Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.

The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3 and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.

The histone demethylase JMJD2B regulates endothelial-to-mesenchymal transition.

Endothelial cells play an important role in maintenance of the vascular system and the repair after injury. Under proinflammatory conditions, endothelial cells can acquire a mesenchymal phenotype by a process named endothelial-to-mesenchymal transition (EndMT), which affects the functional properties of endothelial cells. Here, we investigated the epigenetic control of EndMT. We show that the histone demethylase JMJD2B is induced by EndMT-promoting, proinflammatory, and hypoxic conditions. Silencing of JMJD2B reduced TGF-ß2-induced expression of mesenchymal genes, prevented the alterations in endothelial morphology and impaired endothelial barrier function. Endothelial-specific deletion of JMJD2B in vivo confirmed a reduction of EndMT after myocardial infarction. EndMT did not affect global H3K9me3 levels but induced a site-specific reduction of repressive H3K9me3 marks at promoters of mesenchymal genes, such as Calponin (CNN1), and genes involved in TGF-ß signaling, such as AKT Serine/Threonine Kinase 3 (AKT3) and Sulfatase 1 (SULF1). Silencing of JMJD2B prevented the EndMT-induced reduction of H3K9me3 marks at these promotors and further repressed these EndMT-related genes. Our study reveals that endothelial identity and function is critically controlled by the histone demethylase JMJD2B, which is induced by EndMT-promoting, proinflammatory, and hypoxic conditions, and supports the acquirement of a mesenchymal phenotype.

miR-103/107 regulates left-right asymmetry in zebrafish by modulating Kupffer's vesicle development and ciliogenesis.

In zebrafish, cilia movement within the Kupffer's vesicle (KV) generates a fluid flow responsible for accumulating nodal signals exclusively in the left lateral plate mesoderm, thereby initiating left-right patterning (LRP). Defects in LRP cause devastating congenital disorders including congenital heart malformations due to organ mis-positioning. We identified the miR-103/107 family to be involved in regulating LRP. Depletion of miR-103/107 in zebrafish embryos resulted in malpositioned and malformed visceral organs and hearts due to disturbed LRP gene expression, indicating early defects in LRP. Additionally, loss of miR-103/107 affected KV morphogenesis and cilia formation without disturbing endoderm development. Human fibroblasts depleted of miR-103a/107 often failed to extend cilia or developed shorter cilia, indicating functional conservation between species. We identified arl6, araf and foxH1 as direct targets of miR-103/107 providing a mechanistic link to cilia development and nodal signal titration. We describe a new microRNA family controlling KV development and hence influencing establishment of internal organ asymmetry.

miR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations.

MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent Nkx2.5 enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.

The Zebrafish (Danio rerio) Is a Relevant Model for Studying Sex-Specific Effects of 17ß-Estradiol in the Adult Heart.

Cardiovascular diseases are a major cause of morbidity and mortality, and there are significant sex differences therein. However, the underlying mechanisms are poorly understood. The steroid hormone 17ß-estradiol (E2) is thought to play a major role in cardiovascular sex differences and to be protective, but this may not hold true for males. We aimed at assessing whether the zebrafish is an appropriate model for the study of E2 effects in the heart. We hypothesized that E2 regulates the cardiac contractility of adult zebrafish in a sex-specific manner. Male and female zebrafish were treated with vehicle (control) or E2 and the cardiac contractility was measured 0, 4, 7 and 14 days after treatment initiation using echocardiography. There was no significant effect on the heart rate by E2. Notably, there was a significant decrease in the ejection fraction of male zebrafish treated with E2 compared with controls. By contrast, there was no major difference in the ejection fraction between the two female groups. The dramatic effect in male zebrafish occurred as early as 4 days post treatment initiation. Although there was no significant difference in stroke volume and cardiac output between the two male groups, these were significantly higher in female zebrafish treated with E2 compared with controls. Gene expression analysis revealed that the levels of estrogen receptors were comparable among all groups. In conclusion, our data demonstrate that the adult zebrafish heart robustly responds to E2 and this occurs in a sex-specific manner. Given the benefits of using zebrafish as a model, new targets may be identified for the development of novel cardiovascular therapies for male and female patients. This would contribute towards the realization of personalized medicine.

Induction of cardiac dysfunction in developing and adult zebrafish by chronic isoproterenol stimulation.

Zebrafish is a widely used model to evaluate genetic variants and modifiers that can cause heart muscle diseases. Surprisingly, the ß-adrenergic receptor (ß-AR) pathway in zebrafish is not well characterized, although abnormal ß-AR signaling is a major contributor to human heart failure (HF). Chronic ß-AR activation in the attempt to normalize heart function in the failing heart results in a reduction of the ß-ARs expression and receptor desensitization, largely mediated through G-protein coupled receptor kinase 2 (GRK2) upregulation. This in turn leads to further deterioration of heart function and progression towards HF. This study seeks to systematically characterize the function of the ß-AR signaling in developing and adult zebrafish to ultimately assess the ability to induce HF through chronic ß-AR activation by isoproterenol (ISO) as established in the mouse model. Larval hearts first responded to ISO by 3dpf, in concordance with robust expression of key components of the ß-AR signaling pathway. Although ISO-induced ß1-AR and ß2-AR isoform upregulation persisted, chronic ISO stimulation for 5d caused systolic cardiac dysfunction concurrently with maximal expression of G-protein-coupled receptor kinase-2 (GRK2). More consistent to mammalians, adult zebrafish developed significant heart failure in concert with ß1-AR downregulation, and GRK2 and brain natriuretic peptide (BNP) upregulation in response to prolonged, 14d ISO-stimulation. This was accompanied by significant cell death and inflammation without detectable fibrosis. Our study unveils important characteristics of larvae and adult zebrafish hearts pertaining to ß-AR signaling. A lack of ß-AR responsiveness and atypical ß-AR/GRK2 ratios in larval zebrafish should be considered. Adult zebrafish resembled the mammalian situation on the functional and molecular level more closely, but also revealed differences to dysfunctional mammalian hearts, i.e. lack of fibrosis. Our study establishes the first ISO-inducible HF model in adult zebrafish and present critical characteristics of the zebrafish heart essential to be considered when utilizing the zebrafish as a human disease and future drug discovery model.

Fibronectin mediates mesendodermal cell fate decisions.

Non-cell-autonomous signals often play crucial roles in cell fate decisions during animal development. Reciprocal signaling between endoderm and mesoderm is vital for embryonic development, yet the key signals and mechanisms remain unclear. Here, we show that endodermal cells efficiently promote the emergence of mesodermal cells in the neighboring population through signals containing an essential short-range component. The endoderm-mesoderm interaction promoted precardiac mesoderm formation in mouse embryonic stem cells and involved endodermal production of fibronectin. In vivo, fibronectin deficiency resulted in a dramatic reduction of mesoderm accompanied by endodermal expansion in zebrafish embryos. This event was mediated by regulation of Wnt signaling in mesodermal cells through activation of integrin-ß1. Our findings highlight the importance of the extracellular matrix in mediating short-range signals and reveal a novel function of endoderm, involving fibronectin and its downstream signaling cascades, in promoting the emergence of mesoderm.

Orai1 deficiency leads to heart failure and skeletal myopathy in zebrafish.

Mutations in the store-operated Ca²âº entry pore protein ORAI1 have been reported to cause myopathies in human patients but the mechanism involved is not known. Cardiomyocytes express ORAI1 but its role in heart function is also unknown. Using reverse genetics in zebrafish, we demonstrated that inactivation of the highly conserved zebrafish orthologue of ORAI1 resulted in severe heart failure, reduced ventricular systolic function, bradycardia and skeletal muscle weakness. Electron microscopy of Orai1-deficient myocytes revealed progressive skeletal muscle instability with loss of myofiber integrity and ultrastructural abnormalities of the z-disc in both skeletal and cardiac muscle. Isolated Orai1-deficient cardiomyocytes showed loss of the calcineurin-associated protein calsarcin from the z-discs. Furthermore, we found mechanosignal transduction was affected in Orai1-depleted hearts, indicating an essential role for ORAI1 in establishing the cardiac signaling transduction machinery at the z-disc. Our findings identify ORAI1 as an important regulator of cardiac and skeletal muscle function and provide evidence linking ORAI1-mediated calcium signaling to sarcomere integrity and cardiomyocyte function.

MicroRNA-10 regulates the angiogenic behavior of zebrafish and human endothelial cells by promoting vascular endothelial growth factor signaling.

