Assuntos
Diabetes Mellitus Tipo 2 , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hipoglicemiantes , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/efeitos adversos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/epidemiologia , Alemanha/epidemiologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Feminino , Hipoglicemiantes/uso terapêutico , Hipoglicemiantes/efeitos adversos , Masculino , Pessoa de Meia-Idade , Idoso , Adulto , Fatores Socioeconômicos , Agonistas do Receptor do Peptídeo 1 Semelhante ao GlucagonRESUMO
Patients with idiopathic pulmonary fibrosis show a strongly upregulated expression of chemokine CXCL14, whose target is still unknown. Screening of CXCL14 in a panel of human G protein-coupled receptors (GPCRs) revealed its potent and selective activation of the orphan MAS-related GPCR X2 (MRGPRX2). This receptor is expressed on mast cells and - like CXCL14 - upregulated in bronchial inflammation. CXCL14 induces robust activation of MRGPRX2 and its putative mouse ortholog MRGPRB2 in G protein-dependent and ß-arrestin recruitment assays that is blocked by a selective MRGPRX2/B2 antagonist. Truncation combined with mutagenesis and computational studies identified the pharmacophoric sequence of CXCL14 and its presumed interaction with the receptor. Intriguingly, C-terminal domain sequences of CXCL14 consisting of 4 to 11 amino acids display similar or increased potency and efficacy compared to the full CXCL14 sequence (77 amino acids). These results provide a rational basis for the future development of potential idiopathic pulmonary fibrosis therapies.
Assuntos
Quimiocinas , Fibrose Pulmonar Idiopática , Animais , Humanos , Camundongos , Aminoácidos , Bioensaio , Quimiocinas CXC , Fibrose Pulmonar Idiopática/genética , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G/genética , Receptores de NeuropeptídeosRESUMO
Short-term glycemic variability is associated with the risk of hypoglycemia and hyperglycemia in people living with type 1 diabetes and can potentially affect clinical outcomes. Continuous glucose monitoring (CGM) is of increasing importance to evaluate glycemic variability in greater detail. Specific metrics for assessing glycemic variability were proposed, such as the SD of mean glucose level and associated coefficient of variation, and time in target glucose range to guide study designs, therapy and allow people with diabetes more transparency in interpreting their own CGM data. Randomized controlled trials (RCT) and real-world evidence provide complementary information about the efficacy/effectiveness and safety of interventions. Insulin glargine 300 U/mL (Gla-300) has a longer lasting and less variable action than insulin glargine U100 (Gla-100) with a lower risk of hypoglycemia. While insulin degludec U100 (iDeg-100) was associated with lower glucose values but more time below range in one randomized study compared with Gla-300, Gla-300 was associated with a higher per cent time in range, but also above the therapeutic range. However, a real-world study did not find differences during the day between Gla-300 and iDeg-100. The upcoming InRange RCT is the first head-to-head comparison of Gla-300 with iDeg-100 using CGM in an international population using CGM metrics as the primary endpoint. The non-interventional COMET-T real-world study will determine the real-world effectiveness of Gla-300 using CGM metrics and cover a broad spectrum of clinical practice decisions irrespective of the prior basal insulin.
Assuntos
Diabetes Mellitus Tipo 1 , Hipoglicemia , Glicemia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Humanos , Hipoglicemia/induzido quimicamente , Hipoglicemia/prevenção & controle , Hipoglicemiantes/efeitos adversos , Insulina Glargina/efeitos adversos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10-6 mol L-1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10-6 mol L-1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.
