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To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.
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Coração/crescimento & desenvolvimento , MicroRNAs/genética , Proteínas com Domínio T/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Linhagem Celular , Regulação para Baixo/genética , Feminino , Regulação da Expressão Gênica/genética , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Gravidez , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: T-box genes constitute a large family of transcriptional regulators involved in developmental patterning. Homozygous mutation of tbx5 leads to embryonic lethal cardiac phenotypes and forelimb malformations in vertebrate models. Haploinsufficiency of tbx5 results in Holt-Oram syndrome, a human congenital disease characterized by cardiac and forelimb defects. Homozygous mutation of zebrafish tbx5a leads to lethal defects in cardiac looping morphogenesis, blocks pectoral fin initiation, and impairs outgrowth. Recently, a second zebrafish tbx5 gene was described, termed tbx5b. RESULTS: Our phylogenetic analyses confirm tbx5b as a paralog that likely arose in the teleost-specific whole genome duplication â¼270 MYA. Using morpholino depletion studies, we find that tbx5b is required in the heart for embryonic survival, and influences the timing and morphogenesis of pectoral fin development. Because tbx5a hypomorphic mutations are embryonic lethal, tbx5a and tbx5b functions in the heart must not be completely redundant. Consistent with this hypothesis, simultaneous depletion of both tbx5 paralogs did not lead to more severe phenotypes, and injection of wild-type mRNA from one tbx5 paralog was not sufficient to cross-rescue phenotypes of the paralogous gene. CONCLUSIONS: Collectively, these data indicate that, despite similar spatio-temporal expression patterns, tbx5a and tbx5b have independent functions in heart and fin development.
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Nadadeiras de Animais/embriologia , Coração/embriologia , Proteínas com Domínio T/fisiologia , Peixe-Zebra , Sequência de Aminoácidos , Nadadeiras de Animais/metabolismo , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Embrião não Mamífero , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Especiação Genética , Genoma/genética , Dados de Sequência Molecular , Morfogênese/genética , Filogenia , Isoformas de Proteínas/genética , Homologia de Sequência , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Congenital heart defects arise during the early stages of development, and studies have linked abnormal blood flow and irregular cardiac function to improper cardiac morphogenesis. The embryonic zebrafish offers superb optical access for live imaging of heart development. Here, we build upon previously used techniques to develop a methodology for quantifying cardiac function in the embryonic zebrafish model. Imaging was performed using bright field microscopy at 1500 frames/s at 0.76 µm/pixel. Heart function was manipulated in a wild-type zebrafish at â¼55 h post fertilization (hpf). Blood velocity and luminal diameter were measured at the atrial inlet and atrioventricular junction (AVJ) by analyzing spatiotemporal plots. Control volume analysis was used to estimate the flow rate waveform, retrograde fractions, stroke volume, and cardiac output. The diameter and flow waveforms at the inlet and AVJ are highly repeatable between heart beats. We have developed a methodology for quantifying overall heart function, which can be applied to early stages of zebrafish development.
Assuntos
Coração/embriologia , Coração/fisiologia , Animais , Circulação Coronária , Frequência Cardíaca , Hemodinâmica , Processamento de Imagem Assistida por Computador , Peixe-Zebra/embriologiaRESUMO
BACKGROUND: L-type calcium channels (LTCC) regulate calcium entry into cardiomyocytes. CACNB2 (ß2) LTCC auxiliary subunits traffic the pore-forming CACNA subunit to the membrane and modulate channel kinetics. ß2 is a membrane associated guanylate kinase (MAGUK) protein. A major role of MAGUK proteins is to scaffold cellular junctions and multiprotein complexes. RESULTS: To investigate developmental functions for ß2.1, we depleted it in zebrafish using morpholinos. ß2.1-depleted embryos developed compromised cardiac function by 48 hr postfertilization, which was ultimately lethal. ß2.1 contractility defects were mimicked by pharmacological depression of LTCC, and rescued by LTCC stimulation, suggesting ß2.1 phenotypes are at least in part LTCC-dependent. Morphological studies indicated that ß2.1 contributes to heart size by regulating the rate of ventricle cell proliferation, and by modulating the transition of outer curvature cells to an elongated cell shape during chamber ballooning. In addition, ß2.1-depleted cardiomyocytes failed to accumulate N-cadherin at the membrane, and dissociated easily from neighboring myocytes under stress. CONCLUSIONS: Hence, we propose that ß2.1 may also function in the heart as a MAGUK scaffolding unit to maintain N-cadherin-based adherens junctions and heart tube integrity.
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Canais de Cálcio Tipo L/fisiologia , Coração/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Proliferação de Células , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Coração/fisiologia , Organogênese/fisiologia , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
In the adult heart, acute adaptation of electrical and mechanical activity to changes in mechanical load occurs via feedback processes known as "mechano-electric coupling" and "mechano-mechanical coupling." Whether this occurs during cardiac development is ill-defined, as acutely altering the heart's mechanical load while measuring functional responses in traditional experimental models is difficult, as embryogenesis occurs in utero, making the heart inaccessible. These limitations can be overcome with zebrafish, as larvae develop in a dish and are nearly transparent, allowing for in vivo manipulation and measurement of cardiac structure and function. Here we present a novel approach for the in vivo study of mechano-electric and mechano-mechanical coupling in the developing zebrafish heart. This innovative methodology involves acute in vivo atrial dilation (i.e., increased atrial preload) in larval zebrafish by injection of a controlled volume into the venous circulation immediately upstream of the heart, combined with optical measurement of the acute electrical (change in heart rate) and mechanical (change in stroke area) response. In proof-of-concept experiments, we applied our new method to 48 h post-fertilisation zebrafish, which revealed differences between the electrical and mechanical response to atrial dilation. In response to an acute increase in atrial preload there is a large increase in atrial stroke area but no change in heart rate, demonstrating that in contrast to the fully developed heart, during early cardiac development mechano-mechanical coupling alone drives the adaptive increase in atrial output. Overall, in this methodological paper we present our new experimental approach for the study of mechano-electric and mechano-mechanical coupling during cardiac development and demonstrate its potential for understanding the essential adaptation of heart function to acute changes in mechanical load.
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Heart valve development is governed by both genetic and biomechanical inputs. Prior work has demonstrated that oscillating shear stress associated with blood flow is required for normal atrioventricular (AV) valve development. Cardiac afterload is defined as the pressure the ventricle must overcome in order to pump blood throughout the circulatory system. In human patients, conditions of high afterload can cause valve pathology. Whether high afterload adversely affects embryonic valve development remains poorly understood. Here we describe a zebrafish model exhibiting increased myocardial afterload, caused by vasopressin, a vasoconstrictive drug. We show that the application of vasopressin reliably produces an increase in afterload without directly acting on cardiac tissue in zebrafish embryos. We have found that increased afterload alters the rate of growth of the cardiac chambers and causes remodeling of cardiomyocytes. Consistent with pathology seen in patients with clinically high afterload, we see defects in both the form and the function of the valve leaflets. Our results suggest that valve defects are due to changes in atrioventricular myocyte signaling, rather than pressure directly acting on the endothelial valve leaflet cells. Cardiac afterload should therefore be considered a biomechanical factor that particularly impacts embryonic valve development.
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During development, the heart begins pumping as a valveless multilayered tube capable of driving blood flow throughout the embryonic vasculature. The mechanical properties and how they interface with pumping function are not well-defined at this stage. Here, we evaluate pumping patterns using a fluid-structure interaction computational model, combined with experimental data and an energetic analysis to investigate myocardial mechanical properties. Through this work, we propose that a myocardium modeled as a Neo-Hookean material with a material constant on the order of 10 kPa is necessary for the heart tube to function with an optimal pressure and cardiac output.
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Coração/embriologia , Coração/fisiologia , Hemodinâmica , Contração Miocárdica , Miocárdio/patologia , Peixe-Zebra/embriologia , Animais , Fenômenos Biomecânicos , Débito Cardíaco , Simulação por Computador , Impedância Elétrica , Frequência Cardíaca , Modelos Cardiovasculares , Modelos Teóricos , Peristaltismo , Pressão , Estresse MecânicoRESUMO
Cardiac morphogenesis requires an intricate orchestration of mechanical stress to sculpt the heart as it transitions from a straight tube to a multichambered adult heart. Mechanical properties are fundamental to this process, involved in a complex interplay with function, morphology, and mechanotransduction. In the current work, we propose a pressurization technique applied to the zebrafish atrium to quantify mechanical properties of the myocardium under passive tension. By further measuring deformation, we obtain a pressure-stretch relationship that is used to identify constitutive models of the zebrafish embryonic cardiac tissue. Two-dimensional results are compared with a three-dimensional finite element analysis based on reconstructed embryonic heart geometry. Through these steps, we found that the myocardium of zebrafish results in a stiffness on the order of 10 kPa immediately after the looping stage of development. This work enables the ability to determine how these properties change under normal and pathological heart development.
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Coração/embriologia , Coração/fisiologia , Animais , Embrião não Mamífero , Modelos Biológicos , Estresse Mecânico , Peixe-Zebra/embriologiaRESUMO
Mutations in the T-box transcription factor, TBX5, result in Holt-Oram syndrome (HOS), a human condition in which cardiac development is defective and forelimbs are stunted. Similarly, zebrafish tbx5 morphants and mutants (heartstrings; hst) lack pectoral fins and exhibit a persistently elongated heart that does not undergo chamber looping. Tbx5 is expressed in the developing atrium, ventricle and in pectoral fin fields, but its genetic targets are still being uncovered. In this study, evidence is provided that Tbx5 induces the expression of a specific member of the CaMK-II (the type II multifunctional Ca(2+)/calmodulin-dependent protein kinase) family; this CaMK-II is necessary for proper heart and fin development. Morphants of beta2 CaMK-II (camk2b2), but not the beta1 CaMK-II (camk2b1) paralog, exhibit bradycardia, elongated hearts and diminished pectoral fin development. Normal cardiac phenotypes can be restored by ectopic cytosolic CaMK-II expression in tbx5 morphants. Like tbx5, camk2b2 is expressed in the pectoral fin and looping heart, but this expression is diminished in both tbx5 morphant and hst embryos. Conversely, the introduction of excess Tbx5 into zebrafish embryos and mouse fibroblasts doubles CaMK-II expression. We conclude that beta CaMK-II expression and activity are necessary for proper cardiac and limb morphogenesis. These findings not only identify a morphogenic target for Ca(2+) during heart development, but support implied roles for CaMK-II in adult heart remodeling.
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Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Coração/embriologia , Morfogênese , Proteínas com Domínio T/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Coração/crescimento & desenvolvimento , Camundongos , Dados de Sequência Molecular , Morfogênese/genética , Células NIH 3T3 , Transfecção , Proteínas de Peixe-Zebra/genéticaRESUMO
The vertebrate embryonic heart initially forms with two chambers, a ventricle and an atrium, separated by the atrioventricular junction. Localized genetic and biomechanical information guides the development of valves, which function to ensure unidirectional blood flow. If the valve development process goes awry, pathology associated with congenital valve defects can ensue. Congenital valve defects (CVD) are estimated to affect 1-2% of the population and can often require a lifetime of treatment. Despite significant clinical interest, molecular genetic mechanisms that direct valve development remain incompletely elucidated. Cells in the developing valve must contend with a dynamic hemodynamic environment. A growing body of research supports the idea that cells in the valve are highly sensitive to biomechanical forces, which cue changes in gene expression required for normal development or for maintenance of the adult valve. This review will focus on mechanotransductive pathways involved in valve development across model species. We highlight current knowledge regarding how cells sense physical forces associated with blood flow and pressure in the forming heart, and summarize how these changes are transduced into genetic and developmental responses. Lastly, we provide perspectives on how altered biomechanical cues may lead to CVD pathogenesis.
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NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Ndrg4 knockdown in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth.
