RESUMEN
Type-2 ryanodine receptor (RyR2) is the major Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR) that controls the rhythm and strength of the heartbeat, but its malfunction may generate severe arrhythmia leading to sudden cardiac death or heart failure. S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca2+ currents (ICa), measured either by depolarizations to zero mV or repolarizations from +100 mV to -50 mV, and their activated Ca2+ transients were significantly smaller, despite their larger caffeine-triggered Ca2+ release signals compared to wild type (WT) cells, suggesting ICa-induced Ca2+ release (CICR) was compromised. The larger SR Ca2+ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca2+ sparks and Ca2+ transients and their arrhythmogenic phenotype.
Asunto(s)
Señalización del Calcio , Células Madre Pluripotentes Inducidas , Humanos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Señalización del Calcio/fisiología , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
BACKGROUND INFORMATION: Wnt/ß-catenin signalling, in the microenvironment of pluripotent stem cells (PSCs), plays a critical role in their differentiation and proliferation. Contradictory reports on the role of Wnt/ß-catenin signalling in PSCs self-renewal and differentiation, however, render these mechanisms largely unclear. RESULTS: Wnt/ß-catenin signalling pathway in human-induced pluripotent stem cells (hiPSCs) was activated by inhibiting glycogen synthase kinase 3 (GSK3), driving the cells into a mesodermal/mesenchymal state, exhibiting proliferative, invasive and anchorage-independent growth properties, where over 70% of cell population became CD 44 (+)/CD133 (+). Wnt/ß-catenin signalling activation also altered the metabolic state of hiPSCs from aerobic glycolysis to oxidative metabolism and changed their drug and oxidative stress sensitivities. These effects of GSK3 inhibition were suppressed in HIF1α-stabilised cells. CONCLUSIONS: Persistent activation of Wnt/ß-catenin signalling endows hiPSCs with proliferative/invasive 'teratoma-like' states, shifting their metabolic dependence and allowing HIF1α-stabilisation to inhibit their proliferative/invasive properties. SIGNIFICANCE: The hiPSC potential to differentiate into 'teratoma-like' cells suggest that stem cells may exist in two states with differential metabolic and drug dependency.
Asunto(s)
Células Madre Pluripotentes Inducidas , Vía de Señalización Wnt/fisiología , Diferenciación Celular , Línea Celular , Proliferación Celular , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
How oxygen is sensed by the heart and what mechanisms mediate its sensing remain poorly understood. As recent reports show that low PO2 levels are detected by the cardiomyocytes in a few seconds, the rapid and short applications of low levels of oxygen (acute hypoxia), which avoid multiple effects of chronic hypoxia, may be used to probe the oxygen-sensing pathway of the heart. Here, we explored the oxygen-sensing pathway, focusing primarily on cellular surface membrane proteins that were first exposed to low PO2. Such studies suggest that acute hypoxia primarily targets the cardiac calcium channels, where either the channel itself or moieties closely associated with it, for instance heme-oxygenase-2 (HO-2) interacting through kinase phosphorylation, signal the α-subunit of the channel to the altered levels of PO2. Amino acids 1572-1651, the CaMKII phosphorylation sites (S1487 and S1545), CaM-binding sites (I1624 and Q1625), and Ser1928 of the carboxyl tail of the α-subunit appear to be critical residues that sense oxygen. Future studies on HO-2 knockout mice or CRISPR/Cas9 gene-edited human-induced pluripotent stem-cell-derived cardiomyocytes that reduce CaM-binding affinity are likely to provide deeper insights into the O2-sensing mechanisms.
Asunto(s)
Hipoxia , Oxígeno , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Hemo-Oxigenasa 1/metabolismo , Humanos , Hipoxia/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Oxígeno/metabolismo , FosforilaciónRESUMEN
Ischemic heart disease is an arrhythmogenic condition, accompanied by hypoxia, acidosis, and impaired Ca2+ signaling. Here we report on effects of acute hypoxia and acidification in rat neonatal cardiomyocytes cultures. RESULTS: Two populations of neonatal cardiomyocyte were identified based on inactivation kinetics of L-type ICa: rapidly-inactivating ICa (τ~20ms) myocytes (prevalent in 3-4-day cultures), and slow-inactivating ICa (τ≥40ms) myocytes (dominant in 7-day cultures). Acute hypoxia (pO2<5mmHg for 50-100s) suppressed ICa reversibly in both cell-types to different extent and with different kinetics. This disparity disappeared when Ba2+ was the channel charge carrier, or when the intracellular Ca2+ buffering capacity was increased by dialysis of high concentrations of EGTA and BAPTA, suggesting critical role for calcium-dependent inactivation. Suppressive effect of acute acidosis on ICa (~40%, pH6.7), on the other hand, was not cell-type dependent. Isoproterenol enhanced ICa in both cell-types, but protected only against suppressive effects of acidosis and not hypoxia. Hypoxia and acidosis suppressed global Ca2+ transients by ~20%, but suppression was larger, ~35%, at the RyR2 microdomains, using GCaMP6-FKBP targeted probe. Hypoxia and acidosis also suppressed mitochondrial Ca2+ uptake by 40% and 10%, respectively, using mitochondrial targeted Ca2+ biosensor (mito-GCaMP6). CONCLUSION: Our studies suggest that acute hypoxia suppresses ICa in rapidly inactivating cell population by a mechanism involving Ca2+-dependent inactivation, while compromised mitochondrial Ca2+ uptake seems also to contribute to ICa suppression in slowly inactivating cell population. Proximity of cellular Ca2+ pools to sarcolemmal Ca2+ channels may contribute to the variability of inactivation kinetics of ICa in the two cell populations, while acidosis suppression of ICa appears mediated by proton-induced block of the calcium channel.
Asunto(s)
Acidosis/patología , Señalización del Calcio , Hipoxia/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Acidosis/metabolismo , Enfermedad Aguda , Animales , Animales Recién Nacidos , Bario/metabolismo , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Concentración de Iones de Hidrógeno , Hipoxia/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Isoproterenol/farmacología , Cinética , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Factores de TiempoRESUMEN
Cardiac arrhythmia is a major cause of mortality in cardiovascular pathologies. A host of drugs targeted to sarcolemmal Na+, Ca2+, and K+ channels has had limited success clinically. Recently, Ca2+ signaling has been target of pharmacotherapy based on finding that leaky ryanodine receptors elevate local Ca2+ concentrations causing membrane depolarizations that trigger arrhythmias. In this study, we report that xanthohumol, an antioxidant extracted from hops showing therapeutic effects in other pathologies, suppresses aberrant ryanodine receptor Ca2+ release. The effects of xanthohumol (5-1000 nM) on Ca2+ signaling pathways were probed in isolated rat ventricular myocytes incubated with Fluo-4 AM using the perforated patch-clamp technique. We found that 5-50 nM xanthohumol reduced the frequency of spontaneously occurring Ca2+ sparks (>threefold) and Ca2+ waves in control myocytes and in cells subjected to Ca2+ overload caused by the following: 1) exposure to low K+ solutions, 2) periods of high frequency electrical stimulation, 3) exposures to isoproterenol, or 4) caffeine. At room temperatures, 50-100 nM xanthohumol reduced the rate of relaxation of electrically- or caffeine-triggered Ca2+transients, without suppressing ICa, but this effect was small and reversed by isoproterenol at physiologic temperatures. Xanthohumol also suppressed the Ca2+ content of the SR and its rate of recirculation. The stabilizing effects of xanthohumol on the frequency of spontaneously triggered Ca2+ sparks and waves combined with its antioxidant properties, and lack of significant effects on Na+ and Ca2+ channels, may provide this compound with clinically desirable antiarrhythmic properties.
