Decrease In Amplitude Of Contractions Research Articles

Abstract Mo092: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an In Vitro Model to Evaluate the Efficacy of a Gene Replacement Therapy for Plakophilin-2- Associated Arrhythmogenic Right Ventricular Cardiomyopathy

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene Plakophilin-2 ( PKP2 ) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an in vitro ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic PKP2 variant (c.2146G>C), as a platform to test a PKP2 -gene replacement approach developed at Tenaya Therapeutics for the treatment of PKP2 -associated ARVC patients. Methods: Three isogenic human iPSC lines (wild-type, heterozygous and homozygous PKP2 mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9: PKP2 ) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated. Results: A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9: PKP2 to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function. Conclusions: Our PKP2 human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9: PKP2 restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of PKP2 -ARVC, and the potential for AAV9: PKP2 as a one-time dose to correct the genetic cause of disease in individuals with PKP2 -associated ARVC.

Abstract P2036: Induced Pluripotent Stem Cell- Derived Cardiomyocytes Provide New Insights Of The Pathogenic Consequences Of The Novel Frameshift Variant (c.5769delG) In MYH7 Gene

Background: the genetic basis of hypertrophic cardiomyopathy is pleiotropic; A novel disease causing frameshift variant (c.5769delG, MYH7 ) in the beta-myosin heavy chain gene has been recently described. However, the mechanisms involved have not been adequately defined. We have used iPSC-CMs to investigate the underlying mechanisms. Methods: We have generated patient-specific iPSCs from clinically and genetically defined HCM patients carrying the pathogenic variant (c.5769delG) (n=3) and compared them to iPSCs from healthy controls (n=3). Optical mapping of calcium transients and cell action potential were performed under different stimulation conditions. iPSC-CMs contractility was recorded via widefield microscopy. Gene and protein expression analyses were performed using RT-PCR, immunoblotting and immunocytochemistry. RNA sequencing data from patients’ myocardial tissues was analyzed to validate gene expression data from iPSC-CMs. Results: HCM iPSCs-CMs showed a significant decrease in calcium release duration (i.e., time to peak) with decrease in calcium transient amplitude, compared to controls. This was associated with an accelerated contraction and a decreased contraction amplitude. Nonetheless, action potential analysis showed no significant changes. Transcriptome analysis of iPSC-CMs and patients’ myocardial tissues showed a significant down regulation of SERCA/ATP2A2 , MYH6 and KCNIP2 . Conclusion: iPSC-CMs provided new insights of the pathogenic consequences of the novel variant in MYH7 gene on calcium handling and contractility. Key words: Hypertrophic Cardiomyopathy, Disease model, MYH7 , iPSC-CMs, Calcium Handling, Contractility.

Effects of bisphenol A and bisphenol F on porcine uterus contractility

IntroductionBisphenols, as endocrine disruptors, may cause a wide range of health problems in humans, but so far, not all of them have been confirmed in animals, including pigs. Since animals are also exposed to bisphenols, we hypothesised that these substances may have an effect on uterine contractility in pigs. Therefore, the aim of the study was to investigate the effect of the most-used bisphenol, bisphenol A (BPA), and a selected analogue, bisphenol F (BPF), on the contractile activity of the pig uterus.Material and MethodsThe investigation utilised smooth muscles from immature pigs (n = 6), cyclic pigs on days 12–14 of the oestrous cycle (n = 6) or early pregnant pigs on days 12–16 of pregnancy (n = 6). Strips of the myometrium were exposed to BPA and BPF at concentrations of 10−13–10−1 M. Smooth muscle contractility was determined with equipment for measuring isometric contractions.ResultsBPA caused a significant decrease in contraction amplitude, and frequency and in myometrial tension in all groups examined. BPF caused a decrease in the amplitude and frequency of contractions in all groups and in myometrial tension in the early pregnant group.ConclusionThe obtained results indicate that both BPA and BPF relaxed the porcine myometrium, but these changes, especially in the amplitude and frequency of contractions, were more evident after BPF treatment. The extent of relaxation is dependent on the physiological status of the animals.

Clinical outcomes of sacral neuromodulation in non-neurogenic, non-obstructive dysuria: A 5-year retrospective, multicentre study in China.

