POPDC1 Variants Cause Atrioventricular Node Dysfunction and Arrhythmogenic Changes in Cardiac Electrophysiology and Intracellular Calcium Handling in Zebrafish.

Popeye domain-containing (POPDC) proteins selectively bind cAMP and mediate cellular responses to sympathetic nervous system (SNS) stimulation. The first discovered human genetic variant (POPDC1S201F) is associated with atrioventricular (AV) block, which is exacerbated by increased SNS activity. Zebrafish carrying the homologous mutation (popdc1S191F) display a similar phenotype to humans. To investigate the impact of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling, homozygous popdc1S191F and popdc1 knock-out (popdc1KO) zebrafish larvae and adult isolated popdc1S191F hearts were studied by functional fluorescent analysis. It was found that in popdc1S191F and popdc1KO larvae, heart rate (HR), AV delay, action potential (AP) and calcium transient (CaT) upstroke speed, and AP duration were less than in wild-type larvae, whereas CaT duration was greater. SNS stress by ß-adrenergic receptor stimulation with isoproterenol increased HR, lengthened AV delay, slowed AP and CaT upstroke speed, and shortened AP and CaT duration, yet did not result in arrhythmias. In adult popdc1S191F zebrafish hearts, there was a higher incidence of AV block, slower AP upstroke speed, and longer AP duration compared to wild-type hearts, with no differences in CaT. SNS stress increased AV delay and led to further AV block in popdc1S191F hearts while decreasing AP and CaT duration. Overall, we have revealed that arrhythmogenic effects of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling in zebrafish are varied, but already present in early development, and that AV node dysfunction may underlie SNS-induced arrhythmogenesis associated with popdc1 mutation in adults.

Genomic and physiological analyses of the zebrafish atrioventricular canal reveal molecular building blocks of the secondary pacemaker region.

The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-ß, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs.

Synthesis and Antibacterial Study of Sulfobetaine/Quaternary Ammonium-Modified Star-Shaped Poly[2-(dimethylamino)ethyl methacrylate]-Based Copolymers with an Inorganic Core.

Cationic polymethacrylates are interesting candidates for bacterial disinfectants since they can be made in large-scale by various well-established polymerization techniques such as atom transfer radical polymerization (ATRP). However, they are usually toxic or ineffective in serum and various strategies to improve their biocompatibility or nonfouling property have often resulted in compromised bactericidal activity. Also, star-shaped polymers are less explored than linear polymers for application as antibacterial compounds. In this paper, star polymers with poly[2-(dimethylamino)ethyl methacrylate] (PDMA) as the arms and polyhedral oligomeric silsesquioxane (POSS) as the core (POSS-g-PDMA) were successfully synthesized by ATRP. The minimum inhibition concentrations (MICs) of the synthesized POSS-g-PDMA are in the range of 10-20 µg/mL. POSS-g-PDMA was further modified by various hydrophilization strategies in attempting to reduce hemolysis. With quaternization of POSS-g-PDMA, the antibacterial activities of the obtained quaternary polymers are almost unchanged and the copolymers become relatively nonhemolytic. We also copolymerized sulfobetaine (SB) with POSS-g-PDMA to obtain random and block PDMA-co-PSB arm structures, where the PDMA and poly(sulfobetaine) were the cationic and zwitterionic blocks, respectively. The random cationic-zwitterionic POSS-g-(PDMA-r-PSB) copolymers showed poor antibacterial activity, while the block POSS-g-(PDMA-b-PSB) copolymers retained the antibacterial and hemolytic activity of the pristine POSS-g-PDMA. Further, the block copolymers of POSS-g-(PDMA-b-PSB) showed enhanced antifouling property and serum stability as seen by their nanoparticle size stability in the presence of serum and reduced red blood cell aggregation; the POSS-g-(PDMA-b-PSB) also somewhat retained its MIC in blood unlike the quaternized or random zwitterionic copolymers. The antibacterial kinetics study showed that Escherichia coli can be killed within 30 min by both random and block copolymers of POSS-g-(PDMA-co-PSB). Finally, our POSS star polymers showed low toxicity to zebrafish embryo and could be potentially used in aquaculture antibacterial applications.

Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates.

Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.

Development of the cardiac conduction system in zebrafish.

The cardiac conduction system (CCS) propagates and coordinates the electrical excitation that originates from the pacemaker cells, throughout the heart, resulting in rhythmic heartbeat. Its defects result in life-threatening arrhythmias and sudden cardiac death. Understanding of the factors involved in the formation and function of the CCS remains incomplete. By transposon assisted transgenesis, we have developed enhancer trap (ET) lines of zebrafish that express fluorescent protein in the pacemaker cells at the sino-atrial node (SAN) and the atrio-ventricular region (AVR), termed CCS transgenics. This expression pattern begins at the stage when the heart undergoes looping morphogenesis at 36 h post fertilization (hpf) and is maintained into adulthood. Using the CCS transgenics, we investigated the effects of perturbation of cardiac function, as simulated by either the absence of endothelium or hemodynamic stimulation, on the cardiac conduction cells, which resulted in abnormal compaction of the SAN. To uncover the identity of the gene represented by the EGFP expression in the CCS transgenics, we mapped the transposon integration sites on the zebrafish genome to positions in close proximity to the gene encoding fibroblast growth homologous factor 2a (fhf2a). Fhf2a is represented by three transcripts, one of which is expressed in the developing heart. These transgenics are useful tools for studies of development of the CCS and cardiac disease.

POPDC1(S201F) causes muscular dystrophy and arrhythmia by affecting protein trafficking.

The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.

The cAMP-binding Popdc proteins have a redundant function in the heart.

Popdc (Popeye-domain-containing) genes encode membrane-bound proteins and are abundantly present in cardiac myocytes and in skeletal muscle fibres. Functional analysis of Popdc1 (Bves) and Popdc2 in mice and of popdc2 in zebrafish revealed an overlapping role for proper electrical conduction in the heart and maintaining structural integrity of skeletal muscle. Popdc proteins mediate cAMP signalling and modulate the biological activity of interacting proteins. The two-pore channel TREK-1 interacts with all three Popdc proteins. In Xenopus oocytes, the presence of Popdc proteins causes an enhanced membrane transport leading to an increase in TREK-1 current, which is blocked when cAMP levels are increased. Another important Popdc-interacting protein is caveolin 3, and the loss of Popdc1 affects caveolar size. Thus a family of membrane-bound cAMP-binding proteins has been identified, which modulate the subcellular localization of effector proteins involved in organizing signalling complexes and assuring proper membrane physiology of cardiac myocytes.

The Popeye Domain Containing Genes and cAMP Signaling.

3'-5'-cyclic adenosine monophosphate (cAMP) is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs). Initially, it was thought that protein kinase A (PKA) exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC) and hyperpolarizing cyclic nucleotide-gated (HCN) channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc) genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins.

Popeye domain-containing proteins and stress-mediated modulation of cardiac pacemaking.

An intricate network of ion channels and pumps are involved in generating a diastolic pacemaker potential, which is transmitted to the working myocardium with the help of the cardiac conduction system. The principles of cardiac pacemaking are reasonably well understood, however, the mechanism by which the heart increases its beating frequency in response to adrenergic stimulation has not been fully worked out. The Popeye domain-containing (Popdc) genes encode plasma membrane-localized proteins that are able to bind cAMP with high affinity; mice with null mutations in Popdc1 or 2 have a stress-induced pacemaker dysfunction. The phenotype in both mutants develops in an age-dependent manner and thus may model pacemaker dysfunction in man, as well as provide novel mechanistic insights into the process of pacemaker adaptation to stress.

The Popeye domain containing 2 (popdc2) gene in zebrafish is required for heart and skeletal muscle development.

The Popeye domain containing (Popdc) genes encode a family of transmembrane proteins with an evolutionary conserved Popeye domain. These genes are abundantly expressed in striated muscle tissue, however their function is not well understood. In this study we have investigated the role of the popdc2 gene in zebrafish. Popdc2 transcripts were detected in the embryonic myocardium and transiently in the craniofacial and tail musculature. Morpholino oligonucleotide-mediated knockdown of popdc2 resulted in aberrant development of skeletal muscle and heart. Muscle segments in the trunk were irregularly shaped and craniofacial muscles were severely reduced or even missing. In the heart, pericardial edema was prevalent in the morphants and heart chambers were elongated and looping was abnormal. These pathologies in muscle and heart were alleviated after reducing the morpholino concentration. However the heart still was abnormal displaying cardiac arrhythmia at later stages of development. Optical recordings of cardiac contractility revealed irregular ventricular contractions with a 2:1, or 3:1 atrial/ventricular conduction ratio, which caused a significant reduction in heart frequency. Recordings of calcium transients with high spatiotemporal resolution using a transgenic calcium indicator line (Tg(cmlc2:gCaMP)(s878)) and SPIM microscopy confirmed the presence of a severe arrhythmia phenotype. Our results identify popdc2 as a gene important for striated muscle differentiation and cardiac morphogenesis. In addition it is required for the development of the cardiac conduction system.

RNA-binding protein RBM24 is required for sarcomere assembly and heart contractility.

AIMS: The factors responsible for cardiomyopathy are not fully understood. Our studies of the transcriptome of human embryonic stem cell-derived cardiomyocytes identified novel genes up-regulated during cardiac differentiation, including RBM24. We therefore studied how its deficiency affected heart development. METHODS AND RESULTS: The expression of Rbm24 was detected in mouse cardiomyocytes and embryonic myocardium of zebrafish at the RNA and protein level. The Rbm24 loss-of-function showed that Rbm24 deficiency resulted in a reduction in sarcomeric proteins, Z-disc abnormality, and diminished heart contractility, resulting in the absence of circulation in zebrafish embryos. Gene expression profiling revealed down-regulation of multiple pathways associated with sarcomere assembly and vasculature development in Rbm24 deficiency. CONCLUSION: We identified a novel role of the tissue-specific RNA-binding protein (RBP) Rbm24 involving in the regulation of cardiac gene expression, sarcomeric assembly, and cardiac contractility. This study uncovers a potential novel pathway to cardiomyopathy through down-regulation of the RBP Rbm24.

The Popeye domain containing genes: essential elements in heart rate control.

The Popeye domain containing (Popdc) gene family displays preferential expression in skeletal muscle and heart. Only recently a significant gain in the understanding of the function of Popdc genes in the heart has been obtained. The Popdc genes encode membrane proteins harboring an evolutionary conserved Popeye domain, which functions as a binding domain for cyclic adenosine monophosphate (cAMP). Popdc proteins interact with the two-pore channel TREK-1 and enhance its current. This protein interaction is modulated by cAMP. Null mutations of members of the Popdc gene family in zebrafish and mouse are associated with severe cardiac arrhythmia phenotypes. While in zebrafish an atrioventricular block was prevalent, in mouse a stress-induced sinus bradycardia was observed, which was due to the presence of sinus pauses. Moreover, the phenotype develops in an age-dependent manner, being absent in the young animal and becoming increasingly severe, as the animals grow older. This phenotype is reminiscent of the sick sinus syndrome (SSS), which affects mostly the elderly and is characterized by the poor ability of the cardiac pacemaker to adapt the heart rate to the physiological demand. While being a prevalent disease, which is responsible for a large fraction of pacemaker implantations in Western countries, SSS is poorly understood at the molecular level. It is therefore expected that the study of the molecular basis of the stress-induced bradycardia in Popdc mice will shed new light on the etiology of pacemaker disease.

Fish and Chips: a microfluidic perfusion platform for monitoring zebrafish development.

We have developed a multi-channel microfluidic perfusion platform for culturing zebrafish embryos and capturing live images of various tissues and organs inside the embryo. The Fish and Chips was micro-fabricated in silicon and glass for reproducibility and accuracy of the microfluidic structure. The microfluidic platform consists of three parts: a microfluidic gradient generator, a row of eight fish tanks, in which the fish embryos are individually placed, and eight output channels. The fluidic gradient generator supports dose-dependent drug and chemical studies. A unique perfusion system ensures a uniform and constant flow of media to the fish tank while the wastes are efficiently removed. The fish tanks restrict the embryo movements, except rotationally, for live imaging of internal tissues and organs. The embryos showed developmental abnormalities under the influence of the drug valproic acid (VPA).

Target cell movement in tumor and cardiovascular diseases based on the epithelial-mesenchymal transition concept.

