Superoxide generated at mitochondrial complex III triggers acute responses to hypoxia in the pulmonary circulation

Abstract

Rationale: The role of reactive oxygen species (ROS) signaling in the O(2) sensing mechanism underlying acute hypoxic pulmonary vasoconstriction (HPV) has been controversial. Although mitochondria are important sources of ROS, studies using chemical inhibitors have yielded conflicting results, whereas cellular models using genetic suppression have precluded in vivo confirmation. Hence, genetic animal models are required to test mechanistic hypotheses.

Objectives: We tested whether mitochondrial Complex III is required for the ROS signaling and vasoconstriction responses to acute hypoxia in pulmonary arteries (PA).

Methods: A mouse permitting Cre-mediated conditional deletion of the Rieske iron-sulfur protein (RISP) of Complex III was generated. Adenoviral Cre recombinase was used to delete RISP from isolated PA vessels or smooth muscle cells (PASMC).

Measurements and main results: In PASMC, RISP depletion abolished hypoxia-induced increases in ROS signaling in the mitochondrial intermembrane space and cytosol, and it abrogated hypoxia-induced increases in [Ca(2+)](i). In isolated PA vessels, RISP depletion abolished hypoxia-induced ROS signaling in the cytosol. Breeding the RISP mice with transgenic mice expressing tamoxifen-activated Cre in smooth muscle permitted the depletion of RISP in PASMC in vivo. Precision-cut lung slices from those mice revealed that RISP depletion abolished hypoxia-induced increases in [Ca(2+)](i) of the PA. In vivo RISP depletion in smooth muscle attenuated the acute hypoxia-induced increase in right ventricular systolic pressure in anesthetized mice.

Conclusions: Acute hypoxia induces superoxide release from Complex III of smooth muscle cells. These oxidant signals diffuse into the cytosol and trigger increases in [Ca(2+)](i) that cause acute hypoxic pulmonary vasoconstriction.

Figure 1.

Effects of hypoxia on roGFP oxidation in the cytosol, intermembrane space (IMS), and mitochondrial matrix in wild-type mouse pulmonary arterial smooth muscle cells (PASMC). Averaged results from multiple experiments in wild-type mouse PASMC expressing Cyto-roGFP (A), IMS-roGFP (B), or Mito-roGFP (C) superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. Values are means ± SEM, n = 6 cover slips, 4 to 10 cells/cover slip. *P < 0.05 compared with normoxia.

Figure 2.

Effect of Cre expression on Rieske iron-sulfur protein (RISP) expression in pulmonary arterial smooth muscle cells (PASMC) from conditional knockout mice. (A) Reverse transcriptase–polymerase chain reaction (RT-PCR) blot of mRNA extracted from uninfected (no virus), empty adenovirus–treated (200 pfu), and Cre-expressing adenovirus (200 pfu) PASMC isolated from RISP conditional knockout mice. The RT-PCR product for RISP is approximately 600 bp, whereas that for β-actin (as an internal control) is approximately 540 bp. (B) Western blot of lysates from untransfected (no virus), empty adenovirus–transfected (200 pfu), and Cre-expressing adenovirus–transfected (200 pfu) PASMC isolated from conditional knockout mice probed with antibodies against the RISP subunit or β-actin. (C) Quantified analysis of Western blots with RISP expression normalized to β-actin. Values are means ± SEM, n = 3 Western blots. *P < 0.05 compared with no virus, ^†P < 0.05 compared with empty adenovirus.

Figure 3.

Effect of Rieske iron-sulfur protein (RISP) depletion on hypoxia-induced reactive oxygen species (ROS) signaling in the cytosol, intermembrane space (IMS) and mitochondrial matrix in pulmonary arterial smooth muscle cells (PASMC) from conditional knockout mice. (A–C) PASMC isolated from RISP mice and treated with empty (control) or Cre-expressing adenovirus. Average results from multiple experiments in PASMC expressing Cyto-roGFP (A), IMS-roGFP (B), or Mito-roGFP (C) superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. Values are means ± SEM, n = 6 cover slips, 4 to 10 cells/cover slip. *P < 0.05 compared with normoxic baseline of empty virus–treated PASMC. ^†P < 0.05 compared with normoxic baseline of Cre-expressing PASMC. ^§P < 0.05 compared with empty virus–treated PASMC.

Figure 4.

Effect of Rieske iron-sulfur protein (RISP) depletion on [Ca²⁺]_i in pulmonary arterial smooth muscle cells (PASMC) during hypoxia. PASMC were treated with empty (control) or Cre-expressing adenovirus. (A) Effects of hypoxia on [Ca²⁺]_i in PAMSC assessed by YC2.3. Values are means ± SEM, n = 6 cover slips, 4 to 10 cells/cover slip. *P < 0.05 compared with normoxic baseline of empty virus–treated PASMC. ^†P < 0.05 compared with empty virus–treated PASMC. (B) Effects of exogenous H₂O₂ (50 μmol/L) on [Ca²⁺]_i in RISP-knockout mouse PASMC assessed by the calcium-sensitive, Förster resonance energy transfer (FRET) sensor YC2.3. Values are means ± SEM, n = 6 cover slips, 4 to 10 cells/cover slip. *P < 0.05 compared with baseline of untreated (no virus) PASMC. ^¥P < 0.05 compared with baseline of empty virus–treated PASMC. ^‡P < 0.05 compared with baseline of Cre-expressing PASMC. ^†P < 0.05 compared with empty virus–treated PASMC. ^§P < 0.05 compared with Cre-expressing PASMC.

Figure 5.

Effect of Rieske iron-sulfur protein (RISP) depletion on hypoxia-induced roGFP oxidation in the cytosol of mouse systemic arterial smooth muscle cells (SASMC) isolated from RISP conditional knockout mice. (A, B) SASMC isolated from RISP mice and treated with an empty (control) or Cre-expressing adenovirus. (A) Averaged results from multiple experiments in SASMC expressing Cyto-roGFP superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. (B) Effects of hypoxia on [Ca²⁺]_i in SAMSC assessed by the calcium-sensitive, Förster resonance energy transfer (FRET) sensor YC2.3. Values are means ± SEM, n = 6 cover slips, 4 to 10 cells/cover slip. *P < 0.05 compared with normoxic baseline of empty virus–treated pulmonary arterial smooth muscle cells (PASMC). ^†P < 0.05 compared with normoxic baseline of Cre-expressing PASMC. ^§P < 0.05 compared with empty adenovirus–infected PASMC.

Figure 6.

Effect of Rieske iron-sulfur protein (RISP) depletion on hypoxia-induced roGFP oxidation in the cytosol and mitochondrial matrix of cells in pulmonary arterial (PA) vessels isolated from RISP conditional knockout mice. As part of the isolation procedure for PA smooth muscle cells (PASMC), PA microvessel segments are generated that contain iron particles in their lumen. These iron particles allow for the microvessel segments to be constrained in the flow-through chamber using a magnet, thus allowing for their study under both normoxic and hypoxic conditions. (A) DIC image of PA vessel with iron particles (black objects) in the lumen. (B) Same PA vessel as in (A) demonstrating Cyto-roGFP fluorescence in vascular cells. Average results from multiple experiments in PA vessels expressing Cyto-roGFP (C) and Mito-roGFP (D) superfused with normoxic (21% O₂) or hypoxic (1.5% O₂) media. Values are means ± SEM, n = 6 PA vessels. *P < 0.05 compared with normoxic baseline of empty virus–treated PA vessels. ^†P < 0.05 compared with empty virus–treated PA vessels. DIC = differential interference contrast.

Figure 7.

Effect of Rieske iron-sulfur protein (RISP) depletion on hypoxia-induced increases in [Ca²⁺]_i in smooth muscle cells (SMC) of precision-cut lung slices and right ventricular systolic pressure (RVSP) of SMC- myosin heavy chain (MHC)-Cre/RISP^flox/flox mice. (A) Western blot of lysates from abdominal aorta of untreated (Control), tamoxifen vehicle (Vehicle), or tamoxifen (Tamoxifen) isolated from SMC-MHC-Cre/RISP^flox/flox mice probed with antibodies against the RISP subunit or β-actin. (B) Quantified analysis of Western blots expressed RISP expression normalized to β-actin. Values are means ± SEM, n = 3 Western blots. *P < 0.05 compared with Control, ^†P < 0.05 compared with Vehicle. (C) Hypoxia (1.5% O₂ for 20 min)-induced changes in [Ca²⁺]_i as assessed by calcium-mediated changes in FURA-2 measured in cells within the wall of the pulmonary arteries in precision-cut lung slices. Values are means ± SEM, n = 6 lung slices. *P < 0.05 compared with normoxic baseline for Control and Vehicle, **P < 0.05 compared with Vehicle for Tamoxifen. (D) Hypoxia (5% O₂ for 1 min) increases the RVSP in the RV pressure tracing from a Vehicle (Control) mouse. (E) RISP depletion suppresses the hypoxia (5% O₂ for 1 min)-induced increase in RVSP as observed in the RV pressure tracing from a tamoxifen-treated mouse. (F) Hypoxia (5% O₂ for 1 min)-induced changes in hypoxic pulmonary vasoconstriction as denoted by changes in the RVSP of mice. Normoxic baseline RVSP for the Untreated, Vehicle, and Tamoxifen mice were 17.6 ± 1.5, 17.3 ± 2.3, and 17.0 ± 1.1 mm Hg, respectively. Values are means ± SEM, n = 7–9 animals. *P < 0.05 compared with Untreated. ^†P < 0.05 compared with Vehicle.

Figure 8.

Model depicting mitochondria functioning as the O₂ sensor underlying the acute hypoxic pulmonary vasoconstriction (HPV) response. IMS = intermembrane space; KO = knockout; NAD⁺ = nicotinamide adenine dinucleotide; NADH = NAD⁺ reduced; PASMC = pulmonary arterial smooth muscle cells; ROS = reactive oxygen species; SASMC = systemic arterial smooth muscle cells; YC2.3-FRET = the calcium-sensitive, Förster resonance energy transfer sensor.