OP2113, a new drug for chronic hypoxia-induced pulmonary hypertension treatment in rat

Background and Purpose: Pulmonary hypertension (PH) is a cardiovascular disease characterised by an increase in pulmonary arterial (PA) resistance leading to right ventricular (RV) failure. Reactive oxygen species (ROS) play a major role in PH. OP2113 is a drug with beneficial effects on cardiac injuries that targets mitochondrial ROS. The aim of the study was to address the in vivo therapeutic effect of OP2113 in PH. Experimental Approach: PH was induced by 3 weeks of chronic hypoxia (CH-PH) in rats treated with OP2113


| INTRODUCTION
Pulmonary hypertension (PH) is a multifactorial cardiovascular disease characterised by a progressive increase in both pulmonary arterial resistance (above 2 Wood Units) and pressure (above 20 mmHg) leading to right ventricular (RV) hypertrophy, failure and, ultimately, premature death (Humbert et al., 2022;Vonk Noordegraaf et al., 2019).PH related to chronic hypoxia (CH-PH) is a major risk factor for patients with chronic lung disease or for people living at high altitude, but drugs developed to treat PH are focused on idiopathic pulmonary arterial hypertension (PAH), and these drugs have not been approved for CH-PH (Humbert et al., 2022).So far, although new promising therapies such as sotatercept are under development (Humbert et al., 2021), the main treatments only partially improve symptoms and quality of life, lung transplantation remaining the last option when medication fails (Humbert et al., 2014;Savale et al., 2018).Hyperreactivity due to an imbalance between vasoconstrictor (increase in endothelin-1 and 5-HT) and vasodilator agents (decrease in NO and prostacyclin) and remodelling of the pulmonary vasculature are the main pulmonary arterial (PA) hallmarks in PH (Humbert et al., 2019).Regarding RV remodelling, which is present in all forms of PH, cardiomyocyte hypertrophy is observed in response to pressure or volume overload and further leads to adverse alterations in myocardial performance associated with oxidative stress and metabolic remodelling (Piao et al., 2010;Shults et al., 2018).Currently, although alterations have been observed in left ventricle (LV), especially in mitochondria in PH (Nouette-Gaulain et al., 2011, 2005), approved therapies for the treatment of LV failure are not recommended for RV failure.
Additionally, in PH, metabolic reprogramming has also been highlighted (Agrawal et al., 2020;Freund-Michel et al., 2014).Under physiological conditions, we have previously shown that 5-HT stimulates ROS production such as superoxide anion (O 2 ˙), via the mitochondrial complex I in rat PA smooth muscle cells (PASMCs), and this participates in the contractile response to 5-HT (Billaud et al., 2009;Genet et al., 2017).Several studies have also shown an increase in ROS production in experimental PH, including CH-PH or SUGEN (SU5416)/hypoxia models.ROS then stimulate PA hyperreactivity and remodelling by increasing pulmonary arterial smooth muscle cell proliferation (Freund-Michel et al., 2013;Lee et al., 1998;Liu et al., 2006;Liu & Folz, 2004).A large increase in O 2 ˙has also been shown in the RV from mice with PH (Pak et al., 2018).
Unlike classical antioxidants, which are nonselective ROS scavengers and, hence, can disturb cellular signalling associated with physiological ROS production (Billaud et al., 2009), the specific mechanism of action of OP2113 led the "OP2 Drugs" start-up to develop new applications for this compound, which has already shown efficacy in various cardiac injuries (Dai et al., 2022;Detaille et al., 2019).ATT up-regulates muscarinic receptors (Ukai et al., 1984), which are also known to be (1) involved in the contraction and relaxation of PA in physiological and pathological (PH) conditions (Norel et al., 1996;Orii et al., 2010) and (2) are increased in heart following PH (Morel et al., 2003).
Taking into account (1) the importance of ROS in both PA and RV hallmarks associated with PH and (2) the properties of OP2113 to reduce mitochondrial ROS without altering mitochondrial function, we hypothesized that OP2113 could be a potential new therapeutic option for PH.Using the rat CH-PH model, we evaluated the in vivo effect of OP2113 on the main PH hallmarks both in PA and in the heart.

What is already known
• Pulmonary hypertension is characterised by pulmonary arterial and right ventricular alterations involving reactive oxygen species.
• OP2113 targets mitochondrial reactive oxygen species and has beneficial effects on various cardiac injuries.

What does this study add
• OP2113 has beneficial effects on pulmonary arterial and cardiac alterations in chronic hypoxia-induced pulmonary hypertension.
• OP2113 in vivo treatment has no effect on pulmonary arteries and heart from control rats.

What is the clinical significance
• We show a selective protective effect of OP2113 in hypoxic pulmonary hypertension, encouraging clinical trials.

| Animals
All animal care and experimental procedures were in accordance with the recommendations of the "Comité d'éthique régional d'Aquitaine" (CEEA 50) and the protocol was approved by the same committee (protocol n APAFIS#23042).Animal procedures are declared to be in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020).Male Wistar rats (Janvier Laboratories, Saint Berthevin, France) weighing 300-350 g were randomly and blindly distributed into 43 groups designed to be of equal size for the preventive treatment (18 rats for each group); however, in some instances, groups were unequal due to unexpected loss of animals: Normoxia (N) (16 control rats), chronic hypoxia + vehicle (CH + VEH) (17 rats) and chronic hypoxia + preventive OP2113 treatment (CH + OP2113) (14 rats).For control experiments (normoxia + preventive OP2113 treatment: N + OP2113), we used five rats.For curative treatment, the number of rats used were as follow: normoxia (N) (five control rats), chronic hypoxia + vehicle (CH + VEH) (eight rats) and chronic hypoxia + curative OP2113 treatment (nine rats).Chronic hypoxic PH (CH-PH) was induced by exposing rats to CH using hypobaric chambers (50 kPa) for 21 days (CH groups), while normoxic (N) rats were exposed to animal facility room air (N groups).The CH rat model has been shown to induce a stable PH, reproducing some of the cardiac and pulmonary arterial pathophysiological hallmarks observed in human PH due to high altitude exposure or to hypoxaemic lung diseases and/or hypoxia (Humbert et al., 2022;Singh et al., 2022).
Osmotic mini-pumps (Durect Corporation, Alzet Model 2ML4) were used to subcutaneously treat rats with OP2113 (140 μgÁkg À1 Áday À1 ) or its vehicle (sulfobutylether beta-cyclodextrin 40% w/w in NaCl 0.9%) for the whole protocol duration (preventive treatment) or for the last 14 days of the protocol (curative treatment) at a rate of 2.5 μlÁh À1 (0.7 mgÁml À1 or 2.9 mM of OP2113 in the mini-pump).
Mini-pumps were filled in a sterile environment.Animals were placed on a heating platform in the ventral decubitus position, and minipumps were implanted between the scapulae of animals under isoflurane anaesthesia (3% and 1.5%, for induction and maintenance phase, respectively).Rats underwent a 4-h recovery period after mini-pump implantation before hypobaric chamber housing.Hypobaric chambers were opened three times a week for animal care, cleaning and food/ water supply.Animals were housed in groups of two or three per cage in a temperature-and humidity-monitored animal facility with a 12-h light/dark cycle.For all groups, weight, physical appearance, mobility behaviour and interactions were monitored and scored every 2 days.

