Evidence of endoplasmic reticulum stress and protein synthesis inhibition in the placenta of non-native women at high altitude

Pregnancy at high altitude is associated with a reduction in birth weight of ∼100 g/1000 m of ascent. The underlying mechanisms are unclear but may involve alteration in energy-demanding activities, such as protein synthesis. To test this hypothesis, both in vivo and in vitro approaches were used. Placental tissues from pregnant women residing at 3100 m were studied, and placental cells were incubated under hypoxia. In the 3100-m placentas, we observed dilation of endoplasmic reticulum (ER) cisternae, increased phosphorylation of eukaryotic initiation factor 2 subunit α (P-eIF2α), reduced AKT phosphorylation, and reduced P-4E-BP1 but increased 4E-BP1 protein compared to sea level controls. These findings suggest the presence of ER stress and protein synthesis inhibition. Hypoxia (1% O2) reduced proliferation of trophoblast-like JEG-3 cells, BeWo cells, and placental fibroblasts by ∼40, ∼60, and ∼18%, respectively. Sublethal dosage of salubrinal, an eIF2α phosphatase inhibitor, increased P-eIF2α and reduced BeWo cell and placental fibroblast proliferation by ∼50%. Administration of the PI-3K inhibitor LY294002 also reduced JEG-3 proliferation. Our results demonstrate that exposure to chronic hypobaric hypoxia causes mild placental ER stress, which, in turn, modulates protein synthesis and slows proliferation. These effects may account for the reduced placental villous volume, and contribute to the low birth weight that typifies high-altitude populations.—Yung, H. W., Cox, M., Tissot van Patot, M., Burton, G. J. Evidence of endoplasmic reticulum stress and protein synthesis inhibition in the placenta of non-native women at high altitude

Cross-talk between S-nitrosylation and S-glutathionylation in control of the Na,K-ATPase regulation in hypoxic heart

Oxygen-induced regulation of Na,K-ATPase was studied in rat myocardium. In rat heart Na,K-ATPase responded to hypoxia with a dose-dependent inhibition in hydrolytic activity. Inhibition of Na,K-ATPase in hypoxic rat heart was associated with decrease in NO production and progressive oxidative stress. Accumulation of oxidized glutathione (GSSG) and decrease in NO availability in hypoxic rat heart were followed by a decrease in S-nitrosylation and up-regulation of S-glutathionylation of the catalytic α subunit of the Na,K-ATPase. Induction of S-glutathionylation of the α subunit by treatment of tissue homogenate with GSSG resulted in complete inhibition of the enzyme in rat a myocardial tissue homogenate. Inhibitory effect of GSSG in rat sarcolemma could be significantly decreased upon activation of NO synthases. we have further tested if oxidative stress and suppression of the Na,K-ATPase activity are observed in hypoxic heart of two subterranean hypoxia-tolerant blind mole species (Spalax galili and Spalax judaei). In both hypoxia tolerant Spalax species activity of the enzyme and tissue redox state were maintained under hypoxic conditions. However, localisation of cysteines within the catalytic subunit of the Na,K-ATPase was preserved and induction of S-glutathionylation by GSSG in tissue homogenate inhibited the Spalax ATPase as efficiently as in rat heart. The obtained data indicate that oxygen-induced regulation of the Na,K-ATPase in the heart is mediated by a switch between S-glutathionylation and S-nitrosylation of the regulatory thiol groups localized at the catalytic subunit of the enzyme

MicroRNA-210 expression is induced in hypoxic fibroblasts but not in hypoxic JEG3 cells; RNA and protein expression of COX10 and ISCU 1/2 are downregulated in both fibroblasts and JEG3 cells cultured in hypoxic condition (1% O₂). <i>A, B)</i>

<p>MiR-210 expression in <b><i>A)</i></b> fibroblasts and in <b><i>B)</i></b> JEG3. <b><i>C, D)</i></b> Transcript levels of COX10 and ISCU1/2 in <b><i>C)</i></b> fibroblasts and <i>D)</i> JEG3. <b><i>E, F)</i></b> Protein levels of COX10 and ISCU1/2 in <b><i>E)</i></b> fibroblasts and in <b><i>F)</i></b> JEG3. * p<0.05, ** p<0.01, *** p<0.001 compared with cells cultured at 21% O2; † p<0.05 compared with cells cultured at 10% O2. Minimum of three biological replicates per cell type for each condition were performed.</p

Mitochondrial function and ETS

<p><b>mRNA and protein expression were altered in fibroblasts and JEG3 cells cultured in hypoxic conditions. </b><b><i>A, B</i></b><b>)</b> State 2 and state 3 respiration rates with the complex I substrates, glutamate and malate; and state 3 respiration rates with the complex II substrate, succinate, and complex IV substrates, TMPD and ascorbate in <b><i>A</i></b><b>)</b> fibroblasts and <b><i>B</i></b><b>)</b> JEG3. <b><i>C, D</i></b><b>)</b> Transcript levels of ETS complexes I, IV and V (ATP-synthase) in <b><i>C</i></b><b>)</b> fibroblasts and <b><i>D</i></b><b>)</b> JEG3. <b><i>E, F</i></b><b>)</b> Protein levels of ETS complexes I-IV and V (ATP-synthase) in <b><i>E</i></b><b>)</b> fibroblasts and <b><i>F</i></b><b>)</b> JEG3. * p<0.05, ** p<0.01, *** p<0.001 compared with cells cultured at 21% O2; † p<0.05, †† p<0.01, ††† p<0.001 compared with cells cultured at 10% O2. Three independent experiments were performed in duplicate for each condition; each experiment was carried out in duplicate.</p

Clinical characteristics of sea-level and high-altitude pregnancies; placental samples from non-labored, caesarean deliveries were used for ETS complex protein, mRNA and microRNA analyses in this study.

<p>Clinical characteristics of sea-level and high-altitude pregnancies; placental samples from non-labored, caesarean deliveries were used for ETS complex protein, mRNA and microRNA analyses in this study.</p

Citrate synthase protein levels and mitochondrial DNA copy number in placental fibroblasts and JEG3 cells. <i>A, B)</i>

<p>Protein levels of citrate synthase in <b><i>A)</i></b> fibroblasts and <i>B)</i> JEG3 cells. <b><i>C,D)</i></b> Mitochondrial DNA content in <b><i>C)</i></b> fibroblasts and <b><i>D)</i></b> JEG3. Minimum of four biological replicates per cell type for each condition were performed.</p