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This retrospective begins with Galvani's experiments on frogs at the end of the 18th century and his discovery of 'animal electricity'. It goes on to illustrate the numerous contributions to the field of physical chemistry in the second half of the 19th century (Nernst's equilibrium potential, based on the work of Wilhelm Ostwald, Max Planck's ion electrodiffusion, Einstein's studies of Brownian motion) which led Bernstein to propose his membrane theory in the early 1900s as an explanation of Galvani's findings and cell excitability. These processes were fully elucidated by Hodgkin and Huxley in 1952 who detailed the ionic basis of resting and action potentials, but without addressing the question of where these ions passed. The emerging question of the existence of ion channels, widely debated over the next two decades, was finally accepted and, a decade later, many of them began to be cloned. This led to the possibility of modelling the activity of individual neurons in the brain and then that of simple circuits. Taking advantage of the remarkable advances in computer science in the new millennium, together with a much deeper understanding of brain architecture, more ambitious scientific goals were dreamed of to understand the brain and how it works. The retrospective concludes by reviewing the main efforts in this direction, namely the construction of a digital brain, an in silico copy of the brain that would run on supercomputers and behave just like a real brain.
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Encéfalo , Canales Iónicos , Animales , Humanos , Encéfalo/metabolismo , Encéfalo/fisiología , Canales Iónicos/metabolismo , Historia del Siglo XX , Historia del Siglo XIX , Electricidad , Historia del Siglo XVIII , Modelos NeurológicosRESUMEN
Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.
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Glioblastoma , Humanos , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Glioblastoma/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores Muscarínicos/metabolismoRESUMEN
The malignancy of glioblastoma (GBM), the most aggressive type of human brain tumor, strongly correlates with the presence of hypoxic areas within the tumor mass. Oxygen levels have been shown to control several critical aspects of tumor aggressiveness, such as migration/invasion and cell death resistance, but the underlying mechanisms are still unclear. GBM cells express abundant K+ and Cl- channels, whose activity supports cell volume and membrane potential changes, critical for cell proliferation, migration and death. Volume-regulated anion channels (VRAC), which mediate the swelling-activated Cl- current, and the large-conductance Ca2+-activated K+ channels (BK) are both functionally upregulated in GBM cells, where they control different aspects underlying GBM malignancy/aggressiveness. The functional expression/activity of both VRAC and BK channels are under the control of the oxygen levels, and these regulations are involved in the hypoxia-induced GBM cell aggressiveness. The present review will provide a comprehensive overview of the literature supporting the role of these two channels in the hypoxia-mediated GBM malignancy, suggesting them as potential therapeutic targets in the treatment of GBM.
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Glioblastoma , Humanos , Glioblastoma/patología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Línea Celular Tumoral , Hipoxia/metabolismo , Oxígeno/metabolismoRESUMEN
Calcium ions (Ca2+) enter mitochondria via the mitochondrial Ca2+ uniporter, driven by electrical and concentration gradients. In this regard, transgenic mouse models, such as calsequestrin knockout (CSQ-KO) mice, with higher mitochondrial Ca2+ concentrations ([Ca2+]mito), should display higher cytosolic Ca2+ concentrations ([Ca2+]cyto). However, repeated measurements of [Ca2+]cyto in quiescent CSQ-KO fibers never showed a difference between WT and CSQ-KO. Starting from the consideration that fluorescent Ca2+ probes (Fura-2 and Indo-1) measure averaged global cytosolic concentrations, in this report we explored the role of local Ca2+ concentrations (i.e., Ca2+ microdomains) in regulating mitochondrial Ca2+ in resting cells, using a multicompartmental diffusional Ca2+ model. Progressively including the inward and outward fluxes of sarcoplasmic reticulum (SR), extracellular space, and mitochondria, we explored their contribution to the local Ca2+ distribution within the cell. The model predicts Ca2+ concentration gradients with hot spots or microdomains even at rest, minor but similar to those of evoked Ca2+ release. Due to their specific localization close to Ca2+ release units (CRU), mitochondria could take up Ca2+ directly from high-concentration microdomains, thus sensibly raising [Ca2+]mito, despite minor, possibly undetectable, modifications of the average [Ca2+]cyto.
