RESUMEN
BACKGROUND: A buried penis (BP) is rare in which the penile body is retracted into the prepubic adipose tissue. This research focuses on differences in smooth muscle myosin heavy chain (SMMHC) isoform expressions in the dartos fascia. METHODS: A total of 82 children, 41 of whom had BPs, who applied for circumcision between May and November 2021, were included in the study. The cases were divided into four groups aged ≥6 years (NP6, n = 18) and aged ≤3 years (NP3, n = 17) with normal penile appearance, aged ≥6 years (BP6, n = 23) and aged ≤3 years (BP,n = 24) with a BP. SMMHC isoforms mRNA gene expression analyses were performed by quantitative PCR technique in dartos fascia obtained from foreskin removed by circumcision. RESULTS: Compared to the NP3 group, the SM1 mRNA expressed in the BP6 group was statistically significantly higher (p < 0.005). SM2 mRNA levels expressed in dartos fascia were considerably higher in NP6 and NP3 groups compared to BP6 and BP3 groups (p < 0.001). The SM2/SM1 ratio was 0.85 in the BP6 group and 1.46 in the NP6 group, which was statistically significant (p = 0.006) and increased from 0.87 in the BP3 group to 2.21 in the NP3 group (p < 0.001). CONCLUSION: In a buried penis, there is a difference in the expression of SMMHC isoforms. SM1 is highly expressed, while SM2 decreases, increasing the SM2/SM1 ratio. This causes increased contractility in the smooth muscle, leading to retraction of the penile body. The dartos fascia surrounding it resembles aberrant muscle tissue in boys with a BP. LEVEL OF EVIDENCE: Level III. TYPE OF STUDY: Case-control study.
Asunto(s)
Cadenas Pesadas de Miosina , Pene , Isoformas de Proteínas , Humanos , Masculino , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Niño , Preescolar , Isoformas de Proteínas/genética , Pene/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/análisis , Lactante , Circuncisión Masculina , Enfermedades del Pene/metabolismo , Enfermedades del Pene/genética , Miosinas del Músculo Liso/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/análisisRESUMEN
BACKGROUND: Stent implantation-induced neointima formation is a dominant culprit in coronary artery disease treatment failure after percutaneous coronary intervention. Ferroptosis, an iron-dependent regulated cell death, has been associated with various cardiovascular diseases. However, the effect of ferroptosis on neointima formation remains unclear. METHODS: The mouse common right carotid arteries were ligated for 16 or 30 days, and ligated tissues were collected for further analyses. Primary rat vascular smooth muscle cells (VSMCs) were isolated from the media of aortas of Sprague-Dawley (SD) rats and used for in vitro cell culture experiments. RESULTS: Ferroptosis was positively associated with neointima formation. In vivo, RAS-selective lethal 3 (RSL3), a ferroptosis activator, aggravated carotid artery ligation-induced neointima formation and promoted VSMC phenotypic conversion. In contrast, a ferroptosis inhibitor, ferrostatin-1 (Fer-1), showed the opposite effects in mice. In vitro, RSL3 promoted rat VSMC phenotypic switching from a contractile to a synthetic phenotype, evidenced by increased contractile markers (smooth muscle myosin heavy chain and calponin 1), and decreased synthetic marker osteopontin. The induction of ferroptosis by RSL3 was confirmed by the increased expression level of ferroptosis-associated gene prostaglandin-endoperoxide synthase 2 (Ptgs2). The effect of RSL3 on rat VSMC phenotypic switching was abolished by Fer-1. Moreover, N-acetyl-L-cysteine (NAC), the reactive oxygen species inhibitor, counteracted the effect of RSL3 on the phenotypic conversion of rat VSMCs. CONCLUSIONS: Ferroptosis induces VSMC phenotypic switching and accelerates ligation-induced neointimal hyperplasia in mice. Our findings suggest inhibition of ferroptosis as an attractive strategy for limiting vascular restenosis.
