Pharmacological Characterization of an Antisense Knockdown Zebrafish Model of Dravet Syndrome: Inhibition of Epileptic Seizures by the Serotonin Agonist Fenfluramine

Dravet syndrome (DS) is one of the most pharmacoresistant and devastating forms of childhood epilepsy syndromes. Distinct de novo mutations in the SCN1A gene are responsible for over 80% of DS cases. While DS is largely resistant to treatment with existing anti-epileptic drugs, promising results have been obtained in clinical trials with human patients treated with the serotonin agonist fenfluramine as an add-on therapeutic. We developed a zebrafish model of DS using morpholino antisense oligomers (MOs) targeting scn1Lab, the zebrafish ortholog of SCN1A. Zebrafish larvae with an antisense knockdown of scn1Lab (scn1Lab morphants) were characterized by automated behavioral tracking and high-resolution video imaging, in addition to measuring brain activity through local field potential recordings. Our findings reveal that scn1Lab morphants display hyperactivity, convulsive seizure-like behavior, loss of posture, repetitive jerking and a myoclonic seizure-like pattern. The occurrence of spontaneous seizures was confirmed by local field potential recordings of the forebrain, measuring epileptiform discharges. Furthermore, we show that these larvae are remarkably sensitive to hyperthermia, similar to what has been described for mouse models of DS, as well as for human DS patients. Pharmacological evaluation revealed that sodium valproate and fenfluramine significantly reduce epileptiform discharges in scn1Lab morphants. Our findings for this zebrafish model of DS are in accordance with clinical data for human DS patients. To our knowledge, this is the first study demonstrating effective seizure inhibition of fenfluramine in an animal model of Dravet syndrome. Moreover, these results provide a basis for identifying novel analogs with improved activity and significantly milder or no side effects.     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number

Heart formation is a complex morphogenetic process, and perturbations in cardiac morphogenesis lead to congenital heart disease. NKX2-5 is a key causative gene associated with cardiac birth defects, presumably because of its essential roles during the early steps of cardiogenesis. Previous studies in model organisms implicate NKX2-5 homologs in numerous processes, including cardiac progenitor specification, progenitor proliferation, and chamber morphogenesis. By inhibiting function of the zebrafish NKX2-5 homologs, nkx2.5 and nkx2.7, we show that nkx genes are essential to establish the original dimensions of the linear heart tube. The nkx-deficient heart tube fails to elongate normally: its ventricular portion is atypically short and wide, and its atrial portion is disorganized and sprawling. This atrial phenotype is associated with a surplus of atrial cardiomyocytes, whereas ventricular cell number is normal at this stage. However, ventricular cell number is decreased in nkx-deficient embryos later in development, when cardiac chambers are emerging. Thus, we conclude that nkx genes regulate heart tube extension and exert differential effects on ventricular and atrial cell number. Our data suggest that morphogenetic errors could originate during early stages of heart tube assembly in patients with NKX2-5 mutations.     

Cascade effect of cardiac myogenesis gene expression during cardiac looping in <Emphasis Type="Italic">tbx5</Emphasis> knockdown zebrafish embryos

Zebrafish tbx5 expresses in the heart, pectoral fins and eyes of zebrafish during embryonic development. In zebrafish, injection of tbx5 morpholino antisense RNA caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. We suggested that cardiac myogenesis genes might be responsible for this phenomenon. Morpholino antisense RNA which against the initiation site of tbx5 gene was designed in order to knockdown the expression of tbx5, and the results were analyzed by whole-mount in situ hybridization and quantitative real-time PCR. Expression of cardiac myogenesis genes amhc, vmhc and cmlc2 were expressed constantly at the early embryonic development and reached its highest rate right before cardiac looping initiated. These cardiac myogenesis genes showed insufficient expressions within different heart defect embryos. Moreover, vmhc showed ectopic expression in addition to heart looping defect in heart defective embryos at 36 hpf. Our data suggests that the heart failure caused by the knockdown of tbx5 gene might result from the down-regulation of cardiac myogenesis genes. Jen Her Lu and Jenn Kan Lu contributed equally to this work.     

Embryonic expression of an Nkx2‐5/Cre gene using ROSA26 reporter mice

Summary: Nkx2‐5, one of the earliest cardiac‐specific markers in vertebrate embryos, was used as a genetic locus to knock in the Cre recombinase gene by homologous recombination. Offspring resulting from heterozygous Nkx2‐5/Cre mice mated to ROSA26 (R26R) reporter mice provided a model system for following Nkx2‐5 gene activity by β‐galactosidase (β‐gal) activity. β‐gal activity was initially observed in the early cardiac crescent, cardiomyocytes of the looping heart tube, and in the epithelium of the first pharyngeal arch. In later stage embryos (10.5–13.5 days postcoitum, dpc), β‐gal activity was observed in the stomach and spleen, the dorsum of the tongue, and in the condensing primordium of the tooth. The Nkx2‐5/Cre mouse model should provide a useful genetic resource to elucidate the role of loxP manipulated genetic targets in cardiogenesis and other developmental processes. genesis 31:176–180, 2001. © 2001 Wiley‐Liss, Inc.     

