RESUMEN
The taste is biologically of intrinsic importance. It almost momentarily perceives environmental stimuli for better survival. In the early 2000s, research into taste reception was greatly developed with discovery of the receptors. However, the mechanism of salt taste reception is not fully elucidated yet and many questions still remain. At present, next-generation sequencing and genome-editing technologies are available which would become pivotal tools to elucidate the remaining issues. Here we review current mechanisms of salt taste reception in particular and characterize the properties of transmembrane channel-like 4 as a novel salt taste-related molecule that we found using these sophisticated tools.
Asunto(s)
GustoRESUMEN
The mitochondrial Na+-Ca2+ exchanger, NCLX, was reported to supply Ca2+ to sarcoplasmic reticulum (SR)/endoplasmic reticulum, thereby modulating various cellular functions such as the rhythmicity of cardiomyocytes, and cellular Ca2+ signaling upon antigen receptor stimulation and chemotaxis in B lymphocytes; however, there is little information on the spatial relationships of NCLX with SR Ca2+ handling proteins, and their physiological impact. Here we examined the issue, focusing on the interaction of NCLX with an SR Ca2+ pump SERCA in cardiomyocytes. A bimolecular fluorescence complementation assay using HEK293 cells revealed that the exogenously expressed NCLX was localized in close proximity to four exogenously expressed SERCA isoforms. Immunofluorescence analyses of isolated ventricular myocytes showed that the NCLX was localized to the edges of the mitochondria, forming a striped pattern. The co-localization coefficients in the super-resolution images were higher for NCLX-SERCA2, than for NCLX-ryanodine receptor and NCLX-Na+/K+ ATPase α-1 subunit, confirming the close localization of endogenous NCLX and SERCA2 in cardiomyocytes. The mathematical model implemented with the spatial and functional coupling of NCLX and SERCA well reproduced the NCLX inhibition-mediated modulations of SR Ca2+ reuptake in HL-1 cardiomyocytes. Taken together, these results indicated that NCLX and SERCA are spatially and functionally coupled in cardiomyocytes.
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Retículo Sarcoplasmático , Intercambiador de Sodio-Calcio , Calcio/metabolismo , Células HEK293 , Humanos , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
It has been over 10 years since SLC24A6/SLC8B1, coding the Na+/Ca2+/Li+ exchanger (NCLX), was identified as the gene responsible for mitochondrial Na+-Ca2+ exchange, a major Ca2+ efflux system in cardiac mitochondria. This molecular identification enabled us to determine structure-function relationships, as well as physiological/pathophysiological contributions, and our understandings have dramatically increased. In this review, we provide an overview of the recent achievements in relation to NCLX, focusing especially on its heart-specific characteristics, biophysical properties, and spatial distribution in cardiomyocytes, as well as in cardiac mitochondria. In addition, we discuss the roles of NCLX in cardiac functions under physiological and pathophysiological conditions-the generation of rhythmicity, the energy metabolism, the production of reactive oxygen species, and the opening of mitochondrial permeability transition pores.
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Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/metabolismo , Animales , Metabolismo Energético , Humanos , Mitocondrias Cardíacas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Modelos Moleculares , Conformación Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
"Salty taste" sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.
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Cloruro de Sodio , Gusto , Amilorida , Animales , Canales de Cloruro/genética , Células HEK293 , Humanos , Proteínas de la Membrana , RatonesRESUMEN
NCLX was identified as a mitochondrial Na+-Ca2+ exchanger. However, contribution of NCLX to overall mitochondrial Na+-Ca2+ exchange activity remains unclear, especially in brain mitochondria where plasma membrane Na+-Ca2+ exchanger NCX also exists. We studied the issue using isolated mouse brain mitochondria. The Na+- as well as Li+-dependent Ca2+ efflux from mitochondria was significantly inhibited by a NCLX blocker, but was insensitive to NCX blockers, suggesting that NCLX comprises a major part in forward mode of mitochondrial Na+-Ca2+ exchange activity. On the other hand, the Na+-dependent Ca2+ influx into mitochondria, the reverse mode, was insensitive to all the blockers tested, suggesting unidentified Ca2+ transport systems.
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Encéfalo/metabolismo , Mitocondrias/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Encéfalo/efectos de los fármacos , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Compuestos de Rutenio/farmacología , Intercambiador de Sodio-Calcio/antagonistas & inhibidoresRESUMEN
The electrogenicity of mitochondrial Na+-Ca2+ exchange (NCXm) had been controversial and no membrane current through it had been reported. We succeeded for the first time in recording NCXm-mediated currents using mitoplasts derived from mouse ventricle. Under conditions that K+, Cl-, and Ca2+ uniporter currents were inhibited, extra-mitochondrial Na+ induced inward currents with 1 µM Ca2+ in the pipette. The half-maximum concentration of Na+ was 35.6 mM. The inward current was diminished without Ca2+ in the pipette, and was augmented with 10 µM Ca2+. The Na+-induced inward currents were largely inhibited by CGP-37157, an NCXm blocker. However, the reverse mode of NCXm, which should be detected as an outward current, was hardly induced by extra-mitochondrial application of Ca2+ with Na+ in the pipette. It was concluded that NCXm is electrogenic. This property may be advantageous for facilitating Ca2+ extrusion from mitochondria, which has large negative membrane potential.
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Ventrículos Cardíacos/metabolismo , Corazón/fisiología , Mitocondrias/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
It has been an unsolved question how cardiac mitochondrial energetics is regulated during working transition. Mathematical modelling is a powerful tool for exploring the complicated networks of mitochondrial metabolism. We summarize the recent progress and remaining questions about mitochondrial energetics in heart, especially focusing on approaches utilizing mathematical modelling. Feedback activation by ADP and/or inorganic phosphate is an old but still attractive hypothesis for explaining the regulation mechanisms of cardiac mitochondrial energetics. However, this hypothesis has not been fully validated by experiments because rises of ADP and/or inorganic phosphate concentrations during cardiac workload increase have not been detected in many experiments. The hypothesis of intracellular energetic units is an extended version of feedback activation, which has a similar problem. The each-step activation hypothesis beautifully reproduces metabolite constancy, although such master regulators have not been identified yet. Ca2+ has been the most plausible candidate because some of the mitochondrial dehydrogenases are activated by it. Recent experimental and simulation studies, however, throw doubt on its physiological relevance. Finally, we discuss issues to be solved to obtain a better view of cardiac mitochondrial energetics.
Asunto(s)
Metabolismo Energético , Mitocondrias , Simulación por Computador , Corazón , Mitocondrias/metabolismo , Modelos TeóricosRESUMEN
Roles of mitochondrial Na+-Ca2+ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca2+ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca2+ recycling via NCLX and sarco/endoplasmic reticulum Ca2+-ATPase SERCA, and interaction with store-operated Ca2+ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.
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Linfocitos/metabolismo , Mitocondrias/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Calcio/metabolismo , Movimiento Celular , Humanos , Inmunidad , Linfocitos/citologíaRESUMEN
AIMS: Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4. METHODS AND RESULTS: Patch clamp and biochemical analyses were performed using expression system and an immortalized atrial cardiomyocyte cell line (HL-1), and numerical model simulation was employed. After rapid desensitization, robust reactivation of TRPM4 channels required high micromolar concentrations of Ca2+. However, upon evaluation with a newly devised, ionomycin-permeabilized cell-attached (Iono-C/A) recording technique, submicromolar concentrations of Ca2+ (apparent Kd = â¼500 nM) were enough to activate this channel. Similar submicromolar Ca2+ dependency was also observed with sharp electrode whole-cell recording and in experiments coexpressing TRPM4 and L-type voltage-dependent Ca2+ channels. Numerical simulations using a number of action potential (AP) models (HL-1, Nygren, Luo-Rudy) incorporating the Ca2+- and voltage-dependent gating parameters of TRPM4, as assessed by Iono-C/A recording, indicated that a few-fold increase in TRPM4 activity is sufficient to delay late AP repolarization and further increases (≥ six-fold) evoke early afterdepolarization. These model predictions are consistent with electrophysiological data from angiotensin II-treated HL-1 cells in which TRPM4 expression and activity were enhanced. CONCLUSIONS: These results collectively indicate that the TRPM4 channel is activated by a physiological range of Ca2+ concentrations and its excessive activity can cause arrhythmic changes. Moreover, these results demonstrate potential utility of the first AP models incorporating TRPM4 gating for in silico assessment of arrhythmogenicity in remodelling cardiac tissue.
