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

Thomas Dupas, Thomas Pelé, Justine Dhot, Mélanie Burban, Antoine

Persello, Virginie Aillerie, Angélique Erraud, Angela Tesse, David Stevant, Angélique Blangy-Letheule, et al.

► To cite this version:

Thomas Dupas, Thomas Pelé, Justine Dhot, Mélanie Burban, Antoine Persello, et al. The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model. Oxidative Medicine and Cellular Longevity, 2022, 2022, pp.1-30. 10.1155/2022/7377877. hal-04591804

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Research Article

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

Thomas Dupas ID, Thomas Pelé, Justine Dhot, Mélanie Burban, Antoine Persello ID,
Virginie Aillerie, Angélique Erraud, Angela Tesse ID, David Stevant,
Angélique Blangy-Letheule, Céline Menguy, Vincent Sauzeau ID, Michel De Waard ID,
Bertrand Rozec ID, Chantal Gauthier ID, and Benjamin Lauzier ID

Nantes Université, CHU Nantes, CNRS, INSERM, l'Institut du thorax, F-44000 Nantes, France

Correspondence should be addressed to Benjamin Lauzier; benjamin.lauzier@univ-nantes.fr

Received 21 March 2022; Accepted 27 April 2022; Published 18 May 2022

Academic Editor: Vladimir Jakovljevic

Copyright © 2022 Thomas Dupas et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

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 human β₃-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.

**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 popu-
lation [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 ejec-
tion 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 com-
mon 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 cardiovas-
cular function. The •NO is mainly produced by the endothe-
lial cells *via* the activation of the •NO synthase (NOS) which
exists under three isoforms: the neuronal NOS (nNOS or
Research Article

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

Thomas Dupas ID, Thomas Pelé, Justine Dhot, Mélanie Burban, Antoine Persello ID,
Virginie Aillerie, Angélique Erraud, Angela Tesse ID, David Stevant,
Angélique Blangy-Letheule, Céline Menguy, Vincent Sauzeau ID, Michel De Waard ID,
Bertrand Rozec ID, Chantal Gauthier ID, and Benjamin Lauzier ID

Nantes Université, CHU Nantes, CNRS, INSERM, l'Institut du thorax, F-44000 Nantes, France

Correspondence should be addressed to Benjamin Lauzier; benjamin.lauzier@univ-nantes.fr

Received 21 March 2022; Accepted 27 April 2022; Published 18 May 2022

Academic Editor: Vladimir Jakovljevic

Copyright © 2022 Thomas Dupas et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

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 human β₃-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.

**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 popu-
lation [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 ejec-
tion 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 com-
mon 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 cardiovas-
cular function. The •NO is mainly produced by the endothe-
lial 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 endo-
thelial 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 rep-
resent 20% of the cellular population and the •NO plays a
role in cardiac contractility regulation [6]. The •NO produc-
tion 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 acti-
vation, 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 dia-
stolic dysfunction at 45 weeks of age, characteristic of
HFpEF [10]. The aim of this study is to understand if endo-
thelial 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 connec-
tive 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 integ-
rity, rings were precontracted with phenylephrine (1 µM,
Sigma-Aldrich, P6126) and then exposed to a single concen-
tration of acetylcholine (10 µM, Sigma-Aldrich, A6625). The
tension was recorded on precontracted rings with phenyl-
ephrine (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 evalu-
ated. 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, N<sup>5</sup>- (1-iminoethyl)-L-orni-
thine dihydrochloride, Tocris, 0546); vinyl-L-VNIO, a
NOS1 inhibitor (10 µM, N<sup>5</sup>- (1-imino-3-butenyl)-L-orni-
thine, L-VNIO, Enzo Life Sciences, ALX-270-216-M005);
1400W, a NOS2 inhibitor (10 µM, N-[(3-(aminomethyl)phe-
nyl]methyl]-ethanimidamide dihydrochloride, Tocris, 1415);
and L-NMMA, a nonselective NOS inhibitor (30 µM, NG-
monomethyl-L-arginine acetate, Tocris, 0771) were evalu-
ated. 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
NOS1), the inducible NOS (iNOS or NOS2), and the endo-
thelial 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 rep-
resent 20% of the cellular population and the •NO plays a
role in cardiac contractility regulation [6]. The •NO produc-
tion 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 acti-
vation, 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 dia-
stolic dysfunction at 45 weeks of age, characteristic of
HFpEF [10]. The aim of this study is to understand if endo-
thelial 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 connec-
tive 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 integ-
rity, rings were precontracted with phenylephrine (1 µM,
Sigma-Aldrich, P6126) and then exposed to a single concen-
tration of acetylcholine (10 µM, Sigma-Aldrich, A6625). The
tension was recorded on precontracted rings with phenyl-
ephrine (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 evalu-
ated. 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, N<sup>5</sup>- (1-iminoethyl)-L-orni-
thine dihydrochloride, Tocris, 0546); vinyl-L-VNIO, a
NOS1 inhibitor (10 µM, N<sup>5</sup>- (1-imino-3-butenyl)-L-orni-
thine, L-VNIO, Enzo Life Sciences, ALX-270-216-M005);
1400W, a NOS2 inhibitor (10 µM, N-[(3-(aminomethyl)phe-
nyl]methyl]-ethanimidamide dihydrochloride, Tocris, 1415);
and L-NMMA, a nonselective NOS inhibitor (30 µM, NG-
monomethyl-L-arginine acetate, Tocris, 0771) were evalu-
ated. 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 measure-
ments 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 col-
loid solution containing Na-diethyldithiocarbamate trihy-
drate (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, Ger-
many). 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 fre-
quency; sweep time of 150 s; and 3 scans for •NO measure-
ments 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.

2.6. *Statistical Analysis.* Data were presented as mean ± 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.

3.2. β₃-AR Overexpression in the Long Term Induces Vasodilation Alteration. Concentration-dependent vasodilation to isoproterenol, a nonselective β-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.

CL 316 243, a β₃-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 pro-
duction 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 condi-
tions, 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 Endothelial β₃-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, sug-
gesting 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
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.

2.6. *Statistical Analysis.* Data were presented as mean ± 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. *β<sub>3</sub>-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.

3.2. *β<sub>3</sub>-AR Overexpression in the Long Term Induces Vasodilation Alteration.* Concentration-dependent vasodilation to isoproterenol, a nonselective β-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.

CL 316 243, a β₃-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 Endothelial β<sub>3</sub>-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
<table><caption>TABLE 1: Antibodies used for Western blotting experiments.</caption><thead><tr><th rowspan="2">Protein of interest</th><th colspan="2">Primary antibody</th><th rowspan="2">Dilution</th><th colspan="2">Secondary antibody</th><th rowspan="2">Dilution</th></tr><tr><th>Reference</th><th>Species</th><th>Reference</th><th>Species</th></tr></thead><tbody><tr><td>NOS1</td><td>Cell Signaling (4231)</td><td>Rabbit</td><td>1/500*</td><td>Cell Signaling (7074)</td><td>Goat</td><td rowspan="3">1/10000*</td></tr><tr><td>NOS2</td><td>Millipore (AB5382)</td><td>Rabbit</td><td>1/2000*</td><td>Cell Signaling (7074)</td><td>Goat</td></tr><tr><td>NOS3</td><td>BD Biosciences (610296)</td><td>Mouse</td><td>1/1000*</td><td>Cell Signaling (7076)</td><td>Horse</td></tr><tr><td>p-NOS3</td><td>Cell Signaling (9571)</td><td>Rabbit</td><td>1/1000*</td><td>Cell Signaling (7074)</td><td>Goat</td><td></td></tr></tbody></table>

<figure><img src="image_1.png" /><figcaption>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. *p < 0.05.</figcaption></figure>

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.

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 (Figures 4(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)).

3.3.3. *NO and O₂⁻ Production Is Unchanged in the Tgβ₃ Rats at 15 Weeks. *NO production remained stable on

<footnote>*Dilution carried out in 5% milk.</footnote>
<table><caption>TABLE 1: Antibodies used for Western blotting experiments.</caption><thead><tr><th rowspan="2">Protein of interest</th><th colspan="2">Primary antibody</th><th rowspan="2">Dilution</th><th colspan="2">Secondary antibody</th><th rowspan="2">Dilution</th></tr><tr><th>Reference</th><th>Species</th><th>Reference</th><th>Species</th></tr></thead><tbody><tr><td>NOS1</td><td>Cell Signaling (4231)</td><td>Rabbit</td><td>1/500*</td><td>Cell Signaling (7074)</td><td>Goat</td><td rowspan="3">1/10000*</td></tr><tr><td>NOS2</td><td>Millipore (AB5382)</td><td>Rabbit</td><td>1/2000*</td><td>Cell Signaling (7074)</td><td>Goat</td></tr><tr><td>NOS3</td><td>BD Biosciences (610296)</td><td>Mouse</td><td>1/1000*</td><td>Cell Signaling (7076)</td><td>Horse</td></tr><tr><td>p-NOS3</td><td>Cell Signaling (9571)</td><td>Rabbit</td><td>1/1000*</td><td>Cell Signaling (7074)</td><td>Goat</td><td></td></tr></tbody></table>

<figure><img src="image_2.png" /><figcaption>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. *p < 0.05.</figcaption></figure>

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.

