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BACKGROUND: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. METHODS: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. RESULTS: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. CONCLUSIONS: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.
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Proteínas Co-Represoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Animales , Calcio/metabolismo , Cardiomegalia/metabolismo , Cardiomiopatía Hipertrófica/metabolismo , Humanos , Ratas , VerapamiloRESUMEN
Rapid and cost-effective in vivo assays to screen potential environmental neurodevelopmental toxicants are necessary to address the limitations of in vitro platforms, such as the inability to fully recapitulate the developmental and physiological processes of whole organisms. In the present study, a rapid zebrafish behavioral profiling assay was developed to characterize the neurodevelopmental effects of environmental substances by quantitatively evaluating multiple spontaneous movement features of zebrafish embryos. This video analysis-based assay automatically segmented every embryo and thus was able to accurately quantify spontaneous movement features, including frequency, duration, intensity, interval, and the number of continuous movements. When tested with eight environmental substances known to be neurodevelopmental toxicants, such as chlorpyrifos and bisphenol A, the assay successfully captured frequency alterations that were well-documented in previous studies while also providing additional information. Using an optimized procedure, we further assessed 132 potential neurotoxins that spanned a wide range of molecular targets, many of which were previously detected in environmental waterbodies. The distinct altered behavioral barcodes indicated that the spontaneous movement was impacted by diverse neuroactive substances, and the effects could be effectively evaluated with the developed assay. The web-based tool, named EMAnalysis, is further provided at http://www.envh.sjtu.edu.cn/zebrafish_contraction.jsp. Thus, this assay provides an efficient platform to accelerate the pace of neurotoxic environmental contaminant discoveries.
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Cloropirifos , Pez Cebra , Animales , Bioensayo , Cloropirifos/toxicidad , Embrión no Mamífero , NeurotoxinasRESUMEN
The non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen and diclofenac are highly prescribed worldwide and their presence in aquatic system may pose a potential risk to aquatic organisms. Here, we systematically assessed their cardiovascular disruptive effects in zebrafish (Danio rerio) at environmentally relevant concentrations between 0.04 and 25.0⯵g/L. Ibuprofen significantly increased the cardiac outputs of zebrafish embryos at actual concentrations of 0.91, 4.3 and 21.9⯵g/L. It up-regulated the blood cell velocity, total blood flow and down-regulated the blood cell density at concentrations of 4.3⯵g/L and higher. In comparison, diclofenac led to inhibition of spontaneous muscle contractions and decreased hatching rate of zebrafish embryos at the highest concentration (24.1⯵g/L), while it had negligible effects on the cardiac physiology and hemodynamics. Transcriptional analysis of biomarker genes involved in cardiovascular physiology, such as the significantly up-regulated nppa and nkx2.5 expressions response to ibuprofen but not to diclofenac, is consistent with these observations. In addition, both ibuprofen and diclofenac altered the morphology of intersegmental vessels at high concentrations. Our results revealed unexpected cardiovascular functional alterations of NSAIDs to fish at environmental or slightly higher than surface water concentrations and thus provided novel insights into the understanding of their potential environmental risks.
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Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/toxicidad , Diclofenaco/administración & dosificación , Diclofenaco/toxicidad , Ibuprofeno/administración & dosificación , Ibuprofeno/toxicidad , Organogénesis/efectos de los fármacos , Contaminantes Químicos del Agua/efectos adversos , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Animales , Embrión no Mamífero/efectos de los fármacosRESUMEN
BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS: Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human MYL4 mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafish Myl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.
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Fibrilación Atrial , Mutación , Miocitos Cardíacos , Cadenas Ligeras de Miosina , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Línea Celular , Conexina 43/genética , Conexina 43/metabolismo , Técnicas de Inactivación de Genes , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.
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Canales de Calcio Tipo L/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Proteínas de Unión al ADN/genética , Corazón/embriología , Humanos , Proteínas de la Membrana/genética , Miocitos Cardíacos/enzimología , Ubiquitina-Proteína Ligasas Nedd4/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conejos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitinación , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
NKX2-5 is the most commonly mutated gene associated with human congenital heart defects (CHDs), with a predilection for cardiac pole abnormalities. This homeodomain transcription factor is a central regulator of cardiac development and is expressed in both the first and second heart fields (FHF and SHF). We have previously revealed essential functions of nkx2.5 and nkx2.7, two Nkx2-5 homologs expressed in zebrafish cardiomyocytes, in maintaining ventricular identity. However, the differential roles of these genes in the specific subpopulations of the anterior (aSHF) and posterior (pSHF) SHFs have yet to be fully defined. Here, we show that Nkx genes regulate aSHF and pSHF progenitors through independent mechanisms. We demonstrate that Nkx genes restrict proliferation of aSHF progenitors in the outflow tract, delimit the number of pSHF progenitors at the venous pole and pattern the sinoatrial node acting through Isl1 repression. Moreover, optical mapping highlights the requirement for Nkx gene dose in establishing electrophysiological chamber identity and in integrating the physiological connectivity of FHF and SHF cardiomyocytes. Ultimately, our results may shed light on the discrete errors responsible for NKX2-5-dependent human CHDs of the cardiac outflow and inflow tracts.
