A role of Yes1-associated transcriptional regulator (YAP) and WW domain-containing transcription regulator 1 (TAZ) in vascular and gastrointestinal contractility due to control of myocardin (Myocd) expression, which in turn activates contractile genes, has been demonstrated. Whether this transcriptional hierarchy applies to the urinary bladder is unclear. We found that YAP/TAZ are expressed in human detrusor myocytes and therefore exploited the Itga8-CreERT2 model for the deletion of YAP/TAZ. Recombination occurred in detrusor, and YAP/TAZ transcripts were reduced by >75%. Bladder weights were increased (by ≈22%), but histology demonstrated minimal changes in the detrusor, while arteries in the mucosa were inflamed. Real-time quantitative reverse transcription PCR (RT-qPCR) using the detrusor demonstrated reductions of Myocd (-79 ± 18%) and serum response factor (Srf) along with contractile genes. In addition, the cholinergic receptor muscarinic 2 (Chrm2) and Chrm3 were suppressed (-80 ± 23% and -80 ± 10%), whereas minute increases of Il1b and Il6 were seen. Unlike YAP/TAZ-deficient arteries, SRY (sex-determining region Y)-box 9 (Sox9) did not increase, and no chondrogenic differentiation was apparent. Reductions of smooth muscle myosin heavy chain 11 (Myh11), myosin light-chain kinase gene (Mylk), and Chrm3 were seen at the protein level. Beyond restraining the smooth muscle cell (SMC) program of gene expression, YAP/TAZ depletion silenced SMC-specific splicing, including exon 2a of Myocd. Reduced contractile differentiation was associated with weaker contraction in response to myosin phosphatase inhibition (-36%) and muscarinic activation (reduced by 53% at 0.3 µM carbachol). Finally, short-term overexpression of constitutively active YAP in human embryonic kidney 293 (HEK293) cells increased myocardin (greater than eightfold) along with archetypal target genes, but contractile genes were unaffected or reduced. YAP and TAZ thus regulate myocardin expression in the detrusor, and this is important for SMC differentiation and splicing as well as for contractility.NEW & NOTEWORTHY This study addresses the hypothesis that YAP and TAZ have an overarching role in the transcriptional hierarchy in the smooth muscle of the urinary bladder by controlling myocardin expression. Using smooth muscle-specific and inducible deletion of YAP and TAZ in adult mice, we find that YAP and TAZ control myocardin expression, contractile differentiation, smooth muscle-specific splicing, and bladder contractility. These effects are largely independent of inflammation and chondrogenic differentiation.

Living heart slices have recently emerged as a powerful experimental model for fundamental cardiac research. By retaining the structure and function of the native myocardium while maintaining the simplicity of cell culture models, heart slices can be easily employed in electrophysiological, pharmacological, biochemical, and structural investigations. One single heart yields many slices (>20 slices for rodents, >100 slices for porcine or human hearts), however due to the low throughput of most assays and rapid slice degeneration within 24 h of preparation, many slices remain unused and are discarded at the end of the preparation day. Here we present a novel method to extend viability and functionality of living heart slices, enabling their use in experiments over several consecutive days following preparation. By combining hypothermic conditions with inhibition of myosin II ATPase using 2,3-butanedione monoxime (BDM), slices prepared from the left ventricle of porcine hearts remain viable and exhibit preserved contractile function and morphology for up to 6 days. Electrophysiological function was also confirmed over the 6 days by extracellular field potentials recordings. This simple method not only maximizes the use of slices prepared from one single heart, thus reducing the number of animals required, but also increases data reproducibility by allowing multiple electrophysiological, pharmacological, biochemical, and structural studies to be performed from the same heart.

