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Mechanosensors are emerging players responding to hemodynamic and physical inputs. Their significance in the central nervous system remains relatively uncharted. Using human-derived brain specimens or cells and a pre-clinical model of mesio-temporal lobe epilepsy (MTLE), we examined how the mRNA levels of the mechanosensitive channel PIEZO1 adjust to disease-associated pro-inflammatory trajectories. In brain tissue micro-punches obtained from 18 drug-resistant MTLE patients, PIEZO1 expression positively correlated with pro-inflammatory biomarkers TNFα, IL-1ß, and NF-kB in the epileptogenic hippocampus compared to the adjacent amygdala and temporal cortex tissues. In an experimental MTLE model, hippocampal Piezo1 and cytokine expression levels were increased post-status epilepticus (SE) and during epileptogenesis. Piezo1 expression positively correlated with Tnfα, Il1ß, and Nf-kb in the hippocampal foci. Next, by combining RNAscope with immunohistochemistry, we identified Piezo1 in glio-vascular cells. Post-SE and during epileptogenesis, ameboid IBA1 microglia, hypertrophic GFAP astrocytes, and damaged NG2DsRed pericytes exhibited time-dependent patterns of increased Piezo1 expression. Digital droplet PCR analysis confirmed the Piezo1 trajectory in isolated hippocampal microvessels in the ipsi and contralateral hippocampi. The combined examinations performed in this model showed Piezo1 expression returning towards basal levels after the epileptogenesis-associated peak inflammation. From these associations, we next asked whether pro-inflammatory players directly regulate PIEZO1 expression. We used human-derived brain cells and confirmed that endothelium, astrocytes, and pericytes expressed PIEZO1. Exposure to human recombinant TNFα or IL1ß upregulated NF-kB in all cells. Furthermore, TNFα induced PIEZO1 expression in a dose and time-dependent manner, primarily in astrocytes. This exploratory study describes a spatiotemporal dialogue between PIEZO1 brain cell-mechanobiology and neuro-inflammatory cell remodeling. The precise functional mechanisms regulating this interplay in disease conditions warrant further investigation.
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BACKGROUND: Recent studies have shown the implication of the ROBO-SLIT pathway in heart development. Within this study, we aimed to further assess the implication of the ROBO and SLIT genes mainly in bicuspid aortic valve (BAV) and other human congenital heart defects (CHD). METHODS: We have analyzed a cohort of singleton exome sequencing data comprising 40 adult BAV patients, 20 pediatric BAV patients generated by the Pediatric Cardiac Genomics Consortium, 10 pediatric cases with tetralogy of Fallot (ToF), and one case with coarctation of the aorta. A gene-centered analysis of data was performed. To further advance the interpretation of the variants, we intended to combine more than 5 prediction tools comprising the assessment of protein structure and stability. RESULTS: A total of 24 variants were identified. Only 4 adult BAV patients (10%) had missense variants in the ROBO and SLIT genes. In contrast, 19 pediatric cases carried variants in ROBO or SLIT genes (61%). Three BAV patients with a severe phenotype were digenic. Segregation analysis was possible for two BAV patients. For the homozygous ROBO4: p.(Arg776Cys) variant, family segregation was consistent with an autosomal recessive pattern of inheritance. The ROBO4: c.3001 + 3G > A variant segregates with the affected family members. Interestingly, these variants were also found in two unrelated patients with ToF highlighting that the same variant in the ROBO4 gene may underlie different cardiac phenotypes affecting the outflow tract development. CONCLUSION: Our results further reinforce the implication of the ROBO4 gene not only in BAV but also in ToF hence the importance of its inclusion in clinical genetic testing. The remaining ROBO and SLIT genes may be screened in patients with negative or inconclusive genetic tests.
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Cardiopatias Congênitas , Tetralogia de Fallot , Adulto , Humanos , Criança , Cardiopatias Congênitas/genética , Testes Genéticos , Fenômica , CoraçãoRESUMO
The rhythmical nature of the cardiovascular system constantly generates dynamic mechanical forces. At the centre of this system is the heart, which must detect these changes and adjust its performance accordingly. Mechanoelectric feedback provides a rapid mechanism for detecting even subtle changes in the mechanical environment and transducing these signals into electrical responses, which can adjust a variety of cardiac parameters such as heart rate and contractility. However, pathological conditions can disrupt this intricate mechanosensory system and manifest as potentially life-threatening cardiac arrhythmias. Mechanosensitive ion channels are thought to be the main proponents of mechanoelectric feedback as they provide a rapid response to mechanical stimulation and can directly affect cardiac electrical activity. Here, we demonstrate that the mechanosensitive ion channel PIEZO1 is expressed in zebrafish cardiomyocytes. Furthermore, chemically prolonging PIEZO1 activation in zebrafish results in cardiac arrhythmias. indicating that this ion channel plays an important role in mechanoelectric feedback. This also raises the possibility that PIEZO1 gain of function mutations could be linked to heritable cardiac arrhythmias in humans.
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Arritmias Cardíacas , Canais Iônicos , Animais , Humanos , Arritmias Cardíacas/genética , Doença do Sistema de Condução Cardíaco , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Directional mechanoreception by hair cells is transmitted to the brain via afferent neurons to enable postural control and rheotaxis. Neuronal tuning to individual directions of mechanical flow occurs when each peripheral axon selectively synapses with multiple hair cells of identical planar polarization. How such mechanosensory labeled lines are established and maintained remains unsolved. Here, we use the zebrafish lateral line to reveal that asymmetric activity of the transcription factor Emx2 diversifies hair cell identity to instruct polarity-selective synaptogenesis. Unexpectedly, presynaptic scaffolds and coherent hair cell orientation are dispensable for synaptic selectivity, indicating that epithelial planar polarity and synaptic partner matching are separable. Moreover, regenerating axons recapitulate synapses with hair cells according to Emx2 expression but not global orientation. Our results identify a simple cellular algorithm that solves the selectivity task even in the presence of noise generated by the frequent receptor cell turnover. They also suggest that coupling connectivity patterns to cellular identity rather than polarity relaxes developmental and evolutionary constraints to innervation of organs with differing orientation.
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Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/metabolismo , Mecanotransdução Celular , Coloração e Rotulagem , Animais , Axônios/fisiologia , Polaridade Celular , Células Epiteliais/citologia , Imageamento Tridimensional , Larva/citologia , Sistema da Linha Lateral/citologia , Modelos Biológicos , Regeneração Nervosa , Neurogênese , Sinapses/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
AIMS: During embryogenesis, the onset of circulatory blood flow generates a variety of hemodynamic forces which reciprocally induce changes in cardiovascular development and performance. It has been known for some time that these forces can be detected by as yet unknown mechanosensory systems which in turn promote cardiogenic events such as outflow tract and aortic valve development. PIEZO1 is a mechanosensitive ion channel present in endothelial cells where it serves to detect hemodynamic forces making it an ideal candidate to play a role during cardiac development. We sought to determine whether PIEZO1 is required for outflow tract and aortic valve development. METHODS AND RESULTS: By analysing heart development in zebrafish we have determined that piezo1 is expressed in the developing outflow tract where it serves to detect hemodynamic forces. Consequently, disrupting Piezo1 signalling leads to defective outflow tract and aortic valve development and indicates this gene may be involved in the etiology of congenital heart diseases. Based on these findings, we analysed genomic data generated from patients who suffer from left ventricular outflow tract obstructions (LVOTO) and identified 3 probands who each harboured potentially pathogenic variants in PIEZO1. Subsequent in vitro and in vivo assays indicates that these variants behave as dominant negatives leading to an inhibition of normal PIEZO1 mechanosensory activity. Expressing these dominant negative PIEZO1 variants in zebrafish endothelium leads to defective aortic valve development. CONCLUSION: These data indicate that the mechanosensitive ion channel piezo1 is required for outflow tract and aortic valve development.
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Valva Aórtica/embriologia , Hemodinâmica , Canais Iônicos/genética , Organogênese/genética , Proteínas de Peixe-Zebra/genética , Alelos , Sequência de Aminoácidos , Animais , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Humanos , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
The sense of touch allows an organism to detect and respond to physical environmental stimuli. Mechanosensitive proteins play a crucial role in this process by converting the mechanical cue into a biological response. Recently, the Piezo family of stretch-activated ion channels has been identified as genuine mechanosensitive proteins. We set out to determine whether any of these genes are involved in touch response during zebrafish development. In situ hybridization indicates that piezo2b is specifically expressed in a subset of neurons (Rohon-Beard cells) responsible for detecting light touch. Using morpholino-mediated knockdown, we specifically targeted piezo2b and determined that it is involved in mediating touch-evoked response.
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Canais Iônicos/metabolismo , Tato , Proteínas de Peixe-Zebra/metabolismo , Animais , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Morfolinos/farmacologia , Neurônios/metabolismo , Neurônios/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
Mechanosensitivity is an inherent property of virtually all cell types, allowing them to sense and respond to physical environmental stimuli. Stretch-activated ion channels represent a class of mechanosensitive proteins which allow cells to respond rapidly to changes in membrane tension; however their identity has remained elusive. The piezo genes have recently been identified as a family of stretch-activated mechanosensitive ion channels. We set out to determine the role of piezo1 during zebrafish development. Here we report that morpholino-mediated knockdown of piezo1 impairs erythrocyte survival without affecting hematopoiesis or differentiation. Our results demonstrate that piezo1 is involved in erythrocyte volume homeostasis, disruption of which results in swelling/lysis of red blood cells and consequent anemia.
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Volume de Eritrócitos/genética , Homeostase/genética , Canais Iônicos/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/sangue , Peixe-Zebra/genética , Animais , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Canais Iônicos/metabolismo , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Spatially distributed sensory information is topographically mapped in the brain by point-to-point correspondence of connections between peripheral receptors and central target neurons. In fishes, for example, the axonal projections from the mechanosensory lateral line organize a somatotopic neural map. The lateral line provides hydrodynamic information for intricate behaviors such as navigation and prey detection. It also mediates fast startle reactions triggered by the Mauthner cell. However, it is not known how the lateralis neural map is built to subserve these contrasting behaviors. Here we reveal that birth order diversifies lateralis afferent neurons in the zebrafish. We demonstrate that early- and late-born lateralis afferents diverge along the main axes of the hindbrain to synapse with hundreds of second-order targets. However, early-born afferents projecting from primary neuromasts also assemble a separate map by converging on the lateral dendrite of the Mauthner cell, whereas projections from secondary neuromasts never make physical contact with the Mauthner cell. We also show that neuronal diversity and map topology occur normally in animals permanently deprived of mechanosensory activity. We conclude that neuronal birth order correlates with the assembly of neural submaps, whose combination is likely to govern appropriate behavioral reactions to the sensory context.
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Sistema da Linha Lateral/embriologia , Sistema da Linha Lateral/fisiologia , Neurogênese/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sistema da Linha Lateral/citologia , Mecanorreceptores/fisiologia , Dados de Sequência Molecular , Neurônios Aferentes/fisiologia , Peixe-ZebraRESUMO
BACKGROUND: Hypoxia plays an important role in many biological/pathological processes. In particular, hypoxia is associated with cardiac ischemia. which, although initially inducing a protective response, will ultimately lead to the death of cardiomyocytes and loss of tissue, severely affecting cardiac functionality. Although myocardial damage/loss remains an insurmountable problem for adult mammals, the same is not true for adult zebrafish, which are able to completely regenerate their heart after extensive injury. Myocardial regeneration in zebrafish involves the dedifferentiation and proliferation of cardiomyocytes to replace the damaged/missing tissue; at present, however, little is known about what factors regulate this process. METHODS AND RESULTS: We surmised that ventricular amputation would lead to hypoxia induction in the myocardium of zebrafish and that this may play a role in regulating the regeneration of the missing cardiac tissue. Using a combination of O(2) perturbation, conditional transgenics, in vitro cell culture, and microarray analysis, we found that hypoxia induces cardiomyocytes to dedifferentiate and proliferate during heart regeneration in zebrafish and have identified a number of genes that could play a role in this process. CONCLUSION: These results indicate that hypoxia plays a positive role during heart regeneration, which should be taken into account in future strategies aimed at inducing heart regeneration in humans.
