Physiological increases in lactate inhibit intracellular calcium transients, acidify myocytes and decrease force in term pregnant rat myometrium.
Ontology highlight
ABSTRACT:
SUBMITTER: Hanley JA
PROVIDER: S-EPMC4606541 | biostudies-other | 2015 Oct
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Force measurements in ex vivo and engineered heart tissues are well established. Analysis of calcium transients (CaT) is complementary to force, and the combined analysis is meaningful to the study of cardiomyocyte biology and disease. This article describes a model of human induced pluripotent stem cell cardiomyocyte-derived engineered heart tissues (hiPSC-CM EHTs) transduced with the calcium sensor GCaMP6f followed by sequential analysis of force and CaT. Average peak analysis demonstrated the temporal sequence of the CaT preceding the contraction twitch. The pharmacological relevance of the test system was demonstrated with inotropic indicator compounds. Force-frequency relationship was analyzed in the presence of ivabradine (300 nM), which reduced spontaneous frequency and unmasked a positive correlation of force and CaT at physiological human heart beating frequency with stimulation frequency between 0.75 and 2.5 Hz (force +96%; CaT +102%). This work demonstrates the usefulness of combined force/CaT analysis and demonstrates a positive force-frequency relationship in hiPSC-CM EHTs.
| S-EPMC7013237 | biostudies-literature
Project description:Progesterone (P4), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P4/PR partially exerts these effects by tethering to NF-κB bound to their promot-ers, thereby decreasing NF-κB transcriptional activity. However, the underlying mechanisms whereby P4/PR interaction blocks proinflammatory and CAP gene expression are not fully understood. Herein, we characterized CCR-NOT transcription complex subunit 1 (CNOT1) as a corepressor that also interacts within the same chromatin complex as PR-B. In mouse myome-trium increased expression of CAP genes Oxtr and Cx43 at term coincided with a marked decline in expression and binding of CNOT1 to NF-κB-response elements within the Oxtr and Cx43 promoters. Increased CAP gene expression was accompanied by a pronounced decrease in enrichment of repressive histone marks and increase in enrichment of active histone marks to this genomic region. These changes in histone modification were associated with changes in expression of corresponding histone modifying enzymes. Myometrial tissues from P4-treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. P4 treatment of PR-expressing hTERT-HM cells enhanced CNOT1 expression and its recruitment to PR bound NF-κB-response elements within the CX43 and OXTR promoters. Furthermore, knockdown of CNOT1 significantly increased expression of contractile genes. These novel findings suggest that decreased expression and DNA-binding of the P4/PR-regulated transcriptional corepressor CNOT1 near term and associated changes in histone modifications at the OXTR and CX43 promoters contribute to the induction of myometrial contractility leading to parturition.
| S-EPMC11301068 | biostudies-literature
Project description:K-sensitive microelectrodes were used to measure K(+) within the extracellular space (K(o)) of pregnant rat myometrium. Contractile activity was monitored by measuring either force or bioelectrical signals. Single and double-barreled electrodes were used. Double-barreled electrodes allowed monitoring of electrical activity 15 microns from the site of K(o) measurement. From double-barreled electrode experiments, the bioelectrical burst started first, and then K(o) began to rise 0.6 ± 0.1 seconds later. This delay indicates that K(+) leaves the cells in response to local electrical activity rather than vice versa. Four control experiments were performed to assess the influence of electrical artifacts caused by tissue motion on K(o) values. When observed, artifacts were negative and transient, and hence would result in an underestimation of K(o) rises. Artifacts were minimized when tissue motion was minimized by fixing the tissue at both ends. At 37°C, 7 single barreled experiments and 45 contractions were analyzed. Resting K(o) was within 1 mM of bath K(+) (5 mM) at the beginning and end of the experiments. K(o) rose during the contraction, fell after the completion of the contraction, and normalized before the next contraction began. Peak K(o) values observed during force production were 18.8 ± 5.9 mM, a value high enough to modulate tissue-level electrical activity. K(o) required 15.7 ± 2.8 seconds to normalize halfway (t50). Six experiments expressing 38 contractions were performed at 24°C. The contraction period was longer at 24°C. Values for peak K(o) (26.2 ± 9.9 mM) and t50 (29.8±16.2 sec) were both larger than at 37°C (p<0.0003 for both). The direct relationships between peak K(o), t50 and the contraction period, suggest elevations in K(o) may modulate contraction frequency. The myometrial interstitial space appears to be functionally important, and K(o) metabolism may participate in cell-cell interactions.
| S-EPMC3665820 | biostudies-literature
Project description:The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.
| S-EPMC3761174 | biostudies-literature
Project description:Recently, it has been suggested that progesterone affects the contractile activity of pregnant myometrium via nongenomic pathways; therefore, we aimed to clarify whether progesterone causes and/or inhibits pregnant myometrial contractions via nongenomic pathways. Our in vitro experiments using myometrial strips obtained from rats at 20 days of gestation revealed that progesterone caused myometrial contractions in a concentration- and time-dependent manner at concentrations up to 5 × 10-7 M; however, this effect decreased at concentrations higher than 5 × 10-5 M. Similarly, progesterone enhanced oxytocin-induced contractions up to 5 × 10-7 M and inhibited contractions at concentrations higher than 5 × 10-5 M. Conversely, progesterone did not enhance high-KCl-induced contractions but inhibited contractions in a concentration- and time-dependent manner at concentrations higher than 5 × 10-7 M. We also found that RU486 did not affect progesterone-induced contractions or the progesterone-induced inhibition of high-KCl-induced contractions; however, progesterone-induced contractions were blocked by calcium-free phosphate saline solution, verapamil, and nifedipine. In addition, FPL64176, an activator of L-type voltage-dependent calcium channels, enhanced high-KCl-induced contractions and rescued the decrease in high-KCl-induced contractions caused by progesterone. Together, these results suggest that progesterone exerts conflicting nongenomic effects on the contractions of pregnant myometrium via putative L-type voltage-dependent calcium channels.
| S-EPMC7926872 | biostudies-literature
Project description:Transverse and axial tubules (TATS) are an essential ingredient of the excitation-contraction machinery that allow the effective coupling of L-type Calcium Channels (LCC) and ryanodine receptors (RyR2). They form a regular network in ventricular cells, while their presence in atrial myocytes is variable regionally and among animal species We have studied the effect of variations in the TAT network using a bidomain computational model of an atrial myocyte with variable density of tubules. At each z-line the t-tubule length is obtained from an exponential distribution, with a given mean penetration length. This gives rise to a distribution of t-tubules in the cell that is characterized by the fractional area (F.A.) occupied by the t-tubules. To obtain consistent results, we average over different realizations of the same mean penetration length. To this, in some simulations we add the effect of a network of axial tubules. Then we study global properties of calcium signaling, as well as regional heterogeneities and local properties of sparks and RyR2 openings. In agreement with recent experiments in detubulated ventricular and atrial cells, we find that detubulation reduces the calcium transient and synchronization in release. However, it does not affect sarcoplasmic reticulum (SR) load, so the decrease in SR calcium release is due to regional differences in Ca2+ release, that is restricted to the cell periphery in detubulated cells. Despite the decrease in release, the release gain is larger in detubulated cells, due to recruitment of orphaned RyR2s, i.e, those that are not confronting a cluster of LCCs. This probably provides a safeguard mechanism, allowing physiological values to be maintained upon small changes in the t-tubule density. Finally, we do not find any relevant change in spark properties between tubulated and detubulated cells, suggesting that the differences found in experiments could be due to differential properties of the RyR2s in the membrane and in the t-tubules, not incorporated in the present model. This work will help understand the effect of detubulation, that has been shown to occur in disease conditions such as heart failure (HF) in ventricular cells, or atrial fibrillation (AF) in atrial cells.
| S-EPMC7164608 | biostudies-literature
Project description:Nifedipine, an L-type voltage-gated Ca2+ channel (L-VGCC) blocker, is one of the most used tocolytics to treat preterm labor. In clinical practice, nifedipine efficiently decreases uterine contractions, but its efficacy is limited over time, and repeated or maintained nifedipine-based tocolysis appears to be ineffective in preventing preterm birth. We aimed to understand why nifedipine has short-lasting efficiency for the inhibition of uterine contractions. We used ex vivo term pregnant human myometrial strips treated with cumulative doses of nifedipine. We observed that nifedipine inhibited spontaneous myometrial contractions in tissues with high and regular spontaneous contractions. By contrast, nifedipine appeared to increase contractions in tissues with low and/or irregular spontaneous contractions. To investigate the molecular mechanisms activated by nifedipine in myometrial cells, we used the pregnant human myometrial cell line PHM1-41 that does not express L-VGCC. The in vitro measurement of intracellular Ca2+ showed that high doses of nifedipine induced an important intracellular Ca2+ entry in myometrial cells. The inhibition or downregulation of the genes encoding for store-operated Ca2+ entry channels from the Orai and transient receptor potential-canonical (TRPC) families in PHM1-41 cells highlighted the implication of TRPC1 in nifedipine-induced Ca2+ entry. In addition, the use of 2-APB in combination with nifedipine on human myometrial strips tends to confirm that the pro-contractile effect induced by nifedipine on myometrial tissues may involve the activation of TRPC channels.
| S-EPMC9306527 | biostudies-literature
Project description:We describe here the expression of the ryanodine receptor isoforms RyR2 and RyR3 in human non-pregnant and pregnant (non-labouring) myometrium, and in isolated cultured myometrial cells. The mRNA encoding the RyR3 isoform was found in both non-pregnant and pregnant myometrial tissue samples; however, the mRNA for RyR2 was found only in pregnant samples. It can be speculated that the appearance of this additional isoform in the pregnant myometrium may increase the ability of this tissue to contract at term. Control of expression of the RyR2 gene may therefore be another example of an up-regulated signalling system in pregnancy. Although the mRNA for RyR3 was expressed in cultured myometrial cells, the mRNA for RyR2 could not be detected. Thus cultured myometrial cells appear to be similar to the non-pregnant myometrium. The cytokine transforming growth factor beta (TGF-beta) has been reported to alter RyR mRNA expression in many cell types. After treatment with TGF-beta, both RyR2 and RyR3 mRNAs could be detected in cultured myometrial cells. These observations support the idea that the expression of the RyR2 isoform is up-regulated both in pregnancy and in TGF-beta-treated cultured myometrial cells. Using measurements of 45Ca2+ release, we have further demonstrated that cultured human myometrial cells show a significant augmentation of both the Ca2+-induced Ca2+ release (CICR) mechanism and ryanodine-induced Ca2+ release after treatment with TGF-beta. Additionally, caffeine was able to induce Ca2+ release and sensitize the CICR mechanism to ryanodine. Thus we suggest that the appearance of RyR2 mRNA leads to the expression of this receptor/channel protein with identifiable pharmacological characteristics. These results are discussed in the context of the potential role of gene activation in the process of maturation of the human myometrium during pregnancy.
| S-EPMC1218255 | biostudies-other
Project description:Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1. Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture (p < 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo, as reported previously. Serum supplementation, prostaglandin F2α, phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone (p = 0.0058) and cAMP (p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone/protein deacetylase inhibitors: trichostatin A at 5 (p = 0.0172) and 25 µM (p = 0.0115); suberoylanilide hydroxamic acid at 0.5 (p = 0.0070), 1 (p = 0.0045), 2.5 (p = 0.0181), 5 (p = 0.0066) and 25 µM (p = 0.0014); and suberoyl bis-hydroxamic acid at 5 (p = 0.0480) and 25 µM (p = 0.0238). We propose the inhibition of histone/protein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.
| S-EPMC10784398 | biostudies-literature
Project description:Functional magnetic resonance imaging (fMRI) has revolutionized the study of human brain activity, in both basic and clinical research. The commonly used blood oxygen level dependent (BOLD) signal in fMRI derives from changes in oxygen saturation of cerebral blood flow as a result of brain activity. Beyond the traditional spatial mapping of stimulus-activation correspondences, the detailed waveforms of BOLD responses are of high interest. Especially intriguing are the transient overshoots and undershoots, often, although inconclusively, attributed to the interplay between changes in cerebral blood flow and volume after neuronal activation. While physically simulating the BOLD response in fMRI phantoms, we encountered prominent transient deflections, although the magnetic field inside the phantom varied in a square-wave manner. Detailed analysis and modeling indicated that the transients arise from activation-related partial misalignment of the imaging slices and depend heavily on measurement parameters, such as the time between successive excitations. The results suggest that some transients encountered in normal fMRI recordings may be spurious, potentially compromising the physiological interpretation of BOLD signal overshoots and undershoots.
| S-EPMC2777966 | biostudies-literature