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 21 ] 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 21 ] 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 21 ] i that cause acute hypoxic pulmonary vasoconstriction.Keywords: oxygen sensing; Rieske iron-sulfur protein; reactive oxygen species; roGFP; hypoxic pulmonary vasoconstrictionIn the lung, alveolar hypoxia triggers acute constriction of small pulmonary arteries (PA), a response termed hypoxic pulmonary vasoconstriction (HPV). This response is recapitulated in cultured PA smooth muscle cells (PASMC), indicating that the oxygen-sensing mechanism underlying HPV is intrinsic to the PASMC (1-12). Our previous work has implicated increases in reactive oxygen species (ROS) signaling during hypoxia (9,10, 12). Previous studies using mitochondrial inhibitors and mitochondria-deficient (r 0 ) cells suggested that the electron transport chain (ETC) is required for hypoxia-induced ROS signaling in the pulmonary circulation (9, 11-18). We subsequently assessed ROS signaling in hypoxic PASMC using roGFP, a thiol-containing, redox-sensitive reporter (19-23) targeted to compartments within mitochondria or the cytosol (10). Unlike other methods (24-26), this targeted approach permitted the differentiation of hypoxia-induced ROS changes between mitochondrial subcompartments. During hypoxia, increased oxidation was detected in the mitochondrial intermembrane space (IMS) and the cytoso...

Mitochondria in lung biology and pathology: more than just a powerhouse

“…The acute pulmonary vasoconstriction response was then compared in the knockout and control mice, by measuring the right ventricular systolic pressure (RVSP) response to acute alveolar hypoxia. Control mice exhibited a significant increase in RVSP during 5% O 2 ventilation, which was significantly attenuated in the RISP-deficient mice (93). These findings indicate that a functional complex III is required for the acute vasopressor response to hypoxia in the lung.…”

“…After loss of RISP was confirmed, subsequent roGFP studies demonstrated that the hypoxia-induced changes in redox status in the cytosol, IMS, and the mitochondrial matrix were abolished. These studies therefore demonstrated that a functional complex III was required for the changes in redox observed in different subcellular compartments during hypoxia (93). In parallel studies, it was found that the transient increase in cytosolic Ca 2ϩ in PASMC during acute hypoxia was abrogated in cells where RISP had been deleted (93).…”

“…Accordingly, Waypa et al (93) developed a genetic model by generating a mouse to permit conditional deletion of the Rieske iron-sulfur protein (RISP). This nuclear encoded protein is critical for the function of complex III, where it accepts an electron from ubiquinol, the carrier that moves electrons into complex III from complexes I and II (34).…”

Mitochondria in lung disease

“…While there has been considerable advancement in the development of mitochondria-targeted small molecule antioxidants (3) and alternative approaches to targeting mROS (133)(134)(135)(136)(137) appear promising, such mROS-targeted therapeutic approaches must be used with caution. mROS behave as a cytoprotective agent that leaves cells less susceptible to sub- …”

Endocannabinoid anandamide mediates hypoxic pulmonary vasoconstriction

“…The elevated NAPE-PLD protein expression in PASMCs under hypoxia can explain enhanced AEA levels, even though a contribution of other recently identified enzymes capable of AEA generation such as α/β-hydrolase 4 (Abh4) and glycerophosphodiesterse 1 (GDE1) (27) or protein tyrosine phosphatase, nonreceptor type 22 (PTPn22) (28), cannot be excluded. Moreover, there are recent indications that reactive oxygen species (ROS) are involved in the modulation of pulmonary vascular tone by hypoxia (29). Because AEA has been reported to lead to ROS formation (30), a link between these two pathways also appears possible.…”