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Coração/fisiologia , Hipóxia/fisiopatologia , Regeneração , Peixe-Zebra/fisiologia , Animais , Desdiferenciação Celular , Proliferação de Células , Células Cultivadas , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Miócitos Cardíacos/fisiologiaRESUMO
The polarity of apical stereocilia endows hair cells with directional excitability, which in turn enables animals to determine the vectorial component of a sound. Neuromasts of the lateral line of aquatic vertebrates harbor two populations of hair cells that are oriented at 180 degrees relative to each other. The resulting sensory-vectorial ambiguity is solved by lateralis afferent neurons that discriminate between hair cells of opposite polarities to innervate only those with the same orientation. How neurons select identically oriented hair cells remains unknown. To gain insight into the mechanism that underlies this selection, we devised a simple method to gather dynamic morphometric information about axonal terminals in toto by four-dimensional imaging. Applying this strategy to the zebrafish allowed us to correlate hair cell orientation to single afferent neurons at subcellular resolution. Here we show that in zebrafish with absent hair cell mechanoreception, lateralis afferents arborize profusely in the periphery, display less stability, and make improper target selections. Central axons, however, show no dynamic changes and establish normal contacts with the Mauthner cell, a characteristic second-order target in the hindbrain. We propose that the hardwired developmental mechanisms that underlie peripheral arborization and target recognition are modulated by evoked hair cell activity. This interplay between intrinsic and extrinsic cues is essential for plane-polarized target selection by lateralis afferent neurons.
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Padronização Corporal/fisiologia , Potenciais Evocados/fisiologia , Sistema da Linha Lateral/embriologia , Células Receptoras Sensoriais/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/fisiologia , Surdez/embriologia , Surdez/etiologia , Células Ciliadas Auditivas/fisiologia , Imageamento Tridimensional/métodos , Sistema da Linha Lateral/fisiologia , Mecanotransdução Celular/fisiologia , Microscopia de Fluorescência/métodos , Modelos Biológicos , Transmissão Sináptica/fisiologia , Peixe-Zebra/fisiologiaRESUMO
Myocardial damage caused, for example, by cardiac ischemia leads to ventricular volume overload resulting in increased stretch of the remaining myocardium. In adult mammals, these changes trigger an adaptive cardiomyocyte hypertrophic response which, if the damage is extensive, will ultimately lead to pathological hypertrophy and heart failure. Conversely, in response to extensive myocardial damage, cardiomyocytes in the adult zebrafish heart and neonatal mice proliferate and completely regenerate the damaged myocardium. We therefore hypothesized that in adult zebrafish, changes in mechanical loading due to myocardial damage may act as a trigger to induce cardiac regeneration. Based on this notion we sought to identify mechanosensors which could be involved in detecting changes in mechanical loading and triggering regeneration. Here we show using a combination of knockout animals, RNAseq and in vitro assays that the mechanosensitive ion channel Trpc6a is required by cardiomyocytes for successful cardiac regeneration in adult zebrafish. Furthermore, using a cyclic cell stretch assay, we have determined that Trpc6a induces the expression of components of the AP1 transcription complex in response to mechanical stretch. Our data highlights how changes in mechanical forces due to myocardial damage can be detected by mechanosensors which in turn can trigger cardiac regeneration.
