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.
Assuntos
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
Mutations in Dystrophin, one of the largest proteins in the mammalian body, are causative for a severe form of muscle disease, Duchenne Muscular Dystrophy (DMD), affecting not only skeletal muscle, but also the heart. In particular, exons 45-52 constitute a hotspot for DMD mutations. A variety of molecular therapies have been developed, comprising vectors encoding micro- and minidystrophins as well as utrophin, a protein with partially overlapping functions. With the advent of the CRISPR-Cas9-nuclease, genome editing offers a novel option of correction of the disease-cuasing mutations. Full restoration of the healthy gene by homology directed repair is a rare event. However, non-homologous end-joining (NHEJ) may restore the reading frame by causing exon excision. This approach has first been demonstrated in mice and then translated to large animals (dogs, pigs). This review discusses the potential opportunities and limitations of genome editing in DMD, including the generation of appropriate animal models as well as new developments in genome editing tools.
Assuntos
Distrofia Muscular de Duchenne , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Cães , Edição de Genes , Terapia Genética , Camundongos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , SuínosRESUMO
For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/tendências , Terapia Genética/tendências , Pesquisa Translacional Biomédica/tendências , Animais , Congressos como Assunto , Difusão de Inovações , Modelos Animais de Doenças , Previsões , Predisposição Genética para Doença , Humanos , FenótipoRESUMO
The transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cation channel linked to human cardiac diseases. The human mutation K914R within TRPM4's S4-S5 linker was identified in patients with atrioventricular block. During UV-flash-mediated Ca2+ transients, TRPM4K914R generated a threefold augmented membrane current concomitant with 2 to 3-fold slowed down activation and deactivation kinetics resulting in excessive membrane currents during human cardiac action potentials. Mutagenesis of K914 paired with molecular modeling suggested the importance of the nanoscopic interface between the S4-S5 linker, the MHR4-, and TRP-domain as a major determinant for TRPM4's behavior. Rational mutagenesis of an interacting amino acid (R1062Q) in the TRP domain was able to offset K914R`s gain-of-function by zipping and unzipping of this nanoscopic interface. In conclusion, repulsion and attraction between the amino acids at positions 914 and 1062 alters the flexibility of the nanoscopic interface suggesting a zipping and unzipping mechanism that modulates TRPM4's functions. Pharmacological modulation of this intramolecular mechanism might represent a novel therapeutic strategy for the management of TRPM4-mediated cardiac diseases.
Assuntos
Potenciais de Ação , Sinalização do Cálcio , Cálcio/metabolismo , Sistema de Condução Cardíaco/metabolismo , Cardiopatias/metabolismo , Canais de Cátion TRPM/metabolismo , Substituição de Aminoácidos , Células HEK293 , Sistema de Condução Cardíaco/patologia , Cardiopatias/genética , Cardiopatias/patologia , Humanos , Mutação de Sentido Incorreto , Canais de Cátion TRPM/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
Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Mutação , Células-Tronco Pluripotentes , Potenciais de Ação/genética , Adulto , Células Cultivadas , Canal de Potássio ERG1 , Células-Tronco Embrionárias/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Fenótipo , Células-Tronco Pluripotentes/fisiologia , Transporte Proteico/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND & AIMS: The proportion of HCV-infected patients over age 65 years in Western countries is increasing. This growth and the advent of new antiviral therapy bring into the question the real-world efficacy and safety of the combination of sofosbuvir (SOF) and simeprevir (SMV) plus a flat dose of 800 mg/d ribavirin (RBV) in elderly patients with cirrhosis compared to younger patients. METHODS: Retrospective observational multicentre real-life investigation study of SOF/SMV/RBV for a duration of 12 weeks in HCV genotype 1-infected patients with cirrhosis. RESULTS: Of the 270 patients enrolled in this study, with compensated cirrhosis, 133 (49.2%) were ≥65 years of age. Sustained virological response at 12 weeks (SVR12) was achieved by 94.2% (129/137) of those aged <65 years and 97.7% (130/133) of those ≥65 years. Diabetes was the most common comorbidity in patients ≥65 years compared to younger patients (26.3% vs 12.4% P<.003). The most common adverse event (AE) in elderly patients was a grade 2 anaemia (35.3% vs 19.9% P<.004). CONCLUSIONS: Sofosbuvir/simeprevir plus a daily flat dose of RBV 800 mg for 12 weeks was highly effective and safe in genotype 1 elderly patients with compensated cirrhosis.
