Methods
All experiments were carried out according to the European Community guiding principles
in the care and use of animals (86/609/CEE, CE Off J n°L358, 18 December 1986), the local
ethics committees guidelines and the French decree n°87-848 of October 19, 1987 (J Off
Rép Fr, 20 October 1987, pp. 12,245–12,248). Authorizations to perform animal
experiments according to this decree were obtained from the Ministère Français de
l'Agriculture, de la Pêche et de l'Alimentation (nº D 92-283, December 13, 2012).
Human myocardial tissue. Failing left ventricular (LV) tissues were obtained from patients
with end-stage dilated (DCM) or ischemic cardiomyopathy (ICM) at the time of cardiac
transplantation. For comparison non-failing (NF) human LV tissues were obtained from
organ donors with no known history of cardiac disease who could not be transplanted for
technical reasons. LV tissues (septal “Morrow” resections) also were obtained from adult
patients with symptomatic severe aortic stenosis (AS) undergoing aortic valve replacement
with significant myocardial hypertrophy but normal ejection fraction. Patient characteristics
and medications are summarized in Supplemental Table 1. The study was reviewed and
approved by the Ethical Committee of the University Göttingen and all patients gave written
informed consent.
Tissue from canine model of pacing-induced HF. Rapid ventricular pacing was used to
induce HF in dogs as previously described (1) and kindly provided from Dr. S. Nattel
(Montreal, Quebec, Canada) (2). Briefly, mongrel dogs were instrumented with a corkscrew
electrode in the LV attached to a pacemaker implanted in the neck. Hearts were paced at 240
bpm for 2 weeks. Control dogs were instrumented but not paced. Tachypacing-induced
hemodynamic changes were typical of a dilated cardiomyopathy with failing hearts
demonstrating elevated left ventricular end-diastolic pressure (LVEDP) and decreased
dP/dtmax, typical of this model. Tachypnea, ascites, pulmonary congestion, and pleural
effusion were present. Hearts were removed under anesthesia with morphine (2 mg/kg SC)
and α-chloralose (120 mg/kg, i.v.).
Adenoviruses. The mus musculus PDE2A2 isoform was kindly provided by Dr Joe Beavo
(Washington University, Seattle, USA). This isoform was encoded into a replication1
defective serotype 5 adenovirus (Ad-PDE2) or into a bicistronic replication-defective
serotype 5 adenovirus encoding EGFP (Ad-PDE2-GFP) by GeneCust (Evry, France).
Replication-defective serotype 5 adenovirus encoding β-galactosidase (Ad-βGal) or EGFP
(Ad-GFP) were obtained from the GVPN (Nantes, France). Cells were infected with an MOI
50-100 in standard culture conditions for a period of 48 h.
Rat isoprenaline infusion model. Male Wistar rats (240–300 g) were treated for 4-5 days
with either isoprenaline (2.4 mg/kg/day, ISO) or vehicle (0.9% NaCl, Ctr) administered via
osmotic minipumps (Alzet, USA) (3). The rats were sacrificed by cervical dislocation under
light isoflurane anesthesia. Hearts were rapidly removed and remaining blood was washed
out in ice-cold 0.9% NaCl solution. Ventricular tissue samples were frozen in liquid nitrogen
and stored at -80°C until use or ventricular cardiomyocytes were isolated from these hearts
for FRET experiments, sarcomere shortening and calcium transient measurements.
Isolation and culture of rat cardiomyocytes. Adult rat ventricular myocytes (ARVMs)
were isolated from male Wistar rat heart mounted on a Langendorff apparatus and perfused
through the coronaries with collagenase A as described previously (4). Cells were cultured
in Minimum Essential Medium (MEM) containing 1.2 mM Ca2+, 2.5% fetal bovine serum
(FBS), 1% penicillin-streptomycin and 2% HEPES (pH 7.6) and plated on laminin-coated
culture dishes (10 μg/ml laminin). ARVMs were left to adhere for 2 h in a 95% O 2, 5% CO2
atmosphere at 37°C, before the medium was replaced with FBS-free MEM containing
adenovirus according to the experimental setting.
Immunoblot analysis. Protein samples were separated in denaturating acrylamide gels and
subsequently transferred onto nitrocellulose or PVDF membranes. After blocking the
membranes with 0.5% skimmed milk (AppliChem) or with 3% bovine serum albumin for 1
h, the incubation with anti-ANP (1:200, Santa Cruz), anti-Calsequestrin (1:200, Dianova),
anti-PDE2 (1:200, Santa Cruz), anti-Phosphorylated PLB at Ser16 (1:500, Badrilla) and
anti-total PLB (1:500, Santa Cruz) was carried out over night at 4°C. After incubation with
appropriate secondary antibodies for 1 h, proteins were visualized by enhanced
chemoluminescence and quantified with Quantity one software.
2
Real-Time PCR. The quantitative PCR measurements of cardiac PDE2 and ANP transcripts
were done in left ventricular myocardial tissue. RNA was isolated from ~30 mg frozen
ventricular heart tissue (SV Total RNA Isolation System Kit, Promega) and 500-1000 ng
RNA per sample were taken to generated cDNA (High Capacity cDNA Reverse
Transcription Kits, Applied Biosystems) according to the manufactures guidelines. The
relative amount of PDE2A mRNA transcripts was quantified by real time PCR (SYBR
Green/ROX qPCR Master Mix, Fermentas) with the following primer pairs: ANP:
5`GTGCGGTGTCCAACACAG,
5`GCTTCATCGGTCTGCTCGCTCA;
PDE2:
5`AGTGCTGGGAGAAGAGGTCA, 5`TCATCAGTCGAGCCACTGAC; and GAPDH: 5`ATGTTCCAGTATGACTCTACCCACG, 5`-TGTCGTGGAGTCTACTGGCGTCTTC and
analysed with ABI PRISM 7900HT Sequence Detection System (Applied Biosystems).
cAMP measurements by FRET. ARVMs were infected with Ad-Epac2-camps for 48 h
(5). Thereafter, the cells were washed once and maintained in a physiological buffer
containing (in mM): NaCl 144, KCl 5.4, CaCl2 1.0, MgCl2 1.0, and HEPES 20, pH 7.4 at
room temperature, placed on a Zeiss Axio Observer A1 microscope equipped with PlanApochromat 63x/1.4 oil immersion objective, Polychrome V light source, DV2 DualView
beam splitter and CoolSNAP-HQ2 CCD-camera (Visitron Systems, Pullheim, Germany).
Cells were stimulated with the indicated substances either consecutively or after washout by
continuously superfusion, and the YFP/CFP emission ratio upon 436 nm excitation (filters
YFP 535 ± 15 nm, CFP 480 ± 20 nm) was measured. After each measurement, emission
values were corrected for bleedthrough of CFP into YFP channel and for photobleaching as
recently described (6). The imaging data was acquired by using the VisiView software
(Visitron) and analyzed with Excel and Origin 8.5 (OriginLab) packages.
PDE activity assay. Cyclic AMP- and GMP-degrading activities were measured according
to the method of Thompson and Appleman (7) with some modifications. cAMP-hydrolytic
activity was measured with 1 µM cAMP and 105 cpm 3H-cAMP and cGMP-hydrolytic
activity was measured using 5 µM cGMP and 105 cpm 3H-cGMP. PDE2 activity was
defined as the fraction of cAMP-PDE or cGMP-PDE activity inhibited by 100 nM BAY 607550, a highly specific PDE2 inhibitor.
3
ICa,L current measurements. The whole cell configuration of the patch-clamp technique
was used to record ICa,L. Patch electrodes resistance was between 1–2 MΩ when filled with
internal solution containing (in mM): CsCl 118, EGTA 5, MgCl2 4, Na2phosphocreatine 5,
Na2ATP 3.1, Na2GTP 0.42, CaCl2 0.062 (pCa 8.5), HEPES 10, adjusted to pH 7.3 with
CsOH. Extracellular Cs+-Ringer solution contained (in mM): CaCl2 1.8, MgCl2 1.8, NaCl
107.1, CsCl 20, NaHCO3 4, NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10,
adjusted to pH 7.4 with NaOH. For ICa,L measurement, the cells were depolarized every 8 s
from –50 to 0 mV during 400 ms. The use of –50 mV as holding potential allowed the
inactivation of voltage dependent sodium currents. K+ currents were blocked by replacing all
K+ ions with external and internal Cs+. The cells were voltage-clamped using a patch-clamp
amplifier (model RK-400; Bio-Logic, Claix, France). Currents were analogue filtered at 3
KHz and digitally sampled at 10 KHz using a 16-bits analogue-to-digital converter (DT321;
Data translation, Marlboro, Massachusetts, USA) connected to a PC compatible computer.
The maximal amplitude of whole-cell ICa,L was measured as previously described (4).
Currents were not compensated for capacitance and leak currents.
Measurements of Ca2+ transients and cell shortening. ARVMs were loaded with 5 µM
Fura-2 AM in a Ringer solution containing (in mM): NaCl 121.6, KCl 5.4, MgCl2 1.8,
NaHCO3 4, NaH2PO4 0.8, D-glucose 5, sodium pyruvate 5, HEPES 10 (pH 7.4), and 1 mM
CaCl2 at room temperature for 15 min. The cells were field stimulated (5 V, 4 ms) at a
frequency of 0.5 Hz. Sarcomere length and Fura-2 ratio (measured at 512 nm upon
excitation at 340 nm and 380 nm) were simultaneously recorded using an IonOptix®
System.
Determination of cellular hypertrophy. Twenty four hours after adenoviral infection with
Ad-GFP or Ad-PDE2-GFP, cells were stimulated with 10 µM norepinephrine (NOR) or 10
µM phenylephrine (PHE) for an additional 24 h in FBS-free MEM. Images of GFP positive
cells were taken from different fields in each dish with a cooled charge coupled (CCD)
camera (Sensicam PE; PCO, Kelheim, Germany) using the 20x objective of a Nikon TE 300
inverted microscope and 488 nm illumination. Surface area of individual myocytes was
determined using Image J software (Wayne Rasband, National Institutes of Health, USA).
4
Statistics. To assess differences between two or multiple groups the Student t test (Online
Fig. 1, 2 and 5) or one-way ANOVA followed by Bonferroni’s (Fig 7) were used,
respectively, if a normal distribution could be assumed. In all other comparisons nonparametric analyses were applied. Differences between multiple groups were assessed by
Kruskal-Wallis followed by Dunn's post hoc test (Fig. 4), differences between two groups by
the Mann-Whitney test (Fig. 1, 2, 3 and 6) and differences between pairs were assessed by
the Wilcoxon matched pair analysis (Fig. 4 and 6). Results are presented as means±SEM.
P values of less than 0.05 were considered as statistically significant.
References
1.
Li D, Fareh S, Leung TK, Nattel S. Promotion of atrial fibrillation by heart failure in
dogs: atrial remodeling of a different sort. Circulation 1999;100:87-95.
2.
El-Armouche A, Pohlmann L, Schlossarek S et al. Decreased phosphorylation levels
of cardiac myosin-binding protein-C in human and experimental heart failure.
Journal of molecular and cellular cardiology 2007;43:223-9.
3.
El-Armouche A, Gocht F, Jaeckel E, Wittkopper K, Peeck M, Eschenhagen T. Longterm beta-adrenergic stimulation leads to downregulation of protein phosphatase
inhibitor-1 in the heart. European journal of heart failure 2007;9:1077-80.
4.
Verde I, Vandecasteele G, Lezoualc'h F, Fischmeister R. Characterization of the
cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type
Ca2+ current in rat ventricular myocytes. Br J Pharmacol 1999;127:65-74.
5
5.
Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ. Novel single chain
cAMP
sensors
for
receptor-induced
signal
propagation.
J
Biol
Chem
2004;279:37215-8.
6.
Nikolaev VO, Gambaryan S, Lohse MJ. Fluorescent sensors for rapid monitoring of
intracellular cGMP. Nat Methods 2006;3:23-5.
7.
Thompson WJ, Appleman MM. Multiple cyclic nucleotide phosphodiesterase
activities from rat brain. Biochemistry 1971;10:311-6.
6
Table 1: Clinical characteristics of patients.
Patient ID #
Age
Gender
Diagnosis
Drugs
1
2
3
4
5
6
7*
8
9
10*
11
12
13
14
15
16
17
18*
19
20
21
22
23
24
25
26
27
28
29
30
31
28
34
20
23
n.d.
58
57
40
48
66
43
57
64
49
46
55
52
53
31
47
55
50
58
24
43
80
76
81
60
57
81
M
M
M
F
F
M
F
M
F
M
M
M
M
M
M
M
F
M
F
M
M
M
M
M
M
F
F
F
M
M
F
NF
NF
NF
NF)
NF)
ICM
ICM
ICM
ICM
ICM
ICM
ICM
ICM
ICM
ICM
ICM
ICM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
AS
AS
AS
AS
AS
AS
n.d.
n.d.
n.d.
n.d.
n.d.
ACDOX
ACDX
ACDX
ACDGX
AGDX
n.d.
ADG
ADNO
ADROS
DO
RD
ADRC
DRX
ACDX
n.d.
ADGO
DRGX
ADX
ADXO
ADGXO
AX
D
AX
AX
ACX
AC
NYHA
3-4
n.d.
n.d.
3
3-4
n.d
4
4
4
3
3-4
3
3
3-4
3
3-4
3-4
3-4
4
CI
1.9
n.d.
2.5
2.0
2.4
n.d
3.9
2.1
2.2
1.7
1.7
5.7
n.d
2.1
3.5
3.0
1.9
2.6
1.3
LVEF
n.d.
n.d.
n.d.
n.d.
n.d.
19%
23%
20%
25%
25%
23%
15%
15%
15%
15-20%
20%
25%
37%
34%
28%
20%
25%
25%
19%
20%
50%
60%
60%
65%
65%
60%
Diagnosis: NF: non-failing donor, ICM: ischemic cardiomyopathy, DCM: dilated
cardiomyopathy, AS: aortic stenosis. Drugs: A: angiotensin-converting enzyme inhibitors or
angiotensin receptor blockers, C: calcium-channel-blockers, D: diuretics (including
aldosterone antagonists), G: cardiac glycosides N: nitrates, R: antiarrhythmics (except betablockers), O: dopamine/dobutamine, S: steroids, L: clonidine, V: vasopressin, X: β-Blocker,
n.d.: not determine.*These samples were used for Fig.1 and Supplemental Fig.1.
7
Table 2: Effects of PDE2 inhibition on basal sarcomere shortening and Ca2+ transients
Sarcomere shortening (%)
Fura-2 ratio (%)
Control
2.36 ± 0.74
34.32 ± 2.21
Bay 60-7550
3.09 ± 0.96*
37.63 ± 2.64*
Control
2.04 ± 0.40
36.60 ± 6.02
Bay 60-7550
3.60 ± 0.92*
41.01 ± 6.09*
NaCl (n=17)
Chronic ISO (n=16)
Statistical significance for the differences between Control and Bay 60-7550 was evaluated
using paired t-test because each cell was successively exposed to Control and then to Bay
60-7550, providing an internal control for each cell: *p<0.05.
8
Figure 1
Immunoblot analysis (A) and quantification (B) of PDE2 protein levels in left
ventricular myocardium from patients with end-stage dilated cardiomyopathy (DCM) or
ischemic cardiomyopathy (ICM) treated with beta-blockers (BB) compared to ICM patients
without beta-blocker therapy (n=6). B, PDE2 was normalized to calsequestrin (CSQ).
9
Figure 2
PDE2 activity toward cAMP in LV myocardium from patients with end-stage
dilated (DCM, n=6) or ischemic cardiomyopathy (ICM, n=4) were compared to non-failing
donor hearts (NF, n=6) by radioenzymatic assay and defined as the fraction of total cAMPPDE activity inhibited by 100 nM BAY 60-7550; p=0.0534 for DCM vs. NF.
10
Figure 3
Characterization of PDE2 overexpression in adult rat ventricular
myocytes
Adult rat ventricular myocytes were transduced with the indicated recombinant adenoviruses
for a period of 48 h. (A) Detection of PDE2 protein levels in Ad-EGFP-PDE2-HA and AdPDE2-HA expressing cells by immunoblot analysis. (B) PDE2 hydrolysis activity toward
cAMP and cGMP assessed after treatment with 100 nM Bay 60-7550; n=3.
11
Figure 4
PDE2 overexpression attenuates ISO-induced cAMP generation
Adult rat ventricular myocytes co-expressing Epac2-camps and either βGal or PDE2 were
challenged with a short application of ISO (100 nM, 15s) alone or in the presence of the
PDE2 inhibitor BAY 60-7550 (100 nM). Representative pseudocolor images of the
CFP/YFP ratio of (A) βGal or (B) PDE2 expressing cells. (C) Average effect of ISO
stimulation on βGal (black) and PDE2 (white) expressing cells. (D) Average effect of ISO
stimulation on βGal (grey) and PDE2 (white) in the presence of PDE2 inhibitor BAY 607550 (100 nM). Values are calculated as percentage of maximal response measured in βGal
expressing cells and normalized to the respective CFP/YFP ratios before each ISO
application; n=20-22.
12
Figure 5
PDE2
overexpression
attenuates
ISO-induced
phospholamban
phosphorylation at PKA site in adult rat ventricular myocytes
(A) Representative immunoblots of phospholamban phosphorylated at serine 16 (p-PLBSer16) and total PLB protein from isolated cardiomyocyte preparations, 48 h after infection
with Ad-PDE2 or a control adenovirus encoding β-galactosidase (Ad-βGal) under basal
condition and after ISO application (1 nM, 5 min). (B) Mean intensities of immunoblots
from 4 different isolated cardiomyocyte preparations of p-PLB-Ser16 expressed as the ratio
of p-PLB-Ser16 to total PLB (n=4). *p<0.05.
13