RESUMO
Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2+) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2-) macrophages. Herein, we identified an essential role for CCR2- macrophages in the chronically failing heart. Depletion of CCR2- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Remodelação Ventricular/fisiologia , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Miocárdio/metabolismo , Troponina T/genéticaRESUMO
PGC1α is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor potential vanilloid), a family of ion channels, induced PGC1α expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1α, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.
Assuntos
Metabolismo Energético , Canais de Cátion TRPV/metabolismo , Termogênese , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Feminino , Técnicas de Silenciamento de Genes , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Transativadores/metabolismo , Fatores de Transcrição , Proteína Desacopladora 1RESUMO
BACKGROUND: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca2+ signaling and a potential pharmacological target for treating thrombotic diseases. METHODS: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. RESULTS: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca2+ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca2+-ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca2+ store content and increasing cytosolic Ca2+ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. CONCLUSIONS: Our results suggest that ERO1α acts as a thiol oxidase for Ca2+ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca2+ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.
Assuntos
AVC Isquêmico , Trombose , Animais , Camundongos , Plaquetas/metabolismo , Sinalização do Cálcio , Dissulfetos , AVC Isquêmico/metabolismo , Ativação PlaquetáriaRESUMO
Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 µM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.
Assuntos
Músculo Liso Vascular , Animais , Camundongos , Acetilcolina/farmacologia , Ânions , Proteínas de Membrana/genética , Camundongos Knockout , Fosfatase de Miosina-de-Cadeia-Leve , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologiaRESUMO
Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Xilose/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Oligossacarídeos/metabolismo , Imunidade Vegetal/genética , Regulação da Expressão Gênica de PlantasRESUMO
Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.
Assuntos
Sinalização do Cálcio , Cálcio/imunologia , Neutrófilos/imunologia , Proteína ORAI1/imunologia , Proteína ORAI2/imunologia , Animais , Feminino , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína ORAI1/genética , Proteína ORAI2/genéticaRESUMO
LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L706L707 motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s "bladder." When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors.
Assuntos
Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Osmorregulação/fisiologia , Estresse Fisiológico/fisiologia , Animais , Ânions , Células COS , Sobrevivência Celular/fisiologia , Chlorocebus aethiops , Exocitose , Técnicas de Inativação de Genes , Células HEK293 , Homeostase , Humanos , Proteínas de Membrana/genética , Camundongos , Transcriptoma , VacúolosRESUMO
PURPOSE OF REVIEW: Total ceramide levels in cardiac tissue relate to cardiac dysfunction in animal models. However, emerging evidence suggests that the fatty acyl chain length of ceramides also impacts their relationship to cardiac function. This review explores evidence regarding the relationship between ceramides and left ventricular dysfunction and heart failure. It further explores possible mechanisms underlying these relationships. RECENT FINDINGS: In large, community-based cohorts, a higher ratio of specific plasma ceramides, C16â:â0/C24â:â0, related to worse left ventricular dysfunction. Increased left ventricular mass correlated with plasma C16â:â0/C24â:â0, but this relationship became nonsignificant after adjustment for multiple comparisons. Decreased left atrial function and increased left atrial size also related to C16â:â0/C24â:â0. Furthermore, increased incident heart failure, overall cardiovascular disease (CVD) mortality and all-cause mortality were associated with higher C16â:â0/C24â:â0 (or lower C24â:â0/C16â:â0). Finally, a number of possible biological mechanisms are outlined supporting the link between C16â:â0/C24â:â0 ceramides, ceramide signalling and CVD. SUMMARY: High cardiac levels of total ceramides are noted in heart failure. In the plasma, C16â:â0/C24â:â0 ceramides may be a valuable biomarker of preclinical left ventricular dysfunction, remodelling, heart failure and mortality. Continued exploration of the mechanisms underlying these profound relationships may help develop specific lipid modulators to combat cardiac dysfunction and heart failure.
Assuntos
Insuficiência Cardíaca , Disfunção Ventricular Esquerda , Animais , Biomarcadores , Ceramidas , Coração , HumanosRESUMO
The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain-wide conditional LRRC8A knockout mice (LRRC8A bKO) using NestinCre -driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.
