RESUMO
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and ß-catenin). We detected ES-induced CM maturation as indicated by increased Tnni1 and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Biomarcadores/metabolismo , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Conexinas/genética , Conexinas/metabolismo , Contactina 2/genética , Contactina 2/metabolismo , Estimulação Elétrica , Expressão Gênica , Sistema de Condução Cardíaco/citologia , Sistema de Condução Cardíaco/fisiologia , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Cultura Primária de Células , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Troponina I/genética , Troponina I/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteína alfa-5 de Junções ComunicantesRESUMO
The specialised conducting tissues present in the ventricles are responsible for the fast distribution of the electrical impulse from the atrio-ventricular node to regions in the subendocardial myocardium. Characterisation of anatomical features of the specialised conducting tissues in the ventricles is highly challenging, in particular its most distal section, which is connected to the working myocardium via Purkinje-myocardial junctions. The goal of this work is to characterise the architecture of the distal section of the Purkinje network by differentiating Purkinje cells from surrounding tissue, performing a segmentation of Purkinje fibres at cellular scale, and mathematically describing its morphology and interconnections. Purkinje cells from rabbit hearts were visualised by confocal microscopy using wheat germ agglutinin labelling. A total of 16 3D stacks including labeled Purkinje cells were collected, and semi-automatically segmented. State-of-the-art graph metrics were applied to estimate regional and global features of the Purkinje network complexity. Two types of cell types, tubular and star-like, were characterised from 3D segmentations. The analysis of 3D imaging data confirms the previously suggested presence of two types of Purkinje-myocardium connections, a 2D interconnection sheet and a funnel one, in which the narrow side of a Purkinje fibre connect progressively to muscle fibres. The complex network analysis of interconnected Purkinje cells showed no small-world connectivity or assortativity properties. These results might help building more realistic computational PK systems at high resolution levels including different cell configurations and shapes. Better knowledge on the organisation of the network might help in understanding the effects that several treatments such as radio-frequency ablation might have when the PK system is disrupted locally.
Assuntos
Sistema de Condução Cardíaco/anatomia & histologia , Sistema de Condução Cardíaco/citologia , Ventrículos do Coração/anatomia & histologia , Ventrículos do Coração/citologia , Animais , Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Miocárdio/citologia , Células de Purkinje/citologia , CoelhosRESUMO
The presence of connective tissue as well as interstitial clefts forms a natural barrier to the electrical propagation in the heart. At a microscopic scale, such uncoupling structures change the pattern of the electrical conduction from uniform towards complex and may play a role in the genesis of cardiac arrhythmias. The anatomical diversity of conduction structures and their topology at a microscopic size scale is overwhelming for experimental techniques. Mathematical models have been often employed to study the behavior of the electrical propagation at a sub-cellular level. However, very fine and computationally expensive meshes are required to capture all microscopic details found in the cardiac tissue. In this work, we present a numerical technique based on the finite element method which allows to reproduce the effects of microscopic conduction barriers caused by the presence of uncoupling structures without actually resolving these structures in a high resolution mesh, thereby reducing the computational costs significantly.
Assuntos
Potenciais de Ação/fisiologia , Sistema de Condução Cardíaco/citologia , Sistema de Condução Cardíaco/fisiologia , Modelos Anatômicos , Modelos Cardiovasculares , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Animais , Simulação por Computador , Análise de Elementos Finitos , HumanosRESUMO
Evidence has shown that the sarcoplasmic reticulum (SR) of cardiac cells releases Ca not only during excitation-contraction coupling but also during diastole, albeit at a much lower rate. This diastolic SR Ca release (leak) has also been implicated in the generation of spontaneous depolarization in latent atrial pacemaker cells of the cat right atrium. In the present work, we sought to measure Ca transients in pacemaker and nonpacemaker cells of the cat using the fluorescent Ca indicator indo 1. Atrial latent pacemaker cells develop a slow Ca transient when rested in the presence of both Na- and Ca-free solution and thapsigargin [used to inhibit Na/Ca exchange and SR Ca adenosinetriphosphatase (Ca-ATPase), respectively]. This increase in cytosolic Ca concentration ([Ca]i) is probably caused by the rate of SR Ca leak exceeding the capacity of the remaining Ca transport systems (e.g., sarcolemmal Ca-ATPase and mitochondrial Ca uptake). However, neither cat sinoatrial (SA) node cells nor myocytes from cat atrium or ventricle exhibited a similar increase in [Ca]i during the same protocol. This indicates that SR Ca leak in these cells occurred at a rate low enough to be within the capacity of the slow Ca transporters, as observed previously in rabbit ventricular myocytes. When atrial and ventricular myocytes were stimulated at higher frequencies, sufficient to markedly increase diastolic and systolic [Ca]i and approach Ca overload (and spontaneous activity), they responded to inhibition of SR Ca-ATPase and Na/Ca exchange with a slow Ca transient similar to that normally observed in atrial latent pacemaker cells. Furthermore, the SR Ca depletion by thapsigargin did not affect spontaneous activity of SA node cells, but it prevented or slowed pacemaker activity in the atrial latent pacemaker cells. These findings suggest that enhanced diastolic SR Ca efflux contributes significantly to the generation of spontaneous activity in atrial subsidiary pacemakers under normal conditions and in Ca-overloaded myocytes but not in SA node cells.
Assuntos
Função Atrial , Cálcio/metabolismo , Sistema de Condução Cardíaco/metabolismo , Miocárdio/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Gatos , Diástole , Átrios do Coração , Sistema de Condução Cardíaco/citologia , Ventrículos do Coração , Miocárdio/citologia , Coelhos , Nó Sinoatrial/citologia , Nó Sinoatrial/fisiologiaRESUMO
The authors studied 43 necropsy specimens from adult individual by dissection under a stereomicroscope and by staining methods (Cajal-De Castro and Palmgren silver impregnations, Kluver Barrera and Pal-Weigert for myelinated fibers, and van Gieson and Azan trichromic methods). They suggest the presence of a direct nervous connection between the hepatic plexus (celiac plexus) and the high atrium via the left branches of the hepatic artery and portal vein, venous ligament, left hepatic vein, and v. cava inferior, passing through the foramen for the v. cava. This connection is made by very fine nerves from the upper part of the venous ligament until the left atrium, which generally are only visible on the microscopic level.