RESUMEN
INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart disease with an estimated prevalence in the general population of 0.2% to 0.6%. Clinically, HCM can range from no symptoms to severe symptoms such as heart failure or sudden cardiac death. Currently, the management of HCM involves lifestyle modifications, familial screening, genetic counseling, pharmacotherapy to manage symptoms, sudden cardiac death risk assessment, septal reduction therapy, and heart transplantation for specific patients. Multicenter randomized controlled trials have only recently explored the potential of cardiac myosin inhibitors (CMIs) such as mavacamten as a directed pharmacological approach for managing HCM. AREAS COVERED: We will assess the existing medical treatments for HCM: beta-blockers, calcium channel blockers, disopyramide, and different CMIs. We will also discuss future HCM pharmacotherapy guidelines and underline this patient population's unfulfilled needs. EXPERT OPINION: Mavacamten is the first-in-class CMI approved by the FDA to target HCM pathophysiology specifically. Mavacamten should be incorporated into the standard therapy for oHCM in case of symptom persistence despite using maximally tolerated beta blockers and/or calcium channel blockers. Potential drug-drug interactions should be assessed before initiating this drug. More studies are needed on the use of CMIs in patients with kidney and/or liver failure and pregnant/breastfeeding patients.
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Cardiomiopatía Hipertrófica , Adulto , Humanos , Bencilaminas , Miosinas Cardíacas/genética , Miosinas Cardíacas/antagonistas & inhibidores , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Muerte Súbita Cardíaca/prevención & control , Muerte Súbita Cardíaca/etiología , Interacciones Farmacológicas , Ensayos Clínicos Controlados Aleatorios como Asunto , Uracilo/análogos & derivadosAsunto(s)
Cardiomiopatía Hipertrófica , Fármacos Cardiovasculares , Humanos , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/farmacología , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Cardiomiopatía Hipertrófica/fisiopatología , Fármacos Cardiovasculares/uso terapéutico , Hemodinámica/efectos de los fármacos , Historia del Siglo XX , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiologíaRESUMEN
BACKGROUND: One of the major determinants of exercise intolerance and limiting symptoms among patients with obstructive hypertrophic cardiomyopathy (HCM) is an elevated intracardiac pressure resulting from left ventricular outflow tract obstruction. Aficamten is an oral selective cardiac myosin inhibitor that reduces left ventricular outflow tract gradients by mitigating cardiac hypercontractility. METHODS: In this phase 3, double-blind trial, we randomly assigned adults with symptomatic obstructive HCM to receive aficamten (starting dose, 5 mg; maximum dose, 20 mg) or placebo for 24 weeks, with dose adjustment based on echocardiography results. The primary end point was the change from baseline to week 24 in the peak oxygen uptake as assessed by cardiopulmonary exercise testing. The 10 prespecified secondary end points (tested hierarchically) were change in the Kansas City Cardiomyopathy Questionnaire clinical summary score (KCCQ-CSS), improvement in the New York Heart Association (NYHA) functional class, change in the pressure gradient after the Valsalva maneuver, occurrence of a gradient of less than 30 mm Hg after the Valsalva maneuver, and duration of eligibility for septal reduction therapy (all assessed at week 24); change in the KCCQ-CSS, improvement in the NYHA functional class, change in the pressure gradient after the Valsalva maneuver, and occurrence of a gradient of less than 30 mm Hg after the Valsalva maneuver (all assessed at week 12); and change in the total workload as assessed by cardiopulmonary exercise testing at week 24. RESULTS: A total of 282 patients underwent randomization: 142 to the aficamten group and 140 to the placebo group. The mean age was 59.1 years, 59.2% were men, the baseline mean resting left ventricular outflow tract gradient was 55.1 mm Hg, and the baseline mean left ventricular ejection fraction was 74.8%. At 24 weeks, the mean change in the peak oxygen uptake was 1.8 ml per kilogram per minute (95% confidence interval [CI], 1.2 to 2.3) in the aficamten group and 0.0 ml per kilogram per minute (95% CI, -0.5 to 0.5) in the placebo group (least-squares mean between-group difference, 1.7 ml per kilogram per minute; 95% CI, 1.0 to 2.4; P<0.001). The results for all 10 secondary end points were significantly improved with aficamten as compared with placebo. The incidence of adverse events appeared to be similar in the two groups. CONCLUSIONS: Among patients with symptomatic obstructive HCM, treatment with aficamten resulted in a significantly greater improvement in peak oxygen uptake than placebo. (Funded by Cytokinetics; SEQUOIA-HCM ClinicalTrials.gov number, NCT05186818.).
