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Ítem Disseminated intravascular coagulation phenotype is regulated by the TRPM7 channel during sepsis(2023-12) Jiménez‑Dinamarca, Ivanka; Prado, Yolanda; Tapia, Pablo; Gatica, Sebastian; Alt, Clemens; P. Lin, Charles; Martínez, Cristian Reyes; G. Feijóo, Carmen; Aravena, Cristobal; González‑Canace, Alejandra; Correa, Simón; Varela, Diego; Cabello‑Verrugio, Claudio; Simon, FelipeBackground: Sepsis is an uncontrolled inflammatory response against a systemic infection that results in elevated mortality, mainly induced by bacterial products known as endotoxins, producing endotoxemia. Disseminated intravascular coagulation (DIC) is frequently observed in septic patients and is associated with organ failure and death. Sepsis activates endothelial cells (ECs), promoting a prothrombotic phenotype contributing to DIC. Ion channel-mediated calcium permeability participates in coagulation. The transient reception potential melastatin 7 (TRPM7) non-selective divalent cation channel that also contains an α-kinase domain, which is permeable to divalent cations including Ca2+, regulates endotoxin-stimulated calcium permeability in ECs and is associated with increased mortality in septic patients. However, whether endothelial TRPM7 mediates endotoxemia-induced coagulation is not known. Therefore, our aim was to examine if TRPM7 mediates coagulation during endotoxemia. Results: The results showed that TRPM7 regulated endotoxin-induced platelet and neutrophil adhesion to ECs, dependent on the TRPM7 ion channel activity and by the α-kinase function. Endotoxic animals showed that TRPM7 mediated neutrophil rolling on blood vessels and intravascular coagulation. TRPM7 mediated the increased expression of the adhesion proteins, von Willebrand factor (vWF), intercellular adhesion molecule 1 (ICAM-1), and P-selectin, which were also mediated by the TRPM7 α-kinase function. Notably, endotoxin-induced expression of vWF, ICAM-1 and P-selectin were required for endotoxin-induced platelet and neutrophil adhesion to ECs. Endotoxemic rats showed increased endothelial TRPM7 expression associated with a procoagulant phenotype, liver and kidney dysfunction, increased death events and an increased relative risk of death. Interestingly, circulating ECs (CECs) from septic shock patients (SSPs) showed increased TRPM7 expression associated with increased DIC scores and decreased survival times. Additionally, SSPs with a high expression of TRPM7 in CECs showed increased mortality and relative risk of death. Notably, CECs from SSPs showed significant results from the AUROC analyses for predicting mortality in SSPs that were better than the Acute Physiology and Chronic Health Evaluation II (APACHE II) and the Sequential Organ Failure Assessment (SOFA) scores. Conclusions: Our study demonstrates that sepsis-induced DIC is mediated by TRPM7 in ECs. TRPM7 ion channel activity and α-kinase function are required by DIC-mediated sepsis-induced organ dysfunction and its expression are associated with increased mortality during sepsis. TRPM7 appears as a new prognostic biomarker to predict mortality associated to DIC in SSPs, and as a novel target for drug development against DIC during infectious inflammatory diseases. © 2023, The Author(s).Ítem Endotoxin induces fibrosis in vascular endothelial cells through a mechanism dependent on transient receptor protein melastatin 7 activity(Public Library of Science, 2014-04) Echeverría, Cesar; Montorfano, Ignacio; Hermosilla, Tamara; Armisén, Ricardo; Velásquez, Luis A.; Cabello-Verrugio, Claudio; Varela, Diego; Simon, FelipeThe pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial-to-mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.