RESUMO
The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.
Assuntos
Carcinogênese/patologia , Ciclo Celular , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/patologia , Fígado/enzimologia , Fígado/patologia , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Idoso , Animais , Carcinogênese/efeitos dos fármacos , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/patologia , Humanos , Fígado/cirurgia , Neoplasias Hepáticas/induzido quimicamente , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína Quinase 12 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação , Piridonas/farmacologia , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo , Homologia de Sequência , Especificidade por SubstratoRESUMO
AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.
Assuntos
Síndrome de Brugada , Células-Tronco Pluripotentes Induzidas , Síndrome do QT Longo , Potenciais de Ação/fisiologia , Animais , Síndrome de Brugada/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Espectrina/metabolismo , Espectrina/farmacologiaRESUMO
BACKGROUND: Trunk impairment produces disorders of motor control, balance and gait. Core stability exercises (CSE) are a good strategy to improve local strength of trunk, balance and gait. Methods and analysis: This is a single-blind multicenter randomized controlled trial. Two parallel groups are compared, and both perform the same type of therapy. A control group (CG) (n = 110) performs conventional physiotherapy (CP) (1 h per session) focused on improving balance. An experimental group (EG) (n = 110) performs CSE (30 min) in addition to CP (30 min) (1 h/session in total). EG is divided in two subgroups, in which only half of patients (n = 55) perform CSE plus transcutaneous electrical nerve stimulation (TENS). Primary outcome measures are dynamic sitting, assessed by a Spanish version of Trunk Impairment Scale and stepping, assessed by Brunel Balance Assessment. Secondary outcomes are postural control, assessed by Postural Assessment Scale for Stroke patients; standing balance and risk of fall assessed by Berg Balance Scale; gait speed by BTS G-Walk (accelerometer); rate of falls, lower-limb spasticity by Modified Ashworth Scale; activities of daily living by Barthel Index; and quality of life by EQ-5D-5L. These are evaluated at baseline (T0), at three weeks (T1), at five weeks (end of the intervention) (T2), at 17 weeks (T3) and at 29 weeks (T4). Study duration per patient is 29 weeks (a five-week intervention, followed by a 24-week post-intervention).
Assuntos
Reabilitação do Acidente Vascular Cerebral , Acidente Vascular Cerebral , Atividades Cotidianas , Terapia por Exercício , Marcha , Humanos , Estudos Multicêntricos como Assunto , Equilíbrio Postural , Qualidade de Vida , Ensaios Clínicos Controlados Aleatórios como Assunto , Método Simples-Cego , Postura Sentada , Resultado do TratamentoRESUMO
Synapse-Associated Protein 97 (SAP97) is an anchoring protein that in cardiomyocytes targets to the membrane and regulates Na+ and K+ channels. Here we compared the electrophysiological effects of native (WT) and p.P888L SAP97, a common polymorphism. Currents were recorded in cardiomyocytes from mice trans-expressing human WT or p.P888L SAP97 and in Chinese hamster ovary (CHO)-transfected cells. The duration of the action potentials and the QT interval were significantly shorter in p.P888L-SAP97 than in WT-SAP97 mice. Compared to WT, p.P888L SAP97 significantly increased the charge of the Ca-independent transient outward (Ito,f) current in cardiomyocytes and the charge crossing Kv4.3 channels in CHO cells by slowing Kv4.3 inactivation kinetics. Silencing or inhibiting Ca/calmodulin kinase II (CaMKII) abolished the p.P888L-induced Kv4.3 charge increase, which was also precluded in channels (p.S550A Kv4.3) in which the CaMKII-phosphorylation is prevented. Computational protein-protein docking predicted that p.P888L SAP97 is more likely to form a complex with CaMKII than WT. The Na+ current and the current generated by Kv1.5 channels increased similarly in WT-SAP97 and p.P888L-SAP97 cardiomyocytes, while the inward rectifier current increased in WT-SAP97 but not in p.P888L-SAP97 cardiomyocytes. The p.P888L SAP97 polymorphism increases the Ito,f, a CaMKII-dependent effect that may increase the risk of arrhythmias.
Assuntos
Potenciais de Ação/fisiologia , Arritmias Cardíacas/fisiopatologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Proteína 1 Homóloga a Discs-Large/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio Shal/fisiologia , Animais , Arritmias Cardíacas/genética , Células CHO , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular , Cricetulus , Proteína 1 Homóloga a Discs-Large/genética , Humanos , Canal de Potássio Kv1.5/fisiologia , Camundongos , Técnicas de Patch-Clamp , Fosforilação/fisiologia , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Existe un consenso generalizado en cuanto que la atención del daño cerebral requiere la existencia de unidades y equipos especializados en dar una atención integral.La realidad es que existe una escasez de recursos para la atención de pacientes con daño cerebral, especialmente en cuanto a laexistencia de unidades específicas de neurorehabilitación o unidades de rehabilitación intensiva. A pesar de que la Congregación de Hermanas Hospitalarias cuenta con la Red Menni de daño cerebral no es suficientepara atender a todos los pacientes. Un número importante de pacientes son atendidos actualmente en unidades de convalecencia (centros afines de la Red Menni), los cuales no todos cuentan con un equipo multidisciplinar ni con la tecnología ni robótica necesaria. (AU)
There is a general consensus that carefor brain damage requires the existence ofunits and teamns specialized in providingcomprehensive care. The reality is that there is a shortage of resources for the care of patients with brain, especially regarding the existence of specific units of neurorehabilitation or intensive rehabilitation units.Even though the Congregation of SistersHospitallers has the Menni Network of brain damage not it is enough to take care of all the patiens.A significant number of patients they are currently cared for in convalescent units (related centers of the Red Menni), nota ll of which have a multidisciplinary tea mor the technology no robotics necessary. (AU)
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Lactente , Pré-Escolar , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Necessidades e Demandas de Serviços de Saúde , Assistência ao Convalescente/estatística & dados numéricos , Dano Encefálico Crônico/reabilitação , Dano Encefálico Crônico/epidemiologia , Distribuição por Idade , Espanha/epidemiologiaRESUMO
[This corrects the article DOI: 10.1038/npjregenmed.2016.1.].
RESUMO
Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.
Assuntos
Quimiocina CXCL6/genética , Miocárdio/metabolismo , Neovascularização Fisiológica/genética , Comunicação Parácrina/genética , Proteoma/genética , Receptores de Interleucina-8B/metabolismo , Células-Tronco/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Movimento Celular , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL6/antagonistas & inibidores , Quimiocina CXCL6/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Proteoma/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
Disrupted organ growth leads to disease development. Hypertrophy underlies postnatal heart growth and is triggered after stress, but the molecular mechanisms involved in these processes are largely unknown. Here we show that cardiac activation of p38γ and p38δ increases during postnatal development and by hypertrophy-inducing stimuli. p38γ/δ promote cardiac hypertrophy by phosphorylating the mTORC1 and mTORC2 inhibitor DEPTOR, which leads to its degradation and mTOR activation. Hearts from mice lacking one or both kinases are below normal size, have high levels of DEPTOR, low activity of the mTOR pathway and reduced protein synthesis. The phenotype of p38γ/δ(-/-) mice is reverted by overactivation of mTOR with amino acids, shRNA-mediated knockdown of Deptor, or cardiomyocyte overexpression of active p38γ and p38δ. Moreover, in WT mice, heart weight is reduced by cardiac overexpression of DEPTOR. Our results demonstrate that p38γ/δ control heart growth by modulating mTOR pathway through DEPTOR phosphorylation and subsequent degradation.
Assuntos
Cardiomegalia/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Proteína Quinase 13 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 12 Ativada por Mitógeno/genética , Proteína Quinase 13 Ativada por Mitógeno/genética , Complexos Multiproteicos/genética , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Fosforilação , Proteólise , Serina-Treonina Quinases TOR/genéticaRESUMO
The insulin-like growth factor Ea propeptide (IGF-1Ea) is a powerful enhancer of cardiac muscle growth and regeneration, also blocking age-related atrophy and beneficial in multiple skeletal muscle diseases. The therapeutic potential of IGF-1Ea compared with mature IGF-1 derives from its local action in the area of synthesis. We have developed an adeno-associated virus (AAV) vector for IGF-1Ea delivery to the heart to treat mice after myocardial infarction and examine the reparative effects of local IGF-1Ea production on left ventricular remodelling. A cardiotropic AAV9 vector carrying a cardiomyocyte-specific IGF-1Ea-luciferase bi-cistronic gene expression cassette (AAV9.IGF-1Ea) was administered intravenously to infarcted mice, 5 h after ischemia followed by reperfusion (I/R), as a model of myocardial infarction. Virally encoded IGF-1Ea in the heart improved global left ventricular function and remodelling, as measured by wall motion and thickness, 28 days after delivery, with higher viral titers yielding better improvement. The present study demonstrates that single intravenous AAV9-mediated IGF-1Ea Gene Therapy represents a tissue-targeted therapeutic approach to prevent the adverse remodelling after myocardial infarct.
RESUMO
La agricultura nacional emplea unas 7000 toneladas de plaguicidas por año. En su mayor parte estas sustancias terminan en el suelo, donde habitan muchos organismos y las lombrices de tierra. Las lombrices de tierra ejercen efectos químicos y físicos, directos, y un efecto microbiológico indirecto benefecioso para el suelo, por consiguiente, el impacto negativo de los plaguicidas sobre las lombrices incide en la calidad del suelo. El objetivo principal de este trabajo fue la evaluación del efecto tóxico de 8 plaguicidas de uso común sobre Polypheretima elongata. Para ello empleamos el método de inyección, se midió la mortalidad en función de la concentración de plaguicidas a la 2, 8, 12, 24, 48, 72 y 96 horas. Los resultados indican que de mayor a menor la toxicidad de los plaguicidas sigue el orden de: