RESUMO
BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.
Assuntos
Síndrome do Coração Esquerdo Hipoplásico/genética , Organogênese/genética , Heterogeneidade Genética , HumanosRESUMO
Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.
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Comunicação Celular , Mecanotransdução Celular , Miócitos Cardíacos/metabolismo , Transativadores/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adipogenia , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Proteínas com Homeodomínio LIM/biossíntese , Camundongos , Camundongos SCID , Miócitos Cardíacos/citologia , Transativadores/genética , Fatores de Transcrição/biossíntese , Proteínas WT1/biossíntese , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Arrhythmogenic Right Ventricular cardiomyopathy (ARVC) is an inherited cardiac muscle disease linked to genetic deficiency in components of the desmosomes. The disease is characterized by progressive fibro-fatty replacement of the right ventricle, which acts as a substrate for arrhythmias and sudden cardiac death. The molecular mechanisms underpinning ARVC are largely unknown. Here we propose a mathematical model for investigating the molecular dynamics underlying heart remodeling and the loss of cardiac myocytes identity during ARVC. Our methodology is based on three computational models: firstly, in the context of the Wnt pathway, we examined two different competition mechanisms between ß-catenin and Plakoglobin (PG) and their role in the expression of adipogenic program. Secondly, we investigated the role of RhoA-ROCK pathway in ARVC pathogenesis, and thirdly we analyzed the interplay between Wnt and RhoA-ROCK pathways in the context of the ARVC phenotype. We conclude with the following remark: both Wnt/ß-catenin and RhoA-ROCK pathways must be inactive for a significant increase of PPARγ expression, suggesting that a crosstalk mechanism might be responsible for mediating ARVC pathogenesis.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Adipogenia/genética , Algoritmos , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/patologia , Células Cultivadas , Simulação por Computador , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Teóricos , PPAR gama/genética , PPAR gama/metabolismo , gama Catenina/metabolismoRESUMO
Wnt proteins can activate different intracellular signaling pathways. These pathways need to be tightly regulated for proper cardiogenesis. The canonical Wnt/ß-catenin inhibitor Dkk1 has been shown to be sufficient to trigger cardiogenesis in gain-of-function experiments performed in multiple model systems. Loss-of-function studies however did not reveal any fundamental function for Dkk1 during cardiogenesis. Using Xenopus laevis as a model we here show for the first time that Dkk1 is required for proper differentiation of cardiomyocytes, whereas specification of cardiomyocytes remains unaffected in absence of Dkk1. This effect is at least in part mediated through regulation of non-canonical Wnt signaling via Wnt11. In line with these observations we also found that Isl1, a critical regulator for specification of the common cardiac progenitor cell (CPC) population, acts upstream of Dkk1.
Assuntos
Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Miocárdio/citologia , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Biomarcadores/metabolismo , Padronização Corporal , Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Regulação para Baixo/genética , Embrião não Mamífero/metabolismo , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/metabolismo , Mesoderma/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Organogênese/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismoRESUMO
During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.
Assuntos
Proteínas de Homeodomínio/biossíntese , Proteínas com Homeodomínio LIM/antagonistas & inibidores , Proteínas com Homeodomínio LIM/biossíntese , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Animais , Linhagem da Célula/fisiologia , Células-Tronco Embrionárias/metabolismo , Células HEK293 , Proteína Homeobox Nkx-2.5 , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/fisiologia , XenopusRESUMO
BACKGROUND: Long-QT syndromes are heritable diseases associated with prolongation of the QT interval on an electrocardiogram and a high risk of sudden cardiac death due to ventricular tachyarrhythmia. In long-QT syndrome type 1, mutations occur in the KCNQ1 gene, which encodes the repolarizing potassium channel mediating the delayed rectifier I(Ks) current. METHODS: We screened a family affected by long-QT syndrome type 1 and identified an autosomal dominant missense mutation (R190Q) in the KCNQ1 gene. We obtained dermal fibroblasts from two family members and two healthy controls and infected them with retroviral vectors encoding the human transcription factors OCT3/4, SOX2, KLF4, and c-MYC to generate pluripotent stem cells. With the use of a specific protocol, these cells were then directed to differentiate into cardiac myocytes. RESULTS: Induced pluripotent stem cells maintained the disease genotype of long-QT syndrome type 1 and generated functional myocytes. Individual cells showed a "ventricular," "atrial," or "nodal" phenotype, as evidenced by the expression of cell-typespecific markers and as seen in recordings of the action potentials in single cells. The duration of the action potential was markedly prolonged in "ventricular" and "atrial" cells derived from patients with long-QT syndrome type 1, as compared with cells from control subjects. Further characterization of the role of the R190QKCNQ1 mutation in the pathogenesis of long-QT syndrome type 1 revealed a dominant negative trafficking defect associated with a 70 to 80% reduction in I(Ks) current and altered channel activation and deactivation properties. Moreover, we showed that myocytes derived from patients with long-QT syndrome type 1 had an increased susceptibility to catecholamine-induced tachyarrhythmia and that beta-blockade attenuated this phenotype. CONCLUSIONS: We generated patient-specific pluripotent stem cells from members of a family affected by long-QT syndrome type 1 and induced them to differentiate into functional cardiac myocytes. The patient-derived cells recapitulated the electrophysiological features of the disorder. (Funded by the European Research Council and others.)