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Understanding how certain animals are capable of regenerating their hearts will provide much needed insights into how this process can be induced in humans in order to reverse the damage caused by myocardial infarction. Currently, it is becoming increasingly evident that cardiac interstitial cells play crucial roles during cardiac regeneration. To understand how interstitial cells behave during this process, we performed single-cell RNA sequencing of regenerating zebrafish hearts. Using a combination of immunohistochemistry, chemical inhibition, and novel transgenic animals, we were able to investigate the role of cell type-specific mechanisms during cardiac regeneration. This approach allowed us to identify a number of important regenerative processes within the interstitial cell populations. Here, we provide detailed insight into how interstitial cells behave during cardiac regeneration, which will serve to increase our understanding of how this process could eventually be induced in humans.
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Infarto do Miocárdio , Miócitos Cardíacos , Animais , Humanos , Peixe-Zebra , Animais Geneticamente Modificados , Proliferação de CélulasRESUMO
Bicuspid aortic valve (BAV), the most common cardiovascular malformation occurs in 0.5-1.2% of the population. Although highly heritable, few causal mutations have been identified in BAV patients. Here, we report the targeted sequencing of HOXA1 in a cohort of BAV patients and the identification of rare indel variants in the homopolymeric histidine tract of HOXA1. In vitro analysis shows that disruption of this motif leads to a significant reduction in protein half-life and defective transcriptional activity of HOXA1. In zebrafish, targeting hoxa1a ortholog results in aortic valve defects. In vivo assays indicates that these variants behave as dominant negatives leading abnormal valve development. In mice, deletion of Hoxa1 leads to BAV with a very small, rudimentary non-coronary leaflet. We also show that 17% of homozygous Hoxa1-1His knock-in mice present similar phenotype. Genetic lineage tracing in Hoxa1-/- mutant mice reveals an abnormal reduction of neural crest-derived cells in the valve leaflet, which is caused by a failure of early migration of these cells.
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Doença da Válvula Aórtica Bicúspide , Doenças das Valvas Cardíacas , Proteínas de Homeodomínio , Animais , Camundongos , Valva Aórtica/anormalidades , Doença da Válvula Aórtica Bicúspide/metabolismo , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/metabolismo , Histidina/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Homeodomínio/genéticaAssuntos
Anemia Hemolítica Congênita/genética , Peixe-Zebra , Anemia , Animais , Homozigoto , HumanosRESUMO
Fishes and amphibians localize hydromechanical variations along their bodies using the lateral-line sensory system. This is possible because the spatial distribution of neuromasts is represented in the hindbrain by a somatotopic organization of the lateralis afferent neurons' central projections. The mechanisms that establish lateralis somatotopy are not known. Using BAPTI and neuronal tracing in the zebrafish, we demonstrate growth anisotropy of the posterior lateralis ganglion. We characterized a new transgenic line for in vivo imaging to show that although peripheral growth-cone structure adumbrates somatotopy, the order of neurogenesis represents a more accurate predictor of the position of a neuron's central axon along the somatotopic axis in the hindbrain. We conclude that progressive neurogenesis defines lateralis somatotopy.