Assuntos
Antivirais/administração & dosagem , Hepatite C Crônica/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Ribavirina/administração & dosagem , Simeprevir/administração & dosagem , Sofosbuvir/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/etiologia , Antivirais/efeitos adversos , Esquema de Medicação , Quimioterapia Combinada , Feminino , Hepacivirus/genética , Humanos , Itália , Cirrose Hepática/virologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Retrospectivos , Ribavirina/efeitos adversos , Simeprevir/efeitos adversos , Sofosbuvir/efeitos adversos , Resposta Viral SustentadaRESUMO
PURPOSE OF REVIEW: The promises of human-induced pluripotent stem cells (hiPSCs) for modeling arrhythmogenic disease, but also for drug discovery and toxicity tests, are straightforward and exciting. However, the full potential of this new technology has not been fully realized yet. The purpose of this review is to provide an overview of the state-of-the-art research in arrhythmogenic disease modeling and drug discovery and an outlook of what can be expected from the second decade of hiPSC-based arrhythmia research. RECENT FINDINGS: Remarkable advances in genomic discoveries, stem cell biology, and genome editing via sequence-specific nucleases have been made in recent years. Together, these breakthroughs have allowed us to progress from studying monogenetic diseases with a direct genotype-phenotype relationship to genetically more complex diseases such as arrhythmogenic right ventricular dysplasia and atrial fibrillation. In addition, newly developed tools for arrhythmia research such as optical action potential recordings have facilitated the use of hiPSCs for drug and toxicity screening and their eventual clinical use. These advances in in vitro assay development, genome editing, and stem cell biology will soon enable the implementation of hiPSC-based findings into clinical practice and provide us with unprecedented insights into mechanisms of complex arrhythmogenic diseases.
Assuntos
Arritmias Cardíacas/genética , Descoberta de Drogas , Células-Tronco Pluripotentes Induzidas , Arritmias Cardíacas/fisiopatologia , Displasia Arritmogênica Ventricular Direita/genética , Diferenciação Celular , Técnicas de Reprogramação Celular , Edição de Genes , Humanos , Miócitos Cardíacos/efeitos dos fármacosRESUMO
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
The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3-labeled sense oligonucleotide reporter strands coupled to gold-particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target-specific probe we could successfully demonstrate expression of the GAPDH house-keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence-activated cell sorting. After lentiviral transduction of murine tail-tip fibroblasts Nanog-specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3-fluorescence. The intensity of Nanog-specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high-throughput cell reprogramming and after genomic editing of pluripotent stem cells.
Assuntos
Carbocianinas/farmacologia , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Oligonucleotídeos/farmacologia , Animais , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Transgênicos , SuínosRESUMO
Induced pluripotent stem cells offer the possibility to generate patient-specific stem cell lines from individuals affected by inherited disorders. Cardiomyocytes differentiated from such patient-specific induced pluripotent stem cells lines have been used to study the pathophysiology of arrhythmogenic heart diseases, such as the long-QT syndrome or catecholaminergic polymorphic ventricular tachycardia. Testing for unwanted drug side effects or tailoring medical treatment to the specific needs of individual patients with arrhythmogenic disorders may become future applications of this emerging technology.
Assuntos
Arritmias Cardíacas/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Antiarrítmicos/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/patologia , Miócitos Cardíacos/efeitos dos fármacos , Fenótipo , Medicina de Precisão/métodos , Taquicardia Ventricular/tratamento farmacológico , Taquicardia Ventricular/patologiaRESUMO
Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG), leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS, â¼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG), which encode two repolarizing potassium currents known as I(Ks) and I(Kr). The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I(Kr) by reducing voltage sensitivity of inactivation, not via slowing of deactivation, could more effectively restore normal QT duration if I(Ks) is reduced. Using a unique specific chemical activator for I(Kr) that has a primary effect of causing a right shift of V(1/2) for inactivation, we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed, this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast, an I(Kr) chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent.
Assuntos
Potenciais de Ação/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio KCNQ/metabolismo , Síndrome do QT Longo/genética , Modelos Biológicos , Acetamidas/farmacologia , Animais , Células CHO , Células Cultivadas , Simulação por Computador , Cricetinae , Cricetulus , Primers do DNA/genética , Humanos , Canais de Potássio KCNQ/fisiologia , Síndrome do QT Longo/patologia , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Piridinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.
Assuntos
Técnicas Biossensoriais , Potenciais da Membrana/genética , Miócitos Cardíacos/metabolismo , Potenciais de Ação/genética , Animais , Animais Geneticamente Modificados , Sistema Cardiovascular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Genes Reporter , Humanos , Proteínas Recombinantes de Fusão/genética , Pesquisa , Imagens com Corantes Sensíveis à VoltagemRESUMO
The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSC) by overexpression of a combination of transcription factors bears the potential to spawn a wealth of new applications in both preclinical and clinical cardiovascular research. Disease modeling, which is accomplished by deriving iPSC lines from patients affected by heritable diseases and then studying the pathophysiology of the diseases in somatic cells differentiated from these patient-specific iPSC lines, is the so far most advanced of these applications. Long-QT syndrome and catecholaminergic polymorphic ventricular tachycardia are two heart rhythm disorders that have been already successfully modeled by several groups using this approach, which will likely serve to model other mono- or polygenetic cardiovascular disorders in the future. Test systems based on cells derived from iPSC might prove beneficial to screen for novel cardiovascular drugs or unwanted drug side effects and to individualize medical therapy. The application of iPSC for cell therapy of cardiovascular disorders, albeit promising, will only become feasible if the problem of biological safety of these cells will be mastered.
Assuntos
Doenças Cardiovasculares/terapia , Células-Tronco Pluripotentes Induzidas/citologia , Medicina Regenerativa/métodos , Animais , Pesquisa Biomédica , Técnicas de Cultura de Células , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Desenho de Fármacos , Humanos , Síndrome do QT Longo/fisiopatologia , Síndrome do QT Longo/terapia , Taquicardia Ventricular/fisiopatologia , Taquicardia Ventricular/terapiaRESUMO
Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca(2+) concentration. The analysis of contractile function and Ca(2+) transients is therefore important to discriminate between myofilament responsiveness and changes in Ca(2+) homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca(2+) transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca(2+) and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca(2+) concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca(2+) (EC50: 0.15, 0.39, and 1.05 mM Ca(2+) in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca(2+) sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca(2+) concentration is due to changes in Ca(2+) handling proteins. Finally, this study confirms that fura-2 has Ca(2+) buffering effects and is thereby changing the force response to extracellular Ca(2+).
Assuntos
Sinalização do Cálcio , Microscopia de Fluorescência/métodos , Contração Miocárdica , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Engenharia Tecidual/métodos , Animais , Automação Laboratorial , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/instrumentação , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Ratos , Ratos Endogâmicos Lew , Ratos WistarRESUMO
The mandatory usage of extracellular matrix (ECM) gels in 3D cultures limits antibody penetration and increases background, while the removal of ECM gel causes disruption of morphology and sample loss. These factors pose challenges to effective immune labeling-based staining. Here, we present a protocol for whole-mount immunofluorescence staining of gel-embedded pancreatic organoids. We describe steps for sample fixation, blocking, and antibody incubation. We detail procedures for washing antibodies and mounting.