| Haemodynamic measurements and assessment of right ventricular hypertrophy
After 21 days, rats were anaesthetised with isoflurane (3% and 1.5% for induction and maintenance phases, respectively) (Centravet,

| Measurement of PA remodelling
Rat lungs were fixed in 4% neutral buffered formalin (VWR, Rosny-sous-Bois, France) and dehydrated in increasing grade of ethanol.After delipidation with xylene, lungs were embedded in paraffin and cut into transverse sections (4 μm) which were stained with haematoxylin, eosin and safran (HES) (VWR).Sections were then observed with an optic microscope (Axio scan Z1 ZEISS, Rueil Malmaison, France) (Â20) and analysed using ImageJ software (1.52a, NIH Image, RRID:SCR_003070).PA medial wall remodelling was measured by the percentage medial thickness of intra-acinar arteries with an external diameter of less than 150 μm; 10-20 vessels per rat were analysed in a blinded fashion.The percentage medial wall thickness was calculated as ((external wall area) À (internal wall area)) Â 100/ external wall area and results were shown according to the external diameter (less than 50 μm, 50 to 100 μm or 100 to 150 μm).Intrapulmonary arteries of the first order were dissected in Krebs solution from surrounding connective tissues under binocular microscope and divided into short tubular segments with an external diameter of 1.5 to 2 mm.Arterial rings were then mounted in isolated organ bath systems (EMKA Technologies, Paris, France) containing Krebs solution continuously warmed (37 C) and bubbled (5% CO 2 and 20% O 2 for PA from N rats and 5% CO 2 and 10% O 2 for PA from CH rats).

| Measurement of pulmonary arterial reactivity
As previously described (Rodat et al., 2007), an initial load of 0.8 g (for PA from N rats) or 1.6 g (for PA from CH rats) was applied to arterial rings before isometric tension measurement.Passive and active contractile properties of arterial rings were assessed using transducer systems, coupled to IOX2 software (EMKA Technologies) in order to facilitate data acquisition and analysis.Tissues were allowed to equilibrate for 1 h with Krebs solution washout every 15 min.Viability of arterial rings was assessed using a high concentration of KCl solution (80 mM) to obtain a reference contraction.Arterial rings that produced a contraction less than to 0.1 g were excluded from the analysis.Contractile responsiveness was tested by constructing cumulative concentration-response curves (CCRC) to 5-HT and PGF2α.For acute effects, OP2113 was diluted in DMSO and tissues were preincubated for 30 min with OP2113 (10 μM-final concentration of DMSO was 1/10000) or vehicle, where indicated.The OP2113 concentration used was within the range of its concentration-dependent effects previously observed in vitro (5 to 80 μM) with a half maximal effect equal to 10 μM for succinate-induced reverse electron flow inhibition at the level of mitochondrial complex I (Detaille et al., 2019).
Moreover, such concentrations do not affect cardiac mitochondrial function, namely, neither rotenone-sensitive NADH oxidase activity nor oxidative phosphorylation (Detaille et al., 2019).Similar results were also obtained by Dai et al. (2022) for a concentration of 15 μM.
Contraction was expressed as a percentage of PA maximal contraction in control rats (N rats).The relaxant effect of OP2113 was evaluated by applying OP2113 (10 μM) on phenylephrine (Phe) (30 nM)preconstricted PA rings from N or CH rats.Relaxation was expressed as a percentage of the precontraction to Phe.Endothelial integrity was evaluated using the relaxation induced by carbachol (10 μM) on Phe (30 nM)-preconstricted PA rings.CCRCs to Phe (0.1 nM to 100 μM) were not significantly different in PA from N versus CH rats (Figure S2), demonstrating that CH has no effect on Phe contraction.
CCRCs to 5-HT and carbachol-induced relaxations in PA from CH rats were not changed significantly by a 3-week treatment with vehicle solution (Figure S1e,f respectively).
The experimental details we provided for western blotting experiments conform with BJP Guidelines (Alexander et al., 2018).

| Detection of protein carbonylation
Intrapulmonary arteries of the first order or RV or LV were lysed and homogenised in ice-cold RIPA lysis buffer supplemented with protease inhibitor cocktail, sodium orthovanadate phenylmethylsulfonyl fluoride (1/100) and DTT (50 mM) (Santa Cruz Biotechnology, Clinisciences, Nanterre, France) to prevent protein oxidation that may occur after sample lysis.Due to the presence of high DTT concentration, the protein concentration was determined by using Pierce™ 660 nm Protein Assay (Thermo Scientific, Illkirch, France).To evaluate the protein oxidation status (due to oxygen-derived free radical production), carbonyl groups were detected with the OxyBlot™ Protein Oxidation Detection Kit (Merck, Saint Quentin Fallavier, France).Protein samples were prepared according to the manufacturer's instructions.Briefly, 5 μg of proteins were denatured with SDS 10% and carbonyl groups in the protein were derivatized to 2,4-dinitrophenylhydrazone by reaction with 2-4 dinitrophenylhydrazine (DNPH) for 15 min.2,4-Dinitrophenylhydrazone-derivatized protein samples were then subjected to electrophoresis on a 10% stain free-containing acrylamide gel and transferred to nitrocellulose membranes for western blotting analysis.After 1 h of blocking with TBS supplemented with 0.1% Tween and 5% milk powder, membranes were incubated overnight at 4 C with a primary antibody targeting 2,4-dinitrophenylhydrazone (Merck, part#90451, 1/150).
Membranes were then incubated with the corresponding goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Merck, part#90452, 1/300).Carbonylated proteins were then expressed as percentage of the carbonylated proteins in control conditions (N).

| Preparation of permeabilised cardiac fibres
Cardiac fibres from RV or LV were permeabilised as previously described (Veksler et al., 1987).Freshly removed heart was rinsed with (10 μM) (Sigma), as previously described in the literature (Kim et al., 2014).Mitochondrial outer membrane damage was thus estimated as a percentage of the phosphorylating O 2 flux increase following addition of exogenous cytochrome c.

| Cardiomyocyte isolation
Freshly removed hearts were perfused in the Langendorff configuration (at 12 mlÁmin À1 ) with an isolation buffer (containing [in mM]: 130 NaCl, 5.4 KCl, 1.4 MgCl 2 , 0.4 NaH 2 PO 4 , 5 HEPES, 10 glucose, 10 creatine, 20 taurine, 0.75 CaCl 2 , pH 7.4 -Sigma) for 4 min to wash the tissue.Hearts were then perfused with isolation buffer without CaCl 2 and complemented with 100-μM Na 2 EGTA for approximatively 4 min to stop cardiac contraction.Finally, hearts were perfused with isolation buffer complemented with type II collagenase (1 mgÁml À1 , Worthington, Serlabo, Entraigues sur la sorgues, France) and type XIV protease from Streptomyces (0.1 mgÁml À1 , Sigma) for 10 to 15 min to enzymatically dissociate ventricular cardiomyocytes.RV and LV were then separated, cut in small pieces and agitated in an Erlenmeyer at 37 C for 5 min in isolation buffer complemented with collagenase and protease and filtered using a nylon gauze.Resulting solution was softly centrifuged with a hand-operated centrifuge (JP Selecta, Barcelona, Spain) for 1 min and pellet was suspended in isolation buffer.This process was repeated four or five times, and both cardiomyocyte integrity and number were controlled by optic microscope observation.Cells were stored at 20-23 C and used within 10 h.

| Simultaneous measurement of cardiomyocyte shortening and intracellular calcium transients
To study [Ca 2+ ] i variations, cardiomyocytes were loaded with a Fura-2 AM ratiometric calcium probe (3 μM, Invitrogen, Illkirch, France) for 10 min at room temperature and then softly centrifuged

| Data and statistical analysis
Data and statistical analysis are in accordance with the recommendations of the British Journal of Pharmacology on experimental design and analysis (Curtis et al., 2022).Data analysis was per- Control experiments were present in each set of experiments and results were expressed according to the fold mean of the controls.

| In vivo preventive treatment with OP2113 improved CH-PH without affecting RV hypertrophy
Rats exposed to 3 weeks of CH developed PH characterised with a marked elevation of mPAP (Figure 1a), RVSP (Figure 1b) and associated PVR (Figure 1c).Despite a strong RV hypertrophy characterised by an increase in the Fulton Index (Figure 1d), cardiac output was not affected by CH exposure (Figure 1e).
Preventive treatment with 140 μgÁkg À1 Áday À1 of OP2113 for 3 weeks significantly reduced mPAP and RVSP (reduction of 54% and 57%, respectively [Figure 1a,b]).However, the PVR increase and RV hypertrophy were not significantly affected by OP2113 (Figure 1c,d).Cardiac rate was unchanged in N versus CH rats and OP2113 treatment had no effect (Figure 1f).OP2113 preventive treatment did not change the aforementioned haemodynamic parameters (namely mPAP, RVSP, cardiac output and PVR) as well as RV thickness (Fulton Index) in N rats (Figure S3).

| In vivo preventive treatment with OP2113 improved medial thickening in small PA from CH-PH rats
Since CH induces PA remodelling, we used haematoxylin-eosin staining of lung slices to determine PA wall thickness (Figure 2).CH exposure for 3 weeks significantly increased the PA wall thickness of small vessels with diameters less than 50 μm (Figure 2a,b), between 50 and 100 μm (Figure 2a,c) and between 100 and 150 μm (Figure 2a,d).OP2113 in vivo preventive treatment significantly reduced CH-induced medial thickening of PA whatever the diameter of the vessels (Figure 2a-d).In contrast, medial thickness of small PA in N rats were unaffected by OP2113 whatever the diameter of the vessels (Figure S4).

| In vivo curative treatment with OP2113 improved CH-PH without affecting RV hypertrophy and medial thickening in PA from CH-PH rats
A marked elevation of mPAP (Figure 3a) and Fulton Index (Figure 3b) demonstrated PH development.Cardiac rate was unchanged in N versus CH rats and OP2113 curative treatment had no effect (Figure 3c).
Curative treatment with 140 μgÁkg À1 Áday À1 of OP2113 significantly reduced mPAP (Figure 3a).CH exposure for 3 weeks significantly increased the PA wall thickness of small vessels with diameters less than 50 μm (Figure 3d,e), between 50 and 100 μm (Figure 3d,f) and between 100 and 150 μm (Figure 3d,g).However, OP2113 in vivo curative treatment did not significantly reduce CH-induced medial thickening of PA whatever the diameter of the vessels (Figure 3d-g).

| Preventive and acute OP2113 treatment decreased CH-induced PA hyperreactivity and promoted relaxation
Since OP2113 in vivo treatment reduces CH-induced increase in mPAP and RVSP, we investigated the effect of OP2113 on PA vasoreactivity by measuring isometric tension either on PA rings from rats preventively treated in vivo with OP2113 (140 μgÁkg À1 Áday À1 for 21 days) or on PA rings acutely treated ex vivo with OP2113 (10 μM for 30 min) (Figure 4a).As already demonstrated in the literature (Billaud et al., 2011;Fresquet et al., 2006;Rodat et al., 2007), PA rings show a significant contractile hyperreactivity to cumulative concentrations of 5-HT or PGF2α in CH rats compared to N rats (Figure 4b,d,e).In vivo preventive OP2113 treatment markedly reversed CH-induced PA hyperreactivity to 5-HT, whereas it did not modify 5-HT-induced PA contractions in N rats (Figure 4b).Acute OP2113 treatment also significantly inhibited CH-induced PA hyperreactivity to 5-HT or PGF2α (Figure 4d,e).However, in contrast to the preventive treatment, acute OP2113 treatment strongly reduced PA reactivity in response to 5-HT (but not to PGF2α) in N rats (Figure 4d,e).
These results suggest that OP2113 has short and long term actions on PA reactivity to contractile agonists.
To determine endothelial integrity and function, we examined the relaxation to carbachol (10 μM), an endothelial-and NO-dependent agonist, in PA rings preconstricted with phenylephrine (Phe, 30 nM).
As previously shown (Karamsetty et al., 2001), PA rings from CH rats showed a significant decrease in carbachol-induced relaxation (Figure 4c) and preventive OP2113 treatment did not significantly modify this alteration, suggesting that OP2113 does not improve endothelial dysfunction in CH-induced PH.
However, up to 30-min application of OP2113 (10 μM) on Phe (30 nM)-preconstricted PA rings induced a progressive and significant relaxation in N rats (relaxation of 22%, 40% and 47% of the Phe maximal contraction at 5, 15 and 30 min, respectively) as well as in CH rats (relaxation of 19, 53 and 65% of the Phe maximal contraction at 5, 15 and 30 min, respectively) (Figure 4f,g).Interestingly, this relaxation was significantly higher at 15 and 30 min after OP2113 application in PA from CH compared to N rats (Figure 4f,g).It should be noted that the CCRCs to Phe (0.1 nM to 100 μM) were not significantly different in PA from N versus CH rats (Figure S2) indicating that the difference in OP2113 relaxant effect was not due to a different level of Phe precontraction.

