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# Phosphodiesterases type 2, 3 and 4 promote vascular tone in mesenteric arteries from rats with heart failure

Liting Wang, Fabien Hubert, Sarah Idres, Milia Belacel-Ouari, Valérie Domergue, Séverine Domenichini, Florence Lefebvre, Delphine Mika, Rodolphe Fischmeister, Véronique Leblais, et al.

► To cite this version:

Liting Wang, Fabien Hubert, Sarah Idres, Milia Belacel-Ouari, Valérie Domergue, et al. Phosphodiesterases type 2, 3 and 4 promote vascular tone in mesenteric arteries from rats with heart failure. European Journal of Pharmacology, 2023, pp.175562. 10.1016/j.ejphar.2023.175562. hal-04014181

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# Phosphodiesterases type 2, 3 and 4 promote vascular tone in mesenteric arteries from rats with heart failure

**Running title:** Role of PDEs in a resistance vascular bed in heart failure

**Authors:** Liting Wang*ª, Fabien Hubert*ª, Sarah Idresª, Milia Belacel-Ouariª, Valérie Domergueᵇ, Séverine Domenichiniᵇ, Florence Lefebvreª, Delphine Mikaª, Rodolphe Fischmeisterª, Véronique Leblaisª, Boris Manouryª#.

¹Université Paris-Saclay, Inserm, UMR-S 1180, Orsay, France.

²Université Paris-Saclay, Inserm, Cnrs, Ingénierie et Plateformes au Service de l'Innovation Thérapeutique, Orsay, France.

* Dr Liting Wang and Dr Fabien Hubert contributed equally.

# Correspondence to: Boris Manoury, Université Paris-Saclay, Inserm UMR-S 1180, 17 Avenue des Sciences, 91400 Orsay, France.

E-mail: boris.manoury@universite-paris-saclay.fr; Tel: +33-1-80-00-62-90

## Data availability statement

Data available on request from the authors. The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

## Orcid ID

B.M.: 0000-0001-7305-5633
Abstract

Phosphodiesterases (PDE) type 3 and 4 promote vasoconstriction by hydrolysing cAMP. In experimental heart failure (HF), PDE3 makes PDE4 redundant in aorta, but it is not known if this occurs in resistance vessels, such as mesenteric artery. As PDE2 is increased in the failing myocardium, its possible role in the vasculature also needs to be addressed. Here, the function of PDE2, PDE3 and PDE4 in rat mesenteric arteries was characterized in experimental HF.

Mesenteric arteries were isolated from rats sacrificed 22 weeks after surgical stenosis of the ascending aorta (HF), or Sham surgery. PDE inhibitors were used to probe isoenzyme contributions in enzymatic and isometric tension assays. PDE2 and PDE4 activities, but not PDE3 activity, facilitate contraction produced by the thromboxane analogue U46619 in Sham arteries, while in HF all three isoenzymes contribute to this response. NO synthase inhibition by L-NAME abolished the action of the PDE2 inhibitor. L-NAME eliminated the contribution of PDE4 in HF, but unmasked a contribution for PDE3 in Sham. PDE3 and PDE4 activities attenuated relaxant response to β-adrenergic stimulation in Sham and HF. PDE2 did not participate in cAMP or cGMP-mediated relaxant responses. PDE3 and PDE4 cAMP-hydrolysing activities were smaller in HF mesenteric arteries, while PDE2 activity was scarce in both groups. Endothelial cells and arterial myocytes displayed PDE2 immunolabelling.

We highlight that, by contrast with previous observations in aorta, PDE4 participates equally as PDE3 in contracting mesenteric artery in HF. PDE2 activity emerges as a promoter of contractile response that is preserved in HF.
Key-words

phosphodiesterase inhibitor; vascular tone; mesenteric artery; heart failure; nitric oxide (NO)
# 1. Introduction

Cyclic nucleotides, namely cAMP and cGMP, are intracellular messengers that mediate transduction of various stimuli. In blood vessels, they inhibit smooth muscle cell (SMC) contractility and proliferation, platelet activity and endothelial permeability (Bobin et al., 2016). Phosphodiesterases (PDEs) are enzymes that degrade cyclic nucleotides and as such attenuate associated functions (Baillie et al., 2019; Bobin et al., 2016). PDEs encompass a large group of isoforms displaying various catalytic properties, cell distribution and mechanisms of regulation.

Heart failure (HF) is a severe condition with deterioration in functional, structural and metabolic features of the heart (McDonagh et al., 2021). This is associated with sustained, deleterious neurohormonal activation that increases vasoconstriction of small resistance arteries and thus cardiac afterload (Hartupee & Mann, 2017). Because the cAMP and cGMP signalling pathways are primary targets of the drugs used to alleviate or treat HF (Ghionzoli et al., 2021), it is crucial to clarify how specific PDE families influence vascular tone in this particular pathological context. Abundant work from our laboratory and others has clarified the role of cardiac PDEs in HF (recently reviewed in (Chen & Yan, 2021; Kamel et al., 2022). However, the impact of HF on their ability to promote vasoconstriction has been much less explored. In the vascular smooth muscle, PDE3 and PDE4 families are regarded as the main contributors for cAMP hydrolytic activity, whereas cGMP is mainly degraded by PDE5 and PDE1 (Hubert et al., 2014; Lugnier et al., 1986). PDE2 emerges as an additional mechanism that opposes vasorelaxation (Baliga et al., 2018; Bubb et al., 2014; Haynes et al., 1996; Wang et al., 2021). Because expression of PDE2 in vascular smooth muscle cells is still elusive (Bubb et al., 2014; Komas et al., 1991a; Lugnier et al., 1986; Pauvert et al., 2002; Stephenson et al., 2009; Wallis et al., 1999), how PDE2 may contribute to vasomotor tone remains unclear. Moreover, because specific contribution of PDE2 in the failing myocardium has been controversial (Baliga et al.,
2018; Mehel et al., 2013; Vettel et al., 2016; Zoccarato et al., 2015), it is of interest to clarify
how PDE2 influence vascular tone in this pathological context.

We previously reported that the contribution of PDE4 to vasoconstriction is made redundant by
the competing PDE3 in aorta from rats with HF (Hubert et al., 2014). Because increase in
peripheral vascular resistance is a hallmark of HF in patients (Hartupee & Mann, 2017), it is
also crucial to address whether these modifications of PDE contribution are also valid in small
vascular beds that directly impact cardiac afterload. Mesenteric arteries are a common model
for studying small resistance vessels (Mulvany & Halpern, 1977). Being part of the splanchnic
circulation that normally receives 20-30% of the cardiac output, this vascular bed is a
substantial contributor to peripheral resistance (Fenger-Gron et al., 1995; Takala, 1996). Thus,
we used isolated mesenteric arteries as a relevant model of resistance artery to further explore
the contributions of PDE2, PDE3 and PDE4 to arterial tone in experimental HF.

## 2. Material and Methods

### 2.1 Drugs and reagents

Chemicals and pharmacological reagents were purchased at standard manufacturers. Details on
the suppliers can be found in the online supplement (1.1).

All PDE inhibitors were dissolved in dimethylsulfoxide (DMSO). Final concentration of DMSO did not exceed 0.02%. For each given experiment, amounts of vehicle (water or DMSO) were matched in all groups studied.

### 2.2 Animals and surgical procedures

All animal care and experimental procedures conformed to the European Community guiding
principles in the care and use of animals (Directive 2010/63/EU of the European Parliament)
and authorizations to perform animal experiments according to application decrees were
obtained from the French Ministry of Agriculture, Fisheries and Food (No. C92-019-01, 03
August 2016). All studies involving animals are reported in accordance with the ARRIVE 2.0
guidelines for reporting experiments involving animals (Percie du Sert et al., 2020).

HF was induced in rats by following a surgical procedure mimicking aortic stenosis, as
previously described (De Sousa et al., 1999). The surgery was carried out on 3-week-old rats,
under anaesthesia with a ketamine-xylazine mix (75 mg/kg – 10 mg/kg, respectively, 0.3
mL/100 g, i.p.). Aortic stenosis was mimicked by placing a stainless steel hemoclip (0.6 mm-
internal diameter) on the ascending aorta via thoracic incision, Also, age-matched control
animals underwent the same procedure without placement of the clip (Sham group).
Buprenorphine chlorhydrate (0.03 mg/kg, 0.2 mL/100 g, s.c.) was administered twice daily for
3 days beginning at the end of the surgery.

Animals were housed under standard conditions and were sacrificed 22 weeks after surgery
according the following procedure. Rats were injected pentobarbital (150 mg/kg, i.p.) to obtain
deep anaesthesia, demonstrated by the absence of reaction following toe pinch. The heart, lung,
kidneys, liver and gut associated with the mesenteric vascular bed were quickly isolated and
placed in an ice-cold “Krebs” solution of following composition (in mM): NaCl 119, KCl 4.7,
CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, glucose 11, NaHCO3 25, bubbled with 95% O2 and 5%
CO2 to maintain pH at 7.4. Gut was used for isolation of mesenteric arteries. Heart, lung,
kidneys and liver were used for gravimetric analysis.

In order to comply with the “Reduce” criteria mentioned in the guidelines, some data presented
in this work were obtained using tissues sampled from rats shared with previous studies
published by our group, that were focussing on other vascular beds (Hubert et al., 2014; Idres
et al., 2019). All organs were sampled immediately following the sacrifice, kept in ice-cold
Krebs solutions and frozen or used within 2 hours.
A total of 34 rats with HF sacrificed 22 weeks after surgical aortic stenosis and displaying obvious lung congestion were used (lung weight/tibia length ratio >600 mg/cm). 45 Sham rats were used. In addition, 6 standards, 8-12 week-old, male Wistar rats were used for immunolabelling experiments.

## 2.3 Mesenteric artery preparation

The gut was pinned on a Sylgard®-coated dish filled with cold Krebs solution so that mesenteric vasculature was apparent. Second and third order mesenteric artery branches, displaying thick wall and longitudinal striation, were carefully cleaned from connective and fat tissue and dissected. Dissecting solution was renewed every 10-15 min with cold, bubbled solution. Collected tissue was then frozen in liquid nitrogen and stored at -80°C, or freshly processed, according to the following relevant protocols.

## 2.4 Isometric tension measurement

Mesenteric artery branches were cut into small segments (length 1.5-2 mm) and mounted in the chamber of a small vessel myograph (620 M, Danish Myo Technology A/S, Aarhus, Denmark) using 40 µm tungsten wire, as previously described (Idres et al., 2019). Briefly, vessels underwent a normalization procedure by stretching the vessels stepwise to construct tension-circumference relationship. Rings were eventually set at an internal circumference corresponding to 90% of the circumference the vessel would have if submitted to a transmural pressure of 100 mmHg (13.3 kPa). Working internal diameters were ≈ 150-350 µm. Contractile capacity of the vessels was evaluated by a challenge with a modified Krebs solution containing 60 mM KCl with equimolar substitution of NaCl (depolarizing solution, 60 mM K⁺). Vessels with response less than 2 mN/mm after 5 min were excluded from analysis, unless otherwise indicated. Endothelial function was evaluated in all vessels by measurement of the relaxant effect induced by 10 µM acetylcholine (ACh) or 10 µM carbachol on rings contracted using
depolarizing solution. Vessels with poor response (<20%) were excluded. After washes with Krebs solution, the vessels were submitted to various pharmacological protocols:

(i) Concentration-response curves to the thromboxane analogue U46619 ($10^{-9}$ – $3 \cdot 10^{-6}$ M) were performed by adding the drug in a stepwise, cumulative manner. The influence of specific PDE isoenzyme activity on the contractile response can be inferred by using selective PDE inhibitors used at relevant concentrations (Bobin et al., 2016; Hubert et al., 2014; Lugnier et al., 1986). Thus, this protocol was performed in the presence of Bay-60-7550 (0.1 µM), cilostamide (1 µM) or Ro-20-1724 (10 µM) inhibiting PDE2, PDE3 or PDE4, respectively. Selectivity ratio with relevant references are shown in Supplementary Table S1. Respective vehicle solution contained 0.02% DMSO. Contractile responses were expressed in mN/mm or in percent of the contraction evoked by the depolarizing solution.

(ii) In other experiments, response curves for vasorelaxant agents (ACh, isoprenaline, L858051, rat atrial natriuretic peptide (ANP), diethylamine nonoate (DEA-NO), or Bay-60-7550) were established on $10^{-6}$ M U46619-contracted vessels. Isoprenaline was added in the presence of the α-adrenergic receptor antagonist phentolamine ($10^{-5}$ M). When relevant, Bay-60-7550, cilostamide or Ro-20-1724 were added at least 10 min before contraction. In order to examine the influence of nitric oxide (NO) synthase activity on responses, some experiments were performed in the presence of 300 µM L-NG-Nitroarginine methyl ester (L-NAME), added at least 20 min before contraction. Relaxant responses were expressed in %, relative to the amplitude of the contraction obtained with U46619.

## 2.5 cAMP-PDE activity assay

Cyclic AMP-PDE activity was measured by a two-step radioenzymatic assay as previously described (Hubert et al., 2014; Vandeput et al., 2013). Reactions were carried out with inhibitors of PDEs, namely 1 mM 3-isobutyl-1-methylxanthine (IBMX), as a non-selective PDE inhibitor,
0.1 µM Bay-60-7550 as a selective PDE2 inhibitor, 1 µM cilostamide, as a selective PDE3 inhibitor and 10 µM Ro-20-1724, as a selective PDE4 inhibitor (see Supplementary Table S2), or vehicle. PDE activity was expressed as pmol (of cAMP degraded) /min /mg protein (in the sample). PDE2, PDE3, PDE4 and IBMX-sensitive activities were defined as the fraction of total activity inhibited by 0.1 µM Bay-60-7550, 1 µM cilostamide, 10 µM Ro-20-1724 or 1 mM IBMX, respectively. Each data point was obtained from one single animal and was the average of technical duplicates.

2.6 Analysis of PDE isoforms transcripts

Total RNA was extracted from rat mesenteric arteries following previously described procedure
used for rat aorta (Hubert et al., 2014). cDNA was synthesized from 1 µg total RNA using
iSCRIPT (Bio-Rad, Marnes-la-Coquette, France). Negative RT controls were prepared by using
RNA without addition of the reverse transcriptase. Real-time quantitative PCR (RT-qPCR) was
performed using the SYBR-Green method on a CFX384 TouchTM Real-Time PCR Detection
System (Bio-Rad). Detection of *Pde2a* and *Pde4c* were carried out using SYBR Premix (Takara
Bio Europe, Saint-Germain-en-Laye, France) and SYBR Supermix (Bio-Rad) was used for
*Pde3a*, *Pde3b*, *Pde4a*, *Pde4b* and *Pde4d* detection. For each target gene, a standard curve was
constructed from the analysis of serial dilution of cDNA and was used to determine efficiency
(E). For normalisation, threshold cycle (Ct) for target was subtracted from the mean of Ct for 3
reference genes (ΔCt) to calculate (1+E)ΔCt according to the ΔCt method. *Ywhaz*, *Rpl32* and
*Rplp2* were generally used as reference genes, except for *Pde2a* and *Pde4c* where *Tbp*
substituted for *Rplp2*. Relative expression was expressed as the ratio to the (1+E)ΔCt geometric
average obtained for Sham samples. Each data point was obtained from one single animal and
was the average of technical duplicates. Sequences of the primers used are provided in
Supplementary Table S2.
## 2.7 Western blot analysis

