Basal Contraction Research Articles

Investigation of the effects of tadalafil on the myocardium via electrical and isometric contractions and cardiac markers in Wistar rats

Purpose: Tadalafil, a long-acting phosphodiesterase-5 (PDE-5) inhibitor, is commonly used in the treatment of erectile dysfunction. This study investigates the contribution of tadalafil in cardiac function by measuring isometric and electrical contractions of myocardium in rats. Materials and Methods: Thirty rats were divided into five groups. Basal electrical contractions were recorded via electrocardiogram (ECG) for intervals PR (ms), QRS (ms), QT (ms), Tp (ms), Te (ms), pathological Q and heart beats/min. Then group 1 received saline for 7 days (control); group 2 received tadalafil 1 mg/kg; group 3 received tadalafil 10 mg/kg; group 4 received tadalafil 1 mg/kg for 7 days; group 5 received tadalafil 10 mg/kg for 7 days. After treatments, electrical contractions were re-performed to analyze the differences in ECG. For isometric contractions, hearts were connected to isometric power transducer to determine myocardial contractile forces (g), durations (ms) and frequencies (Hz). Serum samples were collected for cardiac creatine kinase (CK-MB) and cardiac troponin I via ELISA. Results: We found a significant decrease in CK-MB in Group 2 (395.56±124.38 pg/ml) and 3 (377.81±79.61 pg/ml), compared to Group 1 (575.32±83.54 pg/ml). The differences in cardiac contractile forces, contraction durations or frequencies were not statistically significant. Conclusion: Tadalafil did not exert obvious distruption on myocardial electrical or isometrical contractions. It is noteworthy that tadalafil 1 and 10 mg/kg reduced serum CK-MB. Shortening in QT and decrease in heart rate may have important implications on myocardial functions.

Elucidating the spatio-temporal dynamics of the Plasmodium falciparum basal complex.

Asexual replication of Plasmodium falciparum occurs via schizogony, wherein 16-36 daughter cells are produced within the parasite during one semi-synchronized cytokinetic event. Schizogony requires a divergent contractile ring structure known as the basal complex. Our lab has previously identified PfMyoJ (PF3D7_1229800) and PfSLACR (PF3D7_0214700) as basal complex proteins recruited midway through segmentation. Using ultrastructure expansion microscopy, we localized both proteins to a novel basal complex subcompartment. While both colocalize with the basal complex protein PfCINCH upon recruitment, they form a separate, more basal subcompartment termed the posterior cup during contraction. We also show that PfSLACR is recruited to the basal complex prior to PfMyoJ, and that both proteins are removed unevenly as segmentation concludes. Using live-cell microscopy, we show that actin dynamics are dispensable for basal complex formation, expansion, and contraction. We then show that EF-hand containing P. falciparum Centrin 2 partially localizes to this posterior cup of the basal complex and that it is essential for growth and replication, with variable defects in basal complex contraction and synchrony. Finally, we demonstrate that free intracellular calcium is necessary but not sufficient for basal complex contraction in P. falciparum. Thus, we demonstrate dynamic spatial compartmentalization of the Plasmodium falciparum basal complex, identify an additional basal complex protein, and begin to elucidate the unique mechanism of contraction utilized by P. falciparum, opening the door for further exploration of Apicomplexan cellular division.

Myofilament calcium sensitization in non-human primate lower esophageal sphincter and esophagus

Background/Objectives: Peristalsis in the esophagus (ESO) moves ingested contents into the stomach. The lower esophageal sphincter (LES) is located at the distal end of the ESO and generates tone to prevent reflux of gastric contents. Previous studies suggest that the expression and/or phosphorylation of proteins involved in myofilament Ca2+ sensitization differs between phasic and tonic smooth muscles (Brozovich et al., 2016; PMID: 27037223). We hypothesize that differences in expression and/or phosphorylation of these proteins contributes to differences in contractile activity between LES and ESO. The aims were to 1) characterize contractile activity in Cynomolgus monkey LES and ESO, 2) evaluate myofilament Ca2+ sensitization protein expression and phosphorylation, 3) examine changes in phosphorylation when contractile activity is enhanced or inhibited pharmacologically. Methods: Contraction was assessed in strips of LES and ESO by isometric tension recordings. Carbachol (CCh, 1 μM) was used to enhance contractions. ROCK2 (SR3677; 2 μM) or CavL (nifedipine; 1 μM) antagonists were used to inhibit contractions. Tissues were snap frozen in liquid nitrogen after peak response and prepared for protein expression analysis using Jess immunoelectrophoresis. Results: Under basal conditions, the LES exhibited tone whereas the ESO had phasic activity. CCh significantly increased contractions. Nifedipine and SR3677 significantly decreased basal and CCh-induced contractions with nifedipine having the greater effect. MLCK and MYPT1 expression was significantly greater in LES than ESO while LC20 and CPI-17 expression was greater in ESO. ROCK2 expression was similar in LES and ESO. Under basal conditions MYPT1 and LC20 phosphorylation was greater in LES. MYPT1 and LC20 phosphorylation was decreased by SR3677 but not nifedipine in both tissues. CCh increased MYPT1 and LC20 phosphorylation in both tissues though was greater in LES. SR3677 inhibited phosphorylation of both proteins in LES and ESO. Discussion/Conclusions: The differences in expression and phosphorylation of key proteins involved in myofilament Ca2+ sensitization likely contribute to differences in contractile activity. Notably, there is greater LC20 phosphorylation in LES than ESO under basal conditions and with CCh. Furthermore, there is an important role for ROCK2 in regulating basal contractile activity in LES and ESO as both contraction and phosphorylation of MYPT1 and LC20 are reduced by SR3677. While nifedipine significantly reduced contractile activity in LES and ESO, it did not reduce phosphorylation of MYPT1 indicating that the ROCK2 pathway is independent of Ca2+ influx. These findings may improve understanding of the pathophysiology associated with esophageal dysmotility. NIH DK129528 to CAC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation mice†.

