Contractile Tone Research Articles

Inhibiting Ca2+ channels in Alzheimer's disease model mice relaxes pericytes, improves cerebral blood flow and reduces immune cell stalling and hypoxia.

Early in Alzheimer's disease (AD), pericytes constrict capillaries, increasing their hydraulic resistance and trapping of immune cells and, thus, decreasing cerebral blood flow (CBF). Therapeutic approaches to attenuate pericyte-mediated constriction in AD are lacking. Here, using in vivo two-photon imaging with laser Doppler and speckle flowmetry and magnetic resonance imaging, we show that Ca2+ entry via L-type voltage-gated calcium channels (CaVs) controls the contractile tone of pericytes. In AD model mice, we identifed pericytes throughout the capillary bed as key drivers of an immune reactive oxygen species (ROS)-evoked and pericyte intracellular calcium concentration ([Ca2+]i)-mediated decrease in microvascular flow. Blocking CaVs with nimodipine early in disease progression improved CBF, reduced leukocyte stalling at pericyte somata and attenuated brain hypoxia. Amyloid β (Aβ)-evoked pericyte contraction in human cortical tissue was also greatly reduced by CaV block. Lowering pericyte [Ca2+]i early in AD may, thus, offer a therapeutic strategy to enhance brain energy supply and possibly cognitive function in AD.

Relaxant Effects of Piperine and Piperlongumine from Piper longum Fruits on Porcine Coronary Artery.

Since ancient times, Piper longum Linn. fruits have been recognized for exhibiting various effects, including the diaphoretic effects linked to enhanced blood flow. Piperine and piperlongumine coexist in Piper longum Linn. fruits, although the cardiovascular effects of both compounds remain elusive. We investigated their action of piperine and piperlongumine in porcine coronary arteries, comparing them to the Ca2+ channel antagonist diltiazem. Piperlongumine, unlike piperine or diltiazem, concentration-dependently inhibited basal contractile tone in endothelium-denuded coronary arteries. All three compounds inhibit tonic contractions induced by high potassium chloride (KCl) concentrations. The order of relaxation potency indexed by the half-maximal effective concentration (EC50) were as follows: diltiazem > piperlongumine > piperine. These effects were not different between endothelium-intact and -denuded preparations. In endothelial-denuded preparations, pretreatment with these compounds not only inhibited KCl-induced tonic contractions attenuated calcium chloride (CaCl2)-induced ones in a Ca2+-free medium. Histamine-induced phasic contractions in a Ca2+-free medium containing intracellular Ca2+ chelator was completely suppressed by selective inositol trisphosphate receptor antagonist and piperlongumine, whereas piperine or diltiazem do not have the same effect. These findings suggest that piperine and piperlongumine similar to diltiazem cause vasorelaxation by inhibiting both KCl- and CaCl2-induced contractions in coronary arteries, possibly through the inhibition of voltage-dependent Ca2+ channels. Piperlongumine inhibits histamine-induced contractions in a Ca2+-free medium, which is associated with the intracellular Ca2+ signaling pathway, suggesting that the relaxant effect of piperlongumine differs from that of piperine.

Calponin 1 inhibits agonist-induced ERK activation and decreases calcium sensitization in vascular smooth muscle.

Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin-binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC-dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs. Using this new Cnn1 KO mouse, we show that ablation of CNN1 has two effects, depending on the cytosolic free calcium level: (1) in the presence of elevated intracellular calcium caused by agonist stimulation, Cnn1 KO mice display a reduced amplitude of stress and stiffness but an increase in agonist-induced ERK activation; and (2) during intracellular calcium depletion, in the presence of an agonist, Cnn1 KO mice exhibit increased duration of SM tone maintenance. Together, these results suggest that CNN1 plays an important and complex modulatory role in SMC contractile tone amplitude and maintenance.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Noradrenaline released from locus coeruleus axons contracts cerebral capillary pericytes via α2 adrenergic receptors

Noradrenaline (NA) release from locus coeruleus axons generates vascular contractile tone in arteriolar smooth muscle and contractile capillary pericytes. This tone allows neuronal activity to evoke vasodilation that increases local cerebral blood flow (CBF). Much of the vascular resistance within the brain is located in capillaries and locus coeruleus axons have NA release sites closer to pericytes than to arterioles. In acute brain slices, NA contracted pericytes but did not raise the pericyte cytoplasmic Ca2+ concentration, while the α1 agonist phenylephrine did not evoke contraction. Blocking α2 adrenergic receptors (α2Rs, which induce contraction by inhibiting cAMP production), greatly reduced the NA-evoked pericyte contraction, whereas stimulating α2Rs using xylazine (a sedative) or clonidine (an anti-hypertensive drug) evoked pericyte contraction. Noradrenaline-evoked pericyte contraction and capillary constriction are thus mediated via α2Rs. Consequently, α2Rs may not only modulate CBF in health and pathological conditions, but also contribute to CBF changes evoked by α2R ligands administered in research, veterinary and clinical settings.

