RESUMO
T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.
Assuntos
Miócitos Cardíacos , Optogenética , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Membrana Celular , Potenciais da Membrana , Potenciais de Ação/fisiologiaRESUMO
Optogenetics, utilising light-reactive proteins to manipulate tissue activity, are a relatively novel approach in the field of cardiac electrophysiology. We here provide an overview of light-activated transmembrane channels (optogenetic actuators) currently applied in strategies to modulate cardiac activity, as well as newly developed variants yet to be implemented in the heart. In addition, we touch upon genetically encoded indicators (optogenetic sensors) and fluorescent dyes to monitor tissue activity, including cardiac transmembrane potential and ion homeostasis. The combination of the two allows for all-optical approaches to monitor and manipulate the heart without any physical contact. However, spectral congestion poses a major obstacle, arising due to the overlap of excitation/activation and emission spectra of various optogenetic proteins and/or fluorescent dyes, resulting in optical crosstalk. Therefore, optogenetic proteins and fluorescent dyes should be carefully selected to avoid optical crosstalk and consequent disruptions in readouts and/or cellular activity. We here present a novel approach to simultaneously monitor transmembrane potential and cytosolic calcium, while also performing optogenetic manipulation. For this, we used the novel voltage-sensitive dye ElectroFluor 730p and the cytosolic calcium indicator X-Rhod-1 in mouse hearts expressing channelrhodopsin-2 (ChR2). By exploiting the isosbestic point of ElectroFluor 730p and avoiding the ChR2 activation spectrum, we here introduce a novel optical imaging and manipulation approach with minimal crosstalk. Future developments in both optogenetic proteins and fluorescent dyes will allow for additional and more optimised strategies, promising a bright future for all-optical approaches in the field of cardiac electrophysiology.
RESUMO
Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300-1,800 working cardiomyocytes or 90-160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia.
Assuntos
Complexos Cardíacos Prematuros/patologia , Miocárdio/patologia , Optogenética/métodos , Especificidade de Órgãos , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Complexos Cardíacos Prematuros/complicações , Complexos Cardíacos Prematuros/fisiopatologia , Channelrhodopsins , Conexinas/metabolismo , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Fenômenos Eletrofisiológicos , Humanos , Integrases/metabolismo , Ligadura , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ramos Subendocárdicos/metabolismo , Ramos Subendocárdicos/patologia , Ramos Subendocárdicos/fisiopatologia , Proteína alfa-5 de Junções ComunicantesRESUMO
Introduction: Mechanisms underlying cardiac arrhythmias are typically driven by abnormalities in cardiac conduction and/or heterogeneities in repolarization time (RT) across the heart. While conduction slowing can be caused by either electrophysiological defects or physical blockade in cardiac tissue, RT heterogeneities are mainly related to action potential (AP) prolongation or abbreviation in specific areas of the heart. Importantly, the size of the area with altered RT and the difference between the short RT and long RT (RT gradient) have been identified as critical determinators of arrhythmogenicity. However, current experimental methods for manipulating RT gradient rely on the use of ion channel inhibitors, which lack spatial and temporal specificity and are commonly only partially reversible. Therefore, the conditions facilitating sustained arrhythmia upon the presence of RT heterogeneities and/or defects in cardiac conduction remain to be elucidated. Methods: We here employ an approach based on optogenetic stimulation in a low-intensity fashion (sub-threshold illumination), to selectively manipulate cardiac electrical activity in defined areas of the heart. Results: As previously described, subthreshold illumination is a robust tool able to prolong action potentials (AP), decrease upstroke velocity as well as slow cardiac conduction, in a fully reversible manner. By applying a patterned sub-threshold illumination in intact mouse hearts constitutively expressing the light-gated ion channel channelrhodopsin-2 (ChR2), we optically manipulate RT gradients and cardiac conduction across the heart in a spatially selective manner. Moreover, in a proof-of-concept assessment we found that in the presence of patterned sub-threshold illumination, mouse hearts were more susceptible to arrhythmias. Hence, this optogenetic-based approach may be able to mimic conduction slowing and RT heterogeneities present in pathophysiological conditions.
