RESUMEN
BACKGROUND: Atrial fibrillation has an estimated prevalence of 1.5-2%, making it the most common cardiac arrhythmia. The processes that cause and sustain the disease are still not completely understood. An association between atrial fibrillation and systemic, as well as local, inflammatory processes has been reported. However, the exact mechanisms underlying this association have not been established. While it is understood that inflammatory macrophages can influence cardiac electrophysiology, a direct, causative relationship to atrial fibrillation has not been described. This study investigated the pro-arrhythmic effects of activated M1 macrophages on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes, to propose a mechanistic link between inflammation and atrial fibrillation. METHODS: Two hiPSC lines from healthy individuals were differentiated to atrial cardiomyocytes and M1 macrophages and integrated in an isogenic, pacing-free, atrial fibrillation-like coculture model. Electrophysiology characteristics of cocultures were analysed for beat rate irregularity, electrogram amplitude and conduction velocity using multi electrode arrays. Cocultures were additionally treated using glucocorticoids to suppress M1 inflammation. Bulk RNA sequencing was performed on coculture-isolated atrial cardiomyocytes and compared to meta-analyses of atrial fibrillation patient transcriptomes. RESULTS: Multi electrode array recordings revealed M1 to cause irregular beating and reduced electrogram amplitude. Conduction analysis further showed significantly lowered conduction homogeneity in M1 cocultures. Transcriptome sequencing revealed reduced expression of key cardiac genes such as SCN5A, KCNA5, ATP1A1, and GJA5 in the atrial cardiomyocytes. Meta-analysis of atrial fibrillation patient transcriptomes showed high correlation to the in vitro model. Treatment of the coculture with glucocorticoids showed reversal of phenotypes, including reduced beat irregularity, improved conduction, and reversed RNA expression profiles. CONCLUSIONS: This study establishes a causal relationship between M1 activation and the development of subsequent atrial arrhythmia, documented as irregularity in spontaneous electrical activation in atrial cardiomyocytes cocultured with activated macrophages. Further, beat rate irregularity could be alleviated using glucocorticoids. Overall, these results point at macrophage-mediated inflammation as a potential AF induction mechanism and offer new targets for therapeutic development. The findings strongly support the relevance of the proposed hiPSC-derived coculture model and present it as a first of its kind disease model.
Asunto(s)
Fibrilación Atrial , Técnicas de Cocultivo , Células Madre Pluripotentes Inducidas , Macrófagos , Miocitos Cardíacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/metabolismo , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Macrófagos/metabolismo , Fenotipo , Diferenciación Celular , Atrios Cardíacos/patología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/citologíaRESUMEN
This work introduces the first atrial-specific in-silico human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) model, based on a set of phenotype-specific IKur,IKCa and IK1 membrane currents. This model is built on novel in-vitro experimental data recently published by some of the co-authors to simulate the paced action potential of matured atrial-like hiPSC-CMs. The model consists of a system of stiff ordinary differential equations depending on several parameters, which have been tuned by automatic optimization techniques to closely match selected experimental biomarkers. The new model effectively simulates the electronic in-vitro hiPSC-CMs maturation process, transitioning from an unstable depolarized membrane diastolic potential to a stable hyperpolarized resting potential, and exhibits spontaneous firing activity in unpaced conditions. Moreover, our model accurately reflects the experimental rate dependence data at different cycle length and demonstrates the expected response to a specific current blocker. This atrial-specific in-silico model provides a novel computational tool for electrophysiological studies of cardiac stem cells and their applications to drug evaluation and atrial fibrillation treatment.
Asunto(s)
Potenciales de Acción , Atrios Cardíacos , Células Madre Pluripotentes Inducidas , Modelos Cardiovasculares , Miocitos Cardíacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/fisiología , Miocitos Cardíacos/citología , Potenciales de Acción/fisiología , Atrios Cardíacos/citología , Simulación por ComputadorRESUMEN
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling.
