Asunto(s)
Fibrilación Atrial , Mutación con Ganancia de Función , Proteína del Homeodomínio PITX2 , Proteínas de Homeodominio , Factores de Transcripción , Fibrilación Atrial/genética , Fibrilación Atrial/fisiopatología , Fibrilación Atrial/diagnóstico , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Homeodominio/genética , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Animales , Potencial de la Membrana Mitocondrial/genética , Predisposición Genética a la Enfermedad , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , FenotipoAsunto(s)
Proteína Adaptadora de Señalización NOD1 , Nodo Sinoatrial , Animales , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiopatología , Ratones , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD1/genética , Potenciales de Acción , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Atrial fibrillation (AF) is a common heart rhythm disorder that is associated with an increased risk of stroke and heart failure (HF). Initially, an association between AF and ion channel dysfunction was identified, classifying the pathology as a predominantly electrical disease. More recently it has been recognized that fibrosis and structural atrial remodeling play a driving role in the development of this arrhythmia also in these cases. PURPOSE: Understanding the role of fibrosis in genetic determined AF could be important to better comprise the pathophysiology of this arrhythmia and to refine its management also in nongenetic forms. In this review we analyze genetic and epigenetic mechanisms responsible for AF and their link with atrial fibrosis, then we will consider analogies with the pathophysiological mechanism in nongenetic AF, and discuss consequent therapeutic options.
Asunto(s)
Fibrilación Atrial , Insuficiencia Cardíaca , Humanos , Fibrilación Atrial/complicaciones , Atrios Cardíacos , Fibrosis , Canales Iónicos/genética , Canales Iónicos/uso terapéuticoRESUMEN
Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.
Asunto(s)
Caveolina 1 , Caveolina 3 , Adulto , Animales , Cricetinae , Humanos , Caveolina 1/genética , Caveolina 3/genética , Cricetulus , Mutación , Células CHO , Canales IónicosRESUMEN
Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide; however, the underlying causes of AF initiation are still poorly understood, particularly because currently available models do not allow in distinguishing the initial causes from maladaptive remodeling that induces and perpetuates AF. Lately, the genetic background has been proven to be important in the AF onset. iPSC-derived cardiomyocytes, being patient- and mutation-specific, may help solve this diatribe by showing the initial cell-autonomous changes underlying the development of the disease. Transcription factor paired-like homeodomain 2 (PITX2) has been identified as a key regulator of atrial development/differentiation, and the PITX2 genomic locus has the highest association with paroxysmal AF. PITX2 influences mitochondrial activity, and alterations in either its expression or function have been widely associated with AF. In this work, we investigate the activity of mitochondria in iPSC-derived atrial cardiomyocytes (aCMs) obtained from a young patient (24 years old) with paroxysmal AF, carrying a gain-of-function mutation in PITX2 (rs138163892) and from its isogenic control (CTRL) in which the heterozygous point mutation has been reverted to WT. PITX2 aCMs show a higher mitochondrial content, increased mitochondrial activity, and superoxide production under basal conditions when compared to CTRL aCMs. However, increasing mitochondrial workload by FCCP or ß-adrenergic stimulation allows us to unmask mitochondrial defects in PITX2 aCMs, which are incapable of responding efficiently to the higher energy demand, determining ATP deficiency.
RESUMEN
AIMS: Nfix is a transcription factor belonging to the Nuclear Factor I (NFI) family comprising four members (Nfia, b, c, x). Nfix plays important roles in the development and function of several organs. In muscle development, Nfix controls the switch from embryonic to fetal myogenesis by promoting fast twitching fibres. In the adult muscle, following injury, lack of Nfix impairs regeneration, inducing higher content of slow-twitching fibres. Nfix is expressed also in the heart, but its function has been never investigated before. We studied Nfix role in this organ. METHODS: Using Nfix-null and wild type (WT) mice we analyzed: (1) the expression pattern of Nfix during development by qPCR and (2) the functional alterations caused by its absence, by in vivo telemetry and in vitro patch clamp analysis. RESULTS AND CONCLUSIONS: Nfix expression start in the heart from E12.5. Adult hearts of Nfix-null mice show a hearts morphology and sarcomeric proteins expression similar to WT. However, Nfix-null animals show tachycardia that derives form an intrinsic higher beating rate of the sinus node (SAN). Molecular and functional analysis revealed that sinoatrial cells of Nfix-null mice express a significantly larger L-type calcium current (Cacna1d + Cacna1c). Interestingly, downregulation of Nfix by sh-RNA in primary cultures of neonatal rat ventricular cardiomyocytes induced a similar increase in their spontaneous beating rate and in ICaL current. In conclusion, our data provide the first demonstration of a role of Nfix that, increasing the L-type calcium current, modulates heart rate.
RESUMEN
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are commonly used to model arrhythmogenic cardiomyopathy (ACM), a heritable cardiac disease characterized by severe ventricular arrhythmias, fibrofatty myocardial replacement and progressive ventricular dysfunction. Although ACM is inherited as an autosomal dominant disease, incomplete penetrance and variable expressivity are extremely common, resulting in different clinical manifestations. Here, we propose hiPSC-CMs as a powerful in vitro model to study incomplete penetrance in ACM. Six hiPSC lines were generated from blood samples of three ACM patients carrying a heterozygous deletion of exon 4 in the PKP2 gene, two asymptomatic (ASY) carriers of the same mutation and one healthy control (CTR), all belonging to the same family. Whole exome sequencing was performed in all family members and hiPSC-CMs were examined by ddPCR, western blot, Wes™ immunoassay system, patch clamp, immunofluorescence and RNASeq. Our results show molecular and functional differences between ACM and ASY hiPSC-CMs, including a higher amount of mutated PKP2 mRNA, a lower expression of the connexin-43 protein, a lower overall density of sodium current, a higher intracellular lipid accumulation and sarcomere disorganization in ACM compared to ASY hiPSC-CMs. Differentially expressed genes were also found, supporting a predisposition for a fatty phenotype in ACM hiPSC-CMs. These data indicate that hiPSC-CMs are a suitable model to study incomplete penetrance in ACM.
RESUMEN
Discovered some 40 years ago, the If current has since been known as the "pacemaker" current due to its role in the initiation and modulation of the heartbeat and of neuronal excitability. But this is not all, the funny current keeps entertaining the researchers; indeed, several data discovering novel and uncanonical roles of f/HCN channel are quickly accumulating. In the present review, we provide an overview of the expression and cellular functions of HCN/f channels in a variety of systems/organs, and particularly in sour taste transduction, hormones secretion, activation of astrocytes and microglia, inhibition of osteoclastogenesis, renal ammonium excretion, and peristalsis in the gastrointestinal and urine systems. We also analyzed the role of HCN channels in sustaining cellular respiration in mitochondria and their participation to mitophagy under specific conditions. The relevance of HCN currents in undifferentiated cells, and specifically in the control of stem cell cycle and in bioelectrical signals driving left/right asymmetry during zygote development, is also considered. Finally, we present novel data concerning the expression of HCN mRNA in human leukocytes. We can thus conclude that the emerging evidence presented in this review clearly points to an increasing interest and importance of the "funny" current that goes beyond its role in cardiac sinoatrial and neuronal excitability regulation.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales de Potasio , Corazón , Frecuencia Cardíaca , Humanos , NeuronasRESUMEN
AIMS: PCSK9 is secreted into the circulation, mainly by the liver, and interacts with low-density lipoprotein receptor (LDLR) homologous and non-homologous receptors, including CD36, thus favouring their intracellular degradation. As PCSK9 deficiency increases the expression of lipids and lipoprotein receptors, thus contributing to cellular lipid accumulation, we investigated whether this could affect heart metabolism and function. METHODS AND RESULTS: Wild-type (WT), Pcsk9 KO, Liver conditional Pcsk9 KO and Pcsk9/Ldlr double KO male mice were fed for 20 weeks with a standard fat diet and then exercise resistance, muscle strength, and heart characteristics were evaluated. Pcsk9 KO presented reduced running resistance coupled to echocardiographic abnormalities suggestive of heart failure with preserved ejection fraction (HFpEF). Heart mitochondrial activity, following maximal coupled and uncoupled respiration, was reduced in Pcsk9 KO mice compared to WT mice and was coupled to major changes in cardiac metabolism together with increased expression of LDLR and CD36 and with lipid accumulation. A similar phenotype was observed in Pcsk9/Ldlr DKO, thus excluding a contribution for LDLR to cardiac impairment observed in Pcsk9 KO mice. Heart function profiling of the liver selective Pcsk9 KO model further excluded the involvement of circulating PCSK9 in the development of HFpEF, pointing to a possible role locally produced PCSK9. Concordantly, carriers of the R46L loss-of-function variant for PCSK9 presented increased left ventricular mass but similar ejection fraction compared to matched control subjects. CONCLUSION: PCSK9 deficiency impacts cardiac lipid metabolism in an LDLR independent manner and contributes to the development of HFpEF.