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Glioblastoma (GBM), the most lethal form of brain tumors, bases its malignancy on the strong ability of its cells to migrate and invade the narrow spaces of healthy brain parenchyma. Cell migration and invasion are both critically dependent on changes in cell volume and shape driven by the transmembrane transport of osmotically important ions such as K+ and Cl- . However, while the Cl- channels participating in cell volume regulation have been clearly identified, the precise nature of the K+ channels involved is still uncertain. Using a combination of electrophysiological and imaging approaches in GBM U87-MG cells, we found that hypotonic-induced cell swelling triggered the opening of Ca2+ -activated K+ (KCa ) channels of large and intermediate conductance (BKCa and IKCa , respectively), both highly expressed in GBM cells. The influx of Ca2+ mediated by the hypotonic-induced activation of mechanosensitive channels was found to be a key step for opening both the BKCa and the IKCa channels. Finally, the activation of both KCa channels mediated by mechanosensitive channels was found to be essential for the development of the regulatory volume decrease following hypotonic shock. Taken together, these data indicate that KCa channels are the main K+ channels responsible for the volume regulation in U87-MG cells.
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Canales de Calcio , Glioblastoma , Humanos , Calcio , Movimiento Celular , Tamaño de la Célula , Glioblastoma/patología , Canales de Calcio/metabolismoRESUMEN
All cells are capable of secreting extracellular vesicles (EVs), which are not a means to eliminate unneeded cellular compounds but represent a process to exchange material (nucleic acids, lipids and proteins) between different cells. This also happens in the brain, where EVs permit the crosstalk between neuronal and non-neuronal cells, functional to homeostatic processes or cellular responses to pathological stimuli. In brain tumors, EVs are responsible for the bidirectional crosstalk between glioblastoma cells and healthy cells, and among them, astrocytes, that assume a pro-tumoral or antitumoral role depending on the stage of the tumor progression. In this work, we show that astrocyte-derived small EVs (sEVs) exert a defensive mechanism against tumor cell growth and invasion. The effect is mediated by astrocyte-derived EVs (ADEVs) through the transfer to tumor cells of factors that hinder glioma growth. We identified one of these factors, enriched in ADEVs, that is miR124. It reduced both the expression and function of the volume-regulated anion channel (VRAC), that, in turn, decreased the cell migration and invasion of murine glioma GL261 cells.
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Calcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter. By contrast, calsequestrin-1 (CASQ1) ablation induces a compensatory constitutive assembly of CEUs in EDL fibers, resulting in enhanced constitutive and maximum SOCE that counteracts SR Ca2+ depletion during repetitive activity. However, whether CEUs form in slow-twitch fibers, which express both the skeletal CASQ1 and the cardiac CASQ2 isoforms, is unknown. Herein, we compared the structure and function of soleus muscles from WT and knockout mice that lack either CASQ1 (CASQ1-null) or both CASQs (dCASQ-null). Ultrastructural analyses showed that SR/T-tubule junctions at the I band, virtually identical to CEUs in EDL muscle, were present and more frequent in CASQ1-null than WT mice, with dCASQ-null exhibiting the highest incidence. The greater incidence of CEUs in soleus from dCASQ-null mice correlated with increased specific force production during repetitive, high-frequency stimulation, which depended on Ca2+ entry. Consistent with this, Orai1 expression was significantly increased in soleus of CASQ1-null mice, but even more in dCASQ-null mice, compared with WT. Together, these results strengthen the concept that CEU assembly strongly depends on CASQ expression and provides an alternative source of Ca2+ needed to refill SR Ca2+ stores to maintain specific force production during sustained muscle activity.
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Calcio , Calsecuestrina , Animales , Calcio/metabolismo , Calsecuestrina/genética , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Isoformas de Proteínas/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl,swell) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.