Ca2+ signalling in interstitial cells of Cajal contributes to generation and maintenance of tone in mouse and monkey lower oesophageal sphincters.

The lower oesophageal sphincter (LES) generates tone and prevents reflux of gastric contents. LES smooth muscle cells (SMCs) are relatively depolarised, facilitating activation of Cav 1.2 channels to sustain contractile tone. We hypothesised that intramuscular interstitial cells of Cajal (ICC-IM), through activation of Ca2+ -activated Cl- channels (ANO1), set membrane potentials of SMCs favourable for activation of Cav 1.2 channels. In some gastrointestinal muscles, ANO1 channels in ICC-IM are activated by Ca2+ transients, but no studies have examined Ca2+ dynamics in ICC-IM within the LES. Immunohistochemistry and qPCR were used to determine expression of key proteins and genes in ICC-IM and SMCs. These studies revealed that Ano1 and its gene product, ANO1, are expressed in c-Kit+ cells (ICC-IM) in mouse and monkey LES clasp muscles. Ca2+ signalling was imaged in situ, using mice expressing GCaMP6f specifically in ICC (Kit-KI-GCaMP6f). ICC-IM exhibited spontaneous Ca2+ transients from multiple firing sites. Ca2+ transients were abolished by cyclopiazonic acid or caffeine but were unaffected by tetracaine or nifedipine. Maintenance of Ca2+ transients depended on Ca2+ influx and store reloading, as Ca2+ transient frequency was reduced in Ca2+ free solution or by Orai antagonist. Spontaneous tone of LES muscles from mouse and monkey was reduced ∼80% either by Ani9, an ANO1 antagonist or by the Cav 1.2 channel antagonist nifedipine. Membrane hyperpolarisation occurred in the presence of Ani9. These data suggest that intracellular Ca2+ activates ANO1 channels in ICC-IM in the LES. Coupling of ICC-IM to SMCs drives depolarisation, activation of Cav 1.2 channels, Ca2+ entry and contractile tone. KEY POINTS: The lower oesophageal sphincter (LES) generates contractile tone preventing reflux of gastric contents into the oesophagus. LES smooth muscle cells (SMCs) display depolarised membrane potentials facilitating activation of L-type Ca2+ channels. Interstitial cells of Cajal (ICC) express Ca2+ -activated Cl- channels encoded by Ano1 in mouse and monkey LES. Ca2+ signalling in ICC activates ANO1 currents in ICC. ICC displayed spontaneous Ca2+ transients in mice from multiple firing sites in each cell and no entrainment of Ca2+ firing between sites or between cells. Inhibition of ANO1 channels with a specific antagonist caused hyperpolarisation of mouse LES and inhibition of tone in monkey and mouse LES muscles. Our data suggest a novel mechanism for LES tone in which Ca2+ transient activation of ANO1 channels in ICC generates depolarising inward currents that conduct to SMCs to activate L-type Ca2+ currents, Ca2+ entry and contractile tone.

A Role of Caveolae in Trabecular Meshwork Mechanosensing and Contractile Tone.