Epithelial-mesenchymal transition (EMT) is a fundamental mechanism in development driving body plan formation. EMT describes a transition process wherein polarized epithelial cells lose their characteristics and acquire a mesenchymal phenotype. The apico-basal polarity of epithelial cells is replaced by a front-rear polarity in mesenchymal cells which favor cell-extracellular matrix than intercellular adhesion. These events serve as a prerequisite to the context-dependent migratory and invasive functions of mesenchymal cells. In solid tumors, carcinoma cells undergoing EMT not only invade and metastasize but also exhibit cancer stem cell-like properties, providing resistance to conventional and targeted therapies. In cardiovascular systems, epicardial cells engaged in EMT contribute to myocardial regeneration. Conversely, cardiovascular endothelial cells undergoing EMT cause cardiac fibrosis. Growing evidence has shed light on the potential development of novel therapeutics that target cell movement by applying the EMT concept, and this may provide new therapeutic strategies for the treatment of cancer and heart diseases.

Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics.

BACKGROUND: KillerRed (KR) is a novel photosensitizer that efficiently generates reactive oxygen species (ROS) in KR-expressing cells upon intense green or white light illumination in vitro, resulting in damage to their plasma membrane and cell death. RESULTS: We report an in vivo modification of this technique using a fluorescent microscope and membrane-tagged KR (mem-KR)-expressing transgenic zebrafish. We generated several stable zebrafish Tol2 transposon-mediated enhancer-trap (ET) transgenic lines expressing mem-KR (SqKR series), and mapped the transposon insertion sites. As mem-KR accumulates on the cell membrane and/or Golgi, it highlights cell bodies and extensions, and reveals details of cellular morphology. The photodynamic property of KR made it possible to damage cells expressing this protein in a dose-dependent manner. As a proof-of-principle, two zebrafish transgenic lines were used to affect cell viability and function: SqKR2 expresses mem-KR in the hindbrain rhombomeres 3 and 5, and elsewhere; SqKR15 expresses mem-KR in the heart and elsewhere. Photobleaching of KR by intense light in the heart of SqKR15 embryos at lower levels caused a reduction in pumping efficiency of the heart and pericardial edema and at higher levels - in cell death in the hindbrain of SqKR2 and in the heart of SqKR15 embryos. CONCLUSIONS: An intense illumination of tissues expressing mem-KR affects cell viability and function in living zebrafish embryos. Hence, the zebrafish transgenics expressing mem-KR in a tissue-specific manner are useful tools for studying the biological effects of ROS.

Zebrafish cardiac enhancer trap lines: new tools for in vivo studies of cardiovascular development and disease.

Using the transposon-mediated enhancer trap (ET), we generated 18 cardiac enhancer trap (CET) transgenic zebrafish lines. They exhibit EGFP expression in defined cell types--the endocardium, myocardium, and epicardium--or in anatomical regions of the heart--the atrium, ventricle, valves, or bulbus arteriosus. Most of these expression domains are maintained into adulthood. The genomic locations of the transposon insertions were determined by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The expression pattern of EGFP in some CETs is unique and recapitulates expression of genes flanking the transposon insertion site. The CETs enabled us to capture the dynamics of the embryonic heart beating in vivo using fast scanning confocal microscopy coupled with image reconstruction, producing three-dimensional movies in time (4D) illustrating region-specific features of heart contraction. This collection of CET lines represents a toolbox of markers for in vivo studies of heart development, physiology, and drug screening.

Collective cell migration drives morphogenesis of the kidney nephron.

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase-positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow-dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.

Expression of zebrafish nos2b surrounds oral cavity.

Inducible nitric oxide synthase (NOS2) catalyzes the production of nitric oxide (NO), and is one of the factors establishing innate immunity. In zebrafish, Nos2 is represented by nos2a and nos2b. Here, we report the cloning and expression pattern of the zebrafish nos2b gene, which does not seem to participate in induced immune response. nos2b was mapped to zebrafish linkage group 15. The spatial and temporal expression pattern of nos2b in embryonic zebrafish was analyzed by whole-mount in situ hybridization. nos2b is expressed constitutively in two primordia located along the ventral midline. The first group of cells contributes to the neurohypophysis. Initially at the level of the ventral hindbrain, the second group of cells migrates closely with the thyroid primordium to its final position at the basihyal by 3 dpf. Thus, the analysis of expression pattern of nos2b reveals complex morphogenetic movements resulting in its expression surrounding the oral cavity.