Dependent Contraction Research Articles

A Chemomechanobiological Model of the Long-Term Healing Response of Arterial Tissue to a Clamping Injury.

Vascular clamping often causes injury to arterial tissue, leading to a cascade of cellular and extracellular events. A reliable in silico prediction of these processes following vascular injury could help us to increase our understanding thereof, and eventually optimize surgical techniques or drug delivery to minimize the amount of long-term damage. However, the complexity and interdependency of these events make translation into constitutive laws and their numerical implementation particularly challenging. We introduce a finite element simulation of arterial clamping taking into account acute endothelial denudation, damage to extracellular matrix, and smooth muscle cell loss. The model captures how this causes tissue inflammation and deviation from mechanical homeostasis, both triggering vascular remodeling. A number of cellular processes are modeled, aiming at restoring this homeostasis, i.e., smooth muscle cell phenotype switching, proliferation, migration, and the production of extracellular matrix. We calibrated these damage and remodeling laws by comparing our numerical results to in vivo experimental data of clamping and healing experiments. In these same experiments, the functional integrity of the tissue was assessed through myograph tests, which were also reproduced in the present study through a novel model for vasodilator and -constrictor dependent smooth muscle contraction. The simulation results show a good agreement with the in vivo experiments. The computational model was then also used to simulate healing beyond the duration of the experiments in order to exploit the benefits of computational model predictions. These results showed a significant sensitivity to model parameters related to smooth muscle cell phenotypes, highlighting the pressing need to further elucidate the biological processes of smooth muscle cell phenotypic switching in the future.

Modulation of Vascular Smooth Muscle Cell Phenotype by High Mobility Group AT-Hook 1.

ObjectiveThe purpose of this study is to examine the effect of high mobility group AT-hook 1 (HMGA1) on the phenotyptic change of vascular smooth muscle cells (VSMCs).MethodsGene silencing and overexpression of HMGA1 were introduced to evaluate the effect of HMGA1 expression on the phenotypic change of VSMCs. Marker gene expression of VSMCs was measured by promoter assay, quantitative polymerase chain reaction, and western blot analysis. Common left carotid artery ligation model was used to establish in vivo neointima formation.ResultsHMGA1 was expressed strongly in the synthetic type of VSMCs and significantly downregulated during the differentiation of VSMCs. Silencing of HMGA1 in the synthetic type of VSMCs enhanced the expression of contractile marker genes thereby enhanced angiotensin II (Ang II)-dependent contraction, however, significantly suppressed proliferation and migration. Stimulation of contractile VSMCs with platelet-derived growth factor (PDGF) enhanced HMGA1 expression concomitant with the downregulation of marker gene expression which was blocked significantly by the silencing of HMGA1. Silencing of HMGA1 retained the Ang II-dependent contractile function, which was curtailed by PDGF stimulation, however, overexpression of HMGA1 in the contractile type of VSMCs suppressed marker gene expression. Proliferation and migration were enhanced significantly by the overexpression of HMGA1. Furthermore, the Ang II-dependent contraction was reduced significantly by the overexpression of HMGA1. Finally, the expression of HMGA1 was enhanced significantly in the ligated artery, especially in the neointima area.ConclusionHMGA1 plays an essential role in the phenotypic modulation of VSMCs. Therefore, paracrine factors such as PDGF may affect vascular remodeling through the regulation of HMGA1.

Evaluation of Ethyl Acetate, Chloroform and Toluene Fractions of <i>Thevetia peruviana</i> (Pers.) K. Schum Methanolic Leaf Extract for Uterotonic Activity

<i>Thevetia peruviana</i> (Pers.) K. Schum (Apocynacae) leaves have a reputation of abortifacient activity. We investigated traditional claim and found that methanolic leaf extract produce antifertility activity by lowering the progesterone level in rat model. Aim of the present study was to find out the chemical constituent (s) responsible for antifertility activity of methanolic leaf extract of <i>Thevetia peruviana</i> (Pers.). The ethyl acetate, chloroform and toluene fractions of methanolic extract of <i>T. peruviana</i> leaves freed from cardiac glycosides [TPL-Me-G] were selected for phytochemical investigation and <i>in-vitro</i> uterotonic activity. The methanolic extract of <i>T. peruviana</i> leaves (1.103g) was fractionated with toluene (100ml, n=20); chloroform (100ml, n=20); and ethyl acetate (100ml, n=20) in the successive order. These fractions were examined for phytoconstituents and evaluated for <i>in-vitro</i> uterotonic activity. The toluene fraction (TPL-T) was found to have triterpenes, flavonoids and phytosterols. Quercetin (0.8904%) is present in TPL-T. The chloroform fraction (TPL-Ch) was found to contain flavonoids, triterpenes and phytosterols. Presence of alkaloids and flavonoids (quercetin 0.1606%) were observed in ethyl acetate fraction (TPL-Et-Ac). In contrast to TPL-Et-Ac, the TPL-T and TPL-Ch induced dose dependent uterine contraction in the isolated estrogenized rat uterus model. Highest uterotonic activity was found with TPL-Me-G which has kaempferol as phyto-constituent additionally. The <i>in-vitro</i> uterotonic activity is not influenced by quercetin and primary contributor is kaempferol though some unknown phytoconstituent/s also contributes to uterotonic activity and synergizes the action of kaempferol too. So, further research is needed to identify other contributory unknown phytoconstituent/s for antifertility activity of methanolic leaf extract of <i>Thevetia peruviana</i> (Pers.).

Oceanapia magna Sponge Presents Dual Effect on the Gastrointestinal Motility of Rodents: In Vitro and In Vivo Assays.

Oceanapia magna Santos-Neto, Nascimento, Cavalcanti and Pinheiro sponges are distributed across tropical worldwide seas. Some studies of marine products have shown interesting activities in smooth muscle models. Hence, we assessed the effect of the ethanolic extract of Oceanapia magna. (OC-EtOH) on acute toxicity and gastrointestinal motility (in vitro and in vivo) in rodent models. On guinea pig ileum, OC-EtOH induced a concentration dependent contraction on basal tonus, which was not inhibited by atropine, but in the presence of pyrilamine or verapamil, the effect was antagonized. Contrastingly, on KCl- or histamine-induced contractions, OC-EtOH presented a transient contraction followed by a concentration-dependent relaxation. Moreover, OC-EtOH presented a relaxant profile on cumulative curves to CaCl2 and tonic contraction induced by S-(-)-BayK8644, through Cav blockade. The acute toxicity assay showed that OC-EtOH (2,000 mg/kg, p.o.) did not present any sign of toxicity in female mice. Additionally, OC-EtOH presented antidiarrheal effect in mice, increased the intestinal normal transit and reduced the castor oil-induced intestinal transit. Thus, OC-EtOH presented a dual effect on guinea pig ileum promoting contraction through activation of H1 and CaV, and relaxation through CaV blockade, besides the effect on upper gastrointestinal transit in mice, showing a potential medicinal use of this sponge in intestinal diseases such as diarrhea.

Bioengineered Gastrointestinal Tissues with Fibroblast‐Induced Shapes

AbstractBioengineered gastrointestinal (GI) tracts have potential applications in regenerative medicine and disease modeling. Methods for engineering tubular GI tracts containing natural extracellular matrix (ECM) are currently limited. Here, the fabrication of collagen tubes with designed shapes by using lipid bilayer supported droplet networks is reported. Droplets containing cells and collagen are arrayed in lipid‐containing oil to form droplet networks, which undergo thermal gelation to provide continuous collagen tubes. A variety of tubular GI tissues are fabricated. For example, human intestinal organoids embedded in the collagen tubes migrate to the luminal surfaces and fuse to form a continuous epithelial layer, mimicking aspects of intestinal tissue structure. Fibroblasts embedded in the collagen induce a cell density dependent contraction of the tubes. Complex tubular structures are produced by patterning droplets containing different densities of fibroblasts. The fibroblast‐containing collagen tubes are seeded with various epithelial cells at their luminal surfaces to form gastric and colonic tissues, which comprise monolayers or multilayers of epithelial cells and fibroblast‐containing subepithelial layers. The engineered gastric tissues are susceptible to infection withHelicobacter pylori. The versatile technique allows the construction of tubular GI tracts containing ECM and layered structures, with broad potential applications in disease research and regenerative medicine.