Polymorphisms in the CAV1/2 gene loci impart increased risk for primary open-angle glaucoma (POAG). CAV1 encodes caveolin-1 (Cav1), which is required for biosynthesis of plasma membrane invaginations called caveolae. Cav1 knockout mice exhibit elevated intraocular pressure (IOP) and decreased outflow facility, but the mechanistic role of Cav1 in IOP homeostasis is unknown. We hypothesized that caveolae sequester/inhibit RhoA, to regulate trabecular meshwork (TM) mechanosensing and contractile tone. Using phosphorylated myosin light chain (pMLC) as a surrogate indicator for Rho/ROCK activity and contractile tone, we found that pMLC was elevated in Cav1-deficient TM cells compared to control (131 ± 10%, n = 10, p = 0.016). Elevation of pMLC levels following Cav1 knockdown occurred in cells on a soft surface (137 ± 7%, n = 24, p < 0.0001), but not on a hard surface (122 ± 17%, n = 12, p = 0.22). In Cav1-deficient TM cells where pMLC was elevated, Rho activity was also increased (123 ± 7%, n = 6, p = 0.017), suggesting activation of the Rho/ROCK pathway. Cyclic stretch reduced pMLC/MLC levels in TM cells (69 ± 7% n = 9, p = 0.002) and in Cav1-deficient TM cells, although not significantly (77 ± 11% n = 10, p = 0.059). Treatment with the Cav1 scaffolding domain mimetic, cavtratin (1 μM) caused a reduction in pMLC (70 ± 5% n = 7, p = 0.001), as did treatment with the scaffolding domain mutant cavnoxin (1 μM) (82 ± 7% n = 7, p = 0.04). Data suggest that caveolae differentially regulate RhoA signaling, and that caveolae participate in TM mechanotransduction. Cav1 regulation of these key TM functions provide evidence for underlying mechanisms linking polymorphisms in the Cav1/2 gene loci with increased POAG risk.

Ageing causes an aortic contractile dysfunction phenotype by targeting the expression of members of the extracellular signal-regulated kinase pathway.

The extracellular signal‐regulated kinase (ERK) pathway is a well‐known regulator of vascular smooth muscle cell proliferation, but it also serves as a regulator of caldesmon, which negatively regulates vascular contractility. This study examined whether aortic contractile function requires ERK activation and if this activation is regulated by ageing. Biomechanical experiments revealed that contractile responses to the alpha1‐adrenergic agonist phenylephrine are attenuated specifically in aged mice, which is associated with downregulation of ERK phosphorylation. ERK inhibition attenuates phenylephrine‐induced contractility, indicating that the contractile tone is at least partially ERK‐dependent. To explore the mechanisms of this age‐related downregulation of ERK phosphorylation, we transfected microRNAs, miR‐34a and miR‐137 we have previously shown to increase with ageing and demonstrated that in A7r5 cells, both miRs downregulate the expression of Src and paxillin, known regulators of ERK signalling, as well as ERK phosphorylation. Further studies in aortic tissues transfected with miRs show that miR‐34a but not miR‐137 has a negative effect on mRNA levels of Src and paxillin. Furthermore, ERK phosphorylation is decreased in aortic tissue treated with the Src inhibitor PP2. Increases in miR‐34a and miR‐137 with ageing downregulate the expression of Src and paxillin, leading to impaired ERK signalling and aortic contractile dysfunction.

Role of Phospholipases A2 in Vascular Relaxation and Sympatholytic Effects of Five Australian Brown Snake, Pseudonaja spp., Venoms in Rat Isolated Tissues.

Human envenoming by Australian brown snakes (Pseudonaja spp.) may result in potentially life-threatening hypotension and subsequent cardiovascular collapse. There have been relatively few studies of the cardiovascular and sympathetic effects of Pseudonaja spp. venoms. In this study, we have examined the effects of venom from five brown snake species—P. affinis, aspidorhyncha, inframacula, nuchalis, and textilis—on cardiac inotropic and chronotropic responses, vascular tone, and sympathetic nerve-induced vascular contractions in rat isolated tissues. The role of phospholipases A2 (PLA2s) in venom-induced effects was assessed with the sPLA2 inhibitor varespladib. In rat isolated left and right atria, there were no physiologically relevant effects of Pseudonaja venoms (0.1–30 µg/ml) on left atrial force of contraction (inotropy) or right atrial rate (chronotropy). In contrast, in isolated small mesenteric arteries precontracted with a thromboxane mimetic, each of the five brown snake venoms (at 30 µg/ml) caused marked vasorelaxation (−60 to –90% of contractile tone). Pretreatment with varespladib (1 µM) significantly inhibited the vasorelaxation caused by P. aspidorhyncha, P. nuchalis, and P. textilis venoms. Electrically induced sympathetic nerve-mediated contractions of mesenteric arteries were significantly attenuated by only P. textilis, and P. affinis venoms (30 µg/ml) and these sympatholytic effects were inhibited by varespladib (1 µM). Based on their inhibition with the sPLA2 inhibitor varespladib, we conclude that PLA2 toxins in P. aspidorhyncha, P. nuchalis, and P. textilis venoms are involved in brown snake venom-induced vasorelaxation and the sympatholytic effects of P. affinis, and P. textilis venoms. Our study supports the promising potential role of varespladib as an initial (pre-referral) and/or adjunct (in combination with antivenom) therapeutic agent for brown snake envenoming.

The Impact of Monoclonal Antibodies on Airway Smooth Muscle Contractility in Asthma: A Systematic Review

Airway hyperresponsiveness (AHR) represents a central pathophysiological hallmark of asthma, with airway smooth muscle (ASM) being the effector tissue implicated in the onset of AHR. ASM also exerts pro-inflammatory and immunomodulatory actions, by secreting a wide range of cytokines and chemokines. In asthma pathogenesis, the overexpression of several type 2 inflammatory mediators including IgE, IL-4, IL-5, IL-13, and TSLP has been associated with ASM hyperreactivity, all of which can be targeted by humanized monoclonal antibodies (mAbs). Therefore, the aim of this review was to systematically assess evidence across the literature on mAbs for the treatment of asthma with respect to their impact on the ASM contractile tone. Omalizumab, mepolizumab, benralizumab, dupilumab, and tezepelumab were found to be effective in modulating the contractility of the ASM and preventing the AHR, but no available studies concerning the impact of reslizumab on the ASM were identified from the literature search. Omalizumab, dupilumab, and tezepelumab can directly modulate the ASM in asthma, by specifically blocking the interaction between IgE, IL-4, and TSLP, and their receptors are located on the surface of ASM cells. Conversely, mepolizumab and benralizumab have prevalently indirect impacts against AHR by targeting eosinophils and other immunomodulatory effector cells promoting inflammatory processes. AHR has been suggested as the main treatable trait towards precision medicine in patients suffering from eosinophilic asthma, therefore, well-designed head-to-head trials are needed to compare the efficacy of those mAbs that directly target ASM contractility specifically against the AHR in severe asthma, namely omalizumab, dupilumab, and tezepelumab.

Multi-walled carbon nanotubes induce airway hyperresponsiveness in human bronchi by stimulating sensory C-fibers and increasing the release of neuronal acetylcholine

ABSTRACT Objectives The potential of multi-walled carbon nanotubes (MWCNTs) in inducing airway hyperresponsiveness (AHR) was investigated in human airways. Methods Human isolated bronchi were exposed to MWCNTs and the contractility to electrical field stimulation (EFS) was measured. Neuronal acetylcholine (ACh) and cyclic adenosine monophosphate (cAMP) were quantified. Some tissues were desensitized by consecutive administrations of capsaicin. Results MWCNTs (100 ng/ml – 100 µg/ml) induced AHR (overall contractile tone vs. negative control: +83.43 ± 11.13%, P < 0.01). The potency was significantly (P < 0.05) greater when airways were stimulated at low frequency (EFS3Hz) then at medium-to-high frequencies (EFS10Hz and EFS25Hz) (delta potency: +2.13 ± 0.74 and +2.40 ± 0.65 logarithms, respectively). In capsaicin-desensitized airways, the AHR to MWCNTs 100 ng/ml was abolished. MWCNTs increased the release of ACh, an effect prevented by capsaicin-desensitization (−90.17 ± 18.59%, P < 0.05). MWCNTs did not alter the level of cAMP. Conclusion MWCNTs administered at low concentrations elicit AHR in human airways by activating sensory C-fibers and, in turn, increasing the release of neuronal ACh. Our results suggest that work is required to understand the impact of MWCNTs in patients at risk of AHR, such as those suffering from chronic obstructive respiratory disorders.

Involvement of 3',5'-cyclic inosine monophosphate in cystathionine γ-lyase-dependent regulation of the vascular tone.

Background and Purposel‐cysteine or hydrogen sulfide (H2S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 μM followed by vasodilatation at 30–100 μM. Here, we have investigated the signalling involved in the H2S‐induced contraction.Experimental ApproachVascular response to NaHS or l‐cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ‐lyase (CSE−/−), soluble guanylyl cyclase (sGCα1 −/−) and endothelial nitric oxide synthase (eNOS−/−) knock‐out mice. The cAMP, cGMP and inosine 3′,5′‐cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated.Key ResultsCSE‐derived H2S‐induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H2S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell‐permeable analogue of cIMP elicits concentration‐dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE−/− mice, confirms that H2S‐induced contraction involves cIMP.Conclusion and ImplicationsThe endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE‐derived H2S that is mediated by cIMP.

