TGFBR1 Variants Can Associate with Non-Syndromic Congenital Heart Disease without Aortopathy.

BACKGROUND: Congenital heart diseases (CHD) are the most common congenital malformations in newborns and remain the leading cause of mortality among infants under one year old. Molecular diagnosis is crucial to evaluate the recurrence risk and to address future prenatal diagnosis. Here, we describe two families with various forms of inherited non-syndromic CHD and the genetic work-up and resultant findings. METHODS: Next-generation sequencing (NGS) was employed in both families to uncover the genetic cause. In addition, we performed functional analysis to investigate the consequences of the identified variants in vitro. RESULTS: NGS identified possible causative variants in both families in the protein kinase domain of the TGFBR1 gene. These variants occurred on the same amino acid, but resulted in differently substituted amino acids (p.R398C/p.R398H). Both variants co-segregate with the disease, are extremely rare or unique, and occur in an evolutionary highly conserved domain of the protein. Furthermore, both variants demonstrated a significantly altered TGFBR1-smad signaling activity. Clinical investigation revealed that none of the carriers had (signs of) aortopathy. CONCLUSION: In conclusion, we describe two families, with various forms of inherited non-syndromic CHD without aortopathies, associated with unique/rare variants in TGFBR1 that display altered TGF-beta signaling. These findings highlight involvement of TGFBR1 in CHD, and warrant consideration of potential causative TGFBR1 variants also in CHD patients without aortopathies.

Role of Genetic Variation in Transcriptional Regulatory Elements in Heart Rhythm.

Genetic predisposition to cardiac arrhythmias has been a field of intense investigation. Research initially focused on rare hereditary arrhythmias, but over the last two decades, the role of genetic variation (single nucleotide polymorphisms) in heart rate, rhythm, and arrhythmias has been taken into consideration as well. In particular, genome-wide association studies have identified hundreds of genomic loci associated with quantitative electrocardiographic traits, atrial fibrillation, and less common arrhythmias such as Brugada syndrome. A significant number of associated variants have been found to systematically localize in non-coding regulatory elements that control the tissue-specific and temporal transcription of genes encoding transcription factors, ion channels, and other proteins. However, the identification of causal variants and the mechanism underlying their impact on phenotype has proven difficult due to the complex tissue-specific, time-resolved, condition-dependent, and combinatorial function of regulatory elements, as well as their modest conservation across different model species. In this review, we discuss research efforts aimed at identifying and characterizing-trait-associated variant regulatory elements and the molecular mechanisms underlying their impact on heart rate or rhythm.

Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction.

BACKGROUND: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. METHODS: We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). RESULTS: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. CONCLUSIONS: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

Common Genetic Variants Contribute to Risk of Transposition of the Great Arteries.

RATIONALE: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. OBJECTIVE: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. METHODS AND RESULTS: We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. CONCLUSIONS: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.

Neuregulin-1 enhances cell-cycle activity, delays cardiac fibrosis, and improves cardiac performance in rat pups with right ventricular pressure load.

OBJECTIVES: Right ventricular (RV) failure is a leading cause of death in patients with congenital heart disease. RV failure is kept at bay during childhood. Limited proliferation of cardiomyocytes is present in the postnatal heart. We propose that cardiomyocyte proliferation improves RV adaptation to pressure load (PL). We studied adaptation in response to increased RV PL and the role of increased cardiomyocyte cell cycle activity (CCA) in rat pups growing into adulthood. METHODS: We induced RV PL at day of weaning in rats (3 weeks; 30-40 g) by pulmonary artery banding and followed rats into adulthood (300 g). We performed histological analyses and RNA sequencing analysis. To study the effects of increased cardiomyocyte cell cycle activity, we administered neuregulin-1 (NRG1), a growth factor involved in cardiac development. RESULTS: PL induced an increase in CCA, with subsequent decline of CCA (sham/PL at 4 weeks: 0.14%/0.83%; P = .04 and 8 weeks: 0.00%/0.00%; P = .484) and cardiac function (cardiac index: control/PL 4 weeks: 4.41/3.29; P = .468 and 8 weeks: 3.57/1.44; P = .024). RNA sequencing analysis revealed delayed maturation and increased CCA pathways. NRG1 stimulated CCA (PL vehicle/NRG1 at 2 weeks: 0.62%/2.28%; P = .003), improved cardiac function (cardiac index control vs vehicle/NRG1 at 2 weeks: 4.21 vs 3.07/4.17; P = .009/.705) and postponed fibrosis (control vs vehicle/NRG1 at 4 weeks: 1.66 vs 4.82%/2.97%; P = .009/.078) in RV PL rats during childhood. CONCLUSIONS: RV PL during growth induces a transient CCA increase. Further CCA stimulation improves cardiac function and delays fibrosis. This proof-of-concept study shows that stimulation of CCA can improve RV adaptation to PL in the postnatal developing heart and might provide a new approach to preserve RV function in patients with congenital heart disease.

Epigenetic State Changes Underlie Metabolic Switch in Mouse Post-Infarction Border Zone Cardiomyocytes.

Myocardial infarction causes ventricular muscle loss and formation of scar tissue. The surviving myocardium in the border zone, located adjacent to the infarct, undergoes profound changes in function, structure and composition. How and to what extent these changes of border zone cardiomyocytes are regulated epigenetically is not fully understood. Here, we obtained transcriptomes of PCM-1-sorted mouse cardiomyocyte nuclei of healthy left ventricle and 7 days post myocardial infarction border zone tissue. We validated previously observed downregulation of genes involved in fatty acid metabolism, oxidative phosphorylation and mitochondrial function in border zone-derived cardiomyocytes, and observed a modest induction of genes involved in glycolysis, including Slc2a1 (Glut1) and Pfkp. To gain insight into the underlying epigenetic regulatory mechanisms, we performed H3K27ac profiling of healthy and border zone cardiomyocyte nuclei. We confirmed the switch from Mef2- to AP-1 chromatin association in border zone cardiomyocytes, and observed, in addition, an enrichment of PPAR/RXR binding motifs in the sites with reduced H3K27ac signal. We detected downregulation and accompanying epigenetic state changes at several key PPAR target genes including Ppargc1a (PGC-1α), Cpt2, Ech1, Fabpc3 and Vldrl in border zone cardiomyocytes. These data indicate that changes in epigenetic state and gene regulation underlie the maintained metabolic switch in border zone cardiomyocytes.

Variant Intronic Enhancer Controls SCN10A-short Expression and Heart Conduction.

BACKGROUND: Genetic variants in SCN10A, encoding the neuronal voltage-gated sodium channel NaV1.8, are strongly associated with atrial fibrillation, Brugada syndrome, cardiac conduction velocities, and heart rate. The cardiac function of SCN10A has not been resolved, however, and diverging mechanisms have been proposed. Here, we investigated the cardiac expression of SCN10A and the function of a variant-sensitive intronic enhancer previously linked to the regulation of SCN5A, encoding the major essential cardiac sodium channel NaV1.5. METHODS: The expression of SCN10A was investigated in mouse and human hearts. With the use of CRISPR/Cas9 genome editing, the mouse intronic enhancer was disrupted, and mutant mice were characterized by transcriptomic and electrophysiological analyses. The association of genetic variants at SCN5A-SCN10A enhancer regions and gene expression were evaluated by genome-wide association studies single-nucleotide polymorphism mapping and expression quantitative trait loci analysis. RESULTS: We found that cardiomyocytes of the atria, sinoatrial node, and ventricular conduction system express a short transcript comprising the last 7 exons of the gene (Scn10a-short). Transcription occurs from an intronic enhancer-promoter complex, whereas full-length Scn10a transcript was undetectable in the human and mouse heart. Expression quantitative trait loci analysis revealed that the genetic variants in linkage disequilibrium with genetic variant rs6801957 in the intronic enhancer associate with SCN10A transcript levels in the heart. Genetic modification of the enhancer in the mouse genome led to reduced cardiac Scn10a-short expression in atria and ventricles, reduced cardiac sodium current in atrial cardiomyocytes, atrial conduction slowing and arrhythmia, whereas the expression of Scn5a, the presumed enhancer target gene, remained unaffected. In patch-clamp transfection experiments, expression of Scn10a-short-encoded NaV1.8-short increased NaV1.5-mediated sodium current. We propose that noncoding genetic variation modulates transcriptional regulation of Scn10a-short in cardiomyocytes that impacts NaV1.5-mediated sodium current and heart rhythm. CONCLUSIONS: Genetic variants in and around SCN10A modulate enhancer function and expression of a cardiac-specific SCN10A-short transcript. We propose that noncoding genetic variation modulates transcriptional regulation of a functional C-terminal portion of NaV1.8 in cardiomyocytes that impacts on NaV1.5 function, cardiac conduction velocities, and arrhythmia susceptibility.

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system.

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3+/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3+/- mutants. Notably, Tbx3+/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction.

Genome-wide association studies have implicated common genomic variants in the gene desert upstream of TBX3 in cardiac conduction velocity. Whether these noncoding variants affect expression of TBX3 or neighboring genes and how they affect cardiac conduction is not understood. Here, we use high-throughput STARR-seq to test the entire 1.3 Mb human and mouse TBX3 locus, including two cardiac conduction-associated variant regions, for regulatory function. We identified multiple accessible and functional regulatory DNA elements that harbor variants affecting their activity. Both variant regions drove gene expression in the cardiac conduction tissue in transgenic reporter mice. Genomic deletion from the mouse genome of one of the regions caused increased cardiac expression of only Tbx3, PR interval shortening and increased QRS duration. Combined, our findings address the mechanistic link between trait-associated variants in the gene desert, TBX3 regulation and cardiac conduction.

Identification of Functional Variant Enhancers Associated With Atrial Fibrillation.

RATIONALE: Genome-wide association studies have identified a large number of common variants (single-nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located mainly in noncoding regions of the genome and likely include variants that modulate the function of transcriptional regulatory elements (REs) such as enhancers. However, the actual REs modulated by variants and the target genes of such REs remain to be identified. Thus, the biological mechanisms by which genetic variation promotes AF has thus far remained largely unexplored. OBJECTIVE: To identify REs in genome-wide association study loci that are influenced by AF-associated variants. METHODS AND RESULTS: We screened 2.45 Mbp of human genomic DNA containing 12 strongly AF-associated loci for RE activity using self-transcribing active regulatory region sequencing and a recently generated monoclonal line of conditionally immortalized rat atrial myocytes. We identified 444 potential REs, 55 of which contain AF-associated variants (P<10-8). Subsequently, using an adaptation of the self-transcribing active regulatory region sequencing approach, we identified 24 variant REs with allele-specific regulatory activity. By mining available chromatin conformation data, the possible target genes of these REs were mapped. To define the physiological function and target genes of such REs, we deleted the orthologue of an RE containing noncoding variants in the Hcn4 (potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) locus of the mouse genome. Mice heterozygous for the RE deletion showed bradycardia, sinus node dysfunction, and selective loss of Hcn4 expression. CONCLUSIONS: We have identified REs at multiple genetic loci for AF and found that loss of an RE at the HCN4 locus results in sinus node dysfunction and reduced gene expression. Our approach can be broadly applied to facilitate the identification of human disease-relevant REs and target genes at cardiovascular genome-wide association studies loci.

Development of the human heart.

In 2014, an extensive review discussing the major steps of cardiac development focusing on growth, formation of primary and chamber myocardium and the development of the cardiac electrical system, was published. Molecular genetic lineage analyses have since furthered our insight in the developmental origin of the various component parts of the heart, which currently can be unambiguously identified by their unique molecular phenotype. Moreover, genetic, molecular and cell biological analyses have driven insights into the mechanisms underlying the development of the different cardiac components. Here, we build on our previous review and provide an insight into the molecular mechanistic revelations that have forwarded the field of cardiac development. Despite the enormous advances in our knowledge over the last decade, the development of congenital cardiac malformations remains poorly understood. The challenge for the next decade will be to evaluate the different developmental processes using newly developed molecular genetic techniques to further unveil the gene regulatory networks operational during normal and abnormal cardiac development.

An enhancer cluster controls gene activity and topology of the SCN5A-SCN10A locus in vivo.

Mutations and variations in and around SCN5A, encoding the major cardiac sodium channel, influence impulse conduction and are associated with a broad spectrum of arrhythmia disorders. Here, we identify an evolutionary conserved regulatory cluster with super enhancer characteristics downstream of SCN5A, which drives localized cardiac expression and contains conduction velocity-associated variants. We use genome editing to create a series of deletions in the mouse genome and show that the enhancer cluster controls the conformation of a >0.5 Mb genomic region harboring multiple interacting gene promoters and enhancers. We find that this cluster and its individual components are selectively required for cardiac Scn5a expression, normal cardiac conduction and normal embryonic development. Our studies reveal physiological roles of an enhancer cluster in the SCN5A-SCN10A locus, show that it controls the chromatin architecture of the locus and Scn5a expression, and suggest that genetic variants affecting its activity may influence cardiac function.

Identification of atrial fibrillation associated genes and functional non-coding variants.

Disease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target gene expression. To uncover causal genetic variants, variant regulatory elements and their target genes, here we cross-reference human transcriptomic, epigenomic and chromatin conformation datasets. Of 104 genetic variant regions associated with atrial fibrillation candidate target genes are prioritized. We optimize EMERGE enhancer prediction and use accessible chromatin profiles of human atrial cardiomyocytes to more accurately predict cardiac regulatory elements and identify hundreds of sub-threshold variants that co-localize with regulatory elements. Removal of mouse homologues of atrial fibrillation-associated regions in vivo uncovers a distal regulatory region involved in Gja1 (Cx43) expression. Our analyses provide a shortlist of genes likely affected by atrial fibrillation-associated variants and provide variant regulatory elements in each region that link genetic variation and target gene regulation, helping to focus future investigations.

Identification and Characterization of a Transcribed Distal Enhancer Involved in Cardiac Kcnh2 Regulation.

The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying IKr, a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer ∼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers.

Conserved NPPB+ Border Zone Switches From MEF2- to AP-1-Driven Gene Program.

BACKGROUND: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb, encoding B-type natriuretic peptide, after infarction. METHODS: Transgenic reporter mice were used to identify the Nppb-positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing. RESULTS: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB, ANKRD1, DES, UCHL1, JUN, and FOXP1. Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes. CONCLUSIONS: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1-driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.

Chromatin Conformation Links Putative Enhancers in Intracranial Aneurysm-Associated Regions to Potential Candidate Genes.

Background We previously showed that intracranial aneurysm ( IA )-associated single-nucleotide polymorphisms are enriched in promoters and putative enhancers identified in the human circle of Willis, on which IA s develop, suggesting a role for promoters and enhancers in IAs . We further investigated the role of putative enhancers in the pathogenesis of IA by identifying their potential target genes and validating their regulatory activity. Methods and Results Using our previously published circle of Willis chromatin immunoprecipitation and sequencing data, we selected 34 putative enhancers in IA -associated regions from genome-wide association studies. We then used a chromatin conformation capture technique to prioritize target genes and found that 15 putative enhancers interact with the promoters of 6 target genes: SOX 17, CDKN 2B, MTAP , CNNM 2, RPEL 1, and GATA 6. Subsequently, we assessed the activity of these putative enhancers in vivo in zebrafish embryos and confirmed activity for 8 putative enhancers. Last, we found that all 6 target genes are expressed in the circle of Willis, on the basis of RNA sequencing data and in situ hybridization. Furthermore, in situ hybridization showed that these genes are expressed in multiple cell types in the circle of Willis. Conclusions In 4 of 6 IA -associated genome-wide association study regions, we identified 8 putative enhancers that are active in vivo and interact with 6 nearby genes, suggesting that these genes are regulated by the identified putative enhancers. These genes, SOX 17, CDKN 2B, MTAP , CNNM 2, RPEL 1, and GATA 6, are therefore potential candidate genes involved in IA pathogenesis and should be studied using animal models in the future.

Development, Proliferation, and Growth of the Mammalian Heart.

