# The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model

Authors: Thomas Dupas; Thomas Pelé; Justine Dhot; Mélanie Burban; Antoine Persello; Virginie Aillerie; Angélique Erraud; Angela Tesse; David Stevant; Angélique Blangy-Letheule; Céline Menguy; Vincent Sauzeau; Michel De Waard; Bertrand Rozec; Chantal Gauthier; Benjamin Lauzier
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

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## Abstract

50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of humanβ3-adrenoreceptor (Tgβ3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgβ3 rats. The aortic expression of •NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate •NO and O2•- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgβ3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgβ3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00±0.21; Tgβ3: 6.08±2.30; 45 weeks: WT: 1.00±0.12; Tgβ3: 1.55±0.17; p<0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgβ3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00±0.37; Tgβ3: 0.13±0.05; p<0.05). Aortic •NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgβ3 rats (+52% vs. WT). Aortic O2•- production was increased in Tgβ3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p<0.05). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.


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## Body

## 1. Introduction

Over the past decade, cardiovascular diseases have been one of the leading causes of death worldwide [1]. Among these diseases, heart failure (HF) affects 2-3% of the world’s population [2]. There are several forms of HF, including HF with preserved ejection fraction (HFpEF) which has no efficient treatment at present day. This type of HF is characterized by a diastolic dysfunction without major alteration of ejection fraction. The main clinical characteristics of patients with HFpEF are advanced age and female sex [3]. However, the mechanisms leading to HFpEF are not completely understood so far. Few years ago, Paulus and Tschöpe put forward the idea that endothelial function could be the common trigger of all HFpEF etiologies. More specifically, the nitric oxide (•NO) pathway was pointed out [4]. Indeed, the •NO is a major player in the maintenance of cardiovascular function. The •NO is mainly produced by the endothelial cells via the activation of the •NO synthase (NOS) which exists under three isoforms: the neuronal NOS (nNOS or NOS1), the inducible NOS (iNOS or NOS2), and the endothelial NOS (eNOS or NOS3). In physiological conditions, most of the •NO production in the vessels and the heart is mediated by NOS3 [5]. Paracrine action of •NO, produced by endothelial cells, induces a relaxation of smooth muscle cells in the vessels. At the cardiac level, endothelial cells represent 20% of the cellular population and the •NO plays a role in cardiac contractility regulation [6]. The •NO production is partly mediated by the activation of β-adrenoceptors (β-AR) and more specifically the β3-adrenoceptor (β3-AR) through the activation of NOS3 or NOS1 [7, 8]. β3-AR activation, expressed in cardiomyocytes and endothelial cells, induced •NO release [9]. Considering the putative link between HFpEF and endothelial dysfunction and the link between β3-AR and •NO signaling, the purpose of the study is to evaluate the link between endothelial dysfunction and HFpEF development. An animal model that overexpresses the human β3-AR (Tgβ3) on endothelial cells has been developed. Previously, we have shown that Tgβ3 animals overproduced •NO at the cardiac level and develop a diastolic dysfunction at 45 weeks of age, characteristic of HFpEF [10]. The aim of this study is to understand if endothelial dysfunction could be the cause of HFpEF.


## 2. Methods

### 2.1. Experimental Animals

All animal experimental protocols were approved by the Pays de la Loire Ethical Committee and were performed in accordance with the French law on animal welfare, EU Directive 2010/63/EU for animal experiments, the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 2011), and the 1964 Declaration of Helsinki and its later amendments, and all the animals were housed according to standard living conditions. All animals used in the study are male Sprague-Dawley rats. Tgβ3 rats which overexpress human β3-AR in endothelial cells and their control (WT) were used at 15, 30, and 45 weeks of age and fed ad libitum with a soy-free diet (Envigo, #2914C, Huntingdon, United Kingdom) as previously described [10]. Then, animals were euthanized, and the thoracic aorta and superior branch of mesenteric arteries were harvested in order to perform vascular reactivity (thoracic aorta and mesenteric arteries), electronic paramagnetic resonance (EPR) quantification of •NO and O2•- (thoracic aorta), and Western blot protein analysis (thoracic aorta) as described below.


### 2.2. Echocardiography

Echocardiography was performed on anesthetized rats (O2/isoflurane mixture 1.5% with flow rate of 1 L/min) at 15, 30, and 45 weeks of age, using a Vingmed-General Electric ultrasound system (VIVID 7, Horten, Norway) equipped with a 10 MHz imaging probe and offline cine loop analysis software (Echopac TVI, GE-Vingmed Ultrasound) on the Therassay platform of Nantes, as previously described [10].


### 2.3. Vascular Reactivity for Pharmacological Studies

Rats were anesthetized with an O2/isoflurane mixture (induction: 5% isoflurane, flow rate 1 L/min; maintenance: 2% isoflurane, flow rate 0.5 L/min). The thoracic aorta and mesenteric arteries were carefully excised and cleared of fat and connective tissue. Vascular tensions were recorded as previously described [11, 12]. Briefly, aortic and mesenteric rings were mounted on a multichannel isometric myograph (Danish Myo Technology) (95% O2 and 5% CO2) Krebs-Henseleit bicarbonate solution (millimolar concentrations: NaCl 118, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, KCl 4.5, glucose 11, and CaCl2 1.5) at 37°C [13]. To verify the endothelium integrity, rings were precontracted with phenylephrine (1 μM, Sigma-Aldrich, P6126) and then exposed to a single concentration of acetylcholine (10 μM, Sigma-Aldrich, A6625). The tension was recorded on precontracted rings with phenylephrine (1 μM). The vasodilation response to cumulative concentrations of isoproterenol, a nonselective β-AR agonist (1 nM to 100 μM, Sigma-Aldrich, I6504), and of CL 316 243, a β3-AR agonist (1 nM to 100 μM, Tocris, 1499), was evaluated. The vasodilation response to isoproterenol (1 nM to 100 μM) was repeated after 30 min of vessel incubation with NOS inhibitors prior to the phenylephrine precontraction: L-NIO, a NOS3 inhibitor (10 μM, N5-(1-iminoethyl)-L-ornithine dihydrochloride, Tocris, 0546); vinyl-L-VNIO, a NOS1 inhibitor (10 μM, N5-(1-imino-3-butenyl)-L-ornithine, L-VNIO, Enzo Life Sciences, ALX-270-216-M005); 1400W, a NOS2 inhibitor (10 μM, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride, Tocris, 1415); and L-NMMA, a nonselective NOS inhibitor (30 μM, NG-monomethyl-L-arginine acetate, Tocris, 0771) were evaluated. A chamber wire myograph was connected to a digital data recorder (MacLab/4e; AD Instruments), and recordings were analyzed using LabChart v7 software (AD Instruments).


### 2.4. Electronic Paramagnetic Resonance (EPR)

EPR measurements were performed on aortas harvested from rats at 15, 30, and 45 weeks of age, as previously described [14]. Briefly, aortas were incubated 45 min at 37°C in a Krebs-HEPES colloid solution containing Na-diethyldithiocarbamate trihydrate (DETC—Sigma-Aldrich) mixed to FeSO4-7H2O to form Fe2+-(DETC)2 as spin trap for •NO detection in an electromagnetic field. Five conditions were tested: without inhibitors, with L-NMMA (10 μM, negative control), with L-NIO (10 μM) and L-VNIO (10 μM), with L-NIO (10 μM) and 1400W (10 μM), and with L-VNIO (10 μM) and 1400W (10 μM). For O2•- detection, aortas were incubated 45 min at 37°C in a Krebs-HEPES solution containing 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidin (CMH, 500 μM, Noxygen) as spin probe, deferoxamine (25 μM, Sigma-Aldrich) as an iron chelator, and DETC (5 μM) to minimize CMH autooxidation, with or without L-NMMA (10 μM). Then, each sample was snap-frozen in liquid nitrogen and analyzed in a Dewar flask at 77°K using an EPR Miniscope MS5000 (Freiberg Instruments, Germany). The instrument settings were as follows: microwave power of 10 mW; 1 mT or 0.400 mT of amplitude modulation for •NO and O2•-, respectively; 100 kHz modulation frequency; sweep time of 150 s; and 3 scans for •NO measurements or 60 s and 3 scans for O2•- spectra. Signals were quantified by measuring and analyzing the total amplitude of the peaks of the spectra obtained, using the ESRS-Studio software (Freiberg Instruments, Germany) and expressed in arbitrary units (A.U.) and normalized to dry weight of the sample.


### 2.5. Western Blot

Total proteins were extracted from aorta powder as previously described [15]. The amount of protein used for Western blot analysis was 25 μg. Western blots were performed with a migration in 4-15% polyacrylamide stain-free gels in order to evaluate the expression of the NOS1 (#4231S, Cell Signaling, Danvers, USA), NOS2 (AB5382, Millipore, USA), NOS3 (610296, BD Biosciences, San Diego, USA), and phosphorylated Ser1177 NOS3 (p-NOS3; #9571, Cell Signaling, Danvers, USA) (Table 1). Total NOS1, NOS2, and NOS3 expression was expressed as a ratio with stain-free quantification of total proteins. Phosphorylated Ser1177 NOS3 and NOS3 dimers were expressed as a ratio with a NOS3 monomer.Table 1
Antibodies used for Western blotting experiments.
Protein of interestPrimary antibodySecondary antibodyReferenceSpeciesDilutionReferenceSpeciesDilutionNOS1Cell Signaling (4231)Rabbit1/500∗Cell Signaling (7074)Goat1/10000∗NOS2Millipore (AB5382)Rabbit1/2000∗NOS3BD Biosciences (610296)Mouse1/1000∗Cell Signaling (7076)Horsep-NOS3Cell Signaling (9571)Rabbit1/1000∗Cell Signaling (7074)Goat∗Dilution carried out in 5% milk.


### 2.6. Statistical Analysis

Data were presented asmean±SEM of n different rats. For the comparisons involving two groups, animal’s significances were defined using the Mann-Whitney test. For vascular studies, a two-way ANOVA for repeated measures was used with the Bonferroni posttest. For electronic paramagnetic resonance studies, the Kruskal-Wallis test was used followed by an uncorrected Dunn’s test. A value of p<0.05 was considered significant. All statistical calculations and graphs were performed using GraphPad Prism software (version 8.00).


## 2.1. Experimental Animals

All animal experimental protocols were approved by the Pays de la Loire Ethical Committee and were performed in accordance with the French law on animal welfare, EU Directive 2010/63/EU for animal experiments, the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 2011), and the 1964 Declaration of Helsinki and its later amendments, and all the animals were housed according to standard living conditions. All animals used in the study are male Sprague-Dawley rats. Tgβ3 rats which overexpress human β3-AR in endothelial cells and their control (WT) were used at 15, 30, and 45 weeks of age and fed ad libitum with a soy-free diet (Envigo, #2914C, Huntingdon, United Kingdom) as previously described [10]. Then, animals were euthanized, and the thoracic aorta and superior branch of mesenteric arteries were harvested in order to perform vascular reactivity (thoracic aorta and mesenteric arteries), electronic paramagnetic resonance (EPR) quantification of •NO and O2•- (thoracic aorta), and Western blot protein analysis (thoracic aorta) as described below.


