Impairment of IKCa channels contributes to uteroplacental endothelial dysfunction in rat diabetic pregnancy.

Diabetes in rat pregnancy is associated with impaired vasodilation of the maternal uteroplacental vasculature. In the present study, we explored the role of endothelial cell (EC) Ca(2+)-activated K(+) channels of small conductance (SKCa channels) and intermediate conductance (IKCa channels) in diabetes-induced uterine vascular dysfunction. Diabetes was induced by injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with citrate buffer. Changes in smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and vasodilation induced by SKCa/IKCa channel activators were studied in uteroplacental arteries of control and diabetic rats. The impact of diabetes on SKCa- and IKCa-mediated currents was explored in freshly dissociated ECs. NS309 evoked a potent vasodilation that was effectively inhibited by TRAM-34 but not by apamin. NS309-induced smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and dilator responses were significantly diminished by diabetes; N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA)-evoked responses were not affected. Ca(2+)-activated ion currents in ECs were insensitive to paxilline, markedly inhibited by charybdotoxin (ChTX), and diminished by apamin. NS309-induced EC currents were generated mostly due to activation of ChTX-sensitive channels. Maternal diabetes resulted in a significant reduction in ChTX-sensitive currents with no effect on apamin-sensitive or CyPPA-induced currents. We concluded that IKCa channels play a prevalent role over SKCa channels in the generation of endothelial K(+) currents and vasodilation of uteroplacental arteries. Impaired function of IKCa channels importantly contributes to diabetes-induced uterine endothelial dysfunction. Therapeutic restoration of IKCa channel function may be a novel strategy for improvement of maternal uteroplacental blood flow in pregnancies complicated by diabetes.

Role of impaired endothelial cell Ca(2+) signaling in uteroplacental vascular dysfunction during diabetic rat pregnancy.

Diabetes mellitus in pregnancy is associated with impaired endothelium-mediated dilatation of maternal arteries, although the underlying cellular mechanisms remain unknown. In this study, we hypothesized that diabetes during rat gestation attenuates agonist-induced uterine vasodilation through reduced endothelial cell (EC) Ca(2+) elevations and impaired smooth muscle cell (SMC) hyperpolarization and SMC intracellular Ca(2+) concentration ([Ca(2+)]i) responses. Diabetes was induced by an injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with a citrate buffer. Fura-2-based measurements of SMC [Ca(2+)]i or microelectrode recordings of SMC membrane potential were performed concurrently with dilator responses to ACh in uteroplacental arteries from control and diabetic pregnant rats. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations in pressurized vessels and small EC sheets were studied as well. Diabetes reduced ACh-induced vasodilation due to a markedly impaired EDHF-mediated response. Diminished vasodilation to ACh was associated with attenuated SMC hyperpolarization and [Ca(2+)]i responses. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations were significantly reduced by diabetes. In conclusion, these data demonstrate that reduced endothelium-mediated hyperpolarization contributes to attenuated uteroplacental vasodilation and SMC [Ca(2+)]i responses to ACh in diabetic pregnancy. Impaired endothelial Ca(2+) signaling is in part responsible for endothelial dysfunction in the uterine resistance vasculature of diabetic rats. Pharmacological improvement of EC Ca(2+) handling may provide an important strategy for the restoration of endothelial function and enhancement of maternal blood flow in human pregnancies complicated by diabetes.

Allo- and xeno-reassembly of human and rat myometrium from cells and scaffolds.

Neo-myometrium was created by culturing isolated myocytes into decellularized rat and human myometrial scaffolds. The dual purpose of the uterus is to accommodate the growing fetus, and then expel the fetus at term by phasically contracting it. The first function requires physical robustness as well as the ability to expand and remodel. Congenital anomalies or previous surgeries can mechanically compromise the uterus and lead to major complications in pregnancy. The second function utilizes multiple interactions of complex physiological mechanisms that have yet to be fully elucidated, and this knowledge gap contributes to the continuation of serious complications of pregnancy. To address both problems, we reconstructed myometrium from isolated myocytes and scaffold. From both rat and human myometrium, myocytes were isolated using collagenase digestion, and scaffolds were isolated using ethanol/ trypsin protocols. The number of myocytes was amplified using monolayer culture, and then, the myocytes were cultured back into the scaffolds. We called this engineered tissue neo-myometrium, with allo-neo-myometrium being made from components of the same species, and xeno-neo-myometrium from across species. By artificially creating defects in rat scaffold, allo-neo-myometrium was created that demonstrated rapid scaffold remodeling. Xeno-neo myometrium (human myocytes/rat scaffold) was created and demonstrated myocytes occurring in bundles 500 µm deep in the scaffold. These experiments provide proof of principle that modest numbers of myocytes can be amplified and used to create a larger volume of engineered tissue, which may be useful for semi-autologous transplantation to repair structural defects of the human uterus. In isometric contractility experiments, coordinated contractions were observed in xeno-neo-myometrium (human myocytes, rat scaffold), but not allo-neo-myometrium (human myocytes, human scaffold).

Phasic oscillations of extracellular potassium (K(o)) in pregnant rat myometrium.

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.

Mechanotransduction in rat myometrium: coordination of contractions of electrically and chemically isolated tissues.

The generally accepted mechanism for global uterine coordination is propagation of electrical activity. Mechanotransduction mechanisms were briefly considered as a secondary mechanism 40 years ago, but scant data have appeared. Here, we provide evidence that tissue strips are capable of functionally interacting solely by mechanical mechanisms. We mechanically linked, in series, 2 rat myometrial strips of similar size. Strips were placed in separate baths to ensure they were electrically and chemically isolated. A force transducer was used to measure force production. We precisely determined when each tissue contracted by simultaneously measuring each strip's electrical activity using contact electrodes. We observed both in-phase and out-of-phase contraction patterns from the tissues. To determine whether modulation of the electrical properties of the tissue is involved in the mechanotransduction mechanism, we briefly stretched single tissue strips during alternate contractions. This technique provided a control contraction for each test contraction. The duration of the contraction that was stretched measured longer than the control in 33 of 35 pairs (P = .0001, Wilcoxon signed-rank test for paired data). Interestingly, briefly slackening the tissue also prolonged the force-producing phase of that contraction (39 of 42 pairs; P = .0006). Because our data show that mechanotransduction mechanisms coordinate tissue-level contractions, we speculate that mechanotransduction mechanisms may contribute to organ-level coordination of contractions.