Cardiovascular Research (2008) 79, 464–471
doi:10.1093/cvr/cvn113
Inhibition of protein phosphatase 1 by inhibitor-2
exacerbates progression of cardiac failure
in a model with pressure overload
Stephanie Grote-Wessels1, Hideo A. Baba2, Peter Boknik1, Ali El-Armouche3, Larissa Fabritz4,
Hans-Jörg Gillmann5, Dana Kucerova1, Marek Matus1, Frank U. Müller1, Joachim Neumann6,
Martina Schmitz5, Frank Stümpel1, Gregor Theilmeier5,7, Jeremias Wohlschlaeger2,
Wilhelm Schmitz1, and Uwe Kirchhefer1*
1
Institut für Pharmakologie und Toxikologie, Universitätsklinikum Münster, Germany; 2Institut für Pathologie und
Neuropathologie, Universitätsklinikum Essen, Germany; 3Institut für experimentelle und klinische Pharmakologie, Medical
Center Hamburg-Eppendorf, Germany; 4Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster, Germany;
5
Institut für Anatomie and IZKF, Universitätsklinikum Münster, Germany; 6Institut für Pharmakologie und Toxikologie,
Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, Germany; and 7Klinik für Anästhesiologie und
Intensivmedizin, Medical University Hannover, Germany
Received 12 June 2007; revised 15 April 2008; accepted 28 April 2008; online publish-ahead-of-print 3 May 2008
Time for primary review: 47 days
KEYWORDS
Hypertrophy;
Heart failure;
Protein phosphatase;
Transgenic mice;
Pressure overload;
Ca2þ
Aims The progression of human heart failure is associated with increased protein phosphatase 1 (PP1)
activity, which leads to a higher dephosphorylation of cardiac regulatory proteins such as phospholamban. In this study, we tested the hypothesis whether the inhibitor-2 (I-2) of PP1 can mediate cardiac
protection by inhibition of PP1 activity.
Methods and results We induced pressure overload by transverse aortic constriction (TAC) for 28 days in
transgenic (TG) mice with heart-directed overexpression of a constitutively active form of I-2 (TGTAC)
and wild-type littermates (WTTAC). Both groups were compared with sham-operated mice. TAC treatment resulted in comparable ventricular hypertrophy in both groups. However, TGTAC exhibited a
higher atrial mass and an enhanced ventricular mRNA expression of b-myosin heavy chain. The increased
afterload was associated with the development of focal fibrosis in TG. Consistent with signs of overt
heart failure, fractional shortening and diastolic function were impaired in TGTAC as revealed by
Doppler echocardiography. The contractility was reduced in catheterized banded TG mice, which is in
line with a depressed shortening of isolated myocytes. This is due to profoundly abnormal cytosolic
Ca2þ transients and a reduced stimulation of phosphorylation of phospholamban (PLB)Ser16 after TAC
in TG mice. Moreover, administration of isoproterenol was followed by a blunted contractile response
in isolated myocytes of TGTAC mice.
Conclusion These results suggest that cardiac-specific overexpression of a constitutively active form of
I-2 is deleterious for cardiac function under conditions of pressure overload. Thus, the long-term inhibition of PP1 by I-2 is not a therapeutic option in the treatment of heart failure.
1. Introduction
Heart failure and its complications represent the most
important cause of death in Western industrialized
countries. While the positive inotropic effect of catecholamines is a fundamental physiological mechanism regulating
cardiac force of contraction, the chronic stimulation of
* Corresponding author. Institut für Pharmakologie und Toxikologie,
Westfälische Wilhelms-Universität, Domagkstr. 12, 48149 Münster, Germany.
Tel: þ49 251 8355510; fax: þ49 251 8355501.
E-mail address: [email protected]
b-adrenergic receptors (b-ARs) by increased plasma
catecholamines or by positive inotropic cAMP-elevating substances is associated with a worsened prognosis of heart
failure.1,2 Physiologically, the stimulation of the cAMPdependent signalling pathway leads to an activation of the
PKA. This increases cardiac output as a result of a higher
phospholamban (PLB)Ser16 phosphorylation and Ca2þ uptake
into the sarcoplasmic reticulum (SR), and leads to enhanced
cytosolic Ca2þ transients.3 The increased protein kinase A
(PKA)-activated phosphorylation of the ryanodine receptor
(RyR), the troponin inhibitor (TnI), and the L-type Ca2þ
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008.
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465
PP1 inhibitor-2 and heart failure
channel also contributes to the augmented contractility.4–6
The phosphorylation of all these proteins by PKA is reversed
by type 1 and/or 2A of serine/threonine protein phosphatases (PPs).7
In the failing heart, the chronic activation of b-AR leads to
distinct changes in cardiac morphology and function characterized by an imbalance between protein phosphorylation
and dephosphorylation. This is associated with a desensitization of b-AR by b-adrenergic receptor kinase 1-dependent
hyperphosphorylation, a reduced phosphorylation of PLB
and TnI probably due to a higher PP1 activity and/or
reduced cAMP levels, and a PKA-dependent hyperphosphorylation of RyR.8–10 These alterations are paralleled by an
impaired cellular Ca2þ cycling contributing to the progression of cardiac dysfunction.3 A higher activity of PP1
was not only observed in human heart failure11 but also in
animal models with long-term stimulation of b-AR mimicking
characteristic features of the failing heart.12 Overexpression
of PP1 in mouse hearts resulted in diminished contractility,
dilated cardiomyopathy, and premature mortality further
underscoring the pathophysiological role of PP1 in heart
failure.13 It has been concluded from these observations
that the inhibition of increased PP1 may be favourable for
restoring contractility in heart failure and may offer a
new, cAMP-independent therapeutic approach in the treatment of heart failure. Besides exogenous PP inhibitors, the
endogenous heat stable proteins inhibitor-1 (I-1) and I-2
can inhibit PP1 activity.7 The functional role of I-1 in regulating the activity of PP1 was demonstrated both in
cardiac preparations of different species and in a transgenic
mouse model with heart-directed overexpression of I-1.14–16
It was shown by our group that the heart-directed overexpression of a truncated form of I-2 was accompanied by an
improved cardiac performance and Ca2þ handling due to
reduced total PP activity in TG mice.17 Consistently, heart
hypertrophy and cardiac dysfunction in PP1-overexpressing
mice were reversed by co-overexpression of I-2.18
Here we studied whether heart-directed expression of
constitutively active I-2 confers protection in regard to detrimental effects of chronic pressure overload. However, our
data suggest that the long-term inhibition of PP1 by I-2 is
deleterious for cardiac function under conditions of an
increased afterload, resulting in structural remodelling,
contractile depression, and impaired Ca2þ handling, and
does not represent a therapeutic option in the treatment
of heart failure.
2. Methods
2.1 Experimental animals
We used a TG mouse lineage overexpressing 40-fold a COOHterminally truncated form of I-2 as described.17 The truncated
form (aa 1–140) of I-2 cannot be regulated by phosphorylation at
Thr72 and represents a constitutively active inhibitor of PP1.19 All
experiments presented here were performed on 16-week-old TG
mice and wild-type (WT) littermates and conform to the Guide for
the Care and Use of Laboratory Animals published by the US National
Institute of Health (NIH Publication No. 85-23, revised 1996).
2.2 Transverse aortic constriction
Twelve-week-old mice were isoflurane-anaesthetized, placed in
the supine position, and endotracheally intubated using a 27G
cannula connected to a rodent ventilator (Hugo-Sachs minivent,
March-Hugstetten, Germany). After left parasternal transsection
of the clavicula and the upper two ribs, the aortic arch was dissected and ligated over a 27G cannula between brachiocephalic
and left carotid artery as described before.20 The cannula was carefully removed, leaving an aortic constriction. Chest and skin were
closed, and animals were weaned from the ventilator. The mice
recovered from surgery within half an hour. Sham mice underwent
the same procedure except the ligation. After 28 days, mice were
killed with an overdose of avertin (¼tribromoethanol) for removal
of the heart, lung, liver, and kidneys. Organs were weighted and
then frozen for further studies.
2.3 Histological analyses
For 4-chamber view sections, mice were intraperitoneally administered heparin and then anaesthetized. Hearts were perfused with
Tyrode’s solution and fixed by perfusion with formalin. For determination of cell sizes, hearts were excised, immediately fixed in 4%
buffered formalin, dehydrated, and embedded in paraffin. Longitudinal tissue sections of 5 mm thickness were obtained from the
right and left ventricular (LV) free wall and then stained with hematoxylin–eosin or Sirius red reagent. Microscopic images were taken
for 4-chamber views, for determination of cellular morphology,
and for densitometric analysis of heart fibrosis.
2.4 Northern blot analysis
Total RNA from mouse ventricles was isolated as described.21 Ten
micrograms of total RNA was separated by electrophoresis, transferred to nylon membranes, and hybridized with PCR-generated
cDNA fragments of mouse ANF and GAPDH. For the generation of
radioactively labelled cDNA probes, standard protocols were
employed, using cDNAs as template in the presence of
a-32P-labelled dCTP (Amersham Biosciences, Piscataway, NJ, USA).
2.5 Western blot analysis
Fifty milligrams of frozen individual ventricles was homogenized at
48C for 1 min in 0.5 mL of a medium containing 10 mM NaHCO3,
50 mM NaF, 5 mM Na4P2O7, and 4% SDS (pH 7.4) or a medium containing 10% SDS, 20 mM NaHCO3 using a Polytron PT-10 homogenizer
(Kinematica, Lucerne, Switzerland). I-1 was detected in TCA
extracts of Sham-operated TG and WT hearts as previously
described.22 Homogenates were diluted in 5% SDS buffer containing
62.5 mM Tris/HCl (pH 6.8), 5% glycerol, and 40 mM dithiothreitol.
For immunoblot analysis of all proteins, 40–200 mg of homogenates
was electrophoretically separated on SDS–polyacrylamide gels.
After transfer of proteins to nitrocellulose, the blots were incubated
with different antibodies raised against the following proteins:
human I-2 (Transduction Laboratories, Mississauga, Canada), recombinant full-length rat I-1 (custom-made, cross-reaction with mouse,
rabbit, and human I-1, Eurogentec, Brussels, Belgium), PP1ac, PP2A,
GSK3b, phospho-GSK3b, PLB (Upstate, New York, NY, USA), RyR,21
RyRSer2809, PLBSer16, PLBThr17 (Badrilla, Leeds, UK), SERCA2a,21 and
calsequestrin.23 Antibody binding was detected by alkaline
phosphatase-conjugated and horseradish peroxidase-conjugated
antibodies or by 125I-labelled protein A (Amersham Biosciences),
and then quantified using a Storm 860 (Molecular Dynamics, Sunnyvale, CA, USA).
2.6 Real-time Polymerase chain reaction
Quantitative analysis of mRNA expression in hearts was performed
by real-time PCR (RT-PCR) using the LightCycler Detection System
(Roche Diagnostics, Mannheim, Germany) as described previously.24
Specific primers were used for RT-PCR amplification of the following
proteins: I-1, b-myosin heavy chain (MHC), brain natriuretic peptide
(BNP), a-skeletal actin, collagen 1, and collagen 3. All transcripts
were normalized to mouse cyclophilin A.
466
S. Grote-Wessels et al.
2.7 Protein phosphatase assay
Protein phosphatase activity was determined as described previously with 32P-phosphorylase a as substrate.25 Mouse ventricular
tissue was homogenized in a buffer containing 20 mM Tris/HCl (pH
7.4), 5 mM EDTA, 2 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM benzamidine, and 0.5 mM PMSF. Homogenates were centrifuged at
14 000 g for 20 min, and supernatants were used for determination
of phosphorylase phosphatase activity. Five micrograms of supernatants was diluted with 20 mL of 50 mM Tris/HCl (pH 7.4), and
10 mL of okadaic acid was added to give a final concentration of
3 nM. The pre-treated supernatants were then pre-incubated for
10 min at 308C. Twenty microlitres of a final incubation mixture containing 32P-phosphorylase a, 2.5 mM Tris/HCl (pH 7.4), 12.5 mM caffeine, 0.25 mM EDTA, and 0.25% 2-mercaptoethanol was added to
the pre-incubated supernatants. After additional incubation for
30 min at 308C, the reaction was stopped on ice by adding 20 mL
of 50% TCA and 30 mL of 20 mg/mL BSA. Precipitated protein was
sedimented by centrifugation at 10 000 g, and the radioactivity
was determined in an aliquot of the supernatants by liquid scintillation counting.
2.8 Echocardiography and Doppler studies
Transthoracic echocardiographic measurements were performed on
mice which were anaesthetized with 1.5% isoflurane allowing spontaneous breathing. All measurements were performed with a commercially available echocardiographic system (Philips Sonos 5500,
Eindhoven, The Netherlands) equipped with a 15 MHz linear transducer for two-dimensional and M-mode imaging and a 12 MHz transducer for Doppler measurements.26
2.9 Haemodynamic performance
Left ventricular catheterization was performed in closed-chest mice
as described previously.27 Anaesthesia was maintained with 1.5% isoflurane during the measurements. Heart rate, maximum LV pressures (LVPmax), time to 90% relaxation, and the first derivative of
LV pressure development and decline (dP/dtmax and dP/dtmin,
respectively) were monitored continuously. Hearts were quickly
excised within 5 s, freeze-clamped, and stored at 2808C. This procedure nearly preserves the phosphorylation state of PLBSer16, with
the caveat that a short period of hypoxia may contribute to a
minimal dephosphorylation of PLB.28
2.10 Cell shortening and Ca21 transients
Myocytes were enzymatically isolated from ventricles and loaded
with Indo-1/AM (Sigma-Aldrich, St Louis, MO, USA) as described.21
Myocytes were stimulated with 0.5 Hz at 238C and intracellular
Ca2þ transients were determined as reported previously.21 [Ca]i
was estimated by calculating the ratio of fluorescence signals at
405 and 495 nm. The shortening of myocytes was recorded simultaneously using a video edge detection system.29 The response of
myocytes to b-adrenergic stimulation was tested by application of
isoproterenol (Iso, 0.1–1 mM).
2.11 Detection of apoptosis
Tyrode-perfused heart tissue was fixed in formalin, embedded in
paraffin, and cut into 5 mm slices. Fragmented DNA was labelled
with fluorescein-conjugated dUTP using a commercial system
(Promega, Madison, WI, USA). Nuclear density was determined by
counting of DAPI-stained nuclei in 20 fields from each mouse
heart. TUNEL-positive nuclei were identified, counted in the same
fields, and related to the total number of DAPI-stained nuclei.
2.12 Statistical analysis
Data are reported as means + SEM. Statistical differences between
groups were calculated by ANOVA followed by the Student–
Newman–Keuls test. P , 0.05 was considered significant.
3. Results
3.1 Increased atrial hypertrophy and ventricular
fibrosis in TGTAC
TGSham exhibited a lower ventricular weight than WTSham
(Table 1). TAC treatment resulted in cardiac hypertrophy
associated with increased absolute ventricular weights and
enhanced sizes of ventricular myocytes in both groups
(Figure 1A; Table 1). However, the heart/body weight ratio
was higher in TGTAC compared with WTTAC due to increased
left atrial masses in TGTAC (Table 1). These findings were
in line with higher ventricular ANF mRNA levels in TGTAC
(15.9 + 3.2 arbitrary units, n ¼ 7) and WTTAC (13.5 + 2.9,
n ¼ 5) compared with TGSham (2.1 + 0.5, n ¼ 5) and
WTSham (1.3 + 0.4, n ¼ 5, P , 0.05), respectively, suggesting
the initiation of a foetal gene program (Figure 1B). Consistently, TAC led to increased levels of mRNAs encoding BNP
and a-skeletal actin both in TG and WT (data not shown).
GSK3 is an important regulator of cardiac development
and hypertrophy (Figure 1C). Immunoblotting revealed a
higher protein level and phosphorylation state of GSK3b in
Table 1 Gravimetrical and morphometric analysis of myocardium and inner organs
n
HW/BW
AW (mg)
VW (mg)
Lung (mg)
Liver (mg)
Kidney (mg)
Tibia (