During development, the embryonic heart grows by addition of cells from a highly proliferative progenitor pool and by subsequent precisely controlled waves of cardiomyocyte proliferation. In this period, the heart can compensate for cardiomyocyte loss by an increased proliferation rate of the remaining cardiomyocytes. This proliferative capacity is lost soon after birth, with heart growth continuing by an increase in cardiomyocyte volume. The failure of the injured adult heart to regenerate often leads to the development of heart failure, a major cause of death. With the recent observation of a small fraction of cardiomyocytes that appear to have retained the proliferative capacity within the adult heart, as well as the identification of developmental pathways such as the Hippo-signaling pathway that can invoke mature cardiomyocyte proliferation, more studies are taking a knowledge-based mechanistic approach to heart regeneration. A key question being asked is if this knowledge can be used therapeutically to reinitiate cardiomyocyte proliferation after injury such as myocardial infarction. In this respect, uncovering and understanding the mechanisms and conditions that give rise to a fully functional and adaptive heart in the developing embryo could provide us with the answers to many of the questions that are now being asked.

Follistatin-like 1 in development and human diseases.

Follistatin-like 1 (FSTL1) is a secreted glycoprotein displaying expression changes during development and disease, among which cardiovascular disease, cancer, and arthritis. The cardioprotective role of FSTL1 has been intensively studied over the last years, though its mechanism of action remains elusive. FSTL1 is involved in multiple signaling pathways and biological processes, including vascularization and regulation of the immune response, a feature that complicates its study. Binding to the DIP2A, TLR4 and BMP receptors have been shown, but other molecular partners probably exist. During cancer progression and rheumatoid arthritis, controversial data have been reported with respect to the proliferative, apoptotic, migratory, and inflammatory effects of FSTL1. This controversy might reside in the extensive post-transcriptional regulation of FSTL1. The FSTL1 primary transcript also encodes for a microRNA (miR-198) in primates and multiple microRNA-binding sites are present in the 3'UTR. The switch between expression of the FSTL1 protein and miR-198 is an important regulator of tumour metastasis and wound healing. The glycosylation state of FSTL1 is a determinant of biological activity, in cardiomyocytes the glycosylated form promoting proliferation and the non-glycosylated working anti-apoptotic. Moreover, the glycosylation state shows differences between species and tissues which might underlie the differences observed in in vitro studies. Finally, regulation at the level of protein secretion has been described.

Structure and function of the Nppa-Nppb cluster locus during heart development and disease.

Atrial natriuretic factor and brain natriuretic peptide are two important biomarkers in clinical cardiology. These two natriuretic peptide hormones are encoded by the paralogous genes Nppa and Nppb, which are evolutionary conserved. Both genes are predominantly expressed by the heart muscle during the embryonic and fetal stages, and in particular Nppa expression is strongly reduced in the ventricles after birth. Upon cardiac stress, Nppa and Nppb are strongly upregulated in the ventricular myocardium. Much is known about the molecular and physiological ques inducing Nppa and Nppb expression; however, the transcriptional regulatory mechanisms of the Nppa-Nppb cluster in vivo has proven to be quite complex and is not well understood. In this review, we will provide recent insights into the dynamic and complex regulation of Nppa and Nppb during heart development and hypertrophy, and the association of this gene cluster with the cardiomyocyte-intrinsic program of heart regeneration.

Vanadium Chloroperoxidases: The Missing Link in the Formation of Chlorinated Compounds and Chloroform in the Terrestrial Environment?

It is well established that the majority of chlorinated organic substances found in the terrestrial environment are produced naturally. The presence of these compounds in soils is not limited to a single ecosystem. Natural chlorination is also a widespread phenomenon in grasslands and agricultural soils typical for unforested areas. These chlorinated compounds are formed from chlorination of natural organic matter consisting of very complex chemical structures, such as lignin. Chlorination of several lignin model compounds results in the intermediate formation of trichloroacetyl-containing compounds, which are also found in soils. These decay, in general, through a haloform-type reaction mechanism to CHCl3 . Upon release into the atmosphere, CHCl3 will produce chlorine radicals through photolysis, which will, in turn, lead to natural depletion of ozone. There is evidence that fungal chloroperoxidases able to produce HOCl are involved in the chlorination of natural organic matter. The objective of this review is to clarify the role and source of the various chloroperoxidases involved in the natural formation of CHCl3 .