# Cardiac Gene Therapy with PDE2A Limits Remodeling and Arrhythmias in Mouse Models of Heart Failure

Rima Kamel, Aurélia Bourcier, Jean Piero Margaria, Valentin Jin, Audrey Varin, Agnès Hivonnait, Françoise Mercier-Nomé, Delphine Mika, Alessandra Ghigo, Flavien Charpentier, et al.

► To cite this version:

Rima Kamel, Aurélia Bourcier, Jean Piero Margaria, Valentin Jin, Audrey Varin, et al. Cardiac Gene Therapy with PDE2A Limits Remodeling and Arrhythmias in Mouse Models of Heart Failure. Journal of the American Heart Association, 2025, pp.e037343. 10.1161/JAHA.124.037343. hal-04927530

HAL Id: hal-04927530

https://hal.science/hal-04927530v1

Submitted on 3 Feb 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Cardiac Gene Therapy with PDE2A Limits Remodeling and
Arrhythmias in Mouse Models of Heart Failure

Rima Kamel, PhD<fnref n="1" />*, Aurélia Bourcier, PhD<fnref n="1" />*, Jean Piero Margaria, PhD<fnref n="1" />,<fnref n="2" />,<fnref n="5" />*, Valentin Jin, MSc<fnref n="1" />, Audrey Varin, MSc<fnref n="1" />, Agnès Hivonnait, MSc<fnref n="3" />, Françoise Mercier-Nomé, MSc<fnref n="4" />, Delphine Mika, PhD<fnref n="1" />, Alessandra Ghigo, PhD<fnref n="2" />, Flavien Charpentier, PhD<fnref n="3" />, Vincent Algalarrondo, MD, PhD<fnref n="1" />, Emilio Hirsch, PhD<fnref n="2" />, Rodolphe Fischmeister, PhD<fnref n="1" />,§, Grégoire Vandecasteele, PhD<fnref n="1" />, Jérôme Leroy, PhD<fnref n="1" />,§

INSERM UMR-S 1180

Université Paris-Saclay

Faculté de Pharmacie

Bât. Henri Moissan

17, avenue des sciences

91400 Orsay

France

<footnote n="1">¹Université Paris-Saclay, Inserm, Signaling and Cardiovascular Pathophysiology, UMR-S 1180, F-91400 Orsay, France.</footnote>

<footnote n="2">² Molecular Biotechnology Center “Guido Tarone”, Department of Molecular Biotechnology and Health Sciences, University of Torino, I-10126 Torino, Italy.</footnote>

<footnote n="3">³ Nantes Université, CNRS, INSERM, l'institut du thorax, F-44000 Nantes, France.</footnote>

<footnote n="4">⁴ Université Paris-Saclay, Inserm US31, CNRS UMS3679, IPSIT, F-91400 Orsay, France.</footnote>

<footnote n="5">⁵ Cystic Kidney Disorders Unit, Division of Genetics and Cell Biology, Università Vita-Salute San Raffaele, Milan, Italy.</footnote>

<footnote>*these authors contributed equally to this work</footnote>

<footnote>Running title: PDE2 gene therapy to prevent heart failure</footnote>

<footnote>§Correspondence to:</footnote>

<footnote>Jérôme Leroy: jerome.leroy@universite-paris-saclay.fr</footnote>

<footnote>Rodolphe Fischmeister: rodolphe.fischmeister@inserm.fr</footnote>
**BACKGROUND:** Phosphodiesterase 2 (PDE2) is upregulated in human heart failure (HF). Cardiac PDE2-transgenic mice are protected against contractile dysfunction and arrhythmias in HF but whether an acute elevation of PDE2 could be of therapeutic value remains elusive. This hypothesis was tested using cardiac PDE2 gene transfer in preclinical models of HF.

**METHODS AND RESULTS:** C57BL/6 male mice were injected with serotype 9 adeno-associated viruses (AAV9) encoding for PDE2A. This led to a ~10-fold rise of PDE2A protein levels which affected neither cardiac structure nor function in healthy mice. Two weeks after AAV9 inoculation, mice were implanted with minipumps delivering either NaCl, isoproterenol (Iso) (60 mg/kg/day) or Iso and phenylephrine (Iso+Phe, 30 mg/kg/day each) for 2 weeks. In mice injected with AAV9 encoding for luciferase (LUC), Iso or Iso+Phe infusion induced left ventricular hypertrophy, decreased ejection fraction unveiled by echocardiography, promoted fibrosis and apoptosis assessed by Masson's trichrome and Tunel, respectively. Furthermore, inotropic responses to Iso of ventricular cardiomyocytes isolated from Iso+Phe-LUC mice loaded with 1 µmol/L Fura-2AM and stimulated at 1 Hz to record calcium transients and sarcomere shortening were dampened. Spontaneous calcium waves at the cellular level were promoted as well as ventricular arrhythmias evoked in vivo by catheter-mediated ventricular pacing after Iso (1.5 mg/kg) and atropine (1 mg/kg) injection. However, increased PDE2A blunted these adverse outcomes evoked by sympathomimetic amines.

**CONCLUSIONS:** Cardiac gene therapy with PDE2A limits left ventricle remodeling, dysfunction and arrhythmias evoked by catecholamines, providing evidences that increasing PDE2A activity acutely could prevent progression towards HF.

**Key Words:** Heart failure ■ Catecholamines ■ cAMP-Phosphodiesterase ■ Excitation-contraction coupling ■ Arrhythmia ■ Gene therapy
## RESEARCH PERSPECTIVES:

### What is new?

* PDE2A expression is increased in heart failure and cardiac PDE2-transgenic mice are protected against contractile dysfunction and arrhythmia in heart failure. However, it is unknown whether an acute increase in PDE2A activity can prevent maladaptive remodeling and exert anti-arrhythmic effects.

* We show that increased PDE2A expression using AAV9 encoding for this enzyme limits left ventricular (LV) hypertrophy, fibrosis and dysfunction induced by sympathomimetic amines and improves defective β-AR inotropic effects of isolated cardiomyocytes.

* Increasing PDE2A exerts antiarrhythmic effects both at the cellular level and in vivo and is more efficient than increasing PDE4B to prevent ventricular tachycardias.

### What Question Should Be Addressed Next?

* These data suggest that an acute increase in PDE2A activity is of therapeutic potential in heart failure but warrants further testing in larger animal models and clinical studies.
Nonstandard Abbreviations and Acronyms

AAV9 serotype 9 adeno-associated viruses

AC adenylyl cyclase

β-AR β-adrenergic receptor

BW body weight

CaT calcium transients

CMV human cytomegalovirus

ECC excitation-contraction coupling

HF heart failure

Iso isoproterenol

IVSd end-diastolic InterVentricular Septum thickness

LTCC L-type Ca²⁺ channel

LUC luciferase

LV left ventricular

LVIDd Left Ventricular Internal Diameter

LVM left ventricular mass

LVW left ventricular weight

LW lung weight

NO nitric oxide

PDE phosphodiesterase

PDE2 phosphodiesterase 2

PES programmed electrical stimulation

Phe phenylephrine

PLB phospholamban

PKA protein kinase A

PWd Posterior Wall thickness

SCE spontaneous calcium event

SS sarcomere shortening

TL tibia length

VERP ventricular effective refractory periods

VM ventricular myocyte
The "fight-or-flight" response involves a discharge of norepinephrine by the sympathetic nervous system, causing the secretion of catecholamines by the adrenal medulla which act on cardiomyocytes via the β-adrenergic receptor (β-AR)/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) axis to stimulate cardiac rhythm (chronotropy), contractile force (inotropy) and relaxation (lusitropy).¹ PKA phosphorylates many of the proteins critically involved in the excitation-contraction coupling (ECC). These include the small G protein Rad to stimulate the L-type Ca²⁺ channels (LTCCs),² the ryanodine receptor 2 to increase its open probability, the phospholamban to relieve its inhibitory effect on the sarco/endoplasmic reticulum Ca²⁺-ATPase and contractile proteins, such as troponin I and myosin binding protein C.¹ While this acute β-AR activation is favorable allowing the adaptation of the cardiac output to increased body's metabolic demand during a stress or an effort, a chronic overactivation of the sympathetic nervous system is deleterious. In heart failure (HF), the decreased cardiac function leads to a chronic catecholamine elevation³ which elicits maladaptive remodeling comprising cardiac hypertrophy, cardiomyocyte death, and cardiac fibrosis and promotes arrhythmias thus aggravating pump dysfunction. The excessive β-AR stimulation is central in this propelled vicious circle as attested by the beneficial effects of β-AR antagonists which are the first-line therapy to treat chronic HF with reduced ejection fraction.⁴,⁵ However, despite an enhancement in survival rates by the use of β-blockers,⁵⁻⁷ the mortality of HF patients 5 years after diagnosis mainly due to pump failure or ventricular arrhythmias remains unacceptably high at ~50%.⁸ Furthermore, in many patients, intolerance does not allow titration to an effective dosage.⁹ There is thus a need for new therapeutic approaches to treat HF.

Intracellular cAMP levels produced upon β-AR stimulation are counterbalanced by the degradation of the cyclic nucleotide by enzymes called phosphodiesterases (PDEs) which not
only terminate the activation of cAMP effectors but also compartmentalize this second
messenger in discrete subcellular compartments.¹⁰ Among the 6 PDE families expressed in
cardiomyocytes to degrade cAMP, PDE2 has been recently identified as a therapeutic target
to treat HF. PDE2 was shown to inhibit cardiac LTCC activity in various species, including
humans¹¹ and to control ECC even though it exhibits rather low protein expression and its
inhibition has little effect on cAMP hydrolytic activity in healthy cardiomyocytes.¹²⁻¹⁴ In
contrast to PDE3 and PDE4, the two main enzymes degrading cAMP in the heart, expression
and activities of which are generally decreased in pathological hypertrophy and HF,¹⁵⁻¹⁷ PDE2
is increased in HF to blunt β-AR/cAMP signals suggesting a protective mechanism against
increased circulating catecholamines.¹⁸ Accordingly, transgenic (TG) mice with constitutive
cardiac overexpression of PDE2 exhibit improved cardiac function and are protected against
catecholamine-induced arrhythmias after ischemic injury.¹⁹ Moreover, PDE2 hydrolytic
activity is stimulated by 10 to 30-fold by cGMP via binding to the regulatory N-terminal GAF-
B domain²⁰ making PDE2 a unique integrator of cAMP/cGMP signals.¹⁴,²¹,²² Interestingly, a
recent study unveiled that the antiarrhythmic effects of C-type natriuretic peptide occur via
PDE2 stimulation, attenuating spontaneous Ca²⁺ events, limiting CaMKII activation and
decreasing the late Na⁺ current promoted by a β-AR stimulation.²³ However, cardioprotective
effects of increased PDE2 activity is a matter of debate since conflicting results reported that
PDE2 upregulation is pro-hypertrophic.²² Furthermore, its inhibition was found to antagonize
cardiomyocyte growth,²² to protect against apoptotic cell death by promoting mitochondrial
elongation²⁴ and PDE2 blockade with BAY60-7550 hindered cardiac maladaptive remodeling
by enhancing nitric oxide (NO)/guanylyl cyclase/cGMP signaling.²⁵

In an attempt to further delineate the impact of PDE2 upregulation on cardiac function
and its putative beneficial effects in HF, we performed gene therapy with adeno-associated
viruses serotype 9 (AAV9) allowing substantial cardiac overexpression of PDE2A3.²⁶ This strategy allows expression from a definite time point rather than constitutive cardiac expression as previously employed.¹⁹ With this strategy, we demonstrated *in vivo* that PDE2 overexpression protects the heart upon a chronic exposure to sympathomimetic amines (Phe and/or Iso), by reducing remodeling and left ventricule dysfunction. Strikingly, increased cardiac PDE2A expression prevents ventricular arrhythmias upon stress and this correlates with improved ECC at the level of single cardiomyocytes.

# METHODS

## Data Availability Statement

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

## Ethical Approval and Animals

All procedures were carried out at the animal facility and at the laboratory INSERM UMR-S 1180 of the faculty of Pharmacy of the Paris-Saclay University, according to institutional regulations and the local guide for the use and care of laboratory animals. All experiments involving animals were conducted in agreement with the European Community guiding principles in the welfare and use of animals (2010/63/UE) and the French decree no. 2013-118 on the protection of animals used for scientific purposes. The protocol was approved by the local ethics committee (CREEA Ile-de-France Sud) and by the French Ministry of higher
Education and Research (17852-2015051112125554 v4). In this study, 8-week-old male C57BL/6 mice obtained from Janvier LABS were used. 7 days of acclimatization was respected prior the start of all experimental protocols. Mice used in this study were housed in ventilated cages (GM550 Greenline, Tecniplast France) with enrichment and were exposed to 12h light/dark non-reverse cycles (light from 7am to 7pm) in a thermostatically and humidity-controlled room (21-22°C, 40-50% humidity) at our institute's animal facility (ANIMEX IPSIT, approval number B92-019-01). Mice had free access to a standard diet (A03 Safe for breeding, A04 Safe during experimental protocols) and water. Mice were never isolated and were distributed into experimental groups randomly. The animals were monitored daily to evaluate the physical appearance (observation every day) and weight (measurement 3x/week). When a weight loss of 10 to 20% compared to the initial weight was observed, access to food was facilitated. None of the animals included in the experimental protocols reached endpoints such as a weight loss >20%.