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 (Figures 4(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)).

3.3.3. *NO and O₂⁻ Production Is Unchanged in the Tgβ₃ Rats at 15 Weeks. *NO production remained stable on

<footnote>*Dilution carried out in 5% milk.</footnote>
<figure><img src="image_2.png" /><figcaption>FIGURE 2: Continued.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>FIGURE 2: Continued.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>FIGURE 2: Continued.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>FIGURE 2: Continued.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>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.</figcaption></figure>

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.
<figure><img src="image_5.png" /><figcaption>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.</figcaption></figure>

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.
<table><caption>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.</caption><caption>(a)</caption><thead><tr><th rowspan="2">Group</th><th colspan="3">Thoracic aortic arteries—WT</th><th colspan="3">Thoracic aortic arteries—Tgβ<sub>3</sub></th><th colspan="2">Statistics WT vs. Tgβ<sub>3</sub></th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>Emax</th></tr></thead><tbody><tr><td rowspan="4">Isoproterenol</td><td>15 w</td><td>7</td><td>6.53 ± 0.33</td><td>94.5 ± 2.5</td><td>5</td><td>5.93 ± 0.20</td><td>74.1 ± 7.5</td><td>0.239</td><td>0.015</td></tr><tr><td>30 w</td><td>4</td><td>6.73 ± 0.30</td><td>85.6 ± 14.3</td><td>7</td><td>5.49 ± 0.11</td><td>79.0 ± 9.5</td><td>0.002</td><td>0.245</td></tr><tr><td>45 w</td><td>11</td><td>6.56 ± 0.21</td><td>85.9 ± 2.2</td><td>10</td><td>5.82 ± 0.15</td><td>69.2 ± 3.5</td><td>0.023</td><td>0.016</td></tr><tr><td>15 w</td><td>3</td><td>5.18 ± 0.80</td><td>15.0 ± 5.52</td><td>2</td><td>6.79 ± 2.07</td><td>25.7 ± 9.43</td><td>0.408</td><td>0.679</td></tr><tr><td rowspan="4">Isoproterenol+L-NMMA</td><td>30 w</td><td>2</td><td>5.62 ± 0.11</td><td>19.9 ± 5.40</td><td>3</td><td>5.79 ± 0.34</td><td>63.4 ± 18.5</td><td>0.981</td><td>0.080</td></tr><tr><td>45 w</td><td>9</td><td>5.46 ± 0.24</td><td>51.9 ± 7.27</td><td>8</td><td>5.62 ± 0.22</td><td>56.0 ± 6.55</td><td>0.697</td><td>0.760</td></tr><tr><td>15 w</td><td>7</td><td>6.06 ± 0.45</td><td>60.0 ± 12.1</td><td>4</td><td>5.87 ± 0.43</td><td>52.6 ± 7.80</td><td>0.756</td><td>0.491</td></tr><tr><td>15 w</td><td>3</td><td>5.25 ± 0.14</td><td>57.0 ± 22.8</td><td>6</td><td>5.27 ± 0.27</td><td>42.1 ± 14.3</td><td>0.537</td><td>0.647</td></tr><tr><td rowspan="4">Isoproterenol+L-NIO</td><td>30 w</td><td>3</td><td>4.93 ± 0.11</td><td>33.0 ± 4.42</td><td>8</td><td>5.30 ± 0.18</td><td>45.9 ± 5.90</td><td>0.212</td><td>0.278</td></tr><tr><td>45 w</td><td>8</td><td>6.79 ± 0.638</td><td>61.9 ± 16.0</td><td>5</td><td>5.63 ± 0.512</td><td>47.9 ± 21.6</td><td>0.118</td><td>0.449</td></tr><tr><td>15 w</td><td>4</td><td>5.65 ± 0.63</td><td>35.25 ± 11.9</td><td>4</td><td>6.02 ± 0.59</td><td>18.6 ± 8.6</td><td>0.572</td><td>0.530</td></tr><tr><td>15 w</td><td>4</td><td>6.51 ± 0.700</td><td>83.2 ± 7.23</td><td>5</td><td>5.54 ± 0.15</td><td>42.3 ± 2.84</td><td>0.186</td><td>0.702</td></tr><tr><td rowspan="4">Isoproterenol+L-VNIO</td><td>30 w</td><td>4</td><td>6.09 ± 0.41</td><td>45.58 ± 13.7</td><td>7</td><td>5.26 ± 0.32</td><td>63.1 ± 12.6</td><td>0.213</td><td>0.296</td></tr><tr><td>45 w</td><td>5</td><td>4.97 ± 0.14</td><td>33.6 ± 6.15</td><td>6</td><td>5.87 ± 0.43</td><td>52.6 ± 7.80</td><td>0.756</td><td>0.491</td></tr><tr><td>15 w</td><td>4</td><td>6.51 ± 0.700</td><td>83.2 ± 7.23</td><td>4</td><td>6.32 ± 0.656</td><td>65.7 ± 8.77</td><td>0.905</td><td>0.151</td></tr><tr><td>15 w</td><td>3</td><td>5.43 ± 0.46</td><td>58.1 ± 9.44</td><td>8</td><td>5.35 ± 0.13</td><td>53.0 ± 7.52</td><td>0.896</td><td>0.589</td></tr><tr><td rowspan="4">CL 316 243</td><td>30 w</td><td>2</td><td>7.22 ± 0.00</td><td>32.0 ± 0.0</td><td>3</td><td>6.75 ± 0.74</td><td>22.8 ± 2.5</td><td>0.197</td><td>0.533</td></tr><tr><td>45 w</td><td>4</td><td>6.94 ± 0.20</td><td>43.7 ± 1.9</td><td>9</td><td>7.06 ± 0.09</td><td>30.9 ± 3.8</td><td>0.646</td><td>0.044</td></tr></tbody></table>

