RESUMEN
Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.
Asunto(s)
Cardiomiopatías , Proteínas de Microfilamentos , Animales , Adhesión Celular/genética , Proteínas de Microfilamentos/genética , Miocitos Cardíacos/metabolismo , Miofibrillas/metabolismo , Pez Cebra/genética , Transactivadores , Cardiomiopatías/genéticaRESUMEN
AIMS: Mammalian models have been instrumental in investigating adult heart function and human disease. However, electrophysiological differences with human hearts and high costs motivate the need for non-mammalian models. The zebrafish is a well-established genetic model to study cardiovascular development and function; however, analysis of cardiovascular phenotypes in adult specimens is particularly challenging as they are opaque. METHODS AND RESULTS: Here, we optimized and combined multiple imaging techniques including echocardiography, magnetic resonance imaging, and micro-computed tomography to identify and analyse cardiovascular phenotypes in adult zebrafish. Using alk5a/tgfbr1a mutants as a case study, we observed morphological and functional cardiovascular defects that were undetected with conventional approaches. Correlation analysis of multiple parameters revealed an association between haemodynamic defects and structural alterations of the heart, as observed clinically. CONCLUSION: We report a new, comprehensive, and sensitive platform to identify otherwise indiscernible cardiovascular phenotypes in adult zebrafish.
Asunto(s)
Sistema Cardiovascular , Pez Cebra , Animales , Ecocardiografía , Corazón , Humanos , Mamíferos , Microtomografía por Rayos X , Pez Cebra/genéticaRESUMEN
The transcription factor Snai1, a well-known regulator of epithelial-to-mesenchymal transition, has been implicated in early cardiac morphogenesis as well as in cardiac valve formation. However, a role for Snai1 in regulating other aspects of cardiac morphogenesis has not been reported. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required in cardiomyocytes for myocardial wall integrity. Loss of snai1b increases the frequency of cardiomyocyte extrusion away from the cardiac lumen. Extruding cardiomyocytes exhibit increased actomyosin contractility basally as revealed by enrichment of p-myosin and α-catenin epitope α-18, as well as disrupted intercellular junctions. Transcriptomic analysis of wild-type and snai1b mutant hearts revealed the dysregulation of intermediate filament genes, including desmin b (desmb) upregulation. Cardiomyocyte-specific desmb overexpression caused increased cardiomyocyte extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 maintains the integrity of the myocardial epithelium, at least in part by repressing desmb expression.
Asunto(s)
Regulación de la Expresión Génica , Corazón/fisiología , Filamentos Intermedios/genética , Factores de Transcripción de la Familia Snail/genética , Proteínas de Pez Cebra/genética , Pez Cebra/fisiología , Animales , Miocardio/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Tie1 is a receptor tyrosine kinase expressed in endothelial cells, where it modulates Angiopoietin/Tie2 signaling. Previous studies have shown that mouse Tie1 mutants exhibit severe cardiovascular defects; however, much remains to be learned about the role of Tie1, especially during cardiac development. To further understand Tie1 function, we generated a zebrafish tie1 mutant line. Homozygous mutant embryos display reduced endothelial and endocardial cell numbers and reduced heart size. Live imaging and ultrastructural analyses at embryonic stages revealed increased cardiac jelly thickness as well as cardiomyocyte defects, including a loss of sarcomere organization and altered cell shape. Transcriptomic profiling of embryonic hearts uncovered the downregulation of tll1, which encodes a Tolloid-like protease, in tie1-/- compared with wild-type siblings. Using mRNA injections into one-cell stage embryos, we found that tll1 overexpression could partially rescue the tie1 mutant cardiac phenotypes including the endocardial and myocardial cell numbers as well as the cardiac jelly thickness. Altogether, our results indicate the importance of a Tie1-Tolloid-like 1 axis in paracrine signaling during cardiac development.