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Cardiomiopatía Hipertrófica , Fármacos Cardiovasculares , Prueba de Esfuerzo , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Bencilaminas , Miosinas Cardíacas/antagonistas & inhibidores , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Cardiomiopatía Hipertrófica/fisiopatología , Método Doble Ciego , Tolerancia al Ejercicio/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Uracilo/análogos & derivados , Maniobra de Valsalva , Obstrucción del Flujo Ventricular Externo/tratamiento farmacológico , Obstrucción del Flujo Ventricular Externo/fisiopatología , Obstrucción del Flujo Ventricular Externo/etiología , Fármacos Cardiovasculares/farmacología , Fármacos Cardiovasculares/uso terapéutico , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Administración OralRESUMEN
La miocardiopatía hipertrófica (MCH), definida clínicamente como una hipertrofia ventricular izquierda no explicada por sobrecarga hemodinámica ni por infiltración, es la enfermedad miocárdica hereditaria más común. El tratamiento actual se basa en terapias de reducción septal en casos de Miocardiopatía hipertrófica obstructiva (MCHo), prevención de muerte súbita cardíaca mediante desfibrilador automático implantable y en el uso de fármacos para disminuir la obstrucción dinámica del tracto de salida del ventrículo izquierdo. Recientemente se han desarrollado diversos agentes que actúan sobre los mecanismos que, a nivel celular, llevan a la aparición de hipertrofia ventricular. Este artículo se centra en formas de tratamiento farmacológico desarrolladas en los últimos años, en especial con inhibidores de miosina. Se detallan los mecanismos de acción como también los resultados de diversos estudios clínicos de evaluaciones recientes.
Current clinical management of Hypertrophic Cardiomyopathy is mainly based on modifying physiologic mechanisms to decrease left ventricular outflow tract obstruction. Also important are agents that prevent and treat ventricular arrythmias. This review article is focused on the cellular mechanisms that underlie the development of ventricular hypertrophy and the effect of new myosin inhibitors such as mavcamten and aficamten. Promising results of recent clinical trials evaluating the effect of these agents are analyzed. The potential benefits of genetic therapy are also discussed.
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Humanos , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Fármacos Cardiovasculares/uso terapéutico , Miosinas Cardíacas/antagonistas & inhibidores , Cardiomiopatía Hipertrófica/fisiopatología , Terapia GenéticaRESUMEN
BACKGROUND: EXPLORER-HCM (Clinical Study to Evaluate Mavacamten [MYK-461] in Adults With Symptomatic Obstructive Hypertrophic Cardiomyopathy) demonstrated that mavacamten, a cardiac myosin inhibitor, improves symptoms, exercise capacity, and left ventricular outflow tract (LVOT) obstruction in patients with obstructive hypertrophic cardiomyopathy (oHCM). OBJECTIVES: The purpose of this study was to evaluate mavacamten's effect on measures of cardiac structure and function and its association with changes in other clinical measures. METHODS: Key echocardiographic parameters from serial echocardiograms over 30 weeks from 251 symptomatic oHCM patients (mavacamten [n = 123], placebo [n = 128]) were assessed in a core laboratory. RESULTS: More patients on mavacamten (80.9%; n = 76 of 94) vs placebo (34.0%; n = 33 of 97) showed complete resolution of mitral valve systolic anterior motion after 30 weeks (difference, 46.8%; P < 0.0001). Mavacamten also improved measures of diastolic function vs placebo, including left atrial volume index (LAVI) (mean ± SD baseline: 40 ± 12 mL/m2 vs 41 ± 14 mL/m2; mean change from baseline of -7.5 mL/m2 [95% CI: -9.0 to -6.1 mL/m2] vs -0.09 mL/m2 [95% CI: -1.6 to 1.5 mL/m2]; P < 0.0001) and lateral E/e' (baseline, 15 ± 6 vs 15 ± 8; change of -3.8 [95% CI: -4.7 to -2.8] vs 0.04 [95% CI: -0.9 to 1.0]; P < 0.0001). Among mavacamten-treated patients, improvement in resting, Valsalva, and post-exercise LVOT gradients, LAVI, and lateral E/e' was associated with reduction in N-terminal pro-B-type natriuretic peptide (P ≤ 0.03 for all). Reduction in LAVI was associated with improved peak exercise oxygen consumption (P = 0.04). CONCLUSIONS: Mavacamten significantly improved measures of left ventricular diastolic function and systolic anterior motion. Improvement in LVOT obstruction, LAVI, and E/e' was associated with reduction in a biomarker of myocardial wall stress (N-terminal pro-B-type natriuretic peptide). These findings demonstrate improvement in important markers of the pathophysiology of oHCM with mavacamten. (Clinical Study to Evaluate Mavacamten [MYK-461] in Adults With Symptomatic Obstructive Hypertrophic Cardiomyopathy; NCT03470545).