Ítem Excessive release of inorganic polyphosphate by ALS/FTD astrocytes causes non-cell-autonomous toxicity to motoneurons(Cell Press, 2022-05-18) Arredondo, Cristian; Cefaliello, Carolina; Dyrda, Agnieszka; Jury, Nur; Martinez, Pablo; Díaz, Iván; Amaro, Armando; Tran, Helene; Morales, Danna; Pertusa, Maria; Stoica, Lorelei; Fritz, Elsa; Corvalán, Daniela; Abarzúa, Sebastián; Méndez-Ruette, Maxs; Fernández, Paola; Rojas, Fabiola; Kumar, Meenakshi Sundaram; Aguilar, Rodrigo; Almeida, Sandra; Weiss, Alexandra; Bustos, Fernando J.; González-Nilo, Fernando; Otero, Carolina; Tevy, Maria Florencia; Bosco, Daryl A.; Sáez, Juan C.; Kähne, Thilo; Gao, Fen-Biao; Berry, James D.; Nicholson, Katharine; Sena-Esteves, Miguel; Madrid, Rodolfo; Varela, Diego; Montecino, Martin; Brown, Robert H.; van Zundert, BrigitteNon-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD. © 2022 Elsevier Inc.Ítem In Vivo and in vitro antitumor activity of tomatine in hepatocellular carcinoma(Frontiers Media S.A., 2022-09) Echeverría, Cesar; Martin, Aldo; Simon, Felipe; Salas, Cristian O.; Nazal, Mariajesus; Varela, Diego; Pérez-Castro, Ramón A.; Santibanez, Juan F.; Valdés-Valdés, Ricardo O.; Forero-Doria, Oscar; Echeverría, JavierBackground: There is abundant ethnopharmacological evidence the uses of regarding Solanum species as antitumor and anticancer agents. Glycoalkaloids are among the molecules with antiproliferative activity reported in these species. Purpose: To evaluate the anticancer effect of the Solanum glycoalkaloid tomatine in hepatocellular carcinoma (HCC) in vitro (HepG2 cells) and in vivo models. Methods: The resazurin reduction assay was performed to detect the effect of tomatine on cell viability in human HepG2 cell lines. Programmed cell death was investigated by means of cellular apoptosis assays using Annexin V. The expression of cancer related proteins was detected by Western blotting (WB). Reactive oxygen species (ROS) and calcium were determined by 2,7-dichlorodihydrofluorescein diacetate and Fluo-4, respectively. Intrahepatic HepG2 xenograft mouse model was used to elucidate the effect of tomatine on tumor growth in vivo. Results and Discussion: Tomatine reduced HepG2 cell viability and induced the early apoptosis phase of cell death, consistently with caspase-3, -7, Bcl-2 family, and P53 proteins activation. Furthermore, tomatine increased intracellular ROS and cytosolic Ca+2 levels. Moreover, the NSG mouse xenograft model showed that treating mice with tomatine inhibited HepG2 tumor growth. Conclusion: Tomatine inhibits in vitro and in vivo HCC tumorigenesis in part via modulation of p53, Ca+2, and ROS signalling. Thus, the results suggest the potential cancer therapeutic use of tomatine in HCC patients. Copyright © 2022 Echeverría, Martin, Simon, Salas, Nazal, Varela, Pérez-Castro, Santibanez, Valdés-Valdés, Forero-Doria and Echeverría.Ítem OxHDL controls LOX-1 expression and plasma membrane localization through a mechanism dependent on NOX/ROS/NF-κB pathway on endothelial cells(Nature Publishing Group, 2019-03-01) Pérez, Lorena; Vallejos, Alejandro; Echeverria, Cesar; Varela, Diego; Cabello-Verrugio, Claudio; Simon, FelipeSystemic inflammatory diseases enhance circulating oxidative stress levels, which results in the oxidation of circulating high-density lipoprotein (oxHDL). Endothelial cell function can be negatively impacted by oxHDL, but the underlying mechanisms for this remain unclear. Some reports indicate that the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is also a receptor for oxHDL. However, it is unknown if oxHDL induces increased LOX-1 expression at the plasma membrane, as an event that supports endothelial dysfunction. Therefore, the aims of this study were to determine if oxHDL induces plasma-membrane level changes in LOX-1 and, if so, to describe the underlying mechanisms in endothelial cells. Our results demonstrate that the incubation of arterial or vein endothelial cells with oxHDL (and not HDL) induces the increase of LOX-1 expression at the plasma membrane; effect prevented by LOX-1 inhibition. Importantly, same results were observed in endothelial cells from oxHDL-treated rats. Furthermore, the observed oxHDL-induced LOX-1 expression is abolished by the down-regulation of NOX-2 expression with siRNA (and no others NOX isoforms), by the pharmacological inhibition of NAD(P)H oxidase (with DPI or apocynin) or by the inhibition of NF-κB transcription factor. Coherently, LOX-1 expression is augmented by the incubation of endothelial cells with H2O2 or GSSG even in absence of oxHDL, indicating that the NOX-2/ROS/ NF-κB axis is involved. Interestingly, oxHDL incubation also increases TNF-α expression, cytokine that induces LOX-1 expression. Thus, our results suggest a positive feedback mechanism for LOX-1 receptor during inflammatory condition where an oxidative burst will generate oxHDL from native HDL, activating LOX-1 receptor which in turn will increase the expression of NOX-2, TNF-α and LOX-1 receptor at the plasma membrane. In conclusion, oxHDL-induced translocation of LOX-1 to the plasma membrane could constitute an induction mechanism of endothelial dysfunction in systemic inflammatory diseases.Ítem OxHDL controls LOX-1 expression and plasma membrane localization through a mechanism dependent on NOX/ROS/NF-κB pathway on endothelial cells(Nature Publishing Group, 2019-03) Pérez, Lorena; Vallejos, Alejandro; Echeverria, Cesar; Varela, Diego; Cabello-Verrugio, Claudio; Simon, FelipeSystemic inflammatory diseases enhance circulating oxidative stress levels, which results in the oxidation of circulating high-density lipoprotein (oxHDL). Endothelial cell function can be negatively impacted by oxHDL, but the underlying mechanisms for this remain unclear. Some reports indicate that the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is also a receptor for oxHDL. However, it is unknown if oxHDL induces increased LOX-1 expression at the plasma membrane, as an event that supports endothelial dysfunction. Therefore, the aims of this study were to determine if oxHDL induces plasma-membrane level changes in LOX-1 and, if so, to describe the underlying mechanisms in endothelial cells. Our results demonstrate that the incubation of arterial or vein endothelial cells with oxHDL (and not HDL) induces the increase of LOX-1 expression at the plasma membrane; effect prevented by LOX-1 inhibition. Importantly, same results were observed in endothelial cells from oxHDL-treated rats. Furthermore, the observed oxHDL-induced LOX-1 expression is abolished by the down-regulation of NOX-2 expression with siRNA (and no others NOX isoforms), by the pharmacological inhibition of NAD(P)H oxidase (with DPI or apocynin) or by the inhibition of NF-κB transcription factor. Coherently, LOX-1 expression is augmented by the incubation of endothelial cells with H2O2 or GSSG even in absence of oxHDL, indicating that the NOX-2/ROS/ NF-κB axis is involved. Interestingly, oxHDL incubation also increases TNF-α expression, cytokine that induces LOX-1 expression. Thus, our results suggest a positive feedback mechanism for LOX-1 receptor during inflammatory condition where an oxidative burst will generate oxHDL from native HDL, activating LOX-1 receptor which in turn will increase the expression of NOX-2, TNF-α and LOX-1 receptor at the plasma membrane. In conclusion, oxHDL-induced translocation of LOX-1 to the plasma membrane could constitute an induction mechanism of endothelial dysfunction in systemic inflammatory diseases. © 2019, United States & Canadian Academy of Pathology.Ítem Papel de la subunidad CaVβ2 en la respuesta β-adrenérgica de los canales de calcio tipo L(Universidad Andrés Bello, 2017) Acevedo Harnecker, Alejandra Jesús; Varela, Diego; Hermosilla, Tamara; Facultad de Ciencias Biológicas; Escuela de Ingeniería en BiotecnologíaLa respuesta de lucha o huida, particularmente a nivel cardiaco, induce un aumento en la contractibilidad y frecuencia cardiaca producto de la activación de los receptores β-adrenérgicos y del aumento en la actividad de PKA. En el musculo cardiaco, la entrada de calcio (Ca2+) al citoplasma es fundamental para el proceso de contracción, el cual se inicia mediante el