BACKGROUNDManagement of non-neurogenic, non-obstructive dysuria represents one of the most challenging dilemmas in urological practice. The main clinical symptom is the increase in residual urine. Voiding dysfunction is the main cause of dysuria or urinary retention, mainly due to the decrease in bladder contraction (the decrease in contraction amplitude or duration) or the increase in outflow tract resistance. Sacral neuromodulation (SNM) has been used for > 10 years to treat many kinds of lower urinary tract dysfunction. It has become increasingly popular in China in recent years. Consequently, studies focusing on non-neurogenic, non-obstructive dysuria patients treated by SNM are highly desirable.AIMTo assess the outcome of two-stage SNM in non-neurogenic, non-obstructive dysuria.METHODSClinical data of 54 patients (26 men, 28 women) with non-neurogenic, non-obstructive dysuria treated by SNM from January 2012 to December 2016 in ten medical centers in China were retrospectively analyzed. All patients received two or more conservative treatments. The voiding diary, urgency score, and quality of life score before operation, after implantation of tined lead in stage I (test period), and during short-term follow-up (latest follow-up) after implantation of the implanted pulse generator in stage II were compared to observe symptom improvements.RESULTSAmong the 54 study patients, eight refused to implant an implanted pulse generator because of the unsatisfactory effect, and 46 chose to embed the implanted pulse generator at the end of stage I. The conversion rate of stage I to stage II was 85.2%. The average follow-up time was 18.6 mo. There were significant differences between baseline (before stage I) and the test period (after stage I) in residual urine, voiding frequency, average voiding amount, maximum voiding amount, nocturia, urgency score, and quality of life score. The residual urine and urgency score between the test period and the latest follow-up time (after stage II) were also significantly different. No significant differences were observed for other parameters. No wound infection, electrode breakage, or other irreversible adverse events occurred.CONCLUSIONSNM is effective for patients with non-neurogenic, non-obstructive dysuria showing a poor response to traditional treatment. The duration of continuous stimulation may be positively correlated with the improvement of residual urine.

Prolonged nonobstructive urinary retention induced by tibial nerve stimulation in cats.

Nonobstructive urinary retention (NOUR) is a medical condition without an effective drug treatment, but few basic science studies have focused on this condition. In α-chloralose-anesthetized cats, the bladder was cannulated via the dome and infused with saline to induce voiding that could occur without urethral outlet obstruction. A nerve cuff electrode was implanted for tibial nerve stimulation (TNS). The threshold (T) intensity for TNS to induce toe twitch was determined initially. Repeated (6 times) application of 30-min TNS (5 Hz, 0.2 ms, 4-6T) significantly (P < 0.05) increased bladder capacity to 180% of control and reduced the duration of the micturition contraction to 30% of control with a small decrease in contraction amplitude (80% of control), which resulted in urinary retention with a low-voiding efficiency of 30% and a large amount of residual volume equivalent to 130% of control bladder capacity. This NOUR condition persisted for >2 h after the end of repeated TNS. However, lower frequency TNS (1 Hz, 0.2 ms, 4T) applied during voiding partially reversed the NOUR by significantly (P < 0.05) increasing voiding efficiency to 60% and reducing residual volume to 70% of control bladder capacity without changing bladder capacity. These results revealed that tibial nerve afferent input can activate either an excitatory or an inhibitory central nervous system mechanism depending on afferent firing frequencies (1 vs. 5 Hz). This study established the first NOUR animal model that will be useful for basic science research aimed at developing new treatments for NOUR.

Mutation-specific differences in arrhythmias and drug responses in CPVT patients: simultaneous patch clamp and video imaging of iPSC derived cardiomyocytes

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease characterized by arrhythmias under adrenergic stress. Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for CPVT. We characterized electrophysiological properties of CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying different mutations in RYR2 and evaluated effects of carvedilol and flecainide on action potential (AP) and contractile properties of hiPSC-CMs. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion (E3D) and L4115F mutation in RYR2. APs and contractile movement were recorded simultaneously from the same hiPSC-CMs. Differences in AP properties of ventricular like CMs were seen in CPVT and control CMs: APD90 of both E3D (n = 20) and L4115F (n = 25) CPVT CMs was shorter than in control CMs (n = 15). E3D-CPVT CMs had shortest AP duration, lowest AP amplitude, upstroke velocity and more depolarized diastolic potential than controls. Adrenaline had positive and carvedilol and flecainide negative chronotropic effect in all hiPSC CMs. CPVT CMs had increased amount of delayed after depolarizations (DADs) and early after depolarizations (EADs) after adrenaline exposure. E3D CPVT CMs had the most DADs, EADs, and tachyarrhythmia. Discordant negatively coupled alternans was seen in L4115F CPVT CMs. Carvedilol cured almost all arrhythmias in L4115F CPVT CMs. Both drugs decreased contraction amplitude in all hiPSC CMs. E3D CPVT CMs have electrophysiological properties, which render them more prone to arrhythmias. iPSC-CMs provide a unique platform for disease modeling and drug screening for CPVT. Combining electrophysiological measurements, we can gain deeper insight into mechanisms of arrhythmias.