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Neurogênese/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Gânglios Sensitivos/citologia , Gânglios Sensitivos/metabolismo , Neurogênese/genética , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Peixe-Zebra/genéticaRESUMO
The presence of glyphosate represents a debated ecotoxicological and health risk factor. Here, zebrafish larvae were exposed, from 1.5 to 120 h post-fertilization, to a broad concentration range (0.05-10.000 µg/L) of glyphosate to explore its impact on the brain. We evaluated morphology, tracked locomotor behavior and neurophysiological parameters, examined neuro-glio-vascular cell structures, and outlined transcriptomic outcomes by RNA sequencing. At the concentration range tested, glyphosate did not elicit gross morphological changes. Behavioral analysis revealed a significant decrease in locomotor activity following the exposure to 1000 µg/L glyphosate or higher. In parallel, midbrain electrophysiological recordings indicated abnormal, and variable, spike activity in zebrafish larvae exposed to 1000 µg/L glyphosate. Next, we asked whether the observed neurophysiological outcome could be secondary to brain structural modifications. We used transgenic zebrafish and in vivo 2-photon microscopy to examine, at the cellular level, the effects of the behavior-modifying concentration of 1000 µg/L, comparing to low 0.1 µg/L, and control. We ruled out the presence of cerebrovascular and neuronal malformations. However, microglia morphological modifications were visible at the two glyphosate concentrations, specifically the presence of amoeboid cells suggestive of activation. Lastly, RNAseq analysis showed the deregulation of transcript families implicated in neuronal physiology, synaptic transmission, and inflammation, as evaluated at the two selected glyphosate concentrations. In zebrafish larvae, behavioral and neurophysiological defects occur after the exposure to high glyphosate concentrations while cellular and transcript signatures can be detected in response to low dose. The prospective applicability to ecotoxicology and the possible extension to brain-health vulnerability are critically discussed.
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Herbicidas , Peixe-Zebra , Animais , Glicina/análogos & derivados , Herbicidas/toxicidade , Humanos , Larva/genética , Estudos Prospectivos , Peixe-Zebra/genética , GlifosatoRESUMO
Epidemiological indications connect maternal and developmental presence or exposure to pesticides with an increased risk for a spectrum of neurological trajectories. To provide pre-clinical data in support of this hypothesis, we used two distinct experimental models. First, female and male mice were fed immediately prior to mating, and the resulting pregnant dams were continously fed during gestation and lactation periods using chow pellets containing a cocktail of six pesticides at tolerable daily intake levels. Male and female offspring were then tracked for behavioral and in vivo electrophysiological adaptations. Second, a zebrafish model allowed us to screen toxicity and motor-behavior outcomes specifically associated with the developmental exposure to a low-to-high concentration range of the cocktail and of each individual pesticide. Here, we report anxiety-like behavior in aging male mice maternally exposed to the cocktail, as compared to age and gender matched sham animals. In parallel, in vivo electrocorticography revealed a decrease in gamma (40-80 Hz) and an increase of theta (6-9 Hz) waves, delineating a long-term, age-dependent, neuronal slowing. Neurological changes were not accompanied by brain structural malformations. Next, by using zebrafish larvae, we showed an increase of all motor-behavioral parameters resulting from the developmental exposure to 10 µg/L of pesticide cocktail, an outcome that was not associated with midbrain structural or neurovascular modifications as assessed by in vivo 2-photon microscopy. When screening each pesticide, chlorpyrifos elicited modifications of swimming parameters at 0.1 µg/L, while other components provoked changes from 0.5 µg/L. Ziram was the single most toxic component inducing developmental malformations and mortality at 10 µg/L. Although we have employed non-equivalent modalities and timing of exposure in two dissimilar experimental models, these outcomes indicate that presence of a pesticide cocktail during perinatal periods represents an element promoting behavioral and neurophysiological modifications. The study limitations and the possible pertinence of our findings to ecotoxicology and public health are critically discussed.
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Clorpirifos , Praguicidas , Animais , Feminino , Larva , Masculino , Camundongos , Nível de Efeito Adverso não Observado , Praguicidas/toxicidade , Peixe-ZebraRESUMO
The transient receptor potential Melastatin 4 (TRPM4) channel is a calcium-activated non-selective cation channel expressed widely. In the heart, using a knock-out mouse model, the TRPM4 channel has been shown to be involved in multiple processes, including ß-adrenergic regulation, cardiac conduction, action potential duration and hypertrophic adaptations. This channel was recently shown to be involved in stress-induced cardiac arrhythmias in a mouse model overexpressing TRPM4 in ventricular cardiomyocytes. However, the link between TRPM4 channel expression in ventricular cardiomyocytes, the hypertrophic response to stress and/or cellular arrhythmias has yet to be elucidated. In this present study, we induced pathological hypertrophy in response to myocardial infarction using a mouse model of Trpm4 gene invalidation, and demonstrate that TRPM4 is essential for survival. We also demonstrate that the TRPM4 is required to activate both the Akt and Calcineurin pathways. Finally, using two hypertrophy models, either a physiological response to endurance training or a pathological response to myocardial infarction, we show that TRPM4 plays a role in regulating transient calcium amplitudes and leads to the development of cellular arrhythmias potentially in cooperation with the Sodium-calcium exchange (NCX). Here, we report two functions of the TRPM4 channel: first its role in adaptive hypertrophy, and second its association with NCX could mediate transient calcium amplitudes which trigger cellular arrhythmias.