Assuntos
Matriz Extracelular , Imunofluorescência , Organoides , Pâncreas , Organoides/citologia , Organoides/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Pâncreas/citologia , Pâncreas/metabolismo , Imunofluorescência/métodos , Animais , Coloração e Rotulagem/métodos , Humanos , Géis/química , CamundongosRESUMO
OBJECTIVES: Here we investigate Hepatitis D virus (HDV)-prevalence in Italy and its fluctuations over time and we provide an extensive characterization of HDV-infected patients. METHODS: The rate of HDV seroprevalence and HDV chronicity was assessed in 1579 hepatitis B surface antigen (HBsAg)+ patients collected from 2005 to 2022 in Central Italy. RESULTS: In total, 45.3% of HBsAg+ patients received HDV screening with an increasing temporal trend: 15.6% (2005-2010), 45.0% (2011-2014), 49.4% (2015-2018), 71.8% (2019-2022). By multivariable model, factors correlated with the lack of HDV screening were alanine-aminotransferase (ALT) less than two times of upper limit of normality (<2ULN) and previous time windows (P <0.002). Furthermore, 13.4% of HDV-screened patients resulted anti-HDV+ with a stable temporal trend. Among them, 80.8% had detectable HDV-ribonucleic acid (RNA) (median [IQR]:4.6 [3.6-5.6] log copies/ml) with altered ALT in 89.3% (median [IQR]:92 [62-177] U/L). Anti-HDV+ patients from Eastern/South-eastern Europe were younger than Italians (44 [37-54] vs 53 [47-62] years, P <0.0001), less frequently nucleos(t)ide analogs (NUC)-treated (58.5% vs 80%, P = 0.026) with higher HDV-RNA (4.8 [3.6-5.8] vs 3.9 [1.4-4.9] log copies/ml, P = 0.016) and HBsAg (9461 [4159-24,532] vs 4447 [737-13,336] IU/ml, P = 0.032). Phylogenetic analysis revealed the circulation of HDV subgenotype 1e (47.4%) and -1c (52.6%). Notably, subgenotype 1e correlated with higher ALT than 1c (168 [89-190] vs 58 [54-88] U/l, P = 0.015) despite comparable HDV-RNA. CONCLUSIONS: HDV-screening awareness is increasing over time even if some gaps persist to achieve HDV screening in all HBsAg+ patients. HDV prevalence in tertiary care centers tend to scarcely decline in native/non-native patients. Detection of subgenotypes, triggering variable inflammatory stimuli, supports the need to expand HDV molecular characterization.
Assuntos
Hepatite D , Vírus Delta da Hepatite , Humanos , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B , Hepatite D/diagnóstico , Hepatite D/epidemiologia , Vírus Delta da Hepatite/genética , Itália/epidemiologia , Filogenia , Prevalência , RNA , Estudos Soroepidemiológicos , Replicação Viral , Adulto , Pessoa de Meia-IdadeRESUMO
Background and aims: Management of severe thrombocytopenia poses significant challenges in patients with chronic liver disease. Here, we aimed to evaluate the first real-world European post-marketing cohort of cirrhotic patients treated with lusutrombopag, a thrombopoietin receptor agonist, verifying the efficacy and safety of the drug. Methods: In the REAl-world Lusutrombopag treatment in ITalY (REALITY) study, we collected data from consecutive cirrhotic patients treated with lusutrombopag in 19 Italian hepatology centers, mostly joined to the "Club Epatologi Ospedalieri" (CLEO). Primary and secondary efficacy endpoints were the ability of lusutrombopag to avoid platelet transfusions and to raise the platelet count to ≥50,000/µL, respectively. Treatment-associated adverse events were also collected. Results: A total of 66 patients and 73 cycles of treatment were included in the study, since 5 patients received multiple doses of lusutrombopag over time for different invasive procedures. Fourteen patients (19%) had a history of portal vein thrombosis (PVT). Lusutrombopag determined a significant increase in platelet count [from 37,000 (33,000-44,000/µL) to 58,000 (49,000-82,000), p < 0.001]. The primary endpoint was met in 84% of patients and the secondary endpoint in 74% of patients. Baseline platelet count was the only independent factor associated with response in multivariate logistic regression analysis (OR for any 1000 uL of 1.13, CI95% 1.04-1.26, p 0.01), with a good discrimination power (AUROC: 0.78). Notably, a baseline platelet count ≤ 29,000/µL was identified as the threshold for identifying patients unlikely to respond to the drug (sensitivity of 91%). Finally, de novo PVT was observed in four patients (5%), none of whom had undergone repeated treatment, and no other safety or hemorrhagic events were recorded in the entire population analyzed. Conclusions: In this first European real-world series, lusutrombopag demonstrated efficacy and safety consistent with the results of registrational studies. According to our results, patients with baseline platelet counts ≤29,000/µL are unlikely to respond to the drug.