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BACKGROUND/AIMS: Mutations of desmosomal genes are known to cause arrhythmogenic cardiomyopathy characterized by arrhythmias and sudden cardiac death. Previously, we described a novel genetic variant H1684R in desmoplakin gene (DSP), associated with a progressive cardiac conduction disease (PCCD). In the present study, we aimed to investigate an effect of the DSP-H1684R genetic variant on the activity of ion channels. METHODS: We used cardiomyocytes derived from induced pluripotent stem cells (iPSC cardiomyocytes) from a patient with DSP-H1684R genetic variant and from two healthy donors. Immunofluorescent staining and western blot analyses were used to characterize patient-specific cardiomyocytes. By the whole-cell voltage-clamp technique we estimated the activity of voltage-gated sodium, calcium, and potassium channels that are responsible for action potential generation and its shape. Action potentials' parameters were measured using whole-cell current-clamp technique. RESULTS: In patient-specific cardiomyocytes we observed both lower amplitudes of currents through sodium Nav1.5 channels and L-type calcium channels, but higher amplitude of current through transient-outward potassium channels in comparison to donor cardiomyocytes. Current-clamp measurements revealed shortening of action-potential in DSP-H1684R-carrying iPSC cardiomyocytes. Therefore, observed alterations in the channels activity might have a great impact on the properties of action potential and development of PCCD. CONCLUSION: Our results show that desmoplakin genetic variants, besides conduction slowing caused by structural heart remodeling, could affect multiple ion channel activity aggravating arrhythmia manifestation in PCCD.
Assuntos
Doença do Sistema de Condução Cardíaco/genética , Desmoplaquinas/genética , Bloqueio Cardíaco/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Canais Iônicos/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Potenciais de Ação/fisiologia , Canais de Cálcio/fisiologia , Doença do Sistema de Condução Cardíaco/metabolismo , Desmoplaquinas/metabolismo , Imunofluorescência , Bloqueio Cardíaco/metabolismo , Humanos , Canais Iônicos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Canais de Sódio Disparados por Voltagem/fisiologiaRESUMO
Human iPSC cell line FAMRCi010-A was generated from a patient with restrictive cardiomyopathy carrying FLNC p.Gly2011Arg genetic variant. Patient-specific peripheral blood mononuclear cells were reprogrammed using non-integrative Sendai viruses containing OCT4, SOX2, KLF4, and CMYC. FAMRCi010-A was generated and characterized through the study. The reported iPSC line could be useful tool for in vitro modeling of FLNC-associated cardiomyopathies.
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Human iPSC cell line FAMRCi009-A was generated from a patient with restrictive cardiomyopathy and congenital myopathy carrying FLNC p.Val2264Met genetic variant. Patient-specific peripheral blood mononuclear cells were reprogrammed using non-integrative Sendai viruses. Generated iPSC lines showed normal karyotype, expressed pluripotency markers and exhibited trilineage differentiation potential in vitro. The reported iPSC lines could be used for a deeper study of filaminopathies.
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Pathogenic variants in the LMNA gene are known to cause laminopathies, a broad range of disorders with different clinical phenotypes. LMNA genetic variants lead to tissue-specific pathologies affecting various tissues and organs. Common manifestations of laminopathies include cardiovascular system abnormalities, in particular, cardiomyopathies and conduction disorders. In the present study, we used induced pluripotent stem cells from a patient carrying LMNA p.R249Q genetic variant to create an in vitro cardiac model of laminopathy. Induced pluripotent stem cell-derived cardiomyocytes with LMNA p.R249Q genetic variant showed a decreased sodium current density and an impaired sodium current kinetics alongside with changes in transcription levels of cardiac-specific genes. Thus, we obtained compelling in vitro evidence of an association between LMNA p.R249Q genetic variant and cardiac-related abnormalities.
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A-type lamins are the main structural components of the nucleus, which are mainly localized at the nucleus periphery. First of all, A-type lamins, together with B-type lamins and proteins of the inner nuclear membrane, form a stiff structure-the nuclear lamina. Besides maintaining the nucleus cell shape, A-type lamins play a critical role in many cellular events, such as gene transcription and epigenetic regulation. Nowadays it is clear that lamins play a very important role in determining cell fate decisions. Various mutations in genes encoding A-type lamins lead to damages of different types of tissues in humans, collectively known as laminopathies, and it is clear that A-type lamins are involved in the regulation of cell differentiation and stemness. However, the mechanisms of this regulation remain unclear. In this review, we discuss how A-type lamins can execute their regulatory role in determining the differentiation status of a cell. We have summarized recent data focused on lamin A/C action mechanisms in regulation of cell differentiation and identity development of stem cells of different origin. We also discuss how this knowledge can promote further research toward a deeper understanding of the role of lamin A/C mutations in laminopathies.
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Human iPSC cell lines (FAMRCi004-A and FAMRCi004-B) were generated from patient with progressive cardiac conduction disease and sick sinus syndrome carrying DSP p.His1684Arg genetic variant. Patient-specific adipose tissue-derived mesenchymal multipotent stromal cells were reprogrammed using non-integrative Sendai viruses. Established iPSC lines showed normal karyotype, expressed pluripotent markers and were able to differentiate toward three germ layers in vitro. The reported iPSC lines could be useful tool for in vitro modeling of progressive cardiac conduction disease associated with mutations in desmosomal genes.