Omentin: A novel therapeutic approach for the treatment of endothelial dysfunction in type 2 diabetes

BackgroundPerivascular adipose tissue (PVAT) locally influences the functioning of blood vessels and promotes vascular complications associated with diabetes and obesity. The aim of this work was to study the impact of omentin-1 on endothelial function and PVAT in a non-obese type 2 diabetes mellitus animal model, Goto-Kakizaki (GK) rats with or without high fat diet. Material and methodsDiabetic GK rats were divided into four groups: 1) control group; 2) group treated with omentin-1; 3) group of GK rats fed a high fat diet (GKHFD) and 4) group of GKHFD treated with omentin-1. Several in vivo parameters such as adiposity and Lee indexes, lipid profile, fasting glucose levels, glucose and insulin tolerance tests were determined. At the vascular level, endothelial dependent and independent relaxation and contraction studies were performed in aortic rings in the absence (PVAT−) or in the presence (PVAT+) of thoracic PVAT. We also evaluated vascular oxidative stress and determined the pro-inflammatory status of PVAT. RESULTS: Endothelium-dependent relaxation to acetylcholine, assessed by wire myography, was impaired in GK and GKHFD rats and improved by the omentin-1 treatment. In addition, vascular superoxide production was increased in the vascular wall of diabetic rats, accompanied by reduced nitric oxide bioavailability and significantly improved by omentin treatment. PVAT anti-contractile action found under physiological conditions was lost in type 2 diabetes, and partially recovered with omentin-1 administration. In addition, omentin-1 treatment significantly improved proinflammatory and pro-oxidant PVAT phenotype (decreasing C-reactive protein and nitrotyrosine levels). Furthermore, it was observed an improvement in various systemic and metabolic biochemical parameters of diabetic animals treated for one month with omentin. ConclusionsOmentin-1 ameliorates endothelial dysfunction in type 2 diabetes and presents therapeutic potential for the treatment of vascular complications associated with type 2 diabetes.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Study of solidification of menthol for the applications in temporary consolidation of cultural heritage

Abstract Menthol has been employed recently as important temporary consolidant for conservation of cultural heritages. In this paper, a phase field solidification model is proposed and numerical simulations are conducted to investigate the solidification mechanism of menthol. Simulations provide not only the phase transition of menthol during solidification in consistent with experiment, but also how solidification reaction and evolution of heat are mutually interacted. Solidification leads to liberation of latent heat, contributes to the high temperature in un-solidified core and in turn retards solidification. In addition, size effects on solidification quality and mechanical strength are investigated. During solidification, tensile stresses affect solidification and lead to micro defects which are described using relaxation strain in this manuscript, result in size dependent volumetric contraction as well as residual stress after solidification. Operating solidification in smaller size geometries would help lower down the internal stresses and relaxation strain, resulting in denser products of higher mechanical strength. Therefore, multistep casting procedure is suggested rather than one step casting in real consolidations using menthol.

Mechanism of gamete shedding in starfish: Involvement of acetylcholine in extracellular Ca2+-dependent contraction of gonadal walls

Starfish are suitable animals for the study of hormonal regulatory mechanism of oocyte maturation and ovulation. Although contraction of the gonadal walls is essential for the shedding gametes, little was known about the mechanism. When ovaries of starfish Patiria pectinifera were incubated in Ca2+-free seawater in the presence of 1-methyladenine (1-MeAde), the germinal vesicles in oocytes broke down, but no ovulation occurred. Verapamil, a potent inhibitor of voltage-dependent Ca2+ channels, inhibited 1-MeAde-induced ovulation. These results suggest that extracellular Ca2+ and its influx are indispensable for gamete shedding. Furthermore, acetylcholine (ACh) was involved in extracellular Ca2+-dependent contractions of gonadal walls. Although 1-MeAde failed to induce contraction of the gonadal walls in normal seawater containing L-glutamic acid, application of ACh or carbachol, an agonist for ACh receptor, could bring about shedding of mature oocytes. Atropine, a competitive antagonist of the muscarinic ACh receptor, inhibited 1-MeAde-induced ovulation, but a nicotinic ACh receptor antagonist mecamylamine had no effect. Furthermore, ACh was detected in the ovaries and testes in P. pectinifera. These findings suggest that ACh acts on muscarinic ACh receptors in gonadal walls to induce peristaltic contractions caused by Ca2+ influx via Ca2+ channels in the gonadal wall muscle for gamete shedding. The present study also provides new insight into the regulatory mechanism that 1-MeAde acts on secretion of ACh in ovaries and testes.

Follistatin Mitigates Myofibroblast Differentiation and Collagen Synthesis of Fibroblasts from Scar Tissue around Injured Flexor Tendons.

PurposeThe aim of this study was to investigate the effect of FST gene on the inhibition of fibrosis in fibroblastic cells from scar tissue around repaired zone II flexor tendons.Materials and MethodsImmunohistochemistry was conducted on fibroblast cells transfected with adenovirus-LacZ (Ad-LacZ) as a marker gene (control), or with adenovirus-FST (Ad-FST) as a therapeutic gene. Fibroblast cultures without adenoviral exposure served as controls.ResultsFibroblastic cells transfected with Ad-FST demonstrated significant decrease in collagen type I, MMP-1, MMP2, and α-SMA mRNA expressions compared to those transfected with Ad-LacZ. In addition, fibroblastic cells transfected with Ad-FST exhibited significant decrease in MMP-1, TIMP-1, fibronectin, PAI-1, TRPV4, α-SMA, desmin, and PAX7 protein expressions.ConclusionBased on these findings, we conclude that FST may be a novel therapeutic strategy for preventing scar adhesions around repaired tendons by inhibiting fibroblasts from differentiating into myofibroblasts, in addition to producing type I collagen and regulating extracellular matrix turnover via the downregulation of MMP-1 and TIMP-1. FST may also decrease contracture of the scar by inhibiting Ca2+-dependent cell contraction.

Mechanisms underlying the vascular relaxation activities of Zingiber officinale var. rubrum in thoracic aorta of spontaneously hypertensive rats

ObjectiveTo evaluate vasorelaxant and vasoconstriction effects of Zingiber officinale var. rubrum (ZOVR) on live rats and isolated aortic rings of spontaneously hypertensive rats (SHRs). MethodsExtracts of ZOVR were subjected to in-vivo antihypertensive screening using noninvasive blood pressures in SHRs. The most potent extract, ZOVR petroleum ether extract (ZOP) was then fractionated using n-hexane, chloroform and water. Isolated thoracic aortic rings were harvested and subjected to vascular relaxation studies of n-hexane fraction of ZOP (HFZOP) with incubation of different antagonists such as Nω-nitro-l-arginine methyl ester (L-NAME, 10 µmol/L), indomethacin (10 µmol/L), methylene blue (10 µmol/L), atropine (1 µmol/L), glibenclamide (10 µmol/L), prazosin (0.01 µmol/L), and propranolol (1 µmol/L). ResultsDuring the screening of various ZOVR extracts, ZOP produced the most reduction in blood pressures of SHRs and so did HFZOP. HFZOP significantly decreased phenylephrine-induced contraction and enhanced acetylcholine-induced relaxation. L-NAME, indomethacin, methylene blue, atropine, and glibenclamide significantly potentiated the vasorelaxant effects of HFZOP. Propranolol and prazosin did not alter the vasorelaxant effects of HFZOP. HFZOP significantly suppressed the Ca2+-dependent contraction and influenced the ratio of the responses to phenylephrine in Ca2+-free medium. ConclusionThis study demonstrates that ZOP may exert an antihypertensive effect in the SHR model. Its possible vascular relaxation mechanisms involve nitric oxide and prostacyclin release, activation of cGMP-KATP channels, stimulation of muscarinic receptors, and transmembrane calcium channel or Ca2+ release from intracellular stores. Possible active compounds that contribute to the vasorelaxant effects are 6-gingerol, 8-gingerol and 6-shogaol.

Increased inflammation, oxidative stress and a reduction in antioxidant defense enzymes in perivascular adipose tissue contribute to vascular dysfunction in type 2 diabetes

BackgroundPerivascular adipose tissue (PVAT) surrounds most large blood vessels and plays an important role in vascular homeostasis. The present study was conducted to investigate the contribution of PVAT to vascular dysfunction in a rat model of type 2 diabetes. Material and methodsSeveral in vivo parameters such as lipid profile (total cholesterol and triglyceride systemic levels), fasting glucose levels, glucose tolerance and insulin sensitivity (through glucose and insulin tolerance tests, respectively) were determined in Goto-Kakizaki (GK) diabetic rats and compared with control Wistar rats. At the vascular level, endothelial dependent and independent relaxation and contraction studies were performed in aortic rings in the absence (PVAT−) or in the presence (PVAT+) of thoracic PVAT. We also evaluated vascular oxidative stress and performed western blots, PCR and immunohistochemistry analysis of cytokines and various enzymes in PVAT. ResultsEndothelium-dependent relaxation to acetylcholine, assessed by wire myography, was impaired in GK rats and improved by the antioxidant TEMPOL and by the TLR4 inhibitor, CLI-095 suggesting an increase in oxidative stress and inflammation. In addition, vascular superoxide and peroxynitrite production was increased in the vascular wall of diabetic rats, accompanied by reduced nitric oxide bioavailability. The presence of PVAT had an anticontractile effect in response to phenylephrine in Wistar rats that was lost in GK rats. Western blot and immunohistochemistry analysis revealed that PVAT phenotype shifts, under diabetic conditions, towards a proinflammatory (with increment in CRP, CCL2, CD36), pro-oxidant (increased levels of aldose reductase, and reduced levels of antioxidant deference enzymes) and vasoconstriction state. ConclusionOur data suggest that this rat model of type 2 diabetes is associated with perivascular adipose dysfunction that contributes to oxidative stress, inflammation and endothelial dysfunction.

Endocrine regulation of the thymic function in European sea bass: Interactions between environmental factors and the reproductive system development

Starfish are suitable animals for the study of hormonal regulatory mechanism of oocyte maturation and ovulation. Although contraction of the gonadal walls is essential for the shedding gametes, little was known about the mechanism. When ovaries of starfish Patiria pectinifera were incubated in Ca2+-free seawater in the presence of 1-methyladenine (1-MeAde), the germinal vesicles in oocytes broke down, but no ovulation occurred. Verapamil, a potent inhibitor of voltage-dependent Ca2+ channels, inhibited 1-MeAde-induced ovulation. These results suggest that extracellular Ca2+ and its influx are indispensable for gamete shedding. Furthermore, acetylcholine (ACh) was involved in extracellular Ca2+-dependent contractions of gonadal walls. Although 1-MeAde failed to induce contraction of the gonadal walls in normal seawater containing L-glutamic acid, application of ACh or carbachol, an agonist for ACh receptor, could bring about shedding of mature oocytes. Atropine, a competitive antagonist of the muscarinic ACh receptor, inhibited 1-MeAde-induced ovulation, but a nicotinic ACh receptor antagonist mecamylamine had no effect. Furthermore, ACh was detected in the ovaries and testes in P. pectinifera. These findings suggest that ACh acts on muscarinic ACh receptors in gonadal walls to induce peristaltic contractions caused by Ca2+ influx via Ca2+ channels in the gonadal wall muscle for gamete shedding. The present study also provides new insight into the regulatory mechanism that 1-MeAde acts on secretion of ACh in ovaries and testes.

Preliminary Phytochemical Screening and Gastrointestinal Study on the Leaf Extract of Stachytarpheta angustifolia Mill Vahl (Verbenaceae) in Rabbit Jejunum

angustifolia (Verbenaceae) is mostly prescribed by the folkloric healers for various gastrointestinal disorders. This study was carried out to ascertain the gastrointestinal effect of the ethanol leaf extract and other various fractions (CHCl3, EtOAc, n- BuOH and residual aqueous) on rabbit Jejunum. The ethanol, n-butanol and residual aqueous of the extract exhibited dose concentration at (0.1, 0.2, 0.4 and 0.8 mg/ml) dependent contraction of the rabbit Jejunum which was blocked by atropine suggesting that the observed pharmacological actions was mediated through the muscarinic receptors. In contrast, chloroform and ethylacetate fraction of the leaf extract exhibit dose concentration dependent relaxation of the rabbit jejunum. Intreperitoneal LD50 of the extract in mice was found to be 295.8 mg/kg. Preliminary phytochemical screening of the leaf extract revealed the presence of carbohydrates, tannins, saponins, cardiac glycoside, sterols and terpenoids. The result indicated that, the plant extract possesses some pharmacological activity, hence justifying its use traditionally in alleviating gastrointestinal disorder.

UBTD1 is a mechano-regulator controlling cancer aggressiveness.

Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with β-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase β-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.

In vitro vasoactive effects of dexmedetomidine on isolated human umbilical arteries

We aimed to investigate the vasoactive effects of dexmedetomidine on isolated human umbilical arteries and possible mechanisms involved. Human umbilical artery strips were suspended in Krebs-Henseleit solution and dose-response curves were obtained for cumulative dexmedetomidine before and after incubation with different agents; propranolol, atropine, yohimbine, prazosin, indomethacin, verapamil. Effects of calcium on cumulative dexmedetomidine-induced contractions were also studied. Cumulative dexmedetomidine resulted in dose dependent contraction responses. Incubation with propranolol (Emax: 93.3 ± 3.26 %), atropine (Emax: 92.0 ± 6.54 %), or indomethacin (Emax: 94.25 ± 2.62 %), did not attenuate dexmedetomidine-elicited contractions (p > 0.05). There were significant decreases in the contraction responses of cumulative dexmedetomidine with yohimbine (Emax: 12.1 ± 11.9 %), prazosin (Emax: 28.8 ± 4.6 %) and verapamil (Emax: 11.2 ± 13.6 %) (p < 0.05). In Ca+2 free medium contraction responses to cumulative dexmedetomidine was insignificant (Emax: 5.20 ± 3.42 %). Addition of cumulative calcium to the Ca+2 free medium resulted in concentration dependent increase in contractions (Emax: 64.83 ± 37.7 %) (p < 0.05). Dexmedetomidine induces vasoconstriction in endothelial-free umbilical arteries via both, α1- and α2-adrenergic receptors and also extracellular Ca+2 concentrations play a major role. β-adrenergic receptors, muscarinic cholinergic receptors, and inhibition of cyclooxygenase enzyme are not involved in this vasoconstriction (Fig. 3, Ref. 36).

Inhibitory effects of rubratoxin A, a potent inhibitor of protein phosphatase 2, on the Ca2+-dependent contraction of skinned carotid artery from guinea pig.

Rubratoxin A, a potent inhibitor of PP2A, is known to suppress smooth muscle contraction. The inhibitory role of PP2A in smooth muscle contraction is still unclear. In order to clarify the regulatory mechanisms of PP2A on vascular smooth muscle contractility, we examined the effects of rubratoxin A on the Ca2+-induced contraction of β-escin skinned carotid artery preparations from guinea pigs. Rubratoxin A at 1 µM and 10 µM significantly inhibited skinned carotid artery contraction at any Ca2+ concentration. The data fitting to the Hill equation in [Ca2+]-contraction relationship indicated that rubratoxin A decreased Fmax-Ca2+ and increased [Ca2+]50, indices of Ca2+ sensitivity for the force and myosin-actin interaction, respectively. These results suggest that PP2A inhibition causes downregulation of the myosin light chain phosphorylation and direct interference with myosin-actin interaction.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders

Calcium dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium sensitizing properties by possibly stabilizing cTnC in an open conformation. To identify novel drug candidates, we employed a structure-based drug discovery protocol that incorporated the use of a molecular dynamics simuations. In preparation for the virtual screening, cNTnC conformations were identified based on their ability to correctly predict known cNTnC binders using a receiver operating characteristics analysis. Following a virtual screen of the National Cancer Institute’s Developmental Therapeutic Program database, a small number of molecules were experimentally tested using stopped-flow kinetics and steady-state fluorescence titrations. We identified two novel compounds that show increased calcium sensitivity of cTnC in the presence of the regulatory domain of cTnI. The effects of those compounds on the calcium dissociation rate was stronger than that of the known calcium sensitizer bepridil. Additionally, we identified a 3-phenylchromane group as a possible key pharmacophore in the sensitization of cardiac muscle contraction. Finally, we are also developing new computational techniques to quantitatively assess the impact of cardiomyopathy mutations and small molecules on troponin function.

Muscular recruitment is associated with muscular function and swelling following eccentric contractions of human elbow flexors.

This study investigated the relationship between muscle fiber recruitment and the magnitude of muscle damage by isotonic eccentric contractions (ECCs). Ten healthy men (age: 27.2±6.0 y, height: 174. 0±5.3 cm, body mass 70.1±2.1 kg) were recruited in this study. Subjects performed 30 ECCs of the elbow flexors using a dumbbell adjusted to 40% and 80% one repetition maximum of each individual. The dependent variables maximal voluntary isometric contraction (MVC) torque, Range of Motion (ROM), delayed onset muscle soreness (DOMS), cross-sectional area (CSA) and transverse relaxation time (T2) of magnetic resonance imaging (MRI) were measured immediately before, immediately after and 1, 3, and 5 days after each exercise. The decreased MVC and limited ROM were significantly greater for 80% than that 40% (P<0.05) at immediately after ECCs. However, no significant difference between 40% and 80% was found for DOMS. CSA at 5 days after ECCs was significantly higher 80% (P<0.05) than 40% (P<0.05). No significant changes in post T2 (acute T2) was found for 40%, but an increased acute T2 was observed in 80% (P<0.05). We found a significant correlation between the change in T2 at immediately after and MVC at immediately after (r=0.77, P<0.05). In addition, a significant correlation between the change in T2 at immediately after and change in CSA at 3 days after (r=-0.83, P<0.05) was found. We conclude that the muscle strength loss and swelling following ECCs are related to the muscle fiber recruitment.