Assuntos
Astrócitos/patologia , Gliose/mortalidade , Ácido Glutâmico/metabolismo , Proteínas de Membrana/fisiologia , Convulsões/mortalidade , Animais , Astrócitos/metabolismo , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Feminino , Gliose/etiologia , Gliose/patologia , Transporte de Íons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Convulsões/etiologia , Convulsões/patologiaRESUMO
Crystal structures activate innate immune cells, especially macrophages and initiate inflammatory responses. We aimed to understand the role of the mechanosensitive TRPV4 channel in crystal-induced inflammation. Real-time RT-PCR, RNAscope in situ hybridisation, and Trpv4eGFP mice were used to examine TRPV4 expression and whole-cell patch-clamp recording and live-cell Ca2+ imaging were used to study TRPV4 function in mouse synovial macrophages and human peripheral blood mononuclear cells (PBMCs). Both genetic deletion and pharmacological inhibition approaches were used to investigate the role of TRPV4 in NLRP3 inflammasome activation induced by diverse crystals in vitro and in mouse models of crystal-induced pain and inflammation in vivo. TRPV4 was functionally expressed by synovial macrophages and human PBMCs and TRPV4 expression was upregulated by stimulation with monosodium urate (MSU) crystals and in human PBMCs from patients with acute gout flares. MSU crystal-induced gouty arthritis were significantly reduced by either genetic ablation or pharmacological inhibition of TRPV4 function. Mechanistically, TRPV4 mediated the activation of NLRP3 inflammasome by diverse crystalline materials but not non-crystalline NLRP3 inflammasome activators, driving the production of inflammatory cytokine interleukin-1ß which elicited TRPV4-dependent inflammatory responses in vivo. Moreover, chemical ablation of the TRPV1-expressing nociceptors significantly attenuated the MSU crystal-induced gouty arthritis. In conclusion, TRPV4 is a common mediator of inflammatory responses induced by diverse crystals through NLRP3 inflammasome activation in macrophages. TRPV4-expressing resident macrophages are critically involved in MSU crystal-induced gouty arthritis. A neuroimmune interaction between the TRPV1-expressing nociceptors and the TRPV4-expressing synovial macrophages contributes to the generation of acute gout flares.
Assuntos
Artralgia/metabolismo , Artrite/metabolismo , Artropatias por Cristais/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Nociceptores/metabolismo , Canais de Cátion TRPV/genética , Adulto , Animais , Artralgia/imunologia , Artrite/imunologia , Artrite Gotosa/imunologia , Artrite Gotosa/metabolismo , Artropatias por Cristais/imunologia , Gota/imunologia , Gota/metabolismo , Humanos , Inflamassomos/imunologia , Inflamação , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Imagem Óptica , Técnicas de Patch-Clamp , Membrana Sinovial/citologia , Células THP-1 , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Ácido ÚricoRESUMO
TRPM7 (transient receptor potential cation channel subfamily M member 7) regulates gene expression and stress-induced cytotoxicity and is required in early embryogenesis through organ development. Here, we show that the majority of TRPM7 is localized in abundant intracellular vesicles. These vesicles (M7Vs) are distinct from endosomes, lysosomes, and other familiar vesicles or organelles. M7Vs accumulate Zn2+ in a glutathione-enriched, reduced lumen when cytosolic Zn2+ concentrations are elevated. Treatments that increase reactive oxygen species (ROS) trigger TRPM7-dependent Zn2+ release from the vesicles, whereas reduced glutathione prevents TRPM7-dependent cytosolic Zn2+ influx. These observations strongly support the notion that ROS-mediated TRPM7 activation releases Zn2+ from intracellular vesicles after Zn2+ overload. Like the endoplasmic reticulum, these vesicles are a distributed system for divalent cation uptake and release, but in this case the primary divalent ion is Zn2+ rather than Ca2.
Assuntos
Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Vesículas Transportadoras/metabolismo , Zinco/metabolismo , Desenvolvimento Embrionário , Glutationa/metabolismo , Células HEK293 , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Atrial fibrillation (AF) is associated with elevated risk for ischemic stroke and myocardial infarction (MI). The aim of the study is to assess the role of insulin use on the risk of stroke and MI in AF patients with diabetes. METHODS: We identified Medicare beneficiaries with new AF in 2011 to 2013. Primary outcomes were ischemic stroke and MI. Multivariate Cox regression models were used to assess the association between AF and time to stroke and MI. We adjusted for anticoagulant as a time-dependent covariate. RESULTS: Out of 798,592 AF patients, 53,212 (6.7%) were insulin-requiring diabetics (IRD), 250,214 (31.3%) were non-insulin requiring diabetics (NIRD) and 495,166 (62%) were non-diabetics (ND). IRD had a higher risk of stroke when compared to NIRD (adjusted HR: 1.15, 95% CI 1.10-1.21) and ND (aHR 1.24, 95% CI 1.18-1.31) (Pâ¯<â¯.01 for both). The risk of stroke was higher in NIRD compared to ND (aHR 1.08, 95% CI 1.05-1.12). For the outcome of MI, IRD had a higher risk compared to NIRD (aHR 1.24, 95% CI 1.18-1.31) and ND (aHR 1.46, 95% CI 1.38-1.54)]. NIRD had a higher risk compared to ND (aHR 1.17, 95% CI 1.13-1.22). Anticoagulation were most effective at preventing stroke in ND [0.72 (0.69-0.75)], and NIRD [0.88 (0.85-0.92)], but were not associated with significant reduction in stroke in IRD [0.96 (0.89-1.04)]. CONCLUSION: There is an incremental risk of ischemic stroke and MI from non-diabetics to non-insulin diabetics with the highest risk in insulin users. Protective effect of anticoagulation is attenuated with insulin use.
Assuntos
Fibrilação Atrial/complicações , Diabetes Mellitus/tratamento farmacológico , Hipoglicemiantes/efeitos adversos , Insulina/efeitos adversos , Infarto do Miocárdio/etiologia , Acidente Vascular Cerebral/etiologia , Idoso , Idoso de 80 Anos ou mais , Anticoagulantes/uso terapêutico , Diabetes Mellitus/epidemiologia , Angiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/etiologia , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Masculino , Medicare , Infarto do Miocárdio/prevenção & controle , Modelos de Riscos Proporcionais , Risco , Acidente Vascular Cerebral/prevenção & controle , Fatores de Tempo , Estados Unidos/epidemiologiaRESUMO
Sick sinus syndrome and atrioventricular block are common clinical problems, often necessitating permanent pacemaker placement, yet the pathophysiology of these conditions remains poorly understood. Here we show that Transient Receptor Potential Melastatin 7 (TRPM7), a divalent-permeant channel-kinase of unknown function, is highly expressed in embryonic myocardium and sinoatrial node (SAN) and is required for cardiac automaticity in these specialized tissues. TRPM7 disruption in vitro, in cultured embryonic cardiomyocytes, significantly reduces spontaneous Ca(2+) transient firing rates and is associated with robust down-regulation of Hcn4, Cav3.1, and SERCA2a mRNA. TRPM7 knockdown in zebrafish, global murine cardiac Trpm7 deletion (KO(αMHC-Cre)), and tamoxifen-inducible SAN restricted Trpm7 deletion (KO(HCN4-CreERT2)) disrupts cardiac automaticity in vivo. Telemetered and sedated KO(αMHC-Cre) and KO(HCN4-CreERT2) mice show episodes of sinus pauses and atrioventricular block. Isolated SAN from KO(αMHC-Cre) mice exhibit diminished Ca(2+) transient firing rates with a blunted diastolic increase in Ca(2+). Action potential firing rates are diminished owing to slower diastolic depolarization. Accordingly, Hcn4 mRNA and the pacemaker current, I(f), are diminished in SAN from both KO(αMHC-Cre) and KO(HCN4-CreERT2) mice. Moreover, heart rates of KO(αMHC-Cre) mice are less sensitive to the selective I(f) blocker ivabradine, and acute application of the recently identified TRPM7 blocker FTY720 has no effect on action potential firing rates of wild-type SAN cells. We conclude that TRPM7 influences diastolic membrane depolarization and automaticity in SAN indirectly via regulation of Hcn4 expression.