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Potenciales de Acción , Arritmias Cardíacas/metabolismo , Simulación por Computador , Atrios Cardíacos/metabolismo , Frecuencia Cardíaca , Modelos Cardiovasculares , Miocitos Cardíacos/metabolismo , Análisis Numérico Asistido por Computador , Canales Catiónicos TRPM/metabolismo , Potenciales de Acción/efectos de los fármacos , Angiotensina II/farmacología , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Señalización del Calcio , Células HEK293 , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Cinética , Ratones , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-Clamp , Periodo Refractario Electrofisiológico , Canales Catiónicos TRPM/genéticaRESUMEN
INTRODUCTION: Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies. METHOD AND RESULTS: We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 µM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05). CONCLUSIONS: We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs.
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Potenciales de Acción/efectos de los fármacos , Estimulación Eléctrica , Modelos Biológicos , Taquicardia Ventricular/patología , Taquicardia Ventricular/terapia , Tiazepinas/farmacología , Adulto , Animales , Antiasmáticos/química , Antiasmáticos/farmacología , Antiasmáticos/uso terapéutico , Calcio/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Calsecuestrina/genética , Calsecuestrina/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Isoproterenol/farmacología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/trasplante , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Taquicardia Ventricular/tratamiento farmacológico , Tiazepinas/química , Tiazepinas/uso terapéutico , Trasplante HeterólogoRESUMEN
KEY POINTS: The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant during physiological cardiac workload transition. How this is accomplished is not yet clarified, though Ca2+ has been suggested to be one of the possible mechanisms. We constructed a detailed mathematical model of cardiac mitochondria based on experimental data and studied whether known Ca2+ -dependent regulation mechanisms play roles in the metabolite constancy. Model simulations revealed that the Ca2+ -dependent regulation mechanisms have important roles under the in vitro condition of isolated mitochondria where malate and glutamate were mitochondrial substrates, while they have only a minor role and the composition of substrates has marked influence on the metabolite constancy during workload transition under the simulated in vivo condition where many substrates exist. These results help us understand the regulation mechanisms of cardiac energy metabolism during physiological cardiac workload transition. ABSTRACT: The cardiac energy metabolites such as ATP, phosphocreatine, ADP and NADH are kept relatively constant over a wide range of cardiac workload, though the mechanisms are not yet clarified. One possible regulator of mitochondrial metabolism is Ca2+ , because it activates several mitochondrial enzymes and transporters. Here we constructed a mathematical model of cardiac mitochondria, including oxidative phosphorylation, substrate metabolism and ion/substrate transporters, based on experimental data, and studied whether the Ca2+ -dependent activation mechanisms play roles in metabolite constancy. Under the in vitro condition of isolated mitochondria, where malate and glutamate were used as mitochondrial substrates, the model well reproduced the Ca2+ and inorganic phosphate (Pi ) dependences of oxygen consumption, NADH level and mitochondrial membrane potential. The Ca2+ -dependent activations of the aspartate/glutamate carrier and the F1 Fo -ATPase, and the Pi -dependent activation of Complex III were key factors in reproducing the experimental data. When the mitochondrial model was implemented in a simple cardiac cell model, simulation of workload transition revealed that cytoplasmic Ca2+ concentration ([Ca2+ ]cyt ) within the physiological range markedly increased NADH level. However, the addition of pyruvate or citrate attenuated the Ca2+ dependence of NADH during the workload transition. Under the simulated in vivo condition where malate, glutamate, pyruvate, citrate and 2-oxoglutarate were used as mitochondrial substrates, the energy metabolites were more stable during the workload transition and NADH level was almost insensitive to [Ca2+ ]cyt . It was revealed that mitochondrial substrates have a significant influence on metabolite constancy during cardiac workload transition, and Ca2+ has only a minor role under physiological conditions.