<table><caption>(b)</caption><thead><tr><th rowspan="2">Group</th><th colspan="3">Mesenteric arteries—WT</th><th colspan="3">Mesenteric arteries—Tgβ<sub>3</sub></th><th colspan="2">Statistics WT vs. Tgβ<sub>3</sub></th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>Emax</th></tr></thead><tbody><tr><td rowspan="4">Isoproterenol</td><td>15 w</td><td>4</td><td>7.78 ± 0.17</td><td>96.4 ± 3.0</td><td>8</td><td>7.15 ± 0.32</td><td>78.0 ± 8.5</td><td>0.112</td><td>0.151</td></tr><tr><td>30 w</td><td>8</td><td>7.03 ± 0.20</td><td>100.7 ± 4.6</td><td>8</td><td>6.67 ± 0.28</td><td>73.5 ± 10.4</td><td>0.338</td><td>0.022</td></tr><tr><td>45 w</td><td>12</td><td>7.21 ± 0.15</td><td>85.7 ± 3.4</td><td>12</td><td>6.17 ± 0.17</td><td>68.2 ± 5.5</td><td>0.001</td><td>0.061</td></tr><tr><td>15 w</td><td>4</td><td>6.67 ± 0.49</td><td>83.8 ± 14.2</td><td>6</td><td>6.82 ± 0.53</td><td>87.5 ± 8.25</td><td>0.877</td><td>0.920</td></tr><tr><td rowspan="4">Isoproterenol+L-NMMA</td><td>30 w</td><td>7</td><td>6.37 ± 0.50</td><td>92.1 ± 4.60</td><td>6</td><td>5.72 ± 0.15</td><td>86.1 ± 7.93</td><td>0.392</td><td>0.815</td></tr><tr><td>45 w</td><td>9</td><td>6.33 ± 0.47</td><td>5.07 ± 4.53</td><td>8</td><td>6.38 ± 0.47</td><td>-4.03 ± 2.60</td><td>0.918</td><td>0.533</td></tr><tr><td>15 w</td><td>3</td><td>6.04 ± 0.78</td><td>86.1 ± 1.28</td><td>9</td><td>6.89 ± 0.30</td><td>75.4 ± 8.08</td><td>0.300</td><td>0.646</td></tr><tr><td>15 w</td><td>3</td><td>5.91 ± 0.31</td><td>76.9 ± 4.70</td><td>4</td><td>6.66 ± 0.38</td><td>86.8 ± 5.66</td><td>0.195</td><td>0.344</td></tr><tr><td rowspan="4">Isoproterenol+L-NIO</td><td>30 w</td><td>5</td><td>5.83 ± 0.29</td><td>35.1 ± 5.91</td><td>9</td><td>5.65 ± 0.36</td><td>64.0 ± 7.98</td><td>0.818</td><td>0.033</td></tr><tr><td>45 w</td><td>9</td><td>7.08 ± 0.52</td><td>84.1 ± 8.64</td><td>6</td><td>6.24 ± 0.45</td><td>75.8 ± 9.94</td><td>0.221</td><td>0.744</td></tr><tr><td>15 w</td><td>6</td><td>6.29 ± 0.27</td><td>71.5 ± 4.59</td><td>6</td><td>6.78 ± 0.54</td><td>75.1 ± 8.33</td><td>0.491</td><td>0.564</td></tr><tr><td>15 w</td><td>4</td><td>6.41 ± 0.30</td><td>49.6 ± 8.63</td><td>8</td><td>6.82 ± 0.43</td><td>22.79 ± 7.38</td><td>0.392</td><td>0.171</td></tr><tr><td rowspan="4">Isoproterenol+L-VNIO</td><td>30 w</td><td>8</td><td>6.32 ± 0.40</td><td>46.52 ± 10.8</td><td>8</td><td>6.27 ± 0.29</td><td>52.7 ± 13.8</td><td>0.781</td><td>0.325</td></tr><tr><td>45 w</td><td>8</td><td>6.32 ± 0.40</td><td>46.52 ± 10.8</td><td>8</td><td>6.27 ± 0.29</td><td>52.7 ± 13.8</td><td>0.781</td><td>0.325</td></tr><tr><td>15 w</td><td>5</td><td>5.98 ± 0.61</td><td>48.4 ± 19.5</td><td>6</td><td>5.90 ± 0.14</td><td>75.7 ± 5</td><td>0.654</td><td>0.856</td></tr><tr><td>15 w</td><td>3</td><td>6.53 ± 0.64</td><td>75.2 ± 16.8</td><td>4</td><td>6.52 ± 0.65</td><td>93.2 ± 14.5</td><td>0.968</td><td>0.568</td></tr><tr><td rowspan="4">CL 316 243</td><td>30 w</td><td>3</td><td>7.96 ± 0.33</td><td>81.1 ± 7.4</td><td>7</td><td>7.68 ± 0.42</td><td>84.5 ± 5.8</td><td>0.731</td><td>0.737</td></tr><tr><td>45 w</td><td>3</td><td>7.96 ± 0.33</td><td>81.1 ± 7.4</td><td>7</td><td>7.68 ± 0.42</td><td>84.5 ± 5.8</td><td>0.731</td><td>0.737</td></tr></tbody></table>
<table><caption>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.</caption><caption>(a)</caption><thead><tr><th rowspan="2">Group</th><th rowspan="2"></th><th colspan="3">Thoracic aortic arteries—WT</th><th colspan="3">Thoracic aortic arteries—Tgβ<sub>3</sub></th><th colspan="2">Statistics WT vs. Tgβ<sub>3</sub></th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>Emax</th></tr></thead><tbody><tr><td rowspan="4">Isoproterenol</td><td>15 w</td><td>7</td><td>6.53 ± 0.33</td><td>94.5 ± 2.5</td><td>5</td><td>5.93 ± 0.20</td><td>74.1 ± 7.5</td><td>0.239</td><td>0.015</td></tr><tr><td>30 w</td><td>4</td><td>6.73 ± 0.30</td><td>85.6 ± 14.3</td><td>7</td><td>5.49 ± 0.11</td><td>79.0 ± 9.5</td><td>0.002</td><td>0.245</td></tr><tr><td>45 w</td><td>11</td><td>6.56 ± 0.21</td><td>85.9 ± 2.2</td><td>10</td><td>5.82 ± 0.15</td><td>69.2 ± 3.5</td><td>0.023</td><td>0.016</td></tr><tr><td>15 w</td><td>3</td><td>5.18 ± 0.80</td><td>15.0 ± 5.52</td><td>2</td><td>6.79 ± 2.07</td><td>25.7 ± 9.43</td><td>0.408</td><td>0.679</td></tr><tr><td rowspan="4">Isoproterenol+L-NMMA</td><td>30 w</td><td>2</td><td>5.62 ± 0.11</td><td>19.9 ± 5.40</td><td>3</td><td>5.79 ± 0.34</td><td>63.4 ± 18.5</td><td>0.981</td><td>0.080</td></tr><tr><td>45 w</td><td>9</td><td>5.46 ± 0.24</td><td>51.9 ± 7.27</td><td>8</td><td>5.62 ± 0.22</td><td>56.0 ± 6.55</td><td>0.697</td><td>0.760</td></tr><tr><td>15 w</td><td>7</td><td>6.06 ± 0.45</td><td>60.0 ± 12.1</td><td>4</td><td>5.87 ± 0.43</td><td>52.6 ± 7.80</td><td>0.756</td><td>0.491</td></tr><tr><td>15 w</td><td>3</td><td>5.25 ± 0.14</td><td>57.0 ± 22.8</td><td>6</td><td>5.27 ± 0.27</td><td>42.1 ± 14.3</td><td>0.537</td><td>0.647</td></tr><tr><td rowspan="4">Isoproterenol+L-NIO</td><td>30 w</td><td>3</td><td>4.93 ± 0.11</td><td>33.0 ± 4.42</td><td>8</td><td>5.30 ± 0.18</td><td>45.9 ± 5.90</td><td>0.212</td><td>0.278</td></tr><tr><td>45 w</td><td>8</td><td>6.79 ± 0.638</td><td>61.9 ± 16.0</td><td>5</td><td>5.63 ± 0.512</td><td>47.9 ± 21.6</td><td>0.118</td><td>0.449</td></tr><tr><td>15 w</td><td>4</td><td>5.65 ± 0.63</td><td>35.25 ± 11.9</td><td>4</td><td>6.02 ± 0.59</td><td>18.6 ± 8.6</td><td>0.572</td><td>0.530</td></tr><tr><td>15 w</td><td>4</td><td>6.51 ± 0.700</td><td>83.2 ± 7.23</td><td>5</td><td>5.54 ± 0.15</td><td>42.3 ± 2.84</td><td>0.186</td><td>0.702</td></tr><tr><td rowspan="4">Isoproterenol+L-VNIO</td><td>30 w</td><td>4</td><td>6.09 ± 0.41</td><td>45.58 ± 13.7</td><td>7</td><td>5.26 ± 0.32</td><td>63.1 ± 12.6</td><td>0.213</td><td>0.296</td></tr><tr><td>45 w</td><td>5</td><td>4.97 ± 0.14</td><td>33.6 ± 6.15</td><td>6</td><td>5.35 ± 0.13</td><td>53.0 ± 7.52</td><td>0.896</td><td>0.589</td></tr><tr><td>15 w</td><td>4</td><td>5.43 ± 0.46</td><td>58.1 ± 9.44</td><td>8</td><td>6.75 ± 0.74</td><td>22.8 ± 2.5</td><td>0.197</td><td>0.533</td></tr><tr><td>15 w</td><td>3</td><td>6.16 ± 0.67</td><td>17.1 ± 6.9</td><td>4</td><td>6.87 ± 0.45</td><td>26.3 ± 5.0</td><td>0.785</td><td>0.442</td></tr><tr><td rowspan="2">CL 316 243</td><td>30 w</td><td>2</td><td>7.22 ± 0.00</td><td>32.0 ± 0.0</td><td>3</td><td>7.06 ± 0.09</td><td>30.9 ± 3.8</td><td>0.646</td><td>0.044</td></tr><tr><td>45 w</td><td>4</td><td>6.94 ± 0.20</td><td>43.7 ± 1.9</td><td>9</td><td>7.06 ± 0.09</td><td>30.9 ± 3.8</td><td>0.646</td><td>0.044</td></tr></tbody></table>