Role of the PKA regulatory subunit RIα in the contractile tone of the aorta

Protein kinase A (PKA) is a key enzyme activated by binding of cAMP. PKA induces smooth muscle relaxation and vasodilatation. The PKA-RIα knock-in heterozygous mice, [R368X]/[+] (KI), leading to the generation of a truncated RIα and the repression of PKA activation (Le Stunff et al., 2017, J Bone Miner Res ) may help decipher the contribution of PKA-RIα in vascular system which has remained elusive. Our aim is to document the expression of PKA-RIα in mouse aorta and to decipher its role in arterial tone by using wild-type (WT) and KI mice. Aortas were harvested from adult male WT and KI mice. PKA subunits expressions were studied by immunoblot. Vascular reactivity was tested by mounting the aorta on a wire-myograph. The internal circumference (IC) was recorded. Concentration-contractile responses were performed for K + (20–120 mM), U46619 (1 nM–3 μM) and phenylephrine (PE, 1 nM–3 μM). Vasorelaxant responses to acetylcholine (ACh, 1 nM–10 μM) and to L858051 (adenylate cyclase activator, 10 nM–30 μM) were studied in aorta contracted with PE. All groups included 5–6 mice. In WT aorta, the anti-PKA-RIα antibody yielded a signal at 49 kDa, as expected. In KI mice, mutant allele expression was confirmed by an additional signal at 47 kDa. RIIα and Cα expressions were similar in both strains. IC was 20% smaller in KI mice than in WT. The contractile response to K + and PE were similar in both groups, whereas the response to U46619 was stronger in mutant aorta with a higher E max . ACh and L858051 relaxed aortas similarly in both groups. These preliminary data show that PKA-RIα is expressed in mouse aorta. The smaller IC of aorta in [R368X]/[+] mice is consistent with their smaller size (Le Stunff et al.). The stronger constriction to U46619 in mutant mice may be explained by reduced PKA activity compared to WT. The similar relaxation to L858051 in mutant mice maybe due to redundancy of PKA-RIα in mouse aorta.

Amyloid Beta Peptides and Th1 Cytokines Modulate Human Brain Vascular Smooth Muscle Tonic Contractile Capacity In Vitro: Relevance to Alzheimer's Disease?

Alzheimer’s Disease (AD) is a neurodegenerative condition characterized both by the presence of tau protein neurofibrillary tangles and amyloid beta (Aβ) containing extracellular “plaques”. The cleavage of amyloid precursor protein (APP) yields several Aβ peptides. Although Aβ toxicity to neurons has been described extensively, its effects on other components of the neurovasculature such as vascular smooth muscle cells have been less well characterized. AD is now also recognized as a neurovascular disease characterized by cerebral microbleeds and disturbances in autoregulation. AD is also a neuroinflammatory condition in which several proinflammatory cytokines are elevated and may contribute to the intensification of AD severity. Cerebral autoregulation (the mechanism by which brain blood flow is maintained despite changes in perfusion pressure) is extremely tightly controlled in the brain and shows disturbances in AD. The failure of autoregulation in AD may make the brain susceptible to cerebral microbleeds through a reduced capacity to limit blood flow when pressure is increased. Conversely, reduced vasodilation during low flow might could also exacerbate tissue hypoxia. Currently, whether and how Aβ peptides and inflammatory cytokines depress brain smooth muscle cell tonic contraction is not known, but could reveal important targets in the preservation of autoregulation which is disturbed in AD. We used a collagen gel contractility assay to evaluate the influence of Aβ25-35, Aβ1-40 and Aβ1-42 peptides and inflammatory cytokines on the tonic contractility of human brain vascular smooth muscle cells (HBVSMC) as an in vitro model of cerebral autoregulation. We found that 5 and 10 μM Aβ1-42 significantly depressed HBVSM contractility, while Aβ1-40 5–20 μM had no effect on contractility. Conversely, Aβ25-35 (1–50 μM) increased contractility. Interestingly, the inflammatory cytokines TNF-α (20 ng/mL), IL-1β (20 ng/mL) and IFN-γ (1000 U/mL) also depressed HBVSM tonic contractility alone and in combination. These data suggest that both the inflammatory milieu in AD as well as the abundance of Aβ peptides may promote autoregulatory failure and increase brain susceptibility to dysregulated perfusion and microbleeds which are an important and devastating characteristic of AD.