RATIONALE: Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood. OBJECTIVE: To identify microRNAs that regulate angiogenesis. METHODS AND RESULTS: We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating proangiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth in the trunk of zebrafish larvae, whereas overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells. We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters vascular endothelial growth factor, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in human umbilical venous endothelial cells inhibited the angiogenic behavior of endothelial cells largely by antagonizing vascular endothelial growth factor receptor 2 signaling. CONCLUSIONS: Our study provides insights into how FLT1 and vascular endothelial growth factor receptor 2 signaling is titrated in a microRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.

Identification and functional characterization of zebrafish K(2P)10.1 (TREK2) two-pore-domain K(+) channels.

Two-pore-domain potassium (K(2P)) channels mediate K(+) background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K(2P) currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K(2P) channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K(2P)10.1 channel. K(2P)10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK(2P)10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K(2P)10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K(2P)10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K(+) selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK(2P)10.1, induced robust activation of zK(2P)10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK(2P)10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K(2P)10.1 two-pore-domain K(+) channels that exhibit structural and functional properties largely similar to human K(2P)10.1. We conclude that the zebrafish represents a valid model to study K(2P)10.1 function in vivo.

The myosin-interacting protein SMYD1 is essential for sarcomere organization.

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.

A dual role for ErbB2 signaling in cardiac trabeculation.

Cardiac trabeculation is a crucial morphogenetic process by which clusters of ventricular cardiomyocytes extrude and expand into the cardiac jelly to form sheet-like projections. Although it has been suggested that cardiac trabeculae enhance cardiac contractility and intra-ventricular conduction, their exact function in heart development has not been directly addressed. We found that in zebrafish erbb2 mutants, which we show completely lack cardiac trabeculae, cardiac function is significantly compromised, with mutant hearts exhibiting decreased fractional shortening and an immature conduction pattern. To begin to elucidate the cellular mechanisms of ErbB2 function in cardiac trabeculation, we analyzed erbb2 mutant hearts more closely and found that loss of ErbB2 activity resulted in a complete absence of cardiomyocyte proliferation during trabeculation stages. In addition, based on data obtained from proliferation, lineage tracing and transplantation studies, we propose that cardiac trabeculation is initiated by directional cardiomyocyte migration rather than oriented cell division, and that ErbB2 cell-autonomously regulates this process.

Natural genetic variation quantitatively regulates heart rate and dimension.

The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models, Oryzias latipes, and Oryzias sakaizumii. Employing automated quantification of embryonic heart rates as core metric, we profiled phenotype variability across five inbred strains. We observed maximal phenotypic contrast between individuals of the HO5 and the HdrR strain. HO5 showed elevated heart rates associated with embryonic ventricular hypoplasia and impaired adult cardiac function. This contrast served as the basis for genome-wide mapping. In a segregation population of 1192 HO5 x HdrR F2 embryos, we mapped 59 loci (173 genes) associated with heart rate. Experimental validation of the top 12 candidate genes in loss-of-function models revealed their causal and distinct impact on heart rate, development, ventricle size, and arrhythmia. Our study uncovers new diagnostic and therapeutic targets for developmental and electrophysiological cardiac diseases and provides a novel scalable approach to investigate the intricate genetic architecture of the vertebrate heart.

Reconstitution of defective protein trafficking rescues Long-QT syndrome in zebrafish.

Inherited cardiac arrhythmias are caused by genetic defects in ion channels and associated proteins. Mutations in these channels often do not affect their biophysical properties, but rather interfere with their trafficking to the cell membrane. Accordingly, strategies that could reroute the mutated channels to the membrane should be sufficient to restore the electrical properties of the affected cells, thereby suppressing the underlying arrhythmia. We identified here both, embryonic and adult zebrafish breakdance (bre) as a valuable model for human Long-QT syndrome. Electrocardiograms of adult homozygous bre mutants exhibit significant QT prolongation caused by delayed repolarization of the ventricle. We further show that the bre mutation (zERG(I59S)) disrupts ERG protein trafficking, thereby reducing the amount of active potassium channels on the cell membrane. Interestingly, improvement of channel trafficking by cisapride or dimethylsulfoxid is sufficient to reconstitute ERG channels on the cell membrane in a manner that suffices to suppress the Long-QT induced arrhythmia in breakdance mutant zebrafish. In summary, we show for the first time that therapeutic intervention can cure protein trafficking defects and the associated cardiac arrhythmia in vivo.

A single serine in the carboxyl terminus of cardiac essential myosin light chain-1 controls cardiomyocyte contractility in vivo.