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Ventrículos do Coração/metabolismo , Hipertrofia/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Fenômenos Biomecânicos/fisiologia , Calcineurina/metabolismo , Cálcio/metabolismo , Ecocardiografia , Eletrocardiografia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Sódio/metabolismoRESUMO
BACKGROUND: Severe ventricular rhythm disturbances are the hallmark of arrhythmogenic cardiomyopathy (ACM), and are often explained by structural conduction abnormalities. However, comprehensive investigations of ACM cell electrical instability are lacking. This study aimed to elucidate early electrical myogenic signature of ACM. METHODS: We investigated a 41-year-old ACM patient with a missense mutation (c.394C>T) in the DSC2 gene, which encodes desmocollin 2. Pathogenicity of this variant was confirmed using a zebrafish DSC2 model system. Control and DSC2 patient-derived pluripotent stem cells were reprogrammed and differentiated into cardiomyocytes (hiPSC-CM) to examine the specific electromechanical phenotype and its modulation by antiarrhythmic drugs (AADs). Samples of the patient's heart and hiPSC-CM were examined to identify molecular and cellular alterations. RESULTS: A shortened action potential duration was associated with reduced Ca2+ current density and increased K+ current density. This finding led to the elucidation of previously unknown abnormal repolarization dynamics in ACM patients. Moreover, the Ca2+ mobilised during transients was decreased, and the Ca2+ sparks frequency was increased. AAD testing revealed the following: (1) flecainide normalised Ca2+ transients and significantly decreased Ca2+ spark occurrence and (2) sotalol significantly lengthened the action potential and normalised the cells' contractile properties. CONCLUSIONS: Thorough analysis of hiPSC-CM derived from the DSC2 patient revealed abnormal repolarization dynamics, prompting the discovery of a short QT interval in some ACM patients. Overall, these results confirm a myogenic origin of ACM electrical instability and provide a rationale for prescribing class 1 and 3 AADs in ACM patients with increased ventricular repolarization reserve.
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Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/genética , Desmocolinas/genética , Eletrocardiografia/métodos , Canais Iônicos/genética , Adulto , Animais , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Mutação de Sentido Incorreto/genética , Peixe-ZebraRESUMO
Venom peptides are promising drug leads, but their therapeutic use is often limited by stability and bioavailability issues. In this study, we designed cyclic analogues of α-conotoxin CIA, a potent muscle nicotinic acetylcholine receptor (nAChR) blocker with a significantly lower affinity at the neuronal α3ß2 subtype. Remarkably, all analogues retained the low nanomolar activity of native CIA toward muscle-type nAChRs but showed greatly improved resistance to degradation in human serum and, surprisingly, displayed up to 52-fold higher potency for the α3ß2 neuronal nAChR subtype (IC50 1.3 nM). Comparison of nuclear magnetic resonance-derived structures revealed some differences that might explain the gain of potency at α3ß2 nAChRs. All peptides were highly paralytic when injected into adult zebrafish and bath-applied to zebrafish larvae, suggesting barrier-crossing capabilities and efficient uptake. Finally, these cyclic CIA analogues were shown to be unique pharmacological tools to investigate the contribution of the presynaptic α3ß2 nAChR subtype to the train-of-four fade.