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) is one of the most abundant potassium channels in the myometrium. Previous work conducted by our group has identified a link between inflammation, BKCa channels and excitability of myometrial smooth muscle cells. Here, we investigate the role of BKCa channels in spontaneous and lipopolysaccharide (LPS)-stimulated uterine contraction to gain a better understanding of the relationship between the BKCa channel and uterine contraction in basal and inflammatory states. Uteri of C57BL/6J mice on gestational day 18.5 (GD18.5) were obtained and either fixed in formalin or used immediately for tension recording or isolation of primary myocytes for patch-clamp. Paraffin sections were used for immunofluorescenctdetection of BKCa and Toll-like receptor (TLR4). For tension recordings, LPS was administered to determine its effect on uterine contractions. Paxilline, a BKCa inhibitor, was used to dissect the role of BKCa in uterine contraction in basal and inflammatory states. Finally, patch-clamp recordings were performed to investigate the relationship between LPS, the BKCa channel and membrane currents in mouse myometrial smooth muscle cells (mMSMCs). We confirmed the expression of BKCa and TLR4 in the myometrium of GD18.5 mice and found that inhibiting BKCa channels with paxilline suppressed both spontaneous and LPS-stimulated uterine contractions. Furthermore, application of BKCa inhibitors (paxilline or iberiotoxin) after LPS inhibited BKCa channel activity in mMSMCs. Moreover, pretreatment with BKCa inhibitor or the TLR4 inhibitor suppressed LPS-activated BKCa currents. Our study demonstrates that BKCa channels are involved in both basal and LPS-stimulated uterine contraction in pregnant mice.

Chemical composition, in vitro and in silico approaches of the relaxant effect of the jejunum using Thymus algeriensis Boiss. and Reut essential oil

BackgroundGastrointestinal problems are among the most common diseases in Morocco people, who frequently use aromatic and medicinal plants to eliminate these problems. Thymus algeriensis is one of these plants widely used to treat gastrointestinal problems. This research aimed to identify the phytochemical compounds present in Thymus algeriensis essential oil (TaEO), assess its acute toxicity, and investigate its potential myorelaxant and antispasmodic effects. MethodsIn vitro experiments were conducted on rat and rabbit jejunum using an isotonic transducer and in silico docking calculations were conducted on l-type voltage-gated Ca2+and muscarinic receptor active sites. ResultsThe GC–MS analysis of TaEO revealed the presence of 11 compounds, with 89.77 % of the compounds identified being oxygenated monoterpenes, among which Thujone dominates the composition with 32.89 %. The administration of TaEO did not produce any observable signs of toxicity or mortality at 1 g/Kg.bw. TaEO exhibit a dose-dependent decrease in the basal contractions of the rabbit jejunum with an IC50 value of 5.66 ± 1.88 µg/mL. In addition, TaEO induced an antispasmodic effect on KCl and CCh-induced contractions in the rat jejunum. The inhibitory effect demonstrated are similar to those produced by a non-competitive antagonist of voltage-dependent calcium channel and cholinergic receptors. These results are boosted by the docking study when we showed that Thujone could bind to the active site of muscarinic receptor and, l-type voltage-gated Ca2+ with a binding energy equal to -59.401 and, -47.441 Kcal/mol respectively. ConclusionsThese results provide strong confirmation for the traditional use of Thymus algeriensis in Moroccan as an effective antispasmodic remedy and give a new lead to find a phytomedicament against gastrointestinal problems.

IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels.

Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca2+-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca2+ release from sarcoendoplasmic reticulum Ca2+ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca2+ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca2+ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca2+ imaging of LMCs using the genetically encoded Ca2+ sensor GCaMP6f demonstrated an elongation of the Ca2+ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca2+ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation.

Effect of chronic administration of magnesium supplement (magnesium glycinate) on male albino wistar rats’ intestinal (Ileum) motility, body weight changes, food and water intake

Recent researches suggests magnesium as an adjuvant medication for COVID 19 patients. Magnesium relaxes skeletal muscles, an effect when prolonged in intestinal smooth muscles can cause severe discomfort such as bloating, vomiting, constipation and nausea. The objectives of this study was to ascertain if magnesium will cause relaxation of the intestinal (ileum) smooth muscles as it does in skeletal muscles. Also, this research seeks to find out the receptor pathway through which magnesium will alter motility in the gut using acetylcholine, atropine and propranolol. Ten male albino wistar rats (100–150 g) were randomly assigned into two groups (control and magnesium treated) (n = 5). Animals were acclimatized for two weeks before treatment which lasted for 6 weeks. Magnesium treated animals received oral magnesium glycinate (1600 mg/70 kg) daily while control group receive normal saline of equal volume. All animals had free access to food and water ad libitum. Results were analyzed at statistical level of P < 0.05. Body weight changes, food and water intake were not statistically significant. Basal contractions of ileum in magnesium treated group were significantly lower compared to control group. Propranolol significantly increased the percentage relaxation in magnesium treated group compared to the control. Atropine significantly decrease the percentage relaxation in magnesium treated group compared to the control. Higher doses of acetylcholine (10−5 and 10−4) increased the contractions in magnesium treated group. Conclusively, magnesium decreases motility of the intestine through beta adrenergic receptor pathway. Intake of magnesium for long period should be closely monitored to avoid the discomforting symptoms earlier stated.

Increased CaMKII activation and contrast changes of cardiac β1-and β3-Adrenergic signaling pathways in a humanized angiotensinogen model of hypertension

AimsUpregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) contributes to the pathogenesis of cardiovascular disease, including hypertension. Transgenic rats expressing the human angiotensinogen gene [TGR (hAGT)L1623] are a new novel humanized model of hypertension that associates with declines in cardiac contractile function and β-adrenergic receptor (AR) reserve. The molecular mechanisms are unclear. We tested the hypothesis that in TGR (hAGT)L1623 rats, left ventricular (LV) myocyte CaMKIIδ and β3-AR are upregulated, but β1-AR is down-regulated, which are important causes of cardiac dysfunction and β-AR desensitization. Main methodsWe compared LV myocyte CaMKIIδ, CaMKIIδ phosphorylation (at Thr287) (pCaMKIIδ), and β1-and β3-AR expressions and determined myocyte functional and [Ca2+]I transient ([Ca2+]iT) responses to β-AR stimulation with and without pretreatment of myocytes using an inhibitor of CaMKII, KN-93 (10−6 M, 30 min) in male Sprague Dawley (SD; N = 10) control and TGR (hAGT)L1623 (N = 10) adult rats. Key findingsHypertension in TGR (hAGT)L1623 rats was accompanied by significantly increased LV myocyte β3-AR protein levels and reduced β1-AR protein levels. CaMKIIδ phosphorylation (at Thr287), pCaMKIIδ was significantly increased by 35%. These changes were followed by significantly reduced basal cell contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. Isoproterenol (10−8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax, and [Ca2+]iT. Moreover, only in TGR (hAGT)L1623 rats, pretreatment of LV myocytes with KN-93 (10−6 M, 30 min) fully restored normal basal and isoproterenol-stimulated myocyte contraction, relaxation, and [Ca2+]iT. SignificanceLV myocyte CaMKIIδ overactivation with associated contrast changes in β3-AR and β1-AR may be the key molecular mechanism for the abnormal contractile phenotype and β-AR desensitization in this humanized model of hypertension.

Abstract 9703: Cardiac β 3 -Adrenergic Receptor Deficiency Mice Protected from Cardiac Aging: Insights into the Cellular and Molecular Mechanism of Myocardial Aging

Background: We have shown previously that aging-caused declines in cardiac function and β-adrenergic reserve were prevented in β 3 -adrenergic receptor (AR) knockout (β 3 KO) aged mice. The cellular and molecular mechanisms are unclear. We hypothesize that reversal of aging-induced downregulations of cardiac SR Ca 2+ -ATPase (SERCA2a) and β 1 -AR may lead to normalization of myocyte [Ca 2+ ] i regulation, β-AR regulation, and preserving normal myocyte function, thereby preventing aging-induced cardiac dysfunction and eliminating myocardial aging. Methods: We compared protein levels of β 1 - and β 3 -AR, and SERCA2a, myocyte contractile, [Ca 2+ ] i transient ([Ca 2+ ] iT ) and calcium current (I Ca,L ) responses to isoproterenol (ISO, 10 -8 M) in LV myocytes obtained from 2 young (Y) (~6 m) and 2 aged (A) (~28 m) groups (7/group) of wild-type (WT) and β 3 KO mice, respectively. Results: Compared with YWT, AWT myocytes had significantly decreased protein levels of SERCA2a (AWT: 0.24 vs YWT: 0.67), and β 1 -AR (0.35 vs 0.59), but increased β 3 -AR (0.36 vs 0.16) with reduced basal cell contraction (dL/dt max ) (85.3 vs 126.8μm/s), relaxation (dR/dt max ) (66.7 vs 102.1 μm/s), [Ca 2+ ] iT (0.18 vs 0.24), and I Ca,L (4.3 vs 7.6 pA/pF). These changes were accompanied by diminished ISO-stimulated inotropic response. In AWT myocytes, ISO caused significantly less increases in dL/dt max (34% vs 82%), dR/dt max (26% vs 64%), [Ca 2+ ] iT (16% vs 38%) and I Ca,L (15% vs 34%). Versus YWT, Yβ 3 KO did not alter basal myocyte contraction and relaxation and response to ISO stimulation, but had significantly increased protein levels of SERCA2a (1.41 vs 0.61) and unchanged β 1 -AR (0.65 vs 0.62). Aβ 3 KO mice had similar alterations. Importantly, in contrast to AWT, in Aβ 3 KO myocytes, there was increased SERCA2a (1.3) associated with normal basal cell contraction and relaxation, and preserved ISO-stimulated inotropic response. ISO caused similar increases in dL/dt max (Aβ 3 KO: 81% vs Yβ 3 KO: 83%), dR/dt max (68% vs 62%), [Ca 2+ ] iT (31% vs 32%) and I Ca,L (30% vs 32%). Conclusions: β 3 KO prevents aging-caused downregulations of cardiac β 1 -ARs and SERCA2a, leading to the preservation of myocyte function, [Ca 2+ ] iT , I Ca,L and β-adrenergic reserve in aged hearts. Thus, β 3 -AR may provide a new target for cardiac aging.

Prolonged inhibition of bladder function is evoked by low-amplitude electrical stimulation of the saphenous nerve in urethane-anesthetized rats.

To better understand the effects of saphenous nerve (SN) stimulation on bladder function, we investigated the duration of electrical stimulation as a key variable in eliciting urodynamic changes. SN stimulation is a novel approach to electrically modulating bladder function. In previous animal studies, bladder-inhibitory responses were evoked by low-amplitude (25 μA) stimulus pulses applied in short-duration (10min) trials and at frequencies between 10 and 20 Hz. Experiments were performed in urethane-anesthetized rats that were separated into three groups: intravesical saline infusion + SN stimulation (group A), intravesical 0.1% acetic acid infusion + SN stimulation (group B), and intravesical saline infusion + no SN stimulation (group C). Changes in bladder function- basal bladder pressure (P base ), contraction amplitude (ΔP), and inter-contraction interval (T ICI )-were measured in response to stimulation trials applied for different durations (10, 20, and 40 min). Trials were also repeated at frequencies of 10 and 20 Hz. In group A, longer-duration (40 min) stimulation trials applied at 10Hz evoked overflow incontinence (OI) episodes that were characterized by significant changes in P base (122.7 ± 9.1%, p=0.026), ΔP (-60.8 ± 12.8%, p=0.044), and T ICI (-43.2 ± 13.0%, p=0.031). Stimulation-evoked OI was observed in 5 of 8 animals and lasted for 56.5 ± 10.7min. In contrast, no significant changes in bladder function were observed in either group B or group C. Our findings show that longer-duration trials consisting of electrical pulses applied at 10Hz are important stimulation parameters that elicit inhibitory bladder responses in anesthetized rodents.

Women's Orgasms Determined by Autodetection of Pelvic Floor Muscle Contractions Using the Lioness “Smart” Vibrator

ABSTRACT Introduction Bluetooth biofeedback devices have become popular for the detection of physiological responses by individuals. In the past, pelvic floor contractions during sexual self-stimulation in women have been detected using electrodes placed on the surface of the skin. The Lioness biofeedback unit is a new “smart vibrator” self-stimulation device that detects the force of pelvic floor contractions in two sensors at either side of the instrument that collect continuous pressure (in grams force), temperature, accelerometer, and gyroscope measurements at a sampling rate of 12 Hz. It connects by Bluetooth to a secure internet server from which women can download their pelvic floor output during periods of self-stimulation. Objective To validate the ability of the Lioness vibrator to detect the orgasm patterns of individual women reliably relative to a control condition (Lioness inside the vagina but without self-stimulation). Methods A sample of 54 women who use the Lioness Vibrator 2.0 were obtained after informed consent for their participation in the Lioness Sex Research Platform. Women were asked to provide outputs from one control condition and several orgasm conditions. For the orgasm conditions, women were instructed to self-stimulate to one orgasm and then turn the unit off 2 min after orgasm was attained. This allowed a blind rater to determine when orgasms occurred in the outputs and to observe the type of changes in pelvic floor contractions that preceded and followed each orgasm. Results Each woman had a predominant pattern of pelvic floor contractions at orgasm that followed generally one of three patterns or types: A “wave” (a short burst of pelvic contractions that were preceded by an entraining rhythm of pelvic floor tension and release), a “volcano” (orgasm preceded by increasing upward pelvic floor tensions), and an “avalanche” (higher pelvic floor basal contractions maintained throughout self-stimulation, but a downward contraction profile during and after orgasm). In many cases, women's subjective perception of orgasm termination occurred after the cessation of pelvic floor contractions, although prior to smaller contractions (aftershocks) that occurred as the pelvic floor muscles returned to a basal state. These patterns of orgasm are consistent with early reports by Masters and Johnson of three distinct orgasm patterns in women. Conclusions The Lioness allows researchers a new opportunity to examine orgasm patterns in women within more ecologically valid environments that are conducive to sexual responding (e.g., one's home). The device has good test-retest reliability and should be able to detect differences in orgasm patterns in response to treatments for sexual arousal, desire, and orgasm disorders. Disclosure Yes, this is sponsored by industry/sponsor: SmartBod Incorporated DBA Lioness, Oakland CA Clarification Industry funding only - investigator initiated and executed study Any of the authors act as a consultant, employee or shareholder of an industry for: SmartBod Incorporated DBA Lioness

Lippia origanoides essential oil induces tocolytic effect in virgin rat uterus and inhibits writhing in a dysmenorrhea mouse model

Ethnopharmacological relevanceThe species Lippia origanoides Kunth, popularly known as “salva-de-marajó”, is used in Brazilian traditional “quilombola” communities to treat menstrual cramps and uterine inflammation. Aim of the studyEvaluate the spasmolytic activity of Lippia origanoides essential oil (LOO) on experimental models of uterine conditions related to menstrual cramps and investigate its mechanism of action. Materials and methodsVirgin rat-isolated uterus was mounted in the organ bath apparatus to evaluate the spasmolytic effect of LOO on basal tonus and contractions induced by carbachol, KCl, or oxytocin. We used pharmacological agents to verify the relaxation mechanism of LOO. The evaluation of uterine contractility in virgin rats, after treatment with LOO for three consecutive days, was carried out by the construction of a concentration-response curve with oxytocin or carbachol. The primary dysmenorrhea animal model was replicated with an injection of estradiol cypionate in female mice for three consecutive days, followed by intraperitoneal application of oxytocin. ResultsLOO relaxed the rat uterus precontracted with 10−2 IU/mL oxytocin (logEC50 = 1.98 ± 0.07), 1 μM carbachol (logEC50 = 1.42 ± 0.07) or 60 mM KCl (logEC50 = 1.53 ± 0.05). It was also able relax uterus on spontaneous contractions (logEC50 = 0.41 ± 0.05). Preincubation with glibenclamide, propranolol, phentolamine or L-NAME in contractions induced by carbachol did not alter significantly the relaxing effect of LOO. However, in the presence of 4-aminopyridine, CsCl or tetraethylammonium there was a reduction of LOO potency, whereas the blockers methylene blue, ODQ, aminophylline and heparin potentiated the LOO relaxing effect. Preincubation with LOO in a Ca2+ free medium at concentrations of 27 μg/mL or 81 μg/mL reduced the contraction induced by carbachol. The administration of LOO for 3 days did not alter uterus contractility. The treatment with LOO at 30 or 100 mg/kg intraperitoneally, or 100 mg/kg orally, inhibited writhing in female mice. The association of LOO at 10 mg/kg with nifedipine or mefenamic acid potentiated writhing inhibition in mice. ConclusionsThe essential oil of L. origanoides has tocolytic activity in rat isolated uterus pre-contracted with KCl, oxytocin, or carbachol. This effect is possibly related to the opening of potassium channels (Kir, KV, and KCa), cAMP increase, and diminution of intracellular Ca2+. This relaxant effect, probably, contributed to reduce the number of writhings in an animal model of dysmenorrhea being potentiated by nifedipine or mefenamic acid. Taken together, the results here presented indicate that this species has a pharmacological potential for the treatment of primary dysmenorrhea, supporting its use in folk medicine.

Contractility assessment using hiPSC-CMs with alignment

Evaluation of cardiomyocyte contraction using hiPSC-CMs holds great promise to predict drug-induced heart failures in vitro. However, it has been difficult to detect the positive inotropic effects of drug using hiPSC-CMs. Due to the immature property, hiPSC-CMs do not show alignment. In this study, we evaluated whether hiPSC-CM with alignment can detect the drug-induced positive inotropic effects. We used hiPSC-CM (iCell Cardiomyocyte, FCDI), which were cultured using commercially available plates for alignment. After long-term culture, we found that the aligned culture improved the basal contraction velocity, which were measured by motion vector analysis (SI8000, Sony). To understand the molecular mechanisms of the contraction improvement, we performed next-gene sequencing analysis. Gene expression levels of ion channels and metabolism-related factors were up-regulated by alignment. In addition, we observed that isoproterenol enhanced contraction velocity in hiPSC-CMs with alignment in a dose-dependent manner. These results suggests that the aligned culture of hiPSC-CMs facilitates the maturation and the responses to isoproterenol.

Abstract 9052: Upregulation of Cardiac SERCA 2a is a Key Molecular Mechanism of the Protective Effect on Myocardial Aging in Beta3-Adrenergic Receptor Deficiency Mice

Background: We have shown previously that aging-induced cardiomyocyte dysfunction and impaired β-adrenergic regulation were prevented in β 3 -adrenergic receptor (AR) knockout (β 3 KO) aged mice. However, the molecular mechanism is unclear. We hypothesize that reversal of aging-induced down-regulation of SR Ca 2+ -ATPase (SERCA 2a) and β 1 -ARs by β 3 -AR deficiency may play a key role for the protective effect. Methods: We compared cardiac SERCA 2a, phospholamban (PLB), β 1 - and β 3 -AR protein expression and myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to isoproterenol (ISO, 10 -8 M) in left ventriclar (LV) myocytes obtained from 2 young (Y)(~4-6 mo) and 2 aged (A) (~18-24 mo) groups (7/group) of wild-type (WT) and β 3 KO mice, respectively. Results: Compared with YWT, AWT myocytes had significantly decreased SERCA 2a (66%, AWT: 0.21 vs YWT: 0.61), SERCA 2a/PLB ratio (0.76 vs 1.77) and β 1 -AR (33%, 0.39 vs 0.58), but increased β 3 -AR (93%, 0.27 vs 0.14). These changes were associated with reduced basal cell contraction (dL/dt max ) (AWT: 84.3 vs YWT: 124.8 μm/s), relaxation (dR/dt max ) (60.1 vs 89.8 μm/s) and [Ca 2+ ] iT (0.17 vs 0.22). This was accompanied by diminished ISO-stimulated positive inotropic effect; in AWT myocytes, ISO (10 -8 M) caused significantly less increases in dL/dt max (AWT: 33% vs YWT: 81%), dR/dt max (24% vs 58%), and [Ca 2+ ] iT (14% vs 34%). Compared with YWT, Yβ 3 KO did not alter basal contraction and ISO response of myocytes, but SERCA 2a (Yβ 3 KO: 1.2 vs YWT: 0.61) and SERCA 2a/PLB ratio were doubled with relatively unchanged β 1 -AR (0.62 vs 0.58); Aβ 3 KO mice had similar alterations. Importantly, in contrast to AWT, in Aβ 3 KO myocytes, the increased SERCA 2a (1.1) and β 1 -AR (0.61) correlated with normal basal cell contraction and relaxation with preserved ISO-stimulated positive inotropic response. ISO caused similar increases in dL/dt max (84% vs 82%) and [Ca 2+ ] iT (32% vs 34%) compared to Yβ 3 KO mice. Conclusions: Chronic β 3 -AR deficiency upregulates cardiac SERCA 2a, prevents aging-induced downregulation of β 1 -ARs, and leads to the preservation of myocyte function, [Ca 2+ ] iT , and β-adrenergic responsiveness in aged hearts. Thus, antagonizing β 3 -AR could be a new therapeutic strategy for age-related decline in myocardial function.

Abstract 9083: Beta1-Adrenergic cAMP Signaling is Uncoupled after Acute Treatment of Anthrax Lethal Toxin in Cardiomyocytes in Normal Rats

Background: Our recent in vivo study has shown that anthrax lethal toxin (LeTx) causes time-dependent progressive depression of left ventricular (LV) contractility and β-adrenergic reserve. However, the mechanisms are poorly understood. We tested the hypothesis that LeTx-induced altered cardiac subtypes of β-adrenergic receptor (AR) regulation may contribute to this effect. Methods: We examined acute exposure of LeTx caused alterations of myocyte β 1 - and β 3 -AR expression and myocyte basal contraction and relaxation and responses to isoproterenol (ISO), a β-AR agonist, and colforsin (COLF), a cAMP activator. Cardiomyocytes were isolated from eight adult normal rats incubated for 6 hours in study buffer without and with LeTx (10 nM PA and 1 nM LF). Result: Compared with controls, LeTx treatment caused no significant changes on basal myocyte contraction and relaxation. Myocyte β 1 -AR (0.47±0.04 vs 0.46±0.02) and β 3 -AR (0.21±0.02 vs 0.23±0.02) protein levels also remained relatively unchanged. In controls, superfusion with ISO (10 -8 M) caused about 46% increase in cell percent shortening (SA, 14.5 vs 10.0%) and 83% and 44% increases in peak velocity shortening (dL/dt max , 270.0 vs 147.7 μm/s) and relengthening (dR/dt max , 172.2 vs 119.6 μm/s), respectively. However, after LeTx treatment, myocyte positive inotropic functional response to β-AR stimulation was significantly decreased. In LeTx-treated myocytes, ISO produced significantly less increases in SA (25%, 9.9 vs 12.3%), dL/dt max (29%, 130.5 vs 183.6 μm/s) and dR/dt max (32.4%, 87.5 vs 115.8 μm/s). In contrast, superfusion of COLF (10 -6 M) caused similar increases in SA (43.1 vs 43.7%), dL/dt max (77.4 vs 83.2%) and dR/dt max (35.2 vs 30.8%). In addition, compared with normal controls, LeTx-treated myocyte contractile responses to zinterol (ZIN, 10 -5 M), a highly selective β 2 -AR agonist, and BRL-37,344 (BRL, 10 -8 M), a selective β 3 -AR agonist, were also unaffected. Conclusion: Acute treatment of LeTx causes cardiac myocyte β 1 -AR uncoupling from cAMP-mediated signaling and impairs myocyte β-adrenergic reserve. These results suggest that the early changes in cardiac subtypes of β-AR signal transduction system may mediate the connection between the acute and chronic cardiac action of LeTx.

Abstract 9024: Molecular Mechanism of Chronic Beta3-Adrenergic Receptor Antagonist Caused Regression of Diabetic Cardiomyopathy in a Murine Model With Type 2 Diabetes

Background: Diabetic cardiomyopathy (DCM) is a serious complication of diabetes (DM). As yet, no effective therapeutic strategies exist. Recently, we found that DM-induced LV dysfunction was prevented by chronic treatment with a β 3 -adrenergic receptor (AR) antagonist (β 3 ANT). However, the molecular mechanism is unclear. We hypothesized that reversal of DM-induced detrimental alterations on the expressions of cardiac SR Ca 2+ -ATPase (SERCA2a), β-ARs, nitric oxide synthase (NOS) by β 3 ANT may play a key role for its salutary role in DCM. Methods: The protein levels of myocyte β 1 - and β 3 - AR, SERCA2a, NOS and myocyte functional responses were determined in 3 groups (7/group) wild-type female mice over 14 weeks (W): Type 2 DM ( T2 ), 14 W fed a high-fat diet (HFD), but after HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. 5 days); T2β 3 ANT , T2 mice at 10 W received L-748,337, a selective β 3 ANT (10 -7 M/kg/day, mini-pump) for 4 W; and Vehicle controls (C). Results: Versus C, T2DM was induced in mice received HFD and low dose STZ with significantly elevated blood glucose (~340 to 380 mg/dl). Only T2 mice had significantly decreased protein levels of LV myocyte β 1 -AR (T2: 0.35 vs C: 0.49) and SERCA2a (0.19 vs 0.29), but increased β 3 -AR (0.25 vs 0.14) and iNOS (0.25 vs 0.15) without significant changes in eNOS (0.14 vs 0.13) and nNOS. These changes were followed by significantly reduced basal cell contraction (dL/dt max , 75.4 vs 133.7 μm/s), relaxation (dR/dt max , 59.6 vs 113.8 μ m/s) and [Ca 2+ ] iT (0.16 vs 0.21). The isoproterenol (ISO, 10 -8 M)-stimulated increases in dL/dt max (38% vs 58%), dR/dt max (34% vs 55%) and [Ca 2+ ] iT (19% vs 30%) were also significantly reduced. By contrary, versus C, T2β 3 ANT myocytes had similar protein levels of β 1 -AR (0.52), SERCA2a (0.75) and iNOS (0.36) with preserved normal basal cell contractility (127.8 μm/s), relaxation (109.4 μm/s) and [Ca 2+ ] iT (0.21). ISO caused increases in dL/dt max (57%), dL/dt max (52%), and [Ca 2+ ] iT (30%) were also closed to control values. Conclusions: Chronic β 3 ANT prevents DCM-caused downregulations of cardiac β 1 -ARs and SERCA2a, and normalized upregulations of iNOS and β 3 -AR, thus leading to the preservation of normal myocyte contractile function, [Ca 2+ ] iT , and β-adrenergic reserve in a mouse model of T2DM.

Abstract 9026: Endogenous Ca2+/Calmodulin-Dependent Protein Kinase II Overactivation Exacerbates Cardiac Dysfunction and Beta-Adrenergic Desensitization in a Mouse Model With Type 2 Diabetes

Background: Emergent evidence indicates that CaMKII overactivity in diabetes (DM) plays a crucial role in the pathophysiology of diabetic cardiomyopathy (DCM) by targeting a diverse array of proteins involved in cardiac electrical, structural and functional remodeling. CaMKII has been proposed to be a therapeutic target for DCM. However, although chronic inhibition of CaMKII has been associated with cardioprotection in several cardiovascular diseases, the direct cardiac effects of CaMKII activation in type 2 (T2) DM are unclear. Moreover, whether and how DCM alters cardiac responses to CaMKII are undefined. We tested the hypothesis that in DCM, CaMKII is upregulated, which alters cardiac contractile behavior. Endogenous CaMKII activation may produce direct depressions in LV myocyte basal function and β-adrenergic reserve. Acute inactivation of CaMKII with KN93 would have a beneficial impact in DCM. Methods: LV myocyte CaMKIIδ expression and activity and myocyte functional responses were compared in 2 groups (n=9/group) of wild-type female mice: Vehicle Control (C) , animals fed chow for 14 weeks (W); T2DM, i nduced with a high-fat diet (HFD) intake for 14 W, but after fed HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. for 5 days). In T2DM, we further assessed myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses with and without acute treatment of myocytes with KN93 (10 -6 M, 30 min). Results: Versus C, in T2DM myocytes, the CaMKIIδ protein levels (28%, 0.51 vs 0.40) and CaMKIIδ phosphorylation (79%, 0.52 vs 0.29) were significantly increased. These changes were followed by significantly reduced cell contraction (dL/dt max , 76.0 vs 137.3 μm/s), relaxation (dR/dt max , 61.7 vs 116.0 μm/s) and [Ca 2+ ] iT (0.16 vs 0.21). ISO-stimulated increases dL/dt max (39% vs 58%), dR/dt max (35% vs 54%) and [Ca 2+ ] iT (20% vs 30%) were also significantly reduced. Moreover, in T2DM myocytes, superfusion with KN93 significantly improved myocyte basal contraction (104.2 μm/s), relaxation (79.3 μm/s) and [Ca 2+ ] iT (0.21). Conclusions: In T2 diabetes-induced DCM, upregulation of CaMKIIδ has adverse functional effects. Acute endogenous CaMKII activation exacerbates LV myocyte dysfunction. The inhibition of CaMKII has a significant beneficial impact.

Antispasmodic activity of Warionia saharae Benthem ex Benth. &amp; Coss. on the rabbit and rat jejunums

Context: Warionia saharae Benthem ex Benth. &amp; Coss has been widely used for gastrointestinal problems in Morocco and Algeria. Aims: To explore the possible mechanism for its use to such ailments in order to rationalize some of these folkloric uses. We investigated the aqueous extract of this plant (AqWs) for antispasmodic activity on isolated rat and rabbit jejunums. Methods: To investigate the contractile activity of isolated jejunum preparations of rat and rabbit we had used in vitro techniques. Jejunums were suspended in tissue baths filled with KHB culture medium (37°C) and connected to force transducers. AqWs (0.1–3 mg/mL) was used with or without carbachol (10-6 M), KCl (25 mM), yohimbine (10-5 M), prazosin (10-5 M), propranolol (10-5 M), hexamethonium (10-4 M), atropine (10-6 M), methylene blue (10-5 M), L-NAME (10-4 M) and nifedipine (10-6 M). Results: AqWs at 0.1–3 mg/mL produced a relaxation on basal rabbit contractions with an IC50 = 1.55 ± 0.06 mg/mL. This effect was reversible and was not affected by pretreatment with the inhibitors of α and β adrenergic receptors yohimbine, prazosin and propranolol. The extract had an antispasmodic activity with an IC50 = 1.25 ± 0.02 mg/mL on rat jejunum precontracted with KCl rich medium (25 mM). This result suggests that the extract had a Ca2+ antagonist effect. This was fortified when pretreatment of the intestine with the extract induced a rightward shift in the Ca2+ concentration-response curves. AqWs exhibited also antispasmodic effect on carbachol (10-6 M)-induced contractions of rat jejunums with an IC50 = 1.53 ± 0.04 mg/mL. The extract at 3 mg/mL on rat jejunums pre-incubated with hexamethonium (10-4 M), atropine (10-6 M), and methylene blue (10-5 M), then contracted by KCl, decreased the maximum contraction but not totally like KCL alone without these inhibitors. For the jejunum pre-incubated with L-NAME (10-4 M), the spasmolytic effect of AqWs was comparable with KCL. Conclusions: These results suggest that the myorelaxant and antispasmodic effects are mediated possibly via Ca2+ antagonist, anticholinergic, and guanylate cyclase mechanisms but not by adrenergic and nitric oxide pathway.

Takotsubo Cardiomyopathy After mRNA COVID-19 Vaccination

A healthy 63-year-old woman with no cardiovascular risk factors was admitted to the emergency room with new-onset dyspnoea and fever. The symptoms started 1 day after receiving the first of two mRNA-1273 (Moderna, Cambridge, MA, USA) COVID-19 vaccinations. She had no other prior complaints. A retronasal severe acute respiratory syndrome coronavirus 2 polymerase chain reaction test was negative. Laboratory tests revealed normal results for creatinine, creatine kinase, and creatine kinase MB (myocardial type). High-sensitivity troponin T was elevated at 320 ng/L (normal: <14 ng/L), and N-terminal pro-B-type natriuretic peptide was highly elevated at 10,180 pg/mL (normal: <284 pg/mL). C-reactive protein was elevated, but the leucocytes were within the normal range at 8.44 g/L (reference interval [RI]: 3.00–10.5 g/L). Urine and blood cultures were negative. Computed tomography scan of the chest revealed no pulmonary embolism but did show signs of heart failure. An electrocardiogram showed negative T waves over the inferior/anterior leads. Invasive coronary angiography ruled out coronary artery disease, but the ventriculogram (Figure 1) showed mid-ventricular to apical ballooning (asterisk) with preserved basal contraction (blues arrows) and a moderately impaired left ventricular ejection fraction of 40%. Apical ballooning was confirmed by echocardiography and cardiac magnetic resonance imaging (CMR). CMR tissue characterisation further depicted extensive oedema in the mid-ventricular/apical segments (i.e., T2-weighted imaging in the myocardium vs skeletal muscle with a signal intensity ratio of >2.0), elevated T2 mapping at 56 ms (RI: 42–50 ms), elevated T1 mapping at 1,158 ms (RI: 903–1,059 ms), and the extracellular volume fraction was elevated at 35% (RI: 25±4%). There was no late gadolinium enhancement suggesting peri-/myocarditis and no pleural or pericardial effusion. As there were no other stressors that could be determined to be underlying the midventricular/apical oedema and ballooning, the most probable diagnosis of COVID-19 vaccine-induced Takotsubo cardiomyopathy (TCM) was made. Although myocarditis is a possible differential diagnosis after COVID-19 vaccination [[1]Abu Mouch S. Roguin A. Hellou E. Ishai A. Shoshan U. Mahamid L. et al.Myocarditis following COVID-19 mRNA vaccination.Vaccine. 2021; 39: 3790-3793Crossref PubMed Scopus (137) Google Scholar], the clinical and investigative features of this case were strongly suggestive of TCM. There have been rare reports of TCM after influenza vaccination [[2]Singh K. Marinelli T. Horowitz J.D. Takotsubo cardiomyopathy after anti-influenza vaccination: catecholaminergic effects of immune system.Am J Emerg Med. 2013; 31: 1627Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar,[3]Santoro F. Ieva R. Ferraretti A. Carpagnano G. Pappalardo I. De Gennaro L. et al.Tako-Tsubo cardiomyopathy after influenza vaccination.Int J Cardiol. 2013; 167: e51-e52Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar], with an underlying pathophysiology of systemic inflammatory stress reaction after vaccination with a sympathovagal imbalance towards adrenergic predominance [[4]Lanza G.A. Barone L. Scalone G. Pitocco D. Sgueglia G.A. Mollo R. et al.Inflammation-related effects of adjuvant influenza A vaccination on platelet activation and cardiac autonomic function.J Intern Med. 2011; 269: 118-125Crossref PubMed Scopus (29) Google Scholar]. TCM confirmed by CMR after a COVID-19 vaccination has not yet been reported and may be considered as a differential diagnosis besides myocarditis in this clinical setting. Letter to the Editor Regarding: What Triggers Takotsubo After a SARS-CoV-2 Vaccination? Heart Lung Circ. 2021;30(12):e119–20Heart, Lung and CirculationVol. 31Issue 5PreviewWe read with interest the case report by Boscolo Berto et al. about a 63-year-old female who developed the classical type of Takotsubo cardiomyopathy (TTS) one day after her first jab of the Moderna vaccine [1]. The patient completely recovered without any treatment [1]. The study is appealing but raises several comments and concerns. Full-Text PDF

The Circadian Clock Gene Bmal1 Modulates Myometrium Contractile Function in Pregnant Mice

Caesarian section should be avoided unless medically required. Caesarian section is often a result of failed labor enhancement. While most natural births occur at night, labor enhancement is often scheduled during the day. We propose that the disparity between the commonly used timing of labor enhancement in the clinic, from the timing of natural birth, could contribute to the high rate of labor induction failure. Oxytocin receptor agonists are the most used labor enhancing agents. In pregnant non-human primates, oxytocin enhances uterine contractions more efficiently at night than during the day, suggesting a daily change in uterine sensitivity to oxytocin in pregnancy. To identify the molecular mechanisms generating daily changes in uterine function, we here explore the role of the molecular clock gene, Bmal1 (Brain and muscle ARNTL1-like), in the pregnant mouse myometrium. BMAL1 is a transcription factor required to generated circadian rhythms at the cellular level. We hypothesize that Bmal1 in uterine myometrial cells generates circadian rhythms and establishes the daily change in uterine contractile response to oxytocin. To evaluate circadian rhythms ex vivo, we collected myometrium samples from the validated circadian Per2:luciferase reporter mice at gestation day 17-18. We found that the pregnant mouse myometrium possesses circadian rhythms, which are generated by the molecular clock, as triple transgenic Per2:luciferase mice with Bmal1 conditionally deleted in the myometrium (cKO) do not have rhythmic expression of the Per2:luciferase reporter. To determine if BMAL1 is required to establish uterine contractions, we used a myograph to measure ex vivo uterine contractions at gestation day 17-18. In controls, uterine contraction force was significantly higher at ZT15 (3h after lights OFF) versus ZT3 (3h after lights ON). Interestingly, our preliminary data show increased basal contractile force at ZT15 in cKO as compared to controls. In addition, the cKO uterus contracted stronger to oxytocin than controls. Our findings identify Bmal1 as a clock gene modulating basal contractions in the mouse uterus and indicate Bmal1 might be a regulator of uterine sensitivity to oxytocin. Future work will focus on identifying the molecular mechanisms driven by BMAL1 to regulate uterine function in pregnancy. This work has the potential to provide insights into how we can improve labor enhancing treatment strategies in the clinic in the future.