Beclomethasone dipropionate and sodium cromoglycate protect against airway hyperresponsiveness in a human ex vivo model of cow's milk aspiration

BackgroundRecurrent cow's milk (CM) aspiration is often associated with gastroesophageal reflux in infants and toddlers and it seems to be implicated in the etiology of different inflammatory lung disorders. This study aimed to investigate ex vivo the impact of CM aspiration on human airways and whether treatment with beclomethasone dipropionate (BDP) or sodium cromoglycate (SCG) may prevent the potential CM-induced airway hyperresponsiveness (AHR). MethodsHuman isolated bronchi were contracted by electrical field stimulation (EFS10Hz) to mimic the contractile tone induced by the parasympathetic activity and challenged with CM, fat/lactose-free CM, or human breast milk (HM). The effect of pre-treatment with beclomethasone dipropionate (BDP) and sodium cromoglycate (SCG) was also investigated on the AHR induced by CM. ResultsAfter a 60 min-challenge with CM 1:10 v/v and fat/lactose-free CM 1:10 v/v, ASM significantly (P ​< ​0.05) increased compared to control (+67.04 ​± ​17.08% and +77.91 ​± ​1.34%, respectively), a condition that remained stable for 150 ​min post-treatment, whereas HM did not alter ASM contractility. BDP 1 ​μM and 10 ​μM significantly (P ​< ​0.05) reduced the AHR elicited by CM (−52.49 ​± ​10.97% and −66.98 ​± ​7.90%, respectively vs. control). At the same manner, SCG 1 ​μM and 10 ​μM significantly (P ​< ​0.05) inhibited the CM-induced AHR (−59.03 ​± ​9.24% and −73.52 ​± ​7.41%, respectively vs. control). ConclusionCM induces AHR in human ASM by eliciting an increased parasympathetic contractile response. Preventive treatment with nebulized SCG may be indicated in infants or toddlers fed with CM, rather than with BDP due to a superior safety profile.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit.

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC50, the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the β ancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 μM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQ-specific agonist in the tissue.

Extracellular Matrix Rigidity Modulates Human Cervical Smooth Muscle Contractility-New Insights into Premature Cervical Failure and Spontaneous Preterm Birth.

Spontaneous preterm birth (sPTB), a major cause of infant morbidity and mortality, must involve premature cervical softening/dilation for a preterm vaginal delivery to occur. Yet, the mechanism behind premature cervical softening/dilation in humans remains unclear. We previously reported the non-pregnant human cervix contains considerably more cervical smooth muscle cells (CSMC) than historically appreciated and the CSMC organization resembles a sphincter. We hypothesize that premature cervical dilation leading to sPTB may be due to (1) an inherent CSMC contractility defect resulting in sphincter failure and/or (2) altered cervical extracellular matrix (ECM) rigidity which influences CSMC contractility. To test these hypotheses, we utilized immunohistochemistry to confirm this CSMC phenotype persists in the human pregnant cervix and then assessed in vitro arrays of contractility (F:G actin ratios, PDMS pillar arrays) using primary CSMC from pregnant women with and without premature cervical failure (PCF). We show that CSMC from pregnant women with PCF do not have an inherent CSMC contractility defect but that CSMC exhibit decreased contractility when exposed to soft ECM. Given this finding, we used UPLC-ESI-MS/MS to evaluate collagen cross-link profiles in the cervical tissue from non-pregnant women with and without PCF and found that women with PCF have decreased collagen cross-link maturity ratios, which correlates to softer cervical tissue. These findings suggest having soft cervical ECM may lead to decreased CSMC contractile tone and a predisposition to sphincter laxity that contributes to sPTB. Further studies are needed to explore the interaction between cervical ECM properties and CSMC cellular behavior when investigating the pathophysiology of sPTB.