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Bencilaminas/uso terapéutico , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Corazón/efectos de los fármacos , Uracilo/análogos & derivados , Anciano , Bencilaminas/farmacología , Biomarcadores/sangre , Miosinas Cardíacas/antagonistas & inhibidores , Cardiomiopatía Hipertrófica/sangre , Cardiomiopatía Hipertrófica/diagnóstico por imagen , Método Doble Ciego , Ecocardiografía , Tolerancia al Ejercicio/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Uracilo/farmacología , Uracilo/uso terapéuticoRESUMEN
Heart failure (HF) is a syndrome encompassing several important etiologies that lead to the imbalance between oxygen demand and supply. Despite the usage of guideline-directed medical therapy for HF has shown better outcomes, novel therapeutic strategies are desirable, especially for patients with preserved or mildly reduced left ventricular ejection fraction. In this regard, understanding the molecular basis for cardiomyopathies is expected to fill in the knowledge gap and generate new therapies to improve prognosis for HF. This review discusses an evolutionary mechanism designed to regulate cardiac contraction and relaxation through the most often genetically determined cardiomyopathies associated with HF. In addition, both the myosin inhibitor and myosin activator are promising new treatments for cardiomyopathies. A comprehensive review from genetic mutations to the molecular basis of direct sarcomere modulators will help shed light on future studies for a better characterization of HF etiologies and potential therapeutic targets.
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Bencilaminas/uso terapéutico , Miosinas Cardíacas/genética , Insuficiencia Cardíaca/tratamiento farmacológico , Terapia Molecular Dirigida , Uracilo/análogos & derivados , Urea/análogos & derivados , Bencilaminas/farmacología , Miosinas Cardíacas/antagonistas & inhibidores , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Miocitos Cardíacos/patología , Uracilo/farmacología , Uracilo/uso terapéutico , Urea/farmacología , Urea/uso terapéuticoRESUMEN
We have created a novel in-vitro platform to study reverse remodeling of engineered heart tissue (EHT) after mechanical unloading. EHTs were created by seeding decellularized porcine myocardial sections with a mixture of primary neonatal rat ventricular myocytes and cardiac fibroblasts. Each end of the ribbon-like constructs was fixed to a plastic clip, allowing the tissues to be statically stretched or slackened. Inelastic deformation was introduced by stretching tissues by 20% of their original length. EHTs were subsequently unloaded by returning tissues to their original, shorter length. Mechanical characterization of EHTs immediately after unloading and at subsequent time points confirmed the presence of a reverse-remodeling process, through which stress-free tissue length was increased after chronic stretch but gradually decreased back to its original value within 9 days. When a cardiac myosin inhibitor was applied to tissues after unloading, EHTs failed to completely recover their passive and active mechanical properties, suggesting a role for actomyosin contraction in reverse remodeling. Selectively inhibiting cardiomyocyte contraction or fibroblast activity after mechanical unloading showed that contractile activity of both cell types was required to achieve full remodeling. Similar tests with EHTs formed from human induced pluripotent stem cell-derived cardiomyocytes also showed reverse remodeling that was enhanced when treated with omecamtiv mecarbil, a myosin activator. These experiments suggest essential roles for active sarcomeric contraction and fibroblast activity in reverse remodeling of myocardium after mechanical unloading. Our findings provide a mechanistic rationale for designing potential therapies to encourage reverse remodeling in patient hearts.
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Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Miofibroblastos/metabolismo , Sarcómeros/metabolismo , Transducción de Señal/efectos de los fármacos , Ingeniería de Tejidos/métodos , Remodelación Ventricular/efectos de los fármacos , Actomiosina/metabolismo , Animales , Animales Recién Nacidos , Benzamidas/farmacología , Bencilaminas/farmacología , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/metabolismo , Línea Celular , Dioxoles/farmacología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Miofibroblastos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Porcinos , Andamios del Tejido , Uracilo/análogos & derivados , Uracilo/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
BACKGROUND: Cardiac muscle hypercontractility is a key pathophysiological abnormality in hypertrophic cardiomyopathy, and a major determinant of dynamic left ventricular outflow tract (LVOT) obstruction. Available pharmacological options for hypertrophic cardiomyopathy are inadequate or poorly tolerated and are not disease-specific. We aimed to assess the efficacy and safety of mavacamten, a first-in-class cardiac myosin inhibitor, in symptomatic obstructive hypertrophic cardiomyopathy. METHODS: In this phase 3, randomised, double-blind, placebo-controlled trial (EXPLORER-HCM) in 68 clinical cardiovascular centres in 13 countries, patients with hypertrophic cardiomyopathy with an LVOT gradient of 50 mm Hg or greater and New York Heart Association (NYHA) class II-III symptoms were assigned (1:1) to receive mavacamten (starting at 5 mg) or placebo for 30 weeks. Visits for assessment of patient status occurred every 2-4 weeks. Serial evaluations included echocardiogram, electrocardiogram, and blood collection for laboratory tests and mavacamten plasma concentration. The primary endpoint was a 1·5 mL/kg per min or greater increase in peak oxygen consumption (pVO2) and at least one NYHA class reduction or a 3·0 mL/kg per min or greater pVO2 increase without NYHA class worsening. Secondary endpoints assessed changes in post-exercise LVOT gradient, pVO2, NYHA class, Kansas City Cardiomyopathy Questionnaire-Clinical Summary Score (KCCQ-CSS), and Hypertrophic Cardiomyopathy Symptom Questionnaire Shortness-of-Breath subscore (HCMSQ-SoB). This study is registered with ClinicalTrials.gov, NCT03470545. FINDINGS: Between May 30, 2018, and July 12, 2019, 429 adults were assessed for eligibility, of whom 251 (59%) were enrolled and randomly assigned to mavacamten (n=123 [49%]) or placebo (n=128 [51%]). 45 (37%) of 123 patients on mavacamten versus 22 (17%) of 128 on placebo met the primary endpoint (difference +19·4%, 95% CI 8·7 to 30·1; p=0·0005). Patients on mavacamten had greater reductions than those on placebo in post-exercise LVOT gradient (-36 mm Hg, 95% CI -43·2 to -28·1; p<0·0001), greater increase in pVO2 (+1·4 mL/kg per min, 0·6 to 2·1; p=0·0006), and improved symptom scores (KCCQ-CSS +9·1, 5·5 to 12·7; HCMSQ-SoB -1·8, -2·4 to -1·2; p<0·0001). 34% more patients in the mavacamten group improved by at least one NYHA class (80 of 123 patients in the mavacamten group vs 40 of 128 patients in the placebo group; 95% CI 22·2 to 45·4; p<0·0001). Safety and tolerability were similar to placebo. Treatment-emergent adverse events were generally mild. One patient died by sudden death in the placebo group. INTERPRETATION: Treatment with mavacamten improved exercise capacity, LVOT obstruction, NYHA functional class, and health status in patients with obstructive hypertrophic cardiomyopathy. The results of this pivotal trial highlight the benefits of disease-specific treatment for this condition. FUNDING: MyoKardia.
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Bencilaminas/uso terapéutico , Miosinas Cardíacas/antagonistas & inhibidores , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Uracilo/análogos & derivados , Antagonistas Adrenérgicos beta/uso terapéutico , Anciano , Bencilaminas/efectos adversos , Bloqueadores de los Canales de Calcio/uso terapéutico , Cardiomiopatía Hipertrófica/fisiopatología , Fármacos Cardiovasculares/uso terapéutico , Método Doble Ciego , Tolerancia al Ejercicio/fisiología , Femenino , Hemodinámica/fisiología , Humanos , Masculino , Persona de Mediana Edad , Consumo de Oxígeno/fisiología , Evaluación del Resultado de la Atención al Paciente , Uracilo/efectos adversos , Uracilo/uso terapéuticoRESUMEN
The coordination of individual cell behaviors is a crucial step in the assembly and morphogenesis of tissues. Xenopus mesendoderm cells migrate collectively along a fibronectin (FN) substrate at gastrulation, but how the adhesive and mechanical forces required for these movements are generated and transmitted is unclear. Traction force microscopy (TFM) was used to establish that traction stresses are limited primarily to leading edge cells in mesendoderm explants, and that these forces are balanced by intercellular stresses in follower rows. This is further reflected in the morphology of these cells, with broad lamellipodial protrusions, mature focal adhesions and a gradient of activated Rac1 evident at the leading edge, while small protrusions, rapid turnover of immature focal adhesions and lack of a Rac1 activity gradient characterize cells in following rows. Depletion of keratin (krt8) with antisense morpholinos results in high traction stresses in follower row cells, misdirected protrusions and the formation of actin stress fibers anchored in streak-like focal adhesions. We propose that maintenance of mechanical integrity in the mesendoderm by keratin intermediate filaments is required to balance stresses within the tissue to regulate collective cell movements.
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Gastrulación/fisiología , Queratinas/fisiología , Proteínas de Xenopus/fisiología , Xenopus/embriología , Xenopus/fisiología , Actinas/fisiología , Animales , Fenómenos Biomecánicos , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/metabolismo , Movimiento Celular/fisiología , Endodermo/citología , Endodermo/embriología , Endodermo/fisiología , Adhesiones Focales/fisiología , Técnicas de Silenciamiento del Gen , Filamentos Intermedios/fisiología , Queratina-8/antagonistas & inhibidores , Queratina-8/genética , Queratina-8/fisiología , Mesodermo/citología , Mesodermo/embriología , Mesodermo/fisiología , Modelos Biológicos , Morfogénesis/fisiología , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/metabolismo , Transducción de Señal , Estrés Mecánico , Xenopus/genética , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/genética , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C ß2 (PKCß2) at Ser660. Inhibiting (protein kinase C ß) PKCß blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCß2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection.
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Ácido Butírico/farmacología , Miosinas Cardíacas/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteína Quinasa C beta/metabolismo , Uniones Estrechas/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Western Blotting , Células CACO-2 , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Miosinas Cardíacas/antagonistas & inhibidores , Humanos , Inmunoprecipitación , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Uniones Estrechas/metabolismoRESUMEN
The role of myosin light chain II (MLCII) in cellular differentiation of rat mandibular condylar chondrocytes (MCCs) induced by cyclical uniaxial compressive stress (CUCS) remains unclear. In the current study, a fourpoint bending system was used to apply CUCS to primary cultured MCCs from rats. It was identified that CUCS stimulated features of cellular differentiation including morphological alterations, cytoskeleton rearrangement and overproduction of proteoglycans. Furthermore, CUCS promoted runtrelated transcription factor2 (RUNX2) expression at mRNA (P<0.01) and protein levels (P<0.05) and elevated alkaline phosphatase (ALP) activity (P<0.01), which are both markers of osteogenic differentiation. Under conditions of stress, western blotting indicated that the ratio of phosphorylated MLCII to total MLCII was increased significantly (P<0.05). Silencing MLCII by RNA interference reduced ALP activity (P<0.01), and eliminated RUNX2 mRNA expression (P<0.01). Addition of the MLC kinase inhibitor, ML7, reduced the CUCSassociated upregulation of RUNX2 expression (P<0.01) and ALP activity (P<0.01). The data indicated that CUCS promoted cellular differentiation of rat primary MCCs, and this was suggested to be via the phosphorylation of MLCII.
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Miosinas Cardíacas/genética , Condrocitos/citología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Cóndilo Mandibular/crecimiento & desarrollo , Cadenas Ligeras de Miosina/genética , Osteogénesis/genética , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Animales , Azepinas/administración & dosificación , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/biosíntesis , Diferenciación Celular/genética , Condrocitos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cóndilo Mandibular/citología , Cóndilo Mandibular/metabolismo , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/biosíntesis , Naftalenos/administración & dosificación , Fosforilación , Presión , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , RatasRESUMEN
Adropin is a peptide encoded by the energy homeostasis associated gene (Enho) and plays a critical role in the regulation of lipid metabolism, insulin sensitivity, and endothelial function. Little is known of the effects of adropin in the brain and whether this peptide modulates ischemia-induced blood-brain barrier (BBB) injury. Here, we used an in vitro BBB model of rat brain microvascular endothelial cells (RBE4) and hypothesized that adropin would reduce endothelial permeability during ischemic conditions. To mimic ischemic conditions in vitro, RBE4 cell monolayers were subjected to 16h hypoxia/low glucose (HLG). This resulted in a significant increase in paracellular permeability to FITC-labeled dextran (40kDa), a dramatic upregulation of vascular endothelial growth factor (VEGF), and the loss of junction proteins occludin and VE-cadherin. Notably, HLG also significantly decreased Enho expression and adropin levels. Treatment of RBE4 cells with synthetic adropin (1, 10 and 100ng/ml) concentration-dependently reduced endothelial permeability after HLG, but this was not mediated through protection to junction proteins or through reduced levels of VEGF. We found that HLG dramatically increased myosin light chain 2 (MLC2) phosphorylation in RBE4 cells, which was significantly reduced by adropin treatment. We also found that HLG significantly increased Rho-associated kinase (ROCK) activity, a critical upstream effector of MLC2 phosphorylation, and that adropin treatment attenuated that effect. These data indicate that treatment with adropin reduces endothelial cell permeability after HLG insult by inhibition of the ROCK-MLC2 signaling pathway. These promising findings suggest that adropin protects against endothelial barrier dysfunction during ischemic conditions.
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Barrera Hematoencefálica/efectos de los fármacos , Isquemia Encefálica/fisiopatología , Permeabilidad Capilar/efectos de los fármacos , Miosinas Cardíacas/antagonistas & inhibidores , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Quinasas Asociadas a rho/antagonistas & inhibidores , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Animales , Antígenos CD/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Barrera Hematoencefálica/metabolismo , Cadherinas/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Glucosa/metabolismo , Ocludina/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Ratas , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoAsunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Bencilaminas/administración & dosificación , Miosinas Cardíacas/antagonistas & inhibidores , Cardiomiopatía Hipertrófica Familiar/tratamiento farmacológico , Contracción Miocárdica/efectos de los fármacos , Cadenas Pesadas de Miosina/antagonistas & inhibidores , Sarcómeros/efectos de los fármacos , Uracilo/análogos & derivados , Animales , Humanos , MasculinoRESUMEN
Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Bencilaminas/administración & dosificación , Miosinas Cardíacas/antagonistas & inhibidores , Cardiomiopatía Hipertrófica Familiar/tratamiento farmacológico , Contracción Miocárdica/efectos de los fármacos , Cadenas Pesadas de Miosina/antagonistas & inhibidores , Sarcómeros/efectos de los fármacos , Uracilo/análogos & derivados , Animales , Bencilaminas/química , Miosinas Cardíacas/genética , Cardiomiopatía Hipertrófica Familiar/patología , Cardiomiopatía Hipertrófica Familiar/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/patología , Heterocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos , Mutación , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Cadenas Pesadas de Miosina/genética , Ratas , Uracilo/administración & dosificación , Uracilo/químicaRESUMEN
Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (Vil2(kd/kd)) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (Vil2(+/+)) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the Vil2(kd/kd) mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the Vil2(kd/kd) neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the Vil2(kd/kd) neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas de la Membrana/genética , Neurogénesis/fisiología , Neuronas/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Amidas/farmacología , Animales , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Proteínas del Citoesqueleto/metabolismo , Regulación hacia Abajo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/patología , Fosforilación , Transporte de Proteínas , Células Piramidales/fisiología , Piridinas/farmacología , Transducción de Señal , Proteína de Unión al GTP rhoA/antagonistas & inhibidoresRESUMEN
In development and differentiation, morphological changes often accompany mechanical changes [1], but it is unclear whether or when cells in embryos sense tissue elasticity. The earliest embryo is uniformly pliable, while adult tissues vary widely in mechanics from soft brain and stiff heart to rigid bone [2]. However, cell sensitivity to microenvironment elasticity is debated based in part on results from complex three-dimensional culture models [3]. Regenerative cardiology provides strong motivation to clarify any cell-level sensitivities to tissue elasticity because rigid postinfarct regions limit pumping by the adult heart [4]. Here, we focus on the spontaneously beating embryonic heart and sparsely cultured cardiomyocytes, including cells derived from pluripotent stem cells. Tissue elasticity, Et, increases daily for heart to 1-2 kPa by embryonic day 4 (E4), and although this is ~10-fold softer than adult heart, the beating contractions of E4 cardiomyocytes prove optimal at ~Et,E4 both in vivo and in vitro. Proteomics reveals daily increases in a small subset of proteins, namely collagen plus cardiac-specific excitation-contraction proteins. Rapid softening of the heart's matrix with collagenase or stiffening it with enzymatic crosslinking suppresses beating. Sparsely cultured E4 cardiomyocytes on collagen-coated gels likewise show maximal contraction on matrices with native E4 stiffness, highlighting cell-intrinsic mechanosensitivity. While an optimal elasticity for striation proves consistent with the mathematics of force-driven sarcomere registration, contraction wave speed is linear in Et as theorized for excitation-contraction coupled to matrix elasticity. Pluripotent stem cell-derived cardiomyocytes also prove to be mechanosensitive to matrix and thus generalize the main observation that myosin II organization and contractile function are optimally matched to the load contributed by matrix elasticity.
Asunto(s)
Proteínas de la Matriz Extracelular/biosíntesis , Frecuencia Cardíaca , Corazón/embriología , Contracción Miocárdica/fisiología , Miosinas/biosíntesis , Miosinas Cardíacas/antagonistas & inhibidores , Diferenciación Celular , Células Cultivadas , Colágeno/biosíntesis , Colagenasas/farmacología , Elasticidad , Células Madre Embrionarias/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/citología , Miofibrillas/fisiología , Sarcómeros/fisiologíaRESUMEN
The iron-regulated metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), is up-regulated by cellular iron depletion mediated by iron chelators and can inhibit cancer cell migration. However, the mechanism of how NDRG1 achieves this effect remains unclear. In this study, we implemented established and newly constructed NDRG1 overexpression and knockdown models using the DU145, HT29, and HCT116 cancer cell lines to investigate the molecular basis by which NDRG1 exerts its inhibitory effect on cell migration. Using these models, we demonstrated that NDRG1 overexpression inhibits cell migration by preventing actin-filament polymerization, stress fiber assembly and formation. In contrast, NDRG1 knockdown had the opposite effect. Moreover, we identified that NDRG1 inhibited an important regulatory pathway mediated by the Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)/phosphorylated myosin light chain 2 (pMLC2) pathway that modulates stress fiber assembly. The phosphorylation of MLC2 is a key process in inducing stress fiber contraction, and this was shown to be markedly decreased or increased by NDRG1 overexpression or knockdown, respectively. The mechanism involved in the inhibition of MLC2 phosphorylation by NDRG1 was mediated by a significant (P < 0.001) decrease in ROCK1 expression that is a key kinase involved in MLC2 phosphorylation. Considering that NDRG1 is up-regulated after cellular iron depletion, novel thiosemicarbazone iron chelators (e.g., di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone) were demonstrated to inhibit ROCK1/pMLC2-modulated actin-filament polymerization, stress fiber assembly, and formation via a mechanism involving NDRG1. These results highlight the role of the ROCK1/pMLC2 pathway in the NDRG1-mediated antimetastatic signaling network and the therapeutic potential of iron chelators at inhibiting metastasis.
Asunto(s)
Miosinas Cardíacas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quelantes del Hierro/farmacología , Cadenas Ligeras de Miosina/metabolismo , Fibras de Estrés/metabolismo , Quinasas Asociadas a rho/metabolismo , Actinas/genética , Actinas/metabolismo , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HT29 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Terapia Molecular Dirigida , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/genética , Metástasis de la Neoplasia , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tiosemicarbazonas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
We show that in melanoma cells oncogenic BRAF, acting through MEK and the transcription factor BRN2, downregulates the cGMP-specific phosphodiesterase PDE5A. Although PDE5A downregulation causes a small decrease in proliferation, its major impact is to stimulate a dramatic increase in melanoma cell invasion. This is because PDE5A downregulation leads to an increase in cGMP, which induces an increase in cytosolic Ca(2+), stimulating increased contractility and inducing invasion. PDE5A downregulation also this leads to an increase in short-term and long-term colonization of the lungs by melanoma cells. We do not observe this pathway in NRAS mutant melanoma or BRAF mutant colorectal cells. Thus, we show that in melanoma cells oncogenic BRAF induces invasion through downregulation of PDE5A.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/patología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Animales , Calcimicina/farmacología , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Proteínas de Homeodominio/metabolismo , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Melanoma/metabolismo , Ratones , Ratones Desnudos , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/metabolismo , Invasividad Neoplásica/patología , Invasividad Neoplásica/prevención & control , Factores del Dominio POU/metabolismo , Inhibidores de Fosfodiesterasa 5/farmacología , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Unión Proteica/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , ARN Interferente Pequeño/genética , Trasplante Heterólogo/patologíaRESUMEN
Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs.