RESUMO
Barth Syndrome (BTHS) is an inherited cardiomyopathy caused by defects in the mitochondrial transacylase TAFAZZIN (Taz), required for the synthesis of the phospholipid cardiolipin. BTHS is characterized by heart failure, increased propensity for arrhythmias and a blunted inotropic reserve. Defects in Ca2+-induced Krebs cycle activation contribute to these functional defects, but despite oxidation of pyridine nucleotides, no oxidative stress developed in the heart. Here, we investigated how retrograde signaling pathways orchestrate metabolic rewiring to compensate for mitochondrial defects. In mice with an inducible knockdown (KD) of TAFAZZIN, and in induced pluripotent stem cell-derived cardiac myocytes, mitochondrial uptake and oxidation of fatty acids was strongly decreased, while glucose uptake was increased. Unbiased transcriptomic analyses revealed that the activation of the eIF2α/ATF4 axis of the integrated stress response upregulates one-carbon metabolism, which diverts glycolytic intermediates towards the biosynthesis of serine and fuels the biosynthesis of glutathione. In addition, strong upregulation of the glutamate/cystine antiporter xCT increases cardiac cystine import required for glutathione synthesis. Increased glutamate uptake facilitates anaplerotic replenishment of the Krebs cycle, sustaining energy production and antioxidative pathways. These data indicate that ATF4-driven rewiring of metabolism compensates for defects in mitochondrial uptake of fatty acids to sustain energy production and antioxidation.
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Síndrome de Barth , Animais , Camundongos , Síndrome de Barth/genética , Cistina , Antioxidantes , Ácidos Graxos , Glutamatos , GlutationaRESUMO
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are an increasingly employed model in cardiac research and drug discovery. As cellular metabolism plays an integral role in determining phenotype, the characterization of the metabolic profile of hiPSC-CM during maturation is crucial for their translational application. In this study we employ a combination of methods including extracellular flux, 13C-glucose enrichment and targeted metabolomics to characterize the metabolic profile of hiPSC-CM during their maturation in culture from 6 weeks, up to 12 weeks. Results show a progressive remodeling of pathways involved in energy metabolism and substrate utilization along with an increase in sarcomere regularity. The oxidative capacity of hiPSC-CM and particularly their ability to utilize fatty acids increased with time. In parallel, relative glucose oxidation was reduced while glutamine oxidation was maintained at similar levels. There was also evidence of increased coupling of glycolysis to mitochondrial respiration, and away from glycolytic branch pathways at later stages of maturation. The rate of glycolysis as assessed by lactate production was maintained at both stages but with significant alterations in proximal glycolytic enzymes such as hexokinase and phosphofructokinase. We observed a progressive maturation of mitochondrial oxidative capacity at comparable levels of mitochondrial content between these time-points with enhancement of mitochondrial network structure. These results show that the metabolic profile of hiPSC-CM is progressively restructured, recapitulating aspects of early post-natal heart development. This would be particularly important to consider when employing these cell model in studies where metabolism plays an important role.
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Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Células Cultivadas , Metabolismo Energético , Glucose/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Alterations of RNA editing that affect the secondary structure of RNAs can cause human diseases. We therefore studied RNA editing in failing human hearts. Transcriptome sequencing showed that adenosine-to-inosine (A-to-I) RNA editing was responsible for 80% of the editing events in the myocardium. Failing human hearts were characterized by reduced RNA editing. This was primarily attributable to Alu elements in introns of protein-coding genes. In the failing left ventricle, 166 circRNAs were upregulated and 7 circRNAs were downregulated compared to non-failing controls. Most of the upregulated circRNAs were associated with reduced RNA editing in the host gene. ADAR2, which binds to RNA regions that are edited from A-to-I, was decreased in failing human hearts. In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, one of the identified upregulated circRNAs with a high reduction of editing in heart failure, AKAP13, was further characterized. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes. These data show that ADAR2 mediates A-to-I RNA editing in the human heart. A-to-I RNA editing represses the formation of dsRNA structures of Alu elements favoring canonical linear mRNA splicing and inhibiting the formation of circRNAs. The findings are relevant to diseases with reduced RNA editing and increased circRNA levels and provide insights into the human-specific regulation of circRNA formation.
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Células-Tronco Pluripotentes Induzidas , Edição de RNA , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA/química , RNA/genética , RNA/metabolismo , RNA Circular/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Cancer therapies with anthracyclines have been shown to induce cardiovascular complications. The aims of this study were to establish an in vitro induced pluripotent stem cell model (iPSC) of anthracycline-induced cardiotoxicity (ACT) from patients with an aggressive form of B-cell lymphoma and to examine whether doxorubicin (DOX)-treated ACT-iPSC cardiomyocytes (CM) can recapitulate the clinical features exhibited by patients, and thus help uncover a DOX-dependent pathomechanism. ACT-iPSC CM generated from individuals with CD20+ B-cell lymphoma who had received high doses of DOX and suffered cardiac dysfunction were studied and compared to control-iPSC CM from cancer survivors without cardiac symptoms. In cellular studies, ACT-iPSC CM were persistently more susceptible to DOX toxicity including augmented disorganized myofilament structure, changed mitochondrial shape, and increased apoptotic events. Consistently, ACT-iPSC CM and cardiac fibroblasts isolated from fibrotic human ACT myocardium exhibited higher DOX-dependent reactive oxygen species. In functional studies, Ca2+ transient amplitude of ACT-iPSC CM was reduced compared to control cells, and diastolic sarcoplasmic reticulum Ca2+ leak was DOX-dependently increased. This could be explained by overactive CaMKIIδ in ACT CM. Together with DOX-dependent augmented proarrhythmic cellular triggers and prolonged action potentials in ACT CM, this suggests a cellular link to arrhythmogenic events and contractile dysfunction especially found in ACT engineered human myocardium. CamKIIδ inhibition prevented proarrhythmic triggers in ACT. In contrast, control CM upregulated SERCA2a expression in a DOX-dependent manner, possibly to avoid heart failure conditions. In conclusion, we developed the first human patient-specific stem cell model of DOX-induced cardiac dysfunction from patients with B-cell lymphoma. Our results suggest that DOX-induced stress resulted in arrhythmogenic events associated with contractile dysfunction and finally in heart failure after persistent stress activation in ACT patients.
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Cardiopatias , Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Linfoma de Células B , Neoplasias , Cardiotoxicidade/metabolismo , Cardiotoxicidade/patologia , Doxorrubicina/metabolismo , Doxorrubicina/toxicidade , Cardiopatias/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Miócitos Cardíacos/metabolismo , Neoplasias/metabolismoRESUMO
Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20−40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5' splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.
Assuntos
Cardiomiopatia Dilatada , Sequenciamento por Nanoporos , Nanoporos , Humanos , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Cálcio/metabolismo , Virulência , Sítios de Splice de RNA , Mutação , Fenótipo , Linhagem , GenótipoRESUMO
INTRODUCTION: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. METHODS: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. RESULTS: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. CONCLUSION: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.
Assuntos
Doença do Sistema de Condução Cardíaco/genética , Cardiomiopatia Dilatada/genética , Miócitos Cardíacos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células CHO , Linhagem Celular , Cricetulus , Feminino , Predisposição Genética para Doença/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/citologia , Sarcômeros/metabolismo , Sódio/metabolismo , Volume Sistólico/genética , Xenopus laevis/fisiologia , Adulto JovemRESUMO
The ability to generate patient-specific induced pluripotent stem cells (iPSCs) provides a unique opportunity for modeling heart disease in vitro. In this study, we generated iPSCs from a patient with dilated cardiomyopathy (DCM) caused by a missense mutation S635A in RNA-binding motif protein 20 (RBM20) and investigated the functionality and cell biology of cardiomyocytes (CMs) derived from patient-specific iPSCs (RBM20-iPSCs). The RBM20-iPSC-CMs showed abnormal distribution of sarcomeric α-actinin and defective calcium handling compared to control-iPSC-CMs, suggesting disorganized myofilament structure and altered calcium machinery in CMs of the RBM20 patient. Engineered heart muscles (EHMs) from RBM20-iPSC-CMs showed that not only active force generation was impaired in RBM20-EHMs but also passive stress of the tissue was decreased, suggesting a higher visco-elasticity of RBM20-EHMs. Furthermore, we observed a reduced titin (TTN) N2B-isoform expression in RBM20-iPSC-CMs by demonstrating a reduction of exon skipping in the PEVK region of TTN and an inhibition of TTN isoform switch. In contrast, in control-iPSC-CMs both TTN isoforms N2B and N2BA were expressed, indicating that the TTN isoform switch occurs already during early cardiogenesis. Using next generation RNA sequencing, we mapped transcriptome and splicing target profiles of RBM20-iPSC-CMs and identified different cardiac gene networks in response to the analyzed RBM20 mutation in cardiac-specific processes. These findings shed the first light on molecular mechanisms of RBM20-dependent pathological cardiac remodeling leading to DCM. Our data demonstrate that iPSC-CMs coupled with EHMs provide a powerful tool for evaluating disease-relevant functional defects and for a deeper mechanistic understanding of alternative splicing-related cardiac diseases.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Adulto , Animais , Cálcio/metabolismo , Células Cultivadas , Conectina/metabolismo , Feminino , Humanos , Camundongos , Mutação , Fenótipo , Splicing de RNA/genética , Sarcômeros/metabolismo , Transcriptoma/genéticaRESUMO
Filamin C (FLNC) is a highly important actin crosslinker and multi-adaptor protein in striated skeletal and cardiac muscle. Mutations have been linked to a range of cardiomyopathy types. Here, we generated induced pluripotent stem cells (iPSC) from a patient with dilated cardiomyopathy (DCM) harboring a new, unique heterozygous FLNC mutation p.R2187P. From this patient-specific iPSC line, a corresponding isogenic control line was created by CRISPR/Cas9 genome editing. Both, the patient-specific and isogenic-control iPSC maintained full pluripotency, genomic integrity, and in vitro differentiation capacity. All iPSC lines differentiate into iPSC-cardiomyocytes, hence providing the possibility to study the pathogenesis of FLNC-mediated DCM further.
Assuntos
Sistemas CRISPR-Cas , Cardiomiopatia Dilatada , Filaminas , Células-Tronco Pluripotentes Induzidas , Humanos , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Sistemas CRISPR-Cas/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Filaminas/genética , Filaminas/metabolismo , Mutação , Diferenciação Celular , Linhagem Celular , MasculinoRESUMO
RBM20 mutations account for 3 % of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient.
Assuntos
Cardiomiopatias , Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Proteínas de Ligação a RNA , Humanos , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatia Dilatada/genética , Patrimônio Genético , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Mutations in the mitochondrial-DNA or mitochondria related nuclear-encoded-DNA lead to various multisystemic disorders collectively termed mitochondrial diseases. One in three cases of mitochondrial disease affects the heart muscle, which is called mitochondrial cardiomyopathy (MCM) and is associated with hypertrophic, dilated, and noncompact cardiomyopathy. The heart is an organ with high energy demand, and mitochondria occupy 30%-40% of its cardiomyocyte-cell volume. Mitochondrial dysfunction leads to energy depletion and has detrimental effects on cardiac performance. However, disease development and progression in the context of mitochondrial and nuclear DNA mutations, remains incompletely understood. The system of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) is an excellent platform to study MCM since the unique genetic identity to their donors enables a robust recapitulation of the predicted phenotypes in a dish on a patient-specific level. Here, we focus on recent insights into MCM studied by patient-specific iPSC-CM and further discuss research gaps and advances in metabolic maturation of iPSC-CM, which is crucial for the study of mitochondrial dysfunction and to develop novel therapeutic strategies.
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AIMS: Aging entails profound immunological transformations that can impact myocardial homeostasis and predispose to heart failure. However, preclinical research in the immune-cardiology field is mostly conducted in young healthy animals, which potentially weakens its translational relevance. Herein, we sought to investigate how the aging T-cell compartment associates with changes in myocardial cell biology in aged mice. METHODS AND RESULTS: We phenotyped the antigen-experienced effector/memory T cells purified from heart-draining lymph nodes of 2-, 6-, 12-, and 18-month-old C57BL/6J mice using single-cell RNA/T cell receptor sequencing. Simultaneously, we profiled all non-cardiomyocyte cell subsets purified from 2- to 18-month-old hearts and integrated our data with publicly available cardiomyocyte single-cell sequencing datasets. Some of these findings were confirmed at the protein level by flow cytometry. With aging, the heart-draining lymph node and myocardial T cells underwent clonal expansion and exhibited an up-regulated pro-inflammatory transcription signature, marked by an increased interferon-γ (IFN-γ) production. In parallel, all major myocardial cell populations showed increased IFN-γ responsive signature with aging. In the aged cardiomyocytes, a stronger IFN-γ response signature was paralleled by the dampening of expression levels of transcripts related to most metabolic pathways, especially oxidative phosphorylation. Likewise, induced pluripotent stem cells-derived cardiomyocytes exposed to chronic, low grade IFN-γ treatment showed a similar inhibition of metabolic activity. CONCLUSIONS: By investigating the paired age-related alterations in the T cells found in the heart and its draining lymph nodes, we provide evidence for increased myocardial IFN-γ signaling with age, which is associated with inflammatory and metabolic shifts typically seen in heart failure.
Assuntos
Insuficiência Cardíaca , Imunossenescência , Camundongos , Animais , Interferon gama , Camundongos Endogâmicos C57BL , Envelhecimento/genética , Insuficiência Cardíaca/genéticaRESUMO
Gene splicing is a fine-tuned process orchestrated by splice factors including RNA-binding motif 20 (RBM20), and their mutations are linked to the development of cardiac diseases. Here, we provide a step-by-step protocol to transfer RBM20-dependent splicing from rat to human. This protocol describes a PCR-based approach to adapt and quantify RBM20-dependent exon-expression of human target genes. We detail the primer design, the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) for RNA isolation, followed by quantification of splicing products. For complete details on the use and execution of this profile, please refer to Streckfuss-Bömeke et al. (2017).
Assuntos
Splicing de RNA , Proteínas de Ligação a RNA , Animais , Éxons/genética , Humanos , Splicing de RNA/genética , Motivos de Ligação ao RNA , Proteínas de Ligação a RNA/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Atrial fibrillation (AF) can either be a consequence or an underlying mechanism of left ventricular systolic dysfunction. Patients included in the CASTLE-AF (Catheter Ablation vs. Standard Conventional Treatment in Patients With LV Dysfunction and AF) trial who suffered from AF and left ventricular systolic dysfunction benefited from an AF burden <50% after catheter ablation compared with those patients with an AF burden >50%. OBJECTIVES: This analysis tried to explain the clinical findings of the CASTLE-AF trial regarding AF burden in a "back-to-bench" approach. METHODS: To study the ventricular effects of different AF burdens, experiments were performed using human ventricular induced pluripotent stem cell-derived cardiomyocytes undergoing in vitro AF simulation. Epifluorescence microscopy, action potential measurements, and measurements of sarcomere regularity were conducted. RESULTS: Induced pluripotent stem cell-derived cardiomyocytes stimulated with AF burden of 60% or higher displayed typical hallmarks of heart failure. Ca2+ transient amplitude was significantly reduced indicating negative inotropic effects. Action potential duration was significantly prolonged, which represents a potential trigger for arrhythmias. A significant decrease of sarcomere regularity could explain impaired cardiac contractility in patients with high AF burden. These effects were more pronounced after 7 days of AF simulation compared with 48 hours. CONCLUSIONS: Significant functional and structural alterations occurred at the cellular level at a threshold of â¼50% AF burden as it was observed to be harmful in the CASTLE-AF trial. Therefore, these translational results may help to understand the findings of the CASTLE-AF trial.
Assuntos
Fibrilação Atrial , Ablação por Cateter , Insuficiência Cardíaca , Disfunção Ventricular Esquerda , Humanos , Fibrilação Atrial/cirurgia , Ablação por Cateter/métodos , Insuficiência Cardíaca/etiologia , Remodelação Ventricular/fisiologia , Ensaios Clínicos como AssuntoRESUMO
Alternative mRNA splicing is a fundamental process to increase the versatility of the genome. In humans, cardiac mRNA splicing is involved in the pathophysiology of heart failure. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) cause severe forms of cardiomyopathy. To identify novel cardiomyopathy-associated splicing factors, RNA-seq and tissue-enrichment analyses were performed, which identified up-regulated expression of Sam68-Like mammalian protein 2 (SLM2) in the left ventricle of dilated cardiomyopathy (DCM) patients. In the human heart, SLM2 binds to important transcripts of sarcomere constituents, such as those encoding myosin light chain 2 (MYL2), troponin I3 (TNNI3), troponin T2 (TNNT2), tropomyosin 1/2 (TPM1/2), and titin (TTN). Mechanistically, SLM2 mediates intron retention, prevents exon exclusion, and thereby mediates alternative splicing of the mRNA regions encoding the variable proline-, glutamate-, valine-, and lysine-rich (PEVK) domain and another part of the I-band region of titin. In summary, SLM2 is a novel cardiac splicing regulator with essential functions for maintaining cardiomyocyte integrity by binding to and processing the mRNAs of essential cardiac constituents such as titin.
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Conectina/genética , Conectina/metabolismo , Glutamatos , Insuficiência Cardíaca/genética , Humanos , Lisina , Prolina , Fatores de Processamento de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Tropomiosina/metabolismo , Troponina I/metabolismo , Troponina T/metabolismo , ValinaRESUMO
Arrhythmia-induced cardiomyopathy (AIC) is characterized by left-ventricular systolic dysfunction caused by persistent arrhythmia. To date, genetic or pathological drivers causing AIC remain unknown. Here, we generated induced pluripotent stem cells (iPSCs) from an AIC patient. The AIC-iPSCs exhibited full pluripotency and differentiation characteristics and maintained a normal karyotype after reprogramming. The AIC-iPSCs differentiated into functional beating AIC-iPSC-cardiomyocytes (CMs), which represents the cell-type of interest to study molecular, genetic and functional aspects of AIC.
Assuntos
Cardiomiopatias , Células-Tronco Pluripotentes Induzidas , Arritmias Cardíacas/genética , Cardiomiopatias/genética , Diferenciação Celular , Humanos , Miócitos CardíacosRESUMO
RNA binding motif protein 20 (RBM20) is an alternative splicing factor and highly expressed in cardiac tissue. Mutations in the RS domain of RBM20 have been shown to cause different cardiomyopathies. Here, we generated induced pluripotent stem cells (iPSCs) from a dilated cardiomyopathy patient harboring the heterozygous RBM20 mutation p.R634W and consecutively produced isogenic control line using CRISPR/Cas9 genome editing. Patient-specific RBM20 iPSCs and isogenic control line maintained full pluripotency, genomic integrity, and in vitro differentiation capacity. All iPSC-lines were able to differentiate into pure cardiomyocytes, thus providing a valuable tool for studying the pathogenesis of human RBM20-mediated cardiac disease.
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The Takotsubo syndrome (TTS) is characterized by acute transient left ventricular dysfunction in the absence of obstructive coronary lesions. An enhanced ß-adrenergic signaling and higher sensitivity to catecholamine-induced-toxicity were identified as mechanisms associated with TTS. It is still elusive, whether TTS patients with recurrent events show similar underlying signaling pathomechanism. Induced pluripotent stem cell (iPSC)-lines were generated from skin fibroblasts of two independent female Takotsubo syndrome patients with a severe phenotype characterized by recurrent TTS events. For reprogramming, a non-integrative plasmid technique was used. All generated iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro and in vivo differentiation capacity.
Assuntos
Células-Tronco Pluripotentes Induzidas , Cardiomiopatia de Takotsubo , Diferenciação Celular , Feminino , Coração , Humanos , Transdução de SinaisRESUMO
Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30 ± 0.18, Sendai virus: 1.37 ± 0.01, and episomal plasmids: 0.04 ± 0.02; PBMCs: Sendai virus: 0.002 ± 0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32 ± 1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.