stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown. METHODS AND RESULTS: A specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2-hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p-Shox2a) in cell culture as well as in the developing mouse and human SAN. Site-directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal-regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a-S92AS110A, mutant in the developing heart resulted in down-regulation of Nkx2.5 in wild-type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region. CONCLUSIONS: Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.

The role of connexin40 in developing atrial conduction.

Connexin40 (Cx40) is the main connexin expressed in the murine atria and ventricular conduction system. We assess here the developmental role of Cx40 in atrial conduction of the mouse. Cx40 deficiency significantly prolonged activation times in embryonic day 10.5, 12.5 and 14.5 atria during spontaneous activation; the severity decreased with increasing age. In a majority of Cx40 deficient mice the impulse originated from an ectopic focus in the right atrial appendage; in such a case the activation time was even longer due to prolonged activation. Cx40 has thus an important physiological role in the developing atria.

Ion channel-kinase TRPM7 is required for maintaining cardiac automaticity.

Sick sinus syndrome and atrioventricular block are common clinical problems, often necessitating permanent pacemaker placement, yet the pathophysiology of these conditions remains poorly understood. Here we show that Transient Receptor Potential Melastatin 7 (TRPM7), a divalent-permeant channel-kinase of unknown function, is highly expressed in embryonic myocardium and sinoatrial node (SAN) and is required for cardiac automaticity in these specialized tissues. TRPM7 disruption in vitro, in cultured embryonic cardiomyocytes, significantly reduces spontaneous Ca(2+) transient firing rates and is associated with robust down-regulation of Hcn4, Cav3.1, and SERCA2a mRNA. TRPM7 knockdown in zebrafish, global murine cardiac Trpm7 deletion (KO(αMHC-Cre)), and tamoxifen-inducible SAN restricted Trpm7 deletion (KO(HCN4-CreERT2)) disrupts cardiac automaticity in vivo. Telemetered and sedated KO(αMHC-Cre) and KO(HCN4-CreERT2) mice show episodes of sinus pauses and atrioventricular block. Isolated SAN from KO(αMHC-Cre) mice exhibit diminished Ca(2+) transient firing rates with a blunted diastolic increase in Ca(2+). Action potential firing rates are diminished owing to slower diastolic depolarization. Accordingly, Hcn4 mRNA and the pacemaker current, I(f), are diminished in SAN from both KO(αMHC-Cre) and KO(HCN4-CreERT2) mice. Moreover, heart rates of KO(αMHC-Cre) mice are less sensitive to the selective I(f) blocker ivabradine, and acute application of the recently identified TRPM7 blocker FTY720 has no effect on action potential firing rates of wild-type SAN cells. We conclude that TRPM7 influences diastolic membrane depolarization and automaticity in SAN indirectly via regulation of Hcn4 expression.