Asunto(s)
Insuficiencia Cardíaca , Proproteína Convertasa 9 , Animales , Insuficiencia Cardíaca/genética , Masculino , Ratones , Ratones Noqueados , Proproteína Convertasa 9/genética , Receptores de LDL/genética , Volumen SistólicoRESUMEN
Properties of the funny current (If) have been studied in several animal and cellular models, but so far little is known concerning its properties in human pacemaker cells. This work provides a detailed characterization of If in human-induced pluripotent stem cell (iPSC)-derived pacemaker cardiomyocytes (pCMs), at different time points. Patch-clamp analysis showed that If density did not change during differentiation; however, after day 30, it activates at more negative potential and with slower time constants. These changes are accompanied by a slowing in beating rate. If displayed the voltage-dependent block by caesium and reversed (Erev) at - 22 mV, compatibly with the 3:1 K+/Na+ permeability ratio. Lowering [Na+]o (30 mM) shifted the Erev to - 39 mV without affecting conductance. Increasing [K+]o (30 mM) shifted the Erev to - 15 mV with a fourfold increase in conductance. pCMs express mainly HCN4 and HCN1 together with the accessory subunits CAV3, KCR1, MiRP1, and SAP97 that contribute to the context-dependence of If. Autonomic agonists modulated the diastolic depolarization, and thus rate, of pCMs. The adrenergic agonist isoproterenol induced rate acceleration and a positive shift of If voltage-dependence (EC50 73.4 nM). The muscarinic agonists had opposite effects (Carbachol EC50, 11,6 nM). Carbachol effect was however small but it could be increased by pre-stimulation with isoproterenol, indicating low cAMP levels in pCMs. In conclusion, we demonstrated that pCMs display an If with the physiological properties expected by pacemaker cells and may thus represent a suitable model for studying human If-related sinus arrhythmias.
Asunto(s)
Potenciales de Acción/fisiología , Relojes Biológicos/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Potenciales de Acción/efectos de los fármacos , Relojes Biológicos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Electrofisiología/métodos , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Isoproterenol/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp/métodos , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiologíaRESUMEN
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.
Asunto(s)
Potenciales de Acción/genética , Fibrilación Atrial/genética , Canales de Calcio Tipo L/genética , Frecuencia Cardíaca/genética , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Potenciales de Acción/efectos de los fármacos , Antiarrítmicos/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Canales de Calcio Tipo L/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Resistencia a Medicamentos/genética , Predisposición Genética a la Enfermedad , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Persona de Mediana Edad , Hermanos , Secuenciación del ExomaRESUMEN
GNB5 loss-of-function pathogenic variants cause IDDCA, a rare autosomal recessive human genetic disease characterized by infantile onset of intellectual disability, sinus bradycardia, hypotonia, visual abnormalities, and epilepsy. We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a patient with the homozygous c.136delG frameshift variant, and a GNB5 knock-out (KO) line by CRISPR/Cas9 editing. hiPSCs express common pluripotency markers and differentiate into the three germ layers. These lines represent a powerful cellular model to study the molecular basis of GNB5-related disorders as well as offer an in vitro model for drug screening.
Asunto(s)
Línea Celular/metabolismo , Subunidades beta de la Proteína de Unión al GTP/genética , Enfermedades Genéticas Congénitas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Sistemas CRISPR-Cas , Diferenciación Celular , Línea Celular/citología , Células Cultivadas , Niño , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Mutación del Sistema de Lectura , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Edición Génica , Técnicas de Inactivación de Genes , Enfermedades Genéticas Congénitas/metabolismo , Enfermedades Genéticas Congénitas/fisiopatología , Ingeniería Genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Persona de Mediana EdadRESUMEN
Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to ß-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Calcio/metabolismo , Cardiomiopatías/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/metabolismo , Potenciales de Acción/efectos de los fármacos , Biomimética , Cardiomiopatías/genética , Cardiomiopatías/patología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Acoplamiento Excitación-Contracción/efectos de los fármacos , Humanos , Hidrogeles/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Especificidad por SustratoRESUMEN
Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table.
