RESUMEN
Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.
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Miofibrillas , Troponina T , Humanos , Miofibrillas/genética , Miofibrillas/patología , Troponina T/genética , Troponina T/química , Actinas/genética , Mutación , Citoesqueleto de Actina/genética , Miosinas , CalcioRESUMEN
KBTBD13 variants cause nemaline myopathy type 6 (NEM6). The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13 p.R408C). Although KBTBD13 is expressed in cardiac muscle, cardiac involvement in NEM6 is unknown. Here, we constructed pedigrees of three families with the KBTBD13 p.R408C variant. In 65 evaluated patients, 12% presented with left ventricle dilatation, 29% with left ventricular ejection fraction< 50%, 8% with atrial fibrillation, 9% with ventricular tachycardia, and 20% with repolarization abnormalities. Five patients received an implantable cardioverter defibrillator, three cases of sudden cardiac death were reported. Linkage analysis confirmed cosegregation of the KBTBD13 p.R408C variant with the cardiac phenotype. Mouse studies revealed that (1) mice harboring the Kbtbd13 p.R408C variant display mild diastolic dysfunction; (2) Kbtbd13-deficient mice have systolic dysfunction. Hence, (1) KBTBD13 is associated with cardiac dysfunction and cardiomyopathy; (2) KBTBD13 should be added to the cardiomyopathy gene panel; (3) NEM6 patients should be referred to the cardiologist.
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Cardiomiopatías , Proteínas Musculares , Animales , Humanos , Ratones , Arritmias Cardíacas , Cardiomiopatías/genética , Muerte Súbita Cardíaca/etiología , Desfibriladores Implantables , Proteínas Musculares/genética , Volumen Sistólico/fisiología , Función Ventricular IzquierdaRESUMEN
Right-sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline-induced pulmonary hypertensive (MCT-PH) rats. The hypothesis tested was that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular muscle preparations were subjected to 5 Hz sinusoidal length changes at 37°C. Work and suprabasal oxygen consumption ( V Ì O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ ) were measured before and after cross-bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross-sectional area, proton permeability of the inner mitochondrial membrane and monoamine oxidase and glucose 6-phosphate dehydrogenase activities and phosphatidylglycerol/cardiolipin contents were determined. Mechanical efficiency ranged from 23% to 11% in control (n = 6) and from 22% to 1% in MCT-PH (n = 15) and correlated with work (r2 = 0.68, P < 0.0001) but not with V Ì O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ (r2 = 0.004, P = 0.7919). V Ì O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ for cross-bridge cycling was proportional to work (r2 = 0.56, P = 0.0005). Blebbistatin-resistant V Ì O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ (r2 = 0.32, P = 0.0167) and proton permeability of the mitochondrial inner membrane (r2 = 0.36, P = 0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination r2 = 0.79, P = 0.0001). Cytosolic cytochrome c correlated inversely with work (r2 = 0.28, P = 0.0391), but not with efficiency (r2 = 0.20, P = 0.0867). Glucose 6-phosphate dehydrogenase, monoamine oxidase and phosphatidylglycerol/cardiolipin increased in the right ventricular wall of MCT-PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT-PH is a result of activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin-resistant V Ì O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ are discussed. KEY POINTS: Mechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown. Work and oxygen consumption are determined during work loops. Oxygen consumption for activation and cross-bridge cycling confirm the previous heat measurements. Cytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension. Reduced work and mechanical efficiency are related to mitochondrial dysfunction. Upregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is a resiult of the excessive production of reactive oxygen species.
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Hipertensión Pulmonar , Animales , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Glucosa/metabolismo , Monoaminooxidasa/efectos adversos , Monoaminooxidasa/metabolismo , Monocrotalina/efectos adversos , Monocrotalina/metabolismo , Oxidorreductasas/metabolismo , Consumo de Oxígeno/fisiología , Músculos Papilares , Fosfatos/metabolismo , Protones , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. METHODS: To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. RESULTS: Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. INTERPRETATION: Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.
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Actinas/genética , Contracción Muscular/fisiología , Debilidad Muscular/genética , Miopatías Estructurales Congénitas/fisiopatología , Sarcómeros/patología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Debilidad Muscular/fisiopatología , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/genética , Sarcómeros/fisiología , Adulto JovenRESUMEN
RATIONALE: Diaphragm weakness in critically ill patients prolongs ventilator dependency and duration of hospital stay and increases mortality and healthcare costs. The mechanisms underlying diaphragm weakness include cross-sectional fiber atrophy and contractile protein dysfunction, but whether additional mechanisms are at play is unknown. OBJECTIVES: To test the hypothesis that mechanical ventilation with positive end-expiratory pressure (PEEP) induces longitudinal atrophy by displacing the diaphragm in the caudal direction and reducing the length of fibers. METHODS: We studied structure and function of diaphragm fibers of mechanically ventilated critically ill patients and mechanically ventilated rats with normal and increased titin compliance. MEASUREMENTS AND MAIN RESULTS: PEEP causes a caudal movement of the diaphragm, both in critically ill patients and in rats, and this caudal movement reduces fiber length. Diaphragm fibers of 18-hour mechanically ventilated rats (PEEP of 2.5 cm H2O) adapt to the reduced length by absorbing serially linked sarcomeres, the smallest contractile units in muscle (i.e., longitudinal atrophy). Increasing the compliance of titin molecules reduces longitudinal atrophy. CONCLUSIONS: Mechanical ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response that is modulated by the elasticity of the giant sarcomeric protein titin. We postulate that longitudinal atrophy, in concert with the aforementioned cross-sectional atrophy, hampers spontaneous breathing trials in critically ill patients: during these efforts, end-expiratory lung volume is reduced, and the shortened diaphragm fibers are stretched to excessive sarcomere lengths. At these lengths, muscle fibers generate less force, and diaphragm weakness ensues.
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Diafragma/patología , Atrofia Muscular/etiología , Atrofia Muscular/patología , Respiración con Presión Positiva/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biopsia , Diafragma/diagnóstico por imagen , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Atrofia Muscular/diagnóstico por imagen , Ratas , UltrasonografíaRESUMEN
Although hemodilution is attributed as the main cause of microcirculatory impairment during cardiopulmonary bypass (CPB), this relationship has never been investigated. We investigated the distinct effects of hemodilution with or without CPB on microvascular perfusion and subsequent renal tissue injury in a rat model. Male Wistar rats (375-425 g) were anesthetized, prepared for cremaster muscle intravital microscopy, and subjected to CPB (n = 9), hemodilution alone (n = 9), or a sham procedure (n = 6). Microcirculatory recordings were performed at multiple time points and analyzed for perfusion characteristics. Kidney and lung tissue were investigated for mRNA expression for genes regulating inflammation and endothelial adhesion molecule expression. Renal injury was assessed with immunohistochemistry. Hematocrit levels dropped to 0.24 ± 0.03 l/l and 0.22 ± 0.02 l/l after onset of hemodilution with or without CPB. Microcirculatory perfusion remained unaltered in sham rats. Hemodilution alone induced a 13% decrease in perfused capillaries, after which recovery was observed. Onset of CPB reduced the perfused capillaries by 40% (9.2 ± 0.9 to 5.5 ± 1.5 perfused capillaries per microscope field; P < 0.001), and this reduction persisted throughout the experiment. Endothelial and inflammatory activation and renal histological injury were increased after CPB compared with hemodilution or sham procedure. Hemodilution leads to minor and transient disturbances in microcirculatory perfusion, which cannot fully explain impaired microcirculation following cardiopulmonary bypass. CPB led to increased renal injury and endothelial adhesion molecule expression in the kidney and lung compared with hemodilution. Our findings suggest that microcirculatory impairment during CPB may play a role in the development of kidney injury.
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Lesión Renal Aguda/etiología , Lesión Pulmonar Aguda/etiología , Capilares/fisiopatología , Puente Cardiopulmonar/efectos adversos , Hemodilución/efectos adversos , Riñón/irrigación sanguínea , Microcirculación , Lesión Renal Aguda/genética , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Microscopía Intravital , Riñón/metabolismo , Riñón/patología , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Masculino , Modelos Animales , Ratas Wistar , Factores de TiempoRESUMEN
The efficiency (work/oxygen consumption) of isolated papillary muscles from failing hearts is reduced. We investigated whether this can be due to an increase of intrinsic cardiac adrenergic (ICA) cell density. The number of ICA cells in the septum and both ventricular walls was determined by tyrosine hydroxylase immunohistochemistry in rats with monocrotaline-induced pulmonary hypertension. We found that the number of ICA cells is about 200,000 per rat heart. ICA cell density was significantly lower in right ventricular myocardium of hypertrophied hearts (P < 0.01). MAO-A enzyme histochemistry and inhibition experiments with clorgyline in papillary muscles were performed to localize the enzyme and to determine its oxygen consumption. Upregulation of MAO-A was found in the right ventricular wall and papillary muscles of failing hearts (P = 0.018). A positive correlation between ICA cell density and MAO-A activity was absent. Clorgyline (2 µM) decreased the basal rate of oxygen consumption of right ventricular papillary muscles by 65 µM O(2)/s (P = 0.027). This rate can only be maintained for several seconds judging from the catecholamine content of the preparations reported previously. High ICA cell activity rather than density and/or recycling of oxidized catecholamines are discussed as alternative explanations for the low myocardial efficiency in experimental pulmonary hypertension.
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Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/patología , Monoaminooxidasa/metabolismo , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Animales , Recuento de Células , Clorgilina/farmacología , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/metabolismo , Masculino , Inhibidores de la Monoaminooxidasa/farmacología , Norepinefrina/metabolismo , Consumo de Oxígeno , Ratas , Ratas WistarRESUMEN
Because of their ability to eliminate pathogens and to modulate various host immune responses, antimicrobial peptides are considered as candidate agents to fight infections by (antibiotic-resistant) pathogens. We recently reported that hLF1-11 (GRRRRSVQWCA), an antimicrobial peptide derived from the N terminus of human lactoferrin, displays diverse modulatory activities on monocytes, thereby enhancing their actions in innate immune responses. The aim of this study was to identify the cellular target of hLF1-11 that mediates these effects. Results revealed that hLF1-11 binds and subsequently penetrates human monocytes, after which it inhibits the enzymatic activities of myeloperoxidase (MPO). Moreover, a chemical inhibitor of MPO (aminobenzoic acid hydrazide) mimicked the effects of hLF1-11 on the inflammatory response by monocytes and on monocyte-macrophage differentiation. Computer-assisted molecular modeling predicted that hLF1-11 can bind to the edge of and within the crevice of the active site of MPO. Experiments with a set of hLF1-11 peptides with amino acid substitutions identified the stretch of arginines and the cysteine at position 10 as pivotal in these immunomodulatory properties of hLF1-11. We conclude that hLF1-11 may exert its modulatory effects on human monocytes by specific inhibition of MPO activity.
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Factores Inmunológicos/fisiología , Lactoferrina/fisiología , Peroxidasa/antagonistas & inhibidores , Peroxidasa/metabolismo , Antibacterianos , Células Cultivadas , Humanos , Monocitos/enzimología , Monocitos/inmunologíaRESUMEN
Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.
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Diafragma , Contracción Muscular , Miosinas , Respiración Artificial , Músculos Respiratorios , Humanos , Animales , Miosinas/metabolismo , Diafragma/metabolismo , Diafragma/fisiopatología , Músculos Respiratorios/metabolismo , Ratas , Masculino , Unidades de Cuidados Intensivos , Persona de Mediana Edad , Femenino , Anciano , Hibernación/fisiología , Actinas/metabolismoRESUMEN
Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.
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Repetición de Anquirina , Conectina/metabolismo , Sarcómeros , Animales , Conectina/genética , Humanos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares , Ratas , Proteínas Represoras , Sarcómeros/metabolismoRESUMEN
INTRODUCTION: The diaphragm is the main muscle of inspiration, and its dysfunction contributes to adverse clinical outcomes in critically ill patients. We recently reported the infiltration of SARS-CoV-2, and the development of fibrosis, in the diaphragm of critically ill patients with COVID-19. In the current study, we aimed to characterise myofiber structure in the diaphragm of critically ill patients with COVID-19. METHODS: Diaphragm muscle specimens were collected during autopsy from patients who died of COVID-19 in three academic medical centres in the Netherlands in April and May 2020 (n=27). We studied diaphragm myofiber gene expression and structure and compared the findings obtained to those of deceased critically ill patients without COVID-19 (n=10). RESULTS: Myofibers of critically ill patients with COVID-19 showed on average larger cross-sectional area (slow-twitch myofibers: 2441±229 vs 1571±309 µm2; fast-twitch myofibers: 1966±209 vs 1225±222 µm2). Four critically ill patients with COVID-19 showed extremely large myofibers, which were splitting and contained many centralised nuclei. RNA-sequencing data revealed differentially expressed genes involved in muscle regeneration. CONCLUSION: Diaphragm of critically ill patients with COVID-19 has distinct myopathic features compared with critically ill patients without COVID-19, which may contribute to the ongoing dyspnoea and fatigue in the patients surviving COVID-19 infection.
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COVID-19 , Enfermedad Crítica , Diafragma/patología , Anciano , Autopsia , COVID-19/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/patología , Países BajosRESUMEN
The human lactoferrin-derived peptide hLF1-11 displays antimicrobial activities in vitro and is effective against infections with antibiotic-resistant bacteria and fluconazole-resistant Candida albicans in animals. However, the mechanisms underlying these activities remain largely unclear. Since hLF1-11 is ineffective in vitro at physiological salt concentrations, we suggested modulation of the immune system as an additional mechanism of action of the peptide. We investigated whether hLF1-11 affects human monocyte-macrophage differentiation and determined the antimicrobial activities of the resulting macrophages. Monocytes were cultured for 7 days with GM-CSF in the presence of hLF1-11, control peptide, or saline for various intervals. At day 6, the cells were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or heat-killed C. albicans for 24 h. Thereafter, the levels of cytokines in the culture supernatants, the expression of pathogen recognition receptors, and the antimicrobial activities of these macrophages were determined. The results showed that a short exposure of monocytes to hLF1-11 during GM-CSF-driven differentiation is sufficient to direct differentiation of monocytes toward a macrophage subset characterized by both pro- and anti-inflammatory cytokine production and increased responsiveness to microbial structures. Moreover, these macrophages are highly effective against C. albicans and Staphylococcus aureus. In conclusion, hLF1-11 directs GM-CSF-driven differentiation of monocytes toward macrophages with enhanced effector functions.
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Antiinfecciosos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Macrófagos/citología , Macrófagos/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Fragmentos de Péptidos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/inmunología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Humanos , Lactoferrina , Macrófagos/efectos de los fármacos , Monocitos/citología , Fagocitosis/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/inmunologíaRESUMEN
Earlier we reported that the peptide corresponding to the first eleven N-terminal amino acids of human lactoferrin (hLF1-11) is active against multi-drug resistant pathogens in mice. The mechanisms underlying this anti-infective activity remain unclear. Since hLF1-11 is ineffective against pathogens at physiological salt concentrations and hLF1-11 directs differentiation of monocytes toward a macrophage subset with enhanced effector functions, we investigated the effects of hLF1-11 on human and murine monocytes. Results revealed that human and murine monocytes exposed for 1 h to hLF1-11 and then stimulated with the Toll-like receptor (TLR)-ligand LPS for 18 h, displayed enhanced cytokine and chemokine production as compared to control (peptide-treated) monocytes. We also found that expression of mRNA, cell-surface receptor expression, and NF-kappaB activation by hLF1-11-exposed human monocytes were enhanced as compared to control (peptide-treated) monocytes. Furthermore, the kinetics of the cytokine production was unchanged as mRNA levels and protein levels paralleled the enhanced response of hLF1-11-exposed monocytes to LPS. The cytokine production by human monocytes in response to TLR4, TLR5, and TLR7 stimulation, but not to TLR2 stimulation, was elevated by hLF1-11. In concordance, translocation of NF-kappaB subunits to the nucleus was enhanced in hLF1-11-exposed monocytes after TLR stimulation, except for TLR2, as compared to control (peptide-exposed) monocytes. In conclusion, monocytes were primed by hLF1-11 for an enhanced inflammatory response upon TLR4, TLR5, and TLR7 stimulation, but not TLR2 stimulation. Such effects of hLF1-11 on monocyte reactivity should be taken into account when considering the clinical development of this peptide for a therapeutic intervention in patients.
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Lactoferrina/inmunología , Monocitos/inmunología , Fragmentos de Péptidos/inmunología , Receptores Toll-Like/inmunología , Animales , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Femenino , Humanos , Cinética , Lactoferrina/química , Lactoferrina/farmacología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Ratones , Monocitos/efectos de los fármacos , FN-kappa B/inmunología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Receptores de Superficie Celular/biosíntesis , Relación Estructura-ActividadRESUMEN
The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.
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Proteínas Musculares/metabolismo , Relajación Muscular , Miopatías Nemalínicas/metabolismo , Sarcómeros/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Humanos , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/patología , Sarcómeros/patología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The type 2 iodothyronine-deiodinase (D2) enzyme converts T4 to T3, and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T3 derived from T4 in skeletal muscle despite normal circulating T3 levels. Because slow skeletal muscle is particularly susceptible to changes in T3 levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T3-dependent regulator of slow muscle fiber type, was decreased by â¼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes.
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Yoduro Peroxidasa/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Mioblastos/metabolismo , Animales , Línea Celular , Expresión Génica , Yoduro Peroxidasa/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Muscular/genética , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Mioblastos/citología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Tiroxina/metabolismo , Triyodotironina/metabolismo , Yodotironina Deyodinasa Tipo IIRESUMEN
The presence and antimicrobial activity of antimicrobial peptides (AMPs) has been widely recognized as an evolutionary preserved part of the innate immune system. Based on evidence in animal models and humans, AMPs are now positioned as novel anti-infective agents. The current study aimed to evaluate the potential antimicrobial activity of ubiquicidin and small synthetic fragments thereof towards methicillin resistant Staphylococcus aureus (MRSA), as a high priority target for novel antibiotics. In vitro killing of MRSA by synthetic peptides derived from the alpha-helix or beta-sheet domains of the human cationic peptide ubiquicidin (UBI 1-59), allowed selection of AMPs for possible treatment of MRSA infections. The strongest antibacterial activity was observed for the entire peptide UBI 1-59 and for synthetic fragments comprising amino acids 31-38. The availability, chemical synthesis opportunities, and size of these small peptides, combined with their strong antimicrobial activity towards MRSA make these compounds promising candidates for antimicrobial therapy and detection of infections in man.
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Antiinfecciosos/química , Farmacorresistencia Bacteriana Múltiple , Péptidos/farmacología , Proteínas Ribosómicas/química , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antiinfecciosos/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Datos de Secuencia Molecular , Péptidos/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/farmacología , TecnecioRESUMEN
MicroRNAs are involved in many pathologic processes and are a promising target for therapeutic intervention. However, successful, localized delivery of microRNA-based therapeutics is lacking. In this study, cationic ultrasound-responsive microbubbles (MBs) were used to deliver microRNA blockers and mimics in vitro and in vivo. Cationic MBs successfully delivered microRNA blockers to human endothelial cells on ultrasound (US) exposure in vitro. This in vitro US protocol did not successfully deliver microRNA mimics to skeletal muscle of mice, whereas an US protocol that is routinely used for contrast imaging did. Additionally, we used cationic MBs and US to locally deliver antimiR and antagomiR molecules with US causing inertial cavitation. Delivery of antimiR to the extracellular compartments of the muscle was only slightly increased, whereas delivery of antagomiR to the capillaries, myocytes and extracellular space was significantly increased. AntagomiR seems to be a more suitable microRNA blocker than antimiR for use in combination with MBs and US for local delivery.
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Células Endoteliales/fisiología , MicroARNs/genética , Microburbujas , Músculo Esquelético/fisiología , Fonoforesis/métodos , Transfección/métodos , Animales , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/administración & dosificación , Sonicación/métodosRESUMEN
The hLF1-11 peptide comprising the first 11 N-terminal residues of human lactoferrin exerts antimicrobial activity in vivo, enhances the inflammatory response of monocytes and directs monocyte-macrophage differentiation toward cells with enhanced antimicrobial properties. In this study, we investigated the effects of hLF1-11 on human monocyte-dendritic cell (DC) differentiation and subsequent T cell activation. Results revealed that - compared to control (peptide-incubated) DCs - hLF1-11-differentiated DCs displayed enhanced expression of HLA class II antigens and dectin-1, and increased phagocytosis of Candida albicans. In addition, hLF1-11-differentiated DCs produced enhanced amounts of reactive oxygen species, IL-6 and IL-10, but not IL-12p40 and TNF-α, upon stimulation with C. albicans. Moreover, 6-day-cultured hLF1-11-differentiated DCs and control (peptide-incubated) DCs that had been stimulated with a Th17-inducing mix of antigens (including C. albicans) for 24 h were cocultured with autologous CD4+ T cells for 72 h and then the levels of IL-10, IL-17 and IFN-γ production and the percentage of cytokine-producing T cells were assessed. The results revealed that the hLF1-11-differentiated DCs induced an enhanced IL-17, but reduced IFN-γ, production by T cells as compared to control (peptide-incubated) DCs. Collectively, the hLF1-11 peptide drives monocyte-DC differentiation toward DCs that promote antifungal responses and enhance Th17 polarization.