CXCL1 is upregulated during the development of ileus resulting in decreased intestinal contractile activity.

Although the development of ileus is widespread and negatively impacts patient outcomes, the mechanism by which ileus develops remains unclear. The purpose of our study was to examine the contribution of myogenic mechanisms to postoperative ileus development and the involvement of inflammation in mediating intestinal smooth muscle dysfunction. Contractile activity and the effects of CXCL1 were studied in a gut manipulation model. Contraction amplitude in the ileum decreased significantly, while tone increased significantly in response to gut manipulation. Differences in contraction amplitude were affected by tetrodotoxin at earlier time points, but not at later time points. Agonist-induced contractions in the small intestine decreased significantly with ileus development. Intestinal transit slowed significantly after the induction of ileus. Myosin light chain phosphorylation was significantly decreased and edema increased significantly in the intestinal wall. Conditioned media from mechanically activated macrophages depressed intestinal contractile activity. CXCL1 (GroA) was significantly increased in the mechanically activated macrophages and intestinal smooth muscle within 1hour after induction of ileus compared with control cells and sham animals, respectively. Treatment with CXCL1 significantly decreased contraction amplitude and agonist-induced contractile activity and increased tone in the small intestine. In the gut manipulation model, treatment with a CXCR2 antagonist prevented the decrease in agonist-induced contractile activity but not contraction amplitude. These data suggest that CXCL1, released from macrophages during intestinal wall stress, can suppress intestinal contractile activity. CXCL1 is a potential target for preventing or treating ileus in trauma patients.

Sigma‐1 receptor agonists elicit arteriolar dilation via Akt/eNOS activation, and also have endothelial barrier protective properties

Sigma‐1 receptors (σ1) have previously gained considerable attention pertaining to neuroprotection through its actions on calcium homeostasis, and the mitochondria‐associated ER membrane domain. Our laboratory recently showed that the σ1 agonist afobazole causes an endothelial NO‐dependent decrease in contraction amplitude. However, little is known about the mechanisms and functions of σ1 receptor in endothelial cells. In the current study, we investigated how σ1 agonists impact brain arteriolar tone and the Akt/eNOS pathway. Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. The nonselective σ agonist afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM in the presence or absence of the σ1 antagonist BD1047. The selective σ1 agonist, PRE‐084 was used to stimulate human umbilical vein endothelial cells (HUVEC), in which phosphorylation of endothelial NO synthase (eNOS) or Akt phosphorylation on their activation sites (S1177 and S473, respectively) were evaluated by western blotting. NO production was measured by fluorescence microscopy in cells loaded with the indicator dye DAF‐FM. Trans‐endothelial electrical resistance (TER) was used to determine the ability of PRE‐084 to attenuate disruption of endothelial barrier function caused by the the mitochondrial function inhibitors CCCP and antimycin‐A, or the inflammatory cytokine IL‐1β. The results show that afobazole significantly dilated rat brain arterioles, which was inhibited by the σ1 antagonist BD‐1047. Selective activation of σ1 with PRE‐084 significantly increased eNOS phosphorylation on S1177 and this was diminished in the presence of BD‐1047. Akt phosphorylation on S473 following PRE‐084 treatment was also enhanced. In addition, afobazole and PRE‐084 enhanced NO production in endothelial cells. PRE‐084 significantly attenuated decreases in TER caused by IL‐1β or CCCP, but not antimycin‐A, suggesting a potential barrier‐protective role for σ1 receptor, with some limitations that may be related to the source of mitochondrial dysfunction during inflammation. Collectively, our results suggest that σ1 receptor activation can cause vasodilation via the Akt/eNOS pathway in endothelial cells, and that σ1 may enhance endothelial barrier function under inflammatory conditions.Support or Funding InformationSupported by NIH grant R01GM120774.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Kv7 channel modulators on the contractility of diabetic rat stomach in-vitro

Objectives:To study the effect of KV7 channel modulators, Retigabine(KV7 opener) and XE-99 (KV7 blocker), on motility of both fundal and corpal gastric segments in Type I diabetic rats . Method:24 Male Sprague Dawley rats were divided into 2 groups: (I) control& (II) diabetic group (n=12) each were subdivided into 2 subgroups according to the used segment whether fundus or corpus. Each segment group was treated with retigabine and XE-991 on basal and cholinergic stimulated motility. The motility was recorded in-vitro using the Power Lab system. Results: Diabetic stomach significantly showed higher fundal basal amplitude and frequency of contractions when compared with control. Diabetic gastric response to A.ch. was significantly higher as compaired to control. Retigabine, significantly decreased spontaneous contractility of gastric fundal and corpal muscle in diabetic and control rats but diabetic group exhibits significant more decrease in contractility than control. Also, retigabine significantly decreased contractility of cholinergic stimulated fundal and corpal strips with significant more decreased contraction amplitude in diabetic corpus than the control. XE-991, significantly increased spontaneous gastric fundal and corpal contractility in diabetic and control rats but diabetic group exhibits significant more increased contraction in response to XE-991 than the control. Also, XE-991 after A.ch significantly increased the amplitude of fundal and corpal contractility of both diabetic and control groups with significant higher contraction in diabetics , however there is no increase in the tone after A.ch in either groups. Conclusion: KV7 channel modulators could affect gastric activity in type I diabetic and control rats.

Dynamic Action Potential Restitution Contributes to Mechanical Restitution in Right Ventricular Myocytes From Pulmonary Hypertensive Rats.

We investigated the steepened dynamic action potential duration (APD) restitution of rats with pulmonary artery hypertension (PAH) and right ventricular (RV) failure and tested whether the observed APD restitution properties were responsible for negative mechanical restitution in these myocytes. PAH and RV failure were provoked in male Wistar rats by a single injection of monocrotaline (MCT) and compared with saline-injected animals (CON). Action potentials were recorded from isolated RV myocytes at stimulation frequencies between 1 and 9 Hz. Action potential waveforms recorded at 1 Hz were used as voltage clamp profiles (action potential clamp) at stimulation frequencies between 1 and 7 Hz to evoke rate-dependent currents. Voltage clamp profiles mimicking typical CON and MCT APD restitution were applied and cell shortening simultaneously monitored. Compared with CON myocytes, MCT myocytes were hypertrophied; had less polarized diastolic membrane potentials; had action potentials that were triggered by decreased positive current density and shortened by decreased negative current density; APD was longer and APD restitution steeper. APD90 restitution was unchanged by exposure to the late Na+-channel blocker (5 μM) ranolazine or the intracellular Ca2+ buffer BAPTA. Under AP clamp, stimulation frequency-dependent inward currents were smaller in MCT myocytes and were abolished by BAPTA. In MCT myocytes, increasing stimulation frequency decreased contraction amplitude when depolarization duration was shortened, to mimic APD restitution, but not when depolarization duration was maintained. We present new evidence that the membrane potential of PAH myocytes is less stable than normal myocytes, being more easily perturbed by external currents. These observations can explain increased susceptibility to arrhythmias. We also present novel evidence that negative APD restitution is at least in part responsible for the negative mechanical restitution in PAH myocytes. Thus, our study links electrical restitution remodeling to a defining mechanical characteristic of heart failure, the reduced ability to respond to an increase in demand.

Intravesical Botulinum Toxin for Neurogenic Bladder—Can We Just Irrigate the Bladder with Botulinumtoxin?

Historically, patients failing treatment with anti-muscarinic medications were subjected to bladder augmentation or urinary diversion, invasive procedures with significant complications. The approval of botulinum toxin A (BTX-A) intravesical injections revolutionized the treatment of neurogenic bladder (NGB). Ongoing research efforts to find even less invasive methods of treatments are exploring the possibility of intravesical botulinum toxin instillations for the treatment of neurogenic bladder. This paper reviews BTX-A in the treatment of neurogenic bladder, intravesical instillation of other medications in the treatment of this disease, available research on intravesical instillation of BTX-A for neurogenic bladder, and other forms of voiding dysfunction. Initial research has been done on rodents and human subjects. Some studies on BTX-A intravesical instillations have shown preliminary success and others have shown no improvement compared to placebo. Research groups have used a variety of mechanisms to overcome the low permeability of the bladder wall, including protamine sulfate, DMSO, and liposomes. While studies have shown improvements in markers of bladder pain response, a variety of urodynamic study (UDS) parameters, and patient satisfaction scores, none have shown a decrease in contraction amplitude. The most recent development is the use of liposomes as a delivery system for intravesical instillation of BTX. They have seen some preliminary success as some endpoints were met, others not. There is limited research on intravesical instillation of BTX-A for neurogenic detrusor overactivity. While available data suggests some degree of success in modulating symptoms, the data does not show an increase in bladder capacity or decreased voiding pressure. Further research is needed on the impact of BTX-A intravesical instillations in the NGB patient population.

ATP-induced changes in rat skeletal muscle contractility.

Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action.The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors.Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be considered as typical effects of ATP and other purines on skeletal muscles and could not be extrapolated to all warm-blooded animals. Furthermore the role of ATP and its derivatives in the accumulation of vertebrate muscular effort has not been investigated.It is known that in physiological conditions vertebrates may mobilize only up to a third of the maximum muscle force. Why the two-thirds of muscular strength are not used normally but may be used at stress, remains unknown.It is known that the body's adaptive response to stress is a change in the activity of the endocrine system. The leading role in this is given to catechol amines and glucocorticoids, mobilized in significant quantities in blood under stress.We have found previously that incubation of frog sartorius muscle with hydrocortisone resulted in a decrease of contraction amplitude. However, when hydrocortisone was used in combination with ATP, its inhibitory effect on contractile responses disappeared. It is interesting that hydrocortisone had no effect on the inhibitory effect of adenosine. In the following experiments, assessing the effect of hydrocortisone on rat soleus muscle, it was established that hydrocortisone and purines had similar inhibitory effect. When ATP and hydrocortisone were given together the same oppression occurred.To study the effects of ATP and adenosine on contraction parameters of rat skeletal muscle and assess the impact of the catechol amines on these processes.Contractions of rat soleus muscles were recorded isometrically by mechanical sensor Linton FSG-01 (UK) according to standard procedures. The average of muscle parameters received within 30 seconds (30 responses) was treated as one result. Amplitude and time characteristics of the curve reductions were estimated. During all experiments standard Krebs solution flowed through the bath continuously to which agents were added at necessary concentrations. All experimental animals were maintained and prepared for dissection under the European Convention for the Protection of Vertebrate Animals used in scientific experiments. All agents used in the study were supplied by Sigma Chemical Company Ltd. (UK), Tocris Cookson and Research Biochemicals International (USA).The concentration of 100 μM for adenosine is close to saturation [1], and for its predecessor ATP this concentration is created after the passage of a pulse through the synapse [2]. We used this concentration of purines to study the mechanism of action of adenosine and ATP on neuromuscular synapse.The effect of adenosine was partially inhibited in the presence of 100 μM 8-SPT, an antagonist of adenosine receptors. The contraction force of "fast" and "slow" rat skeletal muscles was raised by half in the presence of norepinephrine. In the presence of norepinephrine adenosine exerted its effect fully, but ATP by half reduced its depressor effect on the contraction force of both muscles.1. Norepinephrine increases half times of the reduction of "fast" and "slow" skeletal muscle.2. In the presence of norepinephrine, inhibitory effect of adenosine on contraction force is maintained.3. Inhibitory effect of ATP on contraction force of studied skeletal muscles becomes twice less pronounced in the presence of norepinephrine.We think that reduction of ATP depressive effect on the skeletal muscle by norepinephrine may be an adaptive response to acute stress.

R‐type Ca2+ Channels Contribute to Neural Control of Murine Colonic Motility

BackgroundThe enteric nervous system controls gastrointestinal motility. Multiple subtypes of voltage‐gated Ca2+channels contribute to neurotransmitter release from enteric neurons.ObjectiveWe tested the hypothesis that R‐type Ca2+channels contribute to enteric neurotransmitter release in the mouse colon.MethodsStudies were done in vivo and in vitro using wild type (WT) and a1E Ca2+ channel (R‐type) knockout (KO) mice. Fecal output was measured in vivo. The colonic migrating motor complex (CMMC) was measured in vitro. We used NiCl2 (50 mM) to block R‐type Ca2+ channels , Ω‐conotoxin (CTX, 0.1 mM) to block N‐type Ca2+ channels and tetrodotoxin (TTX, Na+ channel blocker, 0.3 mM) on CMMCs.ResultsThere was no significant difference in fecal output between a1E KO and WT mice. There was no difference in the frequency, propagation speed or amplitude of CMMC contractions between colons from WT and a1E KO mice. NiCl2 slightly decreased contraction amplitude in colons from a1E KO and WT mice. CTX and TTX completely blocked neurogenic contractions and disrupted the CMMC pattern, leaving only spontaneous myogenic contractions behind.ConclusionThese results suggest that either R‐type Ca2+ channels have a minor role in controlling colonic motility, or that there is compensatory upregulation of other Ca2+ types in the constitutive a1E KO mice.*Supported by: R01DK094932

Direct, Differential Effects of Tamoxifen, 4-Hydroxytamoxifen, and Raloxifene on Cardiac Myocyte Contractility and Calcium Handling

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca2+ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca2+ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca2+ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.

Stability of the acetic acid-induced bladder irritation model in alpha chloralose-anesthetized female cats.

Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min “quiet period” (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8th. Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function.

Inter-animal variability in the effects of C-type allatostatin on the cardiac neuromuscular system in the lobster Homarus americanus

Although the global effects of many modulators on pattern generators are relatively consistent among preparations, modulators can induce different alterations in different preparations. We examined the mechanisms that underlie such variability in the modulatory effects of the peptide C-type allatostatin (C-AST; pQIRYHQCYFNPISCF) on the cardiac neuromuscular system of the lobster Homarus americanus. Perfusion of C-AST through the semi-intact heart consistently decreased the frequency of ongoing contractions. However, the effect of C-AST on contraction amplitude varied between preparations, decreasing in some preparations and increasing in others. To investigate this variable effect, we examined the effects of C-AST both peripherally and centrally. When contractions of the myocardium were elicited by controlled stimuli, C-AST did not alter heart contraction at the periphery (myocardium or neuromuscular junction) in any hearts. However, when applied either to the semi-intact heart or to the cardiac ganglion (CG) isolated from hearts that responded to C-AST with increased contraction force, C-AST increased both motor neuron burst duration and the number of spikes per burst by about 25%. In contrast, CG output was increased only marginally in hearts that responded to C-AST with a decrease in contraction amplitude, suggesting that the decrease in amplitude in those preparations resulted from decreased peripheral facilitation. Our data suggest that the differential effects of a single peptide on the cardiac neuromuscular system are due solely to differential effects of the peptide on the pattern generator; the extent to which the peptide induces increased burst duration is crucial in determining its overall effect on the system.

Diabetes is associated with impairment of uterine contractility and high Caesarean section rate

Aims/hypothesisThe prevalence of births worldwide complicated by diabetes mellitus is increasing. In the UK, for example, <25% of diabetic women have a non-instrumental vaginal delivery. Strikingly, more than half the Caesarean sections (CS) in these patients are non-elective, but the reasons for this are not understood. We have tested the hypothesis that poor myometrial contractility as a consequence of the disease contributes to this high CS rate.MethodsWe compared spontaneous, high K depolarisation and oxytocin-induced contractions from diabetic and matched control patients having an elective CS. To investigate the mechanism of any differences we measured intracellular Ca, and performed western blotting and compared the tissues histologically.ResultsThere was significantly decreased contraction amplitude and duration in uteri from diabetic compared with control patients, even when possible confounders such as BMI were analysed. Reduced intracellular calcium signals and expression of calcium entry channels were found in uteruses from diabetic patients, which, along with a reduction in muscle content found on histological examination, could explain the reduced force. Myometrium from diabetic patients was responsive to oxytocin, but still did not reach the levels found in non-diabetic patients.Conclusions/interpretationsThese are the first data investigating myometrium in diabetic patients and they support the hypothesis that there is poorer contractility even in the presence of oxytocin. The underlying mechanism is related to reduced Ca channel expression and intracellular calcium signals and a decrease in muscle mass. We conclude that these factors significantly contribute to the increased emergency CS rate in diabetic patients.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-011-2371-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Feedback From Peripheral Musculature to Central Pattern Generator in the Neurogenic Heart of the Crab Callinectes sapidus: Role of Mechanosensitive Dendrites