## 2.2. Echocardiography

Echocardiography was performed on anesthetized rats (O2/isoflurane mixture 1.5% with flow rate of 1 L/min) at 15, 30, and 45 weeks of age, using a Vingmed-General Electric ultrasound system (VIVID 7, Horten, Norway) equipped with a 10 MHz imaging probe and offline cine loop analysis software (Echopac TVI, GE-Vingmed Ultrasound) on the Therassay platform of Nantes, as previously described [10].


## 2.3. Vascular Reactivity for Pharmacological Studies

Rats were anesthetized with an O2/isoflurane mixture (induction: 5% isoflurane, flow rate 1 L/min; maintenance: 2% isoflurane, flow rate 0.5 L/min). The thoracic aorta and mesenteric arteries were carefully excised and cleared of fat and connective tissue. Vascular tensions were recorded as previously described [11, 12]. Briefly, aortic and mesenteric rings were mounted on a multichannel isometric myograph (Danish Myo Technology) (95% O2 and 5% CO2) Krebs-Henseleit bicarbonate solution (millimolar concentrations: NaCl 118, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, KCl 4.5, glucose 11, and CaCl2 1.5) at 37°C [13]. To verify the endothelium integrity, rings were precontracted with phenylephrine (1 μM, Sigma-Aldrich, P6126) and then exposed to a single concentration of acetylcholine (10 μM, Sigma-Aldrich, A6625). The tension was recorded on precontracted rings with phenylephrine (1 μM). The vasodilation response to cumulative concentrations of isoproterenol, a nonselective β-AR agonist (1 nM to 100 μM, Sigma-Aldrich, I6504), and of CL 316 243, a β3-AR agonist (1 nM to 100 μM, Tocris, 1499), was evaluated. The vasodilation response to isoproterenol (1 nM to 100 μM) was repeated after 30 min of vessel incubation with NOS inhibitors prior to the phenylephrine precontraction: L-NIO, a NOS3 inhibitor (10 μM, N5-(1-iminoethyl)-L-ornithine dihydrochloride, Tocris, 0546); vinyl-L-VNIO, a NOS1 inhibitor (10 μM, N5-(1-imino-3-butenyl)-L-ornithine, L-VNIO, Enzo Life Sciences, ALX-270-216-M005); 1400W, a NOS2 inhibitor (10 μM, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride, Tocris, 1415); and L-NMMA, a nonselective NOS inhibitor (30 μM, NG-monomethyl-L-arginine acetate, Tocris, 0771) were evaluated. A chamber wire myograph was connected to a digital data recorder (MacLab/4e; AD Instruments), and recordings were analyzed using LabChart v7 software (AD Instruments).


## 2.4. Electronic Paramagnetic Resonance (EPR)

EPR measurements were performed on aortas harvested from rats at 15, 30, and 45 weeks of age, as previously described [14]. Briefly, aortas were incubated 45 min at 37°C in a Krebs-HEPES colloid solution containing Na-diethyldithiocarbamate trihydrate (DETC—Sigma-Aldrich) mixed to FeSO4-7H2O to form Fe2+-(DETC)2 as spin trap for •NO detection in an electromagnetic field. Five conditions were tested: without inhibitors, with L-NMMA (10 μM, negative control), with L-NIO (10 μM) and L-VNIO (10 μM), with L-NIO (10 μM) and 1400W (10 μM), and with L-VNIO (10 μM) and 1400W (10 μM). For O2•- detection, aortas were incubated 45 min at 37°C in a Krebs-HEPES solution containing 1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidin (CMH, 500 μM, Noxygen) as spin probe, deferoxamine (25 μM, Sigma-Aldrich) as an iron chelator, and DETC (5 μM) to minimize CMH autooxidation, with or without L-NMMA (10 μM). Then, each sample was snap-frozen in liquid nitrogen and analyzed in a Dewar flask at 77°K using an EPR Miniscope MS5000 (Freiberg Instruments, Germany). The instrument settings were as follows: microwave power of 10 mW; 1 mT or 0.400 mT of amplitude modulation for •NO and O2•-, respectively; 100 kHz modulation frequency; sweep time of 150 s; and 3 scans for •NO measurements or 60 s and 3 scans for O2•- spectra. Signals were quantified by measuring and analyzing the total amplitude of the peaks of the spectra obtained, using the ESRS-Studio software (Freiberg Instruments, Germany) and expressed in arbitrary units (A.U.) and normalized to dry weight of the sample.


## 2.5. Western Blot

Total proteins were extracted from aorta powder as previously described [15]. The amount of protein used for Western blot analysis was 25 μg. Western blots were performed with a migration in 4-15% polyacrylamide stain-free gels in order to evaluate the expression of the NOS1 (#4231S, Cell Signaling, Danvers, USA), NOS2 (AB5382, Millipore, USA), NOS3 (610296, BD Biosciences, San Diego, USA), and phosphorylated Ser1177 NOS3 (p-NOS3; #9571, Cell Signaling, Danvers, USA) (Table 1). Total NOS1, NOS2, and NOS3 expression was expressed as a ratio with stain-free quantification of total proteins. Phosphorylated Ser1177 NOS3 and NOS3 dimers were expressed as a ratio with a NOS3 monomer.Table 1
Antibodies used for Western blotting experiments.
Protein of interestPrimary antibodySecondary antibodyReferenceSpeciesDilutionReferenceSpeciesDilutionNOS1Cell Signaling (4231)Rabbit1/500∗Cell Signaling (7074)Goat1/10000∗NOS2Millipore (AB5382)Rabbit1/2000∗NOS3BD Biosciences (610296)Mouse1/1000∗Cell Signaling (7076)Horsep-NOS3Cell Signaling (9571)Rabbit1/1000∗Cell Signaling (7074)Goat∗Dilution carried out in 5% milk.


## 2.6. Statistical Analysis

Data were presented asmean±SEM of n different rats. For the comparisons involving two groups, animal’s significances were defined using the Mann-Whitney test. For vascular studies, a two-way ANOVA for repeated measures was used with the Bonferroni posttest. For electronic paramagnetic resonance studies, the Kruskal-Wallis test was used followed by an uncorrected Dunn’s test. A value of p<0.05 was considered significant. All statistical calculations and graphs were performed using GraphPad Prism software (version 8.00).


## 3. Results

### 3.1.β3-AR Overexpression in the Long Term Induces Diastolic Dysfunction

HFpEF is characterized by a diastolic dysfunction without major alteration of ejection fraction. In order to validate that our rat develops a diastolic dysfunction as described in Dhot et al. [10], we performed echocardiographic analyses. As shown in Figure 1, heart rate and ejection fraction were similar in both WT and Tgβ3 rats at the same age (Figure 1(a)). The E wave was not changed between WT and Tgβ3 rats (Figure 1(b)). The A wave was decreased in the Tgβ3 group compared to the WT group at 30 and 45 weeks of age (30 weeks of age: WT: 1.02±0.03; Tgβ3: 0.79±0.05; 45 weeks of age: WT: 1.02±0.03; Tgβ3: 0.86±0.04; p<0.05) leading to a significant increase in the E/A ratio in the Tgβ3 group (30 weeks of age: WT: 1.09±0.04; Tgβ3: 1.26±0.06; 45 weeks of age: WT: 1.15±0.01; Tgβ3: 1.33±0.04; p<0.05) (Figure 1(b)). These data validated that our model develops a diastolic dysfunction throughout ageing.Figure 1
Evaluation of cardiac function in WT and Tgβ3 rats at 15, 30, and 45 weeks of age. Evolution of systolic function (a) and diastolic function (b) in WT and Tgβ3 rats at 15, 30, and 45 weeks of age. Values represent mean±SEM. n=3-13. ∗p<0.05.
(a)(b)


### 3.2.β3-AR Overexpression in the Long Term Induces Vasodilation Alteration

Concentration-dependent vasodilation to isoproterenol, a nonselectiveβ-AR agonist, was significantly reduced in aortic rings from Tgβ3 rats at the age of 15 weeks (Emax; WT: 94.5±2.5; Tgβ3: 74.1±7.5; p<0.05), 30 weeks (pD2; WT: 6.73±0.30; Tgβ3: 5.49±0.11; p<0.05), and 45 weeks (pD2; WT: 6.56±0.21; Tgβ3: 5.82±0.15; p<0.05) (Figure 2(a) and Table 2(a)). In mesenteric rings, concentration-dependent vasodilation to isoproterenol was significantly reduced in Tgβ3 rats at 30 weeks of age (Emax; WT: 100.7±4.6; Tgβ3: 73.5±10.4; p<0.05) and at 45 weeks of age (pD2; WT: 7.21±0.15; Tgβ3: 6.17±0.17; p<0.05) (Figure 2(b) and Table 2(b)). β3-AR overexpression seems to alter vasodilation of aortas and mesenteric arteries through the β-AR signaling pathway over ageing.Figure 2
Vasodilation in WT and Tgβ3 rats at 15, 30, and 45 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings (a) and mesenteric artery rings (b). Concentration-relaxation curves to CL 316 243, a β3-AR agonist, on thoracic aortic rings (c) and mesenteric artery rings (d). Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, in the presence of L-NMMA, a nonselective NOS inhibitor, on thoracic aortic rings (e) and mesenteric arteries rings (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=2-12. §p<0.05: WT—15 weeks versus Tgβ3—15 weeks; ∗p<0.05: WT—30 weeks versus Tgβ3—30 weeks; $p<0.05: WT—45 weeks versus Tgβ3—45 weeks.
(a)(b)(c)(d)(e)(f)Table 2
Efficiencies and potencies of isoproterenol with or without the NOS inhibitor and of CL 316 243 on thoracic aortic arteries (a) and mesenteric arteries (b) from WT and Tgβ3 rats at 15, 30, and 45 weeks old.
(a)GroupThoracic aortic arteries—WTThoracic aortic arteries—Tgβ3Statistics WT vs. Tgβ3npD2EmaxnpD2Emaxp pD2p EmaxIsoproterenol15 w76.53±0.3394.5±2.555.93±0.2074.1±7.50.2390.01530 w46.73±0.3085.6±14.375.49±0.1179.0±9.50.0020.24545 w116.56±0.2185.9±2.2105.82±0.1569.2±3.50.0230.016Isoproterenol+L-NMMA15 w35.18±0.8015.0±5.5226.79±2.0725.7±9.430.4080.67930 w25.62±0.1119.9±5.4035.79±0.3463.4±18.50.9810.08045 w95.46±0.2451.9±7.2785.62±0.2256.0±6.550.6970.760Isoproterenol+L-NIO15 w76.06±0.4560.0±12.145.87±0.4352.6±7.800.7560.49130 w35.25±0.1457.0±22.865.27±0.2742.1±14.30.5370.64745 w84.93±0.1133.0±4.4285.30±0.1845.9±5.900.2120.278Isoproterenol+L-VNIO15 w46.79±0.63861.9±16.055.63±0.51247.9±21.60.1180.44930 w45.65±0.6335.25±11.946.02±0.5918.6±8.60.5720.53045 w54.97±0.1433.6±6.1555.54±0.1542.3±2.840.1860.702Isoproterenol+1400W15 w46.51±0.70083.2±7.2346.32±0.65665.7±8.770.9050.15130 w46.09±0.4145.58±13.775.26±0.3263.1±12.60.2130.29645 w75.43±0.4658.1±9.4485.35±0.1353.0±7.520.8960.589CL 316 24315 w36.16±0.6717.1±6.946.75±0.7422.8±2.50.1970.53330 w27.22±0.0032.0±0.036.87±0.4526.3±5.00.7850.44245 w46.94±0.2043.7±1.997.06±0.0930.9±3.80.6460.044(b)GroupMesenteric arteries—WTMesenteric arteries—Tgβ3Statistics WT vs. Tgβ3npD2EmaxnpD2Emaxp pD2p EmaxIsoproterenol15 w47.78±0.1796.4±3.087.15±0.3278.0±8.50.1120.15130 w87.03±0.20100.7±4.686.67±0.2873.5±10.40.3380.02245 w127.21±0.1585.7±3.4126.17±0.1768.2±5.50.0010.061Isoproterenol+L-NMMA15 w46.67±0.4983.8±14.266.82±0.5387.5±8.250.8770.92030 w76.37±0.5092.1±4.6065.72±0.1586.1±7.930.3920.81545 w96.33±0.475.07±4.5386.38±0.47−4.03±2.600.9180.533Isoproterenol+L-NIO15 w36.04±0.7886.1±1.2896.89±0.3075.4±8.080.3000.64630 w55.91±0.3176.9±4.7046.66±0.3886.8±5.660.1950.34445 w95.83±0.2935.1±5.9195.65±0.3664.0±7.980.8180.033Isoproterenol+L-VNIO15 w67.08±0.5284.1±8.6466.24±0.4575.8±9.940.2210.74430 w36.29±0.2771.5±4.5966.78±0.5475.1±8.330.4910.56445 w86.41±0.3049.6±8.6386.82±0.4322.79±7.380.3920.171Isoproterenol+1400W15 w47.34±0.2596.4±2.0276.49±0.3181.8±8.000.0820.51530 w86.64±0.3084.0±10.966.43±0.4492.3±3.230.7590.78945 w86.32±0.4046.52±10.886.27±0.2952.7±13.80.7810.325CL 316 24315 w55.98±0.6148.4±19.565.90±0.1475.7±50.6540.85630 w36.53±0.6475.2±16.846.52±0.6593.2±14.50.9680.56845 w37.96±0.3381.1±7.477.68±0.4284.5±5.80.7310.737CL 316 243, aβ3-AR agonist, produced a concentration-dependent vasodilation in both WT and Tgβ3 vessels. This vasodilation was similar between the two groups of rats at all evaluated ages in both aortic and mesenteric arteries (Figures 2(c) and 2(d) and Tables 2(a) and 2(b)). Interestingly, the difference in vasodilation in response to isoproterenol between the WT and Tgβ3 groups did not seem to be associated with the β3-AR despite its overexpression in Tgβ3 rats.To evaluate the potential involvement of the•NO production through NO synthase (NOS) activity on the observed vascular dysfunction in Tgβ3 rats, vasodilation induced by isoproterenol has been evaluated in the presence of L-NMMA, a nonselective NOS inhibitor. In these conditions, the vasodilation was similar between WT and Tgβ3 at 15, 30, and 45 weeks of age on both thoracic aortic rings (Figure 2(e) and Table 2(a)) and mesenteric artery rings (Figure 2(f) and Table 2(b)). These results indicate that the altered vasodilation observed in Tgβ3 rats at 30 and 45 weeks seems to be due to NOS activity and •NO production. In particular, L-NMMA blunted the vascular relaxation in mesenteric arteries in both WT and Tgβ3 rats at 45 weeks, suggesting a central role of •NO in relaxation in resistance arteries at this age. Subsequently, we were interested in the involvement of different NOS isoforms in the vasodilation in order to highlight the link between β3-AR overexpression and •NO signaling.


