Acute Severe Hypoxia Decreases Mitochondrial Chain Complex II Respiration in Human Peripheral Blood Mononuclear Cells

Abstract

Peripheral blood mononuclear cells' (PBMCs) mitochondrial respiration is impaired and likely involved in myocardial injury and heart failure pathophysiology, but its response to acute and severe hypoxia, often associated with such diseases, is largely unknown in humans. We therefore determined the effects of acute hypoxia on PBMC mitochondrial respiration and ROS production in healthy volunteers exposed to controlled oxygen reduction, achieving an inspired oxygen fraction of 10.5%. We also investigated potential relationships with gene expression of key biomarkers of hypoxia, succinate and inflammation, as hypoxia and inflammation share common mechanisms involved in cardiovascular disease. Unlike global mitochondrial respiration, hypoxemia with a spO₂ ≤ 80% significantly reduced PBMC complex II respiration (from 6.5 ± 1.2 to 3.1 ± 0.5 pmol/s/10⁶ cell, p = 0.04). Complex II activity correlated positively with spO₂ (r = 0.63, p = 0.02) and inversely correlated with the succinate receptor SUCNR1 (r = -0.68), the alpha-subunit of the hypoxia-inducible factor (HIF-1α, r = -0.61), the chemokine ligand-9 (r = -0.68) and interferon-stimulated gene 15 (r = -0.75). In conclusion, severe hypoxia specifically impairs complex II respiration in association with succinate, inflammation and HIF-1α pathway interactions in human PBMCs. These results support further studies investigating whether modulation of complex II activity might modify the inflammatory and metabolic alterations observed in heart failure.

Keywords: PBMCs; complex II; hypoxia; inflammation; mitochondrial respiration; reactive oxygen species; succinate dehydrogenase.

Figure 1

Basal, maximal, coupling (RCR) (A) and complex-linked mitochondrial respiration (B) responses to hypoxia in the entire population at t-start, hypoxia and t + 60′. All the results are expressed as mean ± SEM. OCR: oxygen consumption ratio; OXPHOS: oxidation phosphorylation; RCR: respiratory control ratio.

Figure 2

Complex II mitochondrial respiration is impaired when spO₂ decreases under 80%. (A) Mitochondrial complex II activity (CII OXPHOS) in PBMCs from the healthy volunteers with spO₂ ≤ 80% or spO₂ > 80% at t-start, hypoxia and t + 60′. (B) Positive correlation between the ETC complex II respiration (CII OXPHOS) and the spO₂ values. All the results are expressed as mean ± SEM. * p = 0.04. OXPHOS: oxidation phosphorylation; spO₂: oxygen saturation measured by non-invasive pulse oximetry.

Figure 3

Hypoxia did not modify mitochondrial production of hydrogen peroxide (H₂O₂). H₂O₂ produced by mitochondrial respiration in PBMCs from the 13 healthy volunteers at t-start, hypoxia and t + 60′ in CI, CI+II and CII OXPHOS. All the results are expressed as mean ± SEM. OXPHOS: oxidation phosphorylation.

Figure 4

Gene expression variations in SUCNR1, HIF-1α, ISG15, CXCL9 and STAT3 in PBMCs and correlation with OXPHOS complex II. (A) Comparison of gene expression between t-start, hypoxia and t + 60′ in 8 subjects. (B) Comparison of gene expression between t-start, hypoxia and t + 60′ in the 4 subjects with spO₂ ≤ 80%. Analysis at t + 60′ is missing in one subject. (C) Complex II OXPHOS at t-hypoxia is inversely correlated with SUCNR1, HIF-1α, CXCL9 and ISG15 expression at t + 60′. (D) Correlations between the expressions of HIF-1α and STAT3 and SUCNR1 and the 4 other analyzed genes. Blue dots represent values for subjects with spO₂ ≤ 80% and dark dots represent values for subjects with spO₂ > 80%. β2M: beta-2-microglobulin; CXCL9: chemokine ligand-9; HIF-1α: alpha-subunit of the hypoxia-inducible factor; ISG15: interferon-stimulated gene 15; STAT3: signal transducer and activator of transcription 3; SUCNR1: ligand–receptor pair succinate receptor 1.

Figure 5

Schematic representation of the complex II (succinate dehydrogenase [SDH])–succinate–hypoxia inducible factor (HIF) and ligand–receptor pair succinate receptor 1 (SUCNR1) axis involved in the response to acute hypoxia in PBMCs, extrapolated from our data and the literature. Acute hypoxia induces a decrease in SDH activity, leading to intracellular succinate accumulation and SUCNR1 expression and activation, in synergy with several inflammatory signaling cascades reflected by increased signal transducer and activator of transcription 3 (STAT3), chemokine ligand-9 (CXCL9) and interferon-stimulated gene 15 (ISG15) gene expression. Succinate is transported into the cytoplasm, where it inhibits prolyl hydroxylase domain (PHD) enzyme function, resulting in stabilization of HIF-1α and increasing the expression of genes that have HIF-response elements such as glycolysis genes. Further succinate is exported into the local extracellular environment, where it accumulates and binds and activates SUCNR1, whose signal is in synergy with several inflammatory signaling cascades.

Figure 6

Study design. FiO₂: fraction of inspired oxygen; min: minutes; PBMCs: peripheral blood mononuclear cells.