RESUMO
The Pitx2 gene regulates left-right (L/R) asymmetrical cardiac morphogenesis. Constitutive Pitx2 knock out (ko) mice die before birth and display, among other defects, right atrial isomerism, atrial and ventricular septal defects, and double outlet right ventricle. The myocardial role of the gene has not been dissected. In particular, how Pitx2 regulates the differential L/R cardiac identity program is not clear. Additionally, the relation between Pitx2 ko ventricular defects and the gene expression pattern is not understood. In this article we analyze Pitx2 myocardial function during mouse heart development. By in situ hybridization analysis we show that myocardial Pitx2 expression delineates the remodeling of the left atrioventricular canal, the inner curvature, the ventral part of the interventricular ring, and the ventral portion of the right and left ventricle. By genetic analysis using an allelic series of Pitx2 mutants, among which a myocardial specific ko (ko(myo)) we show it has a crucial role in this process. Pitx2 ko(myo) mutants survive to adulthood, when they present strong cardiac morphological and functional defects. Confocal analysis of embryonic Pitx2 ko(myo) hearts reveals delayed cardiomyocyte development in the ventricular but not in the atrial Pitx2 null areas. Conversely, selective left atrial BMP10 mRNA downregulation which normally occurs at fetal stages is not found in the Pitx2 ko(myo) mice. This is the first evidence for distinct Pitx2 action in mediating L/R atrial identity and asymmetrical ventricular remodeling.
Assuntos
Átrios do Coração/embriologia , Ventrículos do Coração/embriologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Remodelação Ventricular/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Átrios do Coração/metabolismo , Cardiopatias Congênitas/patologia , Ventrículos do Coração/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/patologia , RNA Mensageiro/metabolismo , Nó Sinoatrial/embriologia , Nó Sinoatrial/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteína Homeobox PITX2RESUMO
Calcium stores in neurons are heterogeneous in compartmentalization and molecular composition. Danio rerio (zebrafish) is an animal model with a simply folded cerebellum similar in cellular organization to that of mammals. The aim of the study was to identify new endoplasmic reticulum (ER) calcium store markers in zebrafish adult brain with emphasis on cerebellum and optic tectum. By quantitative polymerase chain reaction, we found three RNA transcripts coding for the intra-ER calcium binding protein calsequestrin: casq1a, casq1b, and casq2. In brain homogenates, two isoforms were detected by mass spectrometry and western blotting. Fractionation experiments of whole brain revealed that Casq1a and Casq2 were enriched in a heavy fraction containing ER microsomes and synaptic membranes. By in situ hybridization, we found the heterogeneous expression of casq1a and casq2 mRNA to be compatible with the cellular localization of calsequestrins investigated by immunofluorescence. Casq1 was expressed in neurogenic differentiation 1 expressing the granule cells of the cerebellum and the periventricular zone of the optic tectum. Casq2 was concentrated in parvalbumin expressing Purkinje cells. At a subcellular level, Casq1 was restricted to granular cell bodies, and Casq2 was localized in cell bodies, dendrites, and axons. Data are discussed in relation to the differential cellular and subcellular distribution of other cerebellum calcium store markers and are evaluated with respect to the putative relevance of calsequestrins in the neuron-specific functional activity.