Asunto(s)
Caveolina 3/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Caveolina 3/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Citometría de Flujo , Humanos , Cariotipo , Mutación/genética , Mutación Missense/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The development of strategies to reduce the load of unwanted bacteria is a fundamental challenge in industrial processing, environmental sciences and medical applications. Here, we report a new method to sequester motile bacteria from a liquid, based on passive, deployable micro-traps that confine bacteria using micro-funnels that open into trapping chambers. Even in low concentrations, micro-traps afford a 70% reduction in the amount of bacteria in a liquid sample, with a potential to reach >90% as shown by modelling improved geometries. This work introduces a new approach to contain the growth of bacteria without chemical means, an advantage of particular importance given the alarming growth of pan-drug-resistant bacteria.
Asunto(s)
Bacterias/aislamiento & purificación , Movimiento Celular , Microbiología del Agua , Bacterias/patogenicidad , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Helicobacter pylori/aislamiento & purificación , Helicobacter pylori/patogenicidad , Humanos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/patogenicidad , Salmonella enterica/aislamiento & purificación , Salmonella enterica/patogenicidad , Vibrio cholerae/aislamiento & purificación , Vibrio cholerae/patogenicidadRESUMEN
AIM: Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na+ current (INa), nifedipine, a blocker of L-type Ca2+ current (ICaL), and E4031, a blocker of the rapid component of delayed rectifier K+ current (IKr). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K+ current (IKs). CONCLUSION: In hiPSC-derived cardiomyocytes of cardiac origin, INa, ICaL, IKr, and IKs were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic cell origins. Further studies are warranted to characterize electrophysiological properties of hiPSC-derived cardiomyocytes generated from CPCs.
Asunto(s)
Canales de Calcio Tipo L/efectos de los fármacos , Diferenciación Celular , Canales de Potasio de Tipo Rectificador Tardío/antagonistas & inhibidores , Fibroblastos/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Miocitos Cardíacos/efectos de los fármacos , Canales de Sodio/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Linaje de la Célula , Células Cultivadas , Reprogramación Celular , Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Potenciales de la Membrana , Miocitos Cardíacos/metabolismo , Fenotipo , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismoRESUMEN
Ten years ago Yamanaka's lab identified a way to reprogram terminally differentiated cells to a pluripotent state, similar to that of embryonic stem cell. This procedure opened the road for the generation of postmitotic human cells, that have completely lost the replication potential. The initial excitement waned when it was observed that the cells produced by this method are somehow immature and do not resemble the adult phenotype. In the absence of cellular markers that recognize the various maturation steps of induced pluripotent stem cell-derived human cardiomyocytes, we propose to follow their maturation looking at their electrophysiological profile. For this reason, we are first reviewing the most common methods of differentiation, from the preliminary complex procedures to the newly-identified two-step protocols and, second, we report the electrical characteristics of the cells, through electrophysiological analysis of ionic currents that give rise to the action potential. We are aware that each protocol leads to the generation of different cardiomyocyte precursors, thus suggesting the need for a wider standardization. The identification of the electrophysiological characteristics of the cells could help in identifying the type and the maturation stage of the obtained cardiomyocyte, thus compensating for the lack of specific markers. Developmental Dynamics 245:1145-1158, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Fenómenos Electrofisiológicos , HumanosRESUMEN
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.
RESUMEN
A critical step in the development of effective therapeutics to treat Parkinson's disease (PD) is the identification of molecular pathogenic mechanisms underlying this chronically progressive neurodegenerative disease. However, while animal models have provided valuable information about the molecular basis of PD, the lack of faithful cellular and animal models that recapitulate human pathophysiology is delaying the development of new therapeutics. The reprogramming of somatic cells to induced pluripotent stem cells (iPSC) using delivery of defined combinations of transcription factors is a groundbreaking discovery that opens great opportunities for modeling human diseases, including PD, since iPSC can be generated from patients and differentiated into disease-relevant cell types, which would capture the patients' genetic complexity. Furthermore, human iPSC-derived neuronal models offer unprecedented access to early stages of the disease, allowing the investigation of the events that initiate the pathologic process in PD. Recently, human iPSC-derived neurons from patients with familial and sporadic PD have been generated and importantly they recapitulate some PD-related cell phenotypes, including abnormal α-synuclein accumulation in vitro, and alterations in the autophagy machinery. This review highlights the current PD iPSC-based models and discusses the potential future research directions of this field.
Asunto(s)
Células Madre Pluripotentes Inducidas/trasplante , Células-Madre Neurales/trasplante , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Trasplante de Células Madre/métodos , Animales , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células-Madre Neurales/inmunología , Células-Madre Neurales/patología , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/inmunología , Células Madre Pluripotentes/inmunología , Células Madre Pluripotentes/patología , Células Madre Pluripotentes/trasplanteRESUMEN
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.