Early View

Original article

Impact of the mitochondria-targeted antioxidant

MitoQ on hypoxia-induced pulmonary

hypertension

Oleg Pak, Susan Scheibe, Azadeh Esfandiary, Mareike Gierhardt, Akylbek Sydykov, Angela Logan,

Athanasios Fysikopoulos, Florian Veit, Matthias Hecker, Florian Kroschel, Karin Quanz, Alexandra Erb,

Katharina Schäfer, Mirja Fassbinder, Nasim Alebrahimdehkordi, Hossein A. Ghofrani, Ralph T.

Schermuly, Ralf P. Brandes, Werner Seeger, Michael P. Murphy, Norbert Weissmann, Natascha

Sommer

Please cite this article as: Pak O, Scheibe S, Esfandiary A et al. Impact of the mitochondria-

targeted antioxidant MitoQ on hypoxia-induced pulmonary hypertension. Eur Respir J 2018; in

press (https://doi.org/10.1183/13993003.01024-2017).

This manuscript has recently been accepted for publication in the European Respiratory Journal. It is

published here in its accepted form prior to copyediting and typesetting by our production team. After

these production processes are complete and the authors have approved the resulting proofs, the article

will move to the latest issue of the ERJ online.

Copyright ©ERS 2018

. Published on February 1, 2018 as doi: 10.1183/13993003.01024-2017ERJ Express

Copyright 2018 by the European Respiratory Society.

Impact of the mitochondria-targeted antioxidant MitoQ on hypoxia-induced pulmonary

hypertension

Oleg Pak1,*, Susan Scheibe1,*, Azadeh Esfandiary1, Mareike Gierhardt1, Akylbek Sydykov1,

Angela Logan2, Athanasios Fysikopoulos1, Florian Veit1, Matthias Hecker1, Florian Kroschel1,

Karin Quanz1, Alexandra Erb1, Katharina Schäfer1, Mirja Fassbinder1, Nasim

Alebrahimdehkordi1, Hossein A. Ghofrani1, Ralph T. Schermuly1, Ralf P. Brandes3, Werner

Seeger1,4, Michael P. Murphy2, Norbert Weissmann1,#, Natascha Sommer1,#

*,# Equal contribution

1Excellence Cluster Cardiopulmonary System, University of Giessen and Marburg Lung Center

(UGMLC), member of the German Center for Lung Research (DZL), Justus-Liebig-University,

35392 Giessen, Germany

2MRC Mitochondrial Biology Unit, CB2 0XY Cambridge, United Kingdom

3Institut für Kardiovaskuläre Physiologie, Goethe-Universität, German Center for

Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main,

Germany.

4Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany

Corresponding author: Prof. Norbert Weissmann

Address: Excellence Cluster Cardiopulmonary System, Aulweg 130, 35392 Giessen, Germany

Email: Norbert.Weissmann@innere.med.uni-giessen.de

Tel.: ++49/641 9942414

Fax.: ++49/641 9942419

Take home message:

The mitochondria-targeted antioxidant MitoQ attenuates acute HPV, and RV remodelling, but

not chronic hypoxia-induced PH.

1

Abstract

Increased mitochondrial reactive oxygen species (ROS), particularly superoxide have been

suggested to mediate hypoxic pulmonary vasoconstriction (HPV), chronic hypoxia-induced

pulmonary hypertension (PH) and right ventricular (RV) remodelling.

We determined ROS in acute, chronic hypoxia and investigated the effect of the mitochondria-

targeted antioxidant MitoQ under these conditions.

The effect of MitoQ or its inactive carrier substance, decyltriphenylphosphonium (TPP+), on

acute HPV (1% O2 for 10 minutes) was investigated in isolated blood-free perfused mouse

lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O2) or after banding of the main

pulmonary artery (PAB) were treated with MitoQ or TPP+ (50 mg/kg/day).

Total cellular superoxide and mitochondrial ROS levels were increased in pulmonary artery

smooth muscle cells (PASMC), but decreased in pulmonary fibroblasts in acute hypoxia. MitoQ

significantly inhibited HPV and acute hypoxia-induced rise in superoxide concentration. ROS

was decreased in PASMC, while it increased in the RV after chronic hypoxia. Correspondingly,

MitoQ did not affect the development of chronic hypoxia-induced PH, but attenuated RV

remodelling after chronic hypoxia as well as after PAB.

Increased mitochondrial ROS of PASMC mediate acute HPV, but not chronic hypoxia-induced

PH. MitoQ may be beneficial under conditions of exaggerated acute HPV.

Keywords: Hypoxic pulmonary vasoconstriction, Pulmonary hypertension, Mitochondria,

MitoQ, Reactive oxygen species, Right ventricular remodelling.

2

Introduction

Hypoxic pulmonary vasoconstriction (HPV) is a reaction of the precapillary pulmonary vessels

to alveolar hypoxia and serves to maintain ventilation-perfusion matching, thereby optimizing

the oxygenation of blood [1]. In contrast, generalized chronic hypoxia (e.g. in chronic

obstructive pulmonary disease) leads to the development of pulmonary hypertension (PH),

which is a progressive disorder characterized by pulmonary vascular remodelling, ultimately

resulting in right heart failure [1]. In addition to vascular remodelling, generalized HPV

contributes to the increased vascular resistance in chronic hypoxia-induced PH.

Pulmonary arterial smooth muscle cells (PASMC) are key players in these responses, as they

react to acute hypoxia with contraction and to chronic hypoxia with proliferation, even when

isolated [2, 3]. Mitochondrial ROS has been suggested to play a crucial role in both processes

by interaction with protein kinases, phospholipases and ion channels inducing intracellular

calcium release, or stabilization of transcription factors [1, 4]. Additionally, ROS may be

involved in the development of right ventricular (RV) remodelling [5]. However, it remains

unclear if ROS are increased or decreased during acute and chronic hypoxia in the pulmonary

vasculature and RV, and which species (superoxide or H2O2) triggers these responses [1, 3, 6].

Application of the unspecific thiol compound N-acetylcysteine which attenuates many ROS

processes could inhibit hypoxia-induced PH [7]. Overexpression of the mitochondrial SOD2

increased and overexpression of mitochondria-targeted catalase decreased pulmonary vascular

remodelling, suggesting that increased mitochondrial H2O2 contributes to hypoxia-induced PH

[8].

MitoQ is an orally available mitochondria-targeted antioxidant, consisting of an ubiquinone

moiety linked to a triphenylphosphonium (TPP+) molecule [9]. The lipophilic TPP+ cation

allows MitoQ to pass through the phospholipid bilayers and to accumulate within the

mitochondrial inner membrane driven by the mitochondrial membrane potential [9].The

3

positively charged residue (TPP+) of MitoQ is adsorbed to the matrix surface, while the

hydrophobic end (ubiquinone) is inserted into the hydrophobic core of the mitochondrial inner

membrane [10]. The active antioxidative form of MitoQ, ubiquinol, is oxidized by ROS to the

inactive form ubiquinone which is continually recycled by complex II of the respiratory chain

to its active ubiquinol form [9]. MitoQ is an effective antioxidant against lipid peroxidation,

peroxynitrite, the hydroperoxyl radical and superoxide, although its reactivity with hydrogen

peroxide (H2O2) is negligible [10-12]. However, under specific conditions, particularly in the

non-membrane bound form, MitoQ also may increase superoxide generation [9, 13], which

probably only occurs in vitro [9]. MitoQ has been demonstrated to be protective against several

ROS mediated pathologies, including cardiovascular diseases [14] and sepsis [15] in animal

models and in humans [16]. As superoxide and H2O2 are suggested to regulate development of

chronic hypoxia-induced PH, MitoQ offers the possibility to specifically target mitochondrial

ROS and oxidative damage therapeutically.

We thus aimed to elucidate the role of mitochondria-derived ROS in HPV, chronic hypoxia-

induced PH and RV remodelling. We hypothesized that the mitochondria-targeted antioxidant

MitoQ is useful 1) to investigate the role of mitochondrial ROS in these process and 2) as a

possible therapeutic tool.

4

Methods

Animal experiments were approved by the governmental authorities. C57BL/6J mice of either

sex were studied. All reagents were from Sigma Aldrich, unless stated otherwise. MitoQ and

decyl-TPP+ was provided by Antipodean Pharmaceuticals Inc.

Isolated blood-free perfused and ventilated mouse lung

Lungs of mice were isolated ventilated and blood-free perfused as described previously [17].

Chronic hypoxic exposure, pulmonary arterial banding and treatment with MitoQ

For induction of chronic hypoxia-induced PH, mice were kept under normobaric hypoxia (10%

O2) for 28 days [18]. Banding of the main pulmonary artery (PAB) was performed as described

[19]. During chronic hypoxic incubation and after PAB, mice were treated with 50mg/kg/day

MitoQ or TPP+ dissolved in H2O by gavage [9].

Development of PH was determined by in vivo hemodynamics and histological analysis [18].

Heart function were measured by transthoracic echocardiography[20].

Isolated pulmonary arterial smooth muscle cells (PASMC) and lung fibroblasts (LF)

PASMC and LF were isolated from precapillary pulmonary arteries as described [21, 22].

Measurement of ROS release by electron spin resonance spectroscopy and fluorescence

approaches

Intracellular and extracellular concentrations of ROS and reactive nitrogen species (RNS) were

determined in PASMC and tissues by a Bruker ESR spectrometer (EMXmicro, Rheinstetten,

Germany), using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine

(CMH, 0.5 mM) [23, 24]. The superoxide portion of the CMH signal was determined by

5

subtracting the ESR signal of the samples incubated with 50 Units/ml polyethylen-glycol

conjugated SOD (pSOD) and CMH from the signal of the CMH-only samples.

Mitochondrial ROS were measured by MitoSOX. The protein-based fluorescent sensor

HyPercyto was used to detect cytosolic H2O2 concentration [25].

MitoQ concentration in the lung and in the heart

MitoQ concentration was measured by reverse phase liquid chromatography and tandem mass

spectrometry [26].

Proliferation assay

Proliferation of PASMC was evaluated by determination of the ratio of proliferating cells

labeled by 5-ethynyl-uridine (EdU) to the total cell number labelled by Hoechst staining [18].

Statistical Methods

Values are given as means±SEM. Statistical significance of the data was calculated by Student's

t test with Welsh's correction in experiments with two experimental groups and by one or two-

way analysis of variance (ANOVA) in experiments with more than two experimental groups.

Two-way ANOVA was used to verify interactions between the factors of treatment (TPP+ vs.

MitoQ) and exposure (normoxia/hypoxia or PAB/sham). A p value less than 0.05 was

considered significant.

Details and additional materials and methods are available in the Supplement Material.

6

Results

MitoQ inhibits acute HPV, as well as the hypoxia-induced increase of superoxide in

PASMC

The strength of HPV was determined as the increase in PAP (∆PAP) during hypoxic ventilation

in isolated mouse lungs. The first hypoxic maneuver in the absence of any substance served as

baseline (100%). Application of 0.5µM MitoQ significantly attenuated the hypoxia-induced

elevation of PAP (4th hypoxic maneuver) compared to ∆PAP in the absence of MitoQ (1st

hypoxic maneuver) or ∆PAP in the presence of 0.5µM of TPP+ during the 4th hypoxic maneuver

(Figure 1A). In contrast, higher doses of MitoQ or TPP+ (1µM) reduced acute HPV to a similar

extent (5th hypoxic maneuver) compared to the control (Figure 1A). Both MitoQ and TPP+ did

not alter U46619-induced vasoconstriction (Figure 1B) displaying the specificity of the MitoQ

effect for HPV. The strength of HPV and U46619-induced pulmonary vasoconstriction was

similar in all experimental groups prior to the application of MitoQ or TPP+ (Figure S1A-B

Supplement).

Exposure of PASMC to 1% O2 but not to 5, 10 and 15% O2 for 5 minutes induced an increase

of the intra- and extracellular superoxide concentration measured by ESR spectroscopy (Figure

1C). 0.1µM MitoQ specifically inhibited the acute hypoxia-induced increase of superoxide,

while no effect of TPP+ in this concentration was detected (Figure 1D). In higher concentrations

(0.5µM) both, MitoQ and TPP+ caused a significant decrease of superoxide concentration in

normoxia and hypoxia.

The values of pO2, pCO2 and pH were monitored during representative experiments (Figure

S2A-F Supplement).

7

Exposure to acute hypoxia induces an increase of cytosolic H2O2, mitochondrial ROS and

total cellular superoxide specifically in PASMC

We further focused on superoxide release in the intact organ and the subcellular distribution of

different ROS. In contrast to isolated PASMC, the ROS/RNS levels in lung homogenate from

intact lungs exposed to acute hypoxic ventilation were decreased as determined by the CMH

probe (Figure 2A). Accordingly, we detected decreased superoxide release from isolated lung

fibroblasts (LF) during hypoxia (Figure 2B). With regard to the intracellular distribution, acute

hypoxia increased mitochondrial ROS in PASMC measured by MitoSOX in fluorescence

microscopy (Figure 2C). Mitochondrial staining pattern of MitoSOX was confirmed by

confocal microscopy (Figure S3 Supplement).

Given that H2O2 has been suggested as downstream messenger during acute hypoxia [1], we

next quantified cytosolic H2O2 by HyPercyto [25]. Acute hypoxia increased the concentration of

H2O2 in PASMC (Figure 2D).

Effect of MitoQ on chronic hypoxia-induced PH, PASMC proliferation and superoxide

release in chronic hypoxia

MitoQ concentration after 4 weeks of treatment with 50 mg/kg/day was in the range previously

demonstrated to have protective effects in the heart [26] (Figure 3A). Exposure of mice to

chronic hypoxia (10% O2, 4 weeks) induced an increase of the right ventricular systolic pressure

(RVSP) to a similar degree in both the MitoQ and TPP+ treated groups (Figure 3B). In

accordance, the degree of pulmonary vascular remodelling was increased after hypoxia, but not

altered by MitoQ or TPP+ treatment (Figure 3C). TPP+ treatment did not have any effect on

RVSP or cardiac output (CO) during chronic hypoxia compared to untreated controls (Figure

S4 Supplement).

8

Moreover, low doses of MitoQ did not prevent hypoxia-induced proliferation of PASMC, while

higher doses showed an unspecific effect on proliferation, as TPP+ also decreased proliferation

in high concentrations (Figure 3D).

Total superoxide concentration, mitochondrial ROS levels and cellular H2O2 were decreased

after 5 days of 1% O2 exposure of PASMC measured by ESR spectroscopy, MitoSOX

fluorescence, and HyPercyto, respectively (Figure 3E-G). Lung homogenate of mice after

chronic hypoxia showed a tendency for decreased superoxide levels which, however, did not

reach significance (Figure 3H). All ROS measurements were performed in a continuous

hypoxic environment, without re-oxygenation (see supplemental methods). Moreover, the level

of 8-hydroxyguanosine, which is an indicator of DNA damage [27], was not changed in isolated

DNA, PASMC lysates and in the medium after chronic hypoxic exposure of PASMC compared

to normoxic values (Figure 3I). Antioxidative enzymes were upregulated on mRNA level in

PASMC after chronic hypoxic exposure, but downregulated in lung homogenate of mice

exposed to hypoxia, with the extracellularly located gluthatione peroxidase (GPX) 3 being the

only enzyme upregulated on mRNA and protein level in PASMC (Figure S5A-D Supplement).

Activity of SODs or catalase and total antioxidant capacity, however, was, unchanged in

chronic hypoxic PASMC (Figure S5E-G Supplement). Mitochondrial DNA amount was

decreased, while expression of enzymes indicating decreased mitochondrial glucose oxidation

(pyruvate dehydrogenase kinase 1, PDK1) and increased anaerobic glycolysis (lactate

dehydrogenase A, LDHA) were increased (Figure S6A-C Supplement). Inhibition of PDK1 by

dichloroactetate could enhance mitochondrial superoxide concentration in normoxia, but not

hypoxia (Figure S6D Supplement).

In low concentrations, in which MitoQ did not inhibit proliferation of PASMC (Fig. 3D), it

decreased the superoxide concentration in normoxia compared to the normoxic untreated

9

control (Fig. 3J). However, there was no difference in hypoxic superoxide concentration in

untreated, TPP+ treated or MitoQ treated PASMC (Fig. 3J).

Effect of MitoQ on RV remodelling in chronic hypoxia and after PAB

In contrast to the development of PH/vascular remodelling, RV remodelling differed after

chronic hypoxia in the MitoQ compared to the TPP+ treated group (Figure 4). The ratio of the

weight of the RV compared to the weight of the LV+septum (RV/LV+septum) was significantly

increased after hypoxic exposure in TPP+, but not in MitoQ treated group (Figure 4A).

Echocardiographically determined right ventricular wall thickness (RVWT) was increased after

chronic hypoxic exposure in both groups, albeit to a significantly lower level in the MitoQ

group (Figure 4C). Moreover, right ventricular internal diameter (RVID) and right ventricular

outflow tract diameter (RVOTD) were not increased after chronic hypoxia in the MitoQ, in

contrast to the TPP+ group, indicating less RV dilatation during chronic hypoxia in the presence

of MitoQ (Figure 4C, D, H). CO and TAPSE were decreased after chronic hypoxia, albeit to a

similar degree in both the MitoQ and TPP+ groups (Figure 4 E, F). Besides these specific effects

of MitQ on the RV,the carrier of MitoQ, TPP+, may have some additional effects on RV

remodeling, as RVWT and weight was decreased in the TPP+ treated group compared to the

untreated group (Figure S4 Supplement).

Furthermore, MitoQ application after PAB, which served as hypoxia-independent stimulus for

RV hypertrophy also improved RV remodelling (Figure 5). RVID was significantly reduced

(Figure 5C), while TAPSE was significantly increased (Figure 5E) after PAB in MitoQ treated

mice compared to TPP+ treated mice.

The level of superoxide concentration was significantly higher in RV homogenate after chronic

hypoxic exposure (Figure 4H Supplement). MitoQ treatment decreased the superoxide

10

concentration after chronic hypoxic exposure compared to the TPP+ group (Figure 4G) and after

PAB compared to the sham group (Figure 5H).

Discussion

This study showed that increased superoxide, most likely originating from mitochondria,

regulates acute, but not chronic hypoxia-induced PH. This conclusion is based on the facts that

1) cellular superoxide and mitochondrial ROS production was increased after 5 minutes of

hypoxic exposure of PASMC and decreased after 5 days of hypoxic exposure, and 2) the

mitochondria-targeted antioxidant MitoQ could inhibit the acute hypoxia-induced increase in

superoxide, but not chronic hypoxia-induced PH. Adaptation of mitochondrial

metabolism/amount may underlie the differential role of ROS acute and chronic hypoxia. .

To the best of our knowledge, this is the first report investigating a specifically mitochondria-

targeted antioxidant with regard to its effects on the response of the pulmonary vasculature to

acute and chronic hypoxia.

Although there is recent evidence that mitochondria-derived superoxide plays an important role

in acute and chronic hypoxic signalling in the pulmonary vasculature [3, 6, 28-30], two

opposing concepts exist suggesting either an increase or decrease of ROS during hypoxia [3, 6,

28, 29]. This controversy is mainly based on the facts that 1) measurement of ROS during

hypoxia is prone to artefacts, 2) localization, time and species of ROS has to be taken into

account and 3) specific pharmacological antioxidants for mitochondria were not available. We

here used three approaches to measure ROS including ESR technology, a fluorescent dye and

a protein-based sensor. Moreover, the experiments in cells and tissue homogenates in this study

were performed under continuous hypoxia, so that artefacts due to reoxygenation of the

measurement sample are excluded. We identified that the concentration of mitochondrial ROS,

as well as cytosolic H2O2, which acts as a possible downstream signalling mediator of

11

superoxide [1], was increased in PASMC during acute hypoxia. Mitochondrially produced

superoxide can either be converted to H2O2 by mitochondrial SODs, which can diffuse into the

cytosol or be converted to water by mitochondrially located gluthatione peroxidases (GPX), or

be released from the mitochondria into the cytosol via voltage-dependent anion channels [31]

where it can be converted to H2O2 by the SOD1. Thus, either H2O2 or local superoxide (e.g. by

co-localization of mitochondria and ion channels) or both can act as downstream mediator in

acute hypoxia in PASMC. Recently, we provided evidence that H2O2 originating from

mitochondrial superoxide is essential for hypoxic signalling and HPV in PASMC [30].

Although the mechanism for the superoxide release during acute hypoxia is still not fully

elucidated, we could show that it depends on the presence of a specific isoform of the

mitochondrial cytochrome c oxidase subunit 4 (Cox4i2) which causes mitochondrial

hyperpolarization in acute hypoxia, a condition well known to promote superoxide release from

complex I or III of the mitochondrial respiratory chain [30]. Downstream targets for H2O2 are

Kv channels [30], but possibly also several other ion channels [3, 32]. In contrast to PASMC,

acute hypoxia decreased superoxide concentration in pulmonary fibroblasts. This finding may

explain why the ROS concentration in the lung homogenates was decreased during acute

hypoxia, although our measurements could not discriminate between different ROS species in

the whole lung. Nevertheless, our results could explain the discrepancy between measurements

of ROS concentration in the whole lung and PASMC by the fact of cell type specific regulation

of ROS. Our findings are consistent with others studies showing increased mitochondrial ROS

concentration in acute hypoxia [33-37]. The relevance of the increased superoxide in PASMC

for HPV was shown, as MitoQ inhibited the superoxide increase and HPV in our study.

The effect that inhibition of HPV reached a significant level at a slightly higher dose of MitoQ

than the inhibition of hypoxia-induced superoxide release in PASMC might be explained by

12

the fact that isolated PASMC may be more sensitive to MitoQ than PASMC in isolated organs,

where the substance has a higher distribution volume and diffusion distance.

At high doses MitoQ and TPP+ have been shown to inhibit oxidative phosphorylation [38] and

interact with the mitochondrial sodium/calcium exchanger [39], thereby likely decreasing

mitochondrial membrane potential. However, low doses of TPP+, which were used in this

current study in vitro, did not alter mitochondrial respiration [40]. Thus, the effect of TPP+ on

acute HPV and the hypoxia-induced increase in superoxide at concentrations beyond 0.5µM

could be related to its property to decrease mitochondrial membrane potential, as mitochondrial

hyperpolarization has been observed during acute HPV [37]. However, as MitoQ inhibited the

acute hypoxic responses in the lower concentrations range, while TPP+ did not, this effect can

be regarded as specific for the antioxidant properties of MitoQ.

In addition to HPV, chronic hypoxia causes PH due to pathological pulmonary vascular

remodelling which is characterized, in part, by proliferation of PASMC of pulmonary vessels

[18]. As in case of HPV, evidence for both a decrease [41] and an increase of cellular ROS

concentration triggering PH development [8] has been provided. Recently, an elegant study

showed that an increase of specifically mitochondrial H2O2 may promote development of

chronic-hypoxia induced PH [8]. In our study, the level of superoxide, mitochondrial ROS and

cytosolic H2O2 measured by ERS spectroscopy, MitoSOX and HyPercyto fluorescence,

respectively, was significantly decreased in the cytosol and mitochondria of PASMC after 5

days of incubation in 1% O2 as well as in lung homogenate of chronically hypoxic mice,

supporting the hypothesis that chronic hypoxia induces a decrease of total cellular ROS.

Additionally, the level of 8-hydroxyguanosine which is an indicator of oxidative-induced DNA

damage[27] was not altered during chronic hypoxia in PASMC. Moreover, oral treatment with

MitoQ reaching lung tissue levels which demonstrated protective effects in a previous study

[26] did not inhibit chronic hypoxia-induced PH or PASMC proliferation. Interestingly, we

13

found downregulation of intracellular antioxidative enzymes but unchanged antioxidative

capacity in chronic hypoxic PASMC. Together with the finding of decreased mitochondrial

glucose oxidation, indicated by decreased mitochondrial DNA amount, increased expression of

PDK1 (which inhibits mitochondrial pyruvate oxidation) and increased expression of LDHA

(which mediates anaerobic pyruvate metabolism), we suggest that adaptation of cellular

metabolism causes the decreased ROS concentration in chronic hypoxia compared to acute

hypoxia. In this regard, it was suggested previously that mitochondrial metabolism may be

downregulated in chronic hypoxia to avoid excessive mitochondrial ROS production [42].

Interestingly, activation of mitochondrial glucose oxidation by DCA, which can inhibit

hypoxia-induced PH and human PAH [43], could increase superoxide release in normoxia, but

not hypoxia, indicating that mitochondrial adaptation to hypoxia-induced ROS levels is

multifactorial and DCA inhibits PH by a superoxide independent mechanism. Thus, our data

suggest that upregulation of mitochondrial ROS does not play a role in chronic hypoxia-induced

pulmonary vascular remodelling due to diverse adaptive mechanisms that decrease superoxide

concentration.

Moreover, decreased mitochondrial ROS in PASMC do not seem to interact with proliferative

pathways in PASMC, as decreased superoxide levels in normoxia during MitoQ treatment did

also not alter pulmonary vascular remodelling.

It cannot be excluded, however, that a cell-type specific and localized subcellular release of

specific ROS may activate proliferation pathways. Upregulation of other ROS sources might

contribute to this finding, as hypoxia-induced ROS release might also originate from specific

isoforms of the NADPH oxidase [4].

Interestingly, in chronic hypoxia MitoQ application decreased the development of RV

hypertrophy and RV dilatation. TPP+ also decreased RV hypertrophy compared to untreated

control animals, albeit to a lesser extent than MitoQ and had almost no effect on RV dilatation,

14

suggesting that mitochondrial ROS specifically can influence RV remodelling. Furthermore,

MitoQ treatment attenuated the hypertrophy and dilatation of the RV as well as prevented

development of RV dysfunction after PAB. Accordingly, MitoQ treatment decreased

superoxide concentration in the RV.

It has been shown previously that RV remodelling may rely on ROS dependent signalling

pathways [5, 44], and that the transition from beneficial adaptive concentric RV hypertrophy to

RV dilatation and failure may depend on the amount of ROS release [45]. Our study does not

answer the question by which mechanisms mitochondrial ROS release is activated in the RV,

but suggest that increased RV afterload and not hypoxia induces the increase in ROS. Moreover,

the therapeutic potential and underlying mechanisms of MitoQ treatment on RV dysfunction in

PH have to be further evaluated.

In conclusion, our study revealed that acute hypoxic exposure initiated an increase of

mitochondrial ROS, most probably superoxide, in PASMC, which regulates HPV, while

chronic hypoxic exposure was associated with a decrease of the concentration of different ROS

in PASMC. Application of the mitochondria-targeted antioxidant, MitoQ, specifically

attenuated HPV, but did not inhibit the development of pulmonary vascular remodelling and

chronic hypoxia-induced PH, while it attenuated RV dilatation (Figure 6).

15

Acknowledgments

The authors thank K. Homberger, I. Breitenborn-Mueller, N. Schupp, M. Wessendorf, and E.

Kappes for technical assistance.

Funding: Supported by the Deutsche Forschungsgemeinschaft AZ WE 1978/4-2 and SFB

1213, projects A06 and CP02, by the Medical Research Council UK (MC_U105663142) and

by a Wellcome Trust Investigator award (110159/Z/15/Z) to MPM.

16

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19

Figure 1.

Effect of MitoQ on hypoxic pulmonary vasoconstriction (HPV) in isolated ventilated and

perfused mouse lungs and on superoxide concentration in pulmonary artery smooth

muscle cells (PASMC) during acute hypoxia

A) Increase of pulmonary arterial pressure (ΔPAP) in isolated ventilated and perfused mouse

lungs during acute hypoxic ventilation (1% O2, 5.3% CO2, balanced with N2) or B) after the

bolus injection of the thromboxane mimetic, U46619, in the absence (Control group) or

presence of the mitochondria-targeted antioxidant (MitoQ group) or the inactive carrier

substance (TPP+ group). n=5-6 isolated lungs each group. ** p<0.01 compared to TPP+ treated

group, †† p<0.01, ††† p<0.001 compared to respective control groups by two-way ANOVA

with Tukey post hoc test.

C) Superoxide concentration in PASMC after 5 minutes of acute exposure to different oxygen

concentrations. The superoxide concentration was measured by ESR spectroscopy as difference

in the CMH signal with or without pegylated superoxide dismutase (∆pSOD). n=3-4 individual

cell isolations per group. *** p<0.001 by one-way ANOVA with Tukey post hoc test.

D) Superoxide concentration in PASMC after either 5 minutes of acute hypoxic (1% O2, rest

N2) or normoxic (21% O2) incubation in presence or absence of the mitochondria-targeted

antioxidant (MitoQ) or the inactive carrier substance (TPP+). n=5-6 individual cell isolations

per group. * p<0.05, ** p<0.01 by two-way ANOVA with Tukey post hoc test. A.U.: arbitrary

units.

Figure 2.

Effect of acute hypoxia on subcellular, cell-type specific ROS concentration

20

A) Total intra- and extracellular ROS/RNS determined by CMH intensity in lung homogenates

from isolated ventilated and perfused lungs after 5 minutes of acute hypoxic (1% O2, 5.3% CO2,

balanced with N2 [5min HOX]) or normoxic ventilation (21% O2, 5.3% CO2, balanced with

N2). n=4 isolated lungs each group. **** p<0.0001 by Student's t test with Welsh's correction.

B) Superoxide concentration in primary mouse lung fibroblasts (LF) after 5 minutes of acute

exposure to different oxygen concentrations. The superoxide concentration was measured by

ESR spectroscopy as the difference in the CMH signal with or without pegylated superoxide

dismutase (∆pSOD). n=3-4 individual cell isolations per group. **** p<0.0001 by one-way

ANOVA with Tukey post hoc test. A.U.: arbitrary units.

C) Mitochondrial ROS concentration in PASMC during perfusion with hypoxic buffer. The

level of mitochondrial ROS concentration is presented as the change in the MitoSox fluorescent

signal compared to baseline and normoxic values. n=4 individual cell isolations. * p<0.05 by

Student's t test with Welsh's correction.

D) Intracellular H2O2 concentration in PASMC during perfusion with hypoxic buffer. The level

of cellular H2O2 is presented as the change in the HyPercyto fluorescent signal compared to

baseline and normoxic values. n=4 individual cell isolations. ** p<0.01, **** p<0.0001 by

Student's t test with Welsh's correction.

Figure 3.

Effect of MitoQ on development of chronic hypoxia induced pulmonary hypertension (4

weeks, 10% O2)

A) MitoQ concentration in mouse lungs and livers after 4 weeks of treatment with MitoQ

(60mg/kg/day) via gavage measured by liquid chromatography and tandem mass spectrometry.

21

B) Right ventricular systolic pressure (RVSP), and C) pulmonary vasculature remodelling in

mice exposed for 4 weeks to chronic hypoxia (10% O2 [4w HOX]) or normoxia (21% O2 ) and

treated with the mitochondria-targeted antioxidant (MitoQ) or the inactive carrier substance

(TPP+). n=8 animals per group. *** p<0.01, ****p<0.0001 by two-way ANOVA with Tukey

post hoc test.

D) Proliferation of PASMC after 5 days of hypoxic (1% O2) or normoxic (21% O2) incubation

in the presence of MitoQ or TPP+. Proliferation is presented as percent of 5-ethynyl-uridine

(EdU) positive proliferating cells compared to the total cell number labeled by Hoechst staining.

n=4 individual cell isolations. *** p<0.001, **** p<0.0001 by two-way ANOVA with Tukey

post hoc test.

E) Superoxide concentration in PASMC after 5 days of chronic hypoxic (1% O2) or normoxic

(21% O2) incubation. Superoxide concentration was measured by ESR spectroscopy as the

difference in the CMH signal with or without pegylated superoxide dismutase (∆pSOD). n=4

individual cell isolations. p<0.001 by Student's t test with Welsh's correction.

F) Mitochondrial ROS concentration determined by MitoSOX fluorescence in PASMC after 5

days of chronic hypoxic (1% O2) or normoxic (21% O2) incubation. n=4 individual cell

isolations. p<0.001 by Student's t test with Welsh's correction.

G) Cytosolic H2O2 concentration determined by HyPercyto fluorescence in PASMC after 5 days

of hypoxic incubation (1% O2). n=4 individual cell isolations. *p<0.05, ***p<0.001,

****p<0.0001 by Student's t test with Welsh's correction.

H) Superoxide concentration per weight (g) in lung homogenate from mice exposed to chronic

hypoxia (10% O2) or normoxia (21% O2) for 4 weeks. The superoxide concentration was

measured by ESR spectroscopy as difference in the CMH signal with or without pegylated

superoxide dismutase (∆pSOD). n=3.

22

I) Concentration of 8-hydroxyguanosine in DNA (diluted in buffer), cell lysate and growth

medium from PASMC exposed to chronic hypoxia (1% O2) or normoxia (21% O2) for 5 days.

J) Superoxide concentration in PASMC exposed to chronic hypoxia (1% O2) or normoxia (21%

O2) for 5 days in presence of 100nM of TPP+ or MitoQ. n=4 individual cell isolations. ** p<0.01

by two-way ANOVA with Tukey post hoc test.

A.U.: arbitrary units.

Figure 4.

Effect of MitoQ on remodelling of the right ventricle (RV) in mice exposed to chronic

hypoxia (10% O2) for 4 weeks

A) Ratio of RV mass to mass of left ventricle plus septum (RV/[LV+Septum]), and

echocardiographic parameters, such as B) right ventricular wall thickness (RVWT), C) right

ventricular internal diameter (RVID), D) right ventricular outflow tract diameter (RVOTD), E)

tricuspid annular plane systolic excursion (TAPSE) as parameter for systolic right heart

function, and F) cardiac output (CO) in mice exposed to chronic hypoxia (10% O2 [4w HOX])

or normoxia (21% O2 ) for 4 weeks and treated with the mitochondria-targeted antioxidant

(MitoQ) or the inactive carrier substance (TPP+). n=8 animals per group, *p<0.05, **p<0.01,

**** p<0.0001 and †p<0.05; †† p<0.01 interaction by two-way ANOVA with Tukey post hoc

test.

G) Superoxide concentration in RV homogenate in mice exposed to chronic hypoxia (10% O2)

or normoxia (21% O2) for 4 weeks and treated with TPP+ or MitoQ. The superoxide

concentration was measured by ESR spectroscopy as difference in the CMH signal with or

23

without pegylated superoxide dismutase (∆pSOD). Data presented in ratio to the organ mass

(g). n=3-5 * p<0.05 by two-way ANOVA with Tukey post hoc test.

H) Representative echocardiographic images of RVWT and RVOTD measurements in the right

parasternal long axis.

A.U.: arbitrary units.

Figure 5. Effect of MitoQ on remodelling of the right ventricle (RV) in mice after

pulmonary artery banding

A) Ratio of RV mass to the mass of left ventricle plus septum (RV/[LV+Septum]), B) right

ventricular wall thickness (RVWT), C) right ventricular internal diameter (RVID), D) right

ventricular outflow tract diameter (RVOTD), E) tricuspid annular plane systolic excursion

(TAPSE), F) right ventricular systolic pressure (RVSP) and G) cardiac output (CO) in mice

after pulmonary artery banding (PAB) or sham operation (Sham). n=8 animals per group,

**p<0.01, **** p<0.0001 and †p<0.05 interaction by two-way ANOVA with Tukey post hoc

test.

H) Superoxide concentration in RV homogenate in mice after sham or PAB operation and

treated with TPP+ or MitoQ. Superoxide concentration was measured by ESR spectroscopy as

difference in the CMH signal with or without pegylated superoxide dismutase (∆pSOD). Data

presented in ratio to the organ mass (g). n=5 * p<0.05 by two-way ANOVA with Tukey post.

I) Representative echocardiographic images of RVWT and RVOTD measurements in the right

parasternal long axis.

Figure 6.

24

Effect of MitoQ in hypoxia

Acute hypoxia increases ROS, while chronic hypoxia decreases ROS in the pulmonary

vasculature, but increases ROS concentration in right ventricle. Mitochondria-targeted

antioxidant MitoQ inhibits acute hypoxia-induced pulmonary vasoconstriction (HPV) and

chronic hypoxia- as well as PAB-induced right ventricular dilatation.

A B

C D

Figure 1

**

Supe

roxi

de

conc

entr

atio

n [A

.U.]

Dose of TPP+, µM

Dose of MitoQ, µM

- 0.500.10

-

-

-

-

-

*Normoxia

5min 1% O2

0.500.10

0.0

0.5

1.0

1.5

Stre

ngth

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PV [%

]

MitoQ group

TPP+ groupControl group

-Hypoxic maneuverDose of TPP+ or MitoQ, µM 0.01 0.10 0.50 1.00

1 2 3 4 5

†††**

††

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n [%

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1 2 3 4

0

50

100

150

200Su

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[A.U

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211 5 10 15Oxygen concentration [%]

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Figure 2

A

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0.06

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Dose of MitoQ, µM

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-

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40

60

80

100

RVS

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20

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40

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600

800

1000

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NOX 4w HOX NOX 4w HOX

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NOX NOX 4w HOX4w HOXMitoQTPP+

NOX NOX 4w HOX4w HOX

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0

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10

15

20

25

TPP+ MitoQ

Normoxia 4w hypoxiaNormoxia 4w hypoxia

RVWT:D=0.200mm

RVOTD:D=1.477mm

RVWT:D=0.292mm

RVOTD:D=1.815mm

RVWT:D=0.195mm

RVOTD:D=1.484mm

RVWT:D=0.246mm

RVOTD:D=1.463mm

Supe

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de c

once

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tion

[A.U

.] /

wei

ght [

g]

*

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20

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Figure 5

E

B

G H

I

CA

F

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TD [m

m]

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TPP+

Sham PAB

MitoQ

*

Sham PAB Sham PAB

TPP+ MitoQ

**** ****

RVS

P [m

mH

g]

0

20

40

60

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RV/

[LV

+ se

ptum

]

Sham PAB Sham PAB

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**** ****

**

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T [m

m]

Sham PAB Sham PAB

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**** ****

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0.1

0.2

0.3

0.4

0.5

Sham PAB Sham PAB

TPP+ MitoQ

RVI

D [m

m]

**** ****

**

† interaction † interaction

†† interaction

0.0

0.5

1.0

1.5

2.0

2.5

Sham PAB Sham PAB

TPP+ MitoQ

Sham PAB Sham PAB

TPP+ MitoQ

**** ****

****

**** ***

†††† interaction

0.0

0.5

1.0

1.5

TAPS

E [m

m]

D

Sham PAB Sham PAB

TPP+ MitoQ

**** ****

CO

[ml/m

in]

0

5

10

15

20

25

0.0

0.5

1.0

1.5

2.0

2.5

TPP+ MitoQ

Sham PAB Sham PAB

RVWT:D=0.215mm

RVOTD:D=1.23mm

RVWT:D=0.425mm

RVOTD:D=1.97mm

RVWT:D=0.421mm

RVOTD:D=1.75mm

RVWT:D=0.22mm

RVOTD:D=1.28mm

Supe

roxi

de c

once

ntra

tion

[A.U

.] /

wei

ght [

g]

0

50

100

150

200

Pulmonary artery HPVPulmonary vascular remodeling/

Pulmonary hypertension

Right ventricular remodeling

Hypertrophy

Heart

Chronic hypoxiaAcute hypoxia

MitoQ

↑ROS

↑ROS

↓ROS

A M E A M EAME

RVLV

LV

RV

Pulmonary arterybanding

Figure 6

Dilatation

Online data Supplement

Methods

Isolated blood-free perfused and ventilated mouse lung

The model of isolated ventilated and blood-free perfused mouse lungs has been described

previously [1]. Briefly, a switch from 21% O2 (5.3% CO2, rest N2) in the ventilator gas supply

to 1% O2 (5.3% 5.3% CO2, rest N2) was used to induce HPV in the blood-free perfused mouse

lungs. Repetitive hypoxic ventilation manoeuvers (10 min) were alternated with normoxic

ventilation periods (15 min). Repetitive hypoxia-independent pulmonary vasoconstriction was

induced by bolus injection of the thromboxane A2 mimetic U46619 (final concentration in the

perfusate 190 nM) into the pulmonary arterial line. Increasing doses 0.01-1.00µM of MitoQ or

decyl-TPP+ as a control were added to the perfusate 5 minutes before each hypoxic ventilation

period, or 5 minutes before bolus injection of U46619 which was used as hypoxia-independent

vasoconstrictive stimulus. ΔPAP (increase in pulmonary arterial pressure) was calculated by

subtracting the peak increase of PAP for each individual intervention (hypoxic ventilation or

application of U46619) from the baseline PAP prior to the intervention. All gas concentrations

were calculated for sea level conditions.

For ROS measurements, CMH was added 5 minutes before the hypoxic ventilation and lung

homogenate taken 5 minutes after start of the hypoxic ventilation.

Isolation of pulmonary artery smooth muscle cells (PASMC)

PASMC were isolated from pulmonary precapillary arteries and cultured as described

previously [2]. Briefly, the pulmonary artery was cannulated by a handmade cannula and M199

(Medium 199, Invitrogen, Carlsbad, USA) growth medium containing 5 mg/ml glow melting

point agarose, 5mg/ml Fe3O4, 1% penicillin, and 1% streptomycin was injected into rats (12

ml) and mice (3 ml). In this mixture, iron particles do not pass through the capillaries and

therefore only precapillary arteries were filled with the rapidly solidifying agarose and iron.

Lung tissue was minced with scissors for 5 min in 1ml PBS (phosphate buffered saline). The

tissue mixture was then suspended in 10ml PBS in a falcon tube, which was placed in a

magnetic holder. The pulmonary arteries containing the iron particles and agarose accumulate

on the tubing walls. The supernatant was aspirated, and the arteries, after rinsing 3 times with

PBS, were transferred into Petri dishes containing 10ml of M199 with 80U/ml collagenase and

were then incubated at 37°C for 60 min. The tissue mixture was disrupted by drawing it through

15 and 18 gauge needles 5-6 times each. The resulting suspension containing the medial layer

of the arteries attached to iron particles was placed in clear plastic tubes and rinsed three times

with M199 containing 10% FCS (fetal calf serum) in the magnetic holder, as described above.

The medial layer of the pulmonary artery attached to iron was finally re-suspended in medium,

transferred to culture flasks and incubated at 37°C in the cell incubator for 4 to 5 days. Upon

reaching 80% confluence, the grown cells were trypsinized and divided into the fresh culture

flasks.

Isolation of primary lung fibroblast (LF)

Primary lung fibroblasts were isolated as described [3]. Briefly, mouse lungs were minced and

incubated in 2mg/ml preheated collagenase P (Hoffmann-La Roche, Basel, Switzerland) for

45min at 37°C. Afterwards, the tissue mixture was disrupted by drawing it through 15 and 18

gauge needles 5-6 times each, filtered through a 40µm strainer and centrifuged at 4°C for 8min

at 400g. The cell pellet was re-suspended in 5 ml 10% FBS, DMEM (Thermo Fisher Scientific,

Waltham, USA). LF from passage 1 were used for experiments.

Measurement of ROS release by electron spin resonance spectroscopy

Intracellular and extracellular concentrations of ROS and reactive nitrogen species (RNS) were

determined in PASMC and tissues by means of an ESR spectrometer (EMXmicro, Bruker,

Rheinstetten, Germany), using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-

tetramethylpyrrolidine (CMH, 0.5 mM) [4, 5] or 1-hydroxy-3-carboxy-2,2,5,5-

tetramethylpyrrolidine (CPH, 0.5mM) (Noxygen, Elzach, Germany). The superoxide portion

of ROS/RNS was determined by subtracting the ESR signal of the samples incubated with 50

Units/ml polyethylen-glycol conjugated superoxide dismutase (pSOD) from the signal of the

CMH-only samples. Incubation and measurements were performed in a specific buffer for ESR

measurements (99.0 mM NaCl, 4.69 mM KCl, 2.5 mM CaCl2 x 2H2O, 1.2 mM MgSO4 x 7H2O,

25 mM NaHCO3, 1.03 mM KH2PO4, 5.6 mM D(+) Glucose, 20 mM Na-HEPES, 25 µM

deferoxamine, 5 µM diethyldithiocarbamate). Approximately 100,000 precapillary mouse

PASMC from passage 1 were used per sample. Incubation was started at the time point

corresponding to the addition of pSOD. After 90 minutes, MitoQ, or alternatively decyl-TPP+

was added in doses of 0.1 and 0.5µM, followed by the addition of CMH after another 5 minutes,

followed by an incubation period of 30 minutes. For the hypoxic samples, hypoxia was applied

by exposing the PASMC for 5 minutes (acute hypoxic experiments) to a 37 °C warm

atmosphere containing 1% O2 (rest N2) within a hypoxic glove chamber (Coy Laboratory

Products, Coy Drive, USA). Normoxic samples, which were handled in parallel, were incubated

in 21% O2 at 37°C. After the end of the incubation period, all samples were immediately frozen

in liquid nitrogen. In order to avoid reoxygenation, the hypoxic samples were frozen directly

within the hypoxic glove chamber.

For chronic hypoxic experiments, PASMC were incubated for 5 days in a 37 °C warm

atmosphere containing 1% O2 (5% CO2, rest N2). Afterwards, the measurement of superoxide

concentration was performed as described above. All manipulations were performed in a

hypoxic glove chamber under continuous hypoxic conditions.

To investigate the superoxide levels within lung and heart tissues in chronic hypoxia, mice

exposed to hypoxia (10% O2) for 4 weeks were sacrificed under hypoxia and superoxide

concentration was measured by ESR microscopy as described above.

For isolated lung experiments only CMH and not the CMH minus CMH/pSOD signal was used,

as perfusion of the lungs with pSOD for 2h prior to measurement was not possible. However,

the CMH signal represents the total ROS/RNS signal without specification for superoxide [4,

5].

X-band (9.65 GHz) ESR spectra of the frozen samples were recorded by the EMXmicro

spectrometer using the following acquisition parameters: G-factor 2.0063, center field ~3366

G, modulation amplitude 2.999 G, receiver gain 50 dB and an attenuation of 20 dB (resulting

in a microwave power of 2mW).

Chronic hypoxic exposure, pulmonary arterial banding, treatment with MitoQ and

determination of PH

For induction of chronic hypoxia-induced PH, mice were kept under normobaric hypoxia (10%

O2) in a ventilated chamber for 28 days. Banding of the main pulmonary artery (PAB) was

performed as described previously [6]. During chronic hypoxic incubation and after PAB, mice

were treated with MitoQ or decyl-TPP+, dissolved in tapped water, at a concentration of

50mg/kg/day by gavage[7]. Control mice were kept in a normoxic chamber and also treated

with MitoQ or TPP+. Development of PH was determined by measurement of right ventricular

systolic pressure (RVSP), pulmonary vasculature remodeling, and right ventricular (RV)

hypertrophy, as described previously[8]. Global and right heart function were measured by

transthoracic echocardiography [9].

ROS measurement by the fluorescent dye MitoSOX and the fluorescent protein HyPercyto

For acute hypoxic experiments, mitochondrial ROS and cytosolic H2O2 concentrations were

measured in PASMC by MitoSOX and HyPercyto, respectively. 5µM MitoSOX (Invitrogen,

Carlsbad, USA) was used for detection of mitochondrial ROS as described [2]. Prior to the

acute hypoxic exposure precapillary PASMC were incubated for 1 h in 5µM MitoSOX diluted

in HRB buffer (HRB; 136.4 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2.2 mM CaCl2, 10 mM

Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 5 mM glucose, pH 7.4) at 37C.

After accumulation in mitochondria, MitoSOX becomes fluorescent upon oxidation by

different ROS [10]. For intracellular H2O2 detection, the coding information for the H2O2-

sensitive, enhanced yellow fluorescent protein variant HyPercyto (cytosolic hydrogen peroxide

sensor) from Evrogen Company (Moscow, Russian Federation) [11] was subcloned under the

control of the EF-1α (human elongation factor-1 alpha) enhancer/promoter into the pWPXL

plasmid (distributed by Addgene, Boston, USA) and packed with a second-generation lentivirus

transduction system with pMD2.G, as the envelope and psPAX2, as a packing vector (Addgene,

Boston, USA). Lentiviral transduction was carried out with a titer of at least 1×107 particles

according to established protocols (see http://tronolab.epfl.ch/ for more details). HyPercyto has

two excitation peaks with maxima at 420 nm and 500 nm, and one emission peak with a

maximum at 516 nm. H2O2 increases the excitation at 500nm and decreases the excitations at

420nm of the HyPercyto construct, allowing ratiometric measurements. Fluorescent signals for

both MitoSOX and Hypercyto were collected from individual PASMC.

Acute hypoxia was induced by switching from normoxic perfusion buffer (with a flow of 1

ml/min) to hypoxic buffer in a closed perfusion chamber (PeCon, Germany). For control

experiments the normoxic perfusion buffer was switched to a second normoxic perfusion

buffer. Fluorescent signals were analyzed using a Polychrome II monochromator and IMAGO

CCD camera (Till Photonics, Germany) coupled to an inverted microscope (IX70; Olympus,

Germany).

To measure the level of mitochondrial ROS concentration in chronic hypoxia, PASMC were

incubated for 4 days in 1% O2, and subsequently split under hypoxia (1% O2 ) within a hypoxic

glove chamber (Coy Laboratory, USA). 30.000 PASMC were seeded into 96 multiwell plates.

The next day, the PASMC were incubated with 5µM MitoSOX (Sigma-Aldrich, Germany) for

20 min at 37 °C. After the incubation, cells were washed twice with PBS under hypoxic

atmosphere. The plates were sealed air-tight and moved to the microplate reader. The

fluorescence signal from wells was measured using a microplate reader (Tecan Group Ltd.,

Männedorf, Swiss). Excitation and emission wavelength was set to 405/580nm. The

experiments were performed in a light protected environment.

To measure H2O2, PASMC were infected with HyPercyto lentivirus and afterwards transferred

into the hypoxic chamber (1% O2) for 5 days. H2O2 was measured in a closed perfusion chamber

(PeCon, Germany) which was assembled and sealed under hypoxia in the hypoxic gloves box.

Excitation and emission parameters of HyPercyto were used as described above.

Proliferation assay

Proliferation of precapillary mouse PASMC was evaluated by determination of the ratio of

proliferating cells labeled by 5-ethynyl-uridine (EdU) to the total cell number labeled by

Hoechst staining as described previously [8]. Chronic hypoxia was simulated by incubation of

PASMCs for 5 days in hypoxic atmosphere (1% O2). Normoxic experiments were conducted

in an atmosphere of 21% O2. MitoQ or TPP+ in doses of 0.1 and 0.5µM were applied during

the whole time period of normoxic/hypoxic incubation.

DNA damage

DNA damage was evaluated by measurement of 8-hydroxyguanosine levels using DNA

Damage Competitive ELISA (Thermo Fisher Scientific, Waltham, USA) according to the

instructions of the manual. 8-hydroxyguanosine, which is the oxidative derivative of guanosine,

is characteristic of DNA damage. PASMC from passage 1 were seeded into a 6-well plate and

incubated for 5 days in 1% O2 (chronic hypoxia) or in 21% O2 (normoxia). Afterwards, cell

growth medium (see above) was collected, PASMC were homogenized in phosphate-buffered

saline with 1x protease inhibitor cocktail (Sigma-Aldrich, Germany) using the bead

homogenizer Precellys24 (Omni International, Kennesaw, USA), and DNA was extracted using

QuickExtract™ DNA Extraction Solution (Epicentre, USA). DNA concentration was

measured by NanoDrop (PeqLab, Erlangen, Germany) and protein concentration was measured

by RC DC™ Protein Assay (Bio-Rad, Hercules, USA) according manual. Afterwards DNA and

protein concentration were normalized.

Determination of the antioxidative capacity

PASMC from passage 1 were seeded into 6-well plates and incubated for 5 min or 5 days in

1% O2 (hypoxia) or in 21% O2 (normoxia). Afterwards, PASMC were homogenized in

phosphate-buffered saline with 1x protease inhibitor cocktail (Sigma-Aldrich, Germany) using

the bead homogenizer Precellys24 (Omni International, Kennesaw, USA). PASMC

homogenates were normalized for protein concentration, which was measured using the RC

DC™ protein assay (Bio-Rad, Hercules, USA). SOD (Thermo Fisher Scientific, Waltham,

USA), Catalase (Abcam, Cambridge, UK) and total antioxidant capacity (Abcam, Cambridge,

UK) were measured according to the manufacturer’s instructions.

Western blot

Cell lysates were separated on a 10 % SDS (sodium dodecyl sulfate) polyacrylamide gel,

followed by electrotransfer to a 0.45µm PVDF (polyvinylidene fluoride) membrane (Pall

Corporation, Dreieich, Germany). After blocking with 5% non-fat dry milk in TBS-T buffer

(Tris Buffer Saline + 0.1% Tween 20) for 1 hour, the membrane was incubated overnight at

room temperature with one of the following antibodies: anti-SOD1 (rabbit antibody, dilution

1:1000, Abcam Cambridge, UK), anti-SOD2 (mouse antibody, dilution 1:1000, Abcam

Cambridge, UK), anti-Catalase (rabbit antibody, dilution 1:1000, Abcam Cambridge, UK),

anti-PDK1 (rabbit antibody, dilution 1:1000, Cell Signaling Technology, Inc. Danvers, USA),

anti-LDHA (rabbit antibody, dilution 1:1000, Cell Signaling Technology, Inc. Danvers, USA),

anti-GPX2, 4 (rabbit antibody, dilution 1:200, Abcam Cambridge, UK), anti-GPX3 (rabbit

antibody, dilution 1:1000, Novus Biologicals, LLC ) and monoclonal mouse anti-β-actin

(dilution 1:50000, Sigma-Aldrich, St. Louis, USA). After washing the membranes in TBS-T

buffer, specific immunoreactive signals were detected by enhanced chemiluminescence (Bi-

Rad, Hercules, USA) using a proprietary secondary antibody coupled to horseradish-peroxidase

diluted 1:5000.

RNA isolation and real-time polymerase chain reaction (PCR)

Total RNA (1µg), extracted from isolated PASMC or lung homogenate by RNeasy Micro Kit

(Qiagen N.V., Hilden, Germany) was reverse-transcribed using the iScript cDNA Synthesis Kit

(Bio-Rad, Hercules, USA). As housekeeping genes β2M (β2-microglobulin) was used.

PCR analysis of Mitochondrial DNA copy number

PASMC from passage 1 were seeded into 6 well plates and incubated for 5 days in 1% O2

(hypoxia) or in 21% O2 (normoxia). DNA was extracted using QuickExtract™ DNA Extraction

Solution (Epicentre, USA). Afterwards, real-time PCR (polymerase chain reaction) was

performed using a master mix for RT PCR (iTaq SYBR Green supermix with ROX, Bio-Rad,

Hercules, USA) and Mx3000P qPCR Systems (Agilent Technologies, Santa Clara, USA).

Primers for mitochondrial NADH-ubiquinone oxidoreductase chain 1 (ND1) and genomic

hexokinase 2 (HK2) were the following:

ND1 f CTAGCAGAAACAAACCGGGC

ND1 r CCGGCTGCGTATTCTACGTT

HK2 f GCCAGCCTCTCCTGATTTTAGTGT

HK2 r GGGAACACAAAAGACCTCTTCTGG

Determination of pO2, pCO2 and pH

Values of pO2, pCO2 and pH were measured in the perfusate of isolated lung experiments,

which was collected from the left ventricle (which corresponds to the outflow line of the lung),

in cell growth medium from PASMC exposed for 5 days to 1% O2 and in ESR buffer after 5

min exposure of PASMC to 1% O2. pO2 was determined by a LICOX CMP tissue oxygen

pressure monitor (GMS, Mielkendorf, Switzerland), pCO2 and pH by a Rapidlab Blood Gas

Analyzer 348 (Siemens, Erlangen, Germany) according to the manufacturer's instructions.

Moreover, pO2 was determined in the closed perfusion chamber by a LICOX CMP tissue

oxygen pressure monitor (GMS, Mielkendorf, Switzerland).

Figure legends

Figure S1

Pulmonary vasoconstriction in isolated mouse lungs in the different experimental groups

prior to substance application

(A) Increase of pulmonary arterial pressure (ΔPAP) during acute hypoxic ventilation (1% O2,

5.3% CO2, rest N2) or (B) after bolus injection of the the thromboxane mimetic U46619 in the

different experimental groups (MitoQ, TPP+, control) in absence of MitoQ or TPP+.

n=5-6 isolated lungs each.

Figure S2

Levels of pO2, pCO2 and pH under different hypoxic conditions

(A) pO2 values, (B) pCO2 values and (C) pH, determined in the perfusate collected from the

left ventricle in isolated ventilated and blood-free perfused mouse lungs during repetitive

ventilation with normoxic (21%, 5% CO2 rest N2) and hypoxic (1% O2, 5% CO2 , rest N2) gas.

D) Time course of pO2 in the closed chamber of the fluorescence microscope, perfused with

Hepes-Ringer buffer pre-gassed with N2.

(E) pO2, pCO2, and (F) pH in the ESR buffer and cell growth medium during exposure of

PASMC to an atmosphere containing 1% O2 (rest N2) for 5 min and to an atmosphere of 1%

O2 (5% CO2, rest N2) for 5 days, respectively.

Figure S3

Representative pictures of PASMC stained with MitoSOX

PASMC were incubated with 5µM MitoSOX diluted in Hepes-Ringer buffer (HRB) for 15 min.

After incubation, PASMC were washed three times with HRB buffer and the fluorescent signal

was analyzed by a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar,

Germany).

Figure S4

Effect of TPP+ on development of pulmonary hypertension

(A) Right ventricular systolic pressure (RVSP), (B) cardiac output (CO), (C) tricuspid annular

plane systolic excursion (TAPSE), (D) ratio of RV mass to the mass of left ventricle plus septum

(RV/[LV+Septum]), (E) right ventricular wall thickness (RVWT), (F) right ventricular internal

diameter (RVID) and (G) right ventricular outflow tract diameter (RVOTD) in TPP+ and

untreated (control) mice exposed for 4 weeks to chronic hypoxia (4w HOX) or normoxia

(NOX). n=5-8 animals per group, *p<0.05, **p<0.01, *** p<0.001, **** p<0.0001 and ††

p<0.01, ††† p<0.001 interaction by two-way ANOVA with Tukey post hoc test.

H) Superoxide concentration in RV homogenate from TPP+ and untreated (control) mice

exposed for 4 weeks to chronic hypoxia (4w HOX) or normoxia (NOX). The superoxide

concentration was measured by ESR spectroscopy as difference in the CMH signal with or

without pegylated superoxide dismutase (∆pSOD). Data are normalized to RV mass (g). n=3-5

* p<0.05 by two-way ANOVA with Tukey post.

Figure S5

Expression and activity of antioxidative enzymes in chronic hypoxic lung tissue or

pulmonary arterial smooth muscle cells

(A) mRNA expression of various enzymes of the antioxidative system in lung homogenate from

mice exposed to 10% O2 for 4 weeks and in (B) PASMC exposed for 5 days to 1% O2. (C)

Protein level of GPX 2, 3, 4 and (D) SOD1/2 in PASMC exposed to 1% O2 for 5 days. (E) SOD

activity and (F) catalase activity in cell lysates from PASMC exposed to 1% O2 for 5min or 5

days. (G) Total antioxidative capacity in cell homogenates from PASMC exposed to 1% O2 for

5 days.

n=4 per group, *p<0.05, **p<0.01, *** p<0.001 and **** p<0.0001 compared to normoxic

control by Student's t test with Welsh's correction.

Figure S6.

Effect of chronic hypoxia on mitochondrial DNA amount and cellular metabolism

(A) The amount of mitochondrial DNA (mtDNA) normalized to genomic DNA (gDNA). (B)

mRNA and (C) protein expression of lactate dehydrogenase A (LDHA) and pyruvate

dehydrogenase kinase 1 (PDK1) in PASMC exposed to 1% O2 for 5 days.

n=4 per group, *p<0.05, **p<0.01, *** p<0.001 and **** p<0.0001 compared to normoxic

control by Student's t test with Welsh's correction.

D) Superoxide concentration in PASMC exposed to chronic hypoxia (1% O2) or normoxia

(21% O2) for 5 days in presence or absence of DCA.

n=4 per group, *p<0.05 and **** p<0.0001 by two-way ANOVA.

References:

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Schermuly RT, Fink L, Seeger W, Grimminger F. Impact of mitochondria and nadph oxidases

on acute and sustained hypoxic pulmonary vasoconstriction. Am J Respir Cell Mol Biol 2006;

34: 505-513.

2. Sommer N, Pak O, Schorner S, Derfuss T, Krug A, Gnaiger E, Ghofrani HA, Schermuly

RT, Huckstorf C, Seeger W, Grimminger F, Weissmann N. Mitochondrial cytochrome redox

states and respiration in acute pulmonary oxygen sensing. Eur Respir J 2010; 36: 1056-1066.

3. Konigshoff M, Kramer M, Balsara N, Wilhelm J, Amarie OV, Jahn A, Rose F, Fink L,

Seeger W, Schaefer L, Gunther A, Eickelberg O. Wnt1-inducible signaling protein-1 mediates

pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J

Clin Invest 2009; 119: 772-787.

4. Berg K, Ericsson M, Lindgren M, Gustafsson H. A high precision method for

quantitative measurements of reactive oxygen species in frozen biopsies. PLoS One 2014; 9:

e90964.

5. Dikalov S, Skatchkov M, Bassenge E. Spin trapping of superoxide radicals and

peroxynitrite by 1-hydroxy-3-carboxy-pyrrolidine and 1-hydroxy-2,2,6, 6-tetramethyl-4-oxo-

piperidine and the stability of corresponding nitroxyl radicals towards biological reductants.

Biochem Biophys Res Commun 1997; 231: 701-704.

6. Janssen W, Schymura Y, Novoyatleva T, Kojonazarov B, Boehm M, Wietelmann A,

Luitel H, Murmann K, Krompiec DR, Tretyn A, Pullamsetti SS, Weissmann N, Seeger W,

Ghofrani HA, Schermuly RT. 5-ht2b receptor antagonists inhibit fibrosis and protect from rv

heart failure. Biomed Res Int 2015; 2015: 438403.

7. Smith RA, Murphy MP. Animal and human studies with the mitochondria-targeted

antioxidant mitoq. Ann N Y Acad Sci 2010; 1201: 96-103.

8. Malczyk M, Veith C, Fuchs B, Hofmann K, Storch U, Schermuly RT, Witzenrath M,

Ahlbrecht K, Fecher-Trost C, Flockerzi V, Ghofrani HA, Grimminger F, Seeger W, Gudermann

T, Dietrich A, Weissmann N. Classical transient receptor potential channel 1 in hypoxia-

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HA, Seeger W, Grimminger F, Brandes RP, Schermuly RT, Weissmann N. Function of nadph

oxidase 1 in pulmonary arterial smooth muscle cells after monocrotaline-induced pulmonary

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739-744.

11. Weissmann N, Sydykov A, Kalwa H, Storch U, Fuchs B, Mederos y Schnitzler M,

Brandes RP, Grimminger F, Meissner M, Freichel M, Offermanns S, Veit F, Pak O, Krause

KH, Schermuly RT, Brewer AC, Schmidt HH, Seeger W, Shah AM, Gudermann T, Ghofrani

HA, Dietrich A. Activation of trpc6 channels is essential for lung ischaemia-reperfusion

induced oedema in mice. Nat Commun 2012; 3: 649.

Figure S1St

reng

th o

f HPV

, ΔPA

P [m

mH

g]

TPP+ group

Control group

MitoQgroup

A

0.0

0.5

1.0

1.5

Stre

ngth

of U

4661

9-in

duce

d v

asoc

onst

rictio

n, Δ

PAP

[mm

Hg]

TPP+ group

Control group

MitoQgroup

B

0.0

0.5

1.0

1.5

Figure S2pO

2 in

buffe

r in

clos

ed c

ham

ber

of fl

uore

scen

ce m

icro

scop

y [m

mH

g]

0 1 2 3 4 5 60

50

100

150

0

2

4

6

8

pCO2 pO2 pCO2 pO2

min

Hypoxia

5m/1% O2 buffer

5m/1% O2 buffer

5d/1% O2 medium

5d/1% O2 medium

pO2 i

n pe

rfus

ate

ofis

olat

ed lu

ng [m

mH

g]

pO2 a

nd p

CO

2 in

buffe

r/med

im w

ith P

ASM

C fo

r ESR

mea

sure

men

t [m

mH

g]

pH in

buf

fer/m

edim

with

PA

SMC

for E

SR m

easu

rem

ent [

mm

Hg]

pH in

per

fusa

te o

fis

olat

ed lu

ng

pCO

2 in

perf

usat

e of

isol

ated

lung

[mm

Hg]

A

D E F

B C

NOX1.H

OXNOX

2.HOX

NOX3.H

OXNOX

4.HOXNOX

5.HOX

NOXNOX

1.HOX

NOX2.H

OXNOX

3.HOX

NOX4.HOX

NOX5.H

OXNOX

NOX1.H

OXNOX

2.HOX

NOX3.H

OXNOX

4.HOXNOX

5.HOX

NOX0

50

100

150

200

0

10

20

30

40

50 pCO2 pO2

30

32

34

36

38

40

7.3

7.4

7.5

50μm

50μm50μm

Figure S3

A

RVS

P [m

mH

g]

B C

CO

[ml/m

in]

D

TAPS

E [m

m]

G

RV/

[LV

+ se

ptum

]

E F

0

10

20

30

40

50

**** ****

0

5

10

15

20

25

***

0.0

0.5

1.0

1.5**** ****

0.0

0.1

0.2

0.3

0.4

0.5

††† interaction

********

TPP+

NOX NOX 4w HOX

Control

4w HOX

TPP+

NOX NOX 4w HOX

Control

4w HOX

TPP+

NOX NOX 4w HOX

Control

4w HOX

TPP+

NOX NOX 4w HOX

Control

4w HOX

RVW

T [m

m]

0.0

0.1

0.2

0.3

0.4

**** ******

TPP+

NOX NOX 4w HOX

Control

4w HOX

RVI

D [m

m]

0.0

0.5

1.0

1.5

2.0†† interaction

***

*

****

TPP+

NOX NOX 4w HOX

Control

4w HOX

RVO

TD [m

m]

0.0

0.5

1.0

1.5

2.0 * *

TPP+

NOX NOX 4w HOX

Control

4w HOX

TPP+

NOX NOX 4w HOX

Control

4w HOX

Figure S4

H

Supe

roxi

de c

once

ntra

tion

[A.U

.] /

wei

ght [

g]

0

10

20

30

Figure S5

SOD1SOD2

SOD3CAT

GPX1GPX2

GPX3GPX4

GPX5

PRDX1

PRDX2

PRDX3

PRDX4

PRDX5

PRDX60

1

2

3

4

* *

***

** *

**

Lung homogenate PASMC

GPX3

GPX4

GPX2

ß-actin

5d HypoxiaNormoxia

GPX2 GPX3 GPX4

** **

0.0

0.2

0.4

0.6

0.8

1.0

NOX 5d HOX

Rel

ativ

e ex

pres

sion

[pro

tein

of i

nter

est/ß

-act

in]

Rel

ativ

e ex

pres

sion

[pro

tein

of i

nter

est/ß

-act

in]

5d HypoxiaNormoxia

SOD1

SOD2

CAT

ß-actin

SOD1 SOD2 CAT

** ***

0

1

2

3

4

5

NOX 5d HOX

mR

NA

exp

ress

ion,

fold

chn

age

[2∆∆

Ct ]

mR

NA

exp

ress

ion,

fold

chn

age

[2∆∆

Ct ]

Tota

l ant

ioxi

dant

cap

acity

/µg

pro

tein

160

180

200

220

240

260

1.8

2.0

2.2

2.4

2.6

2.8

0

2000

4000

6000

8000

SOD

act

ivity

/µg

prot

ein

Cat

alas

e ac

tivity

/µg

prot

ein

NOX 5m HOX 5d HOXNOX

NOX 5m HOX 5d HOXNOX

5d HOXNOX

A B

C D

E

G

SOD1SOD2

SOD3CAT

GPX1GPX2

GPX3GPX4

GPX5

PRDX1

PRDX2

PRDX3

PRDX4

PRDX5

PRDX60.0

0.5

1.0

1.5

*

** ****

*

*

F

5d HypoxiaNormoxia

LDHA

PDK1

ß-actin

**** ***

-4

-2

0

2

NOX 5d HOX NOX 5d HOX

PDK1LDHA

mR

NA

exp

ress

ion

[∆C

t]

0

1

2

3

NOX 5d HOX NOX 5d HOX

PDK1LDHA

* **

Rel

ativ

e ex

pres

sion

[pro

tein

of i

nter

est/ß

-act

in]

BA

C D

Figure S6

***∆C

t [gD

NA

-mtD

NA

]

-9

-8

-7

-6

NOX 5d HOX

5000

10000

15000

20000

25000

NOX

DCA, µM - 0.5 1.00

5d HOX

Mito

chon

dria

l RO

S co

ncen

trat

ion,

Mito

SOX

fluor

esce

nce

[A.U

.]

****

*

0

*** *****