RESUMO
AIMS: Increased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have been associated to total anomalous pulmonary venous return and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease is not understood. To get insight into this problem, we have generated a gain of function ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium. METHODS AND RESULTS: Ankrd1 is expressed non-homogeneously in the embryonic myocardium, with a dynamic nucleo-sarcomeric localization in developing cardiomyocytes. ANKRD1 transgenic mice present sinus venosus defect, which originates during development by impaired remodelling of early embryonic heart. Adult transgenic hearts develop diastolic dysfunction with preserved ejection fraction, which progressively evolves into heart failure, as shown histologically and haemodynamically. Transgenic cardiomyocyte structure, sarcomeric assembly, and stability are progressively impaired from embryonic to adult life. Postnatal transgenic myofibrils also present characteristic functional alterations: impaired compliance at neonatal stage and impaired lusitropism in adult hearts. Altogether, our combined analyses suggest that impaired embryonic remodelling and adult heart dysfunction in ANKRD1 transgenic mice present a common ground of initial cardiomyocyte defects, which are exacerbated postnatally. Molecular analysis showed transient activation of GATA4-Nkx2.5 transcription in early transgenic embryos and subsequent dynamic transcriptional modulation within titin gene. CONCLUSIONS: ANKRD1 is a fine mediator of cardiomyocyte response to haemodynamic load in the developing and adult heart. Increased ANKRD1 levels are sufficient to initiate an altered cellular phenotype, which is progressively exacerbated into a pathological organ response by the high ventricular workload during postnatal life. Our study defines for the first time a unifying picture for ANKRD1 role in heart development and disease and provides the first mechanistic link between ANKRD1 overexpression and cardiac disease onset.
Assuntos
Comunicação Interatrial/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Função Ventricular Esquerda , Animais , Diástole , Feminino , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Comunicação Interatrial/genética , Comunicação Interatrial/patologia , Comunicação Interatrial/fisiopatologia , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Masculino , Camundongos Transgênicos , Proteínas Musculares/genética , Miocárdio/patologia , Proteínas Nucleares/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/genética , Regulação para Cima , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect in which the pulmonary veins fail to enter the left atrium and drain instead into the right atrium or one of its venous tributaries. Although a genetic basis for TAPVR has long been recognized, no single gene involved in the pathogenesis of this disease has been identified to date. We previously reported a TAPVR patient bearing a de novo 10;21 balanced translocation. In this work, we cloned both translocation breakpoints from this patient and mapped the ANKRD1 gene, encoding a cardiac transcriptional regulator, 130 kb proximally to the breakpoint on chromosome 10. In situ hybridization analysis performed on murine embryos showed ANKRD1 expression in the developing pulmonary veins, suggesting a possible role for this gene in TAPVR pathogenesis. Moreover, ANKRD1 expression levels were found to be highly increased in lymphoblastoid cell lines derived from both the translocation-bearing proband and a second independent sporadic TAPVR patient, suggesting that disruption of the normal ANKRD1 expression pattern is associated with TAPVR. Finally, a nonconservative missense mutation in the ANKRD1 gene was found in a third sporadic TAPVR patient. In vitro calpain-mediated degradation assays, coupled to reporter gene analysis in transfected HeLa cells, strongly suggested that this mutation enhances both the stability of the ANKRD1/CARP protein and its transcriptional repression activity upon the cardiac-specific atrial natriuretic factor (ANF) promoter. Taken together, these results define ANKRD1 as a possible candidate gene for TAPVR pathogenesis.
Assuntos
Cardiopatias Congênitas/genética , Proteínas Musculares/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Veias Pulmonares/anormalidades , Proteínas Repressoras/genética , Animais , Sequência de Bases , Linhagem Celular , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 21/genética , DNA/genética , Feminino , Expressão Gênica , Células HeLa , Cardiopatias Congênitas/metabolismo , Humanos , Masculino , Camundongos , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Linhagem , Gravidez , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Transfecção , Translocação GenéticaRESUMO
Goosecoid (Gsc) expression marks the primary embryonic organizer in vertebrates and beyond. While functions have been assigned during later embryogenesis, the role of Gsc in the organizer has remained enigmatic. Using conditional gain-of-function approaches in Xenopus and mouse to maintain Gsc expression in the organizer and along the axial midline, neural tube closure defects (NTDs) arose and dorsal extension was compromised. Both phenotypes represent convergent extension (CE) defects, arising from impaired Wnt/planar cell polarity (PCP) signaling. Dvl2 recruitment to the cell membrane was inhibited by Gsc in Xenopus animal cap assays and key Wnt/PCP factors (RhoA, Vangl2, Prickle, Wnt11) rescued Gsc-mediated NTDs. Re-evaluation of endogenous Gsc functions in MO-mediated gene knockdown frog and knockout mouse embryos unearthed PCP/CE-related phenotypes as well, including cartilage defects in Xenopus and misalignment of inner ear hair cells in mouse. Our results assign a novel function to Gsc as an inhibitor of Wnt/PCP-mediated CE. We propose that in the organizer Gsc represses CE as well: Gsc-expressing prechordal cells, which leave the organizer first, migrate and do not undergo CE like the Gsc-negative notochordal cells, which subsequently emerge from the organizer. In this model, Gsc provides a switch between cell migration and CE, i.e. cell intercalation.
Assuntos
Proteína Goosecoid/metabolismo , Organizadores Embrionários/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Polaridade Celular , Proteínas Desgrenhadas/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário , Genes Reporter , Proteína Goosecoid/deficiência , Proteína Goosecoid/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Transdução de Sinais , Proteínas de Xenopus/genéticaRESUMO
The heart is the first organ to break symmetry in the developing embryo and onset of dextral looping is the first indication of this event. Looping is a complex process that progresses concomitantly to cardiac chamber differentiation and ultimately leads to the alignment of the cardiac regions in their final topology. Generation of cardiac asymmetry is crucial to ensuring proper form and consequent functionality of the heart, and therefore it is a highly regulated process. It has long been known that molecular left/right signals originate far before morphological asymmetry and therefore can direct it. The use of several animal models has led to the characterization of a complex regulatory network, which invariably converges on the Tgf-ß signaling molecule Nodal and its downstream target, the homeobox transcription factor Pitx2. Here, we review current data on the cellular and molecular bases of cardiac looping and laterality, and discuss the contribution of Nodal and Pitx2 to these processes. A special emphasis will be given to the morphogenetic role of Pitx2 and to its modulation of transcriptional and functional properties, which have also linked laterality to atrial fibrillation.
RESUMO
Pitx2 is a bicoid-related homeodomain transcription factor that plays a critical role in directing cardiac asymmetric morphogenesis. Ectopic Pitx2c expression in the developing myocardium correlates with double outlet right ventricle (DORV) in laterality mutants. Pitx2 loss of function experiments cause severe cardiovascular defects, such as atrial isomerism (AI), double inlet left ventricle, transposition of the great arteries (TGA), persistent truncus arteriosus (PTA), and abnormal aortic arch (AAA) remodeling. Current studies suggest that Pitx2-mediated signaling during cardiogenesis is conducted within three different cell types: the myocardium, the cardiac neural crest (CNC) cells, and the pharyngeal arch mesenchyme. Impaired Pitx2 function in discrete myocardial regions seems to lead to DORV, AI, and possibly TGA. On the other hand, impaired Pitx2 expression in the CNC leads preferentially to PTA. AAA remodeling is likely to occur owing to impaired cross-talk of the CNC cells with the pharyngeal arch mesenchyme. Thus, Pitx2 appears to be directing left-right identity to the cardiac venous components (e.g., the atria), whereas it appears to be modeling the morphologic arrangement of distinct myocardial components in the arterial pole. These data suggest that altered left-right signaling underlies the etiology of several common congenital cardiac malformations.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cardiopatias Congênitas/fisiopatologia , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares , Fatores de Transcrição/fisiologia , Animais , Humanos , Proteína Homeobox PITX2RESUMO
OBJECTIVE: The molecular mechanisms that regulate the formation of the conduction system are poorly understood. We studied the developmental expression pattern and functional aspects of the T-box transcription factor Tbx3, a novel marker for the murine central conduction system (CCS). METHODS: The patterns of expression of Tbx3, and of Cx40, Cx43, and Nppa, which are markers for atrial and ventricular chamber-type myocardium in the developing heart, were analyzed in mice by in situ hybridization and three-dimensional reconstruction analysis. The function of Tbx3 in regulating Nppa and Cx40 promoter activity was studied in vitro. RESULTS: In the formed heart, Tbx3 is expressed in the sinoatrial node (SAN), atrioventricular node (AVN), bundle and proximal bundle branches (BBs), as well as the internodal regions and the atrioventricular region. Throughout cardiac development, Tbx3 is expressed in an uninterrupted myocardial domain that extends from the sinoatrial node to the atrioventricular region. This expression domain is present in the looping heart tube from E8.5 onwards. Expression of the chamber-type myocardial markers is specifically absent from the Tbx3 expression domain. Tbx3 is able to repress Nppa and Cx40 promoter activity and abolish the synergistic activation of the Nppa promoter by Tbx5 and Nkx2.5. CONCLUSION: We identified the T-box transcription factor Tbx3 as a novel and accurate marker for the central conduction system. Our analysis implicates a role for Tbx3 in repressing a chamber-specific program of gene expression in regions from which the components of the central conduction system are subsequently formed.