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Corazón/embriología , Proteína Homeótica Nkx-2.5/genética , Proteínas de Homeodominio/genética , Proteínas con Homeodominio LIM/genética , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/metabolismo , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Diferenciación Celular , Proliferación Celular , Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Humanos , MutaciónRESUMEN
Human cardiac myocytes derived from pluripotent stem cells (hCM) have invigorated interest in genetic disease mechanisms and cardiac safety testing; however, the technology to fully assess electrophysiological function in an assay that is amenable to high throughput screening has lagged. We describe a fully contactless system using optical pacing with an infrared (IR) laser and multi-site high fidelity fluorescence imaging to assess multiple electrophysiological parameters from hCM monolayers in a standard 96-well plate. Simultaneous multi-site action potentials (FluoVolt) or Ca2+ transients (Fluo4-AM) were measured, from which high resolution maps of conduction velocity and action potential duration (APD) were obtained in a single well. Energy thresholds for optical pacing were determined for cell plating density, laser spot size, pulse width, and wavelength and found to be within ranges reported previously for reliable pacing. Action potentials measured using FluoVolt and a microelectrode exhibited the same morphology and rate of depolarization. Importantly, we show that this can be achieved accurately with minimal damage to hCM due to optical pacing or fluorescence excitation. Finally, using this assay we demonstrate that hCM exhibit reproducible changes in repolarization and impulse conduction velocity for Flecainide and Quinidine, two well described reference compounds. In conclusion, we demonstrate a high fidelity electrophysiological screening assay that incorporates optical pacing with IR light to control beating rate of hCM monolayers.
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Rayos Infrarrojos , Miocitos Cardíacos/fisiología , Electrofisiología Cardíaca , Células Cultivadas , Humanos , Microelectrodos , Microscopía Fluorescente , Óptica y Fotónica , Técnicas de Placa-ClampRESUMEN
Mechanoelectrical feedback may increase arrhythmia susceptibility, but the molecular mechanisms are incompletely understood. This study showed that mechanical stretch altered the localization, protein levels, and function of the cation-selective transient receptor potential channel (TRPC)-6 in atrial endocardial cells in humans, pigs, and mice. In endocardial/myocardial cross-talk studies, addition of media from porcine atrial endocardium (AE) cells altered the calcium (Ca2+) transient characteristics of human-induced pluripotent stem cell-derived cardiomyocytes. These changes did not occur with media from stretched AE cells. Our data suggested that endocardial TRPC-6-dependent paracrine signaling may modulate myocardial Ca2+ homeostasis under basal conditions and protect against stretch-induced atrial arrhythmias.
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Connexin43 (Cx43) phosphorylation alters gap junction localization and function. In particular, phosphorylation at serine-368 (S368) has been suggested to alter gap junctional conductance, but previous reports have shown inconsistent results for both timing and functional effects of S368 phosphorylation. The objective of this study was to determine the functional effects of isolated S368 phosphorylation. We evaluated wild-type Cx43 (AdCx43) and mutations simulating permanent phosphorylation (Ad368E) or preventing phosphorylation (Ad368A) at S368. Function was assessed by optical mapping of electrical conduction in patterned cultures of neonatal rat ventricular myocytes, under baseline and metabolic stress (MS) conditions. Baseline conduction velocity (CV) was similar for all groups. In the AdCx43 and Ad368E groups, MS moderately decreased CV. Ad368A caused complete conduction block during MS. Triton-X solubility assessment showed no change in Cx43 location during conduction impairment. Western blot analysis showed that Cx43-S368 phosphorylation was present at baseline, and that it decreased during MS. Our data indicate that phosphorylation at S368 does not affect CV under baseline conditions, and that preventing S368 phosphorylation makes Cx43 hypersensitive to MS. These results show the critical role of S368 phosphorylation during stress conditions.
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Conexina 43/metabolismo , Miocitos Cardíacos/metabolismo , Estrés Fisiológico , Potenciales de Acción , Animales , Animales Recién Nacidos , Células Cultivadas , Conexina 43/genética , Mutación , Fosforilación , Ratas Sprague-Dawley , Serina , Transducción de Señal , Factores de Tiempo , Transfección , Imagen de Colorante Sensible al VoltajeRESUMEN
The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction.
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Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Cadherinas/genética , Cadherinas/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Proteínas de Unión a TGF-beta Latente/genética , Proteínas de Unión a TGF-beta Latente/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Elementos Reguladores de la Transcripción/genética , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Cardiac conduction maturation is an important and integral component of heart development. Optical mapping with voltage-sensitive dyes allows sensitive measurements of electrophysiological signals over the entire heart. However, accurate measurements of conduction velocity during early cardiac development is typically hindered by low signal-to-noise ratio (SNR) measurements of action potentials. Here, we present a novel image processing approach based on least squares optimizations, which enables high-resolution, low-noise conduction velocity mapping of smaller tubular hearts. First, the action potential trace measured at each pixel is fit to a curve consisting of two cumulative normal distribution functions. Then, the activation time at each pixel is determined based on the fit, and the spatial gradient of activation time is determined with a two-dimensional (2D) linear fit over a square-shaped window. The size of the window is adaptively enlarged until the gradients can be determined within a preset precision. Finally, the conduction velocity is calculated based on the activation time gradient, and further corrected for three-dimensional (3D) geometry that can be obtained by optical coherence tomography (OCT). We validated the approach using published activation potential traces based on computer simulations. We further validated the method by adding artificially generated noise to the signal to simulate various SNR conditions using a curved simulated image (digital phantom) that resembles a tubular heart. This method proved to be robust, even at very low SNR conditions (SNR = 2-5). We also established an empirical equation to estimate the maximum conduction velocity that can be accurately measured under different conditions (e.g. sampling rate, SNR, and pixel size). Finally, we demonstrated high-resolution conduction velocity maps of the quail embryonic heart at a looping stage of development.
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Two recent studies (Newton-Cheh, C. et al. (2009) Common variants at ten loci influence QT interval duration in the QTGEN Study. Nat. Genet. 41, 399-406 and Pfeufer, A. et al. (2009) Common variants at ten loci modulate the QT interval duration in the QTSCD Study. Nat. Genet. 41, 407-414) identified an association, with genome-wide significance, between a single nucleotide polymorphism within the gene encoding RING finger protein 207 (RNF207) and the QT interval. We sought to determine the role of RNF207 in cardiac electrophysiology. Morpholino knockdown of RNF207 in zebrafish embryos resulted in action potential duration prolongation, occasionally a 2:1 atrioventricular block, and slowing of conduction velocity. Conversely, neonatal rabbit cardiomyocytes infected with RNF207-expressing adenovirus exhibited shortened action potential duration. Using transfections of U-2 OS and HEK293 cells, Western blot analysis and immunocytochemistry data demonstrate that RNF207 and the human ether-a-go-go-related gene (HERG) potassium channel interact and colocalize. Furthermore, RNF207 overexpression significantly elevated total and membrane HERG protein and HERG-encoded current density by â¼30-50%, which was dependent on the intact N-terminal RING domain of RNF207. Finally, coexpression of RNF207 and HSP70 increased HERG expression compared with HSP70 alone. This effect was dependent on the C terminus of RNF207. Taken together, the evidence is strong that RNF207 is an important regulator of action potential duration, likely via effects on HERG trafficking and localization in a heat shock protein-dependent manner.
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Bloqueo Atrioventricular/genética , Canales de Potasio Éter-A-Go-Go/genética , Proteínas HSP70 de Choque Térmico/genética , Corazón/fisiología , Miocitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Potenciales de Acción/genética , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Bloqueo Atrioventricular/metabolismo , Bloqueo Atrioventricular/fisiopatología , Canal de Potasio ERG1 , Embrión no Mamífero , Canales de Potasio Éter-A-Go-Go/metabolismo , Acoplamiento Excitación-Contracción , Regulación de la Expresión Génica , Vectores Genéticos , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Corazón/embriología , Corazón/fisiopatología , Humanos , Datos de Secuencia Molecular , Morfolinos , Miocitos Cardíacos/patología , Estructura Terciaria de Proteína , Conejos , Ubiquitina-Proteína Ligasas/metabolismo , Pez CebraRESUMEN
Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM.
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Cardiomiopatía Dilatada/genética , Trastornos de los Cromosomas/genética , Mapeo Cromosómico/métodos , Proteínas de Unión al ADN/genética , No Compactación Aislada del Miocardio Ventricular/genética , Factores de Transcripción/genética , Animales , Cardiomiopatía Dilatada/patología , Estudios de Casos y Controles , Proliferación Celular , Deleción Cromosómica , Cromosomas Humanos Par 1/genética , Hibridación Genómica Comparativa , Exones , Mutación del Sistema de Lectura , Humanos , Mutación Missense , Contracción Miocárdica , Miocitos Cardíacos , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Altered mechanical loading of the heart leads to hypertrophy, decompensated heart failure and fatal arrhythmias. However, the molecular mechanisms that link mechanical and electrical dysfunction remain poorly understood. Growing evidence suggest that ventricular electrical remodeling (VER) is a process that can be induced by altered mechanical stress, creating persistent electrophysiological changes that predispose the heart to life-threatening arrhythmias. While VER is clearly a physiological property of the human heart, as evidenced by "T wave memory", it is also thought to occur in a variety of pathological states associated with altered ventricular activation such as bundle branch block, myocardial infarction, and cardiac pacing. Animal models that are currently being used for investigating stretch-induced VER have significant limitations. The zebrafish has recently emerged as an attractive animal model for studying cardiovascular disease and could overcome some of these limitations. Owing to its extensively sequenced genome, high conservation of gene function, and the comprehensive genetic resources that are available in this model, the zebrafish may provide new insights into the molecular mechanisms that drive detrimental electrical remodeling in response to stretch. Here, we have established a zebrafish model to study mechano-electrical feedback in the heart, which combines efficient genetic manipulation with high-precision stretch and high-resolution electrophysiology. In this model, only 90 min of ventricular stretch caused VER and recapitulated key features of VER found previously in the mammalian heart. Our data suggest that the zebrafish model is a powerful platform for investigating the molecular mechanisms underlying mechano-electrical feedback and VER in the heart.
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Fenómenos Electrofisiológicos , Retroalimentación Fisiológica , Corazón/fisiología , Fenómenos Mecánicos , Modelos Animales , Pez Cebra/fisiología , Animales , Fenómenos Biomecánicos , Embrión no Mamífero/fisiología , Humanos , Factores de Tiempo , Remodelación VentricularRESUMEN
The heart is a muscular organ with a wrapping, laminar structure embedded with neural and vascular networks, collagen fibrils, fibroblasts, and cardiac myocytes that facilitate contraction. We hypothesized that these non-muscle components may have functional benefit, serving as important structural alignment cues in inter- and intra-cellular organization of cardiac myocytes. Previous studies have demonstrated that alignment of engineered myocardium enhances calcium handling, but how this impacts actual force generation remains unclear. Quantitative assays are needed to determine the effect of alignment on contractile function and muscle physiology. To test this, micropatterned surfaces were used to build 2-dimensional myocardium from neonatal rat ventricular myocytes with distinct architectures: confluent isotropic (serving as the unaligned control), confluent anisotropic, and 20 µm spaced, parallel arrays of multicellular myocardial fibers. We combined image analysis of sarcomere orientation with muscular thin film contractile force assays in order to calculate the peak sarcomere-generated stress as a function of tissue architecture. Here we report that increasing peak systolic stress in engineered cardiac tissues corresponds with increasing sarcomere alignment. This change is larger than would be anticipated from enhanced calcium handling and increased uniaxial alignment alone. These results suggest that boundary conditions (heterogeneities) encoded in the extracellular space can regulate muscle tissue function, and that structural organization and cytoskeletal alignment are critically important for maximizing peak force generation.
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Corazón/fisiología , Contracción Miocárdica , Miocitos Cardíacos/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Calcio/metabolismo , Células Cultivadas , Dimetilpolisiloxanos/química , Fibronectinas/química , Ratas , Ratas Sprague-Dawley , Sarcómeros/fisiología , Sarcómeros/ultraestructuraRESUMEN
Natural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.
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Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Corazón/fisiología , Miocitos Cardíacos/citología , Regeneración , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Muerte CelularRESUMEN
The vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca(2+) handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca(2+) handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4)P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca(2+) handling in the heart through its interaction with phosphatidylinositols.
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Proteínas Musculares/química , Proteínas Musculares/metabolismo , Contracción Miocárdica/fisiología , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Tipificación del Cuerpo , Bradicardia/embriología , Bradicardia/fisiopatología , Señalización del Calcio , Gasto Cardíaco , Recuento de Células , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Corazón/embriología , Corazón/fisiología , Humanos , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Organogénesis , Unión Proteica , Estructura Terciaria de Proteína , Fracciones Subcelulares/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Changes in myocyte cell shape and tissue structure are concurrent with changes in electromechanical function in both the developing and diseased heart. While the anisotropic architecture of cardiac tissue is known to influence the propagation of the action potential, the influence of tissue architecture and its potential role in regulating excitation-contraction coupling (ECC) are less well defined. We hypothesized that changes in the shape and the orientation of cardiac myocytes induced by spatial arrangement of the extracellular matrix (ECM) affects ECC. To test this hypothesis, we isolated and cultured neonatal rat ventricular cardiac myocytes on various micropatterns of fibronectin where they self-organized into tissues with varying degrees of anisotropy. We then measured the morphological features of these engineered myocardial tissues across several hierarchical dimensions by measuring cellular aspect ratio, myocyte area, nuclear density and the degree of cytoskeletal F-actin alignment. We found that when compared with isotropic tissues, anisotropic tissues have increased cellular aspect ratios, increased nuclear densities, decreased myocyte cell areas and smaller variances in actin alignment. To understand how tissue architecture influences cardiac function, we studied the role of anisotropy on intracellular calcium ([Ca(2+)](i)) dynamics by characterizing the [Ca(2+)](i)-frequency relationship of electrically paced tissues. When compared with isotropic tissues, anisotropic tissues displayed significant differences in [Ca(2+)](i) transients, decreased diastolic baseline [Ca(2+)](i) levels and greater [Ca(2+)](i) influx per cardiac cycle. These results suggest that ECM cues influence tissue structure at cellular and subcellular levels and regulate ECC.
Asunto(s)
Calcio/metabolismo , Miocardio/metabolismo , Animales , Forma de la Célula , Acoplamiento Excitación-Contracción , Contracción Miocárdica , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , RatasRESUMEN
To assess the effects during cardiac development of mutations that cause human cardiomyopathy, we modeled a sarcomeric gene mutation in the embryonic zebrafish. We designed morpholino antisense oligonucleotides targeting the exon 13 splice donor site in the zebrafish cardiac troponin T (tnnt2) gene, in order to precisely recapitulate a human TNNT2 mutation that causes hypertrophic cardiomyopathy (HCM). HCM is a disease characterized by myocardial hypertrophy, myocyte and myofibrillar disarray, as well as an increased risk of sudden death. Similar to humans with HCM, the morphant zebrafish embryos displayed sarcomere disarray and there was a robust induction of myocardial hypertrophic pathways. Microarray analysis uncovered a number of shared transcriptional responses between this zebrafish model and a well-characterized mouse model of HCM. However, in contrast to adult hearts, these embryonic hearts developed cardiomyocyte hyperplasia in response to this genetic perturbation. The re-creation of a human disease-causing TNNT2 splice variant demonstrates that sarcomeric mutations can alter cardiomyocyte biology at the earliest stages of heart development with distinct effects from those observed in adult hearts despite shared transcriptional responses.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Corazón/embriología , Mutación/genética , Miocitos Cardíacos/patología , Organogénesis , Pez Cebra/embriología , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Cardiomiopatía Hipertrófica/complicaciones , Cardiomiopatía Hipertrófica/fisiopatología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Embrión no Mamífero/patología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Corazón/efectos de los fármacos , Corazón/fisiopatología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Hiperplasia/complicaciones , Hiperplasia/patología , Ratones , Datos de Secuencia Molecular , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Oligonucleótidos Antisentido/farmacología , Organogénesis/efectos de los fármacos , Sarcómeros/efectos de los fármacos , Sarcómeros/patología , Transcripción Genética/efectos de los fármacos , Troponina T/química , Troponina T/genética , Pez Cebra/genética , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Electrical gradients are critical for many biological processes, including the normal function of excitable tissues, left-right patterning, organogenesis and wound healing. The fundamental mechanisms that regulate the establishment and maintenance of such electrical polarities are poorly understood. Here we identify a gradient of electrical coupling across the developing ventricular myocardium using high-speed optical mapping of transmembrane potentials and calcium concentrations in the zebrafish heart. We excluded a role for differences in cellular excitability, connexin localization, tissue geometry and mechanical inputs, but in contrast we were able to demonstrate that non-canonical Wnt11 signals are required for the genesis of this myocardial electrical gradient. Although the traditional planar cell polarity pathway is not involved, we obtained evidence that Wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane Ca(2+) conductance mediated by the L-type calcium channel. These data reveal a previously unrecognized role for Wnt/Ca(2+) signalling in establishing an electrical gradient in the plane of the developing cardiac epithelium through modulation of ion-channel function. The regulation of cellular coupling through such mechanisms may be a general property of non-canonical Wnt signals.