The neurogenic heart of decapod crustaceans is a very simple, self-contained, model central pattern generator (CPG)-effector system. The CPG, the nine-neuron cardiac ganglion (CG), is embedded in the myocardium itself; it generates bursts of spikes that are transmitted by the CG's five motor neurons to the periphery of the system, the myocardium, to produce its contractions. Considerable evidence suggests that a CPG-peripheral loop is completed by a return feedback pathway through which the contractions modify, in turn, the CG motor pattern. One likely pathway is provided by dendrites, presumably mechanosensitive, that the CG neurons project into the adjacent myocardial muscle. Here we have tested the role of this pathway in the heart of the blue crab, Callinectes sapidus. We performed "de-efferentation" experiments in which we cut the motor neuron axons to the myocardium and "de-afferentation" experiments in which we cut or ligated the dendrites. In the isolated CG, these manipulations had no effect on the CG motor pattern. When the CG remained embedded in the myocardium, however, these manipulations, interrupting either the efferent or afferent limb of the CPG-peripheral loop, decreased contraction amplitude, increased the frequency of the CG motor neuron spike bursts, and decreased the number of spikes per burst and burst duration. Finally, passive stretches of the myocardium likewise modulated the spike bursts, an effect that disappeared when the dendrites were cut. We conclude that feedback through the dendrites indeed operates in this system and suggest that it completes a loop through which the system self-regulates its activity.

Restoration of contractility in hyperhomocysteinemia by cardiac-specific deletion of NMDA-R1

Homocysteine (HCY) activated mitochondrial matrix metalloproteinase-9 and led to cardiomyocyte dysfunction, in part, by inducing mitochondrial permeability (MPT). Treatment with MK-801 [N-methyl-d-aspartate (NMDA) receptor antagonist] ameliorated the HCY-induced decrease in myocyte contractility. However, the role of cardiomyocyte NMDA-receptor 1 (R1) activation in hyperhomocysteinemia (HHCY) leading to myocyte dysfunction was not well understood. We tested the hypothesis that the cardiac-specific deletion of NMDA-R1 mitigated the HCY-induced decrease in myocyte contraction, in part, by decreasing nitric oxide (NO). Cardiomyocyte-specific knockout of NMDA-R1 was generated using cre/lox technology. NMDA-R1 expression was detected by Western blot and confocal microscopy. MPT was determined using a spectrophotometer. Myocyte contractility and calcium transients were studied using the IonOptix video-edge detection system and fura 2-AM loading. We observed that HHCY induced NO production by agonizing NMDA-R1. HHCY induced the MPT by agonizing NMDA-R1. HHCY caused a decrease in myocyte contractile performance, maximal rate of contraction and relaxation, and prolonged the time to 90% peak shortening and 90% relaxation by agonizing NMDA-R1. HHCY decreased contraction amplitude with the increase in calcium concentration. The recovery of calcium transient was prolonged in HHCY mouse myocyte by agonizing NMDA-R1. It was suggested that HHCY increased mitochondrial NO levels and induced MPT, leading to the decline in myocyte mechanical function by agonizing NMDA-R1.

Staircase sign: A newly described uterine contraction pattern seen in rupture of unscarred gravid uterus

Although extremely rare, rupture of an unscarred gravid uterus poses significant morbidity and mortality to both fetus and mother. In the past, loss of uterine contraction was thought to be characteristic of uterine rupture, while recent evidence shows that uterine contraction pattern is not associated with uterine rupture. We report two cases of rupture in the unscarred term uterus. Both patients were multiparous and denied any past medical complications, previous cesarean delivery or myomectomy. Uterine rupture occurred in the latent phase of labor at 1 cm dilation (Case 1) and during an intravenous oxytocin infusion after three doses of intravaginal misoprostol (Case 2). Case 2 required cesarean hysterectomy and blood transfusion. Case 1 was monitored with an external tocodynamometer, while Case 2 was monitored with an internal pressure transducer. External monitoring demonstrated the classic sign of complete loss of uterine tone. In contrast, internal monitoring demonstrated an increase in uterine resting tone. Both techniques revealed a stepwise gradual decrease in contraction amplitude followed by sudden onset of profound and prolonged fetal bradycardia (staircase sign). In cases of uterine rupture, differing baseline characteristics between contraction patterns were dependent on uterine monitoring technique. In both techniques a stepwise gradual decrease in contraction amplitude was followed by prolonged fetal bradycardia.