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Calcio/metabolismo , Metabolismo Energético , Mitocondrias Cardíacas/metabolismo , Modelos Cardiovasculares , Animales , Simulación por Computador , Perros , Potencial de la Membrana Mitocondrial , Mitocondrias Cardíacas/fisiología , Miocardio/metabolismo , NAD/metabolismoRESUMEN
Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.
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Caspasa 8/metabolismo , Tamaño de la Célula , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Apoptosis , Células COS , Caspasa 8/genética , Chlorocebus aethiops , Activación Enzimática , Células HeLa , Humanos , Células MCF-7 , Mutación , Canales de Potasio de Dominio Poro en Tándem/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Transducción de Señal , Especificidad por Sustrato , Factores de Tiempo , Transfección , Xenopus laevisRESUMEN
Lymphocyte chemotaxis plays important roles in immunological reactions, although the mechanism of its regulation is still unclear. We found that the cytosolic Na(+)-dependent mitochondrial Ca(2+) efflux transporter, NCLX, regulates B lymphocyte chemotaxis. Inhibiting or silencing NCLX in A20 and DT40 B lymphocytes markedly increased random migration and suppressed the chemotactic response to CXCL12. In contrast to control cells, cytosolic Ca(2+) was higher and was not increased further by CXCL12 in NCLX-knockdown A20 B lymphocytes. Chelating intracellular Ca(2+) with BAPTA-AM disturbed CXCL12-induced chemotaxis, suggesting that modulation of cytosolic Ca(2+) via NCLX, and thereby Rac1 activation and F-actin polymerization, is essential for B lymphocyte motility and chemotaxis. Mitochondrial polarization, which is necessary for directional movement, was unaltered in NCLX-knockdown cells, although CXCL12 application failed to induce enhancement of mitochondrial polarization, in contrast to control cells. Mouse spleen B lymphocytes were similar to the cell lines, in that pharmacological inhibition of NCLX by CGP-37157 diminished CXCL12-induced chemotaxis. Unexpectedly, spleen T lymphocyte chemotaxis was unaffected by CGP-37157 treatment, indicating that NCLX-mediated regulation of chemotaxis is B lymphocyte-specific, and mitochondria-endoplasmic reticulum Ca(2+) dynamics are more important in B lymphocytes than in T lymphocytes. We conclude that NCLX is pivotal for B lymphocyte motility and chemotaxis.
Asunto(s)
Linfocitos B/citología , Calcio/metabolismo , Quimiotaxis/fisiología , Intercambiador de Sodio-Calcio/metabolismo , Actinas/metabolismo , Animales , Linfocitos B/metabolismo , Línea Celular Tumoral , Quimiocina CXCL12/metabolismo , Clonazepam/análogos & derivados , Clonazepam/farmacología , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Ratones , Mitocondrias/fisiología , Neuropéptidos/metabolismo , Intercambiador de Sodio-Calcio/genética , Bazo/citología , Bazo/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Tiazepinas/farmacología , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Mitochondrial Ca(2+) is known to regulate diverse cellular functions, for example energy production and cell death, by modulating mitochondrial dehydrogenases, inducing production of reactive oxygen species, and opening mitochondrial permeability transition pores. In addition to the action of Ca(2+) within mitochondria, Ca(2+) released from mitochondria is also important in a variety of cellular functions. In the last 5 years, the molecules responsible for mitochondrial Ca(2+) dynamics have been identified: a mitochondrial Ca(2+) uniporter (MCU), a mitochondrial Na(+)-Ca(2+) exchanger (NCLX), and a candidate for a mitochondrial H(+)-Ca(2+) exchanger (Letm1). In this review, we focus on the mitochondrial Ca(2+) release system, and discuss its physiological and pathophysiological significance. Accumulating evidence suggests that the mitochondrial Ca(2+) release system is not only crucial in maintaining mitochondrial Ca(2+) homeostasis but also participates in the Ca(2+) crosstalk between mitochondria and the plasma membrane and between mitochondria and the endoplasmic/sarcoplasmic reticulum.
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Calcio/metabolismo , Mitocondrias/metabolismo , Animales , Antiportadores/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
BACKGROUND: The proliferation of cardiomyocytes is highly restricted after postnatal maturation, limiting heart regeneration. Elucidation of the regulatory machineries for the proliferation and growth arrest of cardiomyocytes is imperative. Chemical biology is efficient to dissect molecular mechanisms of various cellular events and often provides therapeutic potentials. We have been investigating cardiovascular differentiation with pluripotent stem cells. The combination of stem cell and chemical biology can provide novel approaches to investigate the molecular mechanisms and manipulation of cardiomyocyte proliferation. METHODS AND RESULTS: To identify chemicals that regulate cardiomyocyte proliferation, we performed a screening of a defined chemical library based on proliferation of mouse pluripotent stem cell-derived cardiomyocytes and identified 4 chemical compound groups: inhibitors of glycogen synthase kinase-3, p38 mitogen-activated protein kinase, and Ca(2+)/calmodulin-dependent protein kinase II, and activators of extracellular signal-regulated kinase. Several appropriate combinations of chemicals synergistically enhanced proliferation of cardiomyocytes derived from both mouse and human pluripotent stem cells, notably up to a 14-fold increase in mouse cardiomyocytes. We also examined the effects of identified chemicals on cardiomyocytes in various developmental stages and species. Whereas extracellular signal-regulated kinase activators and Ca(2+)/calmodulin-dependent protein kinase II inhibitors showed proliferative effects only on cardiomyocytes in early developmental stages, glycogen synthase kinase-3 and p38 mitogen-activated protein kinase inhibitors substantially and synergistically induced re-entry and progression of cell cycle in neonatal but also as well as adult cardiomyocytes. CONCLUSIONS: Our approach successfully uncovered novel molecular targets and mechanisms controlling cardiomyocyte proliferation in distinct developmental stages and offered pluripotent stem cell-derived cardiomyocytes as a potent tool to explore chemical-based cardiac regenerative strategies.
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Diferenciación Celular , Proliferación Celular , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Animales , Animales Recién Nacidos , Western Blotting , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Inmunohistoquímica , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Mitochondrial Ca(2+) is known to change dynamically, regulating mitochondrial as well as cellular functions such as energy metabolism and apoptosis. The NCLX gene encodes the mitochondrial Na(+)-Ca(2+) exchanger (NCXmit), a Ca(2+) extrusion system in mitochondria. Here we report that the NCLX regulates automaticity of the HL-1 cardiomyocytes. NCLX knockdown using siRNA resulted in the marked prolongation of the cycle length of spontaneous Ca(2+) oscillation and action potential generation. The upstrokes of action potential and Ca(2+) transient were markedly slower, and sarcoplasmic reticulum (SR) Ca(2+) handling were compromised in the NCLX knockdown cells. Analyses using a mathematical model of HL-1 cardiomyocytes demonstrated that blocking NCXmit reduced the SR Ca(2+) content to slow spontaneous SR Ca(2+) leak, which is a trigger of automaticity. We propose that NCLX is a novel molecule to regulate automaticity of cardiomyocytes via modulating SR Ca(2+) handling.