### 3.3. At 15 Weeks of Age, Endothelial Function Is Not Altered by Endothelialβ3-AR Overexpression

As mentioned before, in our Tgβ3 model, the diastolic dysfunction appeared at 30 weeks [10]. We investigated whether rats developed endothelial dysfunction over the time through the NOS expression and activity evaluation.


#### 3.3.1. The Vasodilation Is Predominantly Mediated by NOS1 in Tgβ3 Rats at 15 Weeks

At 15 weeks of age, vasodilation in response to isoproterenol was only altered in aortic rings from WT and Tgβ3 rats with a decrease in maximal effect in the aorta of the Tgβ3 group and a significant reduced response to isoproterenol stimulation (Figure 2(a) and Table 2(a)). To decipher the implication of each NOS isoform, several NOS inhibitors: L-NIO, L-VNIO, and 1400W which inhibit NOS3, NOS1, and NOS2, respectively, have been used.In the presence of L-NIO, the maximal effect was reduced in the aortic rings from WT rats (WT:60.0±12.1; WT+L-NIO: 52.6±7.80; p<0.05) (Figure 3(a) and Table 3(a)). NOS3 inhibition using L-NIO had no significant impact on aorta and mesenteric artery reactivity from Tgβ3 rats, suggesting reduced implication of •NO production from NOS3 in vascular relaxation of 15-week-old Tgβ3 rats compared to WT (Figures 3(a) and 3(b) and Tables 3(a) and 3(b)). In contrast, concentration-dependent vasodilation to isoproterenol in the presence of L-VNIO, a NOS1 inhibitor, was significantly decreased in the aortic rings of Tgβ3 rats (Figure 3(c)), while the difference was not significant on mesenteric arteries (Figure 3(d)). In the presence of 1400W, a NOS2 inhibitor, the vasodilation in response to isoproterenol was not modified on both aortic and mesenteric rings of the two groups of rats (Figures 3(e) and 3(f) and Tables 3(a) and 3(b)). Taken together, these results suggest that vasodilation was predominantly mediated by •NO produced by NOS1 in the aorta from Tgβ3 rats at 15 weeks of age.Figure 3
Evaluation of function of NOS in WT and Tgβ3 rats at 15 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=3-9. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)Table 3
Statistical comparison of the efficiencies and potencies of isoproterenol with or without the NOS inhibitor and of CL 316 243 on thoracic aortic arteries (a) and mesenteric arteries (b) from WT and Tgβ3 rats at 15, 30, and 45 weeks old.
(a)GroupThoracic aortic arteries—WTThoracic aortic arteries—Tgβ3WT vs. WT+isoproterenolTgβ3 vs. Tgβ3+isoproterenolnpD2EmaxnpD2Emaxp pD2p Emaxp pD2p EmaxIsoproterenol15 w76.53±0.3394.5±2.555.93±0.2074.1±7.530 w46.73±0.3085.6±14.375.49±0.1179.0±9.545 w116.56±0.2185.9±2.2105.82±0.1569.2±3.5Isoproterenol+L-NIO15 w76.06±0.4560.0±12.145.87±0.4352.6±7.800.4300.0130.9390.32230 w35.25±0.1457.0±22.865.27±0.2742.1±14.30.0060.0830.6610.08045 w84.93±0.1133.0±4.4285.30±0.1845.9±5.90<0.0001<0.00010.1340.088Isoproterenol+L-VNIO15 w46.79±0.63861.9±16.055.63±0.51247.9±21.60.6200.1250.7990.41430 w45.65±0.6335.25±11.946.02±0.5918.6±8.60.0900.0240.3460.01945 w54.97±0.1433.6±6.1555.54±0.1542.3±2.84<0.0001<0.00010.3770.068Isoproterenol+1400W15 w46.51±0.70083.2±7.2346.32±0.65665.7±8.770.6650.2590.5880.62330 w46.09±0.4145.58±13.775.26±0.3263.1±12.60.1910.0190.9020.32345 w75.43±0.4658.1±9.4485.35±0.1353.0±7.520.0020.0020.1050.265(b)GroupMesenteric arteries—WTMesenteric arteries—Tgβ3WT vs. WT+isoproterenolTgβ3 vs. Tgβ3+isoproterenolnpD2EmaxnpD2Emaxp pD2p Emaxp pD2p EmaxIsoproterenol15 w47.78±0.1796.4±3.087.15±0.3278.0±8.530 w87.03±0.20100.7±4.686.67±0.2873.5±10.445 w127.21±0.1585.7±3.4126.17±0.1768.2±5.5Isoproterenol+L-NIO15 w36.04±0.7886.1±1.2896.89±0.3075.4±8.080.0240.4310.5550.77630 w55.91±0.3176.9±4.7046.66±0.3886.8±5.660.0140.0030.9780.62745 w95.83±0.2935.1±5.9195.65±0.3664.0±7.980.0004<0.00010.4080.640Isoproterenol+L-VNIO15 w67.08±0.5284.1±8.6466.24±0.4575.8±9.940.2900.3610.1270.84430w36.29±0.2771.5±4.5966.78±0.5475.1±8.330.1950.0130.8500.77745 w86.41±0.3049.6±8.6386.82±0.4322.79±7.380.0320.0030.0790.004Isoproterenol+1400W15 w47.34±0.2596.4±2.0276.49±0.3181.8±8.000.4040.9800.1250.78030 w86.64±0.3084.0±10.966.43±0.4492.3±3.230.3780.4180.6800.30745 w86.32±0.4046.52±10.886.27±0.2952.7±13.80.0130.0020.6790.773


#### 3.3.2. NOS2 Expression Is Increased in Tgβ3 Rats at 15 Weeks

The aortic ratio of NOS3 dimer/monomer and p-NOS3/NOS3 monomer protein expression levels and NOS1 expression was not significantly modified at 15 weeks of age (Figures4(a), 4(b), and 4(d)). The overexpression of β3-AR induced a significant 2-fold increase in NOS2 protein expression in the aorta (WT: 1.00±0.14; Tgβ3: 2.06±0.31; p<0.05) (Figure 4(c)).Figure 4
Expression and activity production of NOS in WT and Tgβ3 rats at 15 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, 1400W, and L-VNIO) (e) and O2•- production with or without L-NMMA from WT and Tgβ3 rats (f). Data are expressed as mean±SEM. n=5-14. ∗∗p<0.01, ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


#### 3.3.3.•NO and O2•- Production Is Unchanged in the Tgβ3 Rats at 15 Weeks

•NO production remained stable on thoracic aortas between WT and transgenic rats. With L-NMMA, the •NO production was almost abolished as expected, confirming that NOS-dependent •NO production (negative control). The NOS2-dependent •NO production was not significantly increased in the Tgβ3 rats (+51%; p=0.09). The NOS1- and NOS3-dependent •NO production was not modified between the two groups (Figure 4(e)). The generation of O2•- has been also evaluated under baseline conditions and in the presence of L-NMMA, and no changes on O2•- production with or without L-NMMA were reported between the two groups of rats at this age (Figure 4(f)).


### 3.4.β3-AR Overexpression Induced Endothelial Dysfunction at 30 Weeks

We investigated whether endothelial dysfunction is worsened at 30 weeks of age in Tgβ3 rats.


#### 3.4.1. NOS1 Modulates the Vasodilatation in the Tgβ3 Group

At 30 weeks of age, vasodilation in response to isoproterenol was reduced in aortic rings of Tgβ3 rats associated with a decreased pD2 (WT: 6.73±0.30; Tgβ3: 5.49±0.11; p<0.05) (Figure 2(a) and Table 2(a)).In the presence of L-NIO, the pD2 was reduced in the aortic rings (WT: 6.73±0.30; WT+L-NIO: 5.25±0.14; p<0.05) and mesenteric arteries (WT: 7.03±0.20; WT+L-NIO: 5.91±0.31; p<0.05) from WT rats (Figures 5(a) and 5(b) and Tables 3(a) and 3(b)) suggesting the implication of •NO from NOS3.Figure 5
Evaluation of function of NOS in WT and Tgβ3 rats at 30 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=3-8. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)Vasodilation in response to isoproterenol in the presence of the NOS1 inhibitor (L-VNIO) was blunted with a reduction in maximal effect in the aortic rings from both WT rats (WT:85.6±14.3; WT+L-NIO: 35.25±11.19; p<0.05) and Tgβ3 rats (Tgβ3: 79±9.5; Tgβ3+L-NIO: 18.6±8.6; p<0.05) and a decrease in the maximal effect in the mesenteric arteries from WT rats (WT: 100.7±4.6; WT+L-NIO: 71.5±4.59; p<0.05) (Figures 5(c) and 5(d) and Tables 3(a) and 3(b)). These data suggest an implication of •NO production from NOS1.In the presence of 1400W, the vasodilation in response to isoproterenol was modified on the aorta from WT rats with reduced maximal effect (WT:85.6±14.3; WT+1400W: 45.58±13.7; p<0.05) (Figure 5(e) and Table 3(a)) with no impact on mesenteric arteries (Figure 5(f) and Table 3(b)). At 30 weeks of age, the vasodilation induced by isoproterenol seems to involve all NOS isoforms in WT rats while only NOS1 seems to be involved in the vasodilation of vessels from Tgβ3 rats.


#### 3.4.2.β3-AR Overexpression Is Associated with an Increase in NOS1 Expression

The aortic ratio of the NOS3 dimer/monomer and p-NOS3/NOS3 monomer and NOS2 protein expression levels were not significantly modified between the two groups at 30 weeks (Figures6(a), 6(b), and 6(c)). The β3-AR overexpression induces a significant 6-fold increase in NOS1 protein expression in the aorta (Figure 6(d)) which can explain the decrease in vasodilation in vessels from Tgβ3 with the NOS1 inhibitor.Figure 6
Expression and activity production of NOS in WT and Tgβ3 rats at 30 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, L-VNIO, and 1400W) (e) and O2•- production with or without L-NMMA (f) from WT and Tgβ3 rats. Data are expressed as mean±SEM. n=5-16. ∗p<0.05, ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


#### 3.4.3. Oxidative Stress Is Increased in Tgβ3 Rats at 30 Weeks

At 30 weeks of age,•NO production tends to increase in Tgβ3 rats (+28%). In the presence of L-NMMA, the production of •NO is significantly reduced in WT and Tgβ3 rats. NOS2-dependent •NO production is increased in the Tgβ3 group (+70% vs. WT; p<0.05) (Figure 6(e)).The O2•- production was significantly increased in the Tgβ3 group under baseline conditions (+75% vs. WT; p<0.05) while no change is shown with L-NMMA (Figure 6(f)).The results demonstrated that despite a similar expression of NOS2, NOS2-dependent•NO production is increased in the Tgβ3 group, suggesting that its activity is enhanced in Tgβ3 rats. Interestingly, NOS1 expression which is highly increased in Tgβ3 rats compared to WT does not impact •NO production.


### 3.5.β3-AR Overexpression Maintained the Endothelial Dysfunction at 45 Weeks of Age

At 30 weeks, results indicated an endothelial dysfunction associated with an alteration of the vasodilation. An increase in NOS1 expression could led to endothelial dysfunction and is associated with an increase in O2•- production. Thus, we investigated, at 45 weeks of age, whether endothelial dysfunction was more severe compared to 30 weeks of age.


#### 3.5.1. NOS1 and NOS3 Modulate the Vasodilation in the Tgβ3 Group

At 45 weeks of age, vasodilation in response to isoproterenol was significantly reduced in Tgβ3 rats compared to WT in both aortic and mesenteric arteries (Figures 2(a) and 2(b) and Tables 2(a) and 2(b)). In the presence of L-NIO, 1400W, or L-VNIO, vasodilation was significantly reduced on aortic and mesenteric artery rings from WT rats (Figures 7(a)–7(f) and Tables 3(a) and 3(b)). In Tgβ3 rats, vasodilation was significantly reduced on aortic rings in the presence of L-NIO, while L-VNIO reduced the vasodilation in both aortic and mesenteric rings from Tgβ3 rats (Figures 7(a), 7(c), and 7(d)). Taken together, these results demonstrated that the vasodilation in Tgβ3 rats was altered compared to that in WT rats and seems to depend on NOS1 and NOS3 isoforms.Figure 7
Evaluation of function of NOS in WT and Tgβ3 rats at 45 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=5-12. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)


#### 3.5.2. NOS3 Is Uncoupled in Tgβ3 Rats

In Tgβ3 rats, the aortic NOS3 dimer/NOS3 monomer protein expression ratio and the aortic p-NOS3/NOS3 monomer protein expression ratio were significantly reduced (-87% and -63%, respectively; p<0.05) indicating a potential NOS3 uncoupling (Figures 8(a) and 8(b)). No modification was reported for NOS2 protein expression (Figure 8(c)). The aortic NOS1 protein expression was significantly increased at 45 weeks of age on Tgβ3 rats (+57%; p<0.05) (Figure 8(d)).Figure 8
Expression and activity production of NOS in WT and Tgβ3 rats at 45 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, L-VNIO, and 1400W) (e) and O2•- production with or without L-NMMA (f) from WT and Tgβ3 rats. Data are expressed as mean±SEM. n=5-10. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


#### 3.5.3. O2•- Production Is Increased in Tgβ3 Rats

The production of•NO tends to increase in the aorta in Tgβ3 rats under baseline conditions (+84% vs. WT) (Figure 8(e)). Addition of L-NMMA reduces •NO production in the two groups, indicating that the •NO production from Tgβ3 and WT rats is mainly due to the NOS. The NOS1-dependent •NO production tends to increase in the Tgβ3 rats (+90% vs. WT; p=0.052) (Figure 8(e)). The production of O2•- was significantly increased in Tgβ3 rats (+76% vs. WT; p<0.05). L-NMMA treatment normalized the production with the absence of significant difference between the two groups, suggesting the involvement of NOS in O2•- production from Tgβ3 rats (Figure 8(f)). Results obtained at 45 weeks of age confirm the previous results obtained at 30 weeks of age that Tgβ3 rats present an endothelial dysfunction.


## 3.1.β3-AR Overexpression in the Long Term Induces Diastolic Dysfunction

HFpEF is characterized by a diastolic dysfunction without major alteration of ejection fraction. In order to validate that our rat develops a diastolic dysfunction as described in Dhot et al. [10], we performed echocardiographic analyses. As shown in Figure 1, heart rate and ejection fraction were similar in both WT and Tgβ3 rats at the same age (Figure 1(a)). The E wave was not changed between WT and Tgβ3 rats (Figure 1(b)). The A wave was decreased in the Tgβ3 group compared to the WT group at 30 and 45 weeks of age (30 weeks of age: WT: 1.02±0.03; Tgβ3: 0.79±0.05; 45 weeks of age: WT: 1.02±0.03; Tgβ3: 0.86±0.04; p<0.05) leading to a significant increase in the E/A ratio in the Tgβ3 group (30 weeks of age: WT: 1.09±0.04; Tgβ3: 1.26±0.06; 45 weeks of age: WT: 1.15±0.01; Tgβ3: 1.33±0.04; p<0.05) (Figure 1(b)). These data validated that our model develops a diastolic dysfunction throughout ageing.Figure 1
Evaluation of cardiac function in WT and Tgβ3 rats at 15, 30, and 45 weeks of age. Evolution of systolic function (a) and diastolic function (b) in WT and Tgβ3 rats at 15, 30, and 45 weeks of age. Values represent mean±SEM. n=3-13. ∗p<0.05.
(a)(b)


## 3.2.β3-AR Overexpression in the Long Term Induces Vasodilation Alteration

Concentration-dependent vasodilation to isoproterenol, a nonselectiveβ-AR agonist, was significantly reduced in aortic rings from Tgβ3 rats at the age of 15 weeks (Emax; WT: 94.5±2.5; Tgβ3: 74.1±7.5; p<0.05), 30 weeks (pD2; WT: 6.73±0.30; Tgβ3: 5.49±0.11; p<0.05), and 45 weeks (pD2; WT: 6.56±0.21; Tgβ3: 5.82±0.15; p<0.05) (Figure 2(a) and Table 2(a)). In mesenteric rings, concentration-dependent vasodilation to isoproterenol was significantly reduced in Tgβ3 rats at 30 weeks of age (Emax; WT: 100.7±4.6; Tgβ3: 73.5±10.4; p<0.05) and at 45 weeks of age (pD2; WT: 7.21±0.15; Tgβ3: 6.17±0.17; p<0.05) (Figure 2(b) and Table 2(b)). β3-AR overexpression seems to alter vasodilation of aortas and mesenteric arteries through the β-AR signaling pathway over ageing.Figure 2
Vasodilation in WT and Tgβ3 rats at 15, 30, and 45 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings (a) and mesenteric artery rings (b). Concentration-relaxation curves to CL 316 243, a β3-AR agonist, on thoracic aortic rings (c) and mesenteric artery rings (d). Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, in the presence of L-NMMA, a nonselective NOS inhibitor, on thoracic aortic rings (e) and mesenteric arteries rings (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=2-12. §p<0.05: WT—15 weeks versus Tgβ3—15 weeks; ∗p<0.05: WT—30 weeks versus Tgβ3—30 weeks; $p<0.05: WT—45 weeks versus Tgβ3—45 weeks.
(a)(b)(c)(d)(e)(f)Table 2
Efficiencies and potencies of isoproterenol with or without the NOS inhibitor and of CL 316 243 on thoracic aortic arteries (a) and mesenteric arteries (b) from WT and Tgβ3 rats at 15, 30, and 45 weeks old.
(a)GroupThoracic aortic arteries—WTThoracic aortic arteries—Tgβ3Statistics WT vs. Tgβ3npD2EmaxnpD2Emaxp pD2p EmaxIsoproterenol15 w76.53±0.3394.5±2.555.93±0.2074.1±7.50.2390.01530 w46.73±0.3085.6±14.375.49±0.1179.0±9.50.0020.24545 w116.56±0.2185.9±2.2105.82±0.1569.2±3.50.0230.016Isoproterenol+L-NMMA15 w35.18±0.8015.0±5.5226.79±2.0725.7±9.430.4080.67930 w25.62±0.1119.9±5.4035.79±0.3463.4±18.50.9810.08045 w95.46±0.2451.9±7.2785.62±0.2256.0±6.550.6970.760Isoproterenol+L-NIO15 w76.06±0.4560.0±12.145.87±0.4352.6±7.800.7560.49130 w35.25±0.1457.0±22.865.27±0.2742.1±14.30.5370.64745 w84.93±0.1133.0±4.4285.30±0.1845.9±5.900.2120.278Isoproterenol+L-VNIO15 w46.79±0.63861.9±16.055.63±0.51247.9±21.60.1180.44930 w45.65±0.6335.25±11.946.02±0.5918.6±8.60.5720.53045 w54.97±0.1433.6±6.1555.54±0.1542.3±2.840.1860.702Isoproterenol+1400W15 w46.51±0.70083.2±7.2346.32±0.65665.7±8.770.9050.15130 w46.09±0.4145.58±13.775.26±0.3263.1±12.60.2130.29645 w75.43±0.4658.1±9.4485.35±0.1353.0±7.520.8960.589CL 316 24315 w36.16±0.6717.1±6.946.75±0.7422.8±2.50.1970.53330 w27.22±0.0032.0±0.036.87±0.4526.3±5.00.7850.44245 w46.94±0.2043.7±1.997.06±0.0930.9±3.80.6460.044(b)GroupMesenteric arteries—WTMesenteric arteries—Tgβ3Statistics WT vs. Tgβ3npD2EmaxnpD2Emaxp pD2p EmaxIsoproterenol15 w47.78±0.1796.4±3.087.15±0.3278.0±8.50.1120.15130 w87.03±0.20100.7±4.686.67±0.2873.5±10.40.3380.02245 w127.21±0.1585.7±3.4126.17±0.1768.2±5.50.0010.061Isoproterenol+L-NMMA15 w46.67±0.4983.8±14.266.82±0.5387.5±8.250.8770.92030 w76.37±0.5092.1±4.6065.72±0.1586.1±7.930.3920.81545 w96.33±0.475.07±4.5386.38±0.47−4.03±2.600.9180.533Isoproterenol+L-NIO15 w36.04±0.7886.1±1.2896.89±0.3075.4±8.080.3000.64630 w55.91±0.3176.9±4.7046.66±0.3886.8±5.660.1950.34445 w95.83±0.2935.1±5.9195.65±0.3664.0±7.980.8180.033Isoproterenol+L-VNIO15 w67.08±0.5284.1±8.6466.24±0.4575.8±9.940.2210.74430 w36.29±0.2771.5±4.5966.78±0.5475.1±8.330.4910.56445 w86.41±0.3049.6±8.6386.82±0.4322.79±7.380.3920.171Isoproterenol+1400W15 w47.34±0.2596.4±2.0276.49±0.3181.8±8.000.0820.51530 w86.64±0.3084.0±10.966.43±0.4492.3±3.230.7590.78945 w86.32±0.4046.52±10.886.27±0.2952.7±13.80.7810.325CL 316 24315 w55.98±0.6148.4±19.565.90±0.1475.7±50.6540.85630 w36.53±0.6475.2±16.846.52±0.6593.2±14.50.9680.56845 w37.96±0.3381.1±7.477.68±0.4284.5±5.80.7310.737CL 316 243, aβ3-AR agonist, produced a concentration-dependent vasodilation in both WT and Tgβ3 vessels. This vasodilation was similar between the two groups of rats at all evaluated ages in both aortic and mesenteric arteries (Figures 2(c) and 2(d) and Tables 2(a) and 2(b)). Interestingly, the difference in vasodilation in response to isoproterenol between the WT and Tgβ3 groups did not seem to be associated with the β3-AR despite its overexpression in Tgβ3 rats.To evaluate the potential involvement of the•NO production through NO synthase (NOS) activity on the observed vascular dysfunction in Tgβ3 rats, vasodilation induced by isoproterenol has been evaluated in the presence of L-NMMA, a nonselective NOS inhibitor. In these conditions, the vasodilation was similar between WT and Tgβ3 at 15, 30, and 45 weeks of age on both thoracic aortic rings (Figure 2(e) and Table 2(a)) and mesenteric artery rings (Figure 2(f) and Table 2(b)). These results indicate that the altered vasodilation observed in Tgβ3 rats at 30 and 45 weeks seems to be due to NOS activity and •NO production. In particular, L-NMMA blunted the vascular relaxation in mesenteric arteries in both WT and Tgβ3 rats at 45 weeks, suggesting a central role of •NO in relaxation in resistance arteries at this age. Subsequently, we were interested in the involvement of different NOS isoforms in the vasodilation in order to highlight the link between β3-AR overexpression and •NO signaling.


## 3.3. At 15 Weeks of Age, Endothelial Function Is Not Altered by Endothelialβ3-AR Overexpression

As mentioned before, in our Tgβ3 model, the diastolic dysfunction appeared at 30 weeks [10]. We investigated whether rats developed endothelial dysfunction over the time through the NOS expression and activity evaluation.


### 3.3.1. The Vasodilation Is Predominantly Mediated by NOS1 in Tgβ3 Rats at 15 Weeks

At 15 weeks of age, vasodilation in response to isoproterenol was only altered in aortic rings from WT and Tgβ3 rats with a decrease in maximal effect in the aorta of the Tgβ3 group and a significant reduced response to isoproterenol stimulation (Figure 2(a) and Table 2(a)). To decipher the implication of each NOS isoform, several NOS inhibitors: L-NIO, L-VNIO, and 1400W which inhibit NOS3, NOS1, and NOS2, respectively, have been used.In the presence of L-NIO, the maximal effect was reduced in the aortic rings from WT rats (WT:60.0±12.1; WT+L-NIO: 52.6±7.80; p<0.05) (Figure 3(a) and Table 3(a)). NOS3 inhibition using L-NIO had no significant impact on aorta and mesenteric artery reactivity from Tgβ3 rats, suggesting reduced implication of •NO production from NOS3 in vascular relaxation of 15-week-old Tgβ3 rats compared to WT (Figures 3(a) and 3(b) and Tables 3(a) and 3(b)). In contrast, concentration-dependent vasodilation to isoproterenol in the presence of L-VNIO, a NOS1 inhibitor, was significantly decreased in the aortic rings of Tgβ3 rats (Figure 3(c)), while the difference was not significant on mesenteric arteries (Figure 3(d)). In the presence of 1400W, a NOS2 inhibitor, the vasodilation in response to isoproterenol was not modified on both aortic and mesenteric rings of the two groups of rats (Figures 3(e) and 3(f) and Tables 3(a) and 3(b)). Taken together, these results suggest that vasodilation was predominantly mediated by •NO produced by NOS1 in the aorta from Tgβ3 rats at 15 weeks of age.Figure 3
Evaluation of function of NOS in WT and Tgβ3 rats at 15 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=3-9. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)Table 3
Statistical comparison of the efficiencies and potencies of isoproterenol with or without the NOS inhibitor and of CL 316 243 on thoracic aortic arteries (a) and mesenteric arteries (b) from WT and Tgβ3 rats at 15, 30, and 45 weeks old.
(a)GroupThoracic aortic arteries—WTThoracic aortic arteries—Tgβ3WT vs. WT+isoproterenolTgβ3 vs. Tgβ3+isoproterenolnpD2EmaxnpD2Emaxp pD2p Emaxp pD2p EmaxIsoproterenol15 w76.53±0.3394.5±2.555.93±0.2074.1±7.530 w46.73±0.3085.6±14.375.49±0.1179.0±9.545 w116.56±0.2185.9±2.2105.82±0.1569.2±3.5Isoproterenol+L-NIO15 w76.06±0.4560.0±12.145.87±0.4352.6±7.800.4300.0130.9390.32230 w35.25±0.1457.0±22.865.27±0.2742.1±14.30.0060.0830.6610.08045 w84.93±0.1133.0±4.4285.30±0.1845.9±5.90<0.0001<0.00010.1340.088Isoproterenol+L-VNIO15 w46.79±0.63861.9±16.055.63±0.51247.9±21.60.6200.1250.7990.41430 w45.65±0.6335.25±11.946.02±0.5918.6±8.60.0900.0240.3460.01945 w54.97±0.1433.6±6.1555.54±0.1542.3±2.84<0.0001<0.00010.3770.068Isoproterenol+1400W15 w46.51±0.70083.2±7.2346.32±0.65665.7±8.770.6650.2590.5880.62330 w46.09±0.4145.58±13.775.26±0.3263.1±12.60.1910.0190.9020.32345 w75.43±0.4658.1±9.4485.35±0.1353.0±7.520.0020.0020.1050.265(b)GroupMesenteric arteries—WTMesenteric arteries—Tgβ3WT vs. WT+isoproterenolTgβ3 vs. Tgβ3+isoproterenolnpD2EmaxnpD2Emaxp pD2p Emaxp pD2p EmaxIsoproterenol15 w47.78±0.1796.4±3.087.15±0.3278.0±8.530 w87.03±0.20100.7±4.686.67±0.2873.5±10.445 w127.21±0.1585.7±3.4126.17±0.1768.2±5.5Isoproterenol+L-NIO15 w36.04±0.7886.1±1.2896.89±0.3075.4±8.080.0240.4310.5550.77630 w55.91±0.3176.9±4.7046.66±0.3886.8±5.660.0140.0030.9780.62745 w95.83±0.2935.1±5.9195.65±0.3664.0±7.980.0004<0.00010.4080.640Isoproterenol+L-VNIO15 w67.08±0.5284.1±8.6466.24±0.4575.8±9.940.2900.3610.1270.84430w36.29±0.2771.5±4.5966.78±0.5475.1±8.330.1950.0130.8500.77745 w86.41±0.3049.6±8.6386.82±0.4322.79±7.380.0320.0030.0790.004Isoproterenol+1400W15 w47.34±0.2596.4±2.0276.49±0.3181.8±8.000.4040.9800.1250.78030 w86.64±0.3084.0±10.966.43±0.4492.3±3.230.3780.4180.6800.30745 w86.32±0.4046.52±10.886.27±0.2952.7±13.80.0130.0020.6790.773


### 3.3.2. NOS2 Expression Is Increased in Tgβ3 Rats at 15 Weeks

The aortic ratio of NOS3 dimer/monomer and p-NOS3/NOS3 monomer protein expression levels and NOS1 expression was not significantly modified at 15 weeks of age (Figures4(a), 4(b), and 4(d)). The overexpression of β3-AR induced a significant 2-fold increase in NOS2 protein expression in the aorta (WT: 1.00±0.14; Tgβ3: 2.06±0.31; p<0.05) (Figure 4(c)).Figure 4
Expression and activity production of NOS in WT and Tgβ3 rats at 15 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, 1400W, and L-VNIO) (e) and O2•- production with or without L-NMMA from WT and Tgβ3 rats (f). Data are expressed as mean±SEM. n=5-14. ∗∗p<0.01, ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


### 3.3.3.•NO and O2•- Production Is Unchanged in the Tgβ3 Rats at 15 Weeks

•NO production remained stable on thoracic aortas between WT and transgenic rats. With L-NMMA, the •NO production was almost abolished as expected, confirming that NOS-dependent •NO production (negative control). The NOS2-dependent •NO production was not significantly increased in the Tgβ3 rats (+51%; p=0.09). The NOS1- and NOS3-dependent •NO production was not modified between the two groups (Figure 4(e)). The generation of O2•- has been also evaluated under baseline conditions and in the presence of L-NMMA, and no changes on O2•- production with or without L-NMMA were reported between the two groups of rats at this age (Figure 4(f)).


## 3.3.1. The Vasodilation Is Predominantly Mediated by NOS1 in Tgβ3 Rats at 15 Weeks

At 15 weeks of age, vasodilation in response to isoproterenol was only altered in aortic rings from WT and Tgβ3 rats with a decrease in maximal effect in the aorta of the Tgβ3 group and a significant reduced response to isoproterenol stimulation (Figure 2(a) and Table 2(a)). To decipher the implication of each NOS isoform, several NOS inhibitors: L-NIO, L-VNIO, and 1400W which inhibit NOS3, NOS1, and NOS2, respectively, have been used.In the presence of L-NIO, the maximal effect was reduced in the aortic rings from WT rats (WT:60.0±12.1; WT+L-NIO: 52.6±7.80; p<0.05) (Figure 3(a) and Table 3(a)). NOS3 inhibition using L-NIO had no significant impact on aorta and mesenteric artery reactivity from Tgβ3 rats, suggesting reduced implication of •NO production from NOS3 in vascular relaxation of 15-week-old Tgβ3 rats compared to WT (Figures 3(a) and 3(b) and Tables 3(a) and 3(b)). In contrast, concentration-dependent vasodilation to isoproterenol in the presence of L-VNIO, a NOS1 inhibitor, was significantly decreased in the aortic rings of Tgβ3 rats (Figure 3(c)), while the difference was not significant on mesenteric arteries (Figure 3(d)). In the presence of 1400W, a NOS2 inhibitor, the vasodilation in response to isoproterenol was not modified on both aortic and mesenteric rings of the two groups of rats (Figures 3(e) and 3(f) and Tables 3(a) and 3(b)). Taken together, these results suggest that vasodilation was predominantly mediated by •NO produced by NOS1 in the aorta from Tgβ3 rats at 15 weeks of age.Figure 3
Evaluation of function of NOS in WT and Tgβ3 rats at 15 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=3-9. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)Table 3
Statistical comparison of the efficiencies and potencies of isoproterenol with or without the NOS inhibitor and of CL 316 243 on thoracic aortic arteries (a) and mesenteric arteries (b) from WT and Tgβ3 rats at 15, 30, and 45 weeks old.
(a)GroupThoracic aortic arteries—WTThoracic aortic arteries—Tgβ3WT vs. WT+isoproterenolTgβ3 vs. Tgβ3+isoproterenolnpD2EmaxnpD2Emaxp pD2p Emaxp pD2p EmaxIsoproterenol15 w76.53±0.3394.5±2.555.93±0.2074.1±7.530 w46.73±0.3085.6±14.375.49±0.1179.0±9.545 w116.56±0.2185.9±2.2105.82±0.1569.2±3.5Isoproterenol+L-NIO15 w76.06±0.4560.0±12.145.87±0.4352.6±7.800.4300.0130.9390.32230 w35.25±0.1457.0±22.865.27±0.2742.1±14.30.0060.0830.6610.08045 w84.93±0.1133.0±4.4285.30±0.1845.9±5.90<0.0001<0.00010.1340.088Isoproterenol+L-VNIO15 w46.79±0.63861.9±16.055.63±0.51247.9±21.60.6200.1250.7990.41430 w45.65±0.6335.25±11.946.02±0.5918.6±8.60.0900.0240.3460.01945 w54.97±0.1433.6±6.1555.54±0.1542.3±2.84<0.0001<0.00010.3770.068Isoproterenol+1400W15 w46.51±0.70083.2±7.2346.32±0.65665.7±8.770.6650.2590.5880.62330 w46.09±0.4145.58±13.775.26±0.3263.1±12.60.1910.0190.9020.32345 w75.43±0.4658.1±9.4485.35±0.1353.0±7.520.0020.0020.1050.265(b)GroupMesenteric arteries—WTMesenteric arteries—Tgβ3WT vs. WT+isoproterenolTgβ3 vs. Tgβ3+isoproterenolnpD2EmaxnpD2Emaxp pD2p Emaxp pD2p EmaxIsoproterenol15 w47.78±0.1796.4±3.087.15±0.3278.0±8.530 w87.03±0.20100.7±4.686.67±0.2873.5±10.445 w127.21±0.1585.7±3.4126.17±0.1768.2±5.5Isoproterenol+L-NIO15 w36.04±0.7886.1±1.2896.89±0.3075.4±8.080.0240.4310.5550.77630 w55.91±0.3176.9±4.7046.66±0.3886.8±5.660.0140.0030.9780.62745 w95.83±0.2935.1±5.9195.65±0.3664.0±7.980.0004<0.00010.4080.640Isoproterenol+L-VNIO15 w67.08±0.5284.1±8.6466.24±0.4575.8±9.940.2900.3610.1270.84430w36.29±0.2771.5±4.5966.78±0.5475.1±8.330.1950.0130.8500.77745 w86.41±0.3049.6±8.6386.82±0.4322.79±7.380.0320.0030.0790.004Isoproterenol+1400W15 w47.34±0.2596.4±2.0276.49±0.3181.8±8.000.4040.9800.1250.78030 w86.64±0.3084.0±10.966.43±0.4492.3±3.230.3780.4180.6800.30745 w86.32±0.4046.52±10.886.27±0.2952.7±13.80.0130.0020.6790.773


## 3.3.2. NOS2 Expression Is Increased in Tgβ3 Rats at 15 Weeks

The aortic ratio of NOS3 dimer/monomer and p-NOS3/NOS3 monomer protein expression levels and NOS1 expression was not significantly modified at 15 weeks of age (Figures4(a), 4(b), and 4(d)). The overexpression of β3-AR induced a significant 2-fold increase in NOS2 protein expression in the aorta (WT: 1.00±0.14; Tgβ3: 2.06±0.31; p<0.05) (Figure 4(c)).Figure 4
Expression and activity production of NOS in WT and Tgβ3 rats at 15 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, 1400W, and L-VNIO) (e) and O2•- production with or without L-NMMA from WT and Tgβ3 rats (f). Data are expressed as mean±SEM. n=5-14. ∗∗p<0.01, ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


## 3.3.3.•NO and O2•- Production Is Unchanged in the Tgβ3 Rats at 15 Weeks

•NO production remained stable on thoracic aortas between WT and transgenic rats. With L-NMMA, the •NO production was almost abolished as expected, confirming that NOS-dependent •NO production (negative control). The NOS2-dependent •NO production was not significantly increased in the Tgβ3 rats (+51%; p=0.09). The NOS1- and NOS3-dependent •NO production was not modified between the two groups (Figure 4(e)). The generation of O2•- has been also evaluated under baseline conditions and in the presence of L-NMMA, and no changes on O2•- production with or without L-NMMA were reported between the two groups of rats at this age (Figure 4(f)).


## 3.4.β3-AR Overexpression Induced Endothelial Dysfunction at 30 Weeks

We investigated whether endothelial dysfunction is worsened at 30 weeks of age in Tgβ3 rats.


### 3.4.1. NOS1 Modulates the Vasodilatation in the Tgβ3 Group

At 30 weeks of age, vasodilation in response to isoproterenol was reduced in aortic rings of Tgβ3 rats associated with a decreased pD2 (WT: 6.73±0.30; Tgβ3: 5.49±0.11; p<0.05) (Figure 2(a) and Table 2(a)).In the presence of L-NIO, the pD2 was reduced in the aortic rings (WT: 6.73±0.30; WT+L-NIO: 5.25±0.14; p<0.05) and mesenteric arteries (WT: 7.03±0.20; WT+L-NIO: 5.91±0.31; p<0.05) from WT rats (Figures 5(a) and 5(b) and Tables 3(a) and 3(b)) suggesting the implication of •NO from NOS3.Figure 5
Evaluation of function of NOS in WT and Tgβ3 rats at 30 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=3-8. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)Vasodilation in response to isoproterenol in the presence of the NOS1 inhibitor (L-VNIO) was blunted with a reduction in maximal effect in the aortic rings from both WT rats (WT:85.6±14.3; WT+L-NIO: 35.25±11.19; p<0.05) and Tgβ3 rats (Tgβ3: 79±9.5; Tgβ3+L-NIO: 18.6±8.6; p<0.05) and a decrease in the maximal effect in the mesenteric arteries from WT rats (WT: 100.7±4.6; WT+L-NIO: 71.5±4.59; p<0.05) (Figures 5(c) and 5(d) and Tables 3(a) and 3(b)). These data suggest an implication of •NO production from NOS1.In the presence of 1400W, the vasodilation in response to isoproterenol was modified on the aorta from WT rats with reduced maximal effect (WT:85.6±14.3; WT+1400W: 45.58±13.7; p<0.05) (Figure 5(e) and Table 3(a)) with no impact on mesenteric arteries (Figure 5(f) and Table 3(b)). At 30 weeks of age, the vasodilation induced by isoproterenol seems to involve all NOS isoforms in WT rats while only NOS1 seems to be involved in the vasodilation of vessels from Tgβ3 rats.


### 3.4.2.β3-AR Overexpression Is Associated with an Increase in NOS1 Expression

The aortic ratio of the NOS3 dimer/monomer and p-NOS3/NOS3 monomer and NOS2 protein expression levels were not significantly modified between the two groups at 30 weeks (Figures6(a), 6(b), and 6(c)). The β3-AR overexpression induces a significant 6-fold increase in NOS1 protein expression in the aorta (Figure 6(d)) which can explain the decrease in vasodilation in vessels from Tgβ3 with the NOS1 inhibitor.Figure 6
Expression and activity production of NOS in WT and Tgβ3 rats at 30 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, L-VNIO, and 1400W) (e) and O2•- production with or without L-NMMA (f) from WT and Tgβ3 rats. Data are expressed as mean±SEM. n=5-16. ∗p<0.05, ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


### 3.4.3. Oxidative Stress Is Increased in Tgβ3 Rats at 30 Weeks

At 30 weeks of age,•NO production tends to increase in Tgβ3 rats (+28%). In the presence of L-NMMA, the production of •NO is significantly reduced in WT and Tgβ3 rats. NOS2-dependent •NO production is increased in the Tgβ3 group (+70% vs. WT; p<0.05) (Figure 6(e)).The O2•- production was significantly increased in the Tgβ3 group under baseline conditions (+75% vs. WT; p<0.05) while no change is shown with L-NMMA (Figure 6(f)).The results demonstrated that despite a similar expression of NOS2, NOS2-dependent•NO production is increased in the Tgβ3 group, suggesting that its activity is enhanced in Tgβ3 rats. Interestingly, NOS1 expression which is highly increased in Tgβ3 rats compared to WT does not impact •NO production.


## 3.4.1. NOS1 Modulates the Vasodilatation in the Tgβ3 Group

At 30 weeks of age, vasodilation in response to isoproterenol was reduced in aortic rings of Tgβ3 rats associated with a decreased pD2 (WT: 6.73±0.30; Tgβ3: 5.49±0.11; p<0.05) (Figure 2(a) and Table 2(a)).In the presence of L-NIO, the pD2 was reduced in the aortic rings (WT: 6.73±0.30; WT+L-NIO: 5.25±0.14; p<0.05) and mesenteric arteries (WT: 7.03±0.20; WT+L-NIO: 5.91±0.31; p<0.05) from WT rats (Figures 5(a) and 5(b) and Tables 3(a) and 3(b)) suggesting the implication of •NO from NOS3.Figure 5
Evaluation of function of NOS in WT and Tgβ3 rats at 30 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=3-8. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)Vasodilation in response to isoproterenol in the presence of the NOS1 inhibitor (L-VNIO) was blunted with a reduction in maximal effect in the aortic rings from both WT rats (WT:85.6±14.3; WT+L-NIO: 35.25±11.19; p<0.05) and Tgβ3 rats (Tgβ3: 79±9.5; Tgβ3+L-NIO: 18.6±8.6; p<0.05) and a decrease in the maximal effect in the mesenteric arteries from WT rats (WT: 100.7±4.6; WT+L-NIO: 71.5±4.59; p<0.05) (Figures 5(c) and 5(d) and Tables 3(a) and 3(b)). These data suggest an implication of •NO production from NOS1.In the presence of 1400W, the vasodilation in response to isoproterenol was modified on the aorta from WT rats with reduced maximal effect (WT:85.6±14.3; WT+1400W: 45.58±13.7; p<0.05) (Figure 5(e) and Table 3(a)) with no impact on mesenteric arteries (Figure 5(f) and Table 3(b)). At 30 weeks of age, the vasodilation induced by isoproterenol seems to involve all NOS isoforms in WT rats while only NOS1 seems to be involved in the vasodilation of vessels from Tgβ3 rats.


## 3.4.2.β3-AR Overexpression Is Associated with an Increase in NOS1 Expression

The aortic ratio of the NOS3 dimer/monomer and p-NOS3/NOS3 monomer and NOS2 protein expression levels were not significantly modified between the two groups at 30 weeks (Figures6(a), 6(b), and 6(c)). The β3-AR overexpression induces a significant 6-fold increase in NOS1 protein expression in the aorta (Figure 6(d)) which can explain the decrease in vasodilation in vessels from Tgβ3 with the NOS1 inhibitor.Figure 6
Expression and activity production of NOS in WT and Tgβ3 rats at 30 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, L-VNIO, and 1400W) (e) and O2•- production with or without L-NMMA (f) from WT and Tgβ3 rats. Data are expressed as mean±SEM. n=5-16. ∗p<0.05, ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


## 3.4.3. Oxidative Stress Is Increased in Tgβ3 Rats at 30 Weeks

At 30 weeks of age,•NO production tends to increase in Tgβ3 rats (+28%). In the presence of L-NMMA, the production of •NO is significantly reduced in WT and Tgβ3 rats. NOS2-dependent •NO production is increased in the Tgβ3 group (+70% vs. WT; p<0.05) (Figure 6(e)).The O2•- production was significantly increased in the Tgβ3 group under baseline conditions (+75% vs. WT; p<0.05) while no change is shown with L-NMMA (Figure 6(f)).The results demonstrated that despite a similar expression of NOS2, NOS2-dependent•NO production is increased in the Tgβ3 group, suggesting that its activity is enhanced in Tgβ3 rats. Interestingly, NOS1 expression which is highly increased in Tgβ3 rats compared to WT does not impact •NO production.


## 3.5.β3-AR Overexpression Maintained the Endothelial Dysfunction at 45 Weeks of Age

At 30 weeks, results indicated an endothelial dysfunction associated with an alteration of the vasodilation. An increase in NOS1 expression could led to endothelial dysfunction and is associated with an increase in O2•- production. Thus, we investigated, at 45 weeks of age, whether endothelial dysfunction was more severe compared to 30 weeks of age.


### 3.5.1. NOS1 and NOS3 Modulate the Vasodilation in the Tgβ3 Group

At 45 weeks of age, vasodilation in response to isoproterenol was significantly reduced in Tgβ3 rats compared to WT in both aortic and mesenteric arteries (Figures 2(a) and 2(b) and Tables 2(a) and 2(b)). In the presence of L-NIO, 1400W, or L-VNIO, vasodilation was significantly reduced on aortic and mesenteric artery rings from WT rats (Figures 7(a)–7(f) and Tables 3(a) and 3(b)). In Tgβ3 rats, vasodilation was significantly reduced on aortic rings in the presence of L-NIO, while L-VNIO reduced the vasodilation in both aortic and mesenteric rings from Tgβ3 rats (Figures 7(a), 7(c), and 7(d)). Taken together, these results demonstrated that the vasodilation in Tgβ3 rats was altered compared to that in WT rats and seems to depend on NOS1 and NOS3 isoforms.Figure 7
Evaluation of function of NOS in WT and Tgβ3 rats at 45 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=5-12. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)


### 3.5.2. NOS3 Is Uncoupled in Tgβ3 Rats

In Tgβ3 rats, the aortic NOS3 dimer/NOS3 monomer protein expression ratio and the aortic p-NOS3/NOS3 monomer protein expression ratio were significantly reduced (-87% and -63%, respectively; p<0.05) indicating a potential NOS3 uncoupling (Figures 8(a) and 8(b)). No modification was reported for NOS2 protein expression (Figure 8(c)). The aortic NOS1 protein expression was significantly increased at 45 weeks of age on Tgβ3 rats (+57%; p<0.05) (Figure 8(d)).Figure 8
Expression and activity production of NOS in WT and Tgβ3 rats at 45 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, L-VNIO, and 1400W) (e) and O2•- production with or without L-NMMA (f) from WT and Tgβ3 rats. Data are expressed as mean±SEM. n=5-10. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


### 3.5.3. O2•- Production Is Increased in Tgβ3 Rats

The production of•NO tends to increase in the aorta in Tgβ3 rats under baseline conditions (+84% vs. WT) (Figure 8(e)). Addition of L-NMMA reduces •NO production in the two groups, indicating that the •NO production from Tgβ3 and WT rats is mainly due to the NOS. The NOS1-dependent •NO production tends to increase in the Tgβ3 rats (+90% vs. WT; p=0.052) (Figure 8(e)). The production of O2•- was significantly increased in Tgβ3 rats (+76% vs. WT; p<0.05). L-NMMA treatment normalized the production with the absence of significant difference between the two groups, suggesting the involvement of NOS in O2•- production from Tgβ3 rats (Figure 8(f)). Results obtained at 45 weeks of age confirm the previous results obtained at 30 weeks of age that Tgβ3 rats present an endothelial dysfunction.


## 3.5.1. NOS1 and NOS3 Modulate the Vasodilation in the Tgβ3 Group

At 45 weeks of age, vasodilation in response to isoproterenol was significantly reduced in Tgβ3 rats compared to WT in both aortic and mesenteric arteries (Figures 2(a) and 2(b) and Tables 2(a) and 2(b)). In the presence of L-NIO, 1400W, or L-VNIO, vasodilation was significantly reduced on aortic and mesenteric artery rings from WT rats (Figures 7(a)–7(f) and Tables 3(a) and 3(b)). In Tgβ3 rats, vasodilation was significantly reduced on aortic rings in the presence of L-NIO, while L-VNIO reduced the vasodilation in both aortic and mesenteric rings from Tgβ3 rats (Figures 7(a), 7(c), and 7(d)). Taken together, these results demonstrated that the vasodilation in Tgβ3 rats was altered compared to that in WT rats and seems to depend on NOS1 and NOS3 isoforms.Figure 7
Evaluation of function of NOS in WT and Tgβ3 rats at 45 weeks of age. Concentration-relaxation curves to isoproterenol, a nonselective β-AR agonist, on thoracic aortic rings in the presence or absence of a selective NOS inhibitor: L-NIO (NOS3 inhibitor) (a), 1400W (NOS2 inhibitor) (b), and L-VNIO (NOS1 inhibitor) (c). The same experiments were done on mesenteric artery rings with L-NIO (d), 1400W (e), and L-VNIO (f). Results were expressed as the percentage of relaxation from the maximal contraction induced by phenylephrine. Values represent mean±SEM. n=5-12. ∗p<0.05: WT versus WT+inhibitor; $p<0.05: Tgβ3 versus Tgβ3+inhibitor.
(a)(b)(c)(d)(e)(f)


## 3.5.2. NOS3 Is Uncoupled in Tgβ3 Rats

In Tgβ3 rats, the aortic NOS3 dimer/NOS3 monomer protein expression ratio and the aortic p-NOS3/NOS3 monomer protein expression ratio were significantly reduced (-87% and -63%, respectively; p<0.05) indicating a potential NOS3 uncoupling (Figures 8(a) and 8(b)). No modification was reported for NOS2 protein expression (Figure 8(c)). The aortic NOS1 protein expression was significantly increased at 45 weeks of age on Tgβ3 rats (+57%; p<0.05) (Figure 8(d)).Figure 8
Expression and activity production of NOS in WT and Tgβ3 rats at 45 weeks of age. Effects of the β3-AR overexpression in Tgβ3 rats on aortic protein expression of the NOS3 dimer/monomer ratio (a), p-NOS3/NOS3 monomer (b), NOS2 (c), and NOS1 (d). Evaluation of •NO production on thoracic aortic rings with or without an inhibitor (L-NMMA, L-NIO, L-VNIO, and 1400W) (e) and O2•- production with or without L-NMMA (f) from WT and Tgβ3 rats. Data are expressed as mean±SEM. n=5-10. ∗p<0.05, ∗∗p<0.01, and ∗∗∗p<0.001.
(a)(b)(c)(d)(e)(f)


## 3.5.3. O2•- Production Is Increased in Tgβ3 Rats

The production of•NO tends to increase in the aorta in Tgβ3 rats under baseline conditions (+84% vs. WT) (Figure 8(e)). Addition of L-NMMA reduces •NO production in the two groups, indicating that the •NO production from Tgβ3 and WT rats is mainly due to the NOS. The NOS1-dependent •NO production tends to increase in the Tgβ3 rats (+90% vs. WT; p=0.052) (Figure 8(e)). The production of O2•- was significantly increased in Tgβ3 rats (+76% vs. WT; p<0.05). L-NMMA treatment normalized the production with the absence of significant difference between the two groups, suggesting the involvement of NOS in O2•- production from Tgβ3 rats (Figure 8(f)). Results obtained at 45 weeks of age confirm the previous results obtained at 30 weeks of age that Tgβ3 rats present an endothelial dysfunction.


## 4. Discussion

The aim of this study was to decipher the link between endothelial dysfunction and diastolic dysfunction. We wanted to understand if endothelial dysfunction could be involved in HFpEF development. For that, we investigated cardiac and endothelial function in rats at 15, 30, and 45 weeks old of age. The major finding of this study was thatβ3-AR overexpression led to endothelial dysfunction throughout ageing and that endothelium dysfunction appears prior to diastolic dysfunction.


### 4.1.β3-AR Overexpression Is Associated with Endothelial Dysfunction

Adrb3, the gene coding for the β3 receptor, is the main subtype of the β adrenergic receptor expressed in the whole aorta in the rats. However, smooth muscle cells and endothelial cells are mainly expressing the gene coding for the β2 receptor (Adrb2) [16]. In the literature, β3-AR has a low level of protein expression under physiological conditions, but β3-AR expression is increased in heart failure [17], and many studies demonstrated a cardioprotective role of β3-AR in pathophysiological conditions [18–22]. Also, specific β3-AR overexpression on cardiomyocytes is associated with the cardioprotective effect [23]. In our study however, long-term endothelial overexpression of the β3-AR was associated with deleterious effect such as the development of diastolic dysfunction and an alteration of the vascular function. Vascular reactivity, and specifically vasodilation in response to isoproterenol, was blunted as early as 15 weeks of age, before the apparition of the diastolic dysfunction, suggesting that endothelial dysfunction is a key process in the HFpEF development. Interestingly, altered vasodilation was first detected in the aorta at 15 weeks while it has been detected in both the aorta and mesenteric arteries at 30 weeks. This can be explained in particular by structural differences but also by differences in •NO production between elastic arteries such as the aorta and muscular arteries such as the mesenteric arteries [24].


### 4.2. Endothelial Dysfunction and HFpEF

Endothelial dysfunction has been suggested to be at the center of the HFpEF pathophysiology for about ten years [25–27], yet no study manages to demonstrate that endothelial dysfunction could be the primum movens of HFpEF development.Endothelial dysfunction has been characterized in HFpEF by a decrease in the production of•NO, leading to a decrease in vasodilation, as a consequence of the NOS3 protein loss of function [25]. The decrease in •NO is explained by the decoupling of NOS3, which has been confirmed in patients with HFpEF or in animal models such as ZSF1 (Zucker fatty/spontaneously hypertensive heart failure F1) rats. NOS3 uncoupling leads to a shift of NOS3 from the dimer, which produces •NO, to the monomer, which generates O2•- [28]. This NOS3 uncoupling is also found in a diabetic HFpEF model [29]. In their study, the authors show a decrease in the activity of NOS3 due to uncoupling of this protein and thus a decrease in the production of •NO. Interestingly, Shibata et al. have shown that, by using NOS-knockout mice, the deletion of NOS causes diastolic dysfunction and cardiac hypertrophy [30]. These studies converge to demonstrate that during HFpEF development, the uncoupling of the NOS3 protein induces a decrease in •NO production, which could be at the origin of endothelial dysfunction.NOS1 is largely expressed in the central nervous system, but its expression has also been found in smooth muscle cells and cardiomyocytes. Under physiological conditions, NOS1 expression in smooth muscle cells has been shown to be involved in vascular tone. Cau et al. described that NOS-dependent vasodilation is modified throughout ageing in human and animal models. In fact, NOS3 expression and activity are reduced with age, whereas NOS2 activity is increased and accompanied by peroxynitrite production [31]. These data validate our observations on the involvement of NOS isoforms in vasodilation throughout ageing, with a predominant involvement of NOS3 at 15 weeks of age and all NOS isoforms at 30 and 45 weeks of age. Interestingly, NOS1 has been shown to maintain some degree of vasodilation, when the predominant NOS3 becomes dysfunctional [32, 33] or absent in the knockout mouse model [34, 35]. In our Tgβ3 rat model, we showed that NOS1 expression was increased at 30 and 45 weeks of age and that the NOS1 is the main isoform involved in the vasodilation. These data suggest that NOS1 could compensate for the dysfunction of NOS3 reported at 45 weeks of age.


### 4.3. Oxidative Stress: A Key Player in Endothelial and Diastolic Dysfunction

Oxidative stress in another mechanism recently described a potential trigger to the development of endothelial dysfunction. Indeed, it has been described that the H2O2 concentration in the myocardium of both HFpEF patients and ZSF1 rats is significantly elevated [25]. H2O2 results from the conversion of O2•- by superoxide dismutase (SOD), and the high O2•- concentrations in our model therefore suggest an increase in H2O2 concentration and SOD activity at the vascular and cardiac levels.The endothelial dysfunction is linked to an increase in•NO production by NOS1. In the same time, NOS3 expression is decreased suggesting NOS3 uncoupling leading to O2•- production and oxidative stress as described earlier [9, 36]. Increase in oxidative stress is linked to the genesis of heart failure, but some studies suggested that β3-AR activation inhibited oxidative stress and reactive oxygen production (ROS). Intriguingly, in our model, long-term endothelial β3-AR overexpression did not protect against the ROS production, and the long-term overproduction of •NO is linked to endothelial dysfunction. Several hypotheses can explain this phenomenon. First, the increase in S-nitrosylation could explain the progressive decrease in cardiovascular function. Recently, Schiattarella et al. showed that elevated NOS2 activity leads to S-nitrosylation in multiple proteins and can impair their functions. Furthermore, the study highlighted, in human HFpEF hearts, an increase in NOS2 transcripts [27]. In our study, NOS2 expression was increased as early as 15 weeks of age in the Tgβ3 group suggesting a disruption of protein function. However, nothing can be concluded since the expression of a protein and the enzymatic activity of the latter are not always linked. In the second hypothesis, the increase in the •NO bioavailability associated with an increase in ROS could lead to the production of peroxynitrite (ONOO-). In our study, the trend to increase in •NO levels is associated with the overproduction of O2•- suggesting the production of ONOO-. The production of ONOO- is increased when the NOS3 is uncoupled [37]. In our model, the decrease in O2•- production in the presence of L-NMMA suggested that the NOS uncoupling could be at the origin of a reduced vasodilation. More specifically, the uncoupled NOS3, reported in the Tgβ3 rats, could be at the origin of the diastolic dysfunction. Oxidative stress and more particularly NOS impairment could have a key role in endothelial dysfunction and with ageing could lead to cardiac alteration.


## 4.1.β3-AR Overexpression Is Associated with Endothelial Dysfunction

Adrb3, the gene coding for the β3 receptor, is the main subtype of the β adrenergic receptor expressed in the whole aorta in the rats. However, smooth muscle cells and endothelial cells are mainly expressing the gene coding for the β2 receptor (Adrb2) [16]. In the literature, β3-AR has a low level of protein expression under physiological conditions, but β3-AR expression is increased in heart failure [17], and many studies demonstrated a cardioprotective role of β3-AR in pathophysiological conditions [18–22]. Also, specific β3-AR overexpression on cardiomyocytes is associated with the cardioprotective effect [23]. In our study however, long-term endothelial overexpression of the β3-AR was associated with deleterious effect such as the development of diastolic dysfunction and an alteration of the vascular function. Vascular reactivity, and specifically vasodilation in response to isoproterenol, was blunted as early as 15 weeks of age, before the apparition of the diastolic dysfunction, suggesting that endothelial dysfunction is a key process in the HFpEF development. Interestingly, altered vasodilation was first detected in the aorta at 15 weeks while it has been detected in both the aorta and mesenteric arteries at 30 weeks. This can be explained in particular by structural differences but also by differences in •NO production between elastic arteries such as the aorta and muscular arteries such as the mesenteric arteries [24].


## 4.2. Endothelial Dysfunction and HFpEF

Endothelial dysfunction has been suggested to be at the center of the HFpEF pathophysiology for about ten years [25–27], yet no study manages to demonstrate that endothelial dysfunction could be the primum movens of HFpEF development.Endothelial dysfunction has been characterized in HFpEF by a decrease in the production of•NO, leading to a decrease in vasodilation, as a consequence of the NOS3 protein loss of function [25]. The decrease in •NO is explained by the decoupling of NOS3, which has been confirmed in patients with HFpEF or in animal models such as ZSF1 (Zucker fatty/spontaneously hypertensive heart failure F1) rats. NOS3 uncoupling leads to a shift of NOS3 from the dimer, which produces •NO, to the monomer, which generates O2•- [28]. This NOS3 uncoupling is also found in a diabetic HFpEF model [29]. In their study, the authors show a decrease in the activity of NOS3 due to uncoupling of this protein and thus a decrease in the production of •NO. Interestingly, Shibata et al. have shown that, by using NOS-knockout mice, the deletion of NOS causes diastolic dysfunction and cardiac hypertrophy [30]. These studies converge to demonstrate that during HFpEF development, the uncoupling of the NOS3 protein induces a decrease in •NO production, which could be at the origin of endothelial dysfunction.NOS1 is largely expressed in the central nervous system, but its expression has also been found in smooth muscle cells and cardiomyocytes. Under physiological conditions, NOS1 expression in smooth muscle cells has been shown to be involved in vascular tone. Cau et al. described that NOS-dependent vasodilation is modified throughout ageing in human and animal models. In fact, NOS3 expression and activity are reduced with age, whereas NOS2 activity is increased and accompanied by peroxynitrite production [31]. These data validate our observations on the involvement of NOS isoforms in vasodilation throughout ageing, with a predominant involvement of NOS3 at 15 weeks of age and all NOS isoforms at 30 and 45 weeks of age. Interestingly, NOS1 has been shown to maintain some degree of vasodilation, when the predominant NOS3 becomes dysfunctional [32, 33] or absent in the knockout mouse model [34, 35]. In our Tgβ3 rat model, we showed that NOS1 expression was increased at 30 and 45 weeks of age and that the NOS1 is the main isoform involved in the vasodilation. These data suggest that NOS1 could compensate for the dysfunction of NOS3 reported at 45 weeks of age.


## 4.3. Oxidative Stress: A Key Player in Endothelial and Diastolic Dysfunction

Oxidative stress in another mechanism recently described a potential trigger to the development of endothelial dysfunction. Indeed, it has been described that the H2O2 concentration in the myocardium of both HFpEF patients and ZSF1 rats is significantly elevated [25]. H2O2 results from the conversion of O2•- by superoxide dismutase (SOD), and the high O2•- concentrations in our model therefore suggest an increase in H2O2 concentration and SOD activity at the vascular and cardiac levels.The endothelial dysfunction is linked to an increase in•NO production by NOS1. In the same time, NOS3 expression is decreased suggesting NOS3 uncoupling leading to O2•- production and oxidative stress as described earlier [9, 36]. Increase in oxidative stress is linked to the genesis of heart failure, but some studies suggested that β3-AR activation inhibited oxidative stress and reactive oxygen production (ROS). Intriguingly, in our model, long-term endothelial β3-AR overexpression did not protect against the ROS production, and the long-term overproduction of •NO is linked to endothelial dysfunction. Several hypotheses can explain this phenomenon. First, the increase in S-nitrosylation could explain the progressive decrease in cardiovascular function. Recently, Schiattarella et al. showed that elevated NOS2 activity leads to S-nitrosylation in multiple proteins and can impair their functions. Furthermore, the study highlighted, in human HFpEF hearts, an increase in NOS2 transcripts [27]. In our study, NOS2 expression was increased as early as 15 weeks of age in the Tgβ3 group suggesting a disruption of protein function. However, nothing can be concluded since the expression of a protein and the enzymatic activity of the latter are not always linked. In the second hypothesis, the increase in the •NO bioavailability associated with an increase in ROS could lead to the production of peroxynitrite (ONOO-). In our study, the trend to increase in •NO levels is associated with the overproduction of O2•- suggesting the production of ONOO-. The production of ONOO- is increased when the NOS3 is uncoupled [37]. In our model, the decrease in O2•- production in the presence of L-NMMA suggested that the NOS uncoupling could be at the origin of a reduced vasodilation. More specifically, the uncoupled NOS3, reported in the Tgβ3 rats, could be at the origin of the diastolic dysfunction. Oxidative stress and more particularly NOS impairment could have a key role in endothelial dysfunction and with ageing could lead to cardiac alteration.


## 5. Limits

The aim of the study is to better understand the role of endothelial dysfunction in the development of HFpEF. HFpEF is a disease that primarily affects women. In our model of humanβ3-adrenoreceptor overexpression and as discussed in our previous publication [10], only male rats develop heart failure with preserved ejection fraction. From our results, the involvement of the endothelium in the development of HFpEF could only be linked to the male phenotype.


## 6. Conclusion

Endothelial dysfunction appeared prior to cardiac dysfunction in our HFpEF rat model indicating a potential role of endothelial dysfunction in the development of HFpEF. We have demonstrated that alteration in the NOS function was a potential trigger of HFpEF development via an endothelial dysfunction. The increase in oxidative stress was characterized by an increase in O2•- which was the consequence of NOS deregulation in our model potentially via NOS3 uncoupling. Indeed, in many HFpEF models, the NOS3 protein is described as uncoupled, resulting in a decrease in the production of •NO and an increase in O2•-, yet as a consequence of HFpEF and not as a cause. Our study provides evidence that endothelial dysfunction could be the trigger to develop HFpEF.



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*Source: 7377877-2022-05-18.xml*