PDE isoforms were immunodetected by using standard Western blot procedure described previously (Idres et al., 2019). Briefly, frozen mesenteric arteries were homogenized in a lysis buffer containing (mM): Tris-HCl 50, NaCl 150, EDTA 2, NP40 1%, sodium deoxycholate 0.5%, SDS 0.1%, and protease and phosphatase inhibitor cocktails (Thermofisher Scientific, Les Ulis, France). Membranes were incubated with following primary antibodies: rabbit anti-PDE3A (1/10000; kind gift from Dr. Chen Yan, University of Rochester Medical Center, NY, USA), rabbit anti-PDE4A (1/5000), rabbit anti-PDE4B (1/1000), and mouse anti-PDE4D (1/10000) antibodies, kind gifts from Dr. Marco Conti (University of California, San Francisco, CA, USA). GAPDH (Cell signalling, 14C10) was used for normalisation. Secondary antibodies coupled with horseradish peroxidase were incubated, and chemiluminescent signals were acquired using an iBright FL1000 imaging system (Thermo Fisher Scientific) and quantified using FIJI software (Schindelin et al., 2012; Schneider et al., 2012).

## 2.8 Immunocytochemistry

Freshly isolated vascular cells were obtained using procedures previously described (Antigny et al., 2016; Idres et al., 2019) and detailed in the Supplementary material (1.2.1). Cells were immunolabelled using rabbit anti-PDE2 antibody (PDE2A-101AP, 1:300, Fabgennix, Frisco, TX); rabbit anti-CD31 (1:50, Abcam, Paris, France); rabbit anti-Calponin (1:200, Santa Cruz Biotechnology, Inc., Dallas, TX), then with anti-rabbit secondary antibodies (1:500, Alexa Fluor 488-conjugated or Alexa Fluor 647-conjugated (Invitrogen, Thermofisher Scientific, Les Ulis, France), following a procedure detailed in Supplementary material. The immunolabelled cell preparations were placed under a fluorescent inverted confocal laser scanning microscope Leica TCS SP8 (Leica Microsystems, Germany) visualized using Leica SP8 LAS X software (Version 3.5.5; Leica Microsystems, Germany) then processed and quantified using the FIJI
software (see Supplementary material 1.2.2 for a detailed description). Cells incubated with
secondary antibody, without primary antibody, were included as negative controls.

## 2.9 Data analysis and statistics

Response curves obtained for each vessel were fitted with the Hill equation using Prism 9 software (Graphpad Software, La Jolla, CA, USA) and pharmacological parameters, namely -log (EC₅₀, M) and maximal effect (Eₘₐₓ), were obtained. Data were expressed as mean ± SEM of N experiments, with N representing the number of rats. Where relevant (values normally distributed, equality of variance), group comparisons were performed using one-way ANOVA followed by Holm-Sidak multiple comparison post-test. When above assumptions could not be met, non-parametric Kruskal-Wallis test followed by Dunn's post-test was used. Similarly, either t-test, with or without Welch correction, or Mann-Whitney test were used for 2-group comparison, respectively. Two-group planned comparisons were performed using Mann-Whitney test with Holm correction. Comparisons of response curves were performed using 2-way ANOVA for repeated measures. Values of P<0.05 were considered for statistical significance.

# 3. Results

## 3.1 Characterization of the heart failure model

Twenty-two weeks after surgery, rats with aortic stenosis included in this study (HF group) displayed dramatic increases in heart weight and lung weight compared to Sham rats, indicating cardiac hypertrophy and lung congestion (Suppl. Figure 1). Kidney weight was significantly decreased in HF, without any change in liver weight or body weight. Because some of these rats were shared with other studies focussing on other vascular beds, echocardiographic and hemodynamic data from these series were published previously (Hubert et al., 2014; Idres et
al., 2019). This model was characterized by an increase in left ventricle (LV) mass, LV enlargement, decreased ejection fraction and decreased blood pressure. Overall, these data confirm that all rats included in the HF groups displayed important signs of heart failure.

## 3.2 Basic vascular reactivity of arteries isolated from rats with HF

First, basic vascular reactivity of mesenteric artery from Sham and HF rats was explored (Figure 1). High potassium, depolarizing solution (60 mM K⁺) evoked robust contraction in rings from both HF and Sham rats, although response to depolarizing solution in HF was significantly smaller than in Sham rings (Figure 1.A). The thromboxane A₂ mimetic U46619, a standard vasoconstrictor agonist, increased the tension in a concentration-dependent manner that was not significantly different between Sham and HF (Figure 1.B, see also Suppl. Figure 3.E-F for -log(EC₅₀) and Eₘₐₓ comparisons).

Addition of cumulative concentrations of ACh on rings pre-contracted with U46619 evoked a relaxant response that was significantly inhibited in HF compared to Sham arteries (Figure 1.C). Relaxations evoked by DEA-NO were similar in Sham and HF rings (Figure 1.D). The β-adrenergic receptor agonist isoprenaline (Figure 1.E) and the forskolin analogue L858051, a hydrosoluble direct activator of adenylyl cyclases (Figure 1.F) produced concentration-dependent relaxations which were similar in Sham and HF mesenteric rings. The addition of rat ANP did not produce any clear concentration-dependent relaxation, in Sham nor HF rings (Figure 1.G).

Overall, these results indicate that mesenteric artery rings from HF rats display alterations in contractile responses to the depolarizing solution, but not to a receptor-mediated response. In addition, there was a decrease in the relaxation to ACh without any change in the effect of the NO-donor, an observation compatible with endothelial dysfunction in HF. Other typical
responses mediated by cyclic nucleotides (isoprenaline, forskolin analogue, ANP) were
unchanged in HF.

## 3.3 Effect of PDE inhibitors on the response to the thromboxane analogue U46619 in HF and Sham mesenteric arteries

The effects of PDE2, PDE3 and PDE4 inhibition were first assessed on the contractile response evoked by increasing concentrations of U46619 in rat mesenteric arteries. Compared to vehicle, the selective PDE2 inhibitor Bay-60-7550 (0.1 µM) significantly shifted the response curves to the right and significantly decreased -log(EC₅₀) for U46619 in rings isolated from both Sham and HF rats (Figure 2.A, B, E, F). Consistently, Bay-60-7550 produced a concentration-dependent relaxation of Sham and HF mesenteric arteries contracted with U46619 (Suppl. Figure 2.A, B). These data indicate that PDE2 activity influences the vascular tone induced by U46619 in Sham and HF rat mesenteric arteries.

PDE3 inhibition with 1 µM cilostamide significantly hampered the response to U46619 by decreasing its -log(EC₅₀) in HF, but not in Sham vessels (Figure 2.C, D, E, F). The PDE4 inhibitor Ro-20-1724 used at 10 µM strongly inhibited response to U46619 in both Sham and HF groups and this was associated with a significant decrease in its -log(EC₅₀) values (Figure 2.C, D, E, F). Incubation with PDE inhibitors did not change Eₘₐₓ of the response to U46619 (Suppl. Figure 3.A, B). Overall, these results show that, in the absence of cAMP stimulation, PDE4 activity promotes contractile response to U46619 in both Sham and HF rat mesenteric arteries, while PDE3 influence can only be demonstrated in HF.

## 3.4 cAMP-PDE activity and expression of PDE isoforms in rat mesenteric artery

Cyclic AMP-PDE activity was measured in whole mesenteric artery lysates. Total activity was reduced in HF mesenteric arteries in comparison with Sham (Figure 3.A), and this was mainly
due to a decrease in both PDE3 (cilostamide-sensitive) and PDE4 (Ro-20-1724-sensitive) PDE activities (Figure 3.B). However, the relative contributions of cilostamide-sensitive and Ro-20-1724-sensitive PDE activities were similar in Sham and HF mesenteric arteries, with PDE3 activity accounting for 52 ± 4% and 52 ± 4% of total activity in Sham and HF, respectively, and PDE4 activity for 49 ± 2% and 44 ± 4%, respectively (not significantly different, 2-way ANOVA). Bay-60-7550 inhibited less than 10% of total activity and this effect was not different between Sham and HF (Figure 3.B).

Because HF may be associated with some modification in molecular expression of PDE isoforms, relative mRNA quantification was carried out from whole lysate rat mesenteric artery samples. Figure 3.C shows that expressions of *Pde2a*, *Pde3a*, *Pde3b*, *Pde4a*, *Pde4b* and *Pde4d* were not different in the HF group compared to the Sham group. The *Pde4c* transcript was not detected.

Variants of various sizes, ranging from ≈95 kDa to ≈125 kDa (PDE3A) and ≈70-80 kDa to ≈100 kDa (PDE4D) and ≈98 kDa (PDE4B), were detected in mesenteric artery tissue by Western blot (Figure 3.D), consistent with previous findings in arteries and in various organs (Abi-Gerges et al., 2009; Ding et al., 2005; Idres et al., 2019; Richter et al., 2005). Densitometry analysis did not show any significant difference between Sham and HF groups.

## 3.5 Effect of PDE inhibitors on the response to U46619 in the absence of NO production

The vascular NO-cGMP pathway is known to influence some PDE activities: while cGMP stimulates PDE2, it inhibits PDE3-mediated cAMP hydrolysis (Bobin et al., 2016; Eckly & Lugnier, 1994; Lugnier & Schini, 1990). In addition, the NO-cGMP pathway is reported to be depressed in HF (Drexler & Lu, 1992; Hubert et al., 2014; Katz et al., 1992). Therefore, contraction protocols were repeated in the presence of the NO synthase inhibitor L-NAME (300
µM) in order to reveal a possible action of basal NO release in Sham and HF mesenteric arteries. L-NAME did not produce any tone in mesenteric arteries (data not shown). L-NAME significantly potentiated the response to U46619 in both Sham and HF arteries with -log(EC50) values being significantly increased in both groups (Suppl. Figure 3.E, F). These observations show that tonic production of NO impedes contractile response to U46619 in both Sham and HF arteries.

In the presence of L-NAME, Bay-60-7550 had no effect on the response to U46619 in Sham nor in HF vessels (Figure 4.A, B, E, F). In similar conditions, both cilostamide and Ro-20-1724 inhibited significantly the response in the Sham group (Figure 4.C, E). By contrast, only cilostamide inhibited the response in HF vessels, while Ro-20-1724 had no effect (Figure 4.D, F). When comparing side-by-side these results obtained with (Figure 4) or without (Figure 2) L-NAME, it can be concluded that NO synthase inhibition: i) prevented the ability of PDE2 inhibition to blunt the U46619 response in both Sham and HF rings; ii) revealed an ability of PDE3 inhibition to impede the U46619 response in Sham arteries, while the effect of cilostamide in HF arteries was preserved; iii) did not affect the capacity of PDE4 inhibition to blunt the U46619 response in Sham, but dramatically abolished this effect in HF.

## 3.6 Effect of PDE inhibitors on the response to cAMP stimulation in HF and Sham mesenteric arteries

PDE3 and PDE4 are important repressors of cAMP in the vascular smooth muscle in different species and in various vascular beds, including mesenteric artery (Bobin et al., 2016; Keravis & Lugnier, 2012; Komas et al., 1991a; Komas et al., 1991b; Wallis et al., 1999). Thus, relaxation to the β-adrenergic receptor agonist isoprenaline, a typical cAMP-mediated response (Schoeffter et al., 1987), was studied in Sham and HF mesenteric arteries precontracted with U46619, in the absence or presence of PDE inhibition. In Sham, Bay-60-7550 had no significant
effect on the β-adrenergic response, but cilostamide and Ro-20-1724 significantly increased the
-log(EC50) value of this response, indicating a potentiated relaxation (Figure 5.A, C, E, G). In
HF, both cilostamide and Ro-20-1724 increased the response to isoprenaline and its Emax value,
but Bay-60-7550 had no effect (Figure 5.B, D, F, H).

It can be concluded that PDE2 inhibition does not significantly modify the response to
isoprenaline in rat mesenteric arteries. Influence of PDE3 and PDE4 activity is revealed in both
Sham and HF rings where their respective inhibitors potentiate the β-adrenergic response to
isoprenaline.

Because the β-adrenergic receptor transduction pathway may be complex due to cAMP-
independent pathways (Banquet et al., 2011), we investigated the effect of Bay-60-7550 on a
pure cAMP-mediated response, namely the relaxation to L858051. Pre-incubation with Bay-
60-7550 did not alter this response in Sham nor in HF arteries (Figure 6.A, B). Thus, taken
together, these results show that PDE2 inhibition does not influence the relaxation evoked by
stimulation of cAMP production in rat mesenteric artery.

## 3.7 Effect of PDE2 inhibition in cGMP-mediated vasorelaxation in HF and Sham mesenteric artery

An effect of Bay-60-7550 in cGMP-mediated vasorelaxation had been previously characterized
in rat pulmonary artery (Bubb et al., 2014). Thus, we set out experiments to examine whether
inhibition of PDE2 could also improve the responses to soluble or particulate guanylate cyclase
stimulation in mesenteric artery rings from Sham and HF rats. Pre-incubation with Bay-60-
7550 did not alter the response to the NO donor, DEA-NO, in Sham nor HF precontracted
arteries (Figure 6.C, D), showing that PDE2 inhibition does not influence relaxation evoked by
stimulation with exogenous NO. Similarly, Bay-60-7550 had no effect on the tension when rat
ANP (10<sup>-13</sup> - 10<sup>-8</sup> M) was added to Sham or HF rings (Figure 6.E, F).
## 3.8 Expression of PDE2 in SMCs and ECs freshly isolated from rat mesenteric artery

In order to further address the cell type expressing PDE2 in rat mesenteric artery, isolated EC and SMC preparations were processed for immunolabelling. Our two cell preparations were pure as shown by specific CD31 and calponin labelling (Figure 7.A, B, see Suppl. Figure 4 for negative controls). We then confirmed that the anti-PDE2 antibody stained the recombinant murine PDE2A3 overexpressed in mouse cardiomyocytes (Vettel et al., 2016) (Suppl. Figure 5). A similar protocol with the anti-PDE2 antibody yielded clear staining in ECs and SMCs isolated from rat mesenteric artery, indicating PDE2 expression in both cell types (Figure 7.C). Negative controls when omitting primary antibodies are shown in Suppl. Figure 4. Quantification shows that signal with PDE2 antibody was significantly higher than in negative controls, while mean fluorescence was similar between endothelial and smooth muscle cells (Figure 7.D).

## 4. Discussion

We used a severe model of HF, featured by dramatic LV hypertrophy and severe lung congestion. This was supported by echocardiographic data summarized in previous studies, showing LV systolic dysfunction, LV enlargement and decreased blood pressure (Hubert et al., 2014; Idres et al., 2019). The surgical aortic stenosis model in rat is reminiscent of aortic stenosis in human, that is a frequent, yet increasingly well managed disorder (Joseph et al., 2017). One limitation of the model is the immediate onset of pressure overload, which is in contrast to the slow progression in patients (Riehle & Bauersachs, 2019). Nevertheless, the model displays features of HF with reduced ejection fraction, that still represents a major public health burden (McDonagh et al., 2021; Savarese & Lund, 2017). Thus, we believe that our data help building up an integrated view of the pathophysiological changes occurring in HF.
Mesenteric vessels from rats with HF displayed reduction in contractility together with endothelial dysfunction. Likewise, aorta and coronary arteries demonstrated impaired response to ACh in the same model (Hubert et al., 2014; Idres et al., 2019). By contrast, other groups studying HF models following myocardial infarction in rodents reported no change in responses to K⁺, U46619 nor ACh in mesenteric artery (Bergdahl et al., 1999; Maupoint et al., 2016; Xu et al., 2007). The ability of L-NAME to potentiate U46619 response reveals that a constitutive production of NO hampers contraction. This effect was maintained in HF, suggesting that NO production is functional in this vessels. Interestingly, it was previously observed that the endothelium-derived hyperpolarizing factor was diminished in mesenteric arteries from rat with HF (Xu et al., 2007). Thus, alteration of a NO-independent component may explain the deteriorated response to ACh in this study.

PDE3 and PDE4 are the main cAMP-hydrolysing PDEs in mesenteric artery (Komas et al., 1991a; Wallis et al., 1999). Although responsible for more than 50% of total activity, PDE3 did not contribute significantly to vasoconstriction in Sham under basal cAMP condition. PDE4 activity is relatively large in rat mesenteric arteries (49%) and may hide the effect of PDE3 inhibition in Sham. Also, a constitutive release of endothelial NO may stimulate sufficient cGMP production to repress PDE3 activity (Eckly & Lugnier, 1994). Accordingly, our study shows that inhibition of NO-cGMP signalling with L-NAME promotes PDE3 contribution in Sham arteries.

Variations of PDE expression and activity were previously examined in HF patients and animal models (Hubert et al., 2014; Idres et al., 2019; Kamel et al., 2022). As to cardiac PDEs, several isoforms, including PDE2, are upregulated, while no consensus exists regarding modifications in PDE3 and PDE4 expression in HF (Kamel et al., 2022). As to vasculature, PDE activity was unchanged in aorta from HF rats (Hubert et al., 2014). By contrast, we observed that PDE3 and PDE4 activities were smaller in HF mesenteric arteries. Yet, their respective contributions to
vasoconstriction were robust in HF. This discrepancy suggests that attenuation of whole tissue PDE activity is not a limiting factor for these enzymes to control vasomotor responses. Recent literature revealed how discrete molecular complexes shape cyclic nucleotides signals at a nanometric scale and ensure tight control of cellular function (Bers et al., 2019; Kamel et al., 2022). While characterization of signal compartmentalization in vasculature is only emerging (Lorigo et al., 2021), it may be hypothesized that such a molecular network is preserved in HF and salvages the cAMP pathways involved in vascular tone.

In aorta, the absence of cGMP exacerbates PDE3 and masks PDE4 respective participations in the control of vascular tone (Eckly & Lugnier, 1994; Hubert et al., 2014). Our data suggest that this mechanism is not relevant in Sham mesenteric artery, because the effect of Ro-20-1724 was still observed in the presence of L-NAME. In HF, PDE4 inhibition still hampered contraction, but this effect was clearly NO-dependent, indicating that the NO-cGMP signalling is still active in HF and mitigates PDE3 activity. Overall, our results suggest that, in mesenteric arteries of HF rats, NO bioavailability is necessary for PDE4 activity to compete against PDE3 activity. The reason why PDE4 contribution is more vulnerable to NO synthase inhibition in HF than in Sham is unclear. This may be caused by the absolute smaller PDE4 activity, making it more sensitive to competition with PDE3, which displays above 100-time higher affinity for cAMP (Omori & Kotera, 2007).

When cAMP signalling was stimulated with the β-adrenergic agonist isoprenaline, both PDE3 and PDE4 impeded the relaxant response in Sham and HF mesenteric arteries. PDE3/4 inhibitions appear to be more effective at high isoprenaline concentration, which may not be physiologically relevant (Figure 5). Nevertheless, our data suggest that PDE3/4 inhibitors would rather be potent vasodilators in physiological conditions, namely at low cAMP level and low level of contraction (Figure 2).
Our study is limited to the exploration of PDEs in rat mesenteric artery and our conclusion may apply variably to other species, depending on the relative expressions of PDE3 and PDE4 in this tissue. Interestingly, PDE4 is the main cAMP-PDE in human mesenteric artery (Wallis et al., 1999) and both PDE3 and PDE4 inhibitions relax this vessel (Grzesk et al., 2014; Lindgren et al., 1989). Besides, PDE4 inhibition displays anti-inflammatory properties that were recently shown to be salutary in ischemia-reperfusion (Wan et al., 2022). Thus, inhibition of vascular PDE4 may emerge as a means to relieve the afterload burden on the left ventricle while limiting inflammation associated with HF (Dick & Epelman, 2016). This is supported by a meta-analysis reporting that pentoxyfilline, a PDE inhibitor with vasodilating and anti-inflammatory properties, reduced all-cause mortality in patients with HF (Champion et al., 2014).

By using the selective inhibitor Bay-60-7550 to probe PDE2 contribution to vasomotor tone, we observed that it inhibited contractile response to U46619 in both Sham and HF groups. This is consistent with previous studies reporting that PDE2 inhibition promotes vasodilation in pulmonary and coronary arteries (Baliga et al., 2018; Bubb et al., 2014; Haynes et al., 1996) and decreases arterial elastance in vivo, an indicator proportional to peripheral vascular resistance (Wang et al., 2021). Interestingly, NO synthase inhibition abolished the effect of Bay-60-7550. Similarly, vasodilator effect of Bay-60-7550 in perfused heart was abolished in GC-1α/- mice (Baliga et al., 2018). Together, these results suggest that constitutive NO-cGMP signals are necessary for PDE2 to promote vasoconstriction. The underlying mechanisms may depend on whether PDE2 is localized in vascular SMCs or in ECs. Both options are discussed in the following paragraphs.

Activity and expression of PDE2 were reported in whole vessels or SMCs in various species, (Bubb et al., 2014; Haynes et al., 1996; Pauvert et al., 2002; Sadhu et al., 1999; Saeki & Saito, 1993), including human where PDE2 activity was detected in mesenteric artery (Wallis et al., 1999). Immunostaining, however, revealed weak expression in tunica media (Stephenson et al.,
2009). We showed here that the PDE2 transcript and protein are present in rat whole mesenteric artery and freshly isolated SMCs, respectively. One might expect that PDE2 in SMC would directly mitigate vasorelaxation by limiting cyclic nucleotides signalling. Yet, Bay-60-7550 did not facilitate L858051 or DEA-NO responses. PDE2 expression in the SMCs may not be sufficient to compete against PDE3 or PDE4 activities and influence tone, as suggested by the very low contribution of PDE2 activity to cAMP hydrolysis in tissue homogenates.

Rather, we propose a model where an endothelial PDE2 would limit NO synthesis. In blood vessels, NO is synthetized by endothelial NO synthase, diffuses to SMCs and activates cGMP production by soluble guanylate cyclase which promotes SMC relaxation (Farah et al., 2018). PDE2 expression in ECs is widely documented in human and other species (Favot et al., 2003; Sadhu et al., 1999; Stephenson et al., 2009). We confirmed PDE2 expression in freshly ECs isolated from rat mesenteric artery. Besides, it was demonstrated that PDE inhibitors increase the production of NO in ECs (Hashimoto et al., 2006). PDEs may repress the stimulating effects of cAMP *via* downstream effectors such as PKA or EPAC, which have been shown to stimulate NO synthase (Farah et al., 2018; Garcia-Morales et al., 2017; Iring et al., 2019). We found that NO synthesis is necessary for the PDE2 inhibitor to attenuate contraction evoked by U46619, consistent with the hypothesis where PDE2 limits NO-mediated relaxation of SMCs. Further studies will be needed to establish the modalities of this mechanism in functional arteries.

As to a limitation of our study, it cannot be absolutely ruled out that the vasorelaxant action of Bay-60-7550 originates from an off-target effect. Nevertheless, Bay-60-7550 was used at selective concentration (Suppl. Table S1) and produced effects that were distinct from PDE3 and PDE4 inhibitors, supporting that they can be ascribed to PDE2 inhibition.

Our study limits only to PDE2, PDE3 and PDE4. While other cAMP-PDE families may also influence vascular tone, including PDE1 (Khammy et al., 2017) and PDE11, which are also
expressed in mesenteric artery (Kruse et al., 2009), the development of novel pharmacological
and genetic tools will help to further define the respective roles of these hitherto overlooked
PDEs.

**5. Conclusion**

This study extends the characterization of the effects of PDE2, PDE3 and PDE4 inhibitors on
vascular tone in HF to an archetypical resistance vessel, namely the mesenteric artery. We show
that relative pro-contractile contributions of these PDEs are in general less impacted by HF than
what was observed in aorta (Hubert et al., 2014). While influence of PDE3 is more limited in
Sham than in HF mesenteric artery, the role of PDE4 is more prominent in both normal and
pathological contexts, making this family of enzymes a potential target to address vascular
dysfunction in patients with HF. Our data also revealed a discrete participation of PDE2 to the
contractile response to U46619. This involvement is somewhat related to NO production and is
preserved in HF. This opens new perspectives on how specific PDE activities fine tune the
control of peripheral vascular beds by cyclic nucleotides.

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7. Acknowledgements

We thank the animal core facility for efficient animal care, the MIPSIT microscopy facility and
the ACTAGen transcriptomic facility (IPSIT, Université Paris-Saclay, Châtenay-Malabry,
France) and Audrey Varin (UMR-S 1180) for helpful technical assistance. We thank Pr Alison
Gurney (The University of Manchester) for her comments and wording suggestion. This work
was supported by the Fondation Leducq for the Transatlantic Network of Excellence cycAMP
grant 06CVD02 (to R. F.), the University Paris-Sud (Bonus Attractivité Paris-Sud 2009–2013
to F. H., V. L and B. M.), the Ministère de l'Éducation Nationale, de l'Enseignement Supérieur
et de la Recherche (including PhD fellowship to M. B. O., S. I.). UMR-S1180 is a member of
the Laboratory of Excellence in Research on Medication and Innovative Therapeutics supported
by the Agence Nationale de la Recherche (ANR-10-LABX-33) under the program
“Investissements d'Avenir” (ANR-11-IDEX-0003-01). This work was also funded by grants
from Agence Nationale de la Recherche (ANR-16-CE14-0014 (to D.M.) and
ANR13BSV10003-02) and by the Fédération Française de Cardiologie (to D.M.). L.W was
supported by the China Scholarship Council (PhD fellowship) and by a grant from Pr Zhi-
Cheng Jing (Peking Union Medical College Hospital, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing, China).
8. Figures

<figure><img src="image_2.png" /></figure>

B

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C

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D

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E

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F

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G

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**Figure 1:** Vascular reactivity of mesenteric artery isolated from Sham and HF rats. **A:** Amplitude of response to a depolarizing solution (60 mM K⁺) challenge in Sham and HF rings.

*: P< 0.05 (t-test). Scatter plots with bar indicating mean ± SEM from (N) animals are shown.

**B:** Amplitude of contractile response to U46619 (mN/mm) on Sham and HF rings. **C-G:** Effect of acetylcholine (C, ACh, 10⁻¹⁰ – 3.10⁻⁵ M), DEA-NONOate (D, DEA-NO, 10⁻¹⁰ – 3.10⁻⁵ M), isoprenaline (E, ISO, 10⁻¹⁰ – 10⁻⁶ M), adenylyl cyclase stimulator L858051 (F, 10⁻¹⁰ – 10⁻⁵ M) and rat ANP (G, 10⁻¹³ – 10⁻⁸ M) on Sham and HF rings precontracted with U46619 (1 µM). *:

*: P< 0.05 (two-way ANOVA). **B-G:** All data points are mean ± SEM response of rings from (N) animals. –log(EC₅₀, M) and Emax values are shown. There was no significant difference between Sham and HF groups (t-test or Mann-Whitney test when relevant). Concentration-response relationship to rat ANP were inconsistent and parameters could not be computed.
A

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Sham

B

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HF

C

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D

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E

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F

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**Figure 2:** Effect of PDE2 inhibitor (Bay-60-7550, 0,1 µM), PDE3 inhibitor (cilostamide, 1 µM), PDE4 inhibitor (Ro-20-1724, 10 µM) or vehicle on response curves to U46619 (10⁻⁹ – 3.10⁻⁶ M) in mesenteric artery rings from rat with heart failure (HF) or Sham littermates. **A-D:** response curves represent mean ± SEM of contractile response (mN/mm) of rings from (N) animals. Sham-vehicle and HF-vehicle curves represent the same data for **A** and **C**, and for **B** and **D**, respectively. *: P< 0.05 (repeated measures, two-way ANOVA). **E, F:** Scatter plot and mean ± SEM of -log(EC₅₀, M) calculated for (N) responses in Sham (**E**) and HF (**F**) groups. *: P< 0.05, compared to respective vehicle group (One-way ANOVA followed by Holm-Sidak's post-test).
A

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B

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C

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D

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**Figure 3:** Pattern of PDE activity and expression in mesenteric arteries from rats with heart failure (HF) or Sham (Sh) littermates. **A.** Total PDE activity. Scatter plot and mean ± SEM of N=5 experiments are shown. \*: P<0.05 (Welch's t-test). **B.** PDE activity in Sham and HF mesenteric artery lysates. PDE2, PDE3, PDE4 and IBMX-sensitive activities were defined as the fraction of total activity inhibited by 0.1 µM Bay-60-7550, 1 µM cilostamide, 10 µM Ro-20-1724 or 1 mM IBMX, respectively. Scatter plot and mean ± SEM of N=5 experiments are shown, except in HF / Bay-60-7550 group, where one outlier was excluded (Grubbs' test, α = 0.05). \*: P<0.05 (Welch's t-test with Holm-Sidak correction). **C.** Relative expression of rat *Pde2a*, *Pde3a*, *Pde3b*, *Pde4a*, *Pde4b*, and *Pde4d* mRNA transcripts in Sham and HF rat mesenteric arteries. Scatter plot and mean ± SEM are shown from N=6 rats. **D.** Up: immunoblots showing detection of PDE3A, PDE4D long and short forms and PDE4B in mesenteric artery lysates from Sham (Sh) and rats with heart failure (HF). Bottom: scatter plot and mean ± SEM of N=6 rats are shown. PDE3A and PDE4D were probed successively on the same membrane without stripping, using rabbit and mouse secondary antibodies, respectively. Raw gel images are supplied as supplementary raw material in the Supplementary material (section 4).
Sham + L-NAME

HF + L-NAME

A

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B

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C

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D

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E

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F

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**Figure 4:** Effect of PDE2 inhibitor (Bay-60-7550, 0.1 µM), PDE3 inhibitor (cilostamide, 1 µM), PDE4 inhibitor (Ro-201724, 10 µM) or vehicle on response curves to U46619 (10⁻⁹ – 3.10⁻⁶ M) in the presence of 300 µM L-NAME in mesenteric artery rings from rat with heart failure (HF) or Sham littermates. **A-D:** response curves present mean ± SEM of contractile response (mN/mm) of rings from (N) animals. Sham-vehicle and HF-vehicle curves represent the same data for **A** and **C**, and for **B** and **D**, respectively. *: P< 0.05 (repeated measures, two-way ANOVA). **E, F:** Scatter plot and mean ± SEM of −log(CE₅₀, M) calculated for (N) responses in Sham (**E**) and HF (**F**) groups. *: P< 0.05, compared to the respective vehicle group (one-way ANOVA followed by Holm-Sidak's post-test).
<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

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<figure><img src="image_34.png" /><figcaption>**Figure 5:** Effect of PDE2 inhibitor (Bay-60-7550, 0.1 µM), PDE3 inhibitor (cilostamide, 1 µM), PDE4 inhibitor (Ro-201724, 10 µM) or vehicle on response curves to the β-adrenergic receptor agonist isoprenaline (ISO, 10⁻¹⁰ – 10⁻⁶ M) in mesenteric artery rings from rat with heart failure (HF) or Sham littermates. **A-D:** response curves present mean ± SEM of relaxant response of rings from (N) animals. Sham-vehicle and HF-vehicle curves represent the same data for **A** and **C**, and for **B** and **D**, respectively. \*: P< 0.05 (repeated measures, two-way ANOVA). E-H: Scatter plot or bar graph showing mean ± SEM of –log(EC₅₀, M) (**E**, **F**) and Emax (**G**, **H**) calculated for (N) responses in Sham (**E**, **G**) and HF (**F**, **H**) groups. \*: P< 0.05, compared to respective vehicle group (one-way ANOVA followed by Holm-Sidak's post-test).</figcaption></figure>
Sham

HF

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**Figure 6:** Effect of PDE2 inhibitor (Bay-60-7550) on responses to cAMP and cGMP stimulators in mesenteric arteries from rat with heart failure (HF) or Sham littermates. **A-F:** Response curves to the adenylyl cyclase stimulator L858051 (**A**, **B**, $10^{-10}$ – $10^{-5}$ M), the nitric oxide donor DEA-nonoate (**C**, **D**, DEA-NO, $10^{-10}$ – $3 \cdot 10^{-5}$ M) or rat ANP (**E**, **F**, $10^{-13}$ – $10^{-8}$ M) in mesenteric artery rings. Response curves present mean ± SEM of relaxant response of rings from (N) animals. In **B** (Bay-60-7550 group), one ring contracted less than 2 mN/mm while the response to ACh was normal. Statistical comparison did not yield any significant difference between Sham and HF groups (repeated measures, two-way ANOVA).
<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /><figcaption>**Figure 7:** Immunolabelling of PDE2 in freshly isolated smooth muscle cells (SMC) and endothelial cells (EC) from rat mesenteric artery. **A-C:** Confocal images of isolated SMC or EC sheets immunostained with primary antibodies against the EC marker CD31 (**A**), the SMC marker calponin (**B**) and PDE2 (**C**). All images were obtained at an identical level of laser illumination and have not been altered. DAPI staining indicates nuclei. “Merged” shows overlay of both images. Scale bar: 20 µm is valid for all images. Representative pictures from N=5 independent experiments (PDE2), or N=3 (CD31, calponin) are shown. **D.** Quantification of mean fluorescence obtained with or without the anti-PDE2 antibody. Mean ± SEM obtained from N = 5 independent experiments. For each experiment, 3-9 fields were analysed and averaged. *: P<0.05 vs control condition without the primary antibody (two-way ANOVA followed by Sidak's post hoc test). A.U.: arbitrary unit.</figcaption></figure>
# Supplementary material

## 1. Additional Material and Methods

### 1.1. Drugs and reagents

Cilostamide (Cil) was purchased from Tocris Bioscience (Bristol, UK) and Ro-20-1724 (Ro) from Calbiochem (Merck Chemicals Ltd, Nottingham, UK). 3-isobutyl-1-methylxanthine (IBMX), acetylcholine (ACh), carbamylcholine chloride (carbachol, CCh), isoprenaline (ISO), phentolamine, trypsin inhibitor, bovine serum albumin (BSA), dithiothreitol (DTT), rat atrial natriuretic peptide (ANP) and snake venom from Crotalus atrox were supplied by Sigma-Aldrich (Merck Millipore, Saint Quentin-Fallavier, France). U46619, Bay-60-7550 (Bay-60) were bought from Cayman (Interchim, Montluçon, France) and L858051, diethylamine nonoate (DEA-NONOate), L-NG-Nitroarginine methyl ester (LNAME), from Enzo Life Science (Villeurbanne, France).

### 1.2. Detailed procedure for immunocytochemistry.

#### 1.2.1 Preparation of freshly isolated vascular cells

Rat MA EC sheets were isolated following enzymatic digestion at 37°C for 60 min with papain (0.2 mg/ml) and DTT (0.2 mg/ml) using a modified Hank's balanced salt solution (HBSS) of following composition (in mM): CaCl₂ 1.26, MgCl₂ 0.49, MgSO₄ 0.41, KCl 5.33, KH₂PO₄ 0.44, NaHCO₃ 4.16, NaCl 137.9, Na₂HPO₄ 0.34, D-glucose 5.55, pH=7.4 (Antigny et al., 2016). The SMCs were isolated as previously described (Idres et al., 2019) using serial bathing in dissociation medium (DM, in mM: NaCl 10, KCl 5, HEPES 10, KH₂PO₄ 0.5, NaH₂PO₄ 0.5, NaHCO₃ 10, Taurine 10, EDTA 0.5, D-glucose 10, CaCl₂ 0.16, MgCl₂ 2, Phenol Red 0.03) first containing papain (1.5 mg/mL) and DTT (1 mg/mL) (37 °C, 6 min) and then collagenase (1.6 mg/ml) and trypsin inhibitor (1.6 mg/ml) at 37 °C for 4 min. Vessel segments were subsequently washed 3 times in DM containing 1 mg/mL BSA and gently triturated by pipette to release the SMCs. After smooth centrifugation, SMCs were resuspended in control DM for
immunocytochemistry. Mouse adult cardiac myocytes (ADeCM) were isolated following a standard
procedure described previously by our laboratory (Vettel et al., 2016). Transgenic mice overexpressing
the PDE2A3 variant under the control of αMHC promoter (line TG-4320), and their wild-type
littermates were used (Vettel et al., 2016).

1.2.2 immunolabelling and image analysis

Single drops of suspended cell solutions were added on glass coverslips. The coverslips for ECs were incubated with Corning® Cell-Tak adhesive (Sigma) to improve cell adhesion. Laminin was used for cardiac myocytes. Cells were fixed with 4% paraformaldehyde, washed with PBS, permeabilized with Triton X-100 0.3% in PBS, quenched with NH₄Cl 50 mM in PBS, and blocked with BSA 5% in PBS. Coverslips were then incubated overnight at 4°C with the appropriate primary antibodies: rabbit anti-PDE2A-101AP (1:300, Fabgennix, Frisco, TX); rabbit anti-CD31 (1:50, Abcam, Paris, France); rabbit anti-Calponin (1:200, Santa Cruz Biotechnology, Inc., Dallas, TX), then with anti-rabbit secondary antibodies for 1 h at room temperature (1:500, Alexa Fluor 488-conjugated or Alexa Fluor 647-conjugated; Invitrogen, Thermofisher Scientific, Les Ulis, France). The immunolabelled cell preparations were placed under an inverted confocal laser scanning microscope Leica TCS SP8 (Leica Microsystems, Germany) with an HC PL APO CS2 63x/1.40 oil immersion objective. The microscope was equipped with a 405 nm diode for DAPI (nuclei-blue channel) excitation and a WLL Laser set at 488 nm excitation for AlexaFluor 488 (CD31 and calponin-green channel) or at 647 nm excitation for AlexaFluor 647 (PDE2-magenta channel). Blue, green and magenta channels fluorescence emissions were collected respectively with 413-494, 496-573, 653-751 nm wide emission slits using a sequential mode and with hybrid detector for green and far-red channels. The detection offset was chosen to a small number of zero-value pixels and detector gains were set to optimize the dynamic range while ensuring minimal saturated pixels based on the most fluorescent sample and were fixed for all acquisitions of the same experiment. The pinhole was set at 1.0 Airy unit giving an optical slice thickness of 0.89 µm. Sections labelled by immunofluorescence were visualized using Leica SP8 LAS X software (Version 3.5.5; Leica Microsystems, Germany). Quantification of mean fluorescence intensities (MFI) was performed using the FIJI software (https://imagej.net/Fiji) (Schindelin et al., 2012; Schneider et al.,
2012). Furthermore, bright field (BF) images were present to show relevant cell shape. Cells incubated
with secondary antibody, without primary antibody, were included as negative controls. We have
specifically developed an image processing macro using thresholding based on the intensity of the
AlexaFluor647 fluorescence (shown as the magenta channel) in order to automatically delineate the
regions of interest (cells). Values were exported to Excel and Prism-GraphPad for statistical analyzes
and graphical representations.
## 2. Supplementary Tables

<table><caption>2.1. Table S1: Reference supporting selectivity of pharmacological PDE inhibitors used.</caption><thead><tr><th>Compound</th><th>Concentr. used (µM)</th><th>Target inhibited</th><th>IC50 (µM)</th><th>Selectivity ratio (IC50<sub>PDE</sub> / IC50<sub>target</sub>)</th><th>Reference</th></tr></thead><tbody><tr><td>Bay 60-7550</td><td>0.1</td><td>PDE2</td><td>0.002 - 0.005</td><td>≈ 50 fold (PDE1)<br>≈ 150 fold (PDE5)<br>≈ 350 fold (PDE4B)<br>≈ 850 fold (PDE3B)</td><td>(Boess et al., 2004)</td></tr><tr><td>cilostamide</td><td>1</td><td>PDE3</td><td>0.027 - 0.050</td><td>≈ 250 fold (PDE2)<br>≈ 300 fold (PDE5)<br>≈ 400 fold (PDE7)<br>≈ 1700 fold (PDE4)<br>≈ 6000 fold (PDE1)</td><td>(Sudo et al., 2000)</td></tr><tr><td>Ro-20-1724</td><td>10</td><td>PDE4</td><td>1-2</td><td>≈ 26 fold (PDE3)<br>> 50 fold (PDE2)<br>≈ 100 fold (PDE1)<br>≈ 125 fold (PDE5)</td><td>(Rich et al., 2001)</td></tr></tbody></table>
<table><caption>2.2. Table S2. Primers pairs used in RT-qPCR to quantify mRNA expression.</caption><thead><tr><th>Target (rat)</th><th>Accession Number</th><th>Forward 5'→3', Reverse 5'→3';</th><th>Product size (Bp)</th><th>Annealing temperature (°C)</th></tr></thead><tbody><tr><td>Pde2a</td><td>NM_001143847</td><td>CTGTGCTGGCTGCACTCTAC<br>GAGGATAGCAATGGCCTGAG</td><td>77</td><td>60</td></tr><tr><td>Pde3a</td><td>NM_017337</td><td>ACCTCCCTGCCCTGCATAC<br>CCTCTCTTGTGGTCCCATTCC</td><td>65</td><td>60</td></tr><tr><td>Pde3b</td><td>NM_017229</td><td>GTGGCTACAAATGCACCTCA<br>CTGGGCGAGAAAGATACAGA</td><td>100</td><td>60</td></tr><tr><td>Pde4a</td><td>NM_013101</td><td>CGTCAGTGCTGCGACAGTC<br>CCAGCGTACTCCGACACACA</td><td>190</td><td>60</td></tr><tr><td>Pde4b</td><td>NM_017031</td><td>GATGAGCAGATCAGGGAACC<br>GATGGGATTTCCACATCGTT</td><td>81</td><td>60</td></tr><tr><td>Pde4c</td><td>XM_214325</td><td>GACCCTGTCCTTCCTGTTGA<br>AACCGTCTCAGGATCACACC</td><td>99</td><td>60</td></tr><tr><td>Pde4d</td><td>NM_001113328</td><td>GCCAGCCTTCGAACTGTAAG<br>ATGGATGGTTGGTTGCACAT</td><td>98</td><td>60</td></tr><tr><td>Ywhaz</td><td>NM_013011</td><td>AGA CGG AAG GTG CTG AGA AA<br>GAA GCA TTG GGG ATC AAG AA</td><td>127</td><td>60</td></tr><tr><td>Tbp</td><td>NM_001004198</td><td>AAA GAC CAT TGC ACT TCG TG<br>GCT CCT GTG CAC ACC ATT TT</td><td>132</td><td>60</td></tr><tr><td>Rpl32</td><td>NM_013226</td><td>GCT GCT GAT GTG CAA CAA A<br>GGG ATT GGT GAC TCT GAT GG</td><td>115</td><td>60</td></tr><tr><td>Rplp2</td><td>NM_001030021</td><td>GCT GTG GCT GTT TCT GCT TC</td><td>119</td><td>60</td></tr></tbody></table>
ATG TCG TCA TCC GAC TCC TC
### 3. Supplementary references

Antigny, F., Hautefort, A., Meloche, J., Belacel-Ouari, M., Manoury, B., Rucker-Martin, C., ... Perros, F. (2016). Potassium-Channel Subfamily K-Member 3 (KCNK3) Contributes to the Development of Pulmonary Arterial Hypertension. *Circulation*. doi:10.1161/CIRCULATIONAHA.115.020951

Boess, F. G., Hendrix, M., van der Staay, F. J., Erb, C., Schreiber, R., van Staveren, W., ... Koenig, G. (2004). Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance. *Neuropharmacology*, 47(7), 1081-1092. doi:10.1016/j.neuropharm.2004.07.040

Idres, S., Perrin, G., Domergue, V., Lefebvre, F., Gomez, S., Varin, A., ... Manoury, B. (2019). Contribution of BKCa channels to vascular tone regulation by PDE3 and PDE4 is lost in heart failure. *Cardiovasc Res*, 115(1), 130-144. doi:10.1093/cvr/cvy161

Rich, T. C., Tse, T. E., Rohan, J. G., Schaack, J., & Karpen, J. W. (2001). In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. *J Gen Physiol*, 118(1), 63-78.

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., ... Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. *Nat Methods*, 9(7), 676-682. doi:10.1038/nmeth.2019

Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. *Nat Methods*, 9(7), 671-675. doi:10.1038/nmeth.2089

Sudo, T., Tachibana, K., Toga, K., Tochizawa, S., Inoue, Y., Kimura, Y., & Hidaka, H. (2000). Potent effects of novel anti-platelet aggregatory cilostamide analogues on
recombinant cyclic nucleotide phosphodiesterase isozyme activity. *Biochem Pharmacol*, 59(4), 347-356.

Vettel, C., Lindner, M., Dewenter, M., Lorenz, K., Schanbacher, C., Riedel, M., ... El-Armouche, A. (2016). Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction. *Circ Res*. doi:10.1161/CIRCRESAHA.116.310069
<figure><img src="image_45.png" /><figcaption>**Suppl. Figure 1:** Summary data of organ weight normalized to tibia length and body weight of Sham and HF rats used in this study. Scatter plots with bar indicating mean ± SEM from (N) animals are shown. *: P< 0.05 (t-test, Welsh's t-test or Mann-Whitney test when relevant).</figcaption></figure>
4. Supplementary raw material

<figure><img src="image_46.png" /><figcaption>Mouse Anti-PDE4D</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>Rabbit Anti-GAPDH</figcaption></figure>

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /><figcaption>Rabbit Anti-GAPDH</figcaption></figure>

<figure><img src="image_50.png" /></figure>

**Suppl. Raw material:** Raw images of immunoblots for detection of PDE3A, PDE4B and PDE4D. PDE3A and PDE4D were probed successively on the same membrane without stripping, using rabbit and mouse secondary antibodies, respectively.