Asunto(s)
Corazón/embriología , Metaloproteinasas Similares a Tolloid/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Células Endoteliales/citología , Endotelio Vascular/citología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Cardiopatías Congénitas/genética , Morfogénesis , Mutación , Miocitos Cardíacos/citología , Receptor TIE-1/genética , Receptor TIE-1/fisiología , Metaloproteinasas Similares a Tolloid/genética , Transcriptoma , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The development of the cardiac outflow tract (OFT), which connects the heart to the great arteries, relies on a complex crosstalk between endothelial (ECs) and smooth muscle (SMCs) cells. Defects in OFT development can lead to severe malformations, including aortic aneurysms, which are frequently associated with impaired TGF-ß signaling. To better understand the role of TGF-ß signaling in OFT formation, we generated zebrafish lacking the TGF-ß receptor Alk5 and found a strikingly specific dilation of the OFT: alk5-/- OFTs exhibit increased EC numbers as well as extracellular matrix (ECM) and SMC disorganization. Surprisingly, endothelial-specific alk5 overexpression in alk5-/- rescues the EC, ECM, and SMC defects. Transcriptomic analyses reveal downregulation of the ECM gene fibulin-5, which when overexpressed in ECs ameliorates OFT morphology and function. These findings reveal a new requirement for endothelial TGF-ß signaling in OFT morphogenesis and suggest an important role for the endothelium in the etiology of aortic malformations.
Asunto(s)
Endotelio Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Endotelio Vascular/citología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Proteína smad3/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Cardiac valve disease can lead to severe cardiac dysfunction and is thus a frequent cause of morbidity and mortality. Its main treatment is valve replacement, which is currently greatly limited by the poor recellularization and tissue formation potential of the implanted valves. As we still lack suitable animal models to identify modulators of these processes, here we used adult zebrafish and found that, upon valve decellularization, they initiate a rapid regenerative program that leads to the formation of new functional valves. After injury, endothelial and kidney marrow-derived cells undergo cell cycle re-entry and differentiate into new extracellular matrix-secreting valve cells. The TGF-ß signaling pathway promotes the regenerative process by enhancing progenitor cell proliferation as well as valve cell differentiation. These findings reveal a key role for TGF-ß signaling in cardiac valve regeneration and establish the zebrafish as a model to identify and test factors promoting cardiac valve recellularization and growth.
Asunto(s)
Diferenciación Celular , Endotelio/citología , Válvulas Cardíacas/citología , Riñón/citología , Regeneración , Factor de Crecimiento Transformador beta/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Ciclo Celular , Endotelio/metabolismo , Matriz Extracelular/metabolismo , Válvulas Cardíacas/metabolismo , Riñón/metabolismo , Modelos Animales , Ingeniería de Tejidos/métodos , Pez Cebra/metabolismoRESUMEN
Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of PITX2, a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of pitx2c in zebrafish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of pitx2c+/- fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of pitx2c loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of pitx2c-/- larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart.
Asunto(s)
Arritmias Cardíacas/metabolismo , Proteínas de Homeodominio/genética , Sarcómeros/metabolismo , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Acetilcisteína/farmacología , Animales , Animales Modificados Genéticamente , Antioxidantes/farmacología , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/etiología , Trastorno del Sistema de Conducción Cardíaco/etiología , Trastorno del Sistema de Conducción Cardíaco/genética , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Modelos Animales de Enfermedad , Electrocardiografía , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Larva/efectos de los fármacos , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Sarcómeros/genética , Sarcómeros/patología , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin's cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.
Asunto(s)
Toxinas Botulínicas/genética , Toxinas Botulínicas/metabolismo , Animales , Sitios de Unión , Toxinas Botulínicas Tipo A/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Gangliósidos , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos , Glicoproteínas de Membrana/metabolismo , Ratones , Unión Proteica , Conformación Proteica , Receptores de Neurotransmisores/metabolismo , Serogrupo , Vesículas SinápticasRESUMEN
The emergence of Variola virus-like viruses by natural evolution of zoonotic Orthopoxviruses, like Cowpox virus (CPXV), is a global health threat. The proteasome is essential for poxvirus replication, making the viral components interacting with the ubiquitin-proteasome system attractive antiviral targets. We show that proteasome inhibition impairs CPXV replication by prevention of uncoating, suggesting that uncoating is mediated by proteasomal degradation of viral core proteins. Although Orthopoxvirus particles contain considerable amounts of ubiquitin, distinct modification sites are largely unknown. Therefore, for the first time, we analyzed globally ubiquitination sites in CPXV mature virion proteins using LC-MS/MS. Identification of 137 conserved sites in 54 viral proteins among five CPXV strains revealed extensive ubiquitination of structural core proteins. Moreover, since virions contained primarily K48-linked polyubiquitin, we hypothesized that core proteins are modified accordingly. However, quantitative analysis of ubiquitinated CPXV proteins early in infection showed no proteasomal degradation of core proteins. Instead, our data indicate that the recently suggested proteasomal regulation of the uncoating factor E5 is a prerequisite for uncoating. Expanding our understanding of poxvirus uncoating and elucidating a multitude of novel ubiquitination sites in poxvirus proteins, the present study verifies the major biological significance of ubiquitin in poxvirus infection.