Asunto(s)
Ferroptosis , Neointima , Acetilcisteína/farmacología , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Ciclooxigenasa 2/farmacología , Modelos Animales de Enfermedad , Hiperplasia/metabolismo , Hierro/metabolismo , Hierro/farmacología , Ratones , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Osteopontina/metabolismo , Osteopontina/farmacología , Fenotipo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Miosinas del Músculo Liso/metabolismoRESUMEN
Smooth muscle (SM) is found in most hollow organs of the body. Phasic SM, as found in the gut, contracts to propel content, whereas tonic SM, as found in most blood vessels, maintains tension. This force maintenance is referred to as the latch state and occurs at low levels of myosin activation (myosin light chain [LC20] phosphorylation). Molecular mechanisms have been proposed to explain the latch state but have been studied only at the whole-muscle level because of technological limitations. In the current study, an assay chamber was devised to allow injection of myosin light chain phosphatase (MLCP) during laser trap and in vitro motility assays, without creating bulk flow, to reproduce latch state conditions at the molecular level. Using the laser trap in a single-beam mode, an actin filament was brought in contact with several myosin molecules on a pedestal. Myosin pulled on the actin filament until a plateau force was reached, at which point, MLCP was injected. Force maintenance was observed during LC20 dephosphorylation, the level of which was assessed in a parallel in vitro motility assay performed in the same conditions. Force was maintained longer for myosin purified from tonic SM than from phasic SM. These data support the longstanding dogma of strong bonds caused by dephosphorylated, noncycling cross-bridges. Furthermore, MLCP injection in an in vitro motility mixture assay performed with SM and skeletal muscle myosin suggests that the maintenance of these strong bonds is possible only if no energy is provided by surrounding actively cycling myosin molecules.
Asunto(s)
Músculo Liso , Miosinas del Músculo Liso , Contracción Muscular , Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , Miosinas del Músculo Liso/metabolismoRESUMEN
Airway smooth muscle cell migration plays an essential role in airway development, repair, and remodeling. Smooth muscle myosin II has been traditionally thought to localize in the cytoplasm solely and regulates cell migration by affecting stress fiber formation and focal adhesion assembly. In this study, we unexpectedly found that 20-kDa myosin light chain (MLC20) and myosin-11 (MYH11), important components of smooth muscle myosin, were present at the edge of lamellipodia. The knockdown of MLC20 or MYH11 attenuated the recruitment of c-Abl, cortactinProfilin-1 (Pfn-1), and Abi1 to the cell edge. Moreover, myosin light chain kinase (MLCK) colocalized with integrin ß1 at the tip of protrusion. The inhibition of MLCK attenuated the recruitment of c-Abl, cortactin, Pfn-1, and Abi1 to the cell edge. Furthermore, MLCK localization at the leading edge was reduced by integrin ß1 knockdown. Taken together, our results demonstrate that smooth muscle myosin localizes at the leading edge and orchestrates the recruitment of actin-regulatory proteins to the tip of lamellipodia. Mechanistically, integrin ß1 recruits MLCK to the leading edge, which catalyzes MLC20 phosphorylation. Activated myosin regulates the recruitment of actin-regulatory proteins to the leading edge, and promotes lamellipodial formation and migration.
Asunto(s)
Actinas , Miosinas del Músculo Liso , Actinas/metabolismo , Integrina beta1/metabolismo , Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Miosinas del Músculo Liso/metabolismoRESUMEN
OBJECTIVES: To observe the changes in the bladder of fetal rats with myelomeningocele (MMC) induced by all-trans retinoic acid (atRA) during the embryonic development stages. METHODS: The fetal rat model of MMC was induced by intragastric administration of atRA to pregnant rats on embryonic day 10 (E10). Fetal rats were harvested at E16, E18, E20, and E21 for observation and further testing. Those with MMC were classified as the MMC group, while those without MMC as the RA group. The areas of MMC skin defect, the crown-rump length (CRL), and body weight in different groups were compared. The histopathological changes in the bladder were compared. The expression levels of alpha-smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMMHC), connexin 43 (Cx43), desmin, ß3 tubulin, and vesicular acetylcholine transporter (VAChT) in the bladder were investigated by immunohistochemical staining and Western blotting. Pregnant rats given intragastric administration with olive oil (OIL group) at E10 were set as the blank control group. RESULTS: A total of 415 fetal rats of different gestational ages were harvested with an MMC incidence of 56.05 % (139/248). The incidence rate increased with embryonic days (p < 0.001). Compared with the other two control groups, the CRL and bodyweight of MMC fetal rats were significantly delayed at E21 (p < 0.001). The expression levels of αSMA, SMMHC, Cx43, desmin, ß3 tubulin and VAChT in the bladder of MMC fetal rats were significantly decreased at E21 (p < 0.05). CONCLUSIONS: In atRA-induced MMC fetal rats, there is neural, muscular, and stromal dysplasia in the bladder at an early gestational age. Further investigations on neurogenic bladder secondary to MMC are applicable using this animal model.
Asunto(s)
Meningomielocele , Actinas/metabolismo , Animales , Conexina 43/metabolismo , Desmina/metabolismo , Femenino , Meningomielocele/inducido químicamente , Meningomielocele/metabolismo , Aceite de Oliva , Embarazo , Ratas , Miosinas del Músculo Liso/metabolismo , Tretinoina , Tubulina (Proteína) , Vejiga Urinaria , Proteínas de Transporte Vesicular de AcetilcolinaRESUMEN
The contractile apparatus of smooth muscle is malleable to accommodate stress and strain exerted on the muscle cell and to maintain optimal contractility. Structural lability of smooth muscle myosin filaments is believed to play an important role in the cell's malleability. However, the mechanism and regulation of myosin filament formation is still poorly understood. In the present in vitro study, using a static light scattering method, length distributions were obtained from suspensions of short myosin filaments (SFs) formed by rapid dilution or long ones (LFs) formed by slow dialysis. The distributions indicated the presence of dynamic equilibriums between soluble myosin and the SFs; i.e.: trimers, hexamers and mini filaments, covering the range up to 0.75 µm. The LFs were more stable, exhibiting favorable sizes at about 1.25, 2.4 and 4.5 µm. More distinct distributions were obtained from filaments adsorbed to a glass surface, by evanescent wave scattering and local electric field enhancement. Addition of telokin (TL) to the suspensions of unphosphorylated SFs resulted in widening of the soluble range, while in the case of the LFs this shift was larger, and accompanied by reduced contribution of the soluble myosin species. Such changes were largely absent in the case of phosphorylated myosin. In contrast, the presence of Mg·ATP resulted in elongation of the filaments and clear separation of filaments from soluble myosin species. Thus, TL and Mg·ATP appeared to modify the distribution of myosin filament lengths, i.e., increasing the lengths in preparing for phosphorylation, or reducing it to aid dephosphorylation.
Asunto(s)
Músculo Liso , Miosinas del Músculo Liso , Adenosina Trifosfato/metabolismo , Músculo Liso/metabolismo , Quinasa de Cadena Ligera de Miosina , Miosinas/metabolismo , Fragmentos de Péptidos , Fosforilación , Miosinas del Músculo Liso/metabolismo , SuspensionesRESUMEN
AIMS: During atherosclerosis, smooth muscle cells (SMCs) accumulate in the intima where they switch from a contractile to a synthetic phenotype. From porcine coronary artery, we isolated spindle-shaped (S) SMCs exhibiting features of the contractile phenotype and rhomboid (R) SMCs typical of the synthetic phenotype. S100A4 was identified as a marker of R-SMCs in vitro and intimal SMCs, in pig and man. S100A4 exhibits intra- and extracellular functions. In this study, we investigated the role of extracellular S100A4 in SMC phenotypic transition. METHODS AND RESULTS: S-SMCs were treated with oligomeric recombinant S100A4 (oS100A4), which induced nuclear factor (NF)-κB activation. Treatment of S-SMCs with oS100A4 in combination with platelet-derived growth factor (PDGF)-BB induced a complete SMC transition towards a pro-inflammatory R-phenotype associated with NF-κB activation, through toll-like receptor-4. RNA sequencing of cells treated with oS100A4/PDGF-BB revealed a strong up-regulation of pro-inflammatory genes and enrichment of transcription factor binding sites essential for SMC phenotypic transition. In a mouse model of established atherosclerosis, neutralization of extracellular S100A4 decreased area of atherosclerotic lesions, necrotic core, and CD68 expression and increased α-smooth muscle actin and smooth muscle myosin heavy chain expression. CONCLUSION: We suggest that the neutralization of extracellular S100A4 promotes the stabilization of atherosclerotic plaques. Extracellular S100A4 could be a new target to influence the evolution of atherosclerotic plaques.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Enfermedades de la Aorta/tratamiento farmacológico , Aterosclerosis/tratamiento farmacológico , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Placa Aterosclerótica , Proteína de Unión al Calcio S100A4/antagonistas & inhibidores , Actinas/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Becaplermina/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Proteína de Unión al Calcio S100A4/metabolismo , Proteína de Unión al Calcio S100A4/farmacología , Transducción de Señal , Miosinas del Músculo Liso/metabolismo , Sus scrofa , Receptor Toll-Like 4/metabolismoRESUMEN
Children with Hutchinson-Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.
Asunto(s)
Regulación de la Expresión Génica , Contracción Muscular/fisiología , Músculo Liso Vascular/fisiopatología , Cadenas Pesadas de Miosina/genética , Progeria/genética , Progeria/fisiopatología , Miosinas del Músculo Liso/genética , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/patología , Animales , Aorta/patología , Aorta/fisiopatología , Humanos , Ratones Endogámicos C57BL , Persona de Mediana Edad , Cadenas Pesadas de Miosina/metabolismo , Miosinas del Músculo Liso/metabolismoRESUMEN
Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and ß-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to ß-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or ß-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and ß-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.
Asunto(s)
Actinina/genética , Mecanotransducción Celular , Músculo Liso/metabolismo , Paxillin/genética , Proteínas Proto-Oncogénicas c-akt/genética , Tráquea/metabolismo , Acetilcolina/farmacología , Actinina/metabolismo , Animales , Quimiocina CCL11/genética , Quimiocina CCL11/metabolismo , Perros , Femenino , Regulación de la Expresión Génica , Interleucina-4/genética , Interleucina-4/metabolismo , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/genética , Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Paxillin/metabolismo , Fenotipo , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismo , Tráquea/efectos de los fármacosRESUMEN
Myosin II is a motor protein with two heads and an extended tail that plays an essential role in cell motility. Its active form is a polymer (myosin filament) that pulls on actin to generate motion. Its inactive form is a monomer with a compact structure (10S sedimentation coefficient), in which the tail is folded and the two heads interact with each other, inhibiting activity. This conformation is thought to function in cells as an energy-conserving form of the molecule suitable for storage as well as transport to sites of filament assembly. The mechanism of inhibition of the compact molecule is not fully understood. We have performed a 3-D reconstruction of negatively stained 10S myosin from smooth muscle in the inhibited state using single-particle analysis. The reconstruction reveals multiple interactions between the tail and the two heads that appear to trap ATP hydrolysis products, block actin binding, hinder head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function could explain the high degree of inhibition of the folded form of myosin thought to underlie its energy-conserving function in cells. The reconstruction also suggests a mechanism for unfolding when myosin is activated by phosphorylation.
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Músculo Liso/metabolismo , Miosinas del Músculo Liso/química , Miosinas del Músculo Liso/metabolismo , Animales , Microscopía Electrónica , Unión Proteica , Conformación Proteica , Pliegue de Proteína , PavosRESUMEN
Cytotoxic chemotherapy is the foundation for the treatment of the wide variety of childhood malignancies; however, these therapies are known to have a variety of deleterious side effects. One common chemotherapy used in children, doxorubicin (DOX), is well known to cause cardiotoxicity and cardiomyopathy. Recent studies have revealed that DOX impairs skeletal and smooth muscle function and contributes to fatigue and abnormal intestinal motility in patients. In this study, we tested the hypothesis that systemic DOX administration also affects detrusor smooth muscle (DSM) function in the urinary bladder, especially when administered at a young age. The effects on the DSM and bladder function were assessed in BALB/cJ mice that received six weekly intravenous injections of DOX (3 mg·kg-1·wk-1) or saline for the control group. Systemic DOX administration resulted in DSM hypertrophy, increased voiding frequency, and a significant attenuation of DSM contractility, followed by a slower relaxation compared with the control group. Gene expression analyses revealed that unlike DOX-induced cardiotoxicity, the bladders from DOX-administered animals showed no changes in oxidative stress markers; instead, downregulation of large-conductance Ca2+-activated K+ channels and altered expression of myosin light-chain kinase coincided with reduced myosin light-chain phosphorylation. These results indicate that in vivo DOX exposure caused DSM dysfunction by dysregulation of molecules involved in the detrusor contractile-relaxation mechanisms. Collectively, our findings suggest that survivors of childhood cancer treated with DOX may be at increased risk of bladder dysfunction and benefit from followup surveillance of bladder function.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Doxorrubicina/toxicidad , Síntomas del Sistema Urinario Inferior/inducido químicamente , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Miosinas del Músculo Liso/metabolismo , Enfermedades de la Vejiga Urinaria/inducido químicamente , Vejiga Urinaria/efectos de los fármacos , Urodinámica/efectos de los fármacos , Factores de Edad , Animales , Femenino , Hipertrofia , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Síntomas del Sistema Urinario Inferior/metabolismo , Síntomas del Sistema Urinario Inferior/patología , Síntomas del Sistema Urinario Inferior/fisiopatología , Masculino , Ratones Endogámicos BALB C , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Transducción de Señal , Factores de Tiempo , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Vejiga Urinaria/fisiopatología , Enfermedades de la Vejiga Urinaria/metabolismo , Enfermedades de la Vejiga Urinaria/patología , Enfermedades de la Vejiga Urinaria/fisiopatologíaRESUMEN
The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.
Asunto(s)
Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II/metabolismo , Puntos Cuánticos/metabolismo , Miosinas del Músculo Liso/metabolismo , Coloración y Etiquetado , Animales , Pollos , Cadenas Ligeras de Miosina/ultraestructuraRESUMEN
Smooth muscle contraction is triggered when Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC20). However, blood vessels from Mlck-deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC20 to promote smooth muscle contractility. Active, phosphorylated RSK2 was present in mouse resistance arteries under normal basal tone, and phosphorylation of RSK2 increased with myogenic vasoconstriction or agonist stimulation. Resistance arteries from Rsk2-deficient mice were dilated and showed reduced myogenic tone and RLC20 phosphorylation. RSK2 phosphorylated Ser19 in RLC in vitro. In addition, RSK2 phosphorylated an activating site in the Na+/H+ exchanger (NHE-1), resulting in cytosolic alkalinization and an increase in intracellular Ca2+ that promotes vasoconstriction. NHE-1 activity increased upon myogenic constriction, and the increase in intracellular pH was suppressed in Rsk2-deficient mice. In pressured arteries, RSK2-dependent activation of NHE-1 was associated with increased intracellular Ca2+ transients, which would be expected to increase MLCK activity, thereby contributing to basal tone and myogenic responses. Accordingly, Rsk2-deficient mice had lower blood pressure than normal littermates. Thus, RSK2 mediates a procontractile signaling pathway that contributes to the regulation of basal vascular tone, myogenic vasoconstriction, and blood pressure and may be a potential therapeutic target in smooth muscle contractility disorders.