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Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Potenciales de Acción , Animales , Calcio/metabolismo , Señalización del Calcio , Línea Celular , Simulación por Computador , Técnicas de Silenciamiento del Gen , Cinética , Ratones , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Retículo Sarcoplasmático/metabolismo , TransfecciónRESUMEN
Mitochondria Na(+)-Ca(2+) exchange (NCX(mit)) was first discovered by Carafoli et al. in 1974. Thereafter, the mechanisms and roles of NCX(mit) have been extensively studied. We review NCX(mit) in cardiomyocytes and lymphocytes by presenting our recent studies on it. Studies of NCX(mit) in rat ventricular cells demonstrated that NCX(mit) is voltage dependent and electrogenic. A targeted knockdown and knockout of NCLX in HL-1 cardiomyocytes and B lymphocytes, respectively, significantly reduced the NCX(mit) activity, indicating that NCLX is a major component of NCX(mit) in these cells. The store-operated Ca(2+) entry was greatly attenuated in NCLX knockout lymphocytes, suggesting that substantial amount of Ca(2+) enters into mitochondria and is released to cytosol via NCX(mit). NCX(mit) or NCLX has pivotal roles in Ca(2+) handling in mitochondria and cytoplasm.
Asunto(s)
Linfocitos B/metabolismo , Calcio/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Citoplasma/genética , Citoplasma/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Activación del Canal Iónico/fisiología , Proteínas Mitocondriales/genética , Proteínas Musculares/genética , Ratas , Intercambiador de Sodio-Calcio/genéticaRESUMEN
∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.
Asunto(s)
Cannabis/enzimología , Oxidorreductasas Intramoleculares/química , Psicotrópicos/química , Animales , Berberina/química , Dióxido de Carbono/química , Cristalografía por Rayos X , Cisteína/química , Flavina-Adenina Dinucleótido/química , Ácido Glutámico/química , Histidina/química , Oxidorreductasas Intramoleculares/genética , Mutación , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Tirosina/químicaRESUMEN
We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.
Asunto(s)
Calcio/metabolismo , Guanidinas/farmacología , Hipertrofia Ventricular Izquierda/metabolismo , Miocardio/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Animales , Células Cultivadas , Hipertrofia Ventricular Izquierda/inducido químicamente , Isoproterenol/farmacología , Masculino , Ratas , Ratas Wistar , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) increases upon activation of antigen-receptor in lymphocytes. Mitochondria have been suggested to regulate the [Ca(2+)](i) response, but the molecular mechanisms and the roles are poorly understood. To clarify them, we carried out a combination study of mathematical simulations and knockout or knockdown of NCLX, a gene candidate for the mitochondrial Na(+)-Ca(2+) exchanger (NCX(mit)), in B lymphocytes. A mathematical model of Ca(2+) dynamics in B lymphocytes demonstrated that NCX(mit) inhibition reduces basal Ca(2+) content of endoplasmic reticulum (ER) and suppresses B-cell antigen receptor (BCR)-mediated [Ca(2+)](i) rise. The predictions were validated in DT40 B lymphocytes of heterozygous NCLX knockout (NCLX(+/-)). In NCLX(+/-) cells, mitochondrial Ca(2+) efflux via NCX(mit) was strongly decelerated, suggesting NCLX is a gene responsible for NCX(mit) in B lymphocytes. Consistent with the predictions, ER Ca(2+) content declined and [Ca(2+)](i) hardly rose upon BCR activation in NCLX(+/-) cells. ER Ca(2+) uptake was reduced to â¼58% of the wild-type (WT), while it was comparable to WT when mitochondrial respiration was disturbed. Essentially the same results were obtained by a pharmacological inhibition or knockdown of NCLX by siRNA in A20 B lymphocytes. Unexpectedly, ER Ca(2+) leak was augmented and co-localization of mitochondria with ER was lower in NCLX(+/-) and NCLX silenced cells. Taken together, we concluded that NCLX is a key Ca(2+) provider to ER, and that NCLX-mediated Ca(2+) recycling between mitochondria and ER is pivotal in B cell responses to antigen.