Asunto(s)
Virus de la Viruela Vacuna/genética , Complejo de la Endopetidasa Proteasomal/genética , Ubiquitinación/genética , Proteínas del Núcleo Viral/genética , Proteínas Virales/genética , Línea Celular Tumoral , Replicación del ADN/genética , Células HeLa , Humanos , Poliubiquitina/genética , Ubiquitina/genética , Virión/genéticaRESUMEN
Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 103 PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/inmunología , Orthopoxvirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Orthopoxvirus/genética , Orthopoxvirus/metabolismoRESUMEN
Plastic cell culture dishes that contain a thin bottom of highest optical quality including an imprinted finder grid (µ-Dish Grid-500) are optimally suited for routine correlative light and electron microscopy using chemical fixation. Such dishes allow high-resolution fluorescence and bright-field imaging using fixed and living cells and are compatible with standard protocols for scanning and transmission electron microscopy. Ease of use during cell culture and imaging, as well as a tight cover render the dishes particularly suitable for working with infectious organisms up to the highest biosafety level. Detailed protocols are provided and demonstrated by showing two examples: monitoring the production of virus-like particles of the Human Endogenous Retrovirus HERV-K(HML-2) by HeLa cells and investigation of Rab11-positive membrane-compartments of HeLa cells after infection with Chlamydia trachomatis.
Asunto(s)
Análisis de la Célula Individual/métodos , Chlamydia trachomatis/fisiología , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Retrovirus Endógenos/fisiología , Células HeLa , Humanos , Microscopía Electrónica de Rastreo/métodos , Microscopía Electrónica de Transmisión/métodos , Microscopía Fluorescente/métodos , Microtomía , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Fast and reliable diagnostic assays are required for a resilient detection of clinical infections or biothreat-relevant pathogens. While PCR has proven to be the gold standard for nucleic acid detection, the identification of pathogen particles is still challenging and depends on the availability of well-characterized, chemically stable, and selective recognition molecules. Here, we report the screening of a phage display random peptide library for vaccinia virus-binding peptides. The identified peptide was extensively characterized using peptide-probe ELISA, surface plasmon resonance, nLC-MS/MS, Western Blot, peptide-based immunofluorescence assay, and electron microscopy. Following identification, the phage-free, synthetic peptide, designated αVACVpep05, was shown to bind to vaccinia virus and other orthopoxviruses. We can demonstrate that the highly conserved orthopoxvirus surface protein D8 is the interaction partner of αVACVpep05, thus enabling the peptide to bind to other orthopoxviruses, including cowpox virus and monkeypox virus, viruses that cause clinically relevant zoonotic infections in humans. The process of phage display-mediated peptide identification has been optimized intensively, and we provide recommendations for the identification of peptides suitable for the detection of further pathogens. The peptide described here was critically characterized and seems to be a promising reagent for the development of diagnostic platforms for orthopoxviruses. We believe that our results will help to promote the development of alternative, nonantibody-based synthetic detection molecules for further pathogens.
Asunto(s)
Orthopoxvirus/aislamiento & purificación , Orthopoxvirus/metabolismo , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas/métodosRESUMEN
The emergence of Acinetobacter baumannii as an increasingly multidrug-resistant nosocomial pathogen largely relies on acquisition of resistance genes via horizontal gene transfer. Here, we demonstrate that many clinical isolates of A. baumannii take up DNA while they move along wet surfaces. We show that both motility and DNA uptake are abolished after inactivation of pilT, which putatively encodes the type 4 pilus (T4P) retraction ATPase, and comEC, which putatively encodes the DNA uptake channel, respectively. Inactivation of pilT correlates with an increase in the number and length of pili with an average diameter of 7.2 nm. In the Galleria mellonella infection model, the comEC mutant is significantly attenuated, whereas the pilT mutant is not, dissecting biologically distinct roles of T4P and the DNA uptake channel. Collectively, these findings promote our understanding of the mechanisms of DNA uptake and resistance development in A. baumannii, which may also apply to other important pathogens.