Asunto(s)
Antiarrítmicos/farmacología , Señalización del Calcio/efectos de los fármacos , Flavonoides/farmacología , Ventrículos Cardíacos/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Propiofenonas/farmacología , Animales , Antiarrítmicos/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Flavonoides/metabolismo , Isoproterenol/farmacología , Masculino , Miocitos Cardíacos/metabolismo , Propiofenonas/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , TemperaturaRESUMEN
The sinoatrial (SA) node is the primary site from which the mammalian heart is paced, but the mechanisms underlying the pacemaking still remain clouded. It is generally believed that the hyperpolarization-activated current If, encoded by hyperpolarization-activated cyclic nucleotide-gated (HCN) genes, contributes significantly to pacing, which in tandem with inward current generated by efflux of Ca2+ via the Na+-Ca2+ exchanger (NCX), resulting from the released Ca2+, mediates the diastolic depolarization. Here, we review the data that implicate If as the "pacemaker current" and conclude that there is not only a significant discrepancy between the range of diastolic depolarization potential (-60 to -40 mV) and the activation potential of If (negative to -70 mV), but that also the kinetics of If and its pharmacology are incompatible with the frequency of a heartbeat in rodents and humans. We propose that If serves as a functional insulator, which protects the SA-nodal cells against the large negative electrical sink of atrial tissue connected to it with connexins. We also evaluate the role of If and calcium signaling in mediating the diastolic depolarization in rat neonatal cardiomyocytes (rN-CM), and human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CM), and provide evidence for a possible involvement of mitochondrial Ca2+ in initiating the oscillatory events required for the spontaneous pacing.
Asunto(s)
Relojes Biológicos , Frecuencia Cardíaca , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Nodo Sinoatrial/metabolismo , Potenciales de Acción , Animales , Señalización del Calcio , Diferenciación Celular , Conexinas/metabolismo , Humanos , Mitocondrias Cardíacas/metabolismo , Modelos Cardiovasculares , Ratas , Nodo Sinoatrial/citología , Factores de TiempoRESUMEN
AIMS: CRISPR/Cas9 gene edits of cardiac ryanodine receptor (RyR2) in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide a novel platform for introducing mutations in RyR2 Ca2+-binding residues and examining the resulting excitation contraction (EC)-coupling remodelling consequences. METHODS AND RESULTS: Ca2+-signalling phenotypes of mutations in RyR2 Ca2+-binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and total internal reflection fluorescence-imaged wild type and mutant cardiomyocytes infected with sarcoplasmic reticulum (SR)-targeted ER-GCaMP6 probe. (i) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocytes; (ii) spontaneous beating (Fura-2 Ca2+ transients) persisted in mutant cells without the SR-release signals; (iii) while 5-20â mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant cells, only â¼20% to â¼70% of intact myocytes responded respectively to caffeine; (iv) and 20â mM caffeine transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red. CONCLUSION: Mutating RyR2 Ca2+-binding residues, irrespective of their reported pathogenesis, suppressed both ICa- and caffeine-triggered Ca2+ releases, suggesting interaction between Ca2+- and caffeine-binding sites. Enhanced transmembrane calcium influx and remodelling of EC-coupling pathways may underlie the persistence of spontaneous beating in Ca2+-induced Ca2+ release-suppressed mutant myocytes.
Asunto(s)
Miocitos Cardíacos , Canal Liberador de Calcio Receptor de Rianodina , Humanos , Cafeína/farmacología , Cafeína/metabolismo , Calcio/metabolismo , Fura-2/metabolismo , Miocitos Cardíacos/metabolismo , Mutación Puntual , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
AIMS: Previous studies have identified RyR2 W4645R mutation, located in the caffeine-binding site, to associate with CPVT1 pathology. Caffeine binding to its site is thought to displace the carboxyl-terminal domain to Ca2+-binding, allowing the tryptophan residue (W4645) to regulate Ca2+ sensitivity of RyR2. To gain insights into regulation of RyR2 Ca2+-binding and its interaction with caffeine-binding site, we introduced W4645R-RyR2 point mutation via CRISPR/Cas9 gene-editing in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) and characterized their Ca2+-signaling phenotype compared to WT hiPSCCMs. METHODS AND RESULTS: W4645R-RyR2 cardiomyocytes had: (1) no significant change in ICa magnitude or voltage-dependence; (2) slightly reduced CICR; (3) altered relaxation kinetics of Ca2+-transients with no change in isoproterenol sensitivity; (4) complete loss of caffeine-triggered Ca2+ release; (5) larger SR Ca2+ leak resulting in 40 % lower SR Ca2+ content, as determined by myocytes' response to 4-CmC; (6) lower incidence of calcium sparks and asynchronous spontaneous SR Ca2+ releases. CONCLUSIONS: W4645R-RyR2 mutation induces loss of caffeine-triggered SR Ca2+ release and enhances SR Ca2+ leak that underlie asynchronous spontaneous Ca2+ releases, triggering arrhythmia and impairing cardiac function.
RESUMEN
Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice. Spontaneously occurring Ca(2+) spark frequency, measured at -80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca(2+) transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca(2+) transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na(+)-Ca(2+) exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca(2+) transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2(ADA) mutation produces significant reduction in ICa density and ICa-triggered Ca(2+) release gain, longer but infrequently occurring Ca(2+) sparks, larger sarcoplasmic reticulum Ca(2+) loads, and spontaneous Ca(2+) releases accompanied by activation of large and potentially arrhythmogenic inward INCX.
Asunto(s)
Señalización del Calcio , Calmodulina/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Potenciales de Acción , Animales , Cafeína/farmacología , Ratones , Mutación , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Canal Liberador de Calcio Receptor de Rianodina/genética , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation-contraction coupling. This study aims to determine electrophysiology and Ca(2+)-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment. Measurements of membrane capacitance, dye transfer, expression of connexin 43 (Cx43), and transmission of ionic currents (I(Ca), I(Na)) from one cell to the next suggest that a subset of co-cultured CPCeP and NRCM becomes connected via gap junctions. Unlike NRCM, CPCeP had no significant I(Na), but expressed nifedipine-sensitive I(Ca) that could be measured more consistently with Ba(2+) as permeant ion using ramp-clamp protocols than with Ca(2+) and step-depolarization protocols. The magnitude of I(Ca) in CPCeP increased during culture (4-7 days vs. 1-3 days) and was larger in co-cultures with NRCM and with NRCM-conditioned medium, than in mono-cultured CPCeP. I(Ca) was virtually absent in CPC without engineered expression of Pim-1. Caffeine and KCl-activated Ca(2+)-transients were significantly present in co-cultured CPCeP, but smaller than in NRCM. Conversely, ATP-induced (IP(3)-mediated) Ca(2+) transients were larger in CPCeP than in NRCM. I(NCX) and I(ATP) were expressed in equivalent densities in CPCeP and NRCM. These in vitro studies suggest that CPCeP in co-culture with NRCM: a) develop I(Ca) current and Ca(2+) signaling consistent with cardiac lineage, b) form electrical connections via Cx43 gap junctions, and c) respond to paracrine signals from NRCM. These properties may be essential for durable and functional myocardial regeneration under in vivo conditions.
Asunto(s)
Células Madre Adultas/fisiología , Diferenciación Celular , Miocitos Cardíacos/fisiología , Proteínas Proto-Oncogénicas c-pim-1/genética , Proteínas Recombinantes de Fusión/genética , Adenosina Trifosfato/fisiología , Células Madre Adultas/metabolismo , Animales , Señalización del Calcio , Comunicación Celular , Forma de la Célula , Células Cultivadas , Técnicas de Cocultivo , Conexina 43/metabolismo , Medios de Cultivo Condicionados , Uniones Comunicantes/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Fenotipo , Proteínas Proto-Oncogénicas c-pim-1/biosíntesis , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Acute and chronic hypoxias are common cardiac diseases that lead often to arrhythmia and impaired contractility. At the cellular level it is unclear whether the suppression of cardiac Ca(2+) channels (Ca(V)1.2) results directly from oxygen deprivation on the channel protein or is mediated by intermediary proteins affecting the channel. To address this question we measured the early effects of hypoxia (5-60 s, P(O(2)) < 5 mmHg) on Ca(2+) current (I(Ca)) and tested the involvement of protein kinase A (PKA) phosphorylation, Ca(2+)/calmodulin-mediated signalling and the haem oxygenase (HO) pathway in the hypoxic regulation of Ca(V)1.2 in rat and cat ventricular myocytes and HEK-293 cells. Hypoxic suppression of ICa) and Ca(2+) transients was significant within 5 s and intensified in the following 50 s, and was reversible. Phosphorylation by cAMP or the phosphatase inhibitor okadaic acid desensitized I(Ca) to hypoxia, while PKA inhibition by H-89 restored the sensitivity of I(Ca) to hypoxia. This phosphorylation effect was specific to Ca(2+), but not Ba(2+) or Na(+), permeating through the channel. CaMKII inhibitory peptide and Bay K8644 reversed the phosphorylation-induced desensitization to hypoxia. Mutation of CAM/CaMKII-binding motifs of the α(1c) subunit of Ca(V)1.2 fully desensitized the Ca(2+) channel to hypoxia. Rapid application of HO inhibitors (zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP)) suppressed the channel in a manner similar to acute hypoxia such that: (1) I(Ca) and I(Ba) were suppressed within 5 s of ZnPP application; (2) PKA activation and CaMKII inhibitors desensitized I(Ca), but not I(Ba), to ZnPP; and (3) hypoxia failed to further suppress I(Ca) and I(Ba) in ZnPP-treated myocytes. We propose that the binding of HO to the CaM/CaMKII-specific motifs on Ca(2+) channel may mediate the rapid response of the channel to hypoxia.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hipoxia/metabolismo , Miocardio/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Gatos , Hipoxia de la Célula , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Humanos , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Fosforilación , RatasRESUMEN
Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available. Recent development of CRISPR/Cas9 gene editing system makes it, however, possible to introduce arrhythmia-associated point mutations at the desired loci of the wild-type hiPSCs in relatively short times. This platform was used by us to compare the Ca2+ signaling phenotypes of cardiomyocytes harboring point mutations in cardiac Ca2+ release channel, type-2 ryanodine receptor (RyR2), since over 200 missense mutations in RYR2 gene appear to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1). We have created cardiac myocytes harboring mutations in different domains of RyR2, to study not only their Ca2+ signaling consequences but also their drug and domain specificity as related to CPVT1 pathology. In this chapter, we describe our procedures to establish CRISPR/Cas9 gene-edited hiPSC-derived cardiomyocytes.
Asunto(s)
Células Madre Pluripotentes Inducidas , Canal Liberador de Calcio Receptor de Rianodina , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Sistemas CRISPR-Cas , Calcio/metabolismo , Edición Génica/métodos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Taquicardia VentricularRESUMEN
AIMS: To gain insights into FKBP regulation of cardiac ryanodine receptor (RyR2) and Ca2+ signaling, we introduced the point mutation (N771D-RyR2) corresponding to skeletal muscle mutation (N760D-RyR1) associated with central core disease (CCD) via CRISPR/Cas9 gene-editing in the RyR2 FKBP binding site expressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs). Patients inflicted with CCD and other hereditary skeletal muscle diseases often show higher incidence of atrial or ventricular arrhythmias. METHODS AND RESULTS: Ca2+ imaging of voltage-clamped N771D-RyR2 mutant compared to WT hiPSCCMs showed: (1) â¼30% suppressed ICa with no significant changes in the gating kinetics of ICa; (2) 29% lower SR Ca2+ content and 33% lower RyR2 Ca2+ leak; (3) higher CICR gain and 30-35% increased efficiency of ICa-triggered Ca2±release; (4) higher incidence of aberrant SR Ca2+ releases, DADs, and Ca2+ sparks; (5) no change in fractional Ca2+-release, action potential morphology, sensitivity to isoproterenol, and sarcomeric FKBP-binding pattern. CONCLUSIONS: The more frequent spontaneous Ca2+ releases and longer Ca2+ sparks underlie the increased incidence of DADs and cellular arrhythmogenesis of N771D-RyR2 mutant. The smaller RyR2 Ca2±leak and SR content result from suppressed ICathat is compensated by higher CICR gain.
Asunto(s)
Señalización del Calcio , Células Madre Pluripotentes Inducidas , Canal Liberador de Calcio Receptor de Rianodina , Sitios de Unión , Calcio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
To determine the role of cellularly generated protons in synaptic signaling, we recorded GABA miniature IPSCs (mIPSCs) from cultured rat cerebellar granule cells (CGCs) while varying the extracellular pH buffering capacity. Consistent with previous reports, we found that increasing pH from 7.4 to 8.0 sped mIPSC rise time and suppressed both amplitude of the current and total charge transferred. Conversely, acidification (from pH 7.4 to 6.8) slowed the rise time and increased current amplitude and total charge transferred. In a manner consistent with alkalinization, increasing the buffering capacity from 3 to 24 mm HEPES at pH 7.4 resulted in faster mIPSC rise time, a 37% reduction in amplitude, and a 48% reduction in charge transferred. Supplementing the normal physiological buffers (24 mm HCO(3)(-)/5%CO(2)) with 10 mm HEPES similarly diminished mIPSCs in a manner consistent with alkalinization, resulting in faster rise time, a 39% reduction in amplitude, and a 51% reduction in charge transferred. These findings suggest the existence of an acidifying synaptic force that is overcome by commonly used concentrations (10 mm) of HEPES buffer. Here we show that Na(+)/H(+) exchanger (NHE) activity appears to, in part, contribute to this synaptic acidification because inhibition of NHE by amiloride or lithium under physiological or weak buffering conditions alters mIPSCs in a manner consistent with alkalinization. These results suggest that acidification of the synaptic cleft occurs physiologically during GABAergic transmission and that NHE plays a critical role in generating the acidic nano-environment at the synapse.
Asunto(s)
Potenciales Postsinápticos Inhibidores/fisiología , Receptores de GABA-A/fisiología , Sinapsis/fisiología , Animales , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/fisiología , Femenino , HEPES/farmacología , Concentración de Iones de Hidrógeno , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacosRESUMEN
BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Potenciales de Acción , Calcio/metabolismo , Catecolaminas/metabolismo , Diferenciación Celular , Colforsina/metabolismo , AMP Cíclico/metabolismo , Electrocardiografía , Heterocigoto , Humanos , Células Madre Pluripotentes Inducidas/citología , Cariotipificación , Mutación , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Fenotipo , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologíaRESUMEN
BACKGROUND: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) created from patients with catecholaminergic polymorphic ventricular tachycardia 1 (CPVT1) have been used to study CPVT1 arrhythmia. OBJECTIVE: The purpose of this study was to evaluate the Ca2+ signaling aberrancies and pharmacological sensitivities of 3 CRISPR/Cas9-introduced CPVT1 mutations located in different molecular domains of ryanodine receptor 2 (RyR2). METHODS: CRISPR/Cas9-engineered hiPSC-CMs carrying RyR2 mutations-R420Q, Q4201R, and F2483I-were voltage clamped, and their electrophysiology, pharmacology, and Ca2+ signaling phenotypes measured using total internal reflection fluorescence microscopy. RESULTS: R420Q and Q4201R mutant hiPSC-CMs exhibit irregular, long-lasting, spatially wandering Ca2+ sparks and aberrant Ca2+ releases similar to F2483I unlike the wild-type myocytes. Large sarcoplasmic reticulum (SR) Ca2+ leaks and smaller SR Ca2+ contents were detected in cells expressing Q4201R and F2483I, but not R420Q. Fractional Ca2+ release and calcium-induced calcium release gain were higher in Q4201R than in R420Q and F2483I hiPSC-CMs. JTV519 was equally effective in suppressing Ca2+ sparks, waves, and SR Ca2+ leaks in hiPSC-CMs derived from all 3 mutant lines. Flecainide and dantrolene similarly suppressed SR Ca2+ leaks, but were less effective in decreasing spark frequency and durations. CONCLUSION: CRISPR/Cas9 gene editing of hiPSCs provides a novel approach in studying CPVT1-associated RyR2 mutations and suggests that Ca2+-signaling aberrancies and drug sensitivities may vary depending on the mutation site.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Edición Génica/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Proteína 9 Asociada a CRISPR/metabolismo , Calcio/metabolismo , Señalización del Calcio , Diferenciación Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN/genética , Análisis Mutacional de ADN , Humanos , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/patología , Fenotipo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologíaRESUMEN
The dominant mode of intracellular Ca(2+) release in adult mammalian heart is gated by ryanodine receptors (RyRs), but it is less clear whether inositol 1,4,5-trisphosphate (IP(3))-gated Ca(2+) release channels (IP(3)Rs), which are important during embryogenesis, play a significant role during early postnatal development. To address this question, we measured confocal two-dimensional Ca(2+) dependent fluorescence images in acutely isolated neonatal (days 1 to 2) and juvenile (days 8-10) rat cardiomyocytes, either voltage-clamped or permeabilized, where rapid exchange of solution could be used to selectively activate the two types of Ca(2+) release channel. Targeting RyRs with caffeine produced large and rapid Ca(2+) signals throughout the cells. Application of ATP and endothelin-1 to voltage-clamped, or IP(3) to permeabilized, cells produced smaller and slower Ca(2+) signals that were most prominent in subsarcolemmal regions and were suppressed by either the IP(3)R-blocker 2-aminoethoxydiphenylborate or replacement of the biologically active form of IP(3) with its L-stereoisomer. Such IP(3)R-gated Ca(2+) releases were amplified by Ca(2+)-induced Ca(2+) release (CICR) via RyRs since they were also reduced by compounds that block the RyRs (tetracaine) or deplete the Ca(2+) pools they gate (caffeine, ryanodine). Spatial analysis revealed both subsarcolemmal and perinuclear origins for the IP(3)-mediated Ca(2+) release events RyR- and IP(3)R-gated Ca(2+) signals had larger magnitudes in juvenile than in neonatal cardiomyocytes. Ca(2+) signaling was generally quite similar in atrial and ventricular cardiomyocytes but showed divergent development of IP(3)-mediated regulation in juveniles. Our data suggest that an intermediate stage of Ca(2+) signaling may be present in developing cardiomyocytes, where, in addition to RyR-gated Ca(2+) pools, IP(3)-gated Ca(2+) release is sufficiently large in magnitude and duration to trigger or contribute to activation of CICR and cardiac contraction.
Asunto(s)
Envejecimiento/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/fisiología , Miocitos Cardíacos/fisiología , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Adenosina Trifosfato/metabolismo , Animales , Compuestos de Boro/farmacología , Cafeína/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/efectos de los fármacos , Miocitos Cardíacos/citología , Técnicas de Placa-Clamp , Ratas , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacosRESUMEN
Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) have been extensively used for in vitro modeling of human cardiovascular disease, drug screening and pharmacotherapy, but little rigorous studies have been reported on their biophysical or Ca2+ signaling properties. There is also considerable concern as to the level of their maturity and whether they can serve as reliable models for adult human cardiac myocytes. Ultrastructural difference such as lack of t-tubular network, their polygonal shapes, disorganized sarcomeric myofilament, and their rhythmic automaticity, among others, have been cited as evidence for immaturity of hiPSC-CMs. In this review, we will deal with Ca2+ signaling, its regulation, and its stage of maturity as compared to the mammalian adult cardiomyocytes. We shall summarize the data on functional aspects of Ca2+signaling and its parameters that include: L-type calcium channel (Cav1.2), ICa-induced Ca2+release, CICR, and its parameters, cardiac Na/Ca exchanger (NCX1), the ryanodine receptors (RyR2), sarco-reticular Ca2+pump, SERCA2a/PLB, and the contribution of mitochondrial Ca2+ to hiPSC-CMs excitation-contraction (EC)-coupling as compared with adult mammalian cardiomyocytes. The comparative studies suggest that qualitatively hiPSC-CMs have similar Ca2+signaling properties as those of adult cardiomyocytes, but quantitative differences do exist. This review, we hope, will allow the readers to judge for themselves to what extent Ca2+signaling of hiPSC-CMs represents the adult form of this signaling pathway, and whether these cells can be used as good models of human cardiomyocytes.
Asunto(s)
Señalización del Calcio , Células Madre Pluripotentes Inducidas/citología , Mamíferos/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Adulto , Animales , Diferenciación Celular , Humanos , Modelos BiológicosRESUMEN
Voltage-gated calcium (Ca(2+)) channels are thought to play an important role in epileptogenesis and seizure generation. Here, using the whole cell configuration of patch-clamp techniques, we report on the modifications of biophysical and pharmacological properties of high threshold voltage-activated Ca(2+) channel currents in inferior colliculus (IC) neurons of the genetically epilepsy-prone rats (GEPR-3s). Ca(2+) channel currents were measured by depolarizing pulses from a holding potential of - 80 mV using barium (Ba(2+)) as the charge carrier. We found that the current density of high threshold voltage-activated Ca(2+) channels was significantly larger in IC neurons of seizure-naive GEPR-3s compared to control Sprague-Dawley rats, and that seizure episodes further enhanced the current density in the GEPR-3s. The increased current density was reflected by both a - 20 mV shifts in channel activation and a 25% increase in the non-inactivating fraction of channels in seizure-naive GEPR-3s. Such changes were reduced by seizure episodes in the GEPR-3s. Pharmacological analysis of the current density suggests that upregulation of L-, N- and R-type of Ca(2+) channels may contribute to IC neuronal hyperexcitability that leads to seizure susceptibility in the GEPR-3s.
Asunto(s)
Canales de Calcio/metabolismo , Epilepsia/metabolismo , Colículos Inferiores/metabolismo , Neuronas/metabolismo , Ratas Endogámicas/genética , Animales , Modelos Animales de Enfermedad , Epilepsia/genética , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
AIMS: The effects of acute (100 s) hypoxia and/or acidosis on Ca2+ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time. METHODS AND RESULTS: 1) hiPSC-CMs express two cell populations: rapidly-inactivating ICa myocytes (τi<40 ms, in 4-5 day cultures) and slowly-inactivating ICa (τi ≥ 40 ms, in 6-8 day cultures). 2) Hypoxia suppressed ICa by 10-20% in rapidly- and 40-55% in slowly-inactivating ICa cells. 3) Isoproterenol enhanced ICa in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba2+ was the charge carrier through the calcium channels, implicating Ca2+-dependent inactivation in O2 sensing. 5) Acidosis suppressed ICa by â¼35% and â¼25% in rapidly and slowly inactivating ICa cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was â¼25% in global and â¼37% in RyR2 Ca2+-microdomains in either cell type, whereas with hypoxia suppression was â¼20% and â¼25% respectively in global and RyR2-microdomaine in rapidly and â¼35% and â¼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells. CONCLUSIONS: Variability in ICa inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca2+-signaling in the two-cell types arises from differential Ca2+-dependent inactivation of the Ca2+-channel caused by proximity of Ca2+-release stores to the Ca2+ channels.