## Reagents

Isoproterenol (Iso), phenylephrine (Phe) and atropine were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Bay60-7550 was purchased from Cayman chemical (Ann Arbor, Michigan, USA).
## AAV9-LUC/PDE vectors production, purification, characterization and injection

AAV9-PDE2A holds a PDE2A3 expression cassette flanked by two AAV2 inverted terminal repeats. The expression sequence is pseudotyped with an AAV9 capsid. The cassette contains a CMV promoter, a β-globin intron, a FLAG tag fused in 5' of the complete murine PDE2A3 coding sequence (NCBI: NM_001008548.4) and an hGH polyadenylation signal. AAV9-PDE2A was produced in AAV-293T cells (Stratagene #240073, San Diego, California, USA) with the three-plasmid method and the calcium-chloride transfection. The virus was purified by cesium-chloride gradient. An RT-PCR on the human cytomegalovirus (CMV) promoter was used to determine viral particles titers. An expression cassette encoding firefly luciferase (LUC) was used for control condition. A CMV promoter controlling luciferase expression, was packaged into AAV9 capsids and purified on cesium-chloride gradient to yield AAV9-LUC virus. AAV9-LUC or AAV9-PDE2A were injected via the tail vein at 10<sup>12</sup> viral particles/mouse. AAV9-CMV is the empty control vector used to produce AAV9-PDE2A containing solely the CMV promoter without any open reading frame sequence. The AAV9-PDE4B has been previously described.<sup>15</sup>

## Transthoracic echocardiography

Transthoracic two-dimensional-guided M-mode echocardiography of mice was performed using an echocardiograph with an ML6 linear probe of 15 MHz (Vivid E9, General Electric Healthcare®, Chicago, Illinois, USA) under 1-2% isoflurane gas and 0.4-0.8 L/min oxygen anesthesia. For isoflurane induction, mice were placed for approximately 2 min in an isolation chamber filled with isoflurane (5% in 100% oxygen) at a flow rate of 0.5-1 L/min. Body
temperature was maintained at 37°C by the use of a thermally controlled heating pad (Harvard Apparatus). Measurements from animals with heart frequencies below 400 bpm were discarded. LV cavity and wall thickness dimensions during systole and diastole were determined using two parasternal axes: short and long. The left ventricular weight (LVW) was calculated according to the Penn formula while assuming a spherical LV geometry and validated for the mouse heart (LVM=1.04×[(LVIDd+IVS+PW)³-(LVIDd)³], where 1.04 is the specific gravity of muscle. LVIDd: Left Ventricular Internal Diameter in diastole, IVSd and PWd are end-diastolic InterVentricular Septum and Posterior Wall thicknesses, respectively. Analysis was performed for the echocardiographic images using the EchoPac software (General Electric Healthcare®). For each of the two axes and the measured parameters, three measurements on three different sections were averaged. Analysis of the echocardiography data was blinded to the mice population.

## Sympathomimetic amines infusion models

Schematic representations of the experimental protocols used to treat animals are depicted in Figures 1A, 2A and 3A. Briefly, a first echocardiography to assess cardiac function prior AAV9 injections was performed. Two weeks after, a second echocardiography was realized and sympathomimetic amines administered via osmotic minipumps (2002, Alzet®, Cupertino, California, USA) surgically implanted subcutaneously in the back of the animals as previously described.¹⁵ This allowed to treat the animals for 14 days with either Iso (60 mg/kg/day), Iso+Phe (30 mg/kg/day each) or vehicle (0.9% NaCl). An intermediate echocardiography at day 21 was performed on animals treated with Iso+Phe, a last one at day 28 prior euthanasia after minipump removal to avoid interference on the cardiac function of remaining
sympathomimetic treatment (Figure 3A). Cardiac function was followed at day 28 when the minipump was removed and for two additional weeks for the isoproterenol model by echocardiography performed at days 35 and 42.

Morphological and histological analysis

For euthanasia, mice were anesthetized by an intraperitoneal injection of pentobarbital (Doléthal®, 100 mg/kg). Afterwards, hearts were rapidly excised and remaining blood was washed out in cold Ca²⁺-free phosphate buffer saline solution (155.2 mM NaCl, 1.06 mM KH₂PO₄ and 2.9 mM NaH₂PO₄). Hearts and lungs were weighed and tibia length was measured. For histology, a transversal slice of 3-4 mm width was cut in the middle of the heart and rapidly fixed in 4% paraformaldehyde. The rest of ventricular tissue was frozen in liquid nitrogen and stored at -80°C until use. Histological staining was performed using a standard protocol. After fixation of the hearts for 24 h in 4% paraformaldehyde, they were paraffin embedded and transversely sectioned. Sections (5 or 7 µm) were deparaffinized. Afterwards, samples were subjected to Heat-Induced Epitope Retrieval (HIER) in a 10 mM citrate buffer, pH=6. A Masson's trichrome stain kit (Microm, Brignais, France) was used to assess cardiac fibrosis. To quantify apoptotic myocytes in the heart, autofluorescence was quenched by treating paraffin-embedded sections with PBS/BSA (5%) for 2 h before performing TUNEL staining (Roche, Basel, Switzerland) according to the manufacturer's protocol and using Proteinase K treatment. Sections were counterstained with Alexa Fluor 594-conjugated wheat germ agglutinin (WGA, Invitrogen, Carlsbad, California, USA) at 10 µg/mL for 1 h at room temperature to visualize cell membranes. Slides were scanned by the digital slide scanner NanoZoomer 2.0-RS (Hamamatsu, Japan), which allowed an overall view of the samples and
analysed blinded to the mice population.. Images were digitally captured from the scan slides
using the NDP.view2 software (Hamamatsu, Japan).

ECG recording and intracardiac recording and pacing

The criteria used to measure RR, PR, QRS and QT intervals on recorded ECG have been described elsewhere.²⁷ The QT interval was corrected for heart rate with the Bazett formula adapted to mouse sinus rate, *i.e.* QTc = QT/(RR/100)1/2 with QT and RR, expressed in ms.²⁸ Mice were anesthetized with an intraperitoneal injection of etomidate of 28 mg/kg (Hypnomidate® 2 mg/ml, Janssen-Cilag, Issy les Moulineaux, France) before and after intracardiac and surface ECG recording and pacing. The extremity of a 2F octopolar catheter 1.5 French (Cordis Webster) was placed in the right ventricle through the right internal jugular vein, using intracardiac electrograms as a guide for catheter positioning in the right ventricle. Surface ECG (lead I) and intracardiac electrograms were recorded on a computer through an analog-digital converter (IOX 1.585, Emka Technologies, Paris, France) for monitoring and later analysis (ECG-Auto v3.3.0.5, Emka Technologies, Paris, France). Intracardiac electrograms were filtered between 0.5 and 500 Hz. Pacing was performed with a digital stimulator (DS8000, World Precision Instruments, Sarasota, Florida, USA). The standard pacing protocols were used to determine the ventricular effective refractory periods (VERP) and to induce ventricular arrhythmias were developed as follows: Determination of stimulation threshold, which corresponds to the minimal energy provoking a ventricular depolarization, was done with 1-ms-long pulses with increasing intensity. Afterwards, stimulations were conducted at intensities 1.5 times the excitation threshold. VERP were assessed at baseline by using the programmed electrical stimulation (PES) method. Extrastimuli were delivered following trains
of 20 pulses at a cycle length of 70 ms followed by one, two and then three extrastimuli. The
extrastimulus coupling interval was initially set at 70 ms and then reduced by 2 ms at each
cycle until ventricular refractoriness was reached. The inducibility of ventricular arrhythmias
was assessed in baseline conditions and 3 min after intraperitoneal injection of Iso 1.5 mg/kg
and atropine 1 mg/kg. Ventricular tachycardias were defined as the occurrence of at least 4
consecutive QRS complexes with a morphology different than that seen with a normal sinus
rhythm, after last stimulated beat.

Isolation of mouse cardiomyocytes

Mice were anesthetized by intraperitoneal injection of Doléthal® (100 mg/kg). The heart was quickly removed and placed into a cold Ca²⁺-free Tyrode's solution containing 113 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄-7H₂O, 0.6 mM KH₂PO₄, 0.6 mM NaH₂PO₄, 1.6 mM NaHCO₃, 10 mM HEPES, 30 mM taurine, 20 mM glucose and 17.03 µM insulin adjusted to pH 7.4. The ascending aorta was cannulated, and the heart was perfused at constant pressure (≈ 798 mmHg, flow rate=26 ml/min) with oxygenated Ca²⁺-free Tyrode's solution at 37°C for approximately 4 min using retrograde Langendorff perfusion. Subsequently, the heart was perfused with Ca²⁺-free Tyrode's solution containing Liberase™ research grade (Roche Diagnostics, Basel, Switzerland) for 10 min at 37°C for enzymatic dissociation. Afterwards, the heart was removed and placed into a dish containing a Tyrode's solution supplemented with 0.2 mM CaCl₂ and 5 mg/ml BSA (Sigma-Aldrich, St. Louis, Missouri, USA). Atria were discarded and ventricles were cut into small pieces, and triturated gently with a pipette to disperse the myocytes. Ventricular myocytes were filtered on gauze and allowed to sediment by gravity for approximately 7 min. The supernatant was removed, and cells were suspended in Tyrode's
solution supplemented with 0.5 mM CaCl₂ and 5 mg/ml BSA. Sedimentation was repeated and
cells were finally suspended in a Tyrode's solution with 1 mM CaCl₂. For Ionoptix experiments,
freshly isolated ventricular myocytes were plated for 45 min at a density of 10⁴ cells per dish
in 35-mm culture dishes coated with laminin (10 µg/ml) and stored in a cell incubator at 37°C,
5% CO₂ until use up to 4 hours.

Calcium transients and sarcomere shortening measurements

Isolated cardiomyocytes were loaded with 1 µM fura-2 AM (Invitrogen, Carlsbad, California, USA) at room temperature for 15 min and then washed for 15 additional min with an external Ringer solution containing (in mM): NaCl 121.6, KCl 5.4, NaHCO₃ 4.013, NaH₂PO₄ 0.8, HEPES 10, glucose 5, Na pyruvate 5, MgCl₂ 1.8, and CaCl₂ 1, pH 7.4. The loaded cells were field-stimulated (5 V, 4 ms) at a frequency of 1 Hz. Sarcomere length (SL) and fura-2 ratio (measured at 512 nm upon excitation at 340 nm and 380 nm) were simultaneously recorded using an IonOptix System (IonOptix, Westwood, Massachusetts, USA). Cell contractility was assessed by the percentage of sarcomere shortening. It is the ratio of twitch amplitude (difference of end-diastolic and peak systolic SL) to end-diastolic SL. Ca²⁺ transients were assessed by the percentage of variation of the fura-2 ratio by dividing the twitch amplitude to end-diastolic ratio. The time constant (Tau) obtained from the exponential fit of the Ca²⁺ transient decay kinetics was used as an index of relaxation. All parameters were calculated offline using a dedicated software (IonWizard 6x, Westwood, Massachusetts, USA).
## Western blot assay

Protein extracts were prepared from frozen cardiac tissue which were homogenized using a Precellys Evolution Touch Homogenizer (Bertin Technologie®) during 3 cycles of 10 sec at 6500 rpm in a RIPA buffer containing: Tris HCl (pH 8.0) 50 mM, NaCl 150 mM, EDTA 2 mM, NP40 1%, sodium deoxycholate acid 0.5%, SDS 0.1% supplemented with Complete Protease Inhibitor Tablets and PhosSTOP phosphatase inhibitor tablets (Roche Diagnostics, Basel, Switzerland). Tissue lysates were centrifuged at 15,000 g and 4°C for 20 min, pellets were discarded and supernatants were used. Protein quantification was performed using a BCA protein assay (Thermo Scientific Pierce, #A55865). 20 µg of proteins diluted in a LDS (Lithium Dodecyl Sulfate, Invitrogen, #NP0008) with Sample Reducing Agent (DTT, Invitrogen #NP0009) were heated at 70°C during 10 min and separated in denaturing acrylamide gels. Subsequently, dry transfer (iBlot2) was performed on PVDF membranes (Invitrogen, #IB24001) for 7 min (20V for 1 min, 23V for 4 min and 25V for 2 min). After blocking the membranes with 5% milk buffer for 1 h, the incubation with primary antibodies was carried out over night at 4°C. After incubation with the appropriate secondary antibody (Goat anti-mouse HRP, Invitrogen 31430 or Goat anti-rabbit HRP, Invitrogen 31460, diluted at 1/40,000), proteins were visualized by enhanced chemiluminescence and quantified with ImageJ software. The primary antibodies used were as follows: i) Figure 1: rabbit anti-PDE2A, Fabgennix; PDE2A-101AP diluted at 1/1000; rabbit anti-GAPDH, Cell Signaling, 2118 diluted at 1/1000; ii) Figure 2: goat anti-PDE2A, Santa Cruz, SC-17228 diluted at 1/1000; mouse anti-vinculin, V9131 Sigma-Aldrich diluted at 1/1000; iii) Figure 3: rabbit anti-PDE2A, Fabgennix, PDE2A-101AP diluted at 1/1000; rabbit anti-GAPDH, Cell Signaling, 2118 diluted at 1/1000; iv) Figure S1: rabbit anti-flag L5, Novus Biologicals, NBP1-06712 diluted at 1/500; rabbit anti-
GAPDH, Cell Signaling, 2118 diluted at 1/1000; rabbit anti-PDE2A, Proteintech, 55306-1-AP diluted at 1/1000; rabbit anti-flag L5, Novus Biologicals, NBP1-06712 diluted at 1/500; rabbit anti-GAPDH, Cell Signaling, 2118 diluted at 1/1000; v) Figure S2B: rabbit anti-PDE2A, Proteintech, 55306-1-AP diluted at 1/1000; vi) Figure S3: rabbit anti-PDE2A, Proteintech, 55306-1-AP diluted at 1/1000; vii) Figure S4: rabbit anti-PDE4A AC55 (gift from Dr. Marco Conti, UCSF, San Francisco, CA) diluted at 1/1000; rabbit anti-PDE4B (gift from Dr. Marco Conti) diluted at 1/10,000; mouse anti-PDE4D (gift from Dr. Marco Conti) diluted at 1/1000 and rabbit anti-PDE3A (gift from Dr. Chen Yan, University of Rochester, Rochester, NY) diluted at 1/10,000.

## Quantitative real time polymerase chain reaction assay

Using Trizol reagent (MRCgene, Cincinnati, Ohio, USA), total RNA was extracted from ventricular tissue. Reverse transcription of RNA samples was carried out using iScript cDNA synthesis kit (Bio-Rad, Hercules, California, USA) according to manufacturer's instructions. Real-time PCR reactions were prepared using SYBR Green Supermix (Bio-Rad, Hercules, California, USA) and performed in a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA). The relative amount of mRNA transcripts was quantified using the ΔCt method. *Rplp2* and *Rpl32* housekeeping genes were used as reference for normalization. Sequences of the forward (For) and reverse (Rev) primers used for each studied gene are given below in the 5'- 3' orientation:

*Pde2a* (For) TGGGGAACTCTTTGACTTGG, (Rev) ATGACCTTGCAGGAAAGCTG

*Rplp2* (For) GCT GTG GCT GTT TCT GCT TC, (Rev) ATG TCG TCA TCC GAC TCC TCRpl32 (For) GCT GCT GAT GTG CAA CAA A, (Rev) GGG ATT GGT GAC TCT GAT GG
## Statistics

All results are expressed as mean ± SEM and were analysed using the GraphPad Prism and the R studio softwares. Normal distribution was tested by D'Agostino & Pearson omnibus normality or Shapiro-Wilk normality tests and homogeneity of variance assessed with the Bartlett's and Levene's tests. When normal distribution and homoscedasticity of values were satisfied, unpaired Student's t or paired Wilcoxon's tests were used for two group comparisons, One-way ANOVA or a Two-Way ANOVA (ordinary, repeated measure or mixed effects) followed by a Tukey's post hoc test were used to compare differences between multiple groups. When the data did not follow a normal distribution and/or in case of inhomogeneity of variances, Welch versions of the t-test and ANOVA, Mann-Whitney U test, Kruskal-Wallis test with Dunn's post hoc tests or an aligned rank transform procedure two-way mixed Anova nested test (Artool) (https://cran.r-project.org/web/packages/ARTool/readme/README.html)²⁹ were performed. A Barnard's exact test was used in the analysis of contingency tables. A p value<0.05 was considered statistically significant.
RESULTS

Cardiac PDE2A overexpression in healthy C57BL/6 male mice does not alter the
size, shape and function of the heart

Constructs including a CMV promoter to allow satisfactory expression of the PDE2A or of the luciferase (LUC) as an internal control of overexpression were built (Figure S1A). The AAV9-PDE2A promotes a cAMP-PDE activity which can be repressed by Bay 60-7550 (100 nM), a specific PDE2 inhibitor, demonstrating that the adeno-associated virus allows to overexpress PDE2A specifically (Figure S1B). Prior to testing the cardioprotective potential of enhanced cardiac PDE2A activity using engineered AAV9, we evaluated the effect of PDE2A overexpression on cardiac function and morphology in healthy mice. 10¹² viral particles of AAV9-PDE2A or AAV9-LUC were injected to 8-week-old mice and followed up for 28 days (Figure 1A). This dose of 10¹² viral particles injected per animal was chosen since the dose-response curve demonstrated that it was required to obtain a firefly activity within the myocardium following AAV9-LUC injection (Figure S2A). At this dose, the AAV9-PDE2A allows satisfactory expression of PDE2A in the cardiac tissue which, along with the liver, is substantially transduced when the virus is injected into the tail vein. Peripheral expression is limited, with PDE2A slightly expressed in skeletal muscle, but not in the kidney (Figure S2B) confirming the preferential cardiac tropism of AAV9, a vector of choice for cardiac gene transfer in rodents.³⁰ As expected, cardiac PDE2A expression was significantly increased in AAV9-PDE2A mice with a ≈10-fold rise of PDE2A protein levels compared to AAV9-LUC mice measured by western blotting (Figure 1B). This overexpression neither affected the heart weight over tibia length ratio (HW/TL) nor the lung weight over tibia length ratio (LW/TL) (Figure 1C). Thus, increasing PDE2A activity in the heart did not provoke cardiac hypertrophy
or lung congestion. LV morphology and function evaluated by transthoracic echocardiography remained unaffected by PDE2A with comparable LV weight over body weight ratio (LVW/BW). Importantly, ejection fraction (EF) measured at 14 and 28 days remained identical in animals receiving AAV9-LUC and AAV9-PDE2A injections (Figure 1D and Table S1). Furthermore, heart rate measured in anesthetized mice as well as other morphological and echocardiographic parameters were not modified by PDE2A overexpression (Table S1).

## Cardiac gene therapy with PDE2A limits left ventricle maladaptive remodeling and dysfunction induced by a chronic infusion of sympathomimetic amines

Because the neurohormonal alterations of HF are characterized by marked elevations in norepinephrine and epinephrine circulating concentrations,³¹ we hypothesized that PDE2A overexpression could exert cardioprotective effects in two different mouse models of cardiac dysfunction evoked by chronic infusion with sympathomimetic amines. In both trials, tail vein injections with 10¹² viral particles of AAV9-LUC or AAV9-PDE2A were performed 14 days prior to minipump implantation. We first surmised that increased PDE2A activity may mitigate LV remodeling and dysfunction in response to a chronic β-AR stimulation. Thus, we chose to subject the mice to a 14-day infusion with the non-selective β-AR agonist Iso (60 mg/Kg/day).¹⁵,³² Cardiac function was evaluated *in vivo* by transthoracic echocardiography over 6 weeks (Figure 2A). Analysis of PDE2A mRNA levels (Figure S5) and protein expression revealed a ≈6-fold increase in the harvested hearts from mice injected with AAV9-PDE2A (Figure 2B). Both groups of mice expressing either LUC or PDE2A implanted with Iso minipumps displayed a similar increase in HR at 2 weeks, attesting the adequate infusion of Iso (Figure S6A). This treatment resulted in a marked increase of HW/TL ratio indicating cardiac hypertrophy (p=0.0002), accompanied with lung congestion (LW/TL, p=0.028) (Figure 2C). Echocardiography confirmed LV hypertrophy and revealed a significantly decreased EF after 2
weeks' infusion with Iso (Figure 2D). This LV hypertrophy was accompanied with increased end systolic diameter (LVIDs) evoking contractile dysfunction, and augmented diastolic LV diameter (LVIDd) suggesting left ventricular dilation (Table S2). Moreover, Masson's Trichrome staining revealed a significant increase in fibrosis after the chronic Iso treatment (p=0.005; Figure 2E). While the HW/TL ratio and congestion were only slightly ameliorated in mice overexpressing PDE2A (Figure 2C), LV hypertrophy, systolic dysfunction, myocardial fibrosis and LV dilation induced by Iso were totally prevented (Figure 2 and Table S2).

Since elevated catecholamines in HF activate not only β-AR but also α-adrenoceptors (α-AR), we added phenylephrine (Phe) to Iso in the minipumps at 30 mg/kg/day each. Indeed, the infusion of both amines recapitulates the alterations observed in pressure overload-induced experimental HF and in human hypertrophic cardiomyopathy³³ (Figure 3A). In this set of experiments, a ≈12-fold increase of PDE2A expression in the ventricular tissue was achieved with AAV9 (Figure 3B). Chronic infusion with Iso+Phe induced cardiac hypertrophy and lung congestion as attested by a significant increase in HW/TL and LW/TL ratios (p<0.0001; Figure 3C). These effects were not prevented by PDE2A overexpression despite a trend to diminution (Figure 3C). Similarly, echocardiography revealed a trend for less LV hypertrophy and systolic dysfunction induced by β-AR and α-AR stimulation was attenuated in AAV9-PDE2A mice (Figure 3D and Supplemental Table S3), despite a persistent left ventricular chamber dilation in diastole and systole (Table S3). While fibrosis was not significantly reduced by PDE2A overexpression, apoptosis assessed by the TUNEL assay was clearly diminished (p=0.014; Figure 3E). Also, gene therapy with PDE2A preserved the diastolic function of the treated mice. Indeed, mice treated with Iso+Phe had a clear diminished transmitral blood flow during the left atrial (LA) contraction phase as indicated by the significant decrease of the A wave (p=0.0002; Table S3) analyzed with pulse wave Doppler, while the E wave, thus compliance,
was not improved. This suggests that chronic Iso+Phe treatment altered LA contractility, explaining the trend to increased E/A ratio (Table S3). These alterations of the diastolic function induced by sympathomimetic amines were prevented by PDE2A gene therapy, which preserved the contractility of the left atrium (Table S3).

PDE2A improves defective β-AR inotropic effects of isolated cardiomyocytes after chronic infusion with sympathomimetic amines

To investigate how gene therapy with PDE2A affects the ECC and its β-AR modulation at the cellular level, we measured sarcomere shortening (SS) and calcium transients (CaT) simultaneously in fura-2-loaded ventricular myocytes (VMs) paced at 1 Hz. As shown in Figure 4A-D, baseline CaT and SS amplitudes and time constants for relaxation (τ) were not modified in VMs from Iso+Phe treated mice although CaT decay was slower in cells overexpressing PDE2A (p=0.0134 vs. Iso+Phe-LUC, Figure 4B). When challenged with a submaximal concentration of Iso (3 nM), VMs isolated from NaCl-LUC mice had significantly increased CaT and SS amplitudes by 1.9-3.5-fold respectively (p<0.0001 vs. Baseline, Figure 4A-D and Table S4). Iso also strongly accelerated the relaxation rates of both CaT and SS (p=0.001 vs. Baseline, Figure 4D). These positive inotropic and lusitropic effects were significantly blunted in VMs isolated from LUC mice treated with Iso+Phe. Indeed, Iso had no significant effect on CaT amplitude and kinetics while SS amplitude only increased by 1.6-fold (p=0.0002, vs. NaCl-LUC at baseline; Figure 4A-D). These blunted effects were not due to increased expression of the main cAMP degrading PDEs expressed in cardiomyocytes since neither PDE2A (Figure S3), PDE3A nor PDE4A, PDE4B nor PDE4D protein levels (Figure S4) were significantly affected by chronic Iso+Phe treatment. Interestingly, the positive inotropic effects of Iso were partially restored in VMs from PDE2A-overexpressing animals, although decay times were not improved. This could not be attributed to a remodeling of the expression of the main cAMP-
degrading PDEs since their expression was not changed in VMs from PDE2A-overexpressing animals. Thus, these results demonstrate that gene therapy with PDE2A improves β-AR responsiveness of VMs dampened by chronic infusion with sympathomimetic amines in mice.

## PDE2A cardiac overexpression prevents spontaneous calcium events and ventricular arrhythmias

It has been previously reported that a constitutive cardiac overexpression of PDE2A had antiarrhythmic effects.¹⁹,³⁴ We thus tested whether similar effects could be produced by an overexpression induced from a definite time at adulthood.

First, we investigated the occurrence of pro-arrhythmogenic spontaneous calcium events (SCEs) recorded in VMs isolated from the three groups of mice stimulated with a maximal Iso concentration of 100 nM (Figure 5A). The time to first SCE and the percentage of cells presenting SCEs in each group were similar (Figure S7). However, the number of SCEs recorded during a 3 min period of maximal β-AR stimulation was increased by Iso 100 nM in the Iso+Phe-LUC group, but these cellular pro-arrhythmogenic events were nearly absent in cells isolated from Iso+Phe-PDE2A group (Figure 5B).

Next, we tested the putative antiarrhythmic effect of PDE2A gene therapy *in vivo*. We evaluated the propensity of the treated animals to develop ventricular tachycardia. To do so, intracardiac recordings and pacing were performed. Six-lead ECGs recorded in baseline conditions revealed similar PR interval, QRS complex and QTc interval durations in NaCl-LUC, Iso+Phe-LUC and Iso+Phe-PDE2A animals (Table S5). However, Iso+Phe-LUC mice had a significantly lower RR interval (p=0.0098 vs. NaCl), thus higher heart rate (HR, p=0.0098 vs. NaCl). Interestingly, despite their similar treatment with sympathomimetic amines, mice
injected beforehand with AAV9-PDE2A had a cardiac frequency equivalent to that of control animals (Table S5). Mice were then subjected to catheter-mediated ventricular pacing in baseline conditions which did not evoke cardiac arrhythmias in any animal of the 3 groups. Intraperitoneal injections of Iso+atropine (1.5 mg/kg and 1 mg/kg, respectively) were therefore performed to favor ventricular tachycardia (VT) occurrence. As expected, in the three groups of animals, injection of Iso+atropine significantly increased HR, decreased RR and PR intervals, increased QRS complex durations (but not significantly in NaCl-LUC mice), while QTc duration remained stable (Table S5). While LUC mice treated or not with sympathomimetic amines and animals overexpressing PDE2A had similar ECG parameters following the Iso+atropine injection, they responded differently to ventricular pacing. Indeed, extra-stimuli delivered following trains of 20 pulses triggered arrhythmias in only 1 over 8 NaCl-LUC mice but VT episodes as depicted in Figure 5C were evoked in 5 out of 13 LUC animals chronically infused with sympathomimetic amines (p=0.012 vs. baseline). However, none of the 11 mice overexpressing PDE2A had arrhythmias when applying stimuli in a programmed electrical stimulation (PES) protocol (Figure 5D). This demonstrates that gene therapy with PDE2A protects from ventricular arrhythmias upon stress conditions. To test whether these antiarrhythmic effects are specific to PDE2A overexpression, we evaluated the potency of PDE4B3 to achieve such protective effects, since we previously reported that gene therapy with this enzyme prevents adverse remodeling in HF mice models.¹⁵ Interestingly, a similar treatment with an AAV9 encoding for PDE4B3 did not produce similar anti-arrhythmic effects despite diminished LV hypertrophy and dysfunction (Figure S8A-C, Table S6). This demonstrates that protection against VTs evoked by sympathomimetic amines is better achieved with an increase of PDE2A activity than PDE4B3 in this model.
DISCUSSION

Clinically, PDE inhibition has been considered a promising approach to compensate for the β-adrenergic receptors desensitization that accompanies HF. In that respect, PDE3 inhibitors, such as milrinone or enoximone, have been used clinically to improve systolic function and alleviate the symptoms of acute HF. However, their chronic use has proven to be detrimental, increasing adverse remodeling³⁵ and ventricular arrhythmias.³⁶,³⁷ These adverse effects could potentially be avoided using compartment-specific, PDE isozyme-selective inhibitors.¹⁰ Here, we proposed to test the opposite strategy, i.e. increasing rather than inhibiting PDE activity.

We believe that this strategy, which is reminiscent of the counter-intuitive beneficial effect of beta-blockers in HF, could be therapeutically relevant in HF because it would prevent a deleterious accumulation of cAMP during catecholamines spill over. In that line, we recently demonstrated that constitutive overexpression of PDE4B,¹⁵ one of the main PDE4 isoform expressed in the cardiomyocyte to control the β-AR regulation of the ECC,³⁸ is cardioprotective. We also showed that gene therapy with AAV9-PDE4B exerts cardioprotective effects limiting adverse remodeling evoked by isoproterenol or increased postcharge.¹⁵ Similarly, PDE2A constitutive overexpression exerts anti-hypertrophic effects¹⁸ and transgenic mice overexpressing PDE2A have preserved ejection fraction after myocardial infarction and are protected against sympathomimetic amines-induced ventricular arrhythmia.¹⁹ It was thus important to test whether an increase in PDE2A activity, as obtained during AAV9 gene transfer, could be cardioprotective. Our results are in accordance with a recent study demonstrating that a similar approach not only with PDE4B, but also with PDE2A can prevent pressure-overload-induced HF in mice,³⁹ but we further demonstrate with the present study that PDE2A overexpression is more efficient than PDE4B to prevent polymorphic ventricular tachycardia *in vivo*.
Similarly to what we reported earlier in the transgenic mice,¹⁹ here we found that overexpression of PDE2A obtained after systemic injection of the AAV9 construct did not produce any noticeable change either on the left ventricle size or on its function. Morphometric parameters were not impacted by the overexpression of PDE2A, and contractile parameters were preserved with no congestion.

The safety of this treatment allowed us to move forward and test whether overexpression of PDE2A would counteract the adverse remodeling evoked by catecholamines. Indeed, AAV9 delivery of PDE2A efficiently protected against the detrimental effects of chronic isoproterenol treatment. Chronic sympathomimetic amines infusion models promote cardiomyocyte death and contractile cells are then replaced by interstitial fibrosis.⁴⁰ AAV9-mediated PDE2A overexpression counteracted the systolic dysfunction, attenuated the hypertrophic response, and efficiently prevented fibrosis, a hallmark of pathological remodeling. These cardioprotective effects against β-AR chronic stimulation were as effective as the one obtained by gene therapy with PDE4B,¹⁵ confirming that increasing PDE activity is effective to counteract adverse remodeling evoked by catecholamines. However, elevated catecholamines act not only via β-AR but also via α-AR. Interestingly, gene therapy with PDE2A (and PDE4B, Figure S8) was able to limit LV hypertrophy and dysfunction induced by Iso+Phe challenge, a situation which recapitulates more effectively early transcriptional alterations observed in pressure overload-induced experimental HF and in human hypertrophic cardiomyopathy.³³ Cardioprotective effects of PDE2A overexpression were less pronounced with Iso+Phe than with Iso alone, probably because PDE2A is less efficient in counteracting cAMP-independent effects of α₁-AR stimulation such as apoptosis and fibrosis,³³ than cAMP-signalling emanating from β-AR stimulation. Nevertheless, adverse remodeling and LV dysfunction were reduced by PDE2A overexpression.
Our results demonstrate that gene therapy with PDE2A improves cardiac adaptation to catecholaminergic stress. Indeed, we found that PDE2A cardiac overexpression led to a better responsiveness of the ECC to the β-AR agonist which could be explained by decreased intracellular cAMP levels upon chronic β-AR activation known to promote heterologous desensitization of the receptors by promoting their phosphorylation by PKA.⁴¹ Interestingly, a similar receptor “resensitization” evoked by gene therapy with PDE2A of the β₁-AR/cAMP production was recently reported in ventricular cardiomyocytes isolated from mice in HF induced by increased post-charge.³⁹ This may contribute to the improved ejection fraction we observed in animals subjected to gene therapy with PDE2A. However, overexpression of PDE2A failed to improve the lusitropic effects of Iso in cardiomyocytes. This is likely because, as shown earlier, PDE2 is enriched in the vicinity of the sarco/endoplasmic reticulum Ca²⁺-ATPase, especially under pathological conditions,⁴² and PDE2 gene therapy further increases the activity of the enzyme in this compartment,³⁹ leading to a slower CaT decay upon Iso as observed (Figure 4B). This could explain why compliance of the left ventricle, estimated by the E wave in echocardiography, was not improved by PDE2A. Nonetheless, diastolic dysfunction evoked by the chronic amines' treatment was less pronounced when PDE2A was overexpressed, due to ameliorated atrial contractility estimated by the A wave (Table S3). Prolonged CaT due to decreased SERCA2 activity by PDE2A suggests that this treatment could increase diastolic Ca²⁺ levels and thus, promote arrhythmias. In contrast, myocytes isolated from animals treated with PDE2 gene therapy exhibited fewer spontaneous proarrhythmogenic calcium events upon β-AR stimulation. This is in agreement with what was observed in cells isolated from hearts overexpressing PDE2A, probably by preventing the Epac- and CaMKII-mediated increase of the late sodium current (I<sub>NaL</sub>) and Ca²⁺ leakage via RyR2 as recently described in cardiomyocytes from PDE2A transgenic mice.³⁴ Furthermore, it was
recently demonstrated that gene therapy with PDE2A in HF induced by increased postcharge
could restore partially cAMP compartmentation at the vicinity of the RyR2.³⁹ These
phenomenons could also contribute to improve the cardiac function and decrease the
propensity of these animals to trigger ventricular arrhythmias as we unveil here, in accordance
with the protection conferred by PDE2A constitutive overexpression in the setting of
myocardial infarction previously described.¹⁹

While we showed that PDE2A overexpression is cardioprotective,¹⁸ another group
showed that inhibition rather than increased activity of PDE2 exerts positive outcome in a
pressure-overload model.²⁵ Furthermore, under pathologic conditions PDE2A overexpression
was proposed to increase ventricular cardiomyocyte size, thus to evoke adverse remodeling.²²
Although it was demonstrated that PDE2 regulates cGMP generated by particulate guanylate
cyclases,⁴³ the cardioprotective effects of PDE2 inhibition were attributed to increased cGMP
emanating from the NO stimulated soluble guanylyl cyclase.²⁵ However, PDE2 inhibition by
promoting voltage-gated Ca²⁺ transients in postganglionic neurons from stellate ganglia
increases cardiac neurotransmission,⁴⁴ a mechanism which may also contribute to increase
cardiac function and alleviate cardiac dysfunction. Moreover, global PDE2 inhibition induced
a modest coronary vasodilatation,²⁵ which could have contributed to the beneficial effects
observed.

Here, using AAV9, which has the highest expression in the heart among AAV serotypes,²⁶
we believe that the observed cardioprotective effects are achieved due to a preferential
cardiac overexpression of PDE2. Interestingly, activating mutations in another PDE, PDE3,
responsible for a rare disease characterized by the combination of brachydactyly and
hypertension, confers cardioprotection despite the increase in afterload.⁴⁵ This latter
observation supports our initial postulate and our observations reported here and earlier,¹⁵ that increasing rather than decreasing the activity of PDEs in cardiac tissue is beneficial. However, it remains to be demonstrated which PDE will be the most effective to achieve this goal. We show here that gene therapy with PDE2A is as effective as PDE4B in counteracting the adverse remodelling of the left ventricle and systolic dysfunction induced by chronic catecholamines infusion, but more efficient in counteracting stress-induced ventricular tachycardia. This difference may come from the intrinsic characteristics of the two enzymes. Indeed, PDE2A has a 20-fold lower affinity for cAMP than PDE4B ($K_m=30$ vs. 1.5–4.7 µM), and a ~1000-fold higher hydrolytic activity ($V_{max}=120$ vs. 0.13 µmol/min/mg protein).⁴⁶ Thus, PDE2A would only be turned on when cAMP concentration reaches supra-physiological values, and its high $V_{max}$ would tend to rapidly reduce it to normal. In other words, PDE2A might serve as a safety valve to avoid the cell to be flooded with cAMP. In contrast, PDE4B degrades cAMP at physiological concentrations and, as such, plays a more essential role in the regulation of ECC.³⁸ Its overexpression will thus have different consequences on heart function, notably by reducing the β-AR response of the heart. Of note, and on the contrary to PDE4B, PDE2A is a cGMP-activated PDE⁴⁷ and its cAMP-hydrolytic activity can be increased ~30 fold upon cGMP binding to its GAFB domain. cGMP elevation, which has been shown to be cardioprotective in HF,⁴⁸ could be achieved by activating the particulate guanylyl cyclase localized near PDE2A,⁴³ to constitute a NP/BNP/cGMP-triggered defence mechanism during cardiac stress, in particular during excessive β-AR drive. Interestingly, sacubitril which further elevates NPs improves classical treatments for HF,⁴⁹ and it has been recently demonstrated that NPs exert antiarrhythmic effects via PDE2.²³
## CONCLUSION

Our results demonstrate that increasing PDE2A activity is beneficial in heart failure. They suggest that a gene therapy with PDE2A in cardiac cells, or PDE2A activators yet to be discovered, could be of therapeutic value. We can therefore speculate that further beneficial effects could be obtained with a gene therapy with PDE2 which could constitute a complement to neprilysin inhibitors and actual treatments of chronic HF to limit adverse remodelling and accompanying arrhythmias.

## LIMITATIONS

There are three main limitations to our study. First, only male mice were used here. Sex-dependent heterogeneity in β-AR/cAMP signaling have been shown recently⁵⁰⁻⁵², which may have substantial functional implications in both normal and diseased hearts. Therefore, future experiments should be performed in female mice to examine possible differences in the cardioprotective effect of PDE2A gene therapy between male and female animals. Second, we used here two models of hypertrophy and dysfunction induced by chronic infusion of either Iso or Iso+Phe, a situation which recapitulates early transcriptional alterations observed in pressure overload-induced experimental HF and in human hypertrophic cardiomyopathy³³.

However, since PDEs are part of the signaling pathway downstream of the catecholamine stress, PDE2A overexpression may have interfered with the effectiveness of catecholamines to injure the heart. While this might be one reason for the efficacy of PDE2A gene therapy, other experimental models to induce HF should be tested in the future, such as myocardial infarction or transverse aortic constriction. Finally, it would be important to investigate what
happens if the PDE2 gene therapy is applied after the myocardial dysfunction has taken place
to explore the potential curative effect of the treatment.

Author Contributions

JL, AG, FC, EH, RF, GV and JL contributed to conception and experimental design. RK, AB and JPM performed osmotic pump implantation and echocardiography studies. AB, AH, FC and VA performed and analyzed *in vivo* ECG recording and intracardiac recording and pacing experiments. JPM, AB and AV performed RT-qPCR and Western blot experiments. DM and JPM performed the PDE assays. RK was in charge of cell isolation and performed and analyzed single cell Ca²⁺ transients and contractility experiments. FM-N was in charge of the morphological and histological analysis. RK, AB and JPM prepared the figures and wrote the first draft of the manuscript. All authors contributed to editing the final version of the manuscript.

Sources of Funding

UMR-S1180 is a member of the Laboratory of Excellence LERMIT supported by the French
National Research Agency (ANR-10-LABX-33) under the program "Investissements d'Avenir"
ANR-11-IDEX-0003-01. This work was also funded by grants from the Leducq Foundation for
Cardiovascular Research (19CVD02), ERA-CVD "PDE4HEART", ANR-16-ECVD-0007-01 and EU
MILEAGE project #734931 to RF, the DIM BioConvS of Région Ile-de-France 2023 equip-II-
536836, ANR-19-CE14-0038-02 and ANR-21-CE14-0082-01 to GV, ANR-23-CE14-0051-01 to JL,
Fédération Française de Cardiologie to VA. RK was supported by post-doctoral fellowships
from ERA-CVD and Fondation Lefoulon-Delalande. For the purpose of open access, the authors
have applied a CC-BY public copyright licence to any Author Accepted Manuscript (AAM)
version arising from this submission.

Disclosures

AG and EH are cofounders and shareholders of Kither Biotech, a pharmaceutical product company developing PI3K inhibitors for the treatment of respiratory diseases not in conflict with statements made in this article.

Supplemental Material

Data S1. Supplemental Methods, Figures S1-S8, Tables S1-S6, Reference 53.

REFERENCES

1. Papa A, Kushner J, Marx SO. Adrenergic regulation of calcium channels in the heart. *Annu Rev Physiol*. 2022;84:285-306.

2. Liu G, Papa A, Katchman AN, Zakharov SI, Roybal D, Hennessey JA, Kushner J, Yang L, Chen BX, Kushnir A, et al. Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics. *Nature*. 2020;577:695-700.

3. Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. *The New England journal of medicine*. 1984;311:819-823.

4. Ponikowski P, Voors AA, Anker SD, Bueno H, Cleland JGF, Coats AJS, Falk V, Gonzalez-Juanatey JR, Harjola VP, Jankowska EA, et al. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. *European heart journal*. 2016;37:2129-2200.

5. Packer M, Bristow MR, Cohn JN, Colucci WS, Fowler MB, Gilbert EM, Shusterman NH. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedilol Heart Failure Study Group. *The New England journal of medicine*. 1996;334:1349-1355.
6. Ziaeian B, Fonarow GC. Epidemiology and aetiology of heart failure. *Nat Rev Cardiol*. 2016;13:368-378.

7. investigators M-H. Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF). *Lancet*. 1999;353:2001-2007.

8. Levy D, Kenchaiah S, Larson MG, Benjamin EJ, Kupka MJ, Ho KK, Murabito JM, Vasan RS. Long-term trends in the incidence of and survival with heart failure. *The New England journal of medicine*. 2002;347:1397-1402.

9. Bhatt AS, DeVore AD, DeWald TA, Swedberg K, Mentz RJ. Achieving a maximally tolerated β-blocker dose in heart failure patients: Is there room for improvement? *Journal of the American College of Cardiology*. 2017;69:2542-2550.

10. Kamel R, Leroy J, Vandecasteele G, Fischmeister R. Phosphodiesterases as therapeutic targets in cardiac hypertrophy and heart failure. *Nat Rev Cardiol*. 2023;20:90-108.

11. Rivet-Bastide M, Vandecasteele G, Hatem S, Verde I, Benardeau A, Mercadier JJ, Fischmeister R. cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes. *The Journal of clinical investigation*. 1997;99:2710-2718.

12. Wang YW, Gao QW, Xiao YJ, Zhu XJ, Gao L, Zhang WH, Wang RR, Chen KS, Liu FM, Huang HL, et al. Bay 60-7550, a PDE2 inhibitor, exerts positive inotropic effect of rat heart by increasing PKA-mediated phosphorylation of phospholamban. *Eur J Pharmacol*. 2021;901:174077.

13. Mika D, Bobin P, Pomérance M, Lechêne P, Westenbroek R, Catterall WA, Vandecasteele G, Leroy J, Fischmeister R. Differential regulation of cardiac excitation-contraction coupling by cAMP phosphodiesterase subtypes. *Cardiovascular research*. 2013;100:336-346.

14. Mongillo M, Tocchetti CG, Terrin A, Lissandron V, Cheung YF, Dostmann WR, Pozzan T, Kass DA, Paolocci N, Houslay MD, et al. Compartmentalized phosphodiesterase-2 activity blunts β-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway. *Circulation research*. 2006;98:226-234.

15. Karam S, Margaria JP, Bourcier A, Mika D, Varin A, Bedioune I, Lindner M, Bouadjel K, Dessillons M, Gaudin F, et al. Cardiac overexpression of PDE4B blunts β-adrenergic response and maladaptive remodeling in heart failure. *Circulation*. 2020;142:161-174.

16. Mika D, Bobin P, Lindner M, Boet A, Hodzic A, Lefebvre F, Lechene P, Sadoune M, Samuel JL, Algalarrondo V, et al. Synergic PDE3 and PDE4 control intracellular cAMP and cardiac excitation-contraction coupling in a porcine model. *J Mol Cell Cardiol*. 2019;133:57-66.

17. Abi-Gerges A, Richter W, Lefebvre F, Matéo P, Varin A, Heymes C, Samuel J-L, Lugnier C, Conti M, Fischmeister R, et al. Decreased expression and activity of cAMP phosphodiesterases in cardiac hypertrophy and its impact on β-adrenergic cAMP signals. *Circulation research*. 2009;105:784-792.

18. Mehel H, Emons J, Vettel C, Wittköpper K, Seppelt D, Dewenter M, Lutz S, Sossalla S, Maier LS, Lechêne P, et al. Phosphodiesterase-2 is upregulated in human failing hearts and blunts β-adrenergic responses in cardiomyocytes. *Journal of the American College of Cardiology*. 2013;62:1596-1606.

19. Vettel C, Lindner M, Dewenter M, Lorenz K, Schanbacher C, Riedel M, Lämmle S, Meinecke S, Mason F, Sossalla S, et al. Phosphodiesterase 2 protects against catecholamine-induced arrhythmias and preserves contractile function after myocardial infarction. *Circulation research*. 2017;120:120-132.

20. Martinez SE, Wu AY, Glavas NA, Tang XB, Turley S, Hol WG, Beavo JA. The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding. *Proc Natl Acad Sci USA*. 2002;99:13260-13265.

21. Weber S, Zeller M, Guan K, Wunder F, Wagner M, El-Armouche A. PDE2 at the crossway between cAMP and cGMP signalling in the heart. *Cellular signalling*. 2017;38:76-84.

22. Zoccarato A, Surdo NC, Aronsen JM, Fields LA, Mancuso L, Dodoni G, Stangherlin A, Livie C, Jiang H, Sin YY, et al. Cardiac hypertrophy is inhibited by a local pool of cAMP regulated by phosphodiesterase 2. *Circulation research*. 2015;117:707-719.
23. Cachorro E, Gunscht M, Schubert M, Sadek MS, Siegert J, Dutt F, Bauermeister C, Quickert S, Berning H, Nowakowski F, et al. CNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2. *Circulation research*. 2023;132:400-414.

24. Monterisi S, Lobo MJ, Livie C, Castle JC, Weinberger M, Baillie GS, Surdo NC, Musheshe N, Stangherlin A, Gottlieb E, et al. PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling. *Elife*. 2017;6:e21374.

25. Baliga RS, Preedy MEJ, Dukinfield MS, Chu SM, Aubdool AA, Bubb KJ, Moyes AJ, Tones MA, Hobbs AJ. Phosphodiesterase 2 inhibition preferentially promotes NO/guanylyl cyclase/cGMP signaling to reverse the development of heart failure. *Proc Natl Acad Sci USA*. 2018;115:E7428-E7437.

26. Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. *Mol Ther*. 2008;16:1073-1080.

27. Royer A, van Veen TA, Le Bouter S, Marionneau C, Griol-Charhbili V, Leoni AL, Steenman M, van Rijen HV, Demolombe S, Goddard CA, et al. Mouse model of SCN5A-linked hereditary Lenegre's disease: age-related conduction slowing and myocardial fibrosis. *Circulation*. 2005;111:1738-1746.

28. Mitchell GF, Jeron A, Koren G. Measurement of heart rate and Q-T interval in the conscious mouse. *Am J Physiol*. 1998;274:H747-751.

29. Wobbrock JO, Findlater L, Gergle D, Higgins JJ. The aligned rank transform for nonparametric factorial analyses using only anova procedures. *CHI '11: Proceedings of the SIGCHI Conference on Human Factors in Computing Systems*. 2011:143-146.

30. Bish LT, Morine K, Sleeper MM, Sanmiguel J, Wu D, Gao G, Wilson JM, Sweeney HL. Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat. *Hum Gene Ther*. 2008;19:1359-1368.

31. Lymeropoulos A, Rengo G, Koch WJ. Adrenergic nervous system in heart failure: pathophysiology and therapy. *Circulation research*. 2013;113:739-753.

32. Irie T, Sips PY, Kai S, Kida K, Ikeda K, Hirai S, Moazzami K, Jiramongkolchai P, Bloch DB, Doulias PT, et al. S-nitrosylation of calcium-handling proteins in cardiac adrenergic signaling and hypertrophy. *Circulation research*. 2015;117:793-803.

33. Dewenter M, Pan J, Knodler L, Tz schockel N, Henrich J, Cordero J, Dobreva G, Lutz S, Backs J, Wieland T, et al. Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha1-adrenoceptors to early cardiac stress responses. *Basic research in cardiology*. 2022;117:15.

34. Wagner M, Sadek MS, Dybkova N, Mason FE, Klehr J, Firneburg R, Cachorro E, Richter K, Klapproth E, Kuenzel SR, et al. Cellular mechanisms of the anti-arrhythmic effect of cardiac PDE2 overexpression. *Int J Mol Sci*. 2021;22:4816.

35. Ding B, Abe J, Wei H, Huang Q, Walsh RA, Molina CA, Zhao A, Sadoshima J, Blaxall BC, Berk BC, et al. Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure. *Circulation*. 2005;111:2469-2476.

36. Holmes JR, Kubo SH, Cody RJ, Kligfield P. Milrinone in congestive heart failure: observations on ambulatory ventricular arrhythmias. *American heart journal*. 1985;110:800-806. doi: 10.1016/0002-8703(85)90460-0

37. Packer M, Carver JR, Rodeheffer RJ, Ivanhoe RJ, DiBianco R, Zeldis SM, Hendrix GH, Bommer WJ, Elkayam U, Kukin ML, et al. Effect of oral milrinone on mortality in severe chronic heart failure. The PROMISE Study Research Group. *The New England journal of medicine*. 1991;325:1468-1475.

38. Leroy J, Richter W, Mika D, Castro LRV, Abi-Gerges A, Xie M, Scheitrum C, Lefebvre F, Schittl J, Westenbroek R, et al. Phosphodiesterase 4B in the cardiac L-type Ca²⁺ channel complex regulates Ca²⁺ current and protects against ventricular arrhythmias. *The Journal of clinical investigation*. 2011;121:2651-2661.

39. Pavlaki N, Froese A, Li W, De Jong KA, Geertz B, Subramanian H, Mohagaonkar S, Luo X, Schubert M, Wiegmann R, et al. Gene therapy with phosphodiesterases 2A and 4B
ameliorates heart failure and arrhythmias by improving subcellular cAMP compartmentation.
*Cardiovascular research*. 2024;120:1011-1023.

40. Benjamin IJ, Jalil JE, Tan LB, Cho K, Weber KT, Clark WA. Isoproterenol-induced myocardial fibrosis in relation to myocyte necrosis. *Circulation research*. 1989;65:657-670.

41. Rockman HA, Koch WJ, Lefkowitz RJ. Seven-transmembrane-spanning receptors and heart function. *Nature*. 2002;415:206-212.

42. Sprenger JU, Perera RK, Steinbrecher JH, Lehnart SE, Maier LS, Hasenfuss G, Nikolaev VO. *In vivo* model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease. *Nature communications*. 2015;6:6965.

43. Castro LR, Verde I, Cooper DMF, Fischmeister R. Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. *Circulation*. 2006;113:2221-2228.

44. Wang L, Henrich M, Buckler KJ, McMenamin M, Mee CJ, Sattelle DB, Paterson DJ. Neuronal nitric oxide synthase gene transfer decreases [Ca²⁺]ᵢ in cardiac sympathetic neurons. *J Mol Cell Cardiol*. 2007;43:717-725.

45. Ercu M, Mucke MB, Pallien T, Marko L, Sholokh A, Schachterle C, Aydin A, Kidd A, Walter S, Esmati Y, et al. Mutant phosphodiesterase 3A protects from hypertension-induced cardiac damage. *Circulation*. 2022;146:1758-1778.

46. Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: from molecular regulation to clinical use. *Pharmacol Rev*. 2006;58:488-520.

47. Martins TJ, Mumby MC, Beavo JA. Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from bovine tissues. *J Biol Chem*. 1982;257:1973-1979.

48. Numata G, Takimoto E. Cyclic GMP and PKG signaling in heart failure. *Front Pharmacol*. 2022;13:792798.

49. McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, Rouleau JL, Shi VC, Solomon SD, Swedberg K, et al. Angiotensin-neprilysin inhibition versus enalapril in heart failure. *The New England journal of medicine*. 2014;371:993-1004.

50. Prajapati C, Koivumaki J, Pekkanen-Mattila M, Aalto-Setala K. Sex differences in heart: from basics to clinics. *Eur J Med Res*. 2022;27:241.

51. Caldwell JL, Lee IJ, Ngo L, Wang L, Bahriz S, Xu B, Bers DM, Navedo MF, Bossuyt J, Xiang YK, et al. Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics. *Sci Adv*. 2023;9:eadd5799.

52. Parks RJ, Ray G, Bienvenu LA, Rose RA, Howlett SE. Sex differences in SR Ca release in murine ventricular myocytes are regulated by the cAMP/PKA pathway. *J Mol Cell Cardiol*. 2014;75:162-173.

53. Thompson WJ, Appleman MM. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. *Biochemistry*. 1971;10:311-316.
FIGURE LEGENDS

**Figure 1.** PDE2A overexpression by AAV9 does not alter cardiac function or morphological parameters.

A, Schematic representation of the experimental protocol. Mice were injected in the tail vein with 10¹² viral particles of serotype 9 adeno-associated viruses encoding for Luciferase (AAV9-LUC) or phosphodiesterase 2A3 (AAV9-PDE2A). Twenty-eight days later, mice were euthanized. Cardiac function was assessed throughout the protocol by echocardiography prior, then 2 and 4 weeks after the injection of the recombinant viruses. B, Left panel shows representative blots of PDE2A and GAPDH, the bar graph represents the ratios of PDE2A over GAPDH quantified, expressed as mean ± SEM of fold change in cardiac tissues from AAV9-LUC and AAV9-PDE2A mice. C, Heart weight (HW) and lung weight (LV) over tibia length (TL) ratios expressed as mean ± SEM. D, Time course of ejection fraction and the ratio of calculated left ventricular weight (LVW) over body weight (BW) in AAV9-LUC and AAV9-PDE2A mice. Number of mice is indicated in the brackets. Statistical significance is indicated by the p value determined using: Welch's t-test (B) Mann-Whitney (HW/TL) and unpaired student's t-test (LW/TL) (C), two-way Anova followed by a by a Tukey's test (D).

**Figure 2.** Gene therapy with PDE2A prevents cardiac remodeling and dysfunction induced by chronic isoproterenol infusion.

A, Schematic representation of the experimental protocol. Mice were injected in the tail vein with 10¹² viral particles of a serotype 9 adeno-associated viruses encoding for Luciferase (AAV9-LUC) or phosphodiesterase 2A3 (AAV9-PDE2A). Fourteen days later, mice injected with
AAV9-LUC were implanted subcutaneously with osmotic pumps diffusing either NaCl (NaCl-LUC) or isoproterenol at 60 mg/kg/day (Iso-LUC) and animals injected with AAV9-PDE2A were implanted with pumps delivering isoproterenol at 60 mg/kg/day (Iso-PDE2A). Treatment duration was 14 days (hatched bars). Minipumps were then removed and mice were kept for 2 additional weeks before euthanasia. Cardiac function was evaluated by serial echocardiography before injection of the virus as well as at 14, 28, 35, 42 days. B, Left panel shows representative blots of PDE2A and vinculin. PDE2A protein expression in heart tissues extracts measured by Western blot and the bar graph represents the ratios of PDE2A over vinculin quantified expressed as mean ± SEM in NaCl-LUC, Iso-LUC and Iso-PDE2A groups. C, Heart weight (HW) and lung weight (LW) over tibia length (TL) ratio expressed as mean ± SEM. D, Time course of the normalized ratio of calculated left ventricular weight (LVW) over body weight (BW) and normalized ejection fraction in NaCl-LUC, Iso-LUC and Iso-PDE2A mice. Number of mice is indicated in the brackets. E, Panel on the left, representative images of Masson's trichrome staining (scale bar 100 µm), bar graph on the left represents quantifications of interstitial fibrosis as mean ± SEM in NaCl-LUC (n=9), Iso-LUC (n=9) and Iso+PDE2A (n=9) groups. Statistical significance is indicated by the p value (* vs. NaCl-LUC and † vs. Iso-LUC) determined using: Welch's t-test (B), Kruskal-Wallis followed by a Dunn's test (C panel below and E), two-way Anova followed by a Tukey's test (D), One-way Anova followed by a Tukey's test (C panel above).

<caption>**Figure 3.** PDE2A overexpression limits maladaptive remodeling and cardiac dysfunction induced by chronic isoproterenol and phenylephrine infusion.</caption>

A, Schematic representation of the experimental protocol. Mice were injected with 10¹² viral particles of serotype 9 adeno-associated viruses (AAV9) encoding for Luciferase (AAV9-LUC)
or phosphodiesterase 2A3 (AAV9-PDE2A). Fourteen days later, mice were implanted with osmotic minipumps diffusing either NaCl (NaCl-LUC) or isoproterenol+phenylephrine at 30 mg/kg/day each (Iso+Phe-LUC and Iso+Phe-PDE2A). Minipumps were removed two weeks later and mice were euthanized. Cardiac function was assessed throughout the protocol using echocardiography before the injection of the virus and at 2, 3, 4, 6 weeks. B, Representative blots of PDE2A and GAPDH on the left panel are shown and the bar graph represents ratios of PDE2A over GAPDH quantifications expressed as Mean ± SEM in a subset of the animals treated with NaCl-LUC, Iso+Phe-LUC and Iso+Phe-PDE2A groups. C, Heart weight (HW) and lung weight (LW) over tibia length (TL) ratios expressed as mean ± SEM. D, Time course of the normalized ratios of calculated left ventricular weight (LVW) over body weight (BW) and normalized ejection fraction in NaCl-LUC, Iso+Phe-LUC and Iso+Phe-PDE2A mice. Number of mice is indicated in the brackets. E, Left panel shows representative images of Masson's trichrome staining (scale bar 50 or 200 µm), the right panel depicts representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (scale bar 100 µm) detecting apoptotic nuclei in green co-stained with the glycocalix marker wheat germ agglutinin in red and nuclei in blue colored with Hoechst. Bar graphs below representing mean ± SEM quantifications of interstitial fibrosis and apoptotic nuclei in NaCl-LUC (10), Iso+Phe-LUC (9) and Iso+Phe-PDE2A (9) mice are depicted. Statistical significance indicated by the p value (* vs. NaCl-LUC and † vs. Iso+Phe-LUC) was determined using Welch's t-test (B), Kruskal-Wallis followed by a Dunn's test (C), Two-way Anova followed by a Tukey's test (D), Welch's One-way Anova followed by a Dunnett's test (E).
**Figure 4.** PDE2A overexpression ameliorates the β-AR responsiveness of the excitation-contraction coupling.

A, Representative traces of calcium transients (CaT) and C, sarcomere shortening (SS) obtained simultaneously from ventricular myocytes loaded with fura-2 and paced (1 Hz) isolated from NaCl-LUC (grey), Iso+Phe-LUC (black) or Iso+Phe-PDE2A (green) mice in control conditions and in the presence of 3 nM isoproterenol (Iso). B, bar graph of mean ± SEM of the CaT amplitudes (expressed as the percentage of diastolic fura-2 ratio) (Left panel) and of the kinetics for return to Ca²⁺ diastolic levels (right panel) in control conditions and in the presence of 3 nM Iso. D, bar graph of mean ± SEM of SS amplitude expressed as the percentage of resting sarcomere length (left panel) and of relaxation time constant (tau, right panel) at baseline and in the presence of 3 nM Iso. SS and CaT were measured in 43 to 64 cells from 9 NaCl-LUC (grey bars), 10 Iso+Phe-LUC (black bars) and 8 Iso+Phe-PDE2A (green bars) mice. Numbers are indicated in the brackets as following (n=number of cells; N=Number of mice). Statistical significance indicated by the p value († vs. corresponding group at baseline) was determined after an aligned rank transform procedure (ARTool) using a two-way mixed Anova nested test (B, D).

**Figure 5.** Gene therapy with PDE2A exerts anti-arrhythmic effects at the cellular level and in vivo.

A, Representative traces of spontaneous calcium release events pointed by arrow, expressed as the percentage of diastolic fura-2 ratio in paced (1 Hz) ventricular myocytes isolated from Iso+Phe-LUC mice in the presence of 100 nM isoproterenol. B, SCEs were counted during the 3 min following isoproterenol 100 nM administration. SCEs were measured in 12 to 21 cells isolated from 3 NaCl-LUC (grey), 5 Iso+Phe-LUC (black) and 3 Iso+Phe-PDE2A (green) mice.
Numbers are indicated in the brackets as following (n=number of cells; N=number of mice). C, Representative examples of simultaneous lead 1 ECG and intraventricular electrogram recordings obtained in a mouse subjected to gene therapy with AAV9-LUC and chronically infused with isoproterenol and phenylephrine (Iso+Phe, 30 mg/kg/day each) for 14 days after paced extrasystoles. The protocol consisted of twenty pulses at a cycle length of 70 ms followed by one then two then three closer pulses (cycle length determined by the refractory period). Ventricular tachycardia (VT) was defined as the occurrence after the last paced beat of at least 4 consecutive QRS complexes with a different morphology from that seen in normal sinus rhythm. D, Histogram showing the percentage of mice with arrhythmias in all three groups (NaCl-LUC, Iso+Phe-LUC, Iso+Phe-PDE2A) at baseline and after isoproterenol 1.5 mg/kg + atropine 1 mg/kg injection. The number of animals presenting VTs per group is indicated beneath the histogram. Statistical significance is indicated by the p value († vs. corresponding group at baseline, * Iso+Phe-LUC vs. Iso+Phe-PDE2A) determined using: Wilcoxon's test (B); Barnard's exact test (D).
A

AAV9 injection 10¹²
vp/mouse

Echo
D0

Echo
D14

Echo
D28

Euthanasia

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

B

<figure><img src="image_3.png" /></figure>

PDE2A/GAPDH
(fold change)

<figure><img src="image_4.png" /></figure>

p=0.005

C

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

D

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 1</figcaption></figure>
A

AAV9 injection
$10^{12}$ vp/mouse

Minipump
implantation

Minipump
removed

Euthanasia

<figure><img src="image_9.png" /></figure>

B

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

C

p=0.025

p=0.0002

<figure><img src="image_12.png" /></figure>

D

NaCl-LUC (10)

Iso-LUC (10)

Iso-PDE2A (10)

<figure><img src="image_13.png" /></figure>

E

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

<figure><img src="image_15.png" /></figure>

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

<figure><img src="image_17.png" /><figcaption>Figure 2</figcaption></figure>
A

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

B

PDE2A
GAPDH

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

■ Iso+Phe-LUC (N=9)

■ Iso+Phe-PDE2A (N=10)

PDE2A/GAPDH
(fold change)

p=0.0004

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

C

D

E

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

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

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

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

AAV9-LUC

AAV9-PDE2A

NaCl

Iso+Phe

TUNEL

<figure><img src="image_27.png" /></figure>

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

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

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>
A

<figure><img src="image_41.png" /></figure>

B

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

C

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

D

<figure><img src="image_44.png" /><figcaption>Figure 5</figcaption></figure>
# Cardiac Gene Therapy with PDE2A Limits Remodeling and Arrhythmias in Mouse Models of Heart Failure

Rima Kamel, PhD<fnref n="1" />*, Aurélia Bourcier, PhD<fnref n="1" />*, Jean Piero Margaria, PhD<fnref n="1" />,<fnref n="2" />,<fnref n="5" />*, Valentin Jin, MSc<fnref n="1" />, Audrey Varin, MSc<fnref n="1" />, Agnès Hivonnait, MSc<fnref n="3" />, Françoise Mercier-Nomé, MSc<fnref n="4" />, Delphine Mika, PhD<fnref n="1" />, Alessandra Ghigo, PhD<fnref n="2" />, Flavien Charpentier, PhD<fnref n="3" />, Vincent Algalarrondo, MD, PhD<fnref n="1" />, Emilio Hirsch, PhD<fnref n="2" />, Rodolphe Fischmeister, PhD<fnref n="1" />,§, Grégoire Vandecasteele, PhD<fnref n="1" />, Jérôme Leroy, PhD<fnref n="1" />,§.

*these authors contributed equally to this work

Running title: PDE2 gene therapy to prevent heart failure

*these authors contributed equally to this work

Running title: PDE2 gene therapy to prevent heart failure

§Correspondence to:

Jérôme Leroy: jerome.leroy@universite-paris-saclay.fr

Rodolphe Fischmeister: rodolphe.fischmeister@inserm.fr

INSERM UMR-S 1180
Université Paris-Saclay
Faculté de Pharmacie
Bât. Henri Moissan
17, avenue des sciences
91400 Orsay
France

<footnote n="1">¹Université Paris-Saclay, Inserm, Signaling and Cardiovascular Pathophysiology, UMR-S 1180, F-91400 Orsay, France.</footnote>

<footnote n="2">² Molecular Biotechnology Center “Guido Tarone”, Department of Molecular Biotechnology and Health Sciences, University of Torino, I-10126 Torino, Italy.</footnote>

<footnote n="3">³ Nantes Université, CNRS, INSERM, l'institut du thorax, F-44000 Nantes, France.</footnote>

<footnote n="4">⁴ Université Paris-Saclay, Inserm US31, CNRS UMS3679, IPSIT, F-91400 Orsay, France.</footnote>

<footnote n="5">⁵ Cystic Kidney Disorders Unit, Division of Genetics and Cell Biology, Università Vita-Salute San Raffaele, Milan, Italy.</footnote>
# SUPPLEMENTAL METHODS

## HEK293 cell culture

HEK293T cells were cultured in 10 centimetres dishes and transfected for 48 h with plasmids expressing Flag-tagged exogenous PDE2A. The expression levels of exogenous PDE2A proteins were assessed by immunoblot analysis with the anti-FLAG antibody (Sigma-Aldrich, F3165).

## PDE activity assay

Cyclic AMP-PDE activity was measured according to the method of Thompson and Appleman as previously described⁵³. In brief, samples were assayed for 15 min at 33°C in a 200 µL reaction mixture that contained: 40 mmol/L Tris-HCl (pH 8.0), 1 mmol/L MgCl₂, 1.4 mmol/L β-mercaptoethanol, 1 µmol/L cAMP, 0.75 mg/ml BSA, and 0.1 µCi of [³H]cAMP. The reaction was stopped by heat inactivation in a boiling water bath for 1 min. The PDE reaction produced 5'-AMP which was then hydrolyzed by incubation of the assay mixture with 50 µg of *Crotalus atrox* snake venom for 20 min at 33°C. Finally, the resulting adenosine was separated by anion exchange chromatography using 1 ml AG1-X8 resin (Bio-Rad, Hercules, California, USA) and quantified by scintillation counting.
A

<figure><img src="image_45.png" /></figure>

B

<figure><img src="image_46.png" /><figcaption>Figure S1. AAV9-PDE2A construct allows PDE2A expression to increase PDE2 activity against cAMP in HEK293 cells.</figcaption></figure>

(A) Representative images of western blots to PDE2A, FLAG and GAPDH after AAV9-PDE2A transfection of HEK293 cells. Protein extracts from cells expressing the empty AAV9-CMV did not show any band at ≈100KDa, which is the expected molecular weight for PDE2A, unlike protein extracts from cells expressing either pAdTrack-PDE2A or AAV9-PDE2A. pAdTrack-PDE2A does not contain a CMV promoter controlling PDE2A expression, nor a FLAG tag. (B) Bar graphs showing quantification of PDE activity as mean ± SEM in HEK293 cells transfected with either AAV9-CMV (n=3) or AAV9-PDE2A (n=3) and treated with DMSO (vehicle) or Bay 60-7550 (specific PDE2 inhibitor) at 100 nmol/L. Statistical significance is indicated by the p value determined using a two-way Anova followed by a Tukey's test.
A

<figure><img src="image_47.png" /></figure>

B

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

**Figure S2.** AAV9 mediated transgene expression of luciferase and PDE2A four weeks after systemic delivery by tail vein injection in mouse. A. Firefly activity measured in protein lysates obtained from mice hearts injected with 3 different doses of AAV9-LUC (1x10¹⁰, 1x10¹¹, 1x10¹² viral particles per animal). Bar graphs representing mean ± SEM activity measured in triplicates from one heart. B. Representative images of a western blot of PDE2A in protein extracts from diaphragm (D), liver (L), kidney (K), and heart (H) obtained from mice injected with either AAV9-LUC or AAV9-PDE2A (10¹² viral particles per animals). The bar graphs on the right panel represent the ratios of PDE2A over total protein stain with LI-COR® quantified, expressed as mean ± SEM of fold change in cardiac tissues from AAV9-PDE2A mice compared to tissues from the mouse injected with AAV9-LUC.
A

<figure><img src="image_49.png" /></figure>

B

<figure><img src="image_50.png" /><figcaption>Figure S3. PDE2A protein levels are not affected by chronic isoproterenol-phenylephrine infusion.</figcaption></figure>

A, Representative images of a western blot to PDE2A, in protein extracts from ventricular myocytes isolated from NaCl-LUC, Iso+Phe-LUC and Iso-Phe-PDE2A mice. B, The bar graphs represent the ratios of PDE2A over total protein stain with LI-COR® expressed in NaCl-LUC (grey) or Iso+Phe-LUC (black), as mean ± SEM (n=6 each).
A

<figure><img src="image_51.png" /></figure>

B

<figure><img src="image_52.png" /></figure>

C

<figure><img src="image_53.png" /></figure>

D

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

Figure S4. PDE3A, PDE4A, PDE4B and PDE4D protein levels are not affected by chronic isoproterenol-phenylephrine infusion.

Left, representative images of a western blot of A: PDE3A, B: PDE4A, C: PDE4B and D: PDE4D in protein extracts from ventricular myocytes isolated from NaCl-LUC (grey), Iso+Phe-LUC (black) and Iso-Phe-PDE2A (green) mice. The bar graphs on the right represent the ratios of PDE over total protein stain with LI-COR® expressed in NaCl-LUC (grey), Iso+Phe-LUC (black) and Iso-Phe-PDE2A (green), as mean ± SEM (n=6 each).
<figure><img src="image_59.png" /><figcaption>Figure S5. PDE2A mRNA is increased in cardiac tissues 6 weeks after injection of the recombinant AAV9 encoding for this enzyme.</figcaption></figure>

Bar graphs representing mean ± SEM of relative PDE2A mRNA expression in NaCl-LUC (mice implanted with minipump diffusing saline solution received a tail vein injection with AAV9-LUC), Iso-LUC (mice implanted with isoproterenol minipump received a tail vein injection with AAV9-LUC) and Iso-PDE2A (mice implanted with isoproterenol minipump received a tail vein injection of AAV9-PDE2A). The number of mice per group is indicated in the brackets. Statistical significance is indicated by the p value; Kruskal-Wallis test followed by a Dunn's test.
A

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

B

<figure><img src="image_62.png" /></figure>

<figure><img src="image_63.png" /></figure>

**Figure S6.** Sympathomimetic amines infusion increases heart rate in mice.

A, Time course of the heart rate in NaCl-LUC, Iso-LUC and Iso-PDE2A mice. B, Time course of the heart rate in NaCl-LUC, Iso+Phe-LUC and Iso+Phe-PDE2A mice. Statistical significance is indicated by the p value (* vs. value at day 0 \# vs. NaCl-LUC) determined using a Two-way Anova followed by a Tukey's test.
A

<figure><img src="image_64.png" /></figure>

B

<figure><img src="image_65.png" /><figcaption>**Figure S7. Characterization of the spontaneous calcium events**</figcaption></figure>

A, Representative bar graphs of the median of time in seconds to the first spontaneous calcium event (SCE) in the presence of 100 nM isoproterenol in paced (1 Hz) ventricular myocytes isolated from NaCl-LUC (grey), IP-LUC (black) and IP-PDE2A (green) mice. B, Representative bar graphs of the percentage of cells presenting SCEs at baseline and in the presence of 100 nM isoproterenol in paced (1 Hz) ventricular myocytes isolated from NaCl-LUC, IP-LUC and IP-PDE2A mice.
<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /></figure>

<figure><img src="image_68.png" /></figure>

<figure><img src="image_69.png" /></figure>

Figure S8. PDE4B3 overexpression limits maladaptive cardiac remodelling and dysfunction induced by chronic isoproterenol-phenylephrine infusion but does not limit ventricular arrhythmias induced by stress.

A, Schematic representation of the experimental protocol. Mice were injected with 10¹² viral particles of serotype 9 adeno-associated viruses (AAV9) encoding for Luciferase (LUC) or phosphodiesterase 4B3 (PDE4B). Fourteen days later, mice were implanted with osmotic minipumps diffusing either NaCl (NaCl-LUC) or isoproterenol+phenylephrine at 30 mg/Kg/day each (Iso+Phe-LUC and Iso+Phe-PDE4B3). Minipumps were removed two weeks later and mice were sacrificed. Cardiac function was assessed throughout the protocol using echocardiography before the injection of the virus and at 0, 2, 3, 4 weeks. B, Time course of the normalized ratios of calculated left ventricular weight (LVW) over body weight (BW) and normalized ejection fraction (EF) in NaCl-LUC, Iso+Phe-LUC and Iso+Phe-PDE4B3 mice. Number of mice is indicated in the brackets. C, Bar graph showing the percentage of mice with arrhythmias in Iso+Phe-PDE4B3 mice at baseline and after isoproterenol 1.5 mg/kg + Atropine 1 mg/kg injection. The number of animals presenting VTs per group is indicated beneath the bar graphs. Statistical significance indicated by the p value determined using Two-way Anova followed by a Tukey's test; Barnard's exact test (C).
<caption>Table S1. Echocardiographic parameters of LUC and PDE2A injected mice 28 days after AAV9 injection.</caption>

A=late diastolic transmitral flow velocities, bpm=beats per minute, BW=body weight, Dec. time=E-wave deceleration time, E=early diastolic transmitral flow velocities, EF=ejection fraction, FS=Fractional shortening, HR=heart rate, HW=heart weight, IVSd=end-diastolic interventricular septum, IVSs=end-systolic interventricular septum, LV=left ventricle, LVIDd=end-diastolic left ventricular internal diameter, LVIDs=end-systolic left ventricular internal diameter, LW=lungs weight, PWd=end-diastolic posterior wall, PWs=end-systolic posterior wall, TL=tibia length. No statistical difference among the parameters was found when evaluated using a Mann Whitney test, an unpaired t-test or a Welch's test.

<table><thead><tr><th rowspan="2"></th><th colspan="3">LUC</th><th colspan="3">PDE2A</th></tr><tr><th>Mean</th><th>SEM</th><th>N</th><th>Mean</th><th>SEM</th><th>N</th></tr></thead><tbody><tr><td>HW (mg)</td><td>176.00</td><td>14.31</td><td>5</td><td>176.00</td><td>6.78</td><td>5</td></tr><tr><td>LW (mg)</td><td>130.20</td><td>6.31</td><td>5</td><td>124.00</td><td>5.1</td><td>5</td></tr><tr><td>BW (g)</td><td>27.00</td><td>1.39</td><td>5</td><td>28.00</td><td>0.42</td><td>5</td></tr><tr><td>TL (mm)</td><td>16.52</td><td>0.09</td><td>5</td><td>16.68</td><td>0.18</td><td>5</td></tr><tr><td>HW/BW (mg/g)</td><td>6.58</td><td>0.48</td><td>5</td><td>6.37</td><td>0.22</td><td>5</td></tr><tr><td>LW/BW (mg/g)</td><td>4.87</td><td>0.20</td><td>5</td><td>4.49</td><td>0.18</td><td>5</td></tr><tr><td>HR (bpm)</td><td>405.00</td><td>14.00</td><td>5</td><td>413.00</td><td>13.00</td><td>5</td></tr><tr><td>LV weight (mg)</td><td>102.94</td><td>4.06</td><td>5</td><td>104.11</td><td>1.78</td><td>5</td></tr><tr><td>FS (%)</td><td>30.48</td><td>0.77</td><td>5</td><td>29.69</td><td>0.49</td><td>5</td></tr><tr><td>LVIDd (mm)</td><td>4.40</td><td>0.02</td><td>5</td><td>4.34</td><td>0.03</td><td>5</td></tr><tr><td>IVSd (mm)</td><td>0.67</td><td>0.02</td><td>5</td><td>0.65</td><td>0.02</td><td>5</td></tr><tr><td>PWd (mm)</td><td>0.61</td><td>0.02</td><td>5</td><td>0.67</td><td>0.02</td><td>5</td></tr><tr><td>LVIDs (mm)</td><td>3.06</td><td>0.03</td><td>5</td><td>3.05</td><td>0.03</td><td>5</td></tr><tr><td>IVSs (mm)</td><td>1.04</td><td>0.02</td><td>5</td><td>1.13</td><td>0.02</td><td>5</td></tr><tr><td>PWs (mm)</td><td>1.01</td><td>0.05</td><td>5</td><td>1.03</td><td>0.05</td><td>5</td></tr><tr><td>E (m/s)</td><td>0.64</td><td>0.03</td><td>5</td><td>0.71</td><td>0.03</td><td>5</td></tr><tr><td>Dec. time (ms)</td><td>27.73</td><td>1.86</td><td>5</td><td>25.96</td><td>1.44</td><td>5</td></tr><tr><td>A (m/s)</td><td>0.37</td><td>0.02</td><td>5</td><td>0.41</td><td>0.02</td><td>5</td></tr><tr><td>E/A</td><td>1.77</td><td>0.07</td><td>5</td><td>1.75</td><td>0.05</td><td>5</td></tr></tbody></table>
<caption>Table S2. Echocardiographic parameters of NaCl-LUC, Iso-LUC and Iso-PDE2A mice at 42 days.</caption>

Bpm=beats per minute, BW=body weight, EF=ejection fraction, FS=fractional shortening, HR=heart rate, HW=heart weight, IVSd=end-diastolic interventricular septum, IVSs=end-systolic interventricular septum, LV=left ventricle, LVIDd=end-diastolic left ventricular internal diameter, LVIDs=end-systolic left ventricular internal diameter, LVW=left ventricular weight, LW=lungs weight, PWd=end-diastolic posterior wall, PWs=end-systolic posterior wall, TL=tibia length. Statistical significance indicated by the p value determined (* vs NaCl-LUC and # vs. Iso-LUC), using a One-way ANOVA followed by Tukey's test or a Kruskal-Wallis followed by a Dunn's multiple comparison test.

<table><thead><tr><th rowspan="2"></th><th colspan="3">NaCl-LUC</th><th colspan="3">Iso-LUC</th><th colspan="3">Iso-PDE2A</th></tr><tr><th>Mean</th><th>SEM</th><th>N</th><th>Mean</th><th>SEM</th><th>N</th><th>Mean</th><th>SEM</th><th>N</th></tr></thead><tbody><tr><td>HW (mg)</td><td>157.00</td><td>4.96</td><td>10</td><td>199.00<br>(*p=0.0003)</td><td>8.88</td><td>10</td><td>182.00<br>(*p=0.0315)</td><td>5.12</td><td>10</td></tr><tr><td>LW (mg)</td><td>138.00</td><td>3.27</td><td>10</td><td>155.00</td><td>8.20</td><td>10</td><td>144.00</td><td>3.06</td><td>10</td></tr><tr><td>BW (g)</td><td>28.18</td><td>0.60</td><td>10</td><td>28.00</td><td>0.43</td><td>10</td><td>28.44</td><td>0.67</td><td>10</td></tr><tr><td>TL (mm)</td><td>18.03</td><td>0.07</td><td>10</td><td>17.73</td><td>0.28</td><td>10</td><td>17.83</td><td>0.09</td><td>10</td></tr><tr><td>HW/BW<br>(mg/g)</td><td>5.57</td><td>0.11</td><td>10</td><td>7.11 (*p&lt;0.0013)</td><td>0.30</td><td>10</td><td>6.40 (*p=0.0006)</td><td>0.14</td><td>10</td></tr><tr><td>LW/BW<br>(mg/g)</td><td>4.92</td><td>0.17</td><td>10</td><td>5.55</td><td>0.30</td><td>10</td><td>5.10</td><td>0.17</td><td>10</td></tr><tr><td>HR (bpm)</td><td>508.77</td><td>19.43</td><td>10</td><td>517.42</td><td>22.84</td><td>10</td><td>480.52</td><td>31.49</td><td>10</td></tr><tr><td>LV weight<br>(mg)</td><td>114.67</td><td>6.88</td><td>10</td><td>166.50<br>(*p=0.0014)</td><td>10.99</td><td>10</td><td>129.20</td><td>6.08</td><td>10</td></tr><tr><td>LVW/BW<br>(mg/g)</td><td>3.95</td><td>0.24</td><td>10</td><td>5.72 (*p=0.0005)</td><td>0.40</td><td>10</td><td>4.42 (#p=0.069)</td><td>0.17</td><td>10</td></tr><tr><td>EF (%)</td><td>71.71</td><td>2.34</td><td>10</td><td>40.10 (*p&lt;0.0001)</td><td>3.16</td><td>10</td><td>64.20 (#p&lt;0.0001)</td><td>2.62</td><td>10</td></tr><tr><td>FS (%)</td><td>35.70</td><td>1.76</td><td>10</td><td>16.40<br>(*p&lt;0.0001)</td><td>1.49</td><td>10</td><td>30.30<br>(#p&lt;0.0001)</td><td>1.80</td><td>10</td></tr><tr><td>LVIDd<br>(mm)</td><td>3.46</td><td>0.14</td><td>10</td><td>4.23(*p=0.0001)</td><td>0.14</td><td>10</td><td>3.50(#p&lt;0.0001)</td><td>0.12</td><td>10</td></tr><tr><td>IVSd (mm)</td><td>0.90</td><td>0.06</td><td>10</td><td>0.96</td><td>0.02</td><td>10</td><td>0.86</td><td>0.06</td><td>10</td></tr><tr><td>PWd<br>(mm)</td><td>1.13</td><td>0.11</td><td>10</td><td>0.80(*p=0.0277)</td><td>0.01</td><td>10</td><td>0.87</td><td>0.07</td><td>10</td></tr><tr><td>LVIDs<br>(mm)</td><td>2.31</td><td>0.17</td><td>10</td><td>3.04(*p=0.0001)</td><td>0.22</td><td>10</td><td>2.07(#p&lt;0.0001)</td><td>0.19</td><td>10</td></tr><tr><td>IVSs (mm)</td><td>1.04</td><td>0.07</td><td>10</td><td>0.99</td><td>0.03</td><td>10</td><td>0.98</td><td>0.04</td><td>10</td></tr><tr><td>PWs (mm)</td><td>1.36</td><td>0.10</td><td>10</td><td>1.07(*p=0.0475)</td><td>0.07</td><td>10</td><td>1.02(*p=0.0054)</td><td>0.04</td><td>10</td></tr></tbody></table>
<table><caption>**Table S3. Echocardiographic parameters of NaCl-LUC, Iso+Phe-LUC and Iso+Phe-PDE2A mice at 28 days.**

A=late diastolic transmitral flow velocities, Bpm=beats per minute, BW=body weight, Dec. time=E-wave deceleration time, E=early diastolic transmitral flow velocities, EF=ejection fraction, FS=Fractional shortening, HR=heart rate, HW=heart weight, IVSd=end-diastolic interventricular septum, IVSs=end-systolic interventricular septum, LV=left ventricle, LVIDd=end-diastolic left ventricular internal diameter, LVIDs=end-systolic left ventricular internal diameter, LVW=left ventricular weight, LW=lungs weight, PWd=end-diastolic posterior wall, PWs=end-systolic posterior wall, TL=tibia length. Statistical significance indicated by the p value \*vs. NaCl-LUC, \#vs. Iso+Phe-LUC, determined using a One-way ANOVA followed by a Tukey's test or a Kruskal-Wallis followed by a Dunn's multiple comparison test.</caption><thead><tr><th rowspan="2"></th><th colspan="3">NaCl-LUC</th><th colspan="3">Iso+Phe-LUC</th><th colspan="3">Iso+Phe-PDE2A</th></tr><tr><th>Mean</th><th>SEM</th><th>N</th><th>Mean</th><th>SEM</th><th>N</th><th>Mean</th><th>SEM</th><th>N</th></tr></thead><tbody><tr><td>HW (mg)</td><td>184.00</td><td>3.84</td><td>17</td><td>207.33<br>(*p=0.0016)</td><td>4.65</td><td>20</td><td>204.31(*p=0.0104)</td><td>5.08</td><td>16</td></tr><tr><td>LW (mg)</td><td>143.18</td><td>3.07</td><td>17</td><td>170.25<br>(*p=0.0001)</td><td>4.52</td><td>20</td><td>165.81(*p=0.0026)</td><td>5.25</td><td>16</td></tr><tr><td>BW (g)</td><td>26.26</td><td>0.32</td><td>17</td><td>24.16</td><td>0.46</td><td>20</td><td>25.31</td><td>0.36</td><td>16</td></tr><tr><td>TL (mm)</td><td>16.86</td><td>0.13</td><td>17</td><td>16.60</td><td>0.16</td><td>20</td><td>16.78</td><td>0.14</td><td>16</td></tr><tr><td>HW/BW<br>(mg/g)</td><td>7.02</td><td>0.16</td><td>17</td><td>8.67 (*p&lt;0.0001)</td><td>0.31</td><td>20</td><td>8.35(*p=0.0004)</td><td>0.23</td><td>16</td></tr><tr><td>LW/BW<br>(mg/g)</td><td>5.46</td><td>0.12</td><td>17</td><td>7.11(*p&lt;0.0001)</td><td>0.25</td><td>20</td><td>6.78(*p=0.0001)</td><td>0.23</td><td>16</td></tr><tr><td>HR (bpm)</td><td>477.00</td><td>13.00</td><td>17</td><td>442.00</td><td>13.00</td><td>20</td><td>461.00</td><td>9.00</td><td>16</td></tr><tr><td>LV weight<br>(mg)</td><td>114.78</td><td>4.82</td><td>17</td><td>139.28<br>(*p=0.0024)</td><td>5.28</td><td>20</td><td>128.26</td><td>5.35</td><td>16</td></tr><tr><td>LVW/BW<br>(mg/g)</td><td>4.23</td><td>0.22</td><td>17</td><td>5.66 (*p=0.0002)</td><td>0.24</td><td>20</td><td>5.09 (*p=0.0413)</td><td>0.25</td><td>16</td></tr><tr><td>EF (%)</td><td>69.06</td><td>1.14</td><td>17</td><td>49.85<br>(*p&lt;0.0001)</td><td>1.66</td><td>20</td><td>55.00 (*p&lt;0.0001;<br>#p=0.0361)</td><td>1.30</td><td>16</td></tr><tr><td>FS (%)</td><td>33.70</td><td>0.87</td><td>17</td><td>21.65<br>(*p&lt;0.0001)</td><td>0.92</td><td>20</td><td>24.51<br>(p&lt;0.0001) (#p=0.06)</td><td>0.75</td><td>16</td></tr><tr><td>LVIDd<br>(mm)</td><td>4.29</td><td>0.05</td><td>17</td><td>4.50 (*p=0.082)</td><td>0.07</td><td>20</td><td>4.70 (*p&lt;0.0001;<br>#p=0.0159)</td><td>0.04</td><td>16</td></tr><tr><td>IVSd (mm)</td><td>0.73</td><td>0.03</td><td>17</td><td>0.77</td><td>0.02</td><td>20</td><td>0.67</td><td>0.03</td><td>16</td></tr><tr><td>PWd (mm)</td><td>0.71</td><td>0.03</td><td>17</td><td>0.81(*p=0.0343)</td><td>0.03</td><td>20</td><td>0.72</td><td>0.02</td><td>16</td></tr><tr><td>LVIDs<br>(mm)</td><td>2.85</td><td>0.06</td><td>17</td><td>3.53 (*p&lt;0.0001)</td><td>0.07</td><td>20</td><td>3.55 (*p&lt;0.0001)</td><td>0.05</td><td>16</td></tr><tr><td>IVSs (mm)</td><td>1.24</td><td>0.04</td><td>17</td><td>1.16</td><td>0.03</td><td>20</td><td>1.19</td><td>0.03</td><td>16</td></tr><tr><td>PWs (mm)</td><td>1.14</td><td>0.04</td><td>17</td><td>1.02</td><td>0.04</td><td>20</td><td>1.03</td><td>0.03</td><td>16</td></tr><tr><td>E (m/s)</td><td>0.74</td><td>0.02</td><td>17</td><td>0.66</td><td>0.04</td><td>20</td><td>0.63</td><td>0.03</td><td>16</td></tr><tr><td>Dec. time<br>(ms)</td><td>24.14</td><td>0.85</td><td>17</td><td>22.55</td><td>0.94</td><td>20</td><td>24.22</td><td>0.79</td><td>16</td></tr><tr><td>A (m/s)</td><td>0.43</td><td>0.02</td><td>17</td><td>0.30 (*p=0.0002)</td><td>0.02</td><td>20</td><td>0.36</td><td>0.02</td><td>16</td></tr><tr><td>E/A</td><td>1.78</td><td>0.09</td><td>17</td><td>2.44</td><td>0.28</td><td>20</td><td>1.89</td><td>0.18</td><td>16</td></tr></tbody></table>
<table><caption>**Table S4. Diastolic and systolic sarcomere length and calcium levels under basal conditions and after β-AR stimulation.** The parameters correspond to those obtained when measuring simultaneously the calcium transients and sarcomere shortening in ventricular myocytes loaded with fura-2 and paced (1 Hz), isolated from NaCl-LUC, Iso+Phe-LUC or Iso+Phe-PDE2A mice in control conditions and in the presence of 3 nM isoproterenol (Iso) as presented in Figure 4 of the manuscript.</caption><thead><tr><th rowspan="2">Group</th><th colspan="2">NaCl-LUC</th><th colspan="2">IP-LUC</th><th colspan="2">IP-PDE2A</th></tr><tr><th>CT</th><th>Iso 3nM</th><th>CT</th><th>Iso 3nM</th><th>CT</th><th>Iso 3nM</th></tr></thead><tbody><tr><td>Stimulation</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Diastolic L (μm)</td><td>1.71±0.01</td><td>1.69±0.01</td><td>1.70±0.01</td><td>1.69±0.01</td><td>1.70±0.01</td><td>1.68±0.01</td></tr><tr><td>Peak L (μm)</td><td>1.67±0.01</td><td>1.53±0.01</td><td>1.67±0.01</td><td>1.59±0.01</td><td>1.66±0.01</td><td>1.56±0.01</td></tr><tr><td>ΔL (μm)</td><td>0.036±0.005</td><td>0.161±0.010</td><td>0.037±0.005</td><td>0.094±0.010</td><td>0.040±0.006</td><td>0.117±0.015</td></tr><tr><td>ΔL (%)</td><td>2.10±0.28</td><td>9.55±0.59</td><td>2.17±0.30</td><td>5.58±0.60</td><td>2.33±0.35</td><td>6.95±0.90</td></tr><tr><td>Diastolic Fura-2 ratio</td><td>3.79±0.12</td><td>3.74±0.11</td><td>4.01±0.19</td><td>3.97±0.17</td><td>3.53±0.07</td><td>3.59±0.07</td></tr><tr><td>Systolic Fura-2 ratio</td><td>4.07±0.14</td><td>4.24±0.14</td><td>4.27±0.20</td><td>4.36±0.18</td><td>3.78±0.08</td><td>4.05±0.09</td></tr><tr><td>ΔR</td><td>0.28±0.03</td><td>0.50±0.04</td><td>0.26±0.02</td><td>0.39±0.03</td><td>0.25±0.02</td><td>0.45±0.04</td></tr><tr><td>ΔR (%)</td><td>7.06±0.50</td><td>13.14±0.82</td><td>6.33±0.44</td><td>9.83±0.75</td><td>7.14±0.51</td><td>12.50±1.16</td></tr><tr><td>Number of cells/ Number of mice</td><td>61/9</td><td>61/9</td><td>64/10</td><td>64/10</td><td>43/8</td><td>43/8</td></tr></tbody></table>
<caption>Table S5. Surface ECG parameters before and after a simultaneous injection of Isoproterenol (1.5 mg/Kg) and atropine (1 mg/Kg) in NaCl-LUC, Iso+Phe-LUC and Iso+Phe-PDE2A mice.</caption>

HR=Heart rate. \* Iso+Atropine vs. Basal, † vs. corresponding Baseline NaCl-LUC, # vs. corresponding Baseline ISO+PHE-LUC; . Statistical significance indicated by the p value obtained using, when appropriate, a two-way ANOVA followed by a Tukey's test or a Kruskal-Wallis followed by a Dunn's multiple comparison test.

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="3">Basal</th><th colspan="3">Iso+Atropine</th></tr><tr><th>Mean</th><th>SEM</th><th>N</th><th>Mean</th><th>SEM</th><th>N</th></tr></thead><tbody><tr><td rowspan="5">NaCl-LUC</td><td>RR (ms)</td><td>159.36</td><td>9.40</td><td>11</td><td>116.11(*p=0.0057)</td><td>4.31</td><td>10</td></tr><tr><td>HR (bpm)</td><td>391.06</td><td>22.41</td><td>11</td><td>524.23(*p&lt;0.0001)</td><td>20.10</td><td>10</td></tr><tr><td>PR (ms)</td><td>32.75</td><td>1.35</td><td>11</td><td>26.67(*p=0.0128)</td><td>0.72</td><td>5</td></tr><tr><td>QRS (ms)</td><td>16.55</td><td>1.02</td><td>11</td><td>19.41</td><td>1.19</td><td>10</td></tr><tr><td>QT (ms)</td><td>58.41</td><td>2.76</td><td>11</td><td>58.50</td><td>2.61</td><td>10</td></tr><tr><td rowspan="5">Iso+Phe-LUC</td><td>RR (ms)</td><td>129.49(†p=0.0098)</td><td>3.34</td><td>15</td><td>106.36(*p=0.0032)</td><td>2.46</td><td>14</td></tr><tr><td>HR (bpm)</td><td>467.88(†p=0.0098)</td><td>11.88</td><td>15</td><td>568.78(*p&lt;0.0001)</td><td>12.60</td><td>14</td></tr><tr><td>PR (ms)</td><td>30.71</td><td>0.85</td><td>12</td><td>24.63(*p=0.0003)</td><td>0.53</td><td>13</td></tr><tr><td>QRS (ms)</td><td>17.70</td><td>0.92</td><td>15</td><td>21.11(*p=0.0101)</td><td>1.06</td><td>14</td></tr><tr><td>QT (ms)</td><td>58.85</td><td>2.61</td><td>15</td><td>52.95</td><td>2.74</td><td>14</td></tr><tr><td rowspan="5">Iso+Phe-PDE2A</td><td>RR (ms)</td><td>145.64</td><td>7.75</td><td>11</td><td>108.66(*p=0.002)</td><td>2.85</td><td>11</td></tr><tr><td>HR (bpm)</td><td>423.23</td><td>19.88</td><td>11</td><td>555.86(*p&lt;0.0001)</td><td>13.79</td><td>11</td></tr><tr><td>PR (ms)</td><td>30.99</td><td>1.04</td><td>11</td><td>24.85(*p=0.0008)</td><td>1.53</td><td>10</td></tr><tr><td>QRS (ms)</td><td>16.93</td><td>0.88</td><td>11</td><td>21.58(*p=0.0015)</td><td>1.09</td><td>11</td></tr><tr><td>QT (ms)</td><td>60.32</td><td>3.79</td><td>11</td><td>54.05</td><td>4.53</td><td>11</td></tr></tbody></table>
Table S6. Echocardiographic parameters of IP-LUC and IP-PDE4B injected mice 28 days after AAV9 injection.

A=late diastolic transmitral flow velocities, bpm=beats per minute, BW=body weight, Dec. time=E-wave deceleration time, E=early diastolic transmitral flow velocities, EF=ejection fraction, FS=Fractional shortening, HR=heart rate, IVSd=end-diastolic interventricular septum, IVSs=end-systolic interventricular septum, LV=left ventricle, LVIDd=end-diastolic left ventricular internal diameter, LVIDs=end-systolic left ventricular internal diameter, LW=lungs weight, PWd=end-diastolic posterior wall, PWs=end-systolic posterior wall. Statistical significance indicated by the p value using, when appropriate, a Mann Whitney test, an unpaired t-test or a Welch's test.

<table><thead><tr><th rowspan="2"></th><th colspan="3">IP-LUC</th><th colspan="3">IP-PDE4B</th></tr><tr><th>Mean</th><th>SEM</th><th>N</th><th>Mean</th><th>SEM</th><th>N</th></tr></thead><tbody><tr><td>BW (g)</td><td>25.1</td><td>0.6</td><td>9</td><td>25.4</td><td>0.4</td><td>10</td></tr><tr><td>HR (bpm)</td><td>456.1</td><td>9.3</td><td>9</td><td>473.3</td><td>16.8</td><td>10</td></tr><tr><td>LV weight (mg)</td><td>129.13</td><td>5.88</td><td>9</td><td>117.05 (p=0.09)</td><td>3.39</td><td>10</td></tr><tr><td>LVW (mg)/BW (g)</td><td>5.13</td><td>0.18</td><td>9</td><td>4.61 (p=0.0199)</td><td>0.10</td><td>10</td></tr><tr><td>EF (%)</td><td>53.28</td><td>1.97</td><td>9</td><td>59.66 (p=0.06)</td><td>2.48</td><td>10</td></tr><tr><td>FS (%)</td><td>23.5</td><td>1.2</td><td>9</td><td>27.5 (p=0.05)</td><td>1.7</td><td>10</td></tr><tr><td>LVIDd (mm)</td><td>4.50</td><td>0.14</td><td>9</td><td>4.62</td><td>0.07</td><td>10</td></tr><tr><td>IVSd (mm)</td><td>0.74</td><td>0.02</td><td>9</td><td>0.63 (p=0.0013)</td><td>0.02</td><td>10</td></tr><tr><td>PWd (mm)</td><td>0.75</td><td>0.02</td><td>9</td><td>0.70</td><td>0.03</td><td>10</td></tr><tr><td>LVIDs (mm)</td><td>3.44</td><td>0.11</td><td>9</td><td>3.34</td><td>0.082</td><td>10</td></tr><tr><td>IVSs (mm)</td><td>1.14</td><td>0.04</td><td>9</td><td>1.14</td><td>0.05</td><td>10</td></tr><tr><td>PWs (mm)</td><td>0.95</td><td>0.04</td><td>9</td><td>1.03</td><td>0.05</td><td>10</td></tr><tr><td>E (m/s)</td><td>0.73</td><td>0.075</td><td>9</td><td>0.75</td><td>0.02</td><td>10</td></tr><tr><td>Dec. time (ms)</td><td>21.90</td><td>1.44</td><td>9</td><td>21.73</td><td>0.98</td><td>10</td></tr><tr><td>A (m/s)</td><td>0.28</td><td>0.03</td><td>9</td><td>0.37 (p=0.06)</td><td>0.03</td><td>10</td></tr><tr><td>E/A</td><td>2.88</td><td>0.47</td><td>9</td><td>2.18</td><td>0.19</td><td>10</td></tr></tbody></table>