| In vivo OP2113 treatment improved CHinduced RV cardiomyocyte contractile dynamics
Given that PH development and associated RV failure are linked to an intrinsic dysregulation of cardiomyocyte contractile function and calcium homeostasis (Bernal-Ramirez et al., 2021;Fowler et al., 2018;Medvedev et al., 2021), we then investigated the effects of OP2113 in vivo treatment on contractions and calcium homeostasis in RV and LV cardiomyocytes isolated from CH and N rats.Although sarcomere shortening amplitude was unchanged (Figure 5a), the time to contractile peak (Contraction T100) (Figure 5b) and the time to half relaxation (Relaxation T50) (Figure 5c) were both increased in RV cardiomyocytes from CH rats, suggesting a slowdown of RV contractile dynamics.Similarly to sarcomere shortening amplitude, calcium transient amplitude was not modified between N and CH conditions  (Figure 5d).Although the time to calcium peak (Ca T100) (Figure 5e) and the time to 50% calcium decay (Ca T50) (Figure 5f) were also not modified in RV cardiomyocytes from CH rats, the contractile alterations were accompanied by an increase in the exponential decay time constant of calcium transients (Ca Tau) (Figure 5g).OP2113 in vivo treatment significantly reversed the increase in the Contraction T100 in CH conditions (Figure 5b) without affecting CH-induced increase in the Relaxation T50 (Figure 5c).These results suggest a partial improvement of RV cardiomyocyte contractile dynamics.
Contractile dynamics of LV cardiomyocytes were also impacted by CH exposure (Figure S5).Time to contractile peak (Contrac.T100) (Figure S5b), time to 50% calcium decay (Ca T50) (Figure S5f) and associated Ca Tau exponential decay time constant (Figure S5g) were all significantly increased in LV cardiomyocytes from CH rats compared to N rats.Sarcomere shortening and calcium transient amplitude were not modified between N and CH conditions (Figure S5a and 5d respectively).As in RV cardiomyocytes, the sarcomere shortening Contraction T100 alteration was reversed by OP2113 in vivo treatment in CH rats (Figure S5b).

| In vivo OP2113 treatment had no influence on CH-induced oxidative stress in PA and heart
Although still under debate, CH-PH has been shown to be associated with a dysregulation of mitochondrial ROS production both in PA and in the heart (Bonnet & Boucherat, 2018;Freund-Michel et al., 2013, 2014;Pak et al., 2018).In this context, we have evaluated oxidative stress markers in PA and heart from N and CH rats with or without treatment with OP2113 in vivo (Figures 6 and S6).O 2 ˙levels measured by electronic paramagnetic resonance and lipid peroxidation estimated by 4-HNE expression were not different in PA from CH versus N rats (Figure 6a,c, respectively).In contrast, protein carbonylation was significantly increased in PA from CH rats compared to N rats (Figure 6e) but OP2113 in vivo treatment did not prevent this alteration.
We then evaluated oxidative stress markers in RV and LV (Figure 6 and S6).Regarding RV, O 2 ˙levels and lipid peroxidation were unchanged in CH rats versus N rats, and OP2113 in vivo treatment had no effect (Figure 6b,d).The same findings were observed in LV (Figure S6a and S6b).In contrast to LV, the amount of carbonylated proteins was significantly increased in RV from CH rats (Figure S6c and 6f respectively) suggesting that RV is more sensitive to oxidative stress induced by CH than LV, or that the detoxification system is more efficient in LV than in RV.Similarly to PA from CH rats, CH-induced increase in RV protein carbonylation was not prevented by OP2113 in vivo treatment (Figure 6f) suggesting that, surprisingly, OP2113 does not modify oxidative stress either in PA or in the RV from CH rats.

|
In vivo OP2113 treatment had no effect on CH-induced mitochondrial mass increase in PA whereas it reversed CH-induced mitochondrial mass decrease in RV without affecting mitochondrial respiration Since heart dysfunction observed in CH-PH is associated to structural and functional mitochondrial alterations (Freund-Michel et al., 2014;Nouette-Gaulain et al., 2011, 2005), and since OP2113 is known to directly act on mitochondria (i.e., decrease O 2 ˙production by complex I without affecting mitochondrial respiration) (Detaille et al., 2019;Dias Amoedo et al., 2020), we assessed mitochondrial mass by evaluating expression of VDAC1 (a protein of the mitochondrial outermembrane) and citrate synthase (a protein of the Krebs cycle) by western blotting and we evaluated mitochondrial respiration with an oxygraph system on permeabilised fibres to confirm the absence of OP2113 effects in our CH conditions.Unlike citrate synthase expression, VDAC1 expression was significantly increased in PA whereas both VDAC1 and citrate synthase were reduced in RV from CH rats compared to N rats (Figure 7a-d, respectively).Unlike in PA, alteration of VDAC1 was prevented whereas alteration of citrate synthase expression was only partially improved by OP2113 in vivo treatment in RV from CH rats (Figure 7c,d).In LV from CH rats, VDAC1 expression was not changed but citrate synthase expression was significantly decreased compared to LV from N rats (Figure S7a,b).Since OP2113 F I G U R E 4 Preventive in vivo and acute ex vivo OP2113 treatments modify PA reactivity in both normoxic (N) and chronic hypoxic (CH) rats.(a) Schematic representation of protocols used to study the effects of OP2113 in vivo and ex vivo treatments on pulmonary arterial (PA) reactivity.(b) Cumulative concentration-response curves (CCRCs) to 5-HT are shown for PA from N or CH rats preventively treated with OP2113 (140 μgÁkg À1 Áday À1 ) or its vehicle (VEH).Contraction is expressed as a percentage of the maximal PA contraction in normoxia conditions (N).(c) Carbachol-induced relaxation (10 μM) of Phe-preconstricted (30 nM) PA from N versus CH rats preventively with OP2113 or its VEH.Relaxation is expressed as a percentage of precontraction to Phe.In PA preincubated ex vivo with OP2113 (10 μM) or its VEH for 30 min, CCRCs were performed in response to 5-HT (d) or prostaglandinF2α (PGF2α) (e) in N versus CH rats.Contraction is expressed as a percentage of maximal PA contraction from N rats.(f) OP2113 direct relaxant effect on PA from N (white bars) versus CH rats (grey bars) preconstricted with Phe (30 nM) before OP2113 (10 μM) was applied.Relaxation was measured at 5, 15 and 30 min after OP2113 application and was expressed as a percentage of precontraction to Phe.Typical traces for (f) are shown in (g) for N (top left), N + OP2113 10 μM (bottom left), CH (top right) and CH + OP2113 10 μM (bottom right).N, normoxia; N + OP2113, normoxia + preventive in vivo or acute ex vivo OP2113 treatments; CH, chronic hypoxia; CH + VEH, chronic hypoxia + vehicle preventive in vivo or acute ex vivo treatments; CH + OP2113, chronic hypoxia + preventive in vivo or acute ex vivo OP2113 treatments.Data represent means ± SEM. n represents the number of PA rings.*P < 0.05.
in vivo treatment reversed mitochondrial mass alterations in the RV and alterations of both mitochondrial mass and function have been previously observed in LV (Nouette-Gaulain et al., 2011, 2005), we examined mitochondrial respiration in the RV and the LV.
In permeabilised cardiac fibres and in the presence of glutamate, malate and succinate alone, we recorded basal O 2 flux (BASAL) and this basal flux was significantly decreased in RV fibres from CH rats compared to N rats (Figure 7e).Phosphorylative O 2 flux (oxidative phosphorylation state) measured by ADP addition and maximal oxidative phosphorylation state (MAXIMUM) were also reduced in RV fibres from CH rats compared to N rats (Figure 7e).Inhibition of ATP synthase by oligomycin slightly but not significantly decreased leak respiration (LEAK) in RV fibres from CH rats compared to N rats (Figure 7e).However, none of the aforementioned CH-induced alterations of RV mitochondrial respiratory states were prevented by OP2113 in vivo treatment (Figure 7e).Similarly to RV, LV from CH rats also exhibited mitochondrial respiration alterations such as significant decreases in oxidative phosphorylation state and MAXIMUM associated O 2 flux, with OP2113 in vivo treatment having no effect on these alterations (Figure S7c).The mitochondrial outer membrane was not damaged in RV or LV of CH rats compared to N rats, and OP2113 in vivo treatment had no significant effect on this parameter (Figures 7f and S7d).These results are in accordance with the absence of OP2113 effects on the mitochondrial function, as previously demonstrated by several recent studies (Detaille et al., 2019;Dias Amoedo et al., 2020).
3.8 | In vivo OP2113 treatment had no effect on the expression of M 3 muscarinic receptors in lung, PA and RV ATT up-regulates muscarinic receptors (Ukai et al., 1984) and the M 3 muscarinic receptor is involved in both relaxation and contraction in PA, including in PH conditions (Norel et al., 1996;Orii et al., 2010), as well as in both cardiac hypertrophy and chronic cardiac pressure overload (Grogan et al., 2022).Thus, we assessed the expression of the M 3 muscarinic receptor in lung homogenates, PA and RV from N rats, CH rats treated with vehicle and CH rats preventively treated with OP2113 (Figure 8).In rats with CH-PH, expression of the M 3 receptor was unchanged in lung homogenates (Figure 8a), whereas it was significantly increased in PA (Figure 8b) and RV (Figure 8c).However, preventive treatment with OP2113 did not affect M 3 receptor expression in lung, PA and RV (Figure 8).

| DISCUSSION
Our study demonstrates, for the first time, the protective effect of preventive OP2113 in vivo treatment did not up-regulate the muscarinic M 3 receptors responsible for PA relaxation (Norel et al., 1996;Orii et al., 2010).It is therefore likely that muscarinic M 3 receptors are not involved in the effect of OP2113 on mPAP.
In a previous study, a single intravenous ATT dose of 0.68 mgÁkg À1 or 2.05 mgÁkg À1 was administered in rats (Yu et al., 2011) and the pharmacokinetic results showed a free plasma concentration of ATT of 654.35 and 2626.89ngÁml À1 , respectively.These administered concentrations were much higher than the one we used (140 μgÁkg À1 Áday À1 ).However, the free plasma concentration of ATT is, logically, proportional to the dose intravenously administered.Since 0.68 mgÁkg À1 is 4.85 times higher than 140 μgÁkg À1 , we can approximately estimate the free plasma concentration of ATT at 135 ngÁml À1 in our conditions.However, since our ATT administration is continuous, we may expect that some ATT could also be present in the tissues thus increasing the whole amount of ATT seen by the organs.
Consequently, the OP2113 effects observed in vitro at 10 μM may not closely reflect those observed in vivo.We also observed a direct OP2113 vasorelaxant effect on Pheinduced preconstricted PA rings which was significantly higher in PA from CH-PH rats versus N rats.Interestingly, OP2113 acute administration in PA rings from N rats strongly inhibited PA contraction to 5-HT but not to PGF2α.This effect on 5-HT is particularly relevant in PH since 5-HT is known to be increased in plasma from patients with PAH (Kereveur et al., 2000) and to be strongly involved in human and  Rodat et al., 2007;Rodat-Despoix et al., 2009), cell proliferation and ROS production (Billaud et al., 2009;Ducret et al., 2008;Genet et al., 2017).Liu et al., 2006;Mittal et al., 2007).If the oxidative stress produced in the present animal model is mainly due to NOX activity rather than mitochondria, the OP2113 effect would consequently be independent  Amoedo et al., 2020).However, it should be noted that, in our study, PA remodelling has not been addressed from lungs held under a constant pressure, which could be a limitation of our work.
OP2113 is also an H 2 S-donor (Powell et al., 2018), with H 2 S inducing vessel relaxation, including in PA, via activation of ATPsensitive potassium channels or stimulation of NO synthesis (Roubenne et al., 2021).Moreover, H 2 S reverses the mPAP increase and PA muscularisation in CH-PH rats (Chunyu et al., 2003).Accordingly, the OP2113-induced decrease in mPAP, PA hyperreactivity and remodelling may be due to its H 2 S-releasing properties, but this remains to be further investigated.
Using molecular modelling computer simulation, Dias Amoedo et al. identified ATT interaction with the mitochondrial complex I (Dias Amoedo et al., 2020).We thus investigated whether OP2113 actions may affect mitochondrial mass and/or respiration.We showed that OP2113 significantly reversed the mitochondrial mass decrease in RV from CH-PH rats.In contrast, in PA from CH-PH rats, mitochondrial mass was increased but OP2113 had no effect.Finally, in RV from CH-PH rats, mitochondrial respiration was decreased, including maximum respiration and oxidative phosphorylation, but no damage to the mitochondrial outer membrane was observed.Both the decrease of mitochondrial mass in RV from the rat CH-PH model and the absence of any OP2113 effect on mitochondrial functioning (i.e., respiration) are in accordance with results from previous studies (Dai et al., 2022;Detaille et al., 2019;Nouette-Gaulain et al., 2011, 2005).However, the increase of mitochondrial mass in PA from the CH-PH rat is the opposite of what has been previously observed (Culley & Chan, 2018).Nevertheless, a few recent studies have brought out an up-regulation of the mitochondrial complex I, II and IV in endothelial cells (EC) from PAH patients compared to control human PAEC (Smolders et al., 2022), and an increase of mitochondrial respiration in BMPR2 mutant murine ECs (related to heritable PAH) (Egnatchik et al., 2017).Since we evaluated the mitochondrial mass of whole PA with the endothelium, it cannot be excluded that the increase of the mitochondrial mass may come from the ECs counterbalancing any potential decrease of the mitochondrial biogenesis in the smooth muscle.Although OP2113 prevented the decrease of RV mitochondrial mass in CH-PH, further investigations are needed to determine if such a phenomenon may be responsible for its beneficial effect on RV contractile function.
The function of the RV is of great clinical importance in PH since it determines the outcome of the disease (Humbert et al., 2022).
OP2113 displays cardioprotective effects and reduces the myocardial infarct size in a rat model of myocardial ischemia/reperfusion (Dai et al., 2022;Detaille et al., 2019).We thus explored the effect of OP2113 on heart function.In both RV and LV, as previously shown by Detaille et al. (Detaille et al., 2019), OP2113 ameliorated heart contractility by improving the CH-induced alteration of the contraction (slowdown-increase of Contraction T100) but it had no effect on the amplitude of the contraction.Moreover, intracellular calcium transients were not modified.Heart contraction depends on intracellular calcium, but also on calcium sensitization of the myofilaments due to the balance between kinases and phosphatases, stimulating the myofilament calcium response at a constant intracellular calcium concentration (Kass & Solaro, 2006).For instance, RV failure associated with experimental PH has previously been shown to involve such a desensitisation of the myofilaments to calcium (Fowler et al., 2015).We can thus speculate that OP2113 could affect such processes and these mechanisms need further investigation.
In conclusion, in vivo OP2113 treatment in CH-PH improves haemodynamic parameters, PA remodelling and both PA and heart contraction.Moreover, OP2113 has no effect on the PA and heart in control rats, strengthening the therapeutic interest in OP2113 for CH-PH.OP2113 is a drug in repositioning, it could thus be rapidly used for PH treatment in human.Finally, since (1) PH treatments are currently considered as mono or combination therapies (Humbert et al., 2022) and (2) OP2113 partially improves PH hallmarks, it would be relevant to address the effect of OP2113 in combination with the main drugs currently prescribed to PH patients.Surprisingly, the OP2113 effects are independent of its well-known action on mitochondrial oxidative stress but we can speculate that it could be related to a direct effect on contractile proteins and/or its H 2 S-releasing properties.In parallel to clinical trials, further investigations are also necessary to further document the mechanisms of action of OP2113 in vivo.
Dinan, France) and placed on a heating platform in the dorsal decubitus position.Cardiac output was assessed using the thermodilution method in close-chest animals.Briefly, 150 μl of cold saline solution ($1 C, NaCl 0.9%) was injected into the bloodstream via a 2.5-Fr polyethylene catheter (Biotrol, PE-5) inserted in the right jugular vein.A temperature microprobe (Columbus instruments, FR#1.5 Implantable Microprobe, AD Instruments, Oxford, United Kingdom) inserted into the right carotid artery measured the temperature variations induced by the cold saline injection, and cardiac output (CO) was then automatically calculated using LabChart software (v.8.1.18,AD Instruments, RRID:SCR_001620).Mean pulmonary artery pressure (mPAP) was measured in closed-chest rats by moving a fluid-filled (Heparin 5000 UÁml À1 in NaCl 0.9%) force transducer (Living systems instrumentation, Vermont, United States) from the right jugular vein into the right atrium and right ventricle and then into the main pulmonary artery.Pulmonary vascular resistance (PVR) was calculated as the right ventricle systolic pressure (RVSP)/CO ratio.At the end of the experiment, rats were killed with isoflurane overdose (5%), cardiorespiratory arrest indicating the death of the animal.Heart and lung lobes were then removed for further experimentation as described in next sections.CH-induced right ventricular hypertrophy was estimated using the Fulton index by separating the right ventricle (RV) from the left ventricle (LV) + septum and then calculating the RV/(LV + septum) weight ratio.All haemodynamic parameters were monitored using a Powerlab recording unit (AD Instruments) and LabChart software (v.8.1.18,AD Instruments, RRID:SCR_001620).RVSP, CO, PVR and Fulton index of CH rats were not changed by a 3-week treatment with vehicle solution (Figure S1a,b,c,d, respectively).
μM deferoxamine (Sigma) and 5-mM N,N-diethyldithiocarbamate (DETC, Sigma, Saint Quentin Fallavier, France and Magnettech, Berlin, Germany), Krebs-Hepes bicarbonate (KHB) solution (containing [in mM]: 118 NaCl, 4.7 KCl, 1.2 MgSO 4 , 4 NaHCO 3 , 1.2 KH 2 PO 4 , 2 CaCl 2 , 10 Hepes and 6 D-glucose, pH 7.4 with NaOH -Sigma).The reaction was stopped by snap freezing the samples in liquid nitrogen.Samples were then analysed by electron paramagnetic resonance spectrometry using a tabletop X-band spectrometer miniscope (MS200, Magnettech).Spectra of the oxidised product of CMH (CM•) were recorded at 77K using a Dewar flask.The electron paramagnetic resonance instrumental settings for field scan were as follows: Bo-field: 3339 ± 150 G, microwave frequency: 100 kHz, microwave power: 10 dB, amplitude modulation: 5 G, sweep time: 60 s, gain: 200 and three scans.Signals were quantified by measuring the total amplitude 10 ml of solution A (containing [in mM]: 2.77 CaK 2 EGTA, 7.23 K 2 EGTA, 20 Imidazole, 0.5 DTT, 6.56 MgCl 2 , 53.3 2-morpholinoethanesulfonic acid monohydrate; 20 taurine, 5.3 Na 2 -ATP, 15 Na 2 -phosphocreatine, pH 7.1 -Sigma).RV and LV were separated and cut into pieces in solution A under binocular microscope control.Cardiac fibres were then permeabilised for 30 min with solution A complemented with 50 μM of saponin (Sigma) and washed twice for 10 min with solution B (containing [in mM]: 1.9 CaK 2 EGTA, 8.1 K 2 EGTA, 20 imidazole, 0.5 DTT, 3 KH 2 PO 4 , 4 MgCl 2 , 100 morpholinoethanesulfonic acid monohydrate, 20 taurine, 0.2% BSA, pH 7.1 -Sigma).All procedures were carried out at 4 C. 2.9 | Measurement of mitochondrial respiration Live recording of oxygen consumption by permeabilised cardiac fibres was assessed at 37 C using a temperature-controlled oxygraph (Oxygraph-2K, Oroboros Instrument, Innsbruck, Austria) with a respiration buffer under continuous agitation and containing [in mM]: 0.5 EGTA, 3 MgCl 2 , 60 K-lactobionat, 20 taurine, 10 KH 2 PO 4, 20 HEPES, 110 sucrose, 0.1% BSA, pH 7.1 (Sigma).The different respiratory states (four states) (Detaille et al., 2019) were determined successively on a cumulative basis as follows: State 2 is the basal respiratory state (BASAL) assessed by adding a mixture of glutamate (5 mM), malate (2.5 mM) and succinate (5 mM) (Sigma) to ensure electron supply to complex I and II of the mitochondrial respiratory chain; state 3 is the activation of the oxidative phosphorylation state determined by adding ADP (2 mM) (Sigma) to activate ATP synthase; state 4 is the leak or nonphosphorylating state (LEAK) assessed by adding oligomycin (12 μM) (Sigma) to inhibit ATP synthase; state 5 is the uncoupled or maximum state (MAXIMUM) determined by applying increasing concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.4 to 1.2 μM) (Sigma).Oxygen consumption variations were recorded with DatLab software (DatLab 7.4.0.4) and expressed in pmolÁs À1 Ámg À1 .In parallel, mitochondrial outer membrane permeability to cytochrome c was assessed as an index of a mitochondrial outer membrane damage in the presence of ADP by applying cytochrome c for 1 min.Pellet was suspended in Tyrode solution (containing [in mM]: 137 NaCl, 5.4 KCl, 0.33 NaH 2 PO 4 , 0.5 MgCl 2 , 5 HEPES, 5.6Glucose, 1 CaCl 2 , pH 7.4 with NaOH -Sigma) and agitated for 15 min to allow calcium probe de-esterification.Cells were then placed into an experimental chamber on the stage of an inverted epifluorescence microscope (Â40 magnification, Eclipse Ti-U, Nikon, Champigny sur Marne, France) and continuously perfused with a Tyrode solution (room temperature).Cells were field stimulated using platinum electrodes at a frequency of 1 Hz to investigate [Ca 2+ ] i transients and fractional sarcomere shortening.Cardiomyocytes were alternately excited with excitation light at 340 and 380 nm using a monochromator spectrophotometer system (Optoscan, Cairn Research, Faversham, United Kingdom).Emitted light at 510 nm was collected by a photomultiplier (Hamamatsu, Nikon), and the ratio of emitted light in response to 340-and 380-nm excitation (340/380 ratio) was used as an index of intracellular Ca 2+ signal variations.Cardiomyocyte sarcomere shortening was measured by real-time contrast analysis on the image of the cell provided by a CCD camera (Myocam-S, Ionoptix, Amsterdam, the Netherlands).Signals were recorded and analysed with IonWizard 6.6 (IonOptix).Sarcomere shortening and [Ca 2+ ] i transients signal were monitored simultaneously in the same cell.Such analysis of contraction and calcium transients provided the following parameters: transient amplitude, time to reach the maximal contraction and calcium peak (Contraction T100 and Ca T100, respectively), time to reach 50% relaxation and calcium decay (Relaxtion T50 and Ca T50, respectively) and exponential decay time constant for calcium transients (Ca Tau).
figure legends.The declared sample size (n) is the number of independent values and statistical analysis was done using these independent values.All data were verified for normal distribution using the Kolmogorov-Smirnov normality test.For two group OP2113 and its vehicle were provided by Biocydex firm(Poitiers,   France).Osmotic mini-pumps were purchased from Durect Corporation (Alzet Model 2ML4, Charles River Laboratories, L'Arbresle, France); 5-HT, Phe, carbachol, deferoxamine, DETC, saponin, type XIV protease from Streptomyces were purchased from Sigma.Fura-2 AM ratiometric calcium probe was purchased from Invitrogen.CMH was purchased from Noxygen.PGF2α was purchased from Pfizer.Anti-VDAC1 mouse monoclonal antibodies, anti-4-HNE rabbit polyclonal antibodies and anti-muscarinic acetylcholine receptor M 3 rabbit polyclonal antibodies were purchased from Abcam.Anti-citrate synthase rabbit monoclonal antibody was purchased from Cell Signaling Technology.OxyBlot™ Protein Oxidation Detection Kit was purchased from Merck.Type II collagenase was purchased from Worthington.Information about other products is given in the text.2.14 | Nomenclature of targets and ligandsKey protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22(Alexander, Christopoulos et al., 2021; Alexander, Fabbro,   et al., 2021;Alexander, Kelly, Mathie, Peters, Veale, Armstrong, Faccenda, Harding, Pawson, Southan, Buneman, et al., 2021;   Alexander, Kelly, Mathie, Peters, Veale, Armstrong, Faccenda,   Harding, Pawson, Southan, Davies, et al., 2021 Alexander, Mathie   et al., 2021).

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I G U R E 2 Preventive OP2113 treatment in vivo improved the chronic hypoxia (CH) -induced pulmonary arterial (PA) medial thickening.(a) Representative images of PA stained with HES.All scale bars are 50 μm.The percentage PA medial thickness is shown for vessels with external diameters less than 50 μm (b), between 50 and 100 μm (c), as well as between 100 and 150 μm (d).N, normoxia; CH + VEH, chronic hypoxia + vehicle; CH + OP2113, chronic hypoxia + preventive OP2113 treatment.Data represent means ± SEM. n represents the number of rats.*P < 0.05.

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I G U R E 3 Curative OP2113 treatment in vivo improved chronic hypoxia (CH)-induced increase in mPAP without affecting right ventricular (RV) hypertrophy and pulmonary arterial (PA) medial thickening.(a) Mean pulmonary arterial pressure (mPAP), (b) Fulton Index (right ventricle (RV)/(left ventricle (LV) + septum weight)), (c) cardiac rate expressed in beats per minute (bpm) and (d) representative images of PA stained with HES.All scale bars are 50 μm.The percentage of PA medial thickness is shown for vessels with external diameters less than 50 μm (e), between 50 and 100 μm (f), as well as between 100 and 150 μm (g).N, normoxia; CH + VEH, chronic hypoxia + vehicle; CH + OP2113, chronic hypoxia + curative OP2113 treatment.Data represent means ± SEM. n represents number of rats for haemodynamic parameters results and PA remodelling.*P < 0.05.F I G U R E 4 Legend on next page.

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Effect of a preventive OP2113 in vivo treatment on chronic hypoxia (CH)-induced modifications of contraction and calcium transients, simultaneously recorded in isolated RV cardiomyocytes.Amplitude of sarcomere shortening (a) and calcium transients (d).The time to reach 100% of sarcomere shortening (Contraction T100) (b) and calcium transients (Ca T100) (e).The time to reach 50% of sarcomere relaxation (Relaxation T50) (c) and the calcium transient decay phase (Ca T50) (f).(g) Exponential decay time constant (Ca tau) of calcium transients.N, normoxia; CH + VEH, chronic hypoxia + vehicle in vivo treatment; CH + OP2113, chronic hypoxia + preventive OP2113 in vivo treatment.Data represent means ± SEM and are expressed as a percentage of N condition.n represents the number of cardiomyocytes for five rats.*P < 0.05.F I G U R E 6 Legend on next page.
OP2113 on both PA and heart main hallmarks associated with CH-PH in rats.In particular, haemodynamic parameters (mPAP and RVSP), PA remodelling (wall thickness), PA hyperreactivity, RV and LV contraction (time to peak contraction-Contraction T100) and the decrease of the mitochondrial mass in RV, were all improved by OP2113 in vivo treatment in rats with CH-PH.Interestingly, OP2113 had no effect on PA haemodynamic parameters, both PA and RV wall thickness and both PA contraction and relaxation in N rats treated with OP2113 in vivo.Since ATT is a drug authorised for use in human therapy for its choleretic and sialogogic properties with an excellent drug safety profile, we suggest that OP2113 could be safely used to treat CH-PH.Due to excessive PA vasoconstriction and cell proliferation, pulmonary vascular resistance increases in PH thus increasing RV afterload, leading to RV adaptive remodelling.However, whereas OP2113 has significant beneficial effects on both the elevated mPAP and RVSP, it does not alter RV hypertrophy.Likewise,Smith et al. recently    demonstrated that any attenuation of CH-PH can reduce PA alterations without modifying RV remodelling in mice.Indeed, they showed that RV remodelling and cardiac dysfunction were independent of PA remodelling but directly induced via a hypoxia inducible factor (HIF)-dependent signalling(Smith et al., 2020).Although mice are less susceptible to chronic hypoxia than rats, we thus speculated that OP2113 would act on the PA rather than on the heart by modifying PA contractile activity then inducing reversal of the increased mPAP associated with CH-PH.Such a hypothesis could be supported by our results showing a decrease of the PA hyperreactivity induced by 5-HT following either a preventive OP2113 treatment in vivo (2.9 mM of OP2113 in the mini-pump or 140 μgÁkg À1 Áday À1 for 3 weeks) or an acute OP2113 treatment ex vivo (10 μM for 30 min) on PA from rats with CH-PH.However, it should be pointed out that such in vitro studies at the cellular or tissue levels provide an incomplete insight into what occurs in vivo because of the difference in both concentration and duration of the OP2113 treatment.Moreover,

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I G U R E 6 Effect of a preventive OP2113 in vivo treatment on chronic hypoxia (CH)-induced oxidative stress in pulmonary artery (PA) and right ventricle (RV).(a) Quantification of O 2 ˙production in PA and (b) RV from N versus CH rats treated in vivo with OP2113 (140 μgÁkg À1 Áday À1 ) or its vehicle (VEH).(c) Lipid peroxidation assessed by western blotting targeting 4-hydroxynonenal (4-HNE) in PA and (d) RV from N versus CH rats treated in vivo with OP2113 or its VEH.(e) Protein carbonylation evaluated by western blotting targeting carbonyl groups of carbonylated proteins in PA and (f) RV from N versus CH rats treated in vivo with OP2113 or its VEH.Representative images of western blotting experiments for lipid peroxidation or protein carbonylation are shown in c, d, e and f respectively.N, normoxia; CH + VEH, chronic hypoxia + vehicle in vivo treatment; CH + OP2113, chronic hypoxia + preventive OP2113 in vivo treatment.Data represent means ± SEM.N represents the number of rats.* P < 0.05.
experimental PH and PAH.Under physiological as well as PH/PAH conditions, 5-HT does participate to the contractile status of PA (Ducret et al., 2008; Guibert et al., 2004; Maclean & Dempsie, 2010; F I G U R E 7 Effect of a preventive OP2113 in vivo treatment on modifications of mitochondrial mass and respiration in pulmonary artery (PA) and right ventricle (RV).(a) Quantification of mitochondrial mass assessed by western blotting targeting voltage dependant anion channel 1 (VDAC1) and (b) citrate synthase (CS) in PA from N versus CH rats treated in vivo with OP2113 (140 μgÁkg À1 Áday À1 ) or its vehicle (VEH).(c) Quantification of mitochondrial mass assessed by western blotting targeting VDAC1 and (d) CS in RV from N versus CH rats treated in vivo with OP2113 or its VEH.Representative images of western blotting experiments for VDAC1 and CS expression are shown in a,b and c,d, respectively.(e) Mitochondrial respiration (BASAL respiratory state (BASAL), activation of oxidative phosphorylation state (OXPHOS), leak or non-phosphorylating state (LEAK) and uncoupled or MAXIMUM state (MAXIMUM)) and (f) mitochondrial outer membrane damage, all evaluated in RV from N versus CH rats treated in vivo with OP2113 or its VEH.N, normoxia; CH + VEH, chronic hypoxia + vehicle in vivo treatment; CH + OP2113, chronic hypoxia + preventive OP2113 in vivo treatment.Data represent means ± SEM. n represents the number of rats.*P < 0.05.
Regarding CH-induced oxidative stress, we showed, in both PA and RV, an increase of carbonylated proteins whereas O 2 ˙production and lipid peroxidation remained unchanged.O 2 ˙is very unstable and is not the only ROS produced and responsible for oxidative stress in PH(Freund-Michel et al., 2013), which led us to assess the general effects of oxidative stress such as lipid peroxidation and protein carbonylation.The primary cellular target of oxidative stress depends upon cell type, the nature of the stress imposed (radical or nonradical oxidant), the site of generation (intra or extracellular), the proximity of ROS to a specific target, and the severity of the stress(Dalle-Donne et al., 2003), thus explaining why all markers of oxidative stress (namely O 2 ˙production, lipid peroxidation and protein carbonylation) may not all be switched on in CH-PH.The major advantage of protein carbonylation over lipid peroxidation is that oxidised proteins are generally more stable(Negre-Salvayre et al., 2008): They usually form early and circulate in the blood for longer periods.The resulting increase in protein carbonylation we demonstrated in PA and RV, unlike LV, is thus in agreement with a possible oxidative stress-related protein dysfunction in our model of rat CH-PH.The absence of any OP2113 effect on protein carbonylation may be due to the source and the nature of ROS produced in CH-PH.Indeed, in CH-PH, ROS can be produced not only by mitochondria, but also by NADPHoxidases 1, 2 and 4 (NOX1, 2 and 4), which are present in PA cells(Freund-Michel et al., 2013) and involved in PH(de Jesus et al., 2019; of oxidative stress in CH-PH.Similarly, MitoQ, another mitochondriatargeted antioxidant did not modify O 2 ˙concentration in pulmonary arterial smooth muscle cells exposed to chronic hypoxia (1% O 2 for 5 days)(Pak et al., 2018).However, unlike OP2113, MitoQ did not F I G U R E 8 Effect of a preventive OP2113 in vivo treatment on the expression of the M 3 muscarinic receptor in lung, pulmonary artery (PA) and right ventricle (RV).Quantification of the M 3 muscarinic receptor (M 3 ) assessed by western blotting in lung homogenates (a), PA (b) and RV (c) from N versus CH rats treated in vivo with OP2113 (140 μgÁkg À1 Áday À1 ) or its vehicle (VEH).Representative images of western blotting experiments are shown in a, b and c right panels for lung homogenates, PA and RV respectively.N, normoxia; CH + VEH, chronic hypoxia + vehicle in vivo treatment; CH + OP2113, chronic hypoxia + preventive OP2113 in vivo treatment.Data represent means ± SEM. n represents the number of rats.*P < 0.05.inhibit RVSP and PA remodelling in CH-PH mice (Pak et al., 2018), highlighting a special interest in OP2113 as a new tool to improve CH-PH treatment.In our study, OP2113 significantly reduced PA remodelling.Consistently, OP2113 decreased cell growth and migration in human lung cancer cell lines whereas N-acetylcysteine (NAC), a general bulk antioxidant, had the opposite effect, strengthening again the interest in the properties of OP2113 for PH treatment (Dias