Development and Optimization of a 3D Bioprinted Experimental Model of Skeletal Muscle

BackgroundTraditional cell culture has been the foundation of biomedical research for over 100 years and remains an essential scientific tool. Yet it is becoming increasingly recognized that biological and technical deficiencies limit the relevance of cell culture to health and disease. These deficiencies are especially relevant to muscle tissues; it is difficult to measure contraction in cultured cells, and physical interactions between cells and the physical environment can modulate tissue‐level function. To fill this gap, we used 3D bioprinting technology to create a free‐floating and bioreactor‐free experimental model of skeletal muscle. The model is designed for contraction to be assessed as muscle shortening against a physiologically relevant auxotonic load.MethodsC2C12 mouse skeletal muscle myoblasts were bioprinted at 2.5×107 cells/mL in a bioink containing 0.25–1.25% RGD‐coupled‐alginate, 1–2 mg/mL collagen‐I and 0–20 mg/mL fibrinogen using an Aspect Biosystems RX‐1 bioprinter. Cells were printed as a 10 mm diameter ring, with or without an acellular frame providing an auxotonic load opposing contraction. Cells were thrombin treated to polymerise fibrinogen, cultured with or without the antifibrinolytic agent aprotinin (20 μg/mL), and differentiated on day 1–5 in a low serum media with high glucose and insulin. Tissue structure was assessed by live cell phase contract microscopy and filamentous actin immunofluorescence. Cell phenotype was assessed by qPCR for contraction‐relevant genes.ResultsCells printed in stiff alginate (≥0.5%) did not form cell‐cell contacts required for myotube differentiation. Soft alginate (&lt;0.5%) allowed cell elongation but rings rapidly collapsed, necessitating the use of a very stiff acellular supporting frame (≥1.0% alginate). Early bioinks including only alginate and collagen promoted myotube formation but fibres tore themselves apart under their own contractile tone. Fibrinogen dose dependently improved fibre integrity, and culturing with 20 g/mL aprotinin dramatically improved overall tissue structure (lifespan &gt;10 days) with extensive formation of elongated, multinucleated and f‐actin‐rich myotubes. Final design parameters of: 0.25% cellular alginate; 1 mg/mL collagen; 20 mg/mL fibrinogen; 1% alginate acellular frame; differentiated on day 3; maintained until day 10, resulted in 30‐fold upregulation of myosin heavy chain 1, with a switch from β‐ to α‐actin and reduction in muscle cadherin expression. These outcomes are entirely consistent with the formation of physically stable contractile myotubes.ConclusionOur 3D bioprinted skeletal muscle represents a novel model for studying muscle biology in vitro, incorporating a physiologically relevant 3D structure, realistic mechanical environment, and the ability to directly measure contraction. The standalone structure requires no specialist bioreactor to produce or maintain, making the model easily accessible to a wide range of biomedical researchers, to better replicate health and disease states.Support or Funding InformationFunding: NSERC Discovery Grant (ARW), Research Manitoba Studentship (SS, JO), University of Manitoba Undergraduate Research Award (AK)A freshly 3D bioprinted skeletal muscle construct within a 24 mm diameter ThinCert membrane. The translucent structural (red arrow) and opaque muscle layers (blue arrow) can clearly be seen. The texture of the crosshatching in the structural layer can be seen, providing an auxotonic load opposing muscle contraction.Figure 1Widefield immunofluorescence of 3D bioprinted skeletal muscle constructs showing actin filaments (red) and nuclei (blue) after 7 days of differentiation in a low serum media with high glucose, insulin and 20 ug/mL aprotinin. There is extensive formation of elongated multinucleated cells, corresponding with &gt;30‐fold increase in myosin heavy chain 1 (MYH1) mRNA abundance, and a switch from β‐ to α‐actin expression.Figure 2

Smooth Muscle Tone Contributes to Bladder Wall Stiffness during Filling

Sensations of bladder fullness are conveyed to the central nervous system by sensory nerves as a response to distention of the urinary bladder. This distention is accompanied by non‐voiding transient pressure events and contractions called “micromotions” that are responsible for the majority of sensory outflow during bladder filling. Changes in smooth muscle contractility along with alterations in wall stiffness and sensory feedback are observed in most UB pathologies. Thus, the mechanical properties of bladder wall may play a crucial role in communicating a sense of bladder fullness. We hypothesize that the contractile apparatus of the urinary bladder is actively involved in maintaining bladder wall stiffness during filling.Whole mouse bladders were dissected and mounted in a custom designed pentaplanar reflected image macroscopy (PRIM) system, for simultaneous measurement of bladder wall micromotions, bladder volume, intravesical pressure, bladder wall stress, and bladder wall strain during ex vivo bladder filling. Bladders were filled at a constant rate (30 μl/min) until intravesical pressure reached 25 mmHg. During filling, bladder wall micromotions led to transient pressure events (0–2 mmHg in amplitude). These micromotions began on the lateral sides of the bladder wall near the trigone and expanded upwards as the bladder filled. Baseline bladder pressure, however, remained relatively low over a large infused volume (~350 μL). Next, we derived the stress versus strain relationship of the bladder wall from the pressure‐volume recordings during filling. At baseline pressures &gt;15 mmHg, intravesical pressure rapidly increased and transient pressure events could no longer be distinguished implying that wall compliance is maintained until larger infused volumes, after which bladder wall stress with respect to strain increases rapidly. To determine if smooth muscle tone altered bladder wall stress during filling, bladder was exposed to myosin inhibitor blebbistatin (10 μM) or PI3 kinase inhibitor wortmannin (10 μM). Both blebbistatin and wortmannin effectively diminished the micromotions and shifted the stress vs strain curve to the right, indicating that the smooth muscle contractile apparatus and smooth muscle tone contribute to bladder wall stiffness, but predominantly only at higher filling volumes. These findings suggest that smooth muscle tone directly increases bladder wall stiffness and that changes in this tone could alter sensory feedback during bladder filling.Support or Funding InformationSupported by NIH K01DK103840 (NRT) and R01DK119615 (NRT).

Validation of A 3D Bioprinted Model of Airway Smooth Muscle – A Novel Tool to Study the Effects of Airway Stiffening in Asthma

BackgroundAsthma is a chronic obstructive airway disease characterized by airway wall remodeling and aberrant contraction of airway smooth muscle (ASM), which each contribute to clinical symptoms by restricting airflow. However, the precise link between airway mechanics and ASM contractility remains elusive, because ASM cell biology is routinely studied in 2D models that inadequately replicate the complex in‐vivo 3D microenvironment of healthy or diseased airways. We used a technique called 3D bioprinting to develop an experimental model of ASM in which we could characterize the response of ASM to altered mechanical loads in a realistic 3D structure.MethodsHuman ASM were resuspended at 2.5×107 cells/mL in a bio‐ink consisting of RGD‐coupled‐alginate (0.375% w/v), fibrinogen (5 mg/mL) and collagen‐I (1 mg/mL). Cells were then bioprinted using an Aspect Biosystems RX‐1 Bioprinter, as a ring‐shaped bundle of muscle constrained within a stiffness‐modifiable acellular alginate support (0.75–1.25% w/v). After bioprinting, constructs were thrombin treated (1.25 U/mL, 30 min) to initiate fibrin polymerization and maintained in standard ASM culture conditions. Cell health, viability and morphology were assessed with LDH assays, Hoechst/propidium‐iodide and filamentous‐actin staining respectively. Expression of ASM relevant genes was measured by qPCR. Reduction in lumen area representing contractile tone was tracked by live cell microscopy.ResultsASM bioprinted without acellular supports shortened excessively and rapidly lost structural integrity. Comparatively, including supports provided a mechanical load that controlled baseline shortening; cells supported by 0.75%, 1% and 1.25% alginate exhibited &gt;45%, &gt;25% and &lt;15% lumen reduction respectively. Constructs bioprinted in 1% supports exhibited high cell viability (&gt;80%) for up to 14 days in culture. Histology 4 days after printing revealed development of a highly organized tissue with evidence of cell‐cell contacts and alignment of actin fibres. Nuclei were evenly distributed in all axes indicating formation of a true 3D structure. Compared with 2D, cells in 3D had a higher mRNA abundance of myosin heavy chain, but vimentin and matrix metalloproteinase‐3 were lower (all p&lt;0.05). Importantly, administration of acetylcholine resulted in muscle contraction, while cytochalasin D caused a dramatic decrease in tone for all tissues with structural supports.ConclusionWe have validated a novel tool for studying ASM biomechanics. ASM displayed varying levels of baseline shortening to different mechanical loads, supporting that mechanical cues may have profound effects on cellular function. Further, higher abundance of contractile genes in 3D demonstrates adoption of a physiologically relevant phenotype. Moving forward, contraction, relaxation and calcium dynamics will be quantified across a range of stiffnesses. These data will help unmask mechanisms by which airway mechanics regulate ASM contractility, with important clinical relevance to asthma.Support or Funding InformationFunding: NSERC Discovery Grant (ARW), Research Manitoba Studentship (JO, SS)