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Sistema de Condução Cardíaco/embriologia , Proteínas com Domínio T/genética , Animais , Fator Natriurético Atrial , Células COS , Linhagem Celular , Conexinas/genética , Expressão Gênica , Marcadores Genéticos , Idade Gestacional , Sistema de Condução Cardíaco/química , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Camundongos , Camundongos Endogâmicos , Miocárdio/química , Peptídeo Natriurético Tipo C , Regiões Promotoras Genéticas , Precursores de Proteínas , Proteínas com Domínio T/análise , Proteína alfa-5 de Junções ComunicantesRESUMO
The homeobox transcription factor Pitx2 displays a highly specific expression pattern during embryogenesis. Gain and loss of function experiments have unraveled its pivotal role in left-right signaling. Conditional deletion in mice has demonstrated a complex and intricate role for Pitx2 in distinct aspects of cardiac development and more recently a link to atrial fibrillation has been proposed based on genome-wide association studies. In this review we will revise the role of Pitx2 in the developing heart, starting from the early events of left-right determination followed by its role in cardiac morphogenesis and ending with its role in cardiac arrhythmogenesis.
Assuntos
Arritmias Cardíacas/metabolismo , Coração/embriologia , Proteínas de Homeodomínio/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Coração/fisiopatologia , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Knockout , Morfogênese , Fenótipo , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica , Proteína Homeobox PITX2RESUMO
Connexin40 (Cx40) is the main connexin expressed in the murine atria and ventricular conduction system. We assess here the developmental role of Cx40 in atrial conduction of the mouse. Cx40 deficiency significantly prolonged activation times in embryonic day 10.5, 12.5 and 14.5 atria during spontaneous activation; the severity decreased with increasing age. In a majority of Cx40 deficient mice the impulse originated from an ectopic focus in the right atrial appendage; in such a case the activation time was even longer due to prolonged activation. Cx40 has thus an important physiological role in the developing atria.
Assuntos
Conexinas/metabolismo , Nó Sinoatrial/metabolismo , Animais , Apêndice Atrial/embriologia , Apêndice Atrial/metabolismo , Apêndice Atrial/fisiologia , Conexinas/genética , Coração Fetal/metabolismo , Coração Fetal/fisiologia , Camundongos , Nó Sinoatrial/embriologia , Nó Sinoatrial/fisiologia , Proteína alfa-5 de Junções ComunicantesRESUMO
AIMS: The sinus venous myocardium, comprising the sinoatrial node (SAN) and sinus horns (SH), is a region subject to congenital malformations and cardiac arrhythmias. It differentiates from symmetric bilateral mesenchymal precursors, but morphological, molecular, and functional left/right differences are progressively established through development. The role of the laterality gene Pitx2 in this process is unknown. We aimed to elucidate the molecular events driving left/right patterning in the sinus venosus (SV) myocardium by using a myocardial Pitx2 knockout mouse. METHODS AND RESULTS: We generated a myocardial specific Pitx2 knockout model (cTP mice). cTP embryos present several features of Pitx2 null, including right atrial isomerism with bilateral SANs and symmetric atrial entrance of the systemic veins. By in situ hybridization and optical mapping analysis, we compared throughout development the molecular and functional properties of the SV myocardium in wt and mutant embryos. We observed that Pitx2 prevents the expansion of the left-SAN primordium at the onset of its differentiation into myocardium; Pitx2 promotes expansion of the left SH through development; Pitx2 dose-dependently represses the autorhythmic properties of the left SV myocardium at mid-gestation (E14.5); Pitx2 modulates late foetal gene expression at the left SH-derived superior caval vein. CONCLUSION: Pitx2 drives left/right patterning of the SV myocardium through multiple developmental steps. Overall, Pitx2 plays a crucial functional role by negatively modulating a nodal-type programme in the left SV myocardium.