influjo de este ion a través de los canales de calcio de tipo L (LTCC) ), los cuales están compuestos por una subunidad formadora de poro conocida como CaV1.2 (que permite la entrada de Ca2+ al citoplasma) y dos subunidades auxiliares denominadas CaVα2δ1 y CaVβ2, cuya función está relacionada con el transporte y la permanencia del canal en la membrana. A su vez, la subunidad CaVβ2 posee 5 variantes que difieren en el sitio de inicio de la transcripción (conocidas como variantes CaVβ2–TSS), las cuales sólo difieren en la composición y en la longitud del dominio N-terminal. Estas variantes CaVβ2–TSS, al ser sobre-expresadas en cardiomiocitos de ratas neonatas, incrementan las corrientes de tipo L endógenas de la célula en diferente medida, dependiendo de la subunidad sobre-expresada, además de modificar la cinética de activación/inactivación de los LTCC, otorgando una probabilidad de apertura (Po) diferencial a estos canales. Por otro lado, la activación del sistema nervioso simpático genera la liberación de diversas catecolaminas, las cuales activan a los receptores β-adrenérgicos, los que forman parte de la familia de receptores acoplados a proteína G (GPCR’s). La activación de estos receptores eleva los niveles de cAMP, lo que produce la estimulación de la proteína quinasa A (PKA), la cual fosforila a diversas proteínas, entre ellas, residuos clave en el dominio C-terminal del canal Cav1.2. El canal Cav1.2 sufre una serie de modificaciones post-traduccionales, entre las cuales destaca el procesamiento proteolítico del dominio C- terminal, el cual es luego reasociado al extremo distal del canal, formando un complejo de señalización auto-inhibitorio que es necesario para la correcta regulación de los LTCC. Cabe destacar que para que el canal Cav1.2 sea capaz de responder al estímulo β-adrenérgico es necesario que el extremo C-terminal del canal esté procesado proteolíticamente. La hipótesis más aceptada es que durante la respuesta de lucha o huida, una vez activados los receptores β-adrenérgicos, las fosforilaciones mediadas por PKA en el extremo C-terminal generarían una desestabilización del complejo auto-inhibitorio, desplazándolo y de este modo, generando un incremento de las corrientes de Ca2+ tipo L, como parte de la respuesta de lucha o huida, aumentando fundamentalmente la Po. Como fue mencionado anteriormente, las diferentes variantes CaVβ2–TSS afectan diferencialmente la cinética de activación e inactivación del canal y también la Po. Por lo tanto, el objetivo principal de este estudio fue determinar cómo las diferentes variantes CaVβ2–TSS son capaces de modular los LTCC durante la respuesta β-adrenérgica. Nuestra hipótesis propone que la regulación de los LTCC, durante la respuesta β-adrenérgica, depende tanto de la modulación diferencial de la Po, por parte de las diferentes variantes CaVβ2–TSS, así como también de la cantidad de canales que estén procesados proteolíticamente, de modo que, durante la respuesta β-adrenérgica, el complejo auto-inhibitorio pueda ser desplazado, permitiendo un mayor influjo de Ca2+. Nuestros resultados demuestran por un lado, que la estimulación de cardiomiocitos de ratas neonatas con forskolina genera un aumento, en promedio de 3 veces, en las corrientes de tipo L y además, que la cinética de inactivación del canal Cav1.2 se ve modificada. Asociado a lo anterior, fue observado que la sobre-expresión de las variantes CaVβ2–TSS produce un incremento en las corrientes de tipo L y un desplazamiento del potencial de activación (Va) hacia valores hiperpolarizantes, es decir, hacia potenciales más negativos. Sin embargo, al agregar forskolina y sobre-expresar las variantes CaVβ2–TSS, contrario a lo esperado, no fue observado un aumento significativo de las corrientes ni tampoco cambios en el Va, lo cual sugiere que la sobre-expresión de estas variantes disminuiría la respuesta a forskolina dado que la sobre-expresión per se de las variantes CaVβ2–TSS estaría generando un desplazamiento en el Va, lo