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Edema Encefálico , Quistes , Astrocitos/metabolismo , Edema Encefálico/patología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cloruros/metabolismo , Quistes/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Humanos , Proteínas de la Membrana/metabolismo , Proteómica , Canales Aniónicos Dependientes del Voltaje/metabolismo , Agua/metabolismoRESUMEN
Duchenne muscular dystrophy (DMD), an X-linked disorder caused by loss-of-function mutations in the dystrophin gene, is characterized by progressive muscle degeneration and weakness. Enhanced store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism coordinated by STIM1 sensors of luminal Ca2+ within the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the sarcolemma, is proposed to contribute to Ca2+-mediated muscle damage in DMD. To directly determine the impact of Orai1-dependent SOCE on the dystrophic phenotype, we crossed mdx mice with tamoxifen-inducible, muscle-specific Orai1 knockout mice (mdx-Orai1 KO mice). Both constitutive and SOCE were significantly increased in flexor digitorum brevis fibers from mdx mice, while SOCE was absent in fibers from both Orai1 KO and mdx-Orai1 KO mice. Compared with WT mice, fibers from mdx mice exhibited (1) increased resting myoplasmic Ca2+ levels, (2) reduced total releasable Ca2+ store content, and (3) a prolonged rate of electrically evoked Ca2+ transient decay. These effects were partially normalized in fibers from mdx-Orai1 KO mice. Intact extensor digitorum longus muscles from mdx mice exhibited a significant reduction of maximal specific force, which was rescued in muscles from mdx-Orai1 KO mice. Finally, during exposure to consecutive eccentric contractions, muscles from mdx mice displayed a more pronounced decline in specific force compared with that of WT mice, which was also significantly attenuated by Orai1 ablation. Together, these results indicate that enhanced Orai1-dependent SOCE exacerbates the dystrophic phenotype and that Orai1 deficiency improves muscle pathology by both normalizing Ca2+ homeostasis and promoting sarcolemmal integrity/stability.
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Distrofia Muscular de Duchenne , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Proteína ORAI1/genética , Retículo Sarcoplasmático/metabolismoRESUMEN
Environmental heat-stroke (HS) is a life-threatening response often triggered by hot and humid weather. Several lines of evidence indicate that HS is caused by excessive heat production in skeletal muscle, which in turn is the result of abnormal Ca2+ leak from the sarcoplasmic reticulum (SR) and excessive production of oxidative species of oxygen and nitrogen. As a high fat diet is known to increase oxidative stress, the objective of the present study was to investigate the effects of 3 months of high-fat diet (HFD) on the HS susceptibility of wild type (WT) mice. HS susceptibility was tested in an environmental chamber where 4 months old WT mice were exposed to heat stress (41 °C for 1 h). In comparison with mice fed with a regular diet, mice fed with HFD showed: (a) increased body weight and accumulation of adipose tissue; (b) elevated oxidative stress in skeletal muscles; (c) increased heat generation and oxygen consumption during exposure to heat stress; and finally, (d) enhanced sensitivity to both temperature and caffeine of isolated muscles during in-vitro contracture test. These data (a) suggest that HFD predisposes WT mice to heat stress and (b) could have implications for guidelines regarding food intake during periods of intense environmental heat.
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Dieta Alta en Grasa , Golpe de Calor , Tejido Adiposo , Animales , Dieta Alta en Grasa/efectos adversos , Golpe de Calor/etiología , Respuesta al Choque Térmico/fisiología , Ratones , Músculo Esquelético/fisiologíaRESUMEN
Exertional heat stroke (HS) is a hyperthermic crisis triggered by an excessive accumulation of Ca2+ in skeletal muscle fibers. We demonstrated that exercise leads to the formation of calcium entry units (CEUs), which are intracellular junctions that reduce muscle fatigue by promoting the recovery of extracellular Ca2+ via store-operated Ca2+ entry (SOCE). Here, we tested the hypothesis that exercise-induced assembly of CEUs may increase the risk of HS when physical activity is performed in adverse environmental conditions (high temperature and humidity). Adult mice were: (a) first, divided into three experimental groups: control, trained-1 month (voluntary running in wheel cages), and acutely exercised-1 h (incremental treadmill run); and (b) then subjected to an exertional stress (ES) protocol, a treadmill run in an environmental chamber at 34 °C and 40% humidity. The internal temperature of the mice at the end of the ES was higher in both pre-exercised groups. During an ES ex-vivo protocol, extensor digitorum longus(EDL) muscles from the trained-1 month and exercised-1 h mice generated greater basal tension than in the control and were those that contained a greater number of CEUs, assessed by electron microscopy. The data collected suggest that the entry of Ca2+ from extracellular space via CEUs could contribute to exertional HS when exercise is performed in adverse environmental conditions.