Suppression of Rap1 Impairs Cardiac Myofibrils and Conduction System in Zebrafish

Numerous studies have revealed that Rap1 (Ras-proximate-1 or Ras-related protein 1), a small GTPase protein, plays a crucial role in mediating cAMP signaling in isolated cardiac tissues and cell lines. However, the involvement of Rap1 in the cardiac development in vivo is largely unknown. By injecting anti-sense morpholino oligonucleotides to knock down Rap1a and Rap1b in zebrafish embryos, and in combination with time-lapsed imaging, in situ hybridization, immunohistochemistry and transmission electron microscope techniques, we seek to understand the role of Rap1 in cardiac development and functions. At an optimized low dose of mixed rap1a and rap1b morpholino oligonucleotides, the heart developed essentially normally until cardiac contraction occurred. Morphant hearts showed the myocardium defect phenotypes, most likely due to disrupted myofibril assembly and alignment. In vivo heart electrocardiography revealed prolonged P-R interval and QRS duration, consistent with an adherens junction defect and reduced Connexons in cardiac myocytes of morphants. We conclude that a proper level of Rap1 is crucial for heart morphogenesis and function, and suggest that Rap1 and/or their downstream factor genes are potential candidates for genetic screening for human heart diseases.     

NaV1.6a is required for normal activation of motor circuits normally excited by tactile stimulation

A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non‐active (nav^mi89 and nav^mi130). nav embryos displayed diminished spontaneous and touch‐evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding Na_V1.6a (scn8aa) as a potential candidate for nav. Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in Na_V1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild‐type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav. In‐vivo electrophysiological analysis of the touch‐evoked escape circuit indicated that voltage‐dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that Na_V1.6a was required for touch‐induced activation of the swim locomotor network. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:508–522, 2010     

Voltage-gated ion channel dysfunction precedes cardiomyopathy development in the dystrophic heart

Background

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is associated with severe cardiac complications including cardiomyopathy and cardiac arrhythmias. Recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. It is, however, unknown if the ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiac pathology.

Methodology/Principal Findings

To address this question, we first investigated sodium channel impairments in cardiomyocytes derived from dystrophic neonatal mice prior to cardiomyopahty development, by using the whole cell patch clamp technique. Besides the most common model for DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying an utrophin mutation. In neonatal cardiomyocytes, dystrophin-deficiency generated a 25% reduction in sodium current density. In addition, extra utrophin-deficiency significantly altered sodium channel gating parameters. Moreover, also calcium channel inactivation was considerably reduced in dystrophic neonatal cardiomyocytes, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. To assess developmental changes, we also studied sodium channel impairments in cardiomyocytes derived from dystrophic adult mice, and compared them with the respective abnormalities in dystrophic neonatal cells. Here, we found a much stronger sodium current reduction in adult cardiomyocytes. The described sodium channel impairments slowed the upstroke of the action potential in adult cardiomyocytes, and only in dystrophic adult mice, the QRS interval of the electrocardiogram was prolonged.

Conclusions/Significance

Ion channel impairments precede pathology development in the dystrophic heart, and may thus be considered potential cardiomyopathy triggers.     

Fgf differentially controls cross-antagonism between cardiac and haemangioblast regulators

Fibroblast growth factor (Fgf) has been implicated in the control of heart size during development, although whether this is by controlling cell fate, survival or proliferation has not been clear. Here, we show that Fgf, without affecting survival or proliferation, acts during gastrulation to drive cardiac fate and restrict anterior haemangioblast fate in zebrafish embryos. The haemangioblast programme was thought to be activated before the cardiac programme and is repressive towards it, suggesting that activation by Fgf of the cardiac programme might be via suppression of the haemangioblast programme. However, we show that the cardiac regulator nkx2.5 can also repress the haemangioblast programme and, furthermore, that cardiac specification still requires Fgf signalling even when haemangioblast regulators are independently suppressed. We further show that nkx2.5 and the cloche candidate gene lycat are expressed during gastrulation and regulated by Fgf, and that nkx2.5 overexpression, together with loss of the lycat targets etsrp and scl can stably induce expansion of the heart. We conclude that Fgf controls cardiac and haemangioblast fates by the simultaneous regulation of haemangioblast and cardiac regulators. We propose that elevation of Fgf signalling in the anterior haemangioblast territory could have led to its recruitment into the heart field during evolution, increasing the size of the heart.     

Differentiation of human adipose‐derived stem cells into beating cardiomyocytes

Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca²⁺ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.     

Synergistic effect of bioactive lipid and condition medium on cardiac differentiation of human mesenchymal stem cells from different tissues

Human umbilical cord mesenchymal stem cells (hUCMSCs) and human adipose tissue mesenchymal stem cells (hATMSCs) have the potential to differentiate into cardiomyocytes, making them promising therapeutic candidates for treating damaged cardiac tissues. Currently, however, the differentiated cells induced from hUCMSCs or hATMSCs can hardly display functional characteristics similar to cardiomyocytes. In this study, we have investigated the effects of bioactive lipid sphingosine‐1‐phosphate (S1P) on cardiac differentiations of hUCMSCs and hATMSCs in condition medium composed of cardiac myocytes culture medium or 5‐azacytidine. Cardiac differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. Synergistic effects of S1P and condition medium on cell viability were evaluated by MTT assays. Functional characteristics similar to cardiomyocytes were evaluated through detecting calcium transient. The differentiated hUCMSCs or hATMSCs in each group into cardiomyocytes showed positive expressions of cardiac specific proteins, including α‐actin, connexin‐43 and myosin heavy chain‐6 (MYH‐6). MTT assays showed that suitable differentiation time was 14 days and that the optimal concentration of S1P was 0.5 μM. Moreover, incorporation of S1P and cardiac myocytes culture medium gave rise to calcium transients, an important marker for displaying in vivo electrophysiological properties. This feature was not observed in the S1P‐5‐azacytidine group, indicating the possible lack of cellular stimuli such as transforming growth factor‐beta, TGF‐β. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF‐α–induced injury via inhibiting NF‐κB and JNK signals

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.