Assuntos
Coração/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Canais de Cátion TRPM/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Coração/embriologia , Frequência Cardíaca/genética , Frequência Cardíaca/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nó Sinoatrial/citologia , Nó Sinoatrial/embriologia , Canais de Cátion TRPM/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Transient receptor potential (TRP) channels are a superfamily of broadly expressed ion channels with diverse physiological roles. TRPC1, TRPC3, and TRPC6 are believed to contribute to cardiac hypertrophy in mouse models. Human mutations in TRPM4 have been linked to progressive familial heart block. TRPM7 is a divalent-permeant channel and kinase of unknown function, recently implicated in the pathogenesis of atrial fibrillation; however, its function in ventricular myocardium remains unexplored. METHODS AND RESULTS: We generated multiple cardiac-targeted knockout mice to test the hypothesis that TRPM7 is required for normal ventricular function. Early cardiac Trpm7 deletion (before embryonic day 9; TnT/Isl1-Cre) results in congestive heart failure and death by embryonic day 11.5 as a result of hypoproliferation of the compact myocardium. Remarkably, Trpm7 deletion late in cardiogenesis (about embryonic day 13; αMHC-Cre) produces viable mice with normal adult ventricular size, function, and myocardial transcriptional profile. Trpm7 deletion at an intermediate time point results in 50% of mice developing cardiomyopathy associated with heart block, impaired repolarization, and ventricular arrhythmias. Microarray analysis reveals elevations in transcripts of hypertrophy/remodeling genes and reductions in genes important for suppressing hypertrophy (Hdac9) and for ventricular repolarization (Kcnd2) and conduction (Hcn4). These transcriptional changes are accompanied by action potential prolongation and reductions in transient outward current (Ito; Kcnd2). Similarly, the pacemaker current (If; Hcn4) is suppressed in atrioventricular nodal cells, accounting for the observed heart block. CONCLUSIONS: Trpm7 is dispensable in adult ventricular myocardium under basal conditions but is critical for myocardial proliferation during early cardiogenesis. Loss of Trpm7 at an intermediate developmental time point alters the myocardial transcriptional profile in adulthood, impairing ventricular function, conduction, and repolarization.
Assuntos
Deleção de Genes , Sistema de Condução Cardíaco/fisiologia , Miocárdio/citologia , Miócitos Cardíacos/fisiologia , Canais de Cátion TRPM/deficiência , Função Ventricular/fisiologia , Potenciais de Ação/fisiologia , Fatores Etários , Animais , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Canais de Cátion TRPM/genética , Fatores de TempoRESUMO
Background and objective: Guillain-Barre syndrome (GBS) has been found to have some interesting association with vaccinations. This paper mainly focuses on exploring different associations between COVID-19 vaccination and GBS. Methods: Electronic databases such as PubMed, Google Scholar, Cochrane, and Embase were searched using MESH terms for case reports published till 1 August 2023 from which 70 case reports were documented involving 103 individuals from 23 different countries. Result and discussion: The case reports were from a wide range of individuals aged from 13 to 87 years with an average age of 53±20 interquartile range years along with male predominance. The average time between receiving the vaccine and the onset of symptoms was 13.08±2.14 days. Prominent clinical features included back pain, facial diplegia, weakness, and paraesthesia whereas the main diagnostic studies were cerebrospinal fluid (CSF) analysis and electromagnetic studies. The principal diagnostic clue was albumin-cytological dissociation in CSF while being negative for anti-ganglioside antibodies or SARS-CoV-2. Available treatment options consisted of intravenous immunoglobulin and Plasmapheresis. Patients with comorbidities such as diabetes mellitus, hypertension, dyslipidemia, permanent atrial fibrillation, hypothyroidism, Hashimoto's thyroiditis, Chronic Obstructive Pulmonary Disease, asthma, osteoporosis, migraine, rheumatoid arthritis, osteoarthritis, ulcerative colitis, coeliac disease, seizures, bipolar disorder, endometriosis, multiple sclerosis, bell's palsy, squamous cell carcinoma, prostate cancer were included in our study. Conclusion: Overall, this review evaluated innovative and clinically relevant associations between COVID-19 vaccination and GBS. Understanding of this uncommon potential side effect of COVID-19 vaccination is crucial for prompt diagnosis and appropriate treatment. Importantly, GBS should not be considered a contraindication to vaccination. This underscores the importance of ongoing research to enhance the safety and efficacy of COVID-19 vaccination efforts.
RESUMO
Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.
RESUMO
BACKGROUND: Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing. RESULTS: Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes. CONCLUSIONS: Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.
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Electrical mechanosensing is a process mediated by specialized ion channels, gated directly or indirectly by mechanical forces, which allows cells to detect and subsequently respond to mechanical stimuli. The activation of mechanosensitive (MS) ion channels, intrinsically gated by mechanical forces, or mechanoresponsive (MR) ion channels, indirectly gated by mechanical forces, results in electrical signaling across lipid bilayers, such as the plasma membrane. While the functions of mechanically gated channels within a sensory context (e.g., proprioception and touch) are well described, there is emerging data demonstrating functions beyond touch and proprioception, including mechanoregulation of intracellular signaling and cellular/systemic metabolism. Both MR and MS ion channel signaling have been shown to contribute to the regulation of metabolic dysfunction, including obesity, insulin resistance, impaired insulin secretion, and inflammation. This review summarizes our current understanding of the contributions of several MS/MR ion channels in cell types implicated in metabolic dysfunction, namely, adipocytes, pancreatic ß-cells, hepatocytes, and skeletal muscle cells, and discusses MS/MR ion channels as possible therapeutic targets. © 2024 American Physiological Society. Compr Physiol 14:5269-5290, 2024.
Assuntos
Canais Iônicos , Transdução de Sinais , Humanos , Canais Iônicos/metabolismo , Membrana Celular/metabolismo , Mecanotransdução Celular/fisiologiaRESUMO
Volume-regulated anion channels (VRACs) are a group of ubiquitously expressed outwardly-rectifying anion channels that sense increases in cell volume and act to return cells to baseline volume through an efflux of anions and organic osmolytes, including glutamate. Because cell swelling, increased extracellular glutamate levels, and reduction of the brain extracellular space (ECS) all occur during seizure generation, we set out to determine whether VRACs are dysregulated throughout mesial temporal lobe epilepsy (MTLE), the most common form of adult epilepsy. To accomplish this, we employed the IHKA experimental model of MTLE, and probed for the expression of LRRC8A, the essential pore-forming VRAC subunit, at acute, early-, mid-, and late-epileptogenic time points (1-, 7-, 14-, and 30-days post-IHKA, respectively). Western blot analysis revealed the upregulation of total dorsal hippocampal LRRC8A 14-days post-IHKA in both the ipsilateral and contralateral hippocampus. Immunohistochemical analyses showed an increased LRRC8A signal 7-days post-IHKA in both the ipsilateral and contralateral hippocampus, along with layer-specific changes 1-, 7-, and 30-days post-IHKA bilaterally. LRRC8A upregulation 1 day post-IHKA was observed primarily in astrocytes; however, some upregulation was also observed in neurons. Glutamate-GABA/glutamine cycle enzymes glutamic acid decarboxylase, glutaminase, and glutamine synthetase were also dysregulated at the 7-day timepoint post status epilepticus. The timepoint-dependent upregulation of total hippocampal LRRC8A and the possible subsequent increased efflux of glutamate in the epileptic hippocampus suggest that the dysregulation of astrocytic VRAC may play an important role in the development of epilepsy.
Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Humanos , Adulto , Epilepsia do Lobo Temporal/induzido quimicamente , Epilepsia do Lobo Temporal/metabolismo , Ácido Caínico/toxicidade , Ácido Caínico/metabolismo , Ácido Glutâmico/metabolismo , Epilepsia/metabolismo , Hipocampo/metabolismo , Ânions/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Healthy expansion of adipose tissue is critical for the maintenance of metabolic health, providing an optimized reservoir for energy storage in the form of triacylglycerol-rich lipoproteins. Dysfunctional adipocytes that are unable to efficiently store lipid can result in lipodystrophy and contribute to nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. Leucine-rich repeat containing protein 8a/SWELL1 functionally encodes the volume-regulated anion channel complex in adipocytes, is induced in early obesity, and is required for normal adipocyte expansion during high-fat feeding. Adipose-specific SWELL1 ablation (Adipo KO) leads to insulin resistance and hyperglycemia during caloric excess, both of which are associated with NAFLD. Here, we show that Adipo-KO mice exhibited impaired adipose depot expansion and excess lipolysis when raised on a variety of high-fat diets, resulting in increased diacylglycerides and hepatic steatosis, thereby driving liver injury. Liver lipidomic analysis revealed increases in oleic acid-containing hepatic triacylglycerides and injurious hepatic diacylglyceride species, with reductions in hepatocyte-protective phospholipids and antiinflammatory free fatty acids. Aged Adipo-KO mice developed hepatic steatosis on a regular chow diet, and Adipo-KO male mice developed spontaneous, aggressive hepatocellular carcinomas (HCCs). These data highlight the importance of adipocyte SWELL1 for healthy adipocyte expansion to protect against NAFLD and HCC in the setting of overnutrition and with aging.