RESUMEN
Over the last decades, myocardial infarction and heart failure have accounted every year for millions of deaths worldwide. After a coronary occlusion, the lack of blood supply to downstream muscle leads to cell death and scarring. To date, several pro-angiogenic factors have been tested to stimulate reperfusion of the affected myocardium, VEGFA being one of the most extensively studied. Given the unsuccessful outcomes of clinical trials, understanding how cardiac revascularization takes place in models with endogenous regenerative capacity holds the key to devising more efficient therapies. Here, we summarize the main findings on VEGFA's role during cardiac repair and regeneration, with a particular focus on zebrafish as a regenerative model. Moreover, we provide a comprehensive overview of available tools to modulate Vegfa expression and action in zebrafish regeneration studies. Understanding the role of Vegfa during zebrafish heart regeneration may help devise efficient therapies and circumvent current limitations in using VEGFA for therapeutic angiogenesis approaches.
Asunto(s)
Infarto del Miocardio , Pez Cebra , Animales , Corazón/fisiología , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Ischemic heart disease following the obstruction of coronary vessels leads to the death of cardiac tissue and the formation of a fibrotic scar. In contrast to adult mammals, zebrafish can regenerate their heart after injury, enabling the study of the underlying mechanisms. One of the earliest responses following cardiac injury in adult zebrafish is coronary revascularization. Defects in this process lead to impaired cardiomyocyte repopulation and scarring. Hence, identifying and investigating factors that promote coronary revascularization holds great therapeutic potential. METHODS: We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration. RESULTS: We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1, the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration. CONCLUSIONS: These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.
Asunto(s)
Interleucina-8 , Glicoproteínas de Membrana , Miocitos Cardíacos , Regeneración , Factor C de Crecimiento Endotelial Vascular , Proteínas de Pez Cebra , Pez Cebra , Animales , Proliferación Celular , Células Endoteliales/metabolismo , Corazón/fisiología , Interleucina-8/metabolismo , Glicoproteínas de Membrana/metabolismo , Miocitos Cardíacos/fisiología , Regeneración/fisiología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Musculares/genética , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Estrés Fisiológico/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ventrículos Cardíacos/crecimiento & desarrollo , Ventrículos Cardíacos/lesiones , Ventrículos Cardíacos/metabolismo , Desarrollo de Músculos/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/patología , Proteínas Nucleares/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish.
Asunto(s)
Corazón/embriología , Miocitos Cardíacos/metabolismo , Regeneración/fisiología , Animales , Proliferación Celular , Ventrículos Cardíacos/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/citología , Transducción de Señal , Pez Cebra/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury.
Asunto(s)
Miocitos Cardíacos , Pez Cebra , Animales , Proliferación Celular , Glucólisis , Miocitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pez Cebra/metabolismoRESUMEN
Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.
Asunto(s)
Aminas/análisis , Glucosa/metabolismo , Tuberculosis/metabolismo , Pez Cebra/metabolismo , Aminas/química , Animales , Cromatografía Liquida , Modelos Animales de Enfermedad , Humanos , Larva/metabolismo , Larva/microbiología , Análisis de los Mínimos Cuadrados , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Mycobacterium marinum , Mycobacterium tuberculosis , Pez Cebra/microbiologíaRESUMEN
BACKGROUND: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae. RESULTS: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2+/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2-/- larvae than in tlr2+/-, or tlr2+/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2-/- versus tlr2+/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis. CONCLUSION: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.
Asunto(s)
Enfermedades de los Peces/genética , Regulación del Desarrollo de la Expresión Génica , Infecciones por Mycobacterium no Tuberculosas/genética , Infecciones por Mycobacterium no Tuberculosas/veterinaria , Receptor Toll-Like 2/genética , Pez Cebra/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/inmunología , Quimiocina CXCL11/genética , Quimiocina CXCL11/inmunología , Resistencia a la Enfermedad/genética , Embrión no Mamífero , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Larva/genética , Larva/crecimiento & desarrollo , Larva/inmunología , Larva/microbiología , Linfotoxina-alfa/genética , Linfotoxina-alfa/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Factores de Transcripción Maf/genética , Factores de Transcripción Maf/inmunología , Redes y Vías Metabólicas/genética , Redes y Vías Metabólicas/inmunología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/inmunología , Mycobacterium marinum/patogenicidad , Neutrófilos/inmunología , Neutrófilos/microbiología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/inmunología , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/inmunología , Transcriptoma/inmunología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/inmunología , Pez Cebra/microbiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/inmunologíaRESUMEN
In recent years, there has been increasing interest in the role of lymphatics in organ repair and regeneration, due to their importance in immune surveillance and fluid homeostasis. Experimental approaches aimed at boosting lymphangiogenesis following myocardial infarction in mice, were shown to promote healing of the heart. Yet, the mechanisms governing cardiac lymphatic growth remain unclear. Here, we identify two distinct lymphatic populations in the hearts of zebrafish and mouse, one that forms through sprouting lymphangiogenesis, and the other by coalescence of isolated lymphatic cells. By tracing the development of each subset, we reveal diverse cellular origins and differential response to signaling cues. Finally, we show that lymphatic vessels are required for cardiac regeneration in zebrafish as mutants lacking lymphatics display severely impaired regeneration capabilities. Overall, our results provide novel insight into the mechanisms underlying lymphatic formation during development and regeneration, opening new avenues for interventions targeting specific lymphatic populations.
Asunto(s)
Corazón/fisiología , Linfangiogénesis/fisiología , Vasos Linfáticos/fisiología , Miocardio/metabolismo , Regeneración/fisiología , Transducción de Señal/fisiología , Animales , Animales Modificados Genéticamente , Corazón/embriología , Corazón/crecimiento & desarrollo , Linfangiogénesis/genética , Sistema Linfático/citología , Sistema Linfático/metabolismo , Sistema Linfático/fisiología , Vasos Linfáticos/metabolismo , Ratones Noqueados , Ratones Transgénicos , Mutación , Infarto del Miocardio/fisiopatología , Regeneración/genética , Transducción de Señal/genética , Pez CebraRESUMEN
Defective coronary network function and insufficient blood supply are both cause and consequence of myocardial infarction. Efficient revascularization after infarction is essential to support tissue repair and function. Zebrafish hearts exhibit a remarkable ability to regenerate, and coronary revascularization initiates within hours of injury, but how this process is regulated remains unknown. Here, we show that revascularization requires a coordinated multi-tissue response culminating with the formation of a complex vascular network available as a scaffold for cardiomyocyte repopulation. During a process we term "coronary-endocardial anchoring," new coronaries respond by sprouting (1) superficially within the regenerating epicardium and (2) intra-ventricularly toward the activated endocardium. Mechanistically, superficial revascularization is guided by epicardial Cxcl12-Cxcr4 signaling and intra-ventricular sprouting by endocardial Vegfa signaling. Our findings indicate that the injury-activated epicardium and endocardium support cardiomyocyte replenishment initially through the guidance of coronary sprouting. Simulating this process in the injured mammalian heart should help its healing.
Asunto(s)
Miocitos Cardíacos/fisiología , Neovascularización Fisiológica/fisiología , Regeneración/fisiología , Animales , Proliferación Celular/fisiología , Quimiocina CXCL12/metabolismo , Señales (Psicología) , Endocardio/fisiología , Corazón/fisiología , Ventrículos Cardíacos/metabolismo , Revascularización Miocárdica/métodos , Miocitos Cardíacos/metabolismo , Pericardio/fisiología , Receptores CXCR4/metabolismo , Transducción de Señal/fisiología , Cicatrización de Heridas/fisiología , Pez Cebra/metabolismo , Proteínas de 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
Immediately after cardiac injury, the immune system plays major roles in repair and regeneration as it becomes involved in a number of processes including damage-associated signaling, inflammation, revascularization, cardiomyocyte dedifferentiation and replenishment, and fibrotic scar formation/resolution. Recent studies have revealed that different immune responses occur in the various experimental models capable or incapable of cardiac regeneration, and that harnessing these immune responses might improve cardiac repair. In light of this concept, this review analyzes current knowledge about the immune responses to cardiac injury from a comparative perspective. Insights gained from such comparative analyses may provide ways to modulate the immune response as a potential therapeutic strategy for cardiac disease.
Asunto(s)
Corazón/fisiología , Regeneración , Animales , Proteína HMGB1/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Modelos Animales , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.
Asunto(s)
Recombinación Homóloga , Mutagénesis , Oligonucleótidos , Pez Cebra/genética , Alelos , Animales , Secuencia de Bases , Elementos Transponibles de ADN , Genoma , Intrones , Mutación , Oligonucleótidos/genética , Reproducibilidad de los Resultados , Proteínas de Dominio T Box/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.
Asunto(s)
Repetición de Anquirina , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Condicionamiento Físico Animal , Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Humanos , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Filogenia , Alineación de Secuencia , Estrés Fisiológico , Sintenía , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Linfangiogénesis/genética , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Factor C de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Vasos Sanguíneos/citología , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/metabolismo , Línea Celular , Embrión de Mamíferos , Embrión no Mamífero , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Vasos Linfáticos/citología , Vasos Linfáticos/metabolismo , Ratones , Neovascularización Fisiológica/genética , Proteínas Represoras/deficiencia , Transducción de Señal , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/metabolismoRESUMEN
Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.
Asunto(s)
Lesiones Cardíacas/patología , Inmunidad Celular , Regeneración , Animales , Proliferación Celular , Perfilación de la Expresión Génica , Macrófagos/inmunología , Miocitos Cardíacos/fisiología , Neutrófilos/inmunología , Oryzias , Pez CebraRESUMEN
Macrophages are known to interact with endothelial cells during developmental and pathological angiogenesis but the molecular mechanisms modulating these interactions remain unclear. Here, we show a role for the Hif-1α transcription factor in this cellular communication. We generated hif-1aa;hif-1ab double mutants in zebrafish, hereafter referred to as hif-1α mutants, and find that they exhibit impaired macrophage mobilization from the aorta-gonad-mesonephros (AGM) region as well as angiogenic defects and defective vascular repair. Importantly, macrophage ablation is sufficient to recapitulate the vascular phenotypes observed in hif-1α mutants, revealing for the first time a macrophage-dependent angiogenic process during development. Further substantiating our observations of vascular repair, we find that most macrophages closely associated with ruptured blood vessels are Tnfα-positive, a key feature of classically activated macrophages. Altogether, our data provide genetic evidence that Hif-1α regulates interactions between macrophages and endothelial cells starting with the mobilization of macrophages from the AGM.
Asunto(s)
Vasos Sanguíneos/embriología , Células Endoteliales/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Macrófagos/citología , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Alelos , Animales , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Microscopía Confocal , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxígeno/química , Fenotipo , Tamaño de la Muestra , Transducción de Señal , Pez Cebra/embriologíaRESUMEN
Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization.
Asunto(s)
Corazón/fisiopatología , Revascularización Miocárdica , Regeneración/fisiología , Pez Cebra/fisiología , Animales , Biomarcadores/metabolismo , Proliferación Celular , Supervivencia Celular , Vasos Coronarios/patología , Regulación del Desarrollo de la Expresión Génica , Respuesta al Choque Térmico , Mutación/genética , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica , Pericardio/patología , Conducto Torácico/patología , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Although the responses to many pathogen associated molecular patterns (PAMPs) in cell cultures and extracted organs are well characterized, there is little known of transcriptome responses to PAMPs in whole organisms. To characterize this in detail, we have performed RNAseq analysis of responses of zebrafish embryos to injection of PAMPs in the caudal vein at one hour after exposure. We have compared two ligands that in mammals have been shown to specifically activate the TLR2 and TLR5 receptors: Pam3CSK4 and flagellin, respectively. RESULTS: We identified a group of 80 common genes that respond with high stringency selection to stimulations with both PAMPs, which included several well-known immune marker genes such as il1b and tnfa. Surprisingly, we also identified sets of 48 and 42 genes that specifically respond to either Pam3CSK4 or flagellin, respectively, after a comparative filtering approach. Remarkably, in the Pam3CSK4 specific set, there was a set of transcription factors with more than 2 fold-change, as confirmed by qPCR analyses, including cebpb, fosb, nr4a1 and egr3. We also showed that the regulation of the Pam3CSK4 and flagellin specifically responding sets is inhibited by knockdown of tlr2 or tlr5, respectively. CONCLUSIONS: Our studies show that Pam3CSK4 and flagellin can stimulate the Tlr2 and Tlr5 signaling pathways leading to common and specific responses in the zebrafish embryo system.
Asunto(s)
Inmunidad Innata/genética , Moléculas de Patrón Molecular Asociado a Patógenos/administración & dosificación , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 5/biosíntesis , Animales , Flagelina/genética , Regulación del Desarrollo de la Expresión Génica , Ligandos , Lipopéptidos/genética , Lipopéptidos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 5/genética , Transcriptoma/genética , Transcriptoma/inmunología , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.
Asunto(s)
Encéfalo/metabolismo , Transportador de Glucosa de Tipo 2/metabolismo , Glucosa/metabolismo , Organogénesis/fisiología , Pez Cebra/embriología , Animales , Apoptosis/fisiología , Encéfalo/embriología , Encéfalo/patología , Muerte Celular , Línea Celular , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Técnicas de Silenciamiento del Gen , Transportador de Glucosa de Tipo 2/genética , Células Secretoras de Insulina/metabolismo , Organogénesis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Pez Cebra/metabolismoRESUMEN
Cardiomyopathies-associated metabolic pathologies (e.g., type 2 diabetes and insulin resistance) are a leading cause of mortality. It is known that the association between these pathologies works in both directions, for which heart failure can lead to metabolic derangements such as insulin resistance. This intricate crosstalk exemplifies the importance of a fine coordination between one of the most energy-demanding organs and an equilibrated carbohydrate metabolism. In this light, to assist in the understanding of the role of insulin-regulated glucose transporters (GLUTs) and the development of cardiomyopathies, we have developed a model for glut12 deficiency in zebrafish. GLUT12 is a novel insulin-regulated GLUT expressed in the main insulin-sensitive tissues, such as cardiac muscle, skeletal muscle, and adipose tissue. In this study, we show that glut12 knockdown impacts the development of the embryonic heart resulting in abnormal valve formation. Moreover, glut12-deficient embryos also exhibited poor glycemic control. Glucose measurements showed that these larvae were hyperglycemic and resistant to insulin administration. Transcriptome analysis demonstrated that a number of genes known to be important in cardiac development and function as well as metabolic mediators were dysregulated in these larvae. These results indicate that glut12 is an essential GLUT in the heart where the reduction in glucose uptake due to glut12 deficiency leads to heart failure presumably due to the lack of glucose as energy substrate. In addition, the diabetic phenotype displayed by these larvae after glut12 abrogation highlights the importance of this GLUT during early developmental stages.