Although it is well known that mutations in the cardiac essential myosin light chain-1 (cmlc-1) gene can cause hypertrophic cardiomyopathy, the precise in vivo structural and functional roles of cMLC-1 in the heart are only poorly understood. We have isolated the zebrafish mutant lazy susan (laz), which displays severely reduced contractility of both heart chambers. By positional cloning, we identified a nonsense mutation within the zebrafish cmlc-1 gene to be responsible for the laz phenotype, leading to expression of a carboxyl-terminally truncated cMLC-1. Whereas complete loss of cMLC-1 leads to cardiac acontractility attributable to impaired cardiac sarcomerogenesis, expression of a carboxyl-terminally truncated cMLC-1 in laz mutant hearts is sufficient for normal cardiac sarcomerogenesis but severely impairs cardiac contractility in a cell-autonomous fashion. Whereas overexpression of wild-type cMLC-1 restores contractility of laz mutant cardiomyocytes, overexpression of phosphorylation site serine 195-deficient cMLC-1 (cMLC-1(S195A)) does not reconstitute cardiac contractility in laz mutant cardiomyocytes. By contrast, introduction of a phosphomimetic amino acid on position 195 (cMLC-1(S195D)) rescues cardiomyocyte contractility, demonstrating for the first time an essential role of the carboxyl terminus and especially of serine 195 of cMLC-1 in the regulation of cardiac contractility.

Identification and Tissue-Specific Characterization of Novel SHOX-Regulated Genes in Zebrafish Highlights SOX Family Members Among Other Genes.

SHOX deficiency causes a spectrum of clinical phenotypes related to skeletal dysplasia and short stature, including Léri-Weill dyschondrosteosis, Langer mesomelic dysplasia, Turner syndrome, and idiopathic short stature. SHOX controls chondrocyte proliferation and differentiation, bone maturation, and cellular growth arrest and apoptosis via transcriptional regulation of its direct target genes NPPB, FGFR3, and CTGF. However, our understanding of SHOX-related pathways is still incomplete. To elucidate the underlying molecular mechanisms and to better understand the broad phenotypic spectrum of SHOX deficiency, we aimed to identify novel SHOX targets. We analyzed differentially expressed genes in SHOX-overexpressing human fibroblasts (NHDF), and confirmed the known SHOX target genes NPPB and FGFR among the most strongly regulated genes, together with 143 novel candidates. Altogether, 23 genes were selected for further validation, first by whole-body characterization in developing shox-deficient zebrafish embryos, followed by tissue-specific expression analysis in three shox-expressing zebrafish tissues: head (including brain, pharyngeal arches, eye, and olfactory epithelium), heart, and pectoral fins. Most genes were physiologically relevant in the pectoral fins, while only few genes were also significantly regulated in head and heart tissue. Interestingly, multiple sox family members (sox5, sox6, sox8, and sox18) were significantly dysregulated in shox-deficient pectoral fins together with other genes (nppa, nppc, cdkn1a, cdkn1ca, cyp26b1, and cy26c1), highlighting an important role for these genes in shox-related growth disorders. Network-based analysis integrating data from the Ingenuity pathways revealed that most of these genes act in a common network. Our results provide novel insights into the genetic pathways and molecular events leading to the clinical manifestation of SHOX deficiency.

Cardiac Regeneration and Tumor Growth-What Do They Have in Common?

Acute myocardial infarction is a leading cause of death. Unlike most adult mammals, zebrafish have the capability to almost fully regenerate their hearts after injury. In contrast, ischemic damage in adult human and mouse hearts usually results in scar tissue. mRNA-Sequencing (Seq) and miRNA-Seq analyses of heart regeneration in zebrafish over time showed that the process can be divided into three phases: the first phase represents dedifferentiation and proliferation of cells, the second phase is characterized by migration, and in the third phase cell signals indicate heart development and differentiation. The first two phases seem to share major similarities with tumor development and growth. To gain more insight into these similarities between cardiac regeneration and tumor development and growth, we used patient matched tumor normal ("healthy") RNA-Seq data for several tumor entities from The Cancer Genome Atlas (TCGA). Subsequently, RNA data were processed using the same pipeline for both the zebrafish samples and tumor datasets. Functional analysis showed that multiple Gene Ontology terms (GO terms) are involved in both early stage cardiac regeneration and tumor development/growth across multiple tumor entities. These GO terms are mostly associated with cell cycle processes. Further analysis showed that orthologous genes are the same key players that regulated these changes in both diseases. We also observed that GO terms associated with heart development in the third late phase of cardiac regeneration are downregulated in the tumor entities. Taken together, our analysis illustrates similarities between cardiac remodeling and tumor progression.

Deficient zebrafish ether-à-go-go-related gene channel gating causes short-QT syndrome in zebrafish reggae mutants.

BACKGROUND: Genetic predisposition is believed to be responsible for most clinically significant arrhythmias; however, suitable genetic animal models to study disease mechanisms and evaluate new treatment strategies are largely lacking. METHODS AND RESULTS: In search of suitable arrhythmia models, we isolated the zebrafish mutation reggae (reg), which displays clinical features of the malignant human short-QT syndrome such as accelerated cardiac repolarization accompanied by cardiac fibrillation. By positional cloning, we identified the reg mutation that resides within the voltage sensor of the zebrafish ether-à-go-go-related gene (zERG) potassium channel. The mutation causes premature zERG channel activation and defective inactivation, which results in shortened action potential duration and accelerated cardiac repolarization. Genetic and pharmacological inhibition of zERG rescues recessive reg mutant embryos, which confirms the gain-of-function effect of the reg mutation on zERG channel function in vivo. Accordingly, QT intervals in ECGs from heterozygous and homozygous reg mutant adult zebrafish are considerably shorter than in wild-type zebrafish. CONCLUSIONS: With its molecular and pathophysiological concordance to the human arrhythmia syndrome, zebrafish reg represents the first animal model for human short-QT syndrome.

Biophysical properties of zebrafish ether-à-go-go related gene potassium channels.

The zebrafish is increasingly recognized as an animal model for the analysis of hERG-related diseases. However, functional properties of the zebrafish orthologue of hERG have not been analyzed yet. We heterologously expressed cloned ERG channels in Xenopus oocytes and analyzed biophysical properties using the voltage clamp technique. zERG channels conduct rapidly activating and inactivating potassium currents. However, compared to hERG, the half-maximal activation voltage of zERG current is shifted towards more positive potentials and the half maximal steady-state inactivation voltage is shifted towards more negative potentials. zERG channel activation is delayed and channel deactivation is accelerated significantly. However, time course of zERG conducted current under action potential clamp is highly similar to the human orthologue. In summary, we show that ERG channels in zebrafish exhibit biophysical properties similar to the human orthologue. Considering the conserved channel function, the zebrafish represents a valuable model to investigate human ERG channel related diseases.

Biallelic Variants in ASNA1, Encoding a Cytosolic Targeting Factor of Tail-Anchored Proteins, Cause Rapidly Progressive Pediatric Cardiomyopathy.

BACKGROUND: Pediatric cardiomyopathies are a clinically and genetically heterogeneous group of heart muscle disorders associated with high morbidity and mortality. Although knowledge of the genetic basis of pediatric cardiomyopathy has improved considerably, the underlying cause remains elusive in a substantial proportion of cases. METHODS: Exome sequencing was used to screen for the causative genetic defect in a pair of siblings with rapidly progressive dilated cardiomyopathy and death in early infancy. Protein expression was assessed in patient samples, followed by an in vitro tail-anchored protein insertion assay and functional analyses in zebrafish. RESULTS: We identified compound heterozygous variants in the highly conserved ASNA1 gene (arsA arsenite transporter, ATP-binding, homolog), which encodes an ATPase required for post-translational membrane insertion of tail-anchored proteins. The c.913C>T variant on the paternal allele is predicted to result in a premature stop codon p.(Gln305*), and likely explains the decreased protein expression observed in myocardial tissue and skin fibroblasts. The c.488T>C variant on the maternal allele results in a valine to alanine substitution at residue 163 (p.Val163Ala). Functional studies showed that this variant leads to protein misfolding as well as less effective tail-anchored protein insertion. Loss of asna1 in zebrafish resulted in reduced cardiac contractility and early lethality. In contrast to wild-type mRNA, injection of either mutant mRNA failed to rescue this phenotype. CONCLUSIONS: Biallelic variants in ASNA1 cause severe pediatric cardiomyopathy and early death. Our findings point toward a critical role of the tail-anchored membrane protein insertion pathway in vertebrate cardiac function and disease.