cual posiblemente sería producto de que, al estar estas variantes sobre-expresadas , la razón CaV1.2: CaVβ2 cambia, de modo que la subunidad CaVβ2 podría unirse a un sitio de baja afinidad en el dominio C-terminal del canal CaV1.2 induciendo el desplazamiento del fragmento reasociado y eliminando el efecto auto-inhibitorio, por tanto, al agregar forskolina, no son generados cambios significativos sobre las corrientes puesto que el dominio C-terminal estaría pre-desplazado y por lo tanto, no sería posible un nuevo desplazamiento, lo cual explicaría la ausencia de activación. Cabe destacar que esta situación podría generarse durante estados fisiopatológicos del corazón, donde al disminuir la cantidad de canales presentes en la célula (por ejemplo durante la falla cardiaca), aumentaría la razón de subunidades CaVβ2, disminuyendo la capacidad de respuesta al estimulo β-adrenérgico.Ítem Preventive Leptin Administration Protects Against Sepsis Through Improving Hypotension, Tachycardia, Oxidative Stress Burst, Multiple Organ Dysfunction, and Increasing Survival(Frontiers Media S.A., 2018-12) Vallejos, Alejandro; Olivares, Pedro; Varela, Diego; Echeverria, Cesar; Cabello Verrugio, Claudio; Pérez Leighton, Claudio; Simon, FelipeSepsis syndrome is the most important cause of mortality in critically ill patients admitted to intensive care units (ICUs). However, current therapies for its prevention and treatment are still unsatisfactory, and the mortality rate is still high. Non-septic ICU patients are vulnerable to acquire sepsis syndrome. Thus, a preventive treatment for this population is needed. During sepsis syndrome and endotoxemia, severe hypotension, tachycardia, oxidative and immune response increase, multiple organ dysfunction syndrome (MODS) and decreased survival are observed. Leptin administration protects against negative effects of sepsis syndrome and endotoxemia. Furthermore, it is has been reported that leptin elevates blood pressure mediated by sympathetic nervous system activation. However, whether leptin administration before sepsis induction mediates its protective effects during sepsis through blood pressure regulation is not known. Therefore, we investigated whether pre-treatment of leptin improves blood pressure and MODS, resulting in survival increase during endotoxemia. The results showed that leptin administration before endotoxemia induction reduced both the hypotension and tachycardia characteristically observed during endotoxemia. Notably, this protective effect was observed early and late in the course of endotoxemia. Endotoxemia-induced MODS decreased in leptin-treated rats, which was reflected in normal values for liver and kidney function, inhibition of muscle mass wasting and maintenance of glycemia. Furthermore, leptin pre-treatment decreased the oxidative stress burst in blood and blunted the increased pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 observed during endotoxemia. Remarkably, according to the leptin-induced increase in survival, leptin pre-administration decreased the risk for death associated with sepsis syndrome at early and late times after endotoxemia induction. These results show a potential preventive therapy against sepsis syndrome and endotoxemia in vulnerable patients, based in the beneficial actions of leptin. © Copyright © 2018 Vallejos, Olivares, Varela, Echeverria, Cabello-Verrugio, Pérez-Leighton and Simon.Ítem Regulación β-adrenérgica sobre las cinéticas de inactivación del canal de calcio tipo L durante el potencial de acción cardiaco(Universidad Andrés Bello, 2017) Morales Carrasco, Danna Karyna; Varela, Diego; Facultad de Ciencias Biológicas; Escuela de Ingeniería en BiotecnologíaLa activación de los receptores β-adrenérgicos provoca un aumento en el ritmo cardiaco, la contractibilidad y la frecuencia de la relajación del corazón. En las células contráctiles ventriculares, los cardiomiocitos, la activación de estos receptores induce cambios en el potencial de acción (AP) como consecuencia de la regulación de diversos canales involucrados en este proceso. Entre ellos, el canal de calcio (Ca2+) Cav1.2, lo que genera un aumento en la corriente de Ca2+ tipo L (ICaL). Los canales de Ca2+ dependientes de potencial tipo L (L-VDCC) son la principal vía de entrada de Ca2+ en el corazón; éstos son activados por la despolarización de la membrana durante el AP, y posteriormente inactivados a través de mecanismos dependientes de potencial, y de Ca2+. Ha sido descrito que, bajo estimulación β-adrenérgica, los AP de rata adulta disminuyen su duración, sin embargo, el cómo ocurre este acortamiento no ha sido completamente dilucidado. En particular, cómo los mecanismos de inactivación del canal Cav1.2 influyen en la morfología es desconocido, por lo que la hipótesis de este trabajo es que el estímulo con Isoproterenol sobre los cardiomiocitos de rata neonata modifica el potencial de acción mediante la modulación de las cinéticas de inactivación del canal Cav1.2. Es por ello, que en esta tesis fueron estudiadas las cinéticas de inactivación del canal Cav1.2 durante el AP de cardiomiocitos neonatos bajo estimulación con Isoproterenol en base al uso de técnicas de electrofisiología. Los resultados de este trabajo demuestran que el aumento de la densidad de corrientes de Ca2+, efecto que fue logrado aumentando la concentración de Ca2+ extracelular o induciendo la facilitación del canal mediante el aumento de la frecuencia del estímulo, produce principalmente cambios en la cinética de los AP y no es responsable del acortamiento de los AP observado en cardiomiocitos de ratas neonatas estimuladas con Isoproterenol; por otro lado, la inhibición de la inactivación dependiente de potencial (VDI) o la inactivación dependiente de Ca2+ (CDI) demostraron ser capaces de modificar la duración del AP. Para observar directamente las corrientes de Ca2+ durante el AP fue utilizada la técnica de AP-clamp dinámico. Estos experimentos demuestran que la corriente de Ca2+ tipo L en cardiomiocitos estimulados con Isoproterenol es inactivada más rápido, sin existir diferencia en la cantidad de Ca2+ que ingresa en cada AP. La manipulación de cada tipo de inactivación demostró que la inhibición de cualquiera de ellas produce, paradójicamente, un aumento en la velocidad de decaimiento de la corriente durante el AP, produciendo que la corriente de Ca2+ terminase incluso antes del término del AP. En su conjunto los resultados de esta tesis demuestran que el estímulo β-adrenérgico, sobre cardiomiocitos de ratas neonatas, modula al canal de Ca2+ tipo L durante el AP, aumentando las velocidades de la VDI y de la CDI, siendo el efecto de esta última el que tiene un mayor aporte durante el AP.Ítem TRPM4 is a novel component of the adhesome required for focal adhesion disassembly, migration and contractility(Public Library of Science, 2015-06) Cáceres, Mónica; Ortiz, Liliana; Recabarren, Tatiana; Romero, Anibal; Colombo, Alicia; Leiva-Salcedo, Elías; Varela, Diego; Rivas, José; Silva, Ian; Morales, Diego; Campusano, Camilo; Almarza, Oscar; Simon, Felipe; Toledo, Hector; Park, Kang-Sik; Trimmer, James S.; Cerda, OscarCellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility. © 2015, Public Library of Science. All rights reserved. This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.Ítem TRPM7 mediates kidney injury, endothelial hyperpermeability and mortality during endotoxemia(Springer Nature, 2020-02) Gatica, Sebastian; Villegas, Vicente; Vallejos, Alejandro; Olivares, Pedro; Aballai, Víctor; Lagos-Meza, Felipe; Echeverria, Cesar; Cabello-Verrugio, Claudio; Varela, Diego; Simon, Felipeepsis is the main cause of mortality in patients admitted to intensive care units. During sepsis, endothelial permeability is severely augmented, contributing to renal dysfunction and patient mortality. Ca2+ influx and the subsequent increase in intracellular [Ca2+]i in endothelial cells (ECs) are key steps in the establishment of endothelial hyperpermeability. Transient receptor potential melastatin 7 (TRPM7) ion channels are permeable to Ca2+ and are expressed in a broad range of cell types and tissues, including ECs and kidneys. However, the role of TRPM7 on endothelial hyperpermeability during sepsis has remained elusive. Therefore, we investigated the participation of TRPM7 in renal vascular hyperpermeability, renal dysfunction, and enhanced mortality induced by endotoxemia. Our results showed that endotoxin increases endothelial hyperpermeability and Ca2+ overload through the TLR4/NOX-2/ROS/NF-κB pathway. Moreover, endotoxin exposure was shown to downregulate the expression of VE-cadherin, compromising monolayer integrity and enhancing vascular hyperpermeability. Notably, endotoxin-induced endothelial hyperpermeability was substantially inhibited by pharmacological inhibition and specific suppression of TRPM7 expression. The endotoxin was shown to upregulate the expression of TRPM7 via the TLR4/NOX-2/ROS/NF-κB pathway and induce a TRPM7-dependent EC Ca2+ overload. Remarkably, in vivo experiments performed in endotoxemic animals showed that pharmacological inhibition and specific suppression of TRPM7 expression inhibits renal vascular hyperpermeability, prevents kidney dysfunction, and improves survival in endotoxemic animals. Therefore, our results showed that TRPM7 mediates endotoxemia-induced endothelial hyperpermeability, renal dysfunction, and enhanced mortality, revealing a novel molecular target for treating renal vascular hyperpermeability and kidney dysfunction during endotoxemia, sepsis, and other inflammatory diseases. © 2019, United States & Canadian Academy of Pathology.Ítem TRPM7 mediates kidney injury, endothelial hyperpermeability and mortality during endotoxemia(Springer Nature, 2020-02) Gatica, Sebastian; Villegas, Vicente; Vallejos, Alejandro; Olivares, Pedro; Aballai, Víctor; Lagos-Meza, Felipe; Echeverria, Cesar; Cabello-Verrugio, Claudio; Varela, Diego; Simon, FelipeSepsis is the main cause of mortality in patients admitted to intensive care units. During sepsis, endothelial permeability is severely augmented, contributing to renal dysfunction and patient mortality. Ca2+ influx and the subsequent increase in intracellular [Ca2+]i in endothelial cells (ECs) are key steps in the establishment of endothelial hyperpermeability. Transient receptor potential melastatin 7 (TRPM7) ion channels are permeable to Ca2+ and are expressed in a broad range of cell types and tissues, including ECs and kidneys. However, the role of TRPM7 on endothelial hyperpermeability during sepsis has remained elusive. Therefore, we investigated the participation of TRPM7 in renal vascular hyperpermeability, renal dysfunction, and enhanced mortality induced by endotoxemia. Our results showed that endotoxin increases endothelial hyperpermeability and Ca2+ overload through the TLR4/NOX-2/ROS/NF-κB pathway. Moreover, endotoxin exposure was shown to downregulate the expression of VE-cadherin, compromising monolayer integrity and enhancing vascular hyperpermeability. Notably, endotoxin-induced endothelial hyperpermeability was substantially inhibited by pharmacological inhibition and specific suppression of TRPM7 expression. The endotoxin was shown to upregulate the expression of TRPM7 via the TLR4/NOX-2/ROS/NF-κB pathway and induce a TRPM7-dependent EC Ca2+ overload. Remarkably, in vivo experiments performed in endotoxemic animals showed that pharmacological inhibition and specific suppression of TRPM7 expression inhibits renal vascular hyperpermeability, prevents kidney dysfunction, and improves survival in endotoxemic animals. Therefore, our results showed that TRPM7 mediates endotoxemia-induced endothelial hyperpermeability, renal dysfunction, and enhanced mortality, revealing a novel molecular target for treating renal vascular hyperpermeability and kidney dysfunction during endotoxemia, sepsis, and other inflammatory diseases. © 2019, United States & Canadian Academy of Pathology.