Assuntos
Doença do Sistema de Condução Cardíaco/genética , Desmoplaquinas/genética , Bloqueio Cardíaco/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular , HumanosRESUMO
LMNA mutations are often linked to laminopathies characterized by tissue-specific disorders. We generated two induced pluripotent stem cells lines from patient carrying genetic variant LMNA p.Asp357Val associated with paroxysmal ventricular tachycardia and myopathy. Reprogramming of patient's peripheral blood mononuclear cells was performed using Sendai viruses. Characterization of the FAMRCi005-A and FAMRCi005-B lines revealed that generated iPSC lines expressed pluripotent stem cell markers, had normal karyotype and demonstrated triliniage differentiation ability. Generated cell lines can be used to investigate the molecular links between LMNA genetic variants and cardiac disorders.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Lamina Tipo A/genética , Adulto , Diferenciação Celular , Feminino , HumanosRESUMO
Mutations in LMNA gene are known to cause a broad range of diseases called laminopathies. We have generated two induced pluripotent stem cell lines FAMRCi006-A and FAMRCi006-B from a patient carrying LMNA p. p.Arg527Pro mutation associated with Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. Patient-specific peripheral blood mononuclear cells were reprogrammed to iPSCs using Sendai virus reprogramming system. Characterization of iPSCs had revealed pluripotency marker expression, normal karyotype, ability to differentiate into three embryonic germ layers. The reported iPSC lines could be a useful tool for in vitro modeling of laminopathies associated with LMNA genetic variants.
Assuntos
Cardiomiopatia Dilatada/economia , Cardiomiopatia Dilatada/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Feminino , Humanos , Pessoa de Meia-Idade , MutaçãoRESUMO
BACKGROUND: Mutations in desmosomal genes linked to arrhythmogenic cardiomyopathy are commonly associated with Wnt/ß-catenin signaling abnormalities and reduction of the sodium current density. Inhibitors of GSK3B were reported to restore sodium current and improve heart function in various arrhythmogenic cardiomyopathy models, but mechanisms underlying this effect remain unclear. We hypothesized that there is a crosstalk between desmosomal proteins, signaling pathways, and cardiac sodium channels. METHODS AND RESULTS: To reveal molecular mechanisms of arrhythmogenic cardiomyopathy, we established human iPSC-based model of this pathology. iPSC-derived cardiomyocytes from patient carrying two genetic variants in PKP2 gene demonstrated that PKP2 haploinsufficiency due to frameshift variant, in combination with the missense variant expressed from the second allele, was associated with decreased Wnt/ß-catenin activity and reduced sodium current. Different approaches were tested to restore impaired cardiomyocytes functions, including wild type PKP2 transduction, GSK3B inhibition and Wnt/ß-catenin signaling modulation. Inhibition of GSK3B led to the restoration of both Wnt/ß-catenin signaling activity and sodium current density in patient-specific cardiomyocytes while GSK3B activation led to the reduction of sodium current density. Moreover, we found that upon inhibition GSK3B sodium current was restored through Wnt/ß-catenin-independent mechanism. CONCLUSION: We propose that alterations in GSK3B-Wnt/ß-catenin signaling pathways lead to regulation of sodium current implying its role in molecular pathogenesis of arrhythmogenic cardiomyopathy.
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Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Placofilinas/metabolismo , Sódio/metabolismo , Eletrofisiologia , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Mutação/genética , Técnicas de Patch-Clamp , Placofilinas/genética , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologiaRESUMO
Human iPSC lines were generated from peripheral blood mononuclear cells of patient carrying LMNA mutation associated with Emery-Dreifuss muscular dystrophy accompanied by atrioventricular block and paroxysmal atrial fibrillation. Reprogramming factors OCT4, KLF4, SOX2, CMYC were delivered using Sendai virus transduction. iPSCs were characterized in order to prove the pluripotency markers expression, normal karyotype, ability to differentiate into three embryonic germ layers. Generated iPSC lines would be useful model to investigate disease development associated with genetic variants in LMNA gene.
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Lamin A is involved in many cellular functions due to its ability to bind chromatin and transcription factors and affect their properties. Mutations of LMNA gene encoding lamin A affect the differentiation capacity of stem cells, but the mechanisms of this influence remain largely unclear. We and others have reported recently an interaction of lamin A with Notch pathway, which is among the main developmental regulators of cellular identity. The aim of this study was to explore the influence of LMNA mutations on the proosteogenic response of human cells of mesenchymal origin and to further explore the interaction of LMNA with Notch pathway. Mutations R527C and R471C in LMNA are associated with mandibuloacral dysplasia type A, a highly penetrant disease with a variety of abnormalities involving bone development. We used lentiviral constructs bearing mutations R527C and R471C and explored its influence on proosteogenic phenotype expression and Notch pathway activity in four types of human cells: umbilical vein endothelial cells (HUVEC), cardiac mesenchymal cells (HCMC), aortic smooth muscle cells (HASMC), and aortic valve interstitial cells (HAVIC). The proosteogenic response of the cells was induced by the addition of either LPS or specific effectors of osteogenic differentiation to the culture medium; phenotype was estimated by the expression of osteogenic markers by qPCR; activation of Notch was assessed by expression of Notch-related and Notch-responsive genes by qPCR and by activation of a luciferase CSL-reporter construct. Overall, we observed different reactivity of all four cell lineages to the stimulation with either LPS or osteogenic factors. R527C had a stronger influence on the proosteogenic phenotype. We observed the inhibiting action of LMNA R527C on osteogenic differentiation in HCMC in the presence of activated Notch signaling, while LMNA R527C caused the activation of osteogenic differentiation in HAVIC in the presence of activated Notch signaling. Our results suggest that the effect of a LMNA mutation is strongly dependent not only on a specific mutation itself, but also might be influenced by the intrinsic molecular context of a cell lineage.