Assuntos
Padronização Corporal , Proteínas de Homeodomínio/fisiologia , Nó Sinoatrial/embriologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Nó Sinoatrial/fisiologia , Proteína Homeobox PITX2RESUMO
Individuals with 22q11 deletion syndrome (22q11DS; DiGeorge/velo-cardio-facial syndrome) have multiple congenital malformations, including cardiovascular defects. Most individuals with this syndrome possess 1.5-3.0 Mb hemizygous 22q11.2 deletions. The T-box transcription factor TBX1, lies within the nested 1.5 Mb interval and is a strong candidate for its etiology. Inactivation of Tbx1 in the mouse results in neonatal lethality owing to the presence of a single cardiac outflow tract. One important goal is to understand the molecular pathogenesis of cardiovascular defects in this syndrome. However, the molecular pathways of Tbx1 are still largely unexplored. Here, we show that Tbx1 is co-expressed with the bicoid-like homeodomain transcription factor Pitx2 in secondary heart field cells in the pharyngeal mesenchyme. In situ hybridization studies in Tbx1(-/-) mouse embryos revealed downregulation of Pitx2 in these cells. To test for a possible genetic interaction, we intercrossed Tbx1(+/-) and Pitx2(+/-) mice. Tbx1(+/-); Pitx2(+/-) mice died perinatally with cardiac defects, including double outlet right ventricle, and atrial and ventricular septal defects, all occurring with variable penetrance. An enhancer located between exons 4 and 5 in which a putative T-half site was identified near an Nkx2.5-binding site regulates asymmetric expression of Pitx2. We show using in vitro studies that Tbx1 binds to this site and activates the Pitx2 enhancer with the synergistic action of Nkx2.5. The results presented in this study unravel a novel Tbx1-Pitx2 pathway linking Tbx1 to asymmetric cardiac morphogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Coração/embriologia , Proteínas de Homeodomínio/fisiologia , Miocárdio/citologia , Miocárdio/metabolismo , Proteínas Nucleares/fisiologia , Proteínas com Domínio T/fisiologia , Animais , Elementos Facilitadores Genéticos/fisiologia , Coração/fisiologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Mutantes , Mutação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas com Domínio T/biossíntese , Proteínas com Domínio T/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Proteína Homeobox PITX2RESUMO
The 22q11 deletion syndrome (22q11DS) is characterized by abnormal development of the pharyngeal apparatus. Mouse genetic studies have identified Tbx1 as a key gene in the etiology of the syndrome, in part, via interaction with the fibroblast growth factor (Fgf) genes. Three murine Fgfs, Fgf3, Fgf8 and Fgf10 are coexpressed in different combinations with Tbx1. They are all strongly downregulated in Tbx1-/- embryos, implicating epistatic interactions. Supporting this, Tbx1 and Fgf8 have been shown to genetically interact in the development of the fourth pharyngeal arch artery (PAA) and Fgf10 was identified to be a direct downstream target of Tbx1. To dissect the epistatic relationships of these genes during embryonic development and the molecular pathogenesis of the Tbx1 mutant phenotype, we generated Fgf10+/-;Tbx1+/- and Fgf3-/-;Tbx1+/- mice. Despite strong hypotheses that Fgf10 is the key gene downstream of Tbx1 in the development of the anterior heart field, we do not find evidence for genetic interaction between Tbx1 and Fgf10. Also, the Fgf3-/-;Tbx1+/- mutant mice do not show an additive phenotype. Furthermore, more severe defects do not occur in Fgf8+/-;Tbx1+/- mutants by crossing in the Fgf3 null allele. There is a possible additive effect only in PAA remodeling in the Fgf10+/-;Tbx1+/-;Fgf8+/- embryos. Our findings underscore the importance of potential functional redundancy with additional Fgfs in the development of the pharyngeal apparatus and cardiovascular system via Tbx1. This redundancy should be considered when looking at individual FGF genes as modifiers of 22q11DS.
Assuntos
Síndrome de DiGeorge/genética , Epistasia Genética , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio T/genética , Animais , Anormalidades Craniofaciais/embriologia , Síndrome de DiGeorge/patologia , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Fator 3 de Crescimento de Fibroblastos/genética , Fator 3 de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Cardiopatias Congênitas/embriologia , Humanos , Hibridização In Situ , Camundongos , Faringe/embriologia , Fenótipo , Proteínas com Domínio T/metabolismo , Timo/anormalidades , Timo/embriologia , Glândula Tireoide/anormalidades , Glândula Tireoide/embriologiaRESUMO
Specific regions of the embryonic heart tube differentiate into atrial and ventricular chamber myocardium, whereas the inflow tract, atrioventricular canal, inner curvatures, and outflow tract do not. These regions express Tbx2, a transcriptional repressor. Here, we tested its role in chamber formation. The temporal and spatial pattern of Tbx2 mRNA and protein expression in mouse hearts was found to be complementary to that of chamber myocardium-specific genes Nppa, Cx40, Cx43, and Chisel, and was conserved in human. In vitro, Tbx2 repressed the activity of regulatory fragments of Cx40, Cx43, and Nppa. Hearts of transgenic embryos that expressed Tbx2 in the prechamber myocardium completely failed to form chambers and to express the chamber myocardium-specific genes Nppa, Cx40, and Chisel, whereas other cardiac genes were normally expressed. These findings provide the first evidence that Tbx2 is a determinant in the local repression of chamber-specific gene expression and chamber differentiation.
Assuntos
Coração/embriologia , Proteínas com Domínio T/fisiologia , Fator de Crescimento Transformador beta , Animais , Fator Natriurético Atrial , Proteína Morfogenética Óssea 2 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Conexina 43/genética , Conexinas/genética , Regulação para Baixo , Embrião de Mamíferos/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Coração/anatomia & histologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/metabolismo , Regiões Promotoras Genéticas/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteína alfa-5 de Junções ComunicantesRESUMO
During heart development, chamber myocardium forms locally from the embryonic myocardium of the tubular heart. The atrial natriuretic factor (ANF) gene is specifically expressed in this developing chamber myocardium and is one of the first hallmarks of chamber formation. We investigated the regulatory mechanism underlying this selective expression. Transgenic analysis shows that a small fragment of the ANF gene is responsible for the developmental pattern of endogenous ANF gene expression. Furthermore, this fragment is able to repress cardiac troponin I (cTnI) promoter activity selectively in the embryonic myocardium of the atrioventricular canal (AVC). In vivo inactivation of a T-box factor (TBE)- or NK2-homeobox factor binding element (NKE) within the ANF fragment removed the repression in the AVC without affecting its chamber activity. The T-box family member Tbx2, encoding a transcriptional repressor, is expressed in the embryonic myocardium in a pattern mutually exclusive to ANF, thus suggesting a role in the suppression of ANF. Tbx2 formed a complex with Nkx2.5 on the ANF TBE-NKE, and was able to repress ANF promoter activity. Our data provide a potential mechanism for chamber-restricted gene activity in which the cooperative action of Tbx2 and Nkx2.5 inhibits expression in the AVC.