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Arterias/patología , Músculo Liso/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Miosinas del Músculo Liso/metabolismo , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Actinas/metabolismo , Animales , Aorta/citología , Calcio/metabolismo , Células Cultivadas , Femenino , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Noqueados , Desarrollo de Músculos , Miocitos del Músculo Liso/citología , Miografía , Quinasa de Cadena Ligera de Miosina/metabolismo , Fenilefrina/farmacología , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , VasoconstricciónRESUMEN
PROBLEM: Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. METHODS OF STUDY: Mid-secretory phase endometrial tissues from women with RPL (n = 15) and fertile controls (n = 7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. RESULTS: CD56+ uNK number was significantly higher in women with RPL compared to controls (P < 0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2 = 0.43, P = 0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P < 0.05 and P < 0.0001, respectively), and correlated with the number of uNK cell (r2 = 0.18, P < 0.04, and r2 = 0.65, P < 0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P = 0.0027). CONCLUSION: Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.
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Aborto Habitual/inmunología , Endometrio/irrigación sanguínea , Endometrio/inmunología , Células Asesinas Naturales/inmunología , Neovascularización Patológica/patología , Remodelación Vascular/inmunología , Aborto Habitual/sangre , Adulto , Endometrio/citología , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Recuento de Linfocitos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Miosinas del Músculo Liso/metabolismoRESUMEN
Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non-muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM-A/B) and myosin light chain 17 isoforms (LC17a/b ), and isoform ratios were determined via competitive RT-PCR. SM MHC and NM MHC isoforms (NMMHC-A, NMMHC-B and NMMHC-C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC17a and SM1 but with relatively equal expression of SM-A/SM-B at the mRNA level. Meanwhile, isoforms of NMMHC-A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC-A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre-contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.
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Miosina Tipo II/metabolismo , Próstata/metabolismo , Miosinas del Músculo Liso/metabolismo , Secuencia de Aminoácidos , Animales , Carbacol/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miosina Tipo II/química , Nitroprusiato/farmacología , Especificidad de Órganos , Fenilefrina/farmacología , Cloruro de Potasio/farmacología , Próstata/citología , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratas Sprague-Dawley , Miosinas del Músculo Liso/químicaRESUMEN
Myosin-based motility utilizes catalysis of ATP to drive the relative sliding of F-actin and myosin. The earliest detailed model based on cryo-electron microscopy (cryoEM) and X-ray crystallography postulated that higher actin affinity and lever arm movement were coupled to closure of a feature of the myosin head dubbed the actin-binding cleft. Several studies since then using crystallography of myosin-V and cryoEM structures of F-actin bound myosin-I, -II and -V have provided details of this model. The smooth muscle myosin II interaction with F-actin may differ from those for striated and non-muscle myosin II due in part to different lengths of important surface loops. Here we report a â¼6â¯Å resolution reconstruction of F-actin decorated with the nucleotide-free recombinant smooth muscle myosin-II motor domain (MD) from images recorded using a direct electron detector. Resolution is highest for F-actin and the actin-myosin interface (3.5-4â¯Å) and lowest (â¼6-7â¯Å) for those parts of the MD at the highest radius. Atomic models built into the F-actin density are quite comparable to those previously reported for rabbit muscle actin and show density from the bound ADP. The atomic model of the MD, is quite similar to a recently published structure of vertebrate non-muscle myosin II bound to F-actin and a crystal structure of nucleotide free myosin-V. Larger differences are observed when compared to the cryoEM structure of F-actin decorated with rabbit skeletal muscle myosin subfragment 1. The differences suggest less closure of the 50 kDa domain in the actin bound skeletal muscle myosin structure.
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Actinas/química , Microscopía por Crioelectrón/métodos , Miosinas del Músculo Liso/química , Actinas/metabolismo , Animales , Imagenología Tridimensional , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación Proteica , Dominios Proteicos , Miosinas del Músculo Liso/metabolismoRESUMEN
Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region.
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Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Miosinas del Músculo Liso/metabolismo , Estrés Mecánico , Humanos , Microscopía de Fuerza Atómica , Imagen Individual de MoléculaRESUMEN
Blebbistatin is a potent and specific inhibitor of the motor functions of class II myosins, including striated muscle myosin and nonmuscle myosin-2 (NM2). However, the blebbistatin inhibition of NM2c has not been assessed and remains controversial with respect to its efficacy with smooth muscle myosin (SmM), which is highly homologous to NM2. To clarify these issues, we analyzed the effects of blebbistatin on the motor activities of recombinant SmM and three NM2s (NM2a, -2b, and -2c). We found that blebbistatin potently inhibits the actin-activated ATPase activities of SmM and NM2s with following IC50 values: 6.47 µM for SmM, 3.58 µM for NM2a, 2.30 µM for NM2b, and 1.57 µM for NM2c. To identify the blebbistatin-resistant myosin-2 mutant, we performed mutagenesis analysis of the conserved residues in the blebbistatin-binding site of SmM and NM2s. We found that the A456F mutation renders SmM and NM2s resistant to blebbistatin without greatly altering their motor activities or phosphorylation-dependent regulation, making A456F a useful mutant for investigating the cellular function of NM2s.
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Proteínas Aviares/antagonistas & inhibidores , Proteínas Aviares/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/química , Miosinas del Músculo Liso/antagonistas & inhibidores , Miosinas del Músculo Liso/química , Sustitución de Aminoácidos , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Pollos , Humanos , Ratones , Mutación Missense , Miosina Tipo IIB no Muscular/genética , Miosina Tipo IIB no Muscular/metabolismo , Miosinas del Músculo Liso/genética , Miosinas del Músculo Liso/metabolismoRESUMEN
KEY POINTS: Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. ABSTRACT: The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin.
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Contracción Muscular , Músculo Liso/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Multimerización de Proteína , Miosinas del Músculo Liso/metabolismo , Animales , Células Cultivadas , Perros , Femenino , Masculino , Músculo Liso/fisiología , Mutación , Miosina Tipo IIA no Muscular/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
In early studies on smooth muscle, I described a crude myosin fraction (CMF) in which self-assembly of myosin filaments was observed. For the first time, the 14-nm periodicity stemming from regular arrangement of myosin heads on the filament surface was observed (Sobieszek in J Mol Biol 70:741-744, 1972). In this fraction, we also observed formation of long ribbon-shaped aggregates exhibiting a 5.6-nm periodicity, characteristic of tropomyosin (TM) paracrystals (Sobieszek and Small in Phil Trans R Soc Lond B 265:203-212, 1973). We therefore concluded that these ribbons were made of TM and they might be related to the myosin ribbons observed in electron micrographs (EM) of intact smooth muscle (Lowy and Small in Nature 227:46-51, 1970; Small and Squire in Mol Biol 67:117-149, 1972). Subsequently, Small (J Cell Sci 24:327-349, 1977) concluded that the ribbons observed in the EM sections were an artifact, but their observation in the CMF was not addressed. I have now revisited two aspects of the above studies. Firstly, based on my new multi-angle laser-scattering data and considering the length and stability of the building unit for the filament, a myosin trimer fit better to the previously proposed helical structure. Secondly, after two decades of systematic examinations of protein compositions in multiple smooth muscle extracts and isolated filaments, I concluded that the ribbons were made of caldesmon and not TM. Thirdly, actin-activated ATPase activity measurements indicated that modulation of this activity (by CaD and TM) was synergistic, cooperative and depended on myosin to actin ratio.