RESUMEN
BACKGROUND: Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential face a significantly elevated risk of cardiovascular diseases. Endothelial cells carrying the JAK2V617F mutation have been detected in many patients with MPN. In this study, we investigated the molecular basis for the high incidence of cardiovascular complications in patients with MPN. METHODS: We investigated the impact of endothelial JAK2V617F mutation on cardiovascular disease development using both transgenic murine models and MPN patient-derived induced pluripotent stem cell lines. RESULTS: Our investigations revealed that JAK2V617F mutant endothelial cells promote cardiovascular diseases under stress, which is associated with endothelial-to-mesenchymal transition and endothelial dysfunction. Importantly, we discovered that inhibiting the endothelial TPO (thrombopoietin) receptor MPL (myeloproliferative leukemia virus oncogene) suppressed JAK2V617F-induced endothelial-to-mesenchymal transition and prevented cardiovascular dysfunction caused by mutant endothelial cells. Notably, the endothelial MPL receptor is not essential for the normal physiological regulation of blood cell counts and cardiac function. CONCLUSIONS: JAK2V617F mutant endothelial cells play a critical role in the development of cardiovascular diseases in JAK2V617F-positive MPNs, and endothelial MPL could be a promising therapeutic target for preventing or ameliorating cardiovascular complications in these patients.
Asunto(s)
Enfermedades Cardiovasculares , Células Endoteliales , Células Madre Pluripotentes Inducidas , Janus Quinasa 2 , Mutación , Trastornos Mieloproliferativos , Receptores de Trombopoyetina , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Receptores de Trombopoyetina/genética , Animales , Humanos , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/enzimología , Trastornos Mieloproliferativos/complicaciones , Trastornos Mieloproliferativos/metabolismo , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/enzimología , Enfermedades Cardiovasculares/etiología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/enzimología , Ratones Transgénicos , Transducción de Señal , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , RatonesRESUMEN
AIMS: Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains. METHODS AND RESULTS: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia. CONCLUSION: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.
Asunto(s)
AMP Cíclico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Modelos Animales de Enfermedad , Terapia Genética , Insuficiencia Cardíaca , Miocitos Cardíacos , Canal Liberador de Calcio Receptor de Rianodina , Animales , AMP Cíclico/metabolismo , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/genética , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Humanos , Ratones Endogámicos C57BL , Masculino , Arritmias Cardíacas/enzimología , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/prevención & control , Remodelación Ventricular , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Función Ventricular Izquierda , Señalización del Calcio , Fosforilación , Frecuencia CardíacaRESUMEN
AIMS: Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. METHODS AND RESULTS: We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with ß-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/ß-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. CONCLUSION: Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.
Asunto(s)
Apoptosis , Cardiotoxicidad , Doxorrubicina , Miocitos Cardíacos , Vía de Señalización Wnt , beta Catenina , Doxorrubicina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miocitos Cardíacos/enzimología , Vía de Señalización Wnt/efectos de los fármacos , Humanos , Animales , Apoptosis/efectos de los fármacos , beta Catenina/metabolismo , beta Catenina/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/inducido químicamente , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/enzimología , Cardiomiopatía Dilatada/fisiopatología , Masculino , Transducina/metabolismo , Transducina/genética , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Femenino , Estudios de Casos y Controles , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/toxicidadRESUMEN
AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.
Asunto(s)
Diferenciación Celular , Ácidos Grasos , Miocitos Cardíacos , Miofibrillas , Fosforilación Oxidativa , Proteína 2 de Unión a Retinoblastoma , Humanos , Células Cultivadas , Ácidos Grasos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Histonas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/enzimología , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/genética , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Miofibrillas/metabolismo , Miofibrillas/enzimología , Oxidación-Reducción , Regiones Promotoras Genéticas , Proteína 2 de Unión a Retinoblastoma/metabolismo , Proteína 2 de Unión a Retinoblastoma/genéticaRESUMEN
CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIIδ, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIIδ gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIIδ editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIIδ gene editing may thus represent a permanent and advanced strategy for heart disease therapy.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Edición Génica , Cardiopatías , Animales , Humanos , Ratones , Sistemas CRISPR-Cas , Cardiopatías/genética , Cardiopatías/terapia , Células Madre Pluripotentes Inducidas/enzimología , Miocitos Cardíacos/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genéticaRESUMEN
Human dermal fibroblasts from a Leigh Syndrome (LS) patient harboring the heterozygous NDUFS1 R557X/D618N compound mutation were reprogrammed to generate integration-free induced pluripotent stem cells (iPSCs). The full characterization of IUFi002-A-iPSCs demonstrated that the line is free of exogenous reprogramming genes and maintains the genomic integrity. IUFi002-A-iPSCs' pluripotency was confirmed by the expression of pluripotency markers and embryoid body-based differentiation into cell types representative of each of the three germ layers. The generated iPSC line provides a powerful tool to investigate LS and analyze the molecular mechanisms underlying NDUFS1 mutations-induced pathology.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Leigh , NADH Deshidrogenasa , Humanos , Genómica , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Mutación , NADH Deshidrogenasa/genética , Línea CelularRESUMEN
Cytochrome P450 (CYP) reaction phenotyping has become crucial for predicting drug reactions and side effects. Single nucleotide polymorphisms (SNPs) in CYP genes alter drug metabolism capacity and cause unexpected drug-related reactions. Here, we established two human induced pluripotent stem cell (hiPSC) lines with pharmacologically important SNPs in CYP2D6 in conjunction with CYP2C19 or CYP3A5 genes. These hiPSC lines can serve as valuable resources for expanding our understanding of the relationships between genotypes and drug reactions.