Asunto(s)
Atrios Cardíacos , Ventrículos Cardíacos , Células Madre Pluripotentes Inducidas , Contracción Miocárdica , Miocitos Cardíacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/citología , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/citología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Desarrollo de Medicamentos/métodos , Canales Iónicos/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Carbacol/farmacología , Sistemas MicrofisiológicosRESUMEN
Physiologically, the atria contract first, followed by the ventricles, which is the prerequisite for normal blood circulation. The above phenomenon of atrioventricular sequential contraction results from the characteristically slow conduction of electrical excitation of the atrioventricular node (AVN) between the atria and the ventricles. However, it is not clear what controls the conduction of electrical excitation within AVNs. Here, we find that AVN pacemaker cells (AVNPCs) possess an intact intrinsic GABAergic system, which plays a key role in electrical conduction from the atria to the ventricles. First, along with the discovery of abundant GABA-containing vesicles under the surface membranes of AVNPCs, key elements of the GABAergic system, including GABA metabolic enzymes, GABA receptors, and GABA transporters, were identified in AVNPCs. Second, GABA synchronously elicited GABA-gated currents in AVNPCs, which significantly weakened the excitability of AVNPCs. Third, the key molecular elements of the GABAergic system markedly modulated the conductivity of electrical excitation in the AVN. Fourth, GABAA receptor deficiency in AVNPCs accelerated atrioventricular conduction, which impaired the AVN's protective potential against rapid ventricular frequency responses, increased susceptibility to lethal ventricular arrhythmias, and decreased the cardiac contractile function. Finally, interventions targeting the GABAergic system effectively prevented the occurrence and development of atrioventricular block. In summary, the endogenous GABAergic system in AVNPCs determines the slow conduction of electrical excitation within AVNs, thereby ensuring sequential atrioventricular contraction. The endogenous GABAergic system shows promise as a novel intervention target for cardiac arrhythmias.
Asunto(s)
Nodo Atrioventricular , Atrios Cardíacos , Ventrículos Cardíacos , Receptores de GABA-A , Ácido gamma-Aminobutírico , Animales , Ácido gamma-Aminobutírico/metabolismo , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/citología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/citología , Nodo Atrioventricular/metabolismo , Nodo Atrioventricular/fisiología , Ratones , Receptores de GABA-A/metabolismo , Ratones Endogámicos C57BL , Masculino , Potenciales de Acción , Arritmias Cardíacas/metabolismoRESUMEN
The sinus node (SN) serves as the primary pacemaker of the heart and is the first component of the cardiac conduction system. Due to its anatomical properties and sample scarcity, the cellular composition of the human SN has been historically challenging to study. Here, we employed a novel deep learning deconvolution method, namely Bulk2space, to characterise the cellular heterogeneity of the human SN using existing single-cell datasets of non-human species. As a proof of principle, we used Bulk2Space to profile the cells of the bulk human right atrium using publicly available mouse scRNA-Seq data as a reference. 18 human cell populations were identified, with cardiac myocytes being the most abundant. Each identified cell population correlated to its published experimental counterpart. Subsequently, we applied the deconvolution to the bulk transcriptome of the human SN and identified 11 cell populations, including a population of pacemaker cardiomyocytes expressing pacemaking ion channels (HCN1, HCN4, CACNA1D) and transcription factors (SHOX2 and TBX3). The connective tissue of the SN was characterised by adipocyte and fibroblast populations, as well as key immune cells. Our work unravelled the unique single cell composition of the human SN by leveraging the power of a novel machine learning method.
Asunto(s)
Miocitos Cardíacos , Análisis de la Célula Individual , Nodo Sinoatrial , Humanos , Nodo Sinoatrial/citología , Nodo Sinoatrial/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Análisis de la Célula Individual/métodos , Ratones , Animales , Inteligencia Artificial , Transcriptoma , Atrios Cardíacos/metabolismo , Atrios Cardíacos/citología , Aprendizaje ProfundoRESUMEN
Lipopolysaccharide (LPS) triggers an inflammatory response, causing impairment of cardiomyocyte Ca2+ and Na + regulation. This study aimed to determine whether piscidin-1 (PCD-1), an antimicrobial peptide, improves intracellular Ca2+ and Na + regulation in LPS-challenged atrial cardiomyocytes. Rabbit atrial cardiomyocytes were enzymatically isolated from the left atria. Patch-clamp ionic current recording, intracellular Ca2+ monitoring using Fluo-3, and detection of cytosolic reactive oxygen species production were conducted in control, LPS-challenged, and LPS + PCD-1-treated atrial cardiomyocytes. LPS-challenged cardiomyocytes showed shortened durations of action potential at their 50% and 90% repolarizations, which was reversed by PCD-1 treatment. LPS-challenged cardiomyocytes showed decreased L-type Ca2+ channel currents and larger Na+/Ca2+ exchange currents compared to controls. While LPS did not affect the sodium current, an enhanced late sodium current with increased cytosolic Na+ levels was observed in LPS-challenged cardiomyocytes. These LPS-induced alterations in the ionic current were ameliorated by PCD-1 treatment. LPS-challenged cardiomyocytes displayed lowered Ca2+ transient amplitudes and decreased Ca2+ stores and greater Ca2+ leakage in the sarcoplasmic reticulum compared to the control. Exposure to PCD-1 attenuated LPS-induced alterations in Ca2+ regulation. The elevated reactive oxygen species levels observed in LPS-challenged myocytes were suppressed after PCD-1 treatment. The protein levels of NF-κB and IL-6 increased following LPS treatment. Decreased sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a protein levels were observed in LPS-challenged cardiomyocytes. PCD-1 modulates LPS-induced alterations in inflammatory and Ca2+ regulatory protein levels. Our results suggest that PCD-1 modulates LPS-induced alterations in intracellular Ca2+ and Na + homeostasis, reactive oxygen species production, and the NF-κB inflammatory pathway in atrial cardiomyocytes.
Asunto(s)
Calcio , Atrios Cardíacos , Lipopolisacáridos , Miocitos Cardíacos , Estrés Oxidativo , Especies Reactivas de Oxígeno , Sodio , Animales , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Lipopolisacáridos/farmacología , Conejos , Calcio/metabolismo , Sodio/metabolismo , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/citología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Masculino , Potenciales de Acción/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/efectos de los fármacosRESUMEN
Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and ß-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (ß1 + ß2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (ß1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (ß2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating ß1- and α1-ARs in both human and rabbit, with a ß2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.
Asunto(s)
Canales de Calcio Tipo L , Miocitos Cardíacos , Norepinefrina , Receptores Adrenérgicos alfa , Receptores Adrenérgicos beta , Animales , Humanos , Conejos , Fibrilación Atrial/fisiopatología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Norepinefrina/farmacología , Norepinefrina/fisiología , Prazosina/farmacología , Receptores Adrenérgicos alfa 2 , Atrios Cardíacos/citología , Receptores Adrenérgicos beta/fisiología , Receptores Adrenérgicos alfa/fisiología , Antagonistas Adrenérgicos alfa/farmacología , Antagonistas Adrenérgicos beta/farmacología , Canales de Calcio Tipo L/fisiologíaRESUMEN
Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4-8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.
Asunto(s)
Investigación Biomédica , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Miocitos Cardíacos/citología , Animales , Calcio/farmacología , Separación Celular , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Conejos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
The atrial myocardium demonstrates the highly heterogeneous organization of the transversal-axial tubule system (TATS), although its anatomical distribution and region-specific impact on Ca2+ dynamics remain unknown. Here, we developed a novel method for high-resolution confocal imaging of TATS in intact live mouse atrial myocardium and applied a custom-developed MATLAB-based computational algorithm for the automated analysis of TATS integrity. We observed a twofold higher (P < 0.01) TATS density in the right atrial appendage (RAA) than in the intercaval regions (ICR, the anatomical region between the superior vena cava and atrioventricular junction and between the crista terminalis and interatrial septum). Whereas RAA predominantly consisted of well-tubulated myocytes, ICR showed partially tubulated/untubulated cells. Similar TATS distribution was also observed in healthy human atrial myocardium sections. In both mouse atrial preparations and isolated mouse atrial myocytes, we observed a strong anatomical correlation between TATS distribution and Ca2+ transient synchronization and rise-up time. This region-specific difference in Ca2+ transient morphology disappeared after formamide-induced detubulation. ICR myocytes showed a prolonged action potential duration at 80% of repolarization as well as a significantly lower expression of RyR2 and Cav1.2 proteins but similar levels of NCX1 and Cav1.3 compared with RAA tissue. Our findings provide a detailed characterization of the region-specific distribution of TATS in mouse and human atrial myocardium, highlighting the structural foundation for anatomical heterogeneity of Ca2+ dynamics and contractility in the atria. These results could indicate different roles of TATS in Ca2+ signaling at distinct anatomical regions of the atria and provide mechanistic insight into pathological atrial remodeling.NEW & NOTEWORTHY Mouse and human atrial myocardium demonstrate high variability in the organization of the transversal-axial tubule system (TATS), with more organized TATS expressed in the right atrial appendage. TATS distribution governs anatomical heterogeneity of Ca2+ dynamics and thus could contribute to integral atrial contractility, mechanics, and arrhythmogenicity.
Asunto(s)
Señalización del Calcio , Atrios Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Potenciales de Acción , Animales , Canales de Calcio Tipo L/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiología , Atrios Cardíacos/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.
Asunto(s)
Salud , Células Madre Pluripotentes Inducidas/citología , Adulto , Señalización del Calcio , Diferenciación Celular , Línea Celular , Células Clonales , Etnicidad , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Humanos , Masculino , Persona de Mediana Edad , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Factores de Riesgo , Adulto JovenRESUMEN
Forced expression of core cardiogenic transcription factors can directly reprogram fibroblasts to induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. This cardiac reprogramming approach provides a proof of concept for induced heart regeneration by converting a fibroblast fate to a cardiomyocyte fate. However, it remains elusive whether chamber-specific cardiomyocytes can be generated by cardiac reprogramming. Therefore, we assessed the ability of the cardiac reprogramming approach for chamber specification in vitro and in vivo. We found that in vivo cardiac reprogramming post-myocardial infarction exclusively induces a ventricular-like phenotype, while a major fraction of iCMs generated in vitro failed to determine their chamber identities. Our results suggest that in vivo cardiac reprogramming may have an inherent advantage of generating chamber-matched new cardiomyocytes as a potential heart regenerative approach.
Asunto(s)
Reprogramación Celular/fisiología , Fibroblastos/citología , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Miocitos Cardíacos/citología , Animales , Ratones , Infarto del MiocardioRESUMEN
Human iPSC-derived cardiomyocytes (hiPSC-CMs) are expected to be used in regenerative therapies and drug discovery for heart failure. hiPSC-CMs are a mixture of mainly ventricular CMs (VCMs) and also of atrial CMs (ACMs) and pacemaker cells. Here we describe a method to enrich VCM and ACM differentiation and to characterize these subtypes by gene expression analysis using qRT-PCR and by electrophysiological properties using the patch-clamp method. The differentiated VCMs and ACMs highly express VCM and ACM marker genes, respectively. Furthermore, both subtypes show specific properties of action potentials.
Asunto(s)
Ventrículos Cardíacos/citología , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Potenciales de Acción/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Fenómenos Electrofisiológicos/fisiología , Atrios Cardíacos/citología , Humanos , Técnicas de Placa-Clamp/métodosRESUMEN
Differentiation protocols to direct cell fate decision from pluripotent stem cells to cardiac myocytes normally achieve high purity and quality of cells. Nonetheless, the highly specialized anatomy of the heart enables the possibility that acquisition of terminal somatic differentiation from pluripotency might imply heterogeneity of non-desire cell lineages. Directed cardiac differentiation empowers differentiation of pool of cells commonly reported to contain different proportions of ventricular, atrial, and nodal-like cells. RNA sequencing (RNA-Seq) allows a precise transcriptional profiling, ensuring a quality checking of the cell identity our protocol has derived as a main outcome. Here we describe a workflow methodology on how to adapt RNA sequencing analysis for integration into the R analysis pipeline in order to characterize chamber-specific gene signatures of the major cardiac lineages of myocytes in the heart.
Asunto(s)
Perfilación de la Expresión Génica , Atrios Cardíacos/citología , Ventrículos Cardíacos/citología , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/química , RNA-Seq/métodos , Transcriptoma , Diferenciación Celular/genética , Células Cultivadas , Análisis por Conglomerados , Ontología de Genes , Atrios Cardíacos/química , Ventrículos Cardíacos/química , Humanos , Análisis de Componente Principal , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Flujo de TrabajoRESUMEN
Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Insuficiencia Cardíaca/terapia , Infarto del Miocardio/terapia , Miocitos Cardíacos/trasplante , Células Madre Pluripotentes/fisiología , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Modelos Animales de Enfermedad , Embrión de Mamíferos , Desarrollo Embrionario/fisiología , Atrios Cardíacos/citología , Atrios Cardíacos/embriología , Insuficiencia Cardíaca/patología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/embriología , Ventrículos Cardíacos/patología , Humanos , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Miocitos Cardíacos/fisiología , RatasRESUMEN
Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. IKs (KCNQ1+KCNE1), a slowly activating K+ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the IKs channel. Here, we show that C28 specifically shifted IKs VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of IKs VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Canal de Potasio KCNQ1/genética , Miocitos Cardíacos/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Potenciales de Acción/fisiología , Sustitución de Aminoácidos , Animales , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patología , Calcio/metabolismo , Perros , Furanos/farmacología , Expresión Génica , Cobayas , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/metabolismo , Moxifloxacino/farmacología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Fenetilaminas/farmacología , Potasio/metabolismo , Cultivo Primario de Células , Piridinas/farmacología , Pirimidinas/farmacología , Sodio/metabolismo , Sulfonamidas/farmacología , Transgenes , Xenopus laevisRESUMEN
BACKGROUND: There is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (ICa) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of ß1-adrenoceptor (ß1-AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on ICa and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes. METHODS: Adult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). ICa was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 µM) were used to inhibit PDE3 and PDE4, respectively. ß1-AR were stimulated with the physiological agonist norepinephrine (100 µM). RESULTS: In adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% (n = 10/9, mean ± SEM, number of cells/number patients) and ICa by +10.4 ± 1.5 pA/pF (n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and ICa (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and ICa + 1.4 ± 0.2 pA/pF n = 34/7, p < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and ICa + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on ICa. In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and ICa, were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and ICa + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on ICa (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET. CONCLUSION: Our results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller ICa responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes.
Asunto(s)
AMP Cíclico/metabolismo , Atrios Cardíacos , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Adulto , Células Cultivadas , Medios de Cultivo , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Cultivo Primario de CélulasRESUMEN
Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.
Asunto(s)
Diferenciación Celular/genética , Atrios Cardíacos/crecimiento & desarrollo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Sistemas CRISPR-Cas/genética , Miosinas Cardíacas/genética , Proteínas Fluorescentes Verdes , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/crecimiento & desarrollo , Ventrículos Cardíacos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/citología , Cadenas Ligeras de Miosina/genéticaRESUMEN
MicroRNAs that modulate transcription can regulate other microRNAs and are also up-regulated under pathological stress. MicroRNA-499 (miR-499), microRNA-208a (miR-208a) and B-cell lymphoma 2 (Bcl-2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl-2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts-adult atrial (HCF-aa) was investigated. Using negative pressure, HCF-aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up-regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl-2 was up-regulated by longer stretching times. Pre-treatment with antagomir-499 reversed the miR-208a down-regulation, whereas Bcl-2 expression could be suppressed by miR-208a overexpression. In the HCF-aa under stretching for 1 h, miR-499 overexpression decreased pri-miR-208a luciferase activity; this inhibition of pri-miR-208a luciferase activity with stretching was reversed when the miR-499-5p binding site in pri-miR-208a was mutated. The addition of antagomir-208a reversed the Bcl-2-3'UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre-treatment with miR-499 or antagomir-208a inhibited cellular apoptosis in stretched HCF-aa. In hearts with volume overload, miR-499 overexpression inhibited myocardial miR-208a expression, whereas Bcl-2 expression could be suppressed by the addition of miR-208a. In conclusion, miR-208a mediated the regulation of miR-499 on Bcl-2 expression in stretched HCF-aa and hearts with volume overload.
Asunto(s)
Fibroblastos/metabolismo , Atrios Cardíacos/citología , MicroARNs/genética , Ejercicios de Estiramiento Muscular , Regiones no Traducidas 3' , Animales , Apoptosis/genética , Biomarcadores , Regulación de la Expresión Génica , Humanos , Miocardio/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , RatasRESUMEN
The electrical coupling between myocytes and fibroblasts and the spacial distribution of fibroblasts within myocardial tissues are significant factors in triggering and sustaining cardiac arrhythmias, but their roles are poorly understood. This article describes both direct numerical simulations and an asymptotic theory of propagation and block of electrical excitation in a model of atrial tissue with myocyte-fibroblast coupling. In particular, three idealized fibroblast distributions are introduced: uniform distribution, fibroblast barrier and myocyte strait-all believed to be constituent blocks of realistic fibroblast distributions. Primary action potential biomarkers including conduction velocity, peak potential and triangulation index are estimated from direct simulations in all cases. Propagation block is found to occur at certain critical values of the parameters defining each idealized fibroblast distribution, and these critical values are accurately determined. An asymptotic theory proposed earlier is extended and applied to the case of a uniform fibroblast distribution. Biomarker values are obtained from hybrid analytical-numerical solutions of coupled fast-time and slow-time periodic boundary value problems and compare well to direct numerical simulations. The boundary of absolute refractoriness is determined solely by the fast-time problem and is found to depend on the values of the myocyte potential and on the slow inactivation variable of the sodium current ahead of the propagating pulse. In turn, these quantities are estimated from the slow-time problem using a regular perturbation expansion to find the steady state of the coupled myocyte-fibroblast kinetics. The asymptotic theory gives a simple analytical expression that captures with remarkable accuracy the block of propagation in the presence of fibroblasts.
Asunto(s)
Función Atrial/fisiología , Atrios Cardíacos/citología , Modelos Cardiovasculares , Miocitos Cardíacos/fisiología , Potenciales de Acción/fisiología , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Simulación por Computador , Fenómenos Electrofisiológicos , Fibroblastos/fisiología , Humanos , Conceptos MatemáticosRESUMEN
Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1), and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes, and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death, while overexpressing these genes maintained their expansion in hyperoxia, suggesting that oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1, and other fatty acid synthesis genes in atrial cardiomyocytes may, thus, provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.