<table><caption>(b)</caption><thead><tr><th rowspan="2">Group</th><th rowspan="2"></th><th colspan="3">Mesenteric arteries—WT</th><th colspan="3">Mesenteric arteries—Tgβ<sub>3</sub></th><th colspan="2">Statistics WT vs. Tgβ<sub>3</sub></th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>Emax</th></tr></thead><tbody><tr><td rowspan="4">Isoproterenol</td><td>15 w</td><td>4</td><td>7.78 ± 0.17</td><td>96.4 ± 3.0</td><td>8</td><td>7.15 ± 0.32</td><td>78.0 ± 8.5</td><td>0.112</td><td>0.151</td></tr><tr><td>30 w</td><td>8</td><td>7.03 ± 0.20</td><td>100.7 ± 4.6</td><td>8</td><td>6.67 ± 0.28</td><td>73.5 ± 10.4</td><td>0.338</td><td>0.022</td></tr><tr><td>45 w</td><td>12</td><td>7.21 ± 0.15</td><td>85.7 ± 3.4</td><td>12</td><td>6.17 ± 0.17</td><td>68.2 ± 5.5</td><td>0.001</td><td>0.061</td></tr><tr><td>15 w</td><td>4</td><td>6.67 ± 0.49</td><td>83.8 ± 14.2</td><td>6</td><td>6.82 ± 0.53</td><td>87.5 ± 8.25</td><td>0.877</td><td>0.920</td></tr><tr><td rowspan="4">Isoproterenol+L-NMMA</td><td>30 w</td><td>7</td><td>6.37 ± 0.50</td><td>92.1 ± 4.60</td><td>6</td><td>5.72 ± 0.15</td><td>86.1 ± 7.93</td><td>0.392</td><td>0.815</td></tr><tr><td>45 w</td><td>9</td><td>6.33 ± 0.47</td><td>5.07 ± 4.53</td><td>8</td><td>6.38 ± 0.47</td><td>-4.03 ± 2.60</td><td>0.918</td><td>0.533</td></tr><tr><td>15 w</td><td>3</td><td>6.04 ± 0.78</td><td>86.1 ± 1.28</td><td>9</td><td>6.89 ± 0.30</td><td>75.4 ± 8.08</td><td>0.300</td><td>0.646</td></tr><tr><td>15 w</td><td>3</td><td>5.91 ± 0.31</td><td>76.9 ± 4.70</td><td>4</td><td>6.66 ± 0.38</td><td>86.8 ± 5.66</td><td>0.195</td><td>0.344</td></tr><tr><td rowspan="4">Isoproterenol+L-NIO</td><td>30 w</td><td>5</td><td>5.83 ± 0.29</td><td>35.1 ± 5.91</td><td>9</td><td>5.65 ± 0.36</td><td>64.0 ± 7.98</td><td>0.818</td><td>0.033</td></tr><tr><td>45 w</td><td>9</td><td>7.08 ± 0.52</td><td>84.1 ± 8.64</td><td>6</td><td>6.24 ± 0.45</td><td>75.8 ± 9.94</td><td>0.221</td><td>0.744</td></tr><tr><td>15 w</td><td>6</td><td>6.29 ± 0.27</td><td>71.5 ± 4.59</td><td>6</td><td>6.78 ± 0.54</td><td>75.1 ± 8.33</td><td>0.491</td><td>0.564</td></tr><tr><td>15 w</td><td>4</td><td>6.41 ± 0.30</td><td>49.6 ± 8.63</td><td>8</td><td>6.82 ± 0.43</td><td>22.79 ± 7.38</td><td>0.392</td><td>0.171</td></tr><tr><td rowspan="4">Isoproterenol+L-VNIO</td><td>30 w</td><td>8</td><td>6.32 ± 0.40</td><td>46.52 ± 10.8</td><td>8</td><td>6.27 ± 0.29</td><td>52.7 ± 13.8</td><td>0.781</td><td>0.325</td></tr><tr><td>45 w</td><td>8</td><td>5.98 ± 0.61</td><td>48.4 ± 19.5</td><td>6</td><td>5.90 ± 0.14</td><td>75.7 ± 5</td><td>0.654</td><td>0.856</td></tr><tr><td>15 w</td><td>5</td><td>6.53 ± 0.64</td><td>75.2 ± 16.8</td><td>4</td><td>6.52 ± 0.65</td><td>93.2 ± 14.5</td><td>0.968</td><td>0.568</td></tr><tr><td>15 w</td><td>3</td><td>7.96 ± 0.33</td><td>81.1 ± 7.4</td><td>7</td><td>7.68 ± 0.42</td><td>84.5 ± 5.8</td><td>0.731</td><td>0.737</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>FIGURE 3: Continued.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>FIGURE 3: Continued.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>FIGURE 3: Continued.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>FIGURE 3: Continued.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>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. *p* < 0.05: WT versus WT+inhibitor; $p$ < 0.05: Tgβ₃ versus Tgβ₃+inhibitor.</figcaption></figure>

### 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.

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
<figure><img src="image_8.png" /><figcaption>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.</figcaption></figure>

### 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.

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
<caption>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.</caption>

<table><caption>(a)</caption><thead><tr><th rowspan="2">Group</th><th colspan="3">Thoracic aortic arteries—WT</th><th colspan="3">Thoracic aortic arteries—Tgβ<sub>3</sub></th><th colspan="3">WT vs. WT+isoproterenol</th><th colspan="2">Tgβ<sub>3</sub> vs. Tgβ<sub>3</sub>+isoproterenol</th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>p Emax</th><th>p Emax</th><th>p pD<sub>2</sub></th><th>p Emax</th></tr></thead><tbody><tr><td rowspan="3">Isoproterenol</td><td>15 w</td><td>7</td><td>6.53 ± 0.33</td><td>94.5 ± 2.5</td><td>5</td><td>5.93 ± 0.20</td><td>74.1 ± 7.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>30 w</td><td>4</td><td>6.73 ± 0.30</td><td>85.6 ± 14.3</td><td>7</td><td>5.49 ± 0.11</td><td>79.0 ± 9.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>45 w</td><td>11</td><td>6.56 ± 0.21</td><td>85.9 ± 2.2</td><td>10</td><td>5.82 ± 0.15</td><td>69.2 ± 3.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-NIO</td><td>15 w</td><td>7</td><td>6.06 ± 0.45</td><td>60.0 ± 12.1</td><td>4</td><td>5.87 ± 0.43</td><td>52.6 ± 7.80</td><td>0.430</td><td>0.013</td><td>0.939</td><td>0.322</td><td></td></tr><tr><td>30 w</td><td>3</td><td>5.25 ± 0.14</td><td>57.0 ± 22.8</td><td>6</td><td>5.27 ± 0.27</td><td>42.1 ± 14.3</td><td>0.006</td><td>0.083</td><td>0.661</td><td>0.080</td><td></td></tr><tr><td>45 w</td><td>8</td><td>4.93 ± 0.11</td><td>33.0 ± 4.42</td><td>8</td><td>5.30 ± 0.18</td><td>45.9 ± 5.90</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0.134</td><td>0.088</td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-VNIO</td><td>15 w</td><td>4</td><td>6.79 ± 0.638</td><td>61.9 ± 16.0</td><td>5</td><td>5.63 ± 0.512</td><td>47.9 ± 21.6</td><td>0.620</td><td>0.125</td><td>0.799</td><td>0.414</td><td></td></tr><tr><td>30 w</td><td>4</td><td>5.65 ± 0.63</td><td>35.25 ± 11.9</td><td>4</td><td>6.02 ± 0.59</td><td>18.6 ± 8.6</td><td>0.090</td><td>0.024</td><td>0.346</td><td>0.019</td><td></td></tr><tr><td>45 w</td><td>5</td><td>4.97 ± 0.14</td><td>33.6 ± 6.15</td><td>5</td><td>5.54 ± 0.15</td><td>42.3 ± 2.84</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0.377</td><td>0.068</td><td></td></tr><tr><td rowspan="3">Isoproterenol+1400W</td><td>15 w</td><td>4</td><td>6.51 ± 0.700</td><td>83.2 ± 7.23</td><td>4</td><td>6.32 ± 0.656</td><td>65.7 ± 8.77</td><td>0.665</td><td>0.259</td><td>0.588</td><td>0.623</td><td></td></tr><tr><td>30 w</td><td>4</td><td>6.09 ± 0.41</td><td>45.58 ± 13.7</td><td>7</td><td>5.26 ± 0.32</td><td>63.1 ± 12.6</td><td>0.191</td><td>0.019</td><td>0.902</td><td>0.323</td><td></td></tr><tr><td>45 w</td><td>7</td><td>5.43 ± 0.46</td><td>58.1 ± 9.44</td><td>8</td><td>5.35 ± 0.13</td><td>53.0 ± 7.52</td><td>0.002</td><td>0.002</td><td>0.105</td><td>0.265</td><td></td></tr></tbody></table>

<table><caption>(b)</caption><thead><tr><th rowspan="2">Group</th><th colspan="3">Mesenteric arteries—WT</th><th colspan="3">Mesenteric arteries—Tgβ<sub>3</sub></th><th colspan="3">WT vs. WT+isoproterenol</th><th colspan="2">Tgβ<sub>3</sub> vs. Tgβ<sub>3</sub>+isoproterenol</th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>p Emax</th><th>p Emax</th><th>p pD<sub>2</sub></th><th>p Emax</th></tr></thead><tbody><tr><td rowspan="3">Isoproterenol</td><td>15 w</td><td>4</td><td>7.78 ± 0.17</td><td>96.4 ± 3.0</td><td>8</td><td>7.15 ± 0.32</td><td>78.0 ± 8.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>30 w</td><td>8</td><td>7.03 ± 0.20</td><td>100.7 ± 4.6</td><td>8</td><td>6.67 ± 0.28</td><td>73.5 ± 10.4</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>45 w</td><td>12</td><td>7.21 ± 0.15</td><td>85.7 ± 3.4</td><td>12</td><td>6.17 ± 0.17</td><td>68.2 ± 5.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-NIO</td><td>15 w</td><td>3</td><td>6.04 ± 0.78</td><td>86.1 ± 1.28</td><td>9</td><td>6.89 ± 0.30</td><td>75.4 ± 8.08</td><td>0.024</td><td>0.431</td><td>0.555</td><td>0.776</td><td></td></tr><tr><td>30 w</td><td>5</td><td>5.91 ± 0.31</td><td>76.9 ± 4.70</td><td>4</td><td>6.66 ± 0.38</td><td>86.8 ± 5.66</td><td>0.014</td><td>0.003</td><td>0.978</td><td>0.627</td><td></td></tr><tr><td>45 w</td><td>9</td><td>5.83 ± 0.29</td><td>35.1 ± 5.91</td><td>9</td><td>5.65 ± 0.36</td><td>64.0 ± 7.98</td><td>0.0004</td><td>&lt;0.0001</td><td>0.408</td><td>0.640</td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-VNIO</td><td>15 w</td><td>6</td><td>7.08 ± 0.52</td><td>84.1 ± 8.64</td><td>6</td><td>6.24 ± 0.45</td><td>75.8 ± 9.94</td><td>0.290</td><td>0.361</td><td>0.127</td><td>0.844</td><td></td></tr><tr><td>30 w</td><td>3</td><td>6.29 ± 0.27</td><td>71.5 ± 4.59</td><td>6</td><td>6.78 ± 0.54</td><td>75.1 ± 8.33</td><td>0.195</td><td>0.013</td><td>0.850</td><td>0.777</td><td></td></tr><tr><td>45 w</td><td>8</td><td>6.41 ± 0.30</td><td>49.6 ± 8.63</td><td>8</td><td>6.82 ± 0.43</td><td>22.79 ± 7.38</td><td>0.032</td><td>0.003</td><td>0.079</td><td>0.004</td><td></td></tr><tr><td rowspan="3">Isoproterenol+1400W</td><td>15 w</td><td>4</td><td>7.34 ± 0.25</td><td>96.4 ± 2.02</td><td>7</td><td>6.49 ± 0.31</td><td>81.8 ± 8.00</td><td>0.404</td><td>0.980</td><td>0.125</td><td>0.780</td><td></td></tr><tr><td>30 w</td><td>8</td><td>6.64 ± 0.30</td><td>84.0 ± 10.9</td><td>6</td><td>6.43 ± 0.44</td><td>92.3 ± 3.23</td><td>0.378</td><td>0.418</td><td>0.680</td><td>0.307</td><td></td></tr><tr><td>45 w</td><td>8</td><td>6.32 ± 0.40</td><td>46.52 ± 10.8</td><td>8</td><td>6.27 ± 0.29</td><td>52.7 ± 13.8</td><td>0.013</td><td>0.002</td><td>0.679</td><td>0.773</td><td></td></tr></tbody></table>
<caption>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.</caption>

<table><caption>(a)</caption><thead><tr><th rowspan="2">Group</th><th colspan="3">Thoracic aortic arteries—WT</th><th colspan="3">Thoracic aortic arteries—Tgβ<sub>3</sub></th><th colspan="3">WT vs. WT+isoproterenol</th><th colspan="2">Tgβ<sub>3</sub> vs. Tgβ<sub>3</sub>+isoproterenol</th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>p Emax</th><th>p Emax</th><th>p pD<sub>2</sub></th><th>p Emax</th></tr></thead><tbody><tr><td rowspan="3">Isoproterenol</td><td>15 w</td><td>7</td><td>6.53 ± 0.33</td><td>94.5 ± 2.5</td><td>5</td><td>5.93 ± 0.20</td><td>74.1 ± 7.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>30 w</td><td>4</td><td>6.73 ± 0.30</td><td>85.6 ± 14.3</td><td>7</td><td>5.49 ± 0.11</td><td>79.0 ± 9.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>45 w</td><td>11</td><td>6.56 ± 0.21</td><td>85.9 ± 2.2</td><td>10</td><td>5.82 ± 0.15</td><td>69.2 ± 3.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-NIO</td><td>15 w</td><td>7</td><td>6.06 ± 0.45</td><td>60.0 ± 12.1</td><td>4</td><td>5.87 ± 0.43</td><td>52.6 ± 7.80</td><td>0.430</td><td>0.013</td><td>0.939</td><td>0.322</td><td></td></tr><tr><td>30 w</td><td>3</td><td>5.25 ± 0.14</td><td>57.0 ± 22.8</td><td>6</td><td>5.27 ± 0.27</td><td>42.1 ± 14.3</td><td>0.006</td><td>0.083</td><td>0.661</td><td>0.080</td><td></td></tr><tr><td>45 w</td><td>8</td><td>4.93 ± 0.11</td><td>33.0 ± 4.42</td><td>8</td><td>5.30 ± 0.18</td><td>45.9 ± 5.90</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0.134</td><td>0.088</td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-VNIO</td><td>15 w</td><td>4</td><td>6.79 ± 0.638</td><td>61.9 ± 16.0</td><td>5</td><td>5.63 ± 0.512</td><td>47.9 ± 21.6</td><td>0.620</td><td>0.125</td><td>0.799</td><td>0.414</td><td></td></tr><tr><td>30 w</td><td>4</td><td>5.65 ± 0.63</td><td>35.25 ± 11.9</td><td>4</td><td>6.02 ± 0.59</td><td>18.6 ± 8.6</td><td>0.090</td><td>0.024</td><td>0.346</td><td>0.019</td><td></td></tr><tr><td>45 w</td><td>5</td><td>4.97 ± 0.14</td><td>33.6 ± 6.15</td><td>5</td><td>5.54 ± 0.15</td><td>42.3 ± 2.84</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0.377</td><td>0.068</td><td></td></tr><tr><td rowspan="3">Isoproterenol+1400W</td><td>15 w</td><td>4</td><td>6.51 ± 0.700</td><td>83.2 ± 7.23</td><td>4</td><td>6.32 ± 0.656</td><td>65.7 ± 8.77</td><td>0.665</td><td>0.259</td><td>0.588</td><td>0.623</td><td></td></tr><tr><td>30 w</td><td>4</td><td>6.09 ± 0.41</td><td>45.58 ± 13.7</td><td>7</td><td>5.26 ± 0.32</td><td>63.1 ± 12.6</td><td>0.191</td><td>0.019</td><td>0.902</td><td>0.323</td><td></td></tr><tr><td>45 w</td><td>7</td><td>5.43 ± 0.46</td><td>58.1 ± 9.44</td><td>8</td><td>5.35 ± 0.13</td><td>53.0 ± 7.52</td><td>0.002</td><td>0.002</td><td>0.105</td><td>0.265</td><td></td></tr></tbody></table>

<table><caption>(b)</caption><thead><tr><th rowspan="2">Group</th><th colspan="3">Mesenteric arteries—WT</th><th colspan="3">Mesenteric arteries—Tgβ<sub>3</sub></th><th colspan="3">WT vs. WT+isoproterenol</th><th colspan="2">Tgβ<sub>3</sub> vs. Tgβ<sub>3</sub>+isoproterenol</th></tr><tr><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>n</th><th>pD<sub>2</sub></th><th>Emax</th><th>pD<sub>2</sub></th><th>p Emax</th><th>p Emax</th><th>p pD<sub>2</sub></th><th>p Emax</th></tr></thead><tbody><tr><td rowspan="3">Isoproterenol</td><td>15 w</td><td>4</td><td>7.78 ± 0.17</td><td>96.4 ± 3.0</td><td>8</td><td>7.15 ± 0.32</td><td>78.0 ± 8.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>30 w</td><td>8</td><td>7.03 ± 0.20</td><td>100.7 ± 4.6</td><td>8</td><td>6.67 ± 0.28</td><td>73.5 ± 10.4</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>45 w</td><td>12</td><td>7.21 ± 0.15</td><td>85.7 ± 3.4</td><td>12</td><td>6.17 ± 0.17</td><td>68.2 ± 5.5</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-NIO</td><td>15 w</td><td>3</td><td>6.04 ± 0.78</td><td>86.1 ± 1.28</td><td>9</td><td>6.89 ± 0.30</td><td>75.4 ± 8.08</td><td>0.024</td><td>0.431</td><td>0.555</td><td>0.776</td><td></td></tr><tr><td>30 w</td><td>5</td><td>5.91 ± 0.31</td><td>76.9 ± 4.70</td><td>4</td><td>6.66 ± 0.38</td><td>86.8 ± 5.66</td><td>0.014</td><td>0.003</td><td>0.978</td><td>0.627</td><td></td></tr><tr><td>45 w</td><td>9</td><td>5.83 ± 0.29</td><td>35.1 ± 5.91</td><td>9</td><td>5.65 ± 0.36</td><td>64.0 ± 7.98</td><td>0.0004</td><td>&lt;0.0001</td><td>0.408</td><td>0.640</td><td></td></tr><tr><td rowspan="3">Isoproterenol+L-VNIO</td><td>15 w</td><td>6</td><td>7.08 ± 0.52</td><td>84.1 ± 8.64</td><td>6</td><td>6.24 ± 0.45</td><td>75.8 ± 9.94</td><td>0.290</td><td>0.361</td><td>0.127</td><td>0.844</td><td></td></tr><tr><td>30 w</td><td>3</td><td>6.29 ± 0.27</td><td>71.5 ± 4.59</td><td>6</td><td>6.78 ± 0.54</td><td>75.1 ± 8.33</td><td>0.195</td><td>0.013</td><td>0.850</td><td>0.777</td><td></td></tr><tr><td>45 w</td><td>8</td><td>6.41 ± 0.30</td><td>49.6 ± 8.63</td><td>8</td><td>6.82 ± 0.43</td><td>22.79 ± 7.38</td><td>0.032</td><td>0.003</td><td>0.079</td><td>0.004</td><td></td></tr><tr><td rowspan="3">Isoproterenol+1400W</td><td>15 w</td><td>4</td><td>7.34 ± 0.25</td><td>96.4 ± 2.02</td><td>7</td><td>6.49 ± 0.31</td><td>81.8 ± 8.00</td><td>0.404</td><td>0.980</td><td>0.125</td><td>0.780</td><td></td></tr><tr><td>30 w</td><td>8</td><td>6.64 ± 0.30</td><td>84.0 ± 10.9</td><td>6</td><td>6.43 ± 0.44</td><td>92.3 ± 3.23</td><td>0.378</td><td>0.418</td><td>0.680</td><td>0.307</td><td></td></tr><tr><td>45 w</td><td>8</td><td>6.32 ± 0.40</td><td>46.52 ± 10.8</td><td>8</td><td>6.27 ± 0.29</td><td>52.7 ± 13.8</td><td>0.013</td><td>0.002</td><td>0.679</td><td>0.773</td><td></td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>FIGURE 4: Continued.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>FIGURE 4: Continued.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>FIGURE 4: Continued.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>FIGURE 4: Continued.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>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.</figcaption></figure>