Assuntos
Potenciais de Ação , Células-Tronco Pluripotentes Induzidas/fisiologia , Canal de Potássio KCNQ1/genética , Miócitos Cardíacos/citologia , Síndrome de Romano-Ward/fisiopatologia , Antagonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Cardiotônicos/farmacologia , Criança , Feminino , Fibroblastos/citologia , Expressão Gênica , Humanos , Isoproterenol/farmacologia , Fator 4 Semelhante a Kruppel , Masculino , Mutação de Sentido Incorreto , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Linhagem , Fenótipo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Romano-Ward/tratamento farmacológico , Síndrome de Romano-Ward/genéticaAssuntos
Arritmias Cardíacas/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Potenciais de Ação/fisiologia , Antiarrítmicos/farmacologia , Estudos de Casos e Controles , Cisaprida/farmacologia , Avaliação de Medicamentos/métodos , Humanos , Ivabradina/farmacologia , Síndrome do QT Longo/fisiopatologia , Proteínas Luminescentes/metabolismo , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Bloqueadores dos Canais de Potássio/farmacologiaRESUMO
Domestic pigs (Sus scrofa) share many genetic, anatomical, and physiological traits with humans and therefore constitute an excellent preclinical animal model. Fundamental understanding of the cellular and molecular processes governing early porcine cardiogenesis is critical for developing advanced porcine models used for the study of heart diseases and new regenerative therapies. Here, we provide a detailed characterization of porcine cardiogenesis based on fetal porcine hearts at various developmental stages and cardiac cells derived from porcine expanded pluripotent stem cells (pEPSCs), i.e., stem cells having the potential to give rise to both embryonic and extraembryonic tissue. We notably demonstrate for the first time that pEPSCs can differentiate into cardiovascular progenitor cells (CPCs), functional cardiomyocytes (CMs), epicardial cells and epicardial-derived cells (EPDCs) in vitro. Furthermore, we present an enhanced system for whole-embryo culture which allows continuous ex utero development of porcine post-implantation embryos from the cardiac crescent stage (ED14) up to the cardiac looping (ED17) stage. These new techniques provide a versatile platform for studying porcine cardiac development and disease modeling.
RESUMO
The epicardium, the mesothelial envelope of the vertebrate heart, is the source of multiple cardiac cell lineages during embryonic development and provides signals that are essential to myocardial growth and repair. Here we generate self-organizing human pluripotent stem cell-derived epicardioids that display retinoic acid-dependent morphological, molecular and functional patterning of the epicardium and myocardium typical of the left ventricular wall. By combining lineage tracing, single-cell transcriptomics and chromatin accessibility profiling, we describe the specification and differentiation process of different cell lineages in epicardioids and draw comparisons to human fetal development at the transcriptional and morphological levels. We then use epicardioids to investigate the functional cross-talk between cardiac cell types, gaining new insights into the role of IGF2/IGF1R and NRP2 signaling in human cardiogenesis. Finally, we show that epicardioids mimic the multicellular pathogenesis of congenital or stress-induced hypertrophy and fibrotic remodeling. As such, epicardioids offer a unique testing ground of epicardial activity in heart development, disease and regeneration.