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Proteínas Musculares/metabolismo , Miócitos Cardíacos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Clonagem Molecular , Coração/embriologia , Hibridização In Situ , Proteínas Musculares/genética , Mutação/genética , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/genética , Oligonucleotídeos/genética , Proteínas com Domínio T/metabolismo , Versicanas/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The Ca(2+) channel beta-subunits, encoded by CACNB genes 1-4, are membrane-associated guanylate kinase (MAGUK) proteins. As auxiliary subunits of voltage-gated Ca(2+) channels, the beta-subunits facilitate membrane trafficking of the pore-forming alpha1 subunits and regulate voltage-dependent channel gating. In this report, we investigate whether two zebrafish beta4 genes, beta4.1 and beta4.2, have diverged in structure and function over time. Comparative expression analyses indicated that beta4.1 and beta4.2 were expressed in separable domains within the developing brain and other tissues. Alternative splicing in both genes was subject to differential temporal and spatial regulation, with some organs expressing different subsets of beta4.1 and beta4.2 transcript variants. We used several genomic tools to identify and compare predicted cDNAs for eight teleost and five tetrapod beta4 genes. Teleost species had either one or two beta4 paralogs, whereas each tetrapod species contained only one. Teleost beta4.1 and beta4.2 genes had regions of sequence divergence, but compared with tetrapod beta4s, they exhibited similar exon/intron structure, strong conservation of residues involved in alpha1 subunit binding, and similar 5' alternative splicing. Phylogenetic results are consistent with the duplicate teleost beta4 genes resulting from the teleost whole genome duplication. Following duplication, the beta4.1 genes have evolved faster than beta4.2 genes. We identified disproportionately large second and third introns in several beta4 genes, which we propose may provide regulatory elements contributing to their differential tissue expression. In sum, both mRNA expression data and phylogenetic analysis support the evolutionary divergence of beta4.1 and beta4.2 subunit function.
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Canais de Cálcio/classificação , Canais de Cálcio/genética , Genoma , Processamento Alternativo , Sequência de Aminoácidos , Animais , Canais de Cálcio/metabolismo , Evolução Molecular , Expressão Gênica , Genômica , Humanos , Hibridização In Situ , Íntrons , Dados de Sequência Molecular , Filogenia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Vertebrados , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts. RESULTS: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult. CONCLUSION: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.
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Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Subunidades Proteicas/genética , Tetraodontiformes/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Variação Genética , Genoma , Íntrons/genética , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Subunidades Proteicas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Tetraodontiformes/embriologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/químicaRESUMO
Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.
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OBJECTIVES: The purpose of this study was to assess the phenotype of Filamin C (FLNC) truncating variants in dilated cardiomyopathy (DCM) and understand the mechanism leading to an arrhythmogenic phenotype. BACKGROUND: Mutations in FLNC are known to lead to skeletal myopathies, which may have an associated cardiac component. Recently, the clinical spectrum of FLNC mutations has been recognized to include a cardiac-restricted presentation in the absence of skeletal muscle involvement. METHODS: A population of 319 U.S. and European DCM cardiomyopathy families was evaluated using whole-exome and targeted next-generation sequencing. FLNC truncation probands were identified and evaluated by clinical examination, histology, transmission electron microscopy, and immunohistochemistry. RESULTS: A total of 13 individuals in 7 families (2.2%) were found to harbor 6 different FLNC truncation variants (2 stopgain, 1 frameshift, and 3 splicing). Of the 13 FLNC truncation carriers, 11 (85%) had either ventricular arrhythmias or sudden cardiac death, and 5 (38%) presented with evidence of right ventricular dilation. Pathology analysis of 2 explanted hearts from affected FLNC truncation carriers showed interstitial fibrosis in the right ventricle and epicardial fibrofatty infiltration in the left ventricle. Ultrastructural findings included occasional disarray of Z-discs within the sarcomere. Immunohistochemistry showed normal plakoglobin signal at cell-cell junctions, but decreased signals for desmoplakin and synapse-associated protein 97 in the myocardium and buccal mucosa. CONCLUSIONS: We found FLNC truncating variants, present in 2.2% of DCM families, to be associated with a cardiac-restricted arrhythmogenic DCM phenotype characterized by a high risk of life-threatening ventricular arrhythmias and a pathological cellular phenotype partially overlapping with arrhythmogenic right ventricular cardiomyopathy.
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Cardiomiopatia Dilatada , Filaminas/genética , Mutação/genética , Miocárdio , Arritmias Cardíacas , Cardiomiopatia Dilatada/epidemiologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Adesão Celular/genética , Análise Mutacional de DNA , Europa (Continente) , Humanos , Imuno-Histoquímica , Miocárdio/citologia , Miocárdio/patologia , Polimorfismo de Nucleotídeo Único/genética , Estudos Prospectivos , Estados UnidosRESUMO
BACKGROUND: Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection. RESULTS: After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N6 macronutrient/B5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50-100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical ß-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines. CONCLUSION: The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.
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Normal development of the heart is regulated, in part, by mechanical influences associated with blood flow during early stages of embryogenesis. Specifically, the potential for retrograde flow at the atrioventricular canal (AVC) is particularly important in valve development. However, the mechanisms causing this retrograde flow have received little attention. In this study, a numerical analysis was performed on images of the embryonic zebrafish heart between 48 and 55hpf. During these stages, normal retrograde flow is prevalent. To manipulate this flow, zebrafish were placed in a centrifuge and subjected to a hypergravity environment to alter the cardiac preload at various six-hour intervals between 24 and 48hpf. Parameters of the pumping mechanics were then analyzed through a spatiotemporal analysis of processed image sequences. We find that the loss of retrograde flow in experimentally manipulated embryos occurs in part because of a greater resistance in the form of atrial and AVC contractile closure. Additionally, during retrograde flow, these embryos exhibit significantly greater pressure difference across the AVC based on calculations of expansive and contractile rates of the atrium and ventricle. These results elucidated that the developing heart is highly sensitive to small changes in pumping mechanics as it strives to maintain normal hemodynamic conditions necessary for later cardiac development.
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Desenvolvimento Embrionário , Átrios do Coração/embriologia , Ventrículos do Coração/embriologia , Hemodinâmica , Animais , Desenvolvimento Embrionário/fisiologia , Peixe-Zebra/embriologiaRESUMO
Wilm's tumor 1 interacting protein (Wtip) was identified as an interacting partner of Wilm's tumor protein (WT1) in a yeast two-hybrid screen. WT1 is expressed in the proepicardial organ (PE) of the heart, and mouse and zebrafish wt1 knockout models appear to lack the PE. Wtip's role in the heart remains unexplored. In the present study, we demonstrate that wtip expression is identical in wt1a, tcf21, and tbx18positive PE cells, and that Wtip protein localizes to the basal body of PE cells. We present the first genetic evidence that Wtip signaling in conjunction with WT1 is essential for PE specification in the zebrafish heart. By overexpressing wtip mRNA, we observed ectopic expression of PE markers in the cardiac and pharyngeal arch regions. Furthermore, wtip knockdown embryos showed perturbed cardiac looping and lacked the atrioventricular (AV) boundary. However, the chamberspecific markers amhc and vmhc were unaffected. Interestingly, knockdown of wtip disrupts early leftright (LR) asymmetry. Our studies uncover new roles for Wtip regulating PE cell specification and early LR asymmetry, and suggest that the PE may exert nonautonomous effects on heart looping and AV morphogenesis. The presence of cilia in the PE, and localization of Wtip in the basal body of ciliated cells, raises the possibility of cilia-mediated PE signaling in the embryonic heart.
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Coração/embriologia , Morfogênese/genética , Organogênese/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Miocárdio/metabolismo , Fenótipo , Ligação Proteica , Transdução de Sinais , Proteínas WT1/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: To identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism. BACKGROUND: DCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes. METHODS: We performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model. RESULTS: A novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure. CONCLUSIONS: Using WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM.
RESUMO
Proper blood flow patterns are critical for normal cardiac morphogenesis, a process that occurs rapidly in order to support further development of all tissue and organs. Previously, intracardiac fluid forces have been shown to play a critical role in cardiac morphogenesis. Altered blood flow in early development can result in an array of cardiac defects including ventricular septal defects, valve malformations, and impaired cardiac looping. However, given the dynamic and highly transient nature of cardiac morphogenesis, time dependency of the mechanical environment as an epigenetic factor in relation to intracardiac forces must be significant. Here, we show that abnormal cardiac loading adversely influences cardiac morphology only during certain time windows, thus confirming that mechanical factors are a time-dependent epigenetic factor. To illustrate this, groups of zebrafish embryos were spaced at 6-h increments from 24 to 48 h post-fertilization (hpf) in which embryos were centrifuged to generate a noninvasive alteration of cardiac preload in addition to an overall hypergravity environment. We found that earlier and later treatment groups responded with altered morphology and function, while the group with altered preload from 30 to 36 hpf had no effect. These results demonstrate the inherently time-dependent nature of epigenetic factors as pertaining to intracardiac forces and external mechanical factors. Further, it underscores the highly coupled nature of programmed biology and mechanical forces during cardiac morphogenesis. Future studies with respect to surgical correction during cardiac morphogenesis must consider timing to optimize therapeutic impact.