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 (Figures 6(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.

### 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)).
<figure><img src="image_11.png" /><figcaption>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.</figcaption></figure>

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 (Figures 6(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.

### 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)).
<figure><img src="image_11.png" /><figcaption>FIGURE 5: Continued.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>FIGURE 5: Continued.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>FIGURE 5: Continued.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>FIGURE 5: Continued.</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>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. *p* < 0.05: WT versus WT+inhibitor; $p$ < 0.05: Tgβ₃ versus Tgβ₃+inhibitor.</figcaption></figure>

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
<figure><img src="image_14.png" /></figure>

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. *p* < 0.05: WT versus WT+inhibitor; $p$ < 0.05: Tgβ₃ versus Tgβ₃+inhibitor.

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
<figure><img src="image_14.png" /><figcaption>FIGURE 6: Continued.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>FIGURE 6: Continued.</figcaption></figure>
<figure><img src="image_15.png" /></figure>

NOS2 protein expression

<figure><img src="image_16.png" /></figure>

(c)

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

NOS1 protein expression

<figure><img src="image_19.png" /></figure>

(d)

FIGURE 6: Continued.
<figure><img src="image_16.png" /></figure>

NOS2 protein expression

<figure><img src="image_17.png" /></figure>

(c)

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

NOS1 protein expression

<figure><img src="image_20.png" /></figure>

(d)

FIGURE 6: Continued.
<figure><img src="image_20.png" /><figcaption>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. *p* < 0.05, **p** < 0.01.</figcaption></figure>

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.

### 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><img src="image_21.png" /><figcaption>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. * *p* < 0.05, ** *p* < 0.001.</figcaption></figure>

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.

### 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)).
Vascular reactivity to isoproterenol with L-NIO in aorta

<figure><img src="image_21.png" /></figure>

Vascular reactivity to isoproterenol
with L-NIO in mesenteric arteries

<figure><img src="image_22.png" /></figure>

FIGURE 7: Continued.
Vascular reactivity to isoproterenol with L-NIO in aorta

<figure><img src="image_22.png" /></figure>

Vascular reactivity to isoproterenol
with L-NIO in mesenteric arteries

<figure><img src="image_23.png" /></figure>

FIGURE 7: Continued.
<figure><img src="image_23.png" /><figcaption>FIGURE 7: Continued.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>FIGURE 7: Continued.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>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.</figcaption></figure>

3.5.3. *O*₂\*\* *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 *O*₂\*\* 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 *O*₂\*\* 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.
<figure><img src="image_25.png" /><figcaption>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. *p* = 5-12. \*p* < 0.05: WT versus WT+inhibitor; \*\*p* < 0.05: Tgβ₃ versus Tgβ₃+inhibitor.</figcaption></figure>

3.5.3. *O*₂\*\* Production Is Increased in *Tgβ*₃ 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.
<figure><img src="image_25.png" /><figcaption>FIGURE 8: Continued.</figcaption></figure>
<figure><img src="image_26.png" /><figcaption>FIGURE 8: Continued.</figcaption></figure>
<figure><img src="image_26.png" /><figcaption>FIGURE 8: Continued.</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>FIGURE 8: Continued.</figcaption></figure>
<figure><img src="image_27.png" /><figcaption>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.</figcaption></figure>

## 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 that β₃-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.
<figure><img src="image_28.png" /><figcaption>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. * *p* < 0.05, ** *p* < 0.01, and *** *p* < 0.001.</figcaption></figure>

4. Discussion

The aim of this study was to decipher the link between endo-
thelial 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 that β₃-AR overex-
pression 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 associ- ated with the cardioprotective effect [23]. In our study how- ever, 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 func- tion. 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 dif-
ferences 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 dys-
function 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 human β3-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.

**Data Availability**

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Ethical Approval**

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.

**Conflicts of Interest**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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 human β3-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.

## Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## Ethical Approval

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.

## Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
## Authors' Contributions

All authors have reviewed and approved the manuscript. TD, TP, JD, CG, and BL were responsible for the conceptualization. MB, VA, AE, AT, DS, AP, ABL, CM, and VS were responsible for the investigation. TD, TP, JD, BR, MDW, CG, and BL wrote the original draft. TD, TP, JD, BR, and BL reviewed and edited the paper. VS, BR, MDW, CG, and BL were responsible for the resources and funding acquisition. Thomas Dupas and Thomas Pelé have contributed equally to this work and share the first authorship.

## Acknowledgments

We thank the Therassay platform for their technical assistance. This study was funded by the "Agence Nationale de la Recherche" (ANR-19-CE14-0025, ANR-13-BSV1-0003, and ANR-11-LABX-0015, Paris, France), "Fédération Française de Cardiologie" (Paris, France), "Fondation de l'Avenir pour la Recherche Médicale Appliquée" (Paris, France), "Fondation de France" (Paris, France), "Fondation Genavie" (Nantes, France), and "Institut National de la Santé et de la Recherche Médicale" (Paris, France).

## References

[1] G. A. Roth, G. A. Mensah, C. O. Johnson et al., "Global burden of cardiovascular diseases and risk factors, 1990-2019: update from the GBD 2019 Study," *Journal of the American College of Cardiology*, vol. 76, no. 25, pp. 2982-3021, 2020.

[2] M. Metra and J. R. Teerlink, "Heart failure," *Lancet*, vol. 390, no. 10106, pp. 1981-1995, 2017.

[3] A. A. Oktay, J. D. Rich, and S. J. Shah, "The emerging epidemic of heart failure with preserved ejection fraction," *Current Heart Failure Reports*, vol. 10, no. 4, pp. 401-410, 2013.

[4] W. J. Paulus and C. Tschöpe, "A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation," *Journal of the American College of Cardiology*, vol. 62, no. 4, pp. 263-271, 2013.

[5] I. Fleming, "Molecular mechanisms underlying the activation of ENOS," *Pflügers Archiv - European Journal of Physiology*, vol. 459, no. 6, pp. 793-806, 2010.

[6] V. W. T. Liu and P. L. Huang, "Cardiovascular roles of nitric oxide: a review of insights from nitric oxide synthase gene disrupted mice," *Cardiovascular Research*, vol. 77, no. 1, pp. 19-29, 2008.

[7] C. Gauthier, V. Leblais, L. Kobzik et al., "The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle," *The Journal of Clinical Investigation*, vol. 102, no. 7, pp. 1377-1384, 1998.

[8] D. M. Trappanese, Y. Liu, R. C. McCormick et al., "Chronic B1-adrenergic blockade enhances myocardial B3-adrenergic coupling with nitric oxide-CGMP signaling in a canine model of chronic volume overload: new insight into mechanisms of cardiac benefit with selective B1-blocker therapy," *Basic Research in Cardiology*, vol. 110, no. 1, p. 456, 2015.

[9] A. L. Moens, R. Yang, V. L. Watts, and L. A. Barouch, "Beta 3-adrenoreceptor regulation of nitric oxide in the cardiovascular

system," *Journal of Molecular and Cellular Cardiology*, vol. 48, no. 6, pp. 1088-1095, 2010.

[10] J. Dhot, M. Ferron, V. Prat et al., "Overexpression of endothelial β3-adrenergic receptor induces diastolic dysfunction in rats," *ESC heart failure*, vol. 7, no. 6, pp. 4159-4171, 2020.

[11] T. Tran Quang, B. Rozec, L. Audigane, and C. Gauthier, "Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta," *British Journal of Pharmacology*, vol. 156, no. 4, pp. 601-608, 2009.

[12] V. Sauzeau, M. A. Sevilla, M. J. Montero, and X. R. Bustelo, "The Rho/Rac exchange factor Vav2 controls nitric oxide-dependent responses in mouse vascular smooth muscle cells," *The Journal of Clinical Investigation*, vol. 120, no. 1, pp. 315-330, 2010.

[13] B. Lauzier, P. Sicard, O. Bouchot et al., "After four hours of cold ischemia and cardioplegic protocol, the heart can still be rescued with postconditioning," *Transplantation*, vol. 84, no. 11, pp. 1474-1482, 2007.

[14] A. Agouni, A.-H. Lagrue-Lak-Hal, H. A. Mostefai et al., "Red wine polyphenols prevent metabolic and cardiovascular alterations associated with obesity in Zucker fatty rats (Fa/Fa)," *PLoS One*, vol. 4, no. 5, article e5557, 2009.

[15] T. Dupas, M. Denis, J. Dontaine et al., "ProteinO-GlcNAcylation levels are regulated independently of dietary intake in a tissue and time-specific manner during rat postnatal development," *Acta Physiologica*, vol. 231, no. 3, p. doi:10.1111/apha.13566, 2021.

[16] M. Perez-Aso, N. Flacco, N. Carpena, M. C. Montesinos, P. D'Ocon, and M. D. Ivorra, "β-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and P38," *Vascular Pharmacology*, vol. 61, no. 2-3, pp. 80-89, 2014.

[17] S. Moniotte, L. Kobzik, O. Feron, J. N. Trochu, C. Gauthier, and J. L. Balligand, "Upregulation of β3-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium," *Circulation*, vol. 103, no. 12, pp. 1649-1655, 2001.

[18] Y. Kong, W. Li, and Y. Tian, "Effect of beta3-adrenoreceptors agonist on beta3-adrenoreceptors expression and myocyte apoptosis in a rat model of heart failure," *Zhongguo Wei Zhong Bing Ji Jiu Yi Xue*, vol. 16, no. 3, pp. 142-147, 2004.

[19] X. Niu, V. L. Watts, O. H. Cingolani et al., "Cardioprotective effect of beta-3 adrenergic receptor agonism: role of neuronal nitric oxide synthase," *Journal of the American College of Cardiology*, vol. 59, no. 22, pp. 1979-1987, 2012.

[20] N. Hermida, L. Michel, H. Esfahani et al., "Cardiac myocyte B3-adrenergic receptors prevent myocardial fibrosis by modulating oxidant stress-dependent paracrine signaling," *European Heart Journal*, vol. 39, no. 10, pp. 888-898, 2018.

[21] R. Salie, A. K. H. Alsalhin, E. Marais, and A. Lochner, "Cardio-protective effects of beta3-adrenergic receptor (B3-AR) pre-, per-, and post-treatment in ischemia-reperfusion," *Cardiovascular Drugs and Therapy*, vol. 33, no. 2, pp. 163-177, 2019.

[22] L. Y. M. Michel, C. Farah, and J.-L. Balligand, "The Beta3 adrenergic receptor in healthy and pathological cardiovascular tissues," *Cell*, vol. 9, no. 12, p. 2584, 2020.

[23] C. Belge, J. Hammond, E. Dubois-Deruy et al., "Enhanced expression of B3-adrenoceptors in cardiac myocytes attenuates neurohormone-induced hypertrophic remodeling through nitric oxide synthase," *Circulation*, vol. 129, no. 4, pp. 451-462, 2014.
## Authors' Contributions

All authors have reviewed and approved the manuscript. TD, TP, JD, CG, and BL were responsible for the conceptualization. MB, VA, AE, AT, DS, AP, ABL, CM, and VS were responsible for the investigation. TD, TP, JD, BR, MDW, CG, and BL wrote the original draft. TD, TP, JD, BR, and BL reviewed and edited the paper. VS, BR, MDW, CG, and BL were responsible for the resources and funding acquisition. Thomas Dupas and Thomas Pelé have contributed equally to this work and share the first authorship.

## Acknowledgments

We thank the Therassay platform for their technical assistance. This study was funded by the "Agence Nationale de la Recherche" (ANR-19-CE14-0025, ANR-13-BSV1-0003, and ANR-11-LABX-0015, Paris, France), "Fédération Française de Cardiologie" (Paris, France), "Fondation de l'Avenir pour la Recherche Médicale Appliquée" (Paris, France), "Fondation de France" (Paris, France), "Fondation Genavie" (Nantes, France), and "Institut National de la Santé et de la Recherche Médicale" (Paris, France).

## References

[1] G. A. Roth, G. A. Mensah, C. O. Johnson et al., "Global burden of cardiovascular diseases and risk factors, 1990-2019: update from the GBD 2019 Study," *Journal of the American College of Cardiology*, vol. 76, no. 25, pp. 2982-3021, 2020.

[2] M. Metra and J. R. Teerlink, "Heart failure," *Lancet*, vol. 390, no. 10106, pp. 1981-1995, 2017.

[3] A. A. Oktay, J. D. Rich, and S. J. Shah, "The emerging epidemic of heart failure with preserved ejection fraction," *Current Heart Failure Reports*, vol. 10, no. 4, pp. 401-410, 2013.

[4] W. J. Paulus and C. Tschöpe, "A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation," *Journal of the American College of Cardiology*, vol. 62, no. 4, pp. 263-271, 2013.

[5] I. Fleming, "Molecular mechanisms underlying the activation of ENOS," *Pflügers Archiv - European Journal of Physiology*, vol. 459, no. 6, pp. 793-806, 2010.

[6] V. W. T. Liu and P. L. Huang, "Cardiovascular roles of nitric oxide: a review of insights from nitric oxide synthase gene disrupted mice," *Cardiovascular Research*, vol. 77, no. 1, pp. 19-29, 2008.

[7] C. Gauthier, V. Leblais, L. Kobzik et al., "The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle," *The Journal of Clinical Investigation*, vol. 102, no. 7, pp. 1377-1384, 1998.

[8] D. M. Trappanese, Y. Liu, R. C. McCormick et al., "Chronic B1-adrenergic blockade enhances myocardial B3-adrenergic coupling with nitric oxide-CGMP signaling in a canine model of chronic volume overload: new insight into mechanisms of cardiac benefit with selective B1-blocker therapy," *Basic Research in Cardiology*, vol. 110, no. 1, p. 456, 2015.

[9] A. L. Moens, R. Yang, V. L. Watts, and L. A. Barouch, "Beta 3-adrenoreceptor regulation of nitric oxide in the cardiovascular

system," *Journal of Molecular and Cellular Cardiology*, vol. 48, no. 6, pp. 1088-1095, 2010.

[10] J. Dhot, M. Ferron, V. Prat et al., "Overexpression of endothelial β3-adrenergic receptor induces diastolic dysfunction in rats," *ESC heart failure*, vol. 7, no. 6, pp. 4159-4171, 2020.

[11] T. Tran Quang, B. Rozec, L. Audigane, and C. Gauthier, "Investigation of the different adrenoceptor targets of nebivolol enantiomers in rat thoracic aorta," *British Journal of Pharmacology*, vol. 156, no. 4, pp. 601-608, 2009.

[12] V. Sauzeau, M. A. Sevilla, M. J. Montero, and X. R. Bustelo, "The Rho/Rac exchange factor Vav2 controls nitric oxide-dependent responses in mouse vascular smooth muscle cells," *The Journal of Clinical Investigation*, vol. 120, no. 1, pp. 315-330, 2010.

[13] B. Lauzier, P. Sicard, O. Bouchot et al., "After four hours of cold ischemia and cardioplegic protocol, the heart can still be rescued with postconditioning," *Transplantation*, vol. 84, no. 11, pp. 1474-1482, 2007.

[14] A. Agouni, A.-H. Lagrue-Lak-Hal, H. A. Mostefai et al., "Red wine polyphenols prevent metabolic and cardiovascular alterations associated with obesity in Zucker fatty rats (Fa/Fa)," *PLoS One*, vol. 4, no. 5, article e5557, 2009.

[15] T. Dupas, M. Denis, J. Dontaine et al., "ProteinO-GlcNAcylation levels are regulated independently of dietary intake in a tissue and time-specific manner during rat postnatal development," *Acta Physiologica*, vol. 231, no. 3, p. doi:10.1111/apha.13566, 2021.

[16] M. Perez-Aso, N. Flacco, N. Carpena, M. C. Montesinos, P. D'Ocon, and M. D. Ivorra, "β-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and P38," *Vascular Pharmacology*, vol. 61, no. 2-3, pp. 80-89, 2014.

[17] S. Moniotte, L. Kobzik, O. Feron, J. N. Trochu, C. Gauthier, and J. L. Balligand, "Upregulation of β3-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium," *Circulation*, vol. 103, no. 12, pp. 1649-1655, 2001.

[18] Y. Kong, W. Li, and Y. Tian, "Effect of beta3-adrenoreceptors agonist on beta3-adrenoreceptors expression and myocyte apoptosis in a rat model of heart failure," *Zhongguo Wei Zhong Bing Ji Jiu Yi Xue*, vol. 16, no. 3, pp. 142-147, 2004.

[19] X. Niu, V. L. Watts, O. H. Cingolani et al., "Cardioprotective effect of beta-3 adrenergic receptor agonism: role of neuronal nitric oxide synthase," *Journal of the American College of Cardiology*, vol. 59, no. 22, pp. 1979-1987, 2012.

[20] N. Hermida, L. Michel, H. Esfahani et al., "Cardiac myocyte B3-adrenergic receptors prevent myocardial fibrosis by modulating oxidant stress-dependent paracrine signaling," *European Heart Journal*, vol. 39, no. 10, pp. 888-898, 2018.

[21] R. Salie, A. K. H. Alsalhin, E. Marais, and A. Lochner, "Cardio-protective effects of beta3-adrenergic receptor (B3-AR) pre-, per-, and post-treatment in ischemia-reperfusion," *Cardiovascular Drugs and Therapy*, vol. 33, no. 2, pp. 163-177, 2019.

[22] L. Y. M. Michel, C. Farah, and J.-L. Balligand, "The Beta3 adrenergic receptor in healthy and pathological cardiovascular tissues," *Cell*, vol. 9, no. 12, p. 2584, 2020.

[23] C. Belge, J. Hammond, E. Dubois-Deruy et al., "Enhanced expression of B3-adrenoceptors in cardiac myocytes attenuates neurohormone-induced hypertrophic remodeling through nitric oxide synthase," *Circulation*, vol. 129, no. 4, pp. 451-462, 2014.
[24] A. J. A. Leloup, C. E. Van Hove, A. Heykers, D. M. Schrijvers, G. R. Y. De Meyer, and P. Fransen, "Elastic and muscular arteries differ in structure, basal NO production and voltage-gated Ca2+-channels," *Frontiers in Physiology*, vol. 6, 2015.

[25] C. Franssen, S. Chen, A. Unger et al., "Myocardial microvascular inflammatory endothelial activation in heart failure with preserved ejection fraction," *JACC: Heart Failure*, vol. 4, no. 4, pp. 312-324, 2016.

[26] A. B. Gevaert, H. Shakeri, A. J. Leloup et al., "Endothelial senescence contributes to heart failure with preserved ejection fraction in an aging mouse model," *Circulation: Heart Failure*, vol. 10, no. 6, article e003806, 2017.

[27] G. G. Schiattarella, F. Altamirano, D. Tong et al., "Nitrosative stress drives heart failure with preserved ejection fraction," *Nature*, vol. 568, no. 7752, pp. 351-356, 2019.

[28] G. A. Silberman, T.-H. M. Fan, H. Liu et al., "Uncoupled cardiac nitric oxide synthase mediates diastolic dysfunction," *Circulation*, vol. 121, no. 4, pp. 519-528, 2010.

[29] K. K. Galougahi, C.-C. Liu, C. Gentile et al., "Glutathionylation mediates angiotensin II-induced ENOS uncoupling, amplifying NADPH oxidase-dependent endothelial dysfunction," *Journal of the American Heart Association*, vol. 3, no. 2, article e000731, 2014.

[30] K. Shibata, Y. Yatera, Y. Furuno et al., "Spontaneous development of left ventricular hypertrophy and diastolic dysfunction in mice lacking all nitric oxide synthases," *Circulation Journal*, vol. 74, no. 12, pp. 2681-2692, 2010.

[31] S. B. A. Cau, F. S. Carneiro, and R. C. Tostes, "Differential modulation of nitric oxide synthases in aging: therapeutic opportunities," *Frontiers in Physiology*, vol. 3, p. 218, 2012.

[32] N. Melikian, M. D. Seddon, B. Casadei, P. J. Chowienczyk, and A. M. Shah, "Neuronal nitric oxide synthase and human vascular regulation," *Trends in Cardiovascular Medicine*, vol. 19, no. 8, pp. 256-262, 2009.

[33] U. Förstermann and W. C. Sessa, "Nitric oxide synthases: regulation and function," *European Heart Journal*, vol. 33, no. 7, pp. 829-837, 2012.

[34] W. Meng, J. Ma, C. Ayata et al., "ACh dilates Pial arterioles in endothelial and neuronal NOS knockout mice by NO-dependent mechanisms," *The American Journal of Physiology*, vol. 271, 3 Part 2, pp. H1145-H1150, 1996.

[35] K. G. Lamping, D. W. Nuno, E. G. Shesely, N. Maeda, and F. M. Faraci, "Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice," *American Journal of Physiology. Heart and Circulatory Physiology*, vol. 279, no. 4, pp. H1906-H1912, 2000.

[36] S. Battault, F. Singh, S. Gayrard, J. Zoll, C. Reboul, and G. Meyer, "Endothelial function does not improve with high-intensity continuous exercise training in SHR: implications of ENOS uncoupling," *Hypertension Research*, vol. 39, no. 2, pp. 70-78, 2016.

[37] J. C. Sullivan and J. S. Pollock, "Coupled and uncoupled NOS: separate but equal? Uncoupled NOS in endothelial cells is a critical pathway for intracellular signaling," *Circulation Research*, vol. 98, no. 6, pp. 717-719, 2006.
[24] A. J. A. Leloup, C. E. Van Hove, A. Heykers, D. M. Schrijvers, G. R. Y. De Meyer, and P. Fransen, "Elastic and muscular arteries differ in structure, basal NO production and voltage-gated Ca2+-channels," *Frontiers in Physiology*, vol. 6, 2015.

[25] C. Franssen, S. Chen, A. Unger et al., "Myocardial microvascular inflammatory endothelial activation in heart failure with preserved ejection fraction," *JACC: Heart Failure*, vol. 4, no. 4, pp. 312-324, 2016.

[26] A. B. Gevaert, H. Shakeri, A. J. Leloup et al., "Endothelial senescence contributes to heart failure with preserved ejection fraction in an aging mouse model," *Circulation: Heart Failure*, vol. 10, no. 6, article e003806, 2017.

[27] G. G. Schiattarella, F. Altamirano, D. Tong et al., "Nitrosative stress drives heart failure with preserved ejection fraction," *Nature*, vol. 568, no. 7752, pp. 351-356, 2019.

[28] G. A. Silberman, T.-H. M. Fan, H. Liu et al., "Uncoupled cardiac nitric oxide synthase mediates diastolic dysfunction," *Circulation*, vol. 121, no. 4, pp. 519-528, 2010.

[29] K. K. Galougahi, C.-C. Liu, C. Gentile et al., "Glutathionylation mediates angiotensin II-induced ENOS uncoupling, amplifying NADPH oxidase-dependent endothelial dysfunction," *Journal of the American Heart Association*, vol. 3, no. 2, article e000731, 2014.

[30] K. Shibata, Y. Yatera, Y. Furuno et al., "Spontaneous development of left ventricular hypertrophy and diastolic dysfunction in mice lacking all nitric oxide synthases," *Circulation Journal*, vol. 74, no. 12, pp. 2681-2692, 2010.

[31] S. B. A. Cau, F. S. Carneiro, and R. C. Tostes, "Differential modulation of nitric oxide synthases in aging: therapeutic opportunities," *Frontiers in Physiology*, vol. 3, p. 218, 2012.

[32] N. Melikian, M. D. Seddon, B. Casadei, P. J. Chowienczyk, and A. M. Shah, "Neuronal nitric oxide synthase and human vascular regulation," *Trends in Cardiovascular Medicine*, vol. 19, no. 8, pp. 256-262, 2009.

[33] U. Förstermann and W. C. Sessa, "Nitric oxide synthases: regulation and function," *European Heart Journal*, vol. 33, no. 7, pp. 829-837, 2012.

[34] W. Meng, J. Ma, C. Ayata et al., "ACh dilates Pial arterioles in endothelial and neuronal NOS knockout mice by NO-dependent mechanisms," *The American Journal of Physiology*, vol. 271, 3 Part 2, pp. H1145-H1150, 1996.

[35] K. G. Lamping, D. W. Nuno, E. G. Shesely, N. Maeda, and F. M. Faraci, "Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice," *American Journal of Physiology. Heart and Circulatory Physiology*, vol. 279, no. 4, pp. H1906-H1912, 2000.

[36] S. Battault, F. Singh, S. Gayrard, J. Zoll, C. Reboul, and G. Meyer, "Endothelial function does not improve with high-intensity continuous exercise training in SHR: implications of ENOS uncoupling," *Hypertension Research*, vol. 39, no. 2, pp. 70-78, 2016.

[37] J. C. Sullivan and J. S. Pollock, "Coupled and uncoupled NOS: separate but equal? Uncoupled NOS in endothelial cells is a critical pathway for intracellular signaling," *Circulation Research*, vol. 98, no. 6, pp. 717-719, 2006.