RESUMO
AIMS: Atrial fibrillation (AF) is the most common type of cardiac arrhythmias, whose incidence is likely to increase with the aging of the population. It is considered a progressive condition, frequently observed as a complication of other cardiovascular disorders. However, recent genetic studies revealed the presence of several mutations and variants linked to AF, findings that define AF as a multifactorial disease. Due to the complex genetics and paucity of models, molecular mechanisms underlying the initiation of AF are still poorly understood. Here we investigate the pathophysiological mechanisms of a familial form of AF, with particular attention to the identification of putative triggering cellular mechanisms, using patient's derived cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: Here we report the clinical case of three siblings with untreatable persistent AF whose whole-exome sequence analysis revealed several mutated genes. To understand the pathophysiology of this multifactorial form of AF we generated three iPSC clones from two of these patients and differentiated these cells towards the cardiac lineage. Electrophysiological characterization of patient-derived CMs (AF-CMs) revealed that they have higher beating rates compared to control (CTRL)-CMs. The analysis showed an increased contribution of the If and ICaL currents. No differences were observed in the repolarizing current IKr and in the sarcoplasmic reticulum calcium handling. Paced AF-CMs presented significantly prolonged action potentials and, under stressful conditions, generated both delayed after-depolarizations of bigger amplitude and more ectopic beats than CTRL cells. CONCLUSIONS: Our results demonstrate that the common genetic background of the patients induces functional alterations of If and ICaL currents leading to a cardiac substrate more prone to develop arrhythmias under demanding conditions. To our knowledge this is the first report that, using patient-derived CMs differentiated from iPSC, suggests a plausible cellular mechanism underlying this complex familial form of AF.
Assuntos
Potenciais de Ação/genética , Fibrilação Atrial/genética , Canais de Cálcio Tipo L/genética , Frequência Cardíaca/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Canais de Cálcio Tipo L/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Resistência a Medicamentos/genética , Predisposição Genética para Doença , Frequência Cardíaca/efeitos dos fármacos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Pessoa de Meia-Idade , Irmãos , Sequenciamento do ExomaRESUMO
BACKGROUND: Three functional ferritin genes have been identified so far in mammals, and they encode the cytosolic Heavy (FTH) and Light chain (FTL) and the mitochondrial ferritin. The expression of a transcript by a fourth ferritin-like gene (Ferritin-Heavy-Polypeptide-Like-17, FTHL17) on the X chromosome was reported in mouse spermatogonia and in early embryonic cells. METHODS: The intronless human FTHL17 gene encodes a protein with 64% identity to human FTH with substitution of key residues of the ferroxidase center. The gene was cloned into vectors for expression in Escherichia coli and mammalian cells, linked to a flag-tag. RESULTS: The recombinant FTHL17 from E. coli purified as an assembled 24-mer ferritin devoid of ferroxidase activity and with a reduced physical stability. When transiently expressed in mammalian cells the flag-FTHL17 assembled in ferritin shells that showed reduced stability to denaturants compared with flag H and L ferritins. Immunocytochemistry with anti-flag antibody decorated the nuclei of flag-FTHL17 transfected COS cells, but not those of the cells transfected with flag-FTH or flag-FTL. CONCLUSIONS: We concluded that FTHL17 encodes a ferritin-like protein without ferroxidase activity. Its restricted embryonic expression and partial nuclear localization suggest that this novel ferritin type may have functions other than iron storage. GENERAL SIGNIFICANCE: The work confirms the presence of a fourth functional human ferritin gene with properties distinct from the canonical cytosolic ones.
Assuntos
Apoferritinas/metabolismo , Núcleo Celular/metabolismo , Sequência de Aminoácidos , Animais , Apoferritinas/química , Apoferritinas/genética , Células COS , Diferenciação Celular , Chlorocebus aethiops , Células-Tronco Embrionárias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Hep G2 , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Desnaturação Proteica , Estabilidade Proteica , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.
RESUMO
Fibroblast growth factor receptor 1 (Fgfr1) gene knockout impairs cardiomyocyte differentiation in murine embryonic stem cells (mESC). Here, various chemical compounds able to enhance cardiomyocyte differentiation in mESC [including dimethylsulfoxide, ascorbic acid (vitC), free radicals and reactive oxygen species] were tested for their ability to rescue the cardiomyogenic potential of Fgfr1(-/-) mESC. Among them, only the reduced form of vitC, l-ascorbic acid, was able to recover beating cell differentiation in Fgfr1(-/-) mESC. The appearance of contracting cells was paralleled by the expression of early and late cardiac gene markers, thus suggesting their identity as cardiomyocytes. In the attempt to elucidate the mechanism of action of vitC on Fgfr1(-/-) mESC, we analyzed several parameters related to the intracellular redox state, such as reactive oxygen species content, Nox4 expression, and superoxide dismutase activity. The results did not show any relationship between the antioxidant capacity of vitC and cardiomyocyte differentiation in Fgfr1(-/-) mESC. No correlation was found also for the ability of vitC to modulate the expression of pluripotency genes. Then, we tested the hypothesis that vitC was acting as a prolyl hydroxylase cofactor by maintaining iron in a reduced state. We first analyze hypoxia inducible factor (HIF)-1α mRNA and protein levels that were found to be slightly upregulated in Fgfr1(-/-) cells. We treated mESC with Fe(2+) or the HIF inhibitor CAY10585 during the first phases of the differentiation process and, similar to vitC, the two compounds were able to rescue cardiomyocyte formation in Fgfr1(-/-) mESC, thus implicating HIF-1α modulation in Fgfr1-dependent cardiomyogenesis.
Assuntos
Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Animais , Antioxidantes/farmacologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The basic idea of displaying peptides on a phage, introduced by George P. Smith in 1985, was greatly developed and improved by McCafferty and colleagues at the MRC Laboratory of Molecular Biology and, later, by Barbas and colleagues at the Scripps Research Institute. Their approach was dedicated to building a system for the production of antibodies, similar to a naïve B cell repertoire, in order to by-pass the standard hybridoma technology that requires animal immunization. Both groups merged the phage display technology with an antibody library to obtain a huge number of phage variants, each of them carrying a specific antibody ready to bind its target molecule, allowing, later on, rare phage (one in a million) to be isolated by affinity chromatography. Here, we will briefly review the basis of the technology and the therapeutic application of phage-derived bioactive molecules when addressed against key players in tumor development and progression: growth factors and their tyrosine kinase receptors.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Técnicas de Visualização da Superfície Celular , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Neoplasias/tratamento farmacológico , Receptores Proteína Tirosina Quinases/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Proteínas do Capsídeo/imunologia , Humanos , Biblioteca de PeptídeosRESUMO
Fibroblast growth factor receptor-1 (Fgfr1) gene knockout impairs cardiac and haematopoietic development in murine embryonic stem cells (mESC). In FGFR1, tyrosine residues Y653 and Y654 are responsible for its tyrosine kinase (TK) activity whereas phosphorylated Y463 and Y766 represent docking sites for intracellular substrates. Aim of this study was the characterization of FGFR1 signalling requirements necessary for cardiomyocyte differentiation in mESC. To this purpose, fgfr1(-/-) mESC were infected with lentiviral vectors harbouring human wild-type hFGFR1 or the Y653/654F, Y463F and Y766F hFGFR1 mutants. The resulting embryonic stem (ES) cell lines were differentiated as embryoid bodies (EBs) and beating foci formation was evaluated. In order to appraise the presence of cells belonging to cardiovascular and haematopoietic lineages, specific markers were analysed by quantitative PCR, whole mount in situ hybridization and immunofluorescence. Transduction with TK(+) hFGFR1 or the TK(+) Y766F-hFGFR1 mutant rescued cardiomyocyte beating foci formation in fgfr1(-/-) EBs whereas the TK(-) Y653/654F-hFGFR1 mutant and the TK(+) Y463F-hFGFR1 mutant were both ineffective. Analysis of the expression of early and late cardiac markers in differentiating EBs confirmed these observations. At variance with cardiomyocyte differentiation, all the transduced TK(+) FGFR1 forms were able to rescue haematopoietic differentiation in EBs originated by infected fgfr1(-/-) mESC, only the TK(-) Y653/654F-hFGFR1 mutant being ineffective. In keeping with these observations, treatment with different signalling pathway inhibitors indicates that protein kinase C and ERK activation are essential for cardiomyocyte but not for haematopoietic differentiation in EBs generated by fgfr1(+/-) approximately mESC. In conclusion, our results suggest that, although FGFR1 kinase activity is necessary for both cardiac and haematopoietic lineage maturation in mESC, phosphorylation of Y463 in the intracellular domain of the receptor is a specific requirement for cardiomyocyte differentiation.