Intrauterine Nitric Oxide Deficiency Weakens Differentiation of Vascular Smooth Muscle in Newborn Rats.

Nitric oxide (NO) deficiency during pregnancy is a key reason for preeclampsia development. Besides its important vasomotor role, NO is shown to regulate the cell transcriptome. However, the role of NO in transcriptional regulation of developing smooth muscle has never been studied before. We hypothesized that in early ontogeny, NO is important for the regulation of arterial smooth muscle-specific genes expression. Pregnant rats consumed NO-synthase inhibitor L-NAME (500 mg/L in drinking water) from gestational day 10 till delivery, which led to an increase in blood pressure, a key manifestation of preeclampsia. L-NAME reduced blood concentrations of NO metabolites in dams and their newborn pups, as well as relaxations of pup aortic rings to acetylcholine. Using qPCR, we demonstrated reduced abundances of the smooth muscle-specific myosin heavy chain isoform, α-actin, SM22α, and L-type Ca2+-channel mRNAs in the aorta of newborn pups from the L-NAME group compared to control pups. To conclude, the intrauterine NO deficiency weakens gene expression specific for a contractile phenotype of arterial smooth muscle in newborn offspring.

Intrauterine L-NAME Exposure Weakens the Development of Sympathetic Innervation and Induces the Remodeling of Arterial Vessels in Two-Week-Old Rats.

Nitric oxide (NO) has been shown to stimulate differentiation and increase the survival of ganglionic sympathetic neurons. The proportion of neuronal NOS-immunoreactive sympathetic preganglionic neurons is particularly high in newborn rats and decreases with maturation. However, the role of NO in the development of vascular sympathetic innervation has never been studied before. We tested the hypothesis that intrauterine NO deficiency weakened the development of vascular sympathetic innervation and thereby changed the contractility of peripheral arteries and blood pressure level in two-week-old offspring. Pregnant rats consumed NOS inhibitor L-NAME (250 mg/L in drinking water) from gestational day 10 until delivery. Pups in the L-NAME group had a reduced body weight and blood level of NO metabolites at 1-2 postnatal days. Saphenous arteries from two-week-old L-NAME offspring demonstrated a lower density of sympathetic innervation, a smaller inner diameter, reduced maximal active force and decreased α-actin/ß-actin mRNA expression ratio compared to the controls. Importantly, pups in the L-NAME group exhibited decreased blood pressure levels before, but not after, ganglionic blockade with chlorisondamine. In conclusion, intrauterine L-NAME exposure is followed by the impaired development of the sympathetic nervous system in early postnatal life, which is accompanied by the structural and functional remodeling of arterial blood vessels.

Antenatal/early postnatal hypothyroidism increases the contribution of Rho-kinase to contractile responses of mesenteric and skeletal muscle arteries in adult rats.

BACKGROUND: Maternal thyroid deficiency can increase Rho-kinase procontractile influence in arteries of 2-week-old progeny. Here we hypothesized that augmented role of Rho-kinase persists in arteries from adult progeny of hypothyroid rats. METHODS: Dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%) during pregnancy and 2 weeks postpartum; control (CON) females received PTU-free water. At the age of 10-12-weeks, serum T3/T4 levels did not differ between PTU and CON male offspring. Cutaneous (saphenous), mesenteric, and skeletal muscle (sural) arteries were studied by wire myography, qPCR, and Western blotting. RESULTS: Saphenous arteries of PTU and CON groups showed similar responses to α1-adrenoceptor agonist methoxamine and were equally suppressed by Rho-kinase inhibitor Y27632. Responses of mesenteric arteries also did not differ between PTU and CON, but the effects of Y27632 were more prominent in the PTU group. Sural arteries of PTU rats compared to CON demonstrated augmented responses to methoxamine, increased RhoA mRNA contents and higher levels of MYPT1 phosphorylation at Thr855. Intergroup differences in contractile responses and phospho-MYPT1-Thr855 were eliminated by Y27632. CONCLUSION: Rho-kinase contribution to contractile responses of mesenteric and especially sural arteries is augmented in adult PTU rats. Therefore, maternal thyroid deficiency may have long-term detrimental consequences for vasculature in adult offspring.

NO-mediated anticontractile effect of the endothelium is abolished in coronary arteries of adult rats with antenatal/early postnatal hypothyroidism.

INTRODUCTION: Thyroid hormones are essential for proper development of many systems and organs, including circulatory system. Thyroid deficiency during pregnancy may affect the cardiovascular function in children early on and later in adulthood. However, long-term effects of early thyroid deficiency are poorly understood. We hypothesized that antenatal/early postnatal hypothyroidism will influence anticontractile effect of NO in coronary arteries of adult rats. DESIGN AND METHODS: To model antenatal/early postnatal hypothyroidism dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%, w/v) from the first day of pregnancy till 2 weeks after delivery. Control females were supplied with pure water. Their male offspring was grown up till the age of 10-12 weeks. Systolic blood pressure was measured using tail cuff method. Septal coronary arteries were isolated and studied in wire myograph. Blood serum thyroid hormones concentrations (ELISA) and NO metabolites level (Griess method) were evaluated. RESULTS: At the age of 10-12 weeks thyroid hormones, TSH concentrations, NO metabolites and systolic blood pressure level didn't differ between groups. Arterial responses to acetylcholine and exogenous NO-donor DEA/NO were similar in control and PTU groups. Along with that, in control rats endothelium denudation strongly potentiated basal tone of arteries and their contractile responses to thromboxane A2 receptor agonist U46619. The effects of endothelium denudation were absent in PTU rats indicating that anticontractile effect of endothelium is abolished in their arteries. Further, NO-synthase inhibitor L-NNA (100 µM) caused significant elevation of basal tone and increased U46619-induced contraction of endothelium-intact arteries only in control rats, while had no effect in PTU group. CONCLUSIONS: Our data demonstrate that NO-mediated anticontractile effect of endothelium is eliminated in coronary arteries of adult rats, which suffered from antenatal/early postnatal hypothyroidism. Therefore, maternal thyroid hormones deficiency may have detrimental consequences in adult offspring including coronary circulation pathologies, despite normal blood levels of thyroid hormones.

Region-specific effects of antenatal/early postnatal hypothyroidism on endothelial NO-pathway activity in systemic circulation.

BACKGROUND: Antenatal/early postnatal hypothyroidism weakens NO-mediated anticontractile influence of endothelium in coronary arteries of adult rats, but it remains unclear whether this occurs in other vascular regions. We hypothesized that developmental thyroid deficiency is followed by region-specific changes in the endothelial NO-pathway activity in systemic vasculature. To explore this, we estimated the effects of antenatal/early postnatal hypothyroidism on NO-pathway activity and its potential local control mechanisms in rat mesenteric and skeletal muscle (sural) arteries. METHODS: Dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%) during pregnancy and 2 weeks postpartum; control (CON) females received PTU-free water. Adult offspring (10-12-weeks) arteries were studied by wire myography, qPCR, and Western blotting. RESULTS: Endothelium removal or inhibition of NO-synthase with L-NNA augmented contractile responses to α1-adrenoceptor agonist methoxamine. In PTU compared to CON group, these effects were stronger in sural arteries, but did not differ in mesenteric arteries. The responses of both arteries to NO-donor DEA/NO were similar in CON and PTU rats. mRNA contents of deiodinase 2 and thyroid hormone receptor α were similar in mesenteric arteries of two groups but were elevated in sural arteries of PTU group compared to CON. The abundance of eNOS protein was higher in sural arteries of PTU compared to CON rats. CONCLUSION: Antenatal/early postnatal hypothyroidism is followed by an increase in NO-mediated anticontractile influence in sural, but not in mesenteric arteries of adult animals. The diversity of hypothyroidism effects may be due to different alterations of local T3 synthesis/reception in different vascular beds.

Voluntary wheel exercise training affects locomotor muscle, but not the diaphragm in the rat.

Introduction: Functional tests and training regimens intensity-controlled by an individual are used in sport practice, clinical rehabilitation, and space medicine. The model of voluntary wheel running in rats can be used to explore molecular mechanisms of such training regimens in humans. Respiratory and locomotor muscles demonstrate diverse adaptations to treadmill exercise, but the effects of voluntary exercise training on these muscle types have not been compared yet. Therefore, this work aimed at the effects of voluntary ET on rat triceps brachii and diaphragm muscles with special attention to reactive oxygen species, which regulate muscle plasticity during exercise. Methods: Male Wistar rats were distributed into exercise trained (ET) and sedentary (Sed) groups. ET group had free access to running wheels, running activity was continuously recorded and analyzed using the original hardware/software complex. After 8 weeks, muscle protein contents were studied using Western blotting. Results: ET rats had increased heart ventricular weights but decreased visceral/epididymal fat weights and blood triglyceride level compared to Sed. The training did not change corticosterone, testosterone, and thyroid hormone levels, but decreased TBARS content in the blood. ET rats demonstrated higher contents of OXPHOS complexes in the triceps brachii muscle, but not in the diaphragm. The content of SOD2 increased, and the contents of NOX2 and SOD3 decreased in the triceps brachii muscle of ET rats, while there were no such changes in the diaphragm. Conclusion: Voluntary wheel running in rats is intensive enough to govern specific adaptations of muscle fibers in locomotor, but not respiratory muscle.

Thyroxine Induces Acute Relaxation of Rat Skeletal Muscle Arteries via Integrin αvß3, ERK1/2 and Integrin-Linked Kinase.

Aim: Hyperthyroidism is associated with a decreased peripheral vascular resistance, which could be caused by the vasodilator genomic or non-genomic effects of thyroid hormones (TH). Non-genomic, or acute, effects develop within several minutes and involve a wide tissue-specific spectrum of molecular pathways poorly studied in vasculature. We aimed to investigate the mechanisms of acute effects of TH on rat skeletal muscle arteries. Methods: Sural arteries from male Wistar rats were used for isometric force recording (wire myography) and phosphorylated protein content measurement (Western blotting). Results: Both triiodothyronine (T3) and thyroxine (T4) reduced contractile response of sural arteries to α1-adrenoceptor agonist methoxamine. The effect of T4 was more prominent than T3 and not affected by iopanoic acid, an inhibitor of deiodinase 2. Endothelium denudation abolished the effect of T3, but not T4. Integrin αvß3 inhibitor tetrac abolished the effect of T4 in endothelium-denuded arteries. T4 weakened methoxamine-induced elevation of phospho-MLC2 (Ser19) content in arterial samples. The effect of T4 in endothelium-denuded arteries was abolished by inhibiting ERK1/2 activation with U0126 as well as by ILK inhibitor Cpd22 but persisted in the presence of Src- or Rho-kinase inhibitors (PP2 and Y27632, respectively). Conclusion: Acute non-genomic relaxation of sural arteries induced by T3 is endothelium-dependent and that induced by T4 is endothelium-independent. The effect of T4 on α1-adrenergic contraction is stronger compared to T3 and involves the suppression of extracellular matrix signaling via integrin αvß3, ERK1/2 and ILK with subsequent decrease of MLC2 (Ser19) phosphorylation.

Intrauterine growth restriction weakens anticontractile influence of NO in coronary arteries of adult rats.

Intrauterine growth restriction (IUGR) is one of the most common pathologies of pregnancy. The cardiovascular consequences of IUGR do not disappear in adulthood and can manifest themselves in pathological alterations of vasomotor control. The hypothesis was tested that IUGR weakens anticontractile influence of NO and augments procontractile influence of Rho-kinase in arteries of adult offspring. To model IUGR in the rat, dams were 50% food restricted starting from the gestational day 11 till delivery. Mesenteric and coronary arteries of male offspring were studied at the age of 3 months using wire myography, qPCR, and Western blotting. Contractile responses of mesenteric arteries to α1-adrenoceptor agonist methoxamine as well as influences of NO and Rho-kinase did not differ between control and IUGR rats. However, coronary arteries of IUGR rats demonstrated elevated contraction to thromboxane A2 receptor agonist U46619 due to weakened anticontractile influence of NO and enhanced role of Rho-kinase in the endothelium. This was accompanied by reduced abundance of SODI protein and elevated content of RhoA protein in coronary arteries of IUGR rats. IUGR considerably changes the regulation of coronary vascular tone in adulthood and, therefore, can serve as a risk factor for the development of cardiac disorders.

Trophic sympathetic influence weakens pro-contractile role of Cl- channels in rat arteries during postnatal maturation.

Membrane transporters and their functional contribution in vasculature change during early postnatal development. Here we tested the hypothesis that the contribution of Cl- channels to arterial contraction declines during early postnatal development and this decline is associated with the trophic sympathetic influence. Endothelium-denuded saphenous arteries from 1- to 2-week-old and 2- to 3-month-old male rats were used. Arterial contraction was assessed in the isometric myograph, in some experiments combined with measurements of membrane potential. mRNA and protein levels were determined by qPCR and Western blot. Sympathectomy was performed by treatment with guanethidine from the first postnatal day until 8-9-week age. Cl- substitution in the solution as well as Cl--channel blockers (MONNA, DIDS) had larger suppressive effect on the methoxamine-induced arterial contraction and methoxamine-induced depolarization of smooth muscle cells in 1- to 2-week-old compared to 2- to 3-month-old rats. Vasculature of younger group demonstrated elevated expression levels of TMEM16A and bestrophin 3. Chronic sympathectomy increased Cl- contribution to arterial contraction in 2-month-old rats that was associated with an increased TMEM16A expression level. Our study demonstrates that contribution of Cl- channels to agonist-induced arterial contraction and depolarization decreases during postnatal development. This postnatal decline is associated with sympathetic nerves development.

Antenatal/early postnatal hypothyroidism alters arterial tone regulation in 2-week-old rats.

The mechanisms of vascular alterations resulting from early thyroid hormones deficiency are poorly understood. We tested the hypothesis that antenatal/early postnatal hypothyroidism would alter the activity of endothelial NO pathway and Rho-kinase pathway, which are specific for developing vasculature. Dams were treated with propylthiouracil (PTU, 7 ppm) in drinking water during gestation and 2 weeks after delivery, and their progeny had normal body weight but markedly reduced blood levels of thyroid hormones (ELISA). Small arteries from 2-week-old male pups were studied using wire myography, qPCR and Western blotting. Mesenteric arteries of PTU pups, compared to controls, demonstrated smaller maximum response to α1-adrenergic agonist methoxamine and reduced mRNA contents of smooth muscle differentiation markers α-actin and SERCA2A. Inhibition of basal NO synthesis by l-NNA led to tonic contraction of mesenteric arteries and augmented their contractile responses to methoxamine; both l-NNA effects were impaired in PTU pups. PTU pups demonstrated lower blood level of NO metabolites compared to control group (Griess reaction). Rho-kinase inhibitor Y27632 strongly reduced mesenteric arteries responses to methoxamine in PTU pups, that was accompanied by elevated Rho-kinase content in their arteries in comparison to control ones. Unlike mesenteric, saphenous arteries of PTU pups, compared to controls, had no changes in α-actin and SERCA2A contents and in responses to l-NNA and Y27632. In conclusion, thyroid hormones deficiency suppresses the anticontractile effect of NO and potentiates the procontractile Rho-kinase effects in mesenteric arteries of 2-week-old pups. Such alterations disturb perinatal cardiovascular homeostasis and might lead to cardiovascular pathologies in adulthood.