Rabbit duodenum has been used for examining the ability of motilin to cause muscle contraction in vitro. A motilin-related peptide, ghrelin, is known to be involved in the regulation of gastrointestinal (GI) motility in various animals, but its ability to cause rabbit GI contraction have not been well examined. The aim of this study is to clarify the action of rat ghrelin and its interaction with motilin in the rabbit duodenum. The mRNA expression of ghrelin and motilin receptors was also examined using RT-PCR. Rat ghrelin (10-9-10-6 M) did not change the contractile activity of the duodenum measured by the mean muscle tonus and area under the curve of contraction waves. In agreement with this result, the distribution of ghrelin receptor mRNA in the rabbit GI tract varied depending on the GI region from which the samples were taken; the expression level in the duodenum was negligible, but that in the esophagus or stomach was significant. On the other hand, motilin (10-10-10-6 M) caused a concentration-dependent contraction by means of increased mean muscle tonus, and consistently, motilin receptor mRNA was expressed heterogeneously depending on the GI region (esophagus = stomach = colon = rectum < duodenum = jejunum = ileum < cecum). Expression level of motilin receptor was comparable to that of ghrelin receptor in the esophagus and stomach. Pretreatment with ghrelin (10-6 M) prior to motilin did not affect the contractile activity of motilin in the duodenum. In conclusion, ghrelin does not affect muscle contractility or motilin-induced contraction in the rabbit duodenum, which is due to the lack of ghrelin receptors. The present in vitro results suggest that ghrelin might not be a regulator of intestinal motility in rabbits.

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment due to oil and diesel fuel spills is a serious threat to Arctic fish populations. PAHs produce multiple toxic effects in fish, but disturbance of electrical and contractile activity of the heart seems to be the most negative effect. Our study focused on the effects of fluorene, a tricyclic PAH resembling the well-investigated tricyclic phenanthrene, on major ionic currents and action potential (AP) waveform in isolated ventricular myocytes and on contractile activity in isolated whole hearts of polar navaga cod (Eleginus nawaga). Among the studied currents, the repolarizing rapid delayed rectifier K+ current IKr demonstrated the highest sensitivity to fluorene with IC50 of 0.54 μM. The depolarizing inward currents, INa and ICaL, were inhibited with 10 μM fluorene by 20.2 ± 2.8 % and 27.9 ± 8.4 %, respectively, thereby being much less sensitive to fluorene than IKr. Inward rectifier IK1 current was insensitive to fluorene (up to 10 μM). While 3 μM fluorene prolonged APs, 10 μM also slowed the AP upstroke. Resting membrane potential was not affected by any tested concentrations. In isolated heart experiments 10 μM fluorene caused modest depression of ventricular contractile activity. Thus, we have demonstrated that fluorene, a tricyclic PAH present in high quantities in crude oil, strongly impacts electrical activity with only slight effects on contractile activity in the heart of the polar fish, the navaga cod.

BACKGROUND: Impaired interstitial cells of Cajal (ICCs) are central to the pathophysiology of acute cholecystitis (AC). Common bile duct ligation is a common model of AC, producing acute inflammatory changes and decrease in gallbladder contractility.
OBJECTIVE: To investigate the origin of slow wave (SW) in the gallbladder and the effect of ICCs on gallbladder contractions during the process of AC.
METHODS: Methylene blue (MB) with light was used to establish selective impaired ICCs gallbladder tissue. Gallbladder motility was assessed using the frequency of SW and gallbladder muscle contractility in vitro in normal control (NC), AC12h, AC24h, and AC48h groups of guinea pigs. Hematoxylin and eosin and Masson-stained gallbladder tissues were scored for inflammatory changes. ICCs pathological changes alterations were estimated using immunohistochemistry and transmission electron microscopy. The alterations of c-Kit, α-SMA, cholecystokinin A receptor (CCKAR), and connexin 43 (CX43) were assessed using Western blot.
RESULTS: Impaired ICCs muscle strips resulted in the decrease in gallbladder SW frequency and contractility. The frequency of SW and gallbladder contractility were significantly lower in the AC12h group. Compared with the NC group, the density and ultrastructure of ICCs were remarkably impaired in the AC groups, especially in the AC12h group. The protein expression levels of c-Kit were significantly decreased in the AC12h group, while CCKAR and CX43 protein expression levels were significantly decreased in the AC48h group.
CONCLUSIONS: Loss ICCs could lead to a decrease in gallbladder SW frequency and contractility. The density and ultrastructure of ICCs were clearly impaired in the early stage of AC, while CCKAR and CX43 were significantly reduced at end stage.

(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca2+. In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (-)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.

Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract.
Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms.
Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program.
Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Fine particulate matter (PM2.5) exposure correlates with airway obstruction, but the mechanism remains to be fully elucidated. We aim to investigate the role of exosomal circular RNAs (circRNAs)-mediated communication between airway epithelial cells and airway smooth muscle cells in PM2.5-induced airway obstruction. RNA sequencing revealed that acute PM2.5 exposure altered the expression profiles of 2904 exosomal circRNAs. Among them, exosomal hsa_circ_0029069 (spliced from CLIP1, thus termed circCLIP1 hereafter) with a loop structure was upregulated by PM2.5 exposure and mainly encapsulated in exosomes. Then, the biological functions and the underlying mechanisms were explored by Western blot, RNA immunoprecipitation and RNA pull-down, etc. Phenotypically, exosomal circCLIP1 entered recipient cells, inducing mucus secretion in recipient HBE cells and contractility of sensitive HBSMCs. Mechanistically, circCLIP1 was upregulated by METTL3-mediated N6-methyladenine (m6A) modification in PM2.5-treated producer HBE cells and exosomes, then enhancing the expression of SEPT10 in recipient HBE cells and sensitive HBSMCs. Our study revealed that exosomal circCLIP1 played a critical role in PM2.5-induced airway obstruction and provided a new potential biomarker for the assessment of PM2.5-related adverse effects.

Measuring myocardial contractility is of great value in exploring cardiac pathogenesis and quantifying drug efficacy. Among the biosensing platforms developed for detecting the weak contractility of a single layer of cardiomyocytes (CMs), thin brittle metal membrane sensors with microcracks are highly sensitive. However, their poor stability limits the application in long-term measurement. Here, we report a high stability crack sensor fabricated by deposition of a 105 nm thick Ag/Cr with microcracks onto a carbon nanotubes-polydimethylsiloxane (CNT-PDMS) layer. This brittle-tough bilayer crack sensor achieved high sensitivity (gauge factor: 108 241.7), a wide working range (0.01-44%), and high stability (stable period >2 000 000 cycles under the strain caused by a monolayer of CMs). During 14-day continuously monitoring CMs culturing and drug treatment testings, the device demonstrated high sensitivity and stability to record the dynamic change caused by contractility of the CMs.

The purpose of this study was to investigate the effects of oxymatrine and matrine on integrated cardiac function in rats using pressure-volume loop analysis. A pressure-volume loop catheter was advanced into the left ventricle in anesthetized rats. Steady-state hemodynamic and load-independent parameters were recorded before and after oxymatrine or matrine injection. Oxymatrine (200 mg/kg) and matrine (50, 100 mg/kg) significantly increased the preload recruitable stroke work, slope of maximal systolic pressure increase (dP/dtmax) - end-diastolic volume relationship, end-systolic elastance and volume axis intercept (V0), which are load-independent parameters. Furthermore, the observed increased cardiac efficiency, along with the decreased ventricular arterial coupling, pressure volume area and potential energy, reflect improved mechanoenergetics in oxymatrine (200 mg/kg) and matrine (25, 50 or 100 mg/kg) treated rats respectively. In addition, matrine (25, 50 mg/kg) decreased end-systolic volume and end-diastolic volume, and increased ejection fraction; matrine at 100 mg/kg further decreased end-systolic volume, end-diastolic volume, stroke volume and stroke work, shortened the time constant of left ventricular pressure decay, and increased dP/dtmax, and heart rate. These results suggest that both oxymatrine and matrine enhance left ventricular contractility and improve cardiac mechanical function. As the dose of matrine was much lower than that of oxymatrine, the effect of matrine on myocardial contractility was stronger than that of oxymatrine.