MicroRNA-210 Mediates Hypoxia-Induced Repression of Spontaneous Transient Outward Currents in Sheep Uterine Arteries During Gestation

Hypoxia during pregnancy is a major contributor to the pathogenesis of preeclampsia and intrauterine growth restriction. Our recent studies revealed that pregnancy-induced uterine vascular adaptation depended on the enhanced Ca 2+ spark/spontaneous transient outward current (STOC) coupling and hypoxia during gestation diminished this adaption. In the present study, we test the hypothesis of a mechanistic link of microRNA-210 (miR-210) in hypoxia-impaired Ca 2+ spark/STOC coupling in uterine arteries. Pregnant ewes acclimatized to high-altitude (3801 m) hypoxia for ≈110 days significantly increased circulation levels of miR-210 in both the ewe and her fetus. Treatment of uterine arteries from high-altitude animals with the antagomir miR-210-LNA recovered hypoxia-repressed STOCs in pregnant ewes and restored the hormonal regulation of STOCs in nonpregnant animals. In uterine arteries from low-altitude control animals, miR-210 mimic suppressed STOCs in pregnant ewes and inhibited the hormonal regulation of STOCs in nonpregnant animals. Mechanistically, miR-210 directly targeted and downregulated type 2 ryanodine receptor and large-conductance Ca 2+ -activated K + channel β1 subunit, resulting in significant decreases in Ca 2+ sparks and STOCs in uterine arteries. In addition, miR-210 indirectly decreased STOCs by targeting ten-eleven translocation methylcytosine dioxygenase. Together, the present study revealed a mechanistic link of miR-210 in hypoxia-induced repression of Ca 2+ spark/STOC coupling in uterine arteries during gestation, providing novel insights into the understanding of pregnancy complications associated with hypoxia and the potential therapeutic targets.

Gestational Hypoxia Inhibits Pregnancy-Induced Upregulation of Ca 2+ Sparks and Spontaneous Transient Outward Currents in Uterine Arteries Via Heightened Endoplasmic Reticulum/Oxidative Stress

Hypoxia during pregnancy profoundly affects uterine vascular adaptation and increases the risk of pregnancy complications, including preeclampsia and fetal intrauterine growth restriction. We recently demonstrated that increases in Ca 2+ sparks and spontaneous transient outward currents (STOCs) played an essential role in pregnancy-induced uterine vascular adaptation. In the present study, we hypothesize that gestational hypoxia suppresses Ca 2+ sparks/STOCs coupling leading to increased uterine vascular tone via enhanced endoplasmic reticulum (ER)/oxidative stress. Uterine arteries were obtained from nonpregnant and near-term pregnant sheep residing in low altitude or acclimatizing to high-altitude (3801 m) hypoxia for ≈110 days. High-altitude hypoxia suppressed pregnancy-induced upregulation of RyR1 and RyR2 (ryanodine receptor 1 and 2) protein abundance, Ca 2+ sparks, and STOCs in uterine arteries. Inhibition of Ca 2+ sparks/STOCs with the RyR inhibitor ryanodine significantly increased pressure-dependent myogenic tone in uterine arteries from low-altitude normoxic pregnant animals but not those from high-altitude hypoxic pregnant animals. Gestational hypoxia significantly increased ER/oxidative stress in uterine arteries. Of importance, the hypoxia-mediated suppression of Ca 2+ sparks/STOCs and increase in myogenic tone in uterine arteries of pregnant animals were reversed by inhibiting ER/oxidative stress. Of great interest, the impaired sex hormonal regulation of STOCs in high-altitude animals was annulled by scavenging reactive oxygen species but not by inhibiting ER stress. Together, the findings reveal the differential mechanisms of ER and oxidative stresses in suppressing Ca 2+ sparks/STOCs and increasing myogenic tone of uterine arteries in hypoxia during gestation, providing new insights into the understanding of pregnancy complications associated with hypoxia.

Ryanodine receptor subtypes regulate Ca2+ sparks/spontaneous transient outward currents and myogenic tone of uterine arteries in pregnancy

Aims Our recent study demonstrated that increased Ca2+ sparks and spontaneous transient outward currents (STOCs) played an important role in uterine vascular tone and haemodynamic adaptation to pregnancy. The present study examined the role of ryanodine receptor (RyR) subtypes in regulating Ca2+ sparks/STOCs and myogenic tone in uterine arterial adaptation to pregnancy. Methods and results Uterine arteries isolated from non-pregnant and near-term pregnant sheep were used in the present study. Pregnancy increased the association of α and β1 subunits of large-conductance Ca2+-activated K+ (BKCa) channels and enhanced the co-localization of RyR1 and RyR2 with the β1 subunit in the uterine artery. In contrast, RyR3 was not co-localized with BKCa β1 subunit. Knockdown of RyR1 or RyR2 in uterine arteries of pregnant sheep downregulated the β1 but not α subunit of the BKCa channel and decreased the association of α and β1 subunits. Unlike RyR1 and RyR2, knockdown of RyR3 had no significant effect on either expression or association of BKCa subunits. In addition, knockdown of RyR1 or RyR2 significantly decreased Ca2+ spark frequency, suppressed STOCs frequency and amplitude, and increased pressure-dependent myogenic tone in uterine arteries of pregnant animals. RyR3 knockdown did not affect Ca2+ sparks/STOCs and myogenic tone in the uterine artery. Conclusion Together, the present study demonstrates a novel mechanistic paradigm of RyR subtypes in the regulation of Ca2+ sparks/STOCs and uterine vascular tone, providing new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

Intraluminal pressure triggers myogenic response via activation of calcium spark and calcium-activated chloride channel in rat renal afferent arteriole

Myogenic contraction of renal arterioles is an important regulatory mechanism for renal blood flow autoregulation. We have previously demonstrated that integrin-mediated mechanical force increases the occurrence of Ca2+ sparks in freshly isolated renal vascular smooth muscle cells (VSMCs). To further test whether the generation of Ca2+ sparks is a downstream signal of mechanotransduction in pressure-induced myogenic constriction, the relationship between Ca2+ sparks and transmural perfusion pressure was investigated in intact VSMCs of pressurized rat afferent arterioles. Spontaneous Ca2+ sparks were found in VSMCs when afferent arterioles were perfused at 80 mmHg. The spark frequency was significantly increased when perfusion pressure was increased to 120 mmHg. A similar increase of spark frequency was also observed in arterioles stimulated with β1-integrin-activating antibody. Moreover, spark frequency was significantly higher in arterioles of spontaneous hypertensive rats at 80 and 120 mmHg. Spontaneous membrane current recorded using whole cell perforated patch in renal VSMCs showed predominant activity of spontaneous transient inward currents instead of spontaneous transient outward currents when holding potential was set close to physiological resting membrane potential. Real-time PCR and immunohistochemistry confirmed the expression of Ca2+-activated Cl− channel (ClCa) TMEM16A in renal VSMCs. Inhibition of TMEM16A with T16Ainh-A01 impaired the pressure-induced myogenic contraction in perfused afferent arterioles. Our study, for the first time to our knowledge, detected Ca2+ sparks in VSMCs of intact afferent arterioles, and their frequencies were positively modulated by the perfusion pressure. Our results suggest that Ca2+ sparks may couple to ClCa channels and trigger pressure-induced myogenic constriction via membrane depolarization.

Intracellular cAMP: the “switch” that triggers on “spontaneous transient outward currents” generation in freshly isolated myocytes from thoracic aorta

Spontaneous transient outward currents (STOCs) have been reported in resistance and small arteries but have not yet been found in thoracic aorta. Do thoracic aorta myocytes possess cellular machinery that generates STOCs? It was found that the majority of aortic myocytes do not generate STOCs. STOCs were generated in 8.7% of freshly isolated aortic myocytes. Myocytes that did not generate STOCs we have called “silent” myocytes and myocytes with STOCs have been called “active.” STOCs recorded in active myocytes were voltage dependent and were inhibited by ryanodine, caffeine, and charybdotoxin. Forskolin was reported to increase STOCs frequency in myocytes isolated from resistance arteries. Forskolin (10 μM) triggered STOCs generation in 35.1% of silent aortic myocytes. In 36.8% percent of silent myocytes, forskolin did not trigger STOCs but increased the amplitude of charybdotoxin-sensitive outward net current to 136.1 ± 8.5% at 0 mV. Membrane-permeable 8BrcAMP triggered STOCs generation in 38.7% of silent myocytes. Forskolin- or 8BrcAMP-triggered STOCs were inhibited by charybdotoxin. 8BrcAMP also increased open probability of BKCa channels in BAPTA-AM-pretreated cells. Our data demonstrate that, in contrast to resistance arteries, STOCs are present just in the minority of myocytes in the thoracic aorta. However, cellular machinery that generates STOCs can be “switched” on by cAMP. Such an inactive cellular mechanism could modulate the contractility of the thoracic aorta in response to physiological demand.