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Temperatura Corporal , Músculo Esquelético , Animales , Calcio , Ratones , Fatiga Muscular , Fibras Musculares Esqueléticas , Músculo Esquelético/fisiologíaRESUMEN
Regulatory volume decrease (RVD), a homeostatic process responsible for the re-establishment of the original cell volume upon swelling, is critical in controlling several functions, including migration. RVD is mainly sustained by the swelling-activated Cl- current (ICl,swell ), which can be modulated by cytoplasmic Ca2+ . Cell swelling also activates mechanosensitive channels, including the ubiquitously expressed Ca2+ -permeable channel Piezo1. We hypothesized that, by controlling cytoplasmic Ca2+ and in turn ICl,swell , Piezo1 is involved in the fine regulation of RVD and cell migration. We compared RVD and ICl,swell in wild-type (WT) HEK293T cells, which express endogenous levels of Piezo1, and in cells overexpressing (OVER) or knockout (KO) for Piezo1. Compared to WT, RVD was markedly increased in OVER, while virtually absent in KO cells. Consistently, ICl,swell amplitude was highest in OVER and lowest in KO cells, with WT cells displaying an intermediate level, suggesting a Ca2+ -dependent modulation of the current by Piezo1 channels. Indeed, in the absence of external Ca2+ , ICl,swell in both WT and OVER cells, as well as the RVD probed in OVER cells, were significantly lower than in the presence of Ca2+ and no longer different compared to KO cells. However, the Piezo-mediated Ca2+ influx was ineffective in enhancing ICl,swell in the absence of releasable Ca2+ from intracellular stores. The different expression levels of Piezo1 affected also cell migration which was strongly enhanced in OVER, while reduced in KO cells, as compared to WT. Taken together, our data indicate that Piezo1 controls RVD and migration in HEK293T cells by modulating ICl,swell through Ca2+ influx.
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Calcio , Tamaño de la Célula , Canales de Cloruro , Canales Iónicos , Calcio/metabolismo , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Canales Iónicos/genéticaRESUMEN
Ageing is associated with an increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. Effective contraction and relaxation of cardiomyocytes depend on efficient production of ATP (handled by mitochondria) and on proper Ca2+ supply to myofibrils during excitation-contraction (EC) coupling (handled by Ca2+ release units, CRUs). Here, we analyzed mitochondria and CRUs in hearts of adult (4 months old) and aged (≥24 months old) mice. Analysis by confocal and electron microscopy (CM and EM, respectively) revealed an age-related loss of proper organization and disposition of both mitochondria and EC coupling units: (a) mitochondria are improperly disposed and often damaged (percentage of severely damaged mitochondria: adults 3.5 ± 1.1%; aged 16.5 ± 3.5%); (b) CRUs that are often misoriented (longitudinal) and/or misplaced from the correct position at the Z line. Immunolabeling with antibodies that mark either the SR or T-tubules indicates that in aged cardiomyocytes the sarcotubular system displays an extensive disarray. This disarray could be in part caused by the decreased expression of Cav-3 and JP-2 detected by western blot (WB), two proteins involved in formation of T-tubules and in docking SR to T-tubules in dyads. By WB analysis, we also detected increased levels of 3-NT in whole hearts homogenates of aged mice, a product of nitration of protein tyrosine residues, recognized as marker of oxidative stress. Finally, a detailed EM analysis of CRUs (formed by association of SR with T-tubules) points to ultrastructural modifications, i.e., a decrease in their frequency (adult: 5.1 ± 0.5; aged: 3.9 ± 0.4 n./50 µm2) and size (adult: 362 ± 40 nm; aged: 254 ± 60 nm). The changes in morphology and disposition of mitochondria and CRUs highlighted by our results may underlie an inefficient supply of Ca2+ ions and ATP to the contractile elements, and possibly contribute to cardiac dysfunction in ageing.
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Calcio/metabolismo , Mitocondrias Cardíacas/ultraestructura , Miocitos Cardíacos/ultraestructura , Envejecimiento , Animales , Senescencia Celular , Acoplamiento Excitación-Contracción , Masculino , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patologíaRESUMEN
Skeletal muscle contraction relies on both high-fidelity calcium (Ca2+) signals and robust capacity for adenosine triphosphate (ATP) generation. Ca2+ release units (CRUs) are highly organized junctions between the terminal cisternae of the sarcoplasmic reticulum (SR) and the transverse tubule (T-tubule). CRUs provide the structural framework for rapid elevations in myoplasmic Ca2+ during excitation-contraction (EC) coupling, the process whereby depolarization of the T-tubule membrane triggers SR Ca2+ release through ryanodine receptor-1 (RyR1) channels. Under conditions of local or global depletion of SR Ca2+ stores, store-operated Ca2+ entry (SOCE) provides an additional source of Ca2+ that originates from the extracellular space. In addition to Ca2+, skeletal muscle also requires ATP to both produce force and to replenish SR Ca2+ stores. Mitochondria are the principal intracellular organelles responsible for ATP production via aerobic respiration. This review provides a broad overview of the literature supporting a role for impaired Ca2+ handling, dysfunctional Ca2+-dependent production of reactive oxygen/nitrogen species (ROS/RNS), and structural/functional alterations in CRUs and mitochondria in the loss of muscle mass, reduction in muscle contractility, and increase in muscle damage in sarcopenia and a wide range of muscle disorders including muscular dystrophy, rhabdomyolysis, central core disease, and disuse atrophy. Understanding the impact of these processes on normal muscle function will provide important insights into potential therapeutic targets designed to prevent or reverse muscle dysfunction during aging and disease.
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BACKGROUND: Body mass index (BMI), age, left atrium (LA) dimension and left ventricular ejection fraction (LVEF) have been linked to post-operative atrial fibrillation (POAF) after cardiac surgery. The aim of this study was to better define the role of these risk factors. METHODS: This retrospective cohort study evaluated 249 patients (without prior atrial dysrhythmia) undergoing cardiac or aortic surgery. Prior to surgery, the following data were collected: age, BMI, LA diameter, LA area, LVEF, thyroid stimulating hormone (TSH), creatinine and the presence of arterial hypertension (AH) and diabetes. Intraoperative data such as operation time, total clamp time, cardiopulmonary bypass time, and presence of pericardial/pleural effusion were also collected. Only patients without pre- and post-surgery prophylactic anti-arrhythmic therapy were included. RESULTS: Patients with (N = 127, 51%) and without POAF (N = 122, 49%) were compared. No difference was observed for sex, LA diameter, LA area, LVEF, TSH, diabetes and use of ACE inhibitors or statins prior to intervention. Moreover, no difference was observed in terms of operation time, total clamp time, cardiopulmonary bypass time, and presence of pericardial/pleural effusion. However, patients with POAF were older (70.6 ± 10.7 vs. 60.4 ± 16.4 years, p = 0.001), had higher BMI (26.8 ± 4.5 vs. 24.9 ± 3.6 kg/m2, p = 0.001), higher baseline creatinine (1.06 ± 0.91 vs. 0.88 ± 0.32 mg/dL, p = 0.038) and a higher frequency of arterial hypertension (73.2% vs. 50%, p = 0.001) and Bentall procedure (24.4% vs. 9.8%, p = 0.023). Multivariate analysis showed that the only independent predictors of POAF were age (OR = 1.05, 95%CI 1.02-1.07, p = 0.001) and BMI (OR = 1.11 95%CI 1.03-1.2,p = 0.006). CONCLUSIONS: These findings suggest that advanced age and a higher BMI are strong risk factors for POAF in patients without previous AF even in the presence of comparable LA dimensions and LVEF.
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Fibrilación Atrial/etiología , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Función del Atrio Izquierdo , Índice de Masa Corporal , Estudios de Cohortes , Femenino , Humanos , Italia , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/etiología , Estudios Retrospectivos , Factores de Riesgo , Volumen Sistólico , Función Ventricular Izquierda , Adulto JovenRESUMEN
Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx mechanism triggered by depletion of Ca2+ stores from the endoplasmic/sarcoplasmic reticulum (ER/SR). We recently reported that acute exercise in WT mice drives the formation of Ca2+ entry units (CEUs), intracellular junctions that contain STIM1 and Orai1, the two key proteins mediating SOCE. The presence of CEUs correlates with increased constitutive- and store-operated Ca2+ entry, as well as sustained Ca2+ release and force generation during repetitive stimulation. Skeletal muscle from mice lacking calsequestrin-1 (CASQ1-null), the primary Ca2+-binding protein in the lumen of SR terminal cisternae, exhibits significantly reduced total Ca2+ store content and marked SR Ca2+ depletion during high-frequency stimulation. Here, we report that CEUs are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of sedentary CASQ1-null mice. The higher density of CEUs in EDL (39.6 ± 2.1/100 µm2 versus 2.0 ± 0.3/100 µm2) and FDB (16.7 ± 1.0/100 µm2 versus 2.7 ± 0.5/100 µm2) muscles of CASQ1-null compared with WT mice correlated with enhanced constitutive- and store-operated Ca2+ entry and increased expression of STIM1, Orai1, and SERCA. The higher ability to recover Ca2+ ions via SOCE in CASQ1-null muscle served to promote enhanced maintenance of peak Ca2+ transient amplitude, increased dependence of luminal SR Ca2+ replenishment on BTP-2-sensitive SOCE, and increased maintenance of contractile force during repetitive, high-frequency stimulation. Together, these data suggest that muscles from CASQ1-null mice compensate for the lack of CASQ1 and reduction in total releasable SR Ca2+ content by assembling CEUs to promote constitutive and store-operated Ca2+ entry.
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Calcio , Calsecuestrina , Músculo Esquelético , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio , Calsecuestrina/fisiología , Iones , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético/fisiología , Proteína ORAI1 , Molécula de Interacción Estromal 1RESUMEN
Tubular aggregates (TAs) in skeletal muscle fibers are unusual accumulation of sarcoplasmic reticulum (SR) tubes that are found in different disorders including TA myopathy (TAM). TAM is a muscular disease characterized by muscle pain, cramping, and weakness that has been recently linked to mutations in STIM1 and ORAI1. STIM1 and ORAI1 are the two main proteins mediating store-operated Ca2+ entry (SOCE), a mechanism activated by depletion of intracellular Ca2+ stores (e.g., SR) that allows recovery of Ca2+ from the extracellular space during repetitive muscle activity. We have recently shown that exercise triggers the formation of unique intracellular junctions between SR and transverse tubules named Ca 2+ entry units (CEUs). CEUs promote colocalization of STIM1 with ORAI1 and improve muscle function in presence of external Ca2+. TAs virtually identical to those of TAM patients are also found in fast-twitch fibers of aging male mice. Here, we used a combination of electron and confocal microscopy, Western blotting, and ex vivo stimulation protocols (in presence or absence of external Ca2+) to evaluate the presence of TAs, STIM1-ORAI1 localization and expression and fatigue resistance of intact extensor digitorum longus (EDL) muscles in wild-type male adult (4-month-old) and aged (24-month-old) mice and in mice trained in wheel cages for 15 months (from 9 to 24 months of age). The results collected indicate that (i) aging causes STIM1 and ORAI1 to accumulate in TAs and (ii) long-term exercise significantly reduced formation of TAs. In addition, (iii) EDL muscles from aged mice exhibited a faster decay of contractile force than adult muscles, likely caused by their inability to refill intracellular Ca2+ stores, and (iv) exercise in wheel cages restored the capability of aged EDL muscles to use external Ca2+ by promoting maintenance of CEUs. In conclusion, exercise prevented improper accumulation of STIM1 and ORAI1 in TAs during aging, maintaining the capability of aged muscle to refill intracellular Ca2+ stores via SOCE.
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Mice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca2+ leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca2+ uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca2+ concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca2+ release from SR during repetitive electrical stimulation or caffeine administration. Also mitochondrial ROS production associated with contractile activity (detected with Mitosox probe) was much higher in YS fibers than in WT. Importantly, the inhibition of mitochondrial Ca2+ uptake achieved by silencing MCU reduced ROS accumulation in the matrix and Ca2+ release from SR. Finally, inhibition of mitochondrial ROS accumulation using Mitotempo reduced SR Ca2+ release in YS fibers exposed to caffeine. The present results support the view that mitochondria take up larger amounts of Ca2+ in YS than in WT fibers and that mitochondrial ROS production substantially contributes to the increased caffeine-sensitivity and to the enhanced Ca2+ release from SR in YS fibers.
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Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca2+ entry (SOCE). Here, we report that SOCE, peak Ca2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca2+ entry coincident with a modest decrease in total releasable Ca2+ store content. Importantly, this decrease in releasable Ca2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca2+ dynamics and muscle contractile function during acute exercise.