Assuntos
Artérias Mesentéricas/fisiologia , Guanilil Ciclase Solúvel/fisiologia , Acetilcolina/farmacologia , Fatores Etários , Animais , Aorta/metabolismo , GMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Guanilato Ciclase/metabolismo , Masculino , Artérias Mesentéricas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Kidney fibrosis presents a hallmark of chronic kidney disease. With ever-increasing patient numbers and limited treatment options available, novel strategies for therapeutic intervention in kidney disease are warranted. Fibrosis commonly results from a wound healing response to repeated or chronic tissue damage, irrespective of the underlying etiology, and can occur in virtually any solid organ or tissue. In order to identify targets relevant for kidney fibrosis, we aimed to employ CRISPR screening in primary human kidney fibroblasts. We demonstrate that CRISPR technology can be applied in primary kidney fibroblasts and can furthermore be used to conduct arrayed CRISPR screening using a high-content imaging readout in a whole genome-wide manner. Hits coming out of this screen were validated using orthogonal approaches and present starting points for validation of novel targets relevant to kidney disease.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Fibrose/genética , Nefropatias/genética , Rim/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose/tratamento farmacológico , Fibrose/patologia , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Terapia de Alvo Molecular/tendênciasRESUMO
BACKGROUND: The aim of the current study was to determine whether the time to insulin therapy initiation in patients with type 2 diabetes in primary care in Germany has changed in recent years. METHODS: Longitudinal data from general practices in Germany (Disease Analyzer) were analyzed. These data included information of 7128 patients (age: 68.5 [SD: 11.5] years; 54.4% male) receiving incident insulin therapy in 2010/2011 and 8216 patients (age: 69.1 [SD: 11.9] years; 54.9% male) receiving incident insulin therapy in 2016/2017. Changes in time to insulin initiation in the practices and the last HbA1c value before the start of insulin therapy were analyzed, stratified by index date. To analyze the impact of covariables on the time to insulin initiation, a multivariate regression analysis was performed, adjusted for age, sex, diabetologist care, and HbA1c as independent variables. RESULTS: Median time from T2D diagnosis to insulin therapy in the Disease Analyzer database increased from 1717 days in 2010/2011 to 1917 days in 2016/2017 (P < .001). The proportion of patients with a HbA1c value ≥9% before insulin initiation was high in both groups (2010/2011: 33.0%, 2016/2017: 34.2%, P = .347). The time to insulin initiation in DPP-4i patients was 112 days longer, and in SGLT2 patients 346 days longer than in patients treated with sulfonylurea. CONCLUSIONS: The present analysis confirms an increasing delay of the insulin therapy initiation as a consequence of the more frequent use of newer oral antidiabetics. However, the rather moderate increase of time to insulin might display insufficient long-term glycemic control using these agents. Still, more than one-third of patients receive insulin only when HbA1c levels exceed 9%.
Assuntos
Glicemia/análise , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Padrões de Prática Médica , Idoso , Bases de Dados Factuais , Feminino , Alemanha , Hemoglobinas Glicadas/análise , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Atenção Primária à Saúde , Tempo para o TratamentoRESUMO
Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N2, 5% CO2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O2, 5% CO2). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels KCa3.1 and KCa2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.
RESUMO
AIMS: Women with aortic stenosis develop a more concentric form of LV hypertrophy than men. However, the molecular factors underlying sex differences in LV remodelling are incompletely understood. We took an unbiased approach to identify sex-specific patterns in gene expression and pathway regulation, and confirmed the most prominent findings in human hearts. METHODS AND RESULTS: Echocardiography was performed in 104 patients (53.8% women) with aortic stenosis before aortic valve replacement. LV mass, LV end-diastolic diameter, and relative wall thickness were included in a factor analysis to generate an index classifying LV remodelling as adaptive or maladaptive. Maladaptive remodelling was present in 64.6% of male and in 32.7% of female patients (P < 0.01). Genome-wide expression profiling of LV samples was performed in a representative subgroup of 19 patients (52.6% women) compared with samples from healthy controls (n = 18). Transcriptome characterization revealed that fibrosis-related genes/pathways were induced in male overloaded ventricles, while extracellular matrix-related and inflammatory genes/pathways were repressed in female overloaded ventricles (adjusted P < 0.05). We confirmed gene regulation by quantitative real-time reverse transcription-polymerase chain reaction and immunoblotting analysis, and we further demonstrate the relevance of our findings by histological documentation of higher fibrosis in men than in women. CONCLUSION: We conclude that in pressure overload distinct molecular processes are regulated between men and women. Maladaptive LV remodelling occurs more frequently in men and is associated with greater activation of profibrotic and inflammatory markers. Collectively, sex-specific regulation of these processes may contribute to sex differences in the progression to heart failure.
Assuntos
Estenose da Valva Aórtica/patologia , Hipertrofia Ventricular Esquerda/patologia , Remodelação Ventricular , Idoso , Estenose da Valva Aórtica/diagnóstico por imagem , Estenose da Valva Aórtica/genética , Biópsia , Ecocardiografia , Feminino , Fibrose/diagnóstico por imagem , Fibrose/genética , Fibrose/patologia , Perfilação da Expressão Gênica , Humanos , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/genética , Immunoblotting , Inflamação/diagnóstico por imagem , Inflamação/genética , Inflamação/patologia , Masculino , RNA/análise , Reação em Cadeia da Polimerase em Tempo Real , Fatores Sexuais , Análise Serial de Tecidos , Transcriptoma/genética , Remodelação Ventricular/genéticaRESUMO
In vertebrates, SLC22A13 is an evolutionarily conserved transport protein of the plasma membrane. In humans and rat, it is principally expressed in the kidney. The precise localization and physiological function are unknown. In the present study, immunohistochemistry revealed that expression of SLC22A13 is confined to the basolateral membrane of type A intercalated cells in rat kidney. Double-staining confirmed that SLC22A13 co-localizes with anion exchanger 1. LC-MS difference shading showed that heterologous expression of human and rat SLC22A13 in HEK (human embryonic kidney)-293 cells stimulates efflux of guanidinosuccinate, aspartate, glutamate and taurine. Time courses of uptake of [3H]aspartate and [3H]glutamate revealed that SLC22A13 counteracted endogenous uptake. By contrast, OAT2 (organic anion transporter 2), a bidirectional glutamate transporter, increased accumulation of [3H]glutamate. Thus SLC22A13 catalyses unidirectional efflux. Velocity of efflux of standard amino acids was measured by LC-MS/MS. Expression of SLC22A13 strongly stimulated efflux of aspartate, taurine and glutamate. When the intracellular concentrations of aspartate and taurine were increased by pre-incubation, velocities of efflux increased linearly. We propose that in type A intercalated cells, SLC22A13 compensates luminal exit of protons by mediating the basolateral expulsion of the anions aspartate and glutamate. In this context, unidirectional efflux is essential to avoid anion re-entering. Loss of SLC22A13 function could cause distal tubular acidosis.
Assuntos
Ácido Aspártico/metabolismo , Células Epiteliais/metabolismo , Ácido Glutâmico/metabolismo , Túbulos Renais Coletores/metabolismo , Transportadores de Ânions Orgânicos/biossíntese , Animais , Catálise , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos/genética , Transporte Proteico/fisiologia , RatosRESUMO
In human heart failure (HF), reduced cardiac function has, at least partly, been ascribed to altered calcium homeostasis in cardiomyocytes. The effects of the calcium sensitizer levosimendan on diastolic dysfunction caused by reduced removal of calcium from cytosol in early diastole are not well known. In this study, we investigated the effect of long-term levosimendan treatment in a murine model of HF where the sarco(endo)plasmatic reticulum ATPase (Serca) gene is specifically disrupted in the cardiomyocytes, leading to reduced removal of cytosolic calcium. After induction of Serca2 gene disruption, these mice develop marked diastolic dysfunction as well as impaired contractility. SERCA2 knockout (SERCA2KO) mice were treated with levosimendan or vehicle from the time of KO induction. At the 7-wk end point, cardiac function was assessed by echocardiography and pressure measurements. Vehicle-treated SERCA2KO mice showed significantly diminished left-ventricular (LV) contractility, as shown by decreased ejection fraction, stroke volume, and cardiac output. LV pressure measurements revealed a marked increase in the time constant (τ) of isovolumetric pressure decay, showing impaired relaxation. Levosimendan treatment significantly improved all three systolic parameters. Moreover, a significant reduction in τ toward normalization indicated improved relaxation. Gene-expression analysis, however, revealed an increase in genes related to production of the ECM in animals treated with levosimendan. In conclusion, long-term levosimendan treatment improves both contractility and relaxation in a heart-failure model with marked diastolic dysfunction due to reduced calcium transients. However, altered gene expression related to fibrosis was observed.
Assuntos
Cardiotônicos/farmacologia , Diástole/efeitos dos fármacos , Insuficiência Cardíaca/tratamento farmacológico , Hidrazonas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Piridazinas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sístole/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrose , Regulação da Expressão Gênica , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Camundongos , Camundongos Knockout , Miócitos Cardíacos/enzimologia , Recuperação de Função Fisiológica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/deficiência , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Simendana , Volume Sistólico/efeitos dos fármacos , Fatores de Tempo , Disfunção Ventricular Esquerda/tratamento farmacológico , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
Light-sensitive Ca(2+) -regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca(2+) -regulated photoproteins, but a very low degree of sequence identity (27-29%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca(2+) -discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (λ(max) = 491 nm) and a change in pH over the range 6.0-9.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca(2+) -discharged protein (λ(ex) = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca(2+) concentration-effect curve is a sigmoid with a slope on a log-log plot of â¼ 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca(2+) -independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium. Database The nucleotide sequences have been deposited in the GenBankTM/EBI Data Bank with accession numbers: apoberovin cDNA genes, JN673813 (BA1), JN673814 (BA2), JN673815 (BA3), JN673816 (BA4); fragment 18S rRNA, JN673817 (BA-rRNA5).
Assuntos
Cálcio/farmacologia , Ctenóforos/metabolismo , Luz , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Clonagem Molecular , Cricetinae , Concentração de Íons de Hidrogênio , Cinética , Luciferases/metabolismo , Medições Luminescentes , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Homologia de Sequência de AminoácidosRESUMO
Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Glutationa/metabolismo , Potencial da Membrana Mitocondrial , Proteínas do Tecido Nervoso/metabolismo , Linhagem Celular , Doença de Charcot-Marie-Tooth/genética , Humanos , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/genética , Estresse OxidativoRESUMO
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy primarily of the right ventricle characterized through fibrofatty replacement of cardiomyocytes. The genetic etiology in ARVC patients is most commonly caused by dominant inheritance and high genetic heterogeneity. Though histological examinations of ARVC-affected human myocardium reveals fibrolipomatous replacement, the molecular mechanisms leading to loss of cardiomyocytes are largely unknown. We therefore analyzed the transcriptomes of six ARVC hearts and compared our findings to six nonfailing donor hearts (NF). To characterize the ARVC-specific transcriptome, we compared our findings to samples from seven patients with idiopathic dilated cardiomyopathy (DCM). The myocardial DCM and ARVC samples were prepared from hearts explanted during an orthotopic heart transplantation representing myocardium from end-stage heart failure patients (NYHA IV). From each heart, left (LV) and right ventricular (RV) myocardial samples were analyzed by Affymetrix HG-U133 Plus 2.0 arrays, adding up to six sample groups. Unsupervised cluster analyses of the groups revealed a clear separation of NF and cardiomyopathy samples. However, in contrast to the other samples, the analyses revealed no distinct expression pattern in LV and RV of myocardial ARVC samples. We further identified differentially expressed transcripts using t-tests and found transcripts separating diseased and NF ventricular myocardium. Of note, in failing myocardium only ~15-16% of the genes are commonly regulated compared with NF samples. In addition both cardiomyopathies are clearly distinct on the transcriptome level. Comparison of the expression patterns between the failing RV and LV using a paired t-test revealed a lack of major differences between LV and RV gene expression in ARVC hearts. Our study is the first analysis of specific ARVC-related RV and LV gene expression patterns in terminal failing human hearts.
Assuntos
Displasia Arritmogênica Ventricular Direita/genética , Miocárdio/metabolismo , Transcriptoma , Adolescente , Adulto , Idoso , Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/patologia , Estudos de Casos e Controles , Análise por Conglomerados , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Miocárdio/patologia , Transcriptoma/genética , Adulto JovemRESUMO
Store-operated Ca(2+) entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca(2+) concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Doença Crônica , Córtex Entorrinal/citologia , Córtex Entorrinal/fisiopatologia , Epilepsia do Lobo Temporal/fisiopatologia , Hipocampo/citologia , Hipocampo/fisiopatologia , Humanos , Proteínas de Neoplasias/metabolismo , Rede Nervosa/metabolismo , Rede Nervosa/fisiopatologia , Neurônios/citologia , Técnicas de Cultura de Órgãos , Cultura Primária de Células , Ratos , Molécula 1 de Interação Estromal , Molécula 2 de Interação EstromalRESUMO
G-protein-coupled receptor 30 (GPR30) has been reported to act as a membrane-bound estrogen receptor that is involved in the mediation of non-genomic estradiol signalling. In this study, we demonstrated that male, but not female, GPR30-deficient mice suffer from impaired leftventricular cardiac function. Left ventricles from male mutant mice were enlarged. There were no malformations in the valves or outflow tract of the heart. Both the contractility and relaxation capacity of the left ventricle were reduced, leading to increased leftventricular end-diastolic pressure in GPR30-deficient mice. In conclusion, our data support a role for GPR30 in the gender-specific aspects of heart failure.
Assuntos
Deleção de Genes , Ventrículos do Coração/fisiopatologia , Receptores Acoplados a Proteínas G/genética , Função Ventricular Esquerda , Animais , Feminino , Insuficiência Cardíaca/genética , Ventrículos do Coração/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Estrogênio , Fatores SexuaisRESUMO
OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 µl·min⻹·mg of protein⻹) and human (46 µl·min⻹·mg of protein⻹). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [³H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.
Assuntos
Ácido Glutâmico/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ácido Orótico/metabolismo , Alcaloides/metabolismo , Animais , Transporte Biológico Ativo/genética , Domínio Catalítico/genética , Linhagem Celular Transformada , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Ratos , Especificidade por Substrato/genéticaRESUMO
TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca(2+)-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.