Asunto(s)
Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Células Madre Pluripotentes Inducidas , Preparaciones Farmacéuticas , Humanos , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Células Madre Pluripotentes Inducidas/enzimología , Línea Celular , Polimorfismo de Nucleótido Simple , Preparaciones Farmacéuticas/metabolismoRESUMEN
Cancer stem cells (CSCs) are capable of continuous proliferation, self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. We have established a model of CSCs that was originally developed from mouse induced pluripotent stem cells (miPSCs) by proposing miPSCs to the conditioned medium (CM) of cancer derived cells, which is a mimic of carcinoma microenvironment. Further research found that not only PI3K-Akt but also EGFR signaling pathway was activated during converting miPSCs into CSCs. In this study, we tried to observe both of PI3Kγ inhibitor Eganelisib and EGFR inhibitor Gefitinib antitumor effects on the models of CSCs derived from miPSCs (miPS-CSC) in vitro and in vivo. As the results, targeting these two pathways exhibited significant inhibition of cell proliferation, self-renewal, migration and invasion abilities in vitro. Both Eganelisib and Gefitinib showed antitumor effects in vivo while Eganelisib displayed more significant therapeutic efficacy and less side effects than Gefitinib on all miPS-CSC models. Thus, these data suggest that the inhibitiors of PI3K and EGFR, especially PI3Kγ, might be a promising therapeutic strategy against CSCs defeating cancer in the near future.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Receptores ErbB/antagonistas & inhibidores , Gefitinib/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Transducción de Señal , Carga Tumoral/efectos de los fármacosRESUMEN
AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.
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Cardiomiopatía Hipertrófica/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Peroxidasa/antagonistas & inhibidores , Función Ventricular Izquierda/efectos de los fármacos , Animales , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomiopatía Hipertrófica/enzimología , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/fisiopatología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Hipertrofia Ventricular Izquierda/enzimología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Masculino , Ratones Endogámicos C57BL , Mutación Missense , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Peroxidasa/metabolismo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson's disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.
Asunto(s)
Encéfalo/enzimología , Factor de Transcripción COUP I/metabolismo , Neuronas Dopaminérgicas/enzimología , Células Madre Pluripotentes Inducidas/enzimología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Células-Madre Neurales/enzimología , Neurogénesis , Enfermedad de Parkinson/enzimología , Animales , Encéfalo/patología , Factor de Transcripción COUP I/genética , Ciclo Celular , Línea Celular , Proliferación Celular , Supervivencia Celular , Neuronas Dopaminérgicas/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Mutación , Células-Madre Neurales/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Fenotipo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Factores de TiempoRESUMEN
Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson's disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remain largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes and identify the abnormal accumulation of key PD-related proteins within multivesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.
Asunto(s)
Astrocitos/enzimología , Comunicación Celular , Neuronas Dopaminérgicas/enzimología , Exosomas/enzimología , Células Madre Pluripotentes Inducidas/enzimología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Células-Madre Neurales/enzimología , Enfermedad de Parkinson/enzimología , Animales , Astrocitos/ultraestructura , Atrofia , Estudios de Casos y Controles , Línea Celular , Neuronas Dopaminérgicas/patología , Endocitosis , Exosomas/genética , Exosomas/ultraestructura , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Células-Madre Neurales/ultraestructura , Biogénesis de Organelos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patologíaRESUMEN
Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.
Asunto(s)
Proteínas de la Membrana/efectos de los fármacos , Terapia Molecular Dirigida/métodos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Activación Enzimática/efectos de los fármacos , Células Gigantes/enzimología , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Microsomas/enzimología , Simulación de Dinámica Molecular , Estructura Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Fosfatidilcolinas , Dominios Proteicos/efectos de los fármacos , Retículo Sarcoplasmático/enzimología , Bibliotecas de Moléculas Pequeñas/efectos adversos , Bibliotecas de Moléculas Pequeñas/farmacología , Porcinos , AguaRESUMEN
Reducing infarct size (IS) by interfering with mechanisms for cardiomyocyte death remains an elusive goal. DMX-5804, a selective inhibitor of the stress-activated kinase MAP4K4, suppresses cell death in mouse myocardial infarction (MI), human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), and 3D human engineered heart tissue, whose fidelity to human biology is hoped to strengthen the route to clinical success. Here, DMX-10001, a soluble, rapidly cleaved pro-drug of DMX-5804, was developed for i.v. testing in large-mammal MI. Following pharmacodynamic studies, a randomized, blinded efficacy study was performed in swine subjected to LAD balloon occlusion (60 min) and reperfusion (24 h). Thirty-six animals were enrolled; 12 were excluded by pre-defined criteria, death before infusion, or technical issues. DMX-10001 was begun 20 min before reperfusion (30 min, 60 mg/kg/h; 23.5 h, 17 mg/kg/h). At all times tested, beginning 30 min after the start of infusion, DMX-5804 concentrations exceeded > fivefold the levels that rescued hPSC-CMs and reduced IS in mice after oral dosing with DMX-5804 itself. No significant reduction occurred in IS or no-reflow corrected for the area at ischemic risk, even though DMX-10001 reduced IS, expressed in grams or % of LV mass, by 27%. In summary, a rapidly cleaved pro-drug of DMX-5804 failed to reduce IS in large-mammal MI, despite exceeding the concentrations for proven success in both mice and hPSC-CMs.
Asunto(s)
Células Madre Pluripotentes Inducidas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Infarto del Miocardio/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Profármacos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Femenino , Hemodinámica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Profármacos/farmacocinética , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/metabolismo , Sus scrofa , Investigación Biomédica Traslacional , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Lysosomes are critical for maintaining protein homeostasis and cellular metabolism. Lysosomal dysfunction and disrupted protein trafficking contribute to cell death in neurodegenerative disorders, including Parkinson's disease and dementia. We describe three complementary protocols-the use of protein glycosylation, western blotting, immunofluorescence, and hydrolase activity measurement-to analyze the trafficking and activity of lysosomal proteins in patient-derived neurons differentiated from iPSCs. These methods should help to identify lysosomal phenotypes in patient-derived cultures and aid the discovery of therapeutics that augment lysosomal function. For complete details on the use and execution of this protocol, please refer to Cuddy et al. (2019).
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Diferenciación Celular , Hidrolasas/metabolismo , Células Madre Pluripotentes Inducidas/enzimología , Lisosomas/enzimología , Modelos Neurológicos , Enfermedad de Parkinson/enzimología , Humanos , Células Madre Pluripotentes Inducidas/patología , Lisosomas/patología , Enfermedad de Parkinson/patología , Transporte de ProteínasRESUMEN
AIMS: NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. METHODS AND RESULTS: In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by IKs blockade (JNJ303) and ß-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1-A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced ICaL and INaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed 'transient inward current' events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and ICaL larger; NOS1AP and NOS1 expression and co-localization were decreased. CONCLUSION: The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes.
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Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/genética , Frecuencia Cardíaca , Células Madre Pluripotentes Inducidas/enzimología , Canal de Potasio KCNQ1/genética , Mutación , Miocitos Cardíacos/enzimología , Óxido Nítrico Sintasa de Tipo I/genética , Polimorfismo de Nucleótido Simple , Síndrome de Romano-Ward/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Señalización del Calcio , Línea Celular , Predisposición Genética a la Enfermedad , Cobayas , Humanos , Canal de Potasio KCNQ1/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Fenotipo , Síndrome de Romano-Ward/diagnóstico , Síndrome de Romano-Ward/enzimología , Síndrome de Romano-Ward/fisiopatología , Factores de TiempoRESUMEN
Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by ß-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.
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Inductores de las Enzimas del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/genética , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/metabolismo , Adulto , Células CACO-2 , Línea Celular , Células Cultivadas , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismoRESUMEN
Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.
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Reprogramación Celular , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre Pluripotentes Inducidas/enzimología , Animales , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Metilación , RatonesRESUMEN
Recent findings suggest a pathologic role of skeletal muscle in amyotrophic lateral sclerosis (ALS) onset and progression. However, the exact mechanism by which this occurs remains elusive due to limited human-based studies. To this end, phenotypic ALS skeletal muscle models were developed from induced pluripotent stem cells (iPSCs) derived from healthy individuals (WT) and ALS patients harboring mutations in the superoxide dismutase 1 (SOD1) gene. Although proliferative, SOD1 myoblasts demonstrated delayed and reduced fusion efficiency compared to WT. Additionally, SOD1 myotubes exhibited significantly reduced length and cross-section. Also, SOD1 myotubes had loosely arranged myosin heavy chain and reduced acetylcholine receptor expression per immunocytochemical analysis. Functional analysis indicated considerably reduced contractile force and synchrony in SOD1 myotubes. Mitochondrial assessment indicated reduced inner mitochondrial membrane potential (ΔΨm) and metabolic plasticity in the SOD1-iPSC derived myotubes. This work presents the first well-characterized in vitro iPSC-derived muscle model that demonstrates SOD1 toxicity effects on human muscle regeneration, contractility and metabolic function in ALS. Current findings align with previous ALS patient biopsy studies and suggest an active contribution of skeletal muscle in NMJ dysfunction. Further, the results validate this model as a human-relevant platform for ALS research and drug discovery studies.
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Esclerosis Amiotrófica Lateral/patología , Células Madre Pluripotentes Inducidas/metabolismo , Músculo Esquelético/patología , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Amiotrófica Lateral/genética , Linaje de la Célula/genética , Progresión de la Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/enzimología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Mutación/genética , Mioblastos/enzimología , Mioblastos/patologíaRESUMEN
Signal regulators during early cardiogenetic differentiation for the cellular automaticity are largely unknown. Our investigations were designed to clarify the role of transcription factors and their modulators in P19-derived cardiomyocytes to the expression of cardiac pacemaker ion channels. Transcription factors Csx/Nkx2.5 and GATA4 but not MEF2C were markedly inhibited by p38 MAP kinase inhibition in a distinct manner; expression but not phosphorylation of GATA4 was reduced by inhibition of p38 MAP kinase actions. In the presence of an ERK1/2,5 inhibitor PD98059 or a JNK MAP kinase inhibitor SP600125, P19 cells successfully differentiated into cardiomyocytes displaying spontaneous beatings with expression of three types of pacemaker ion channels. We demonstrate that acquisition of cellular automaticity and the expression of pacemaker ion channels are regulated by the transcription factors, Csx/Nkx2.5 and GATA4, through intracellular signals including p38 MAP kinase in the process of P19-derived pluripotent cells differentiation into cardiomyocytes.
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Relojes Biológicos , Diferenciación Celular , Frecuencia Cardíaca , Células Madre Pluripotentes Inducidas/enzimología , Canales Iónicos/metabolismo , Miocitos Cardíacos/enzimología , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Potenciales de Acción , Animales , Línea Celular Tumoral , Factor de Transcripción GATA4/metabolismo , Proteína Homeótica Nkx-2.5/metabolismo , Factores de Transcripción MEF2/metabolismo , Ratones , Fenotipo , Fosforilación , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
Human-induced pluripotent stem cells (hiPSCs) are invaluable sources for drug screening and toxicity tests because of their differentiation potential and proliferative capacity. Recently, the CRISPR-Cas9-mediated homologous recombination system has enabled reporter knock-ins at desired loci in hiPSCs, and here, we generated a hiPSC reporter line expressing mCherry-tagged cytochrome P450 1A1 (CYP1A1), which can be utilized to screen for the modulators of aryl hydrocarbon receptor (AHR) in live cells. CYP1A1-mCherry hiPSCs exhibited typical characteristics of pluripotent stem cells such as marker expression, differentiation potential, and normal karyotype. After differentiation into hepatocyte-like cells (HLCs), CYP1A1-mCherry fusion protein was expressed and localized at the endoplasmic reticulum, and induced by AHR agonists. We obtained 23 hits modulating CYP1A1 expression from high-content screening with 241 hepatotoxicity chemicals and nuclear receptor ligands, and identified three upregulating chemicals and two downregulating compounds. Responses of hiPSC-HLCs against an AHR agonist were more similar to human primary hepatocytes than of HepG2 hepatocellular carcinoma cells. This platform has the advantages of live-cell screening without sacrificing cells (unlike previously available CYP1A1 reporter cell lines), as well as an indefinite supply of cells, and can be utilized in a wide range of screening related to AHR- and CYP1A1-associated diseases in desired cell types.