Microelectrode array analysis of mouse uterine smooth muscle electrical activity†.
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ABSTRACT:
SUBMITTER: Ma X
PROVIDER: S-EPMC7124962 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Myometrium plays critical roles in multiple processes such as embryo spacing through peristalsis during mouse implantation, indicating vital roles of smooth muscle in the successful establishment and quality of implantation. Actin, a key element of cytoskeleton structure, plays an important role in the movement and contraction of smooth muscle cells (SMCs). However, the function of peri-implantation uterine smooth muscle and the regulation mechanism of muscle tension are still unclear. This study focused on the molecular mechanism of actin assembly regulation on implantation in smooth muscle. Phalloidin is a highly selective bicyclic peptide used for staining actin filaments (also known as F-actin). Phalloidin staining showed that F-actin gradually weakened in the CD-1 mouse myometrium from day 1 to day 4 of early pregnancy. More than 3 mice were studied for each group. Jasplakinolide (Jasp) used to inhibit F-actin depolymerization promotes F-actin polymerization in SMCs during implantation window and consequently compromises embryo implantation quality. Transcriptome analysis following Jasp treatment in mouse uterine SMCs reveals significant molecular changes associated with actin assembly. Tagln is involved in the regulation of the cell cytoskeleton and promotes the polymerization of G-actin to F-actin. Our results show that Tagln expression is gradually reduced in mouse uterine myometrium from day 1 to 4 of pregnancy. Furthermore, progesterone inhibits the expression of Tagln through the progesterone receptor. Using siRNA to knock down Tagln in day 3 SMCs, we found that phalloidin staining is decreased, which confirms the critical role of Tagln in F-actin polymerization. In conclusion, our data suggested that decreases in actin assembly in uterine smooth muscle during early pregnancy is critical to optimal embryo implantation. Tagln, a key molecule involved in actin assembly, regulates embryo implantation by controlling F-actin aggregation before implantation, suggesting moderate uterine contractility is conducive to embryo implantation. This study provides new insights into how the mouse uterus increases its flexibility to accommodate implanting embryos in the early stage of pregnancy.
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Project description:In mammalian visceral organs, vascular smooth muscle cells (VSMCs) originate from an epithelial-to-mesenchymal transition (EMT) of embryonic mesothelial cells (MCs). The ability of adult MCs to recapitulate EMT and to acquire smooth muscle (SM) markers upon provasculogenic culture suggested they might retain embryonic vasculogenic differentiation potential. However, it remains unknown whether adult MCs-derived SM-like cells may acquire specific vascular SM lineage markers and the functionality of differentiated contractile VSMCs. Here, we describe how a gentle trypsinization of adult mouse uterine cords could selectively detach their outermost uterine mesothelial layer cells. As other MCs; uterine MCs (UtMCs) uniformly expressed the epithelial markers ?-catenin, ZO-1, E-cadherin, CD54, CD29, and CK18. When cultured in a modified SM differentiation media (SMDM) UtMCs initiated a loss of epithelial characteristics and gained markers expression of EMT (Twist, Snail, and Slug), stem and progenitor (Nanog, Sox2, C-kit, Gata-4, Isl-1, and nestin), SM (?-SMA, calponin, caldesmon, SM22?, desmin, SM-MHC, and smoothelin-B) and cardiac (BMP2, BMP4, ACTC1, sACTN, cTnI, cTnT, ANF, Cx43, and MLC2a). UtMCs repeatedly subcultured in SMDM acquired differentiated VSM-like characteristics and expressed smoothelin-B in the typical stress-fiber pattern expression of contractile VSMCs. Relevantly, UtMCs-derived VSM-like cells could generate "mechanical force" to compact collagen lattices and displayed in diverse degree voltage (K(+)) and receptor (endothelin-1, oxytocin, norepinephrine, carbachol and vasopressin)-induced [Ca(2+)](i) rises and contraction. Thus, we show for the first time that UtMCs could recapitulate in vitro differentiative events of early cardiovascular differentiation and transdifferentiate in cells exhibiting molecular and functional characteristics of VSMCs.
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Project description:The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not completely known. Nitric oxide relaxes uterine smooth muscle in a manner disparate from that for other smooth muscles because global elevation of cGMP after activation of soluble guanylyl cyclase does not relax the muscle. S-Nitrosylation, the covalent addition of an nitric oxide (NO) group to a cysteine thiol is a likely mechanism to explain the ability of NO to relax myometrium. This work is the first to describe the myometrial S-nitrosylproteome in both pregnant and nonpregnant tissue states. Using the guinea pig model, we show that specific sets of proteins involved in contraction and relaxation are S-nitrosylated in laboring and nonlaboring muscle and that many of these proteins are uniquely S-nitrosylated in only one state of the tissue. In particular, we show that S-nitrosylation of the intermediate filament protein desmin is significantly increased (5.7-fold, p < 0.005) in pregnancy and that this increase cannot be attributed solely to the increase in protein expression (1.8-fold, p < 0.005) that accompanies pregnancy. Elucidation of the myometrial S-nitrosylproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction/relaxation.
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Project description:IntroductionHydrochlorothiazide has a negative influence on penile erection but little is known about the mechanism(s) involved.AimsTo characterize the effects of this diuretic on mouse corpus cavernosum (CC) smooth muscle in vitro and ex vivo.MethodsCC strips of C57BL/6 mice (12-16 weeks old) were mounted in organ baths containing Krebs-Henseleit solution and tissue reactivity was evaluated. Expression of genes encoding diuretic targets and enzymes involved in penile erection were evaluated by polymerase chain reaction.Main outcome measuresStimulation-response curves to phenylephrine (10 nmol/L-100 ?mol/L) or to electrical field stimulation (1-32 Hz) were constructed, with or without hydrochlorothiazide. Strips of CC from mice after long-term hydrochlorothiazide treatment (6 mg/kg/day for 4 weeks) with or without amiloride (0.6 mg/kg/day for 4 weeks) in vivo also were studied. Nitric oxide and Rho-kinase pathways were evaluated.ResultsHydrochlorothiazide (100 ?mol/L) increased the maximum response to phenylephrine by 64% in vitro. This effect was unaffected by the addition of indomethacin (5 ?mol/L) but was abolished by N((?))-nitro-L-arginine methyl ester (100 ?mol/L). Hydrochlorothiazide (100 ?mol/L) potentiated electrical field stimulation-induced contraction in vitro, but not ex vivo. Long-term treatment with hydrochlorothiazide increased the maximum response to phenylephrine by 60% and resulted in a plasma concentration of 500 ± 180 nmol/L. Amiloride (100?mol/L) caused rightward shifts in concentration-response curves to phenylephrine in vitro. Long-term treatment with hydrochlorothiazide plus amiloride did not significantly increase the maximum response to phenylephrine (+13%). Reverse transcriptase polymerase chain reaction did not detect the NaCl cotransporter in mouse CC. Hydrochlorothiazide did not change Rho-kinase activity, whereas amiloride decreased it in vitro and ex vivo (approximately 18% and 24% respectively). A 40% decrease in Rock1 expression also was observed after long-term treatment with hydrochlorothiazide plus amiloride.ConclusionHydrochlorothiazide potentiates contraction of smooth muscle from mouse CC. These findings could explain why diuretics such as hydrochlorothiazide are associated with erectile dysfunction.
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Project description:S-nitrosoproteome of human unterine smooth muscle in different states of pregnancy (Labor, non labor, and preterm labor). Samples were isolated by the biotin switch and streptavidin pulldown before being analyzed by MS/MS on an Orbitrap instrument. Database Searching and bioinformatics: Tandem mass spectra were extracted; charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, version v.27, rev. 11). Sequest was initiated to search the database containing ipi.HUMAN.v3.87, the Global Proteome Machine cRAP v.2012.01.01, and random decoy sequences (183158 entries) assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine, iodoacetamide derivative of cysteine, and n-ethylmaleimide on cysteine were specified in Sequest as variable modifications. Criteria for Protein Identification-PROTEOIQ (V2.6, www.nusep.com) was used to validate MS/MS based peptide and protein identifications. Peptides were parsed before analysis with a minimum Xcorr value of 1.5 and a minimum length of 6 amino acids. There were no matches to the concatenated decoy database and therefore a false discovery value is not applicable or "0". Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides with 5 spectra per peptide. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
2014-03-02 | PXD000226 | Pride
Project description:Uterine smooth muscle tumors (USMTs) consist of a group of histologically heterogeneous and clinically diverse diseases ranging from malignant leiomyosarcoma (LMS) to benign leiomyoma (ULM). The genetic alterations in LMS are complex, with some genetic alterations present in both LMS and other atypical histologic variants of USMT. In this study, we reviewed 119 USMTs with a diagnosis of LMS, smooth muscle tumor of uncertain malignant potential, atypical leiomyomas/leiomyoma with bizarre nuclei, and cellular leiomyoma, as well as 46 ULMs and 60 myometrial controls. We selected 17 biomarkers highly relevant to LMS in 4 tumorigenic pathways including steroid hormone receptors (estrogen receptor [ER] and progesterone receptor [PR]), cell cycle/tumor suppressor genes, AKT pathway markers, and associated oncogenes. ER and PR expression was significantly lower in LMS than smooth muscle tumor of uncertain malignant potential, atypical leiomyomas/leiomyoma with bizarre nuclei, cellular leiomyoma, and ULM (P?<?.01). Sixty-five percent of LMSs showed complete loss of ER, and 75% of LMSs showed complete loss of PR. All cell cycle genes were differentially expressed in different types of tumor, but significant overlap was noted. More than 75% of LMSs had Ki-67 index greater than 33%, and only 5% in all other types of USMT. Expression of the selected oncogenes varied widely among different types of USMT. PR positivity and p53 had a borderline association with progression-free survival (P?=?.055 for PR and P?=?.0847 for p53). Furthermore, high PR expression was significantly associated with a longer overall survival (P?=?.0163, hazard ratio 0.198). Cell proliferative indices (Ki-67) and sex steroid hormone receptors were the most valuable markers in differentiating LMS from other USMT variants.
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Project description:Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca2+ -activated Cl- channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg-1 ). Whole-cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl- equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)-A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+- ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3 R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L-type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection.
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