Assuntos
Coração , Pericárdio , Humanos , Pericárdio/metabolismo , Miocárdio , Diferenciação Celular/genética , Linhagem da Célula/genética , BiologiaRESUMO
Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.
Assuntos
Células-Tronco Pluripotentes , Tretinoína , Humanos , Animais , Camundongos , Tretinoína/farmacologia , Coração , Miocárdio , Diferenciação Celular , Miócitos CardíacosRESUMO
Rhoh is a hematopoietic system-specific GTPase. Rhoh-deficient T cells have been shown to have a defect in TCR signaling manifested during their thymic development. Our aims were to investigate the phenotype of peripheral Rhoh-deficient T cells and to explore in vivo the potential benefit of Rhoh deficiency in a clinically relevant situation, in which T-cell inhibition is desirable. In murine allogenic kidney transplantation, Rhoh deficiency caused a significant 75% reduction of acute and chronic transplant rejection accompanied by 75% lower alloantigen-specific antibody levels and significantly better graft function. This effect was independent of the lower T-cell numbers in Rhoh-deficient recipients, because injection of equal numbers of Rhoh-deficient or control T cells into kidney transplanted mice with SCID led again to a significant 60% reduction of rejection. Mixed lymphocyte reaction revealed that the weaker alloreactivity was associated with a 85% lower cytotoxicity and a 50-80% lower cytokine release in Rhoh-deficient T cells without an influence on the secretion itself. Antigen uptake and presentation in DC were unaffected by Rhoh deficiency. These findings stress the importance of Rhoh for the function of peripheral T cells.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Rim/imunologia , Linfócitos T/imunologia , Fatores de Transcrição/imunologia , Proteínas rho de Ligação ao GTP/imunologia , Doença Aguda , Animais , Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Doença Crônica , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Rejeição de Enxerto/genética , Isoantígenos/imunologia , Transplante de Rim/patologia , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Camundongos SCID , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
TRPM4 is a Ca2+-activated channel mediating the transport of monovalent cations across the cell membrane. Mutations in the TRPM4 gene have been associated with cardiac arrhythmias in humans. Using CRISPR/Cas9 gene editing technology, we established two TRPM4 knockout human iPSC lines - one heterozygous (MRli003-A-3) and one homozygous (MRli003-A-4) - by inserting a frameshift mutation in exon 2 of the TRPM4 gene. Both lines maintained pluripotency, a normal karyotype, parental cell morphology, and the ability to differentiate into the three germ layers.
Assuntos
Células-Tronco Pluripotentes Induzidas , Canais de Cátion TRPM , Sistemas CRISPR-Cas/genética , Edição de Genes , Heterozigoto , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) represent an excellent in vitro model in cardiovascular research. Changes in their action potential (AP) dynamics convey information that is essential for disease modeling, drug screening and toxicity evaluation. High-throughput optical AP recordings utilizing intramolecular Förster resonance energy transfer (FRET) of the voltage-sensitive fluorescent protein (VSFP) have emerged as a substitute or complement to the resource-intensive patch clamp technique. Here, we functionally validated our recently generated voltage indicator hiPSC lines stably expressing CAG-promoter-driven VSFP in the AAVS1 safe harbor locus. By combining subtype-specific cardiomyocyte differentiation protocols, we established optical AP recordings in ventricular, atrial, and nodal CMs in 2D monolayers using fluorescence microscopy. Moreover, we achieved high-throughput optical AP measurements in single hiPSC-derived CMs in a 3D context. Overall, this system greatly expands the spectrum of possibilities for high-throughput, non-invasive and long-term AP analyses in cardiovascular research and drug discovery.
RESUMO
Assessment of the electrophysiological properties of cardiomyocytes is necessary for phenotyping cardiac disorders and for drug screening. Optical action potential imaging using a genetically encoded voltage-sensing fluorescent protein (VSFP) allows for high-throughput functional characterization of cardiomyocytes, which offers an advantage over the traditional patch-clamp technique. Here, we knocked VSFP into the AAVS1 safe harbor locus of human iPSCs, generating two stable voltage indicator lines - one heterozygous (MRIi003-A-5) and the other homozygous (MRI003-A-6). Both lines can be used for optical membrane potential recordings and provide a powerful platform for a wide range of applications in cardiovascular biomedicine.
Assuntos
Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the de novo coenzyme A (CoA) synthesis starting from pantothenate. Mutations in PPCS cause autosomal-recessive dilated cardiomyopathy, often fatal, without apparent neurodegeneration, whereas pathogenic variants in PANK2 and COASY, two other genes involved in the CoA synthesis, cause Neurodegeneration with Brain Iron Accumulation (NBIA). PPCS-deficiency is a relatively new disease with unclear pathogenesis and no targeted therapy. Here, we report the generation of induced pluripotent stem cells from fibroblasts of two PPCS-deficient patients. These cellular models could represent a platform for pathophysiological studies and testing of therapeutic compounds for PPCS-deficiency.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Coenzima A , Fibroblastos , Humanos , Mutação/genéticaRESUMO
Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current Iks. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.
Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/metabolismo , MicroRNAs/metabolismo , Arritmias Cardíacas/genética , Perfilação da Expressão Gênica , Células HEK293 , Ventrículos do Coração/fisiopatologia , Humanos , Síndrome do QT Longo/genética , MicroRNAs/genética , Miocárdio , Miócitos CardíacosRESUMO
Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host-graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.
Assuntos
Proteínas do Tecido Nervoso , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Cicatriz/patologia , Cicatriz/prevenção & controle , Fibrose , Humanos , Miocárdio/patologia , Miócitos Cardíacos/patologia , Células-Tronco Pluripotentes/patologia , Receptores Imunológicos , SuínosRESUMO
Myosin-10, also known as non-muscle myosin IIB, is a cytoskeletal protein implicated in cardiac development and disease. In humans, it is encoded by the MYH10 gene. Using CRISPR/Cas9 gene editing technology, we generated two MYH10 knockout human iPSC lines - one heterozygous (MRli003-A-1) and one homozygous (MRli003-A-2) - by introducing a frameshift deletion in exon 2. We then verified that both lines had maintained pluripotency, parental cell morphology, trilineage differentiation potential and a normal karyotype.
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Bio-engineered myocardium has great potential to substitute damaged myocardium and for studies of myocardial physiology and disease, but structural and functional immaturity still implies limitations. Current protocols of engineered heart tissue (EHT) generation fall short of simulating the conditions of postnatal myocardial growth, which are characterized by tissue expansion and increased mechanical load. To investigate whether these two parameters can improve EHT maturation, we developed a new approach for the generation of cardiac tissues based on biomimetic stimulation under application of continuously increasing stretch. Methods: EHTs were generated by assembling cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) at high cell density in a low collagen hydrogel. Maturation and growth of the EHTs were induced in a custom-made biomimetic tissue culture system that provided continuous electrical stimulation and medium agitation along with progressive stretch at four different increments. Tissues were characterized after a three week conditioning period. Results: The highest rate of stretch (S3 = 0.32 mm/day) increased force development by 5.1-fold compared to tissue with a fixed length, reaching contractility of 11.28 mN/mm². Importantly, intensely stretched EHTs developed physiological length-dependencies of active and passive forces (systolic/diastolic ratio = 9.47 ± 0.84), and a positive force-frequency relationship (1.25-fold contractility at 180 min-1). Functional markers of stretch-dependent maturation included enhanced and more rapid Ca2+ transients, higher amplitude and upstroke velocity of action potentials, and pronounced adrenergic responses. Stretch conditioned hiPSC-CMs displayed structural improvements in cellular volume, linear alignment, and sarcomere length (2.19 ± 0.1 µm), and an overall upregulation of genes that are specifically expressed in adult cardiomyocytes. Conclusions: With the intention to simulate postnatal heart development, we have established techniques of tissue assembly and biomimetic culture that avoid tissue shrinkage and yield muscle fibers with contractility and compliance approaching the properties of adult myocardium. This study demonstrates that cultivation under progressive stretch is a feasible way to induce growth and maturation of stem cell-derived myocardium. The novel tissue-engineering approach fulfills important requirements of disease modelling and therapeutic tissue replacement.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio , Miócitos Cardíacos/citologia , Estresse Mecânico , Técnicas de Cultura de Tecidos , Engenharia Tecidual , Materiais Biomiméticos , Reatores Biológicos , Tamanho Celular , Diástole , Estimulação Elétrica , Acoplamento Excitação-Contração , Humanos , Hidrogéis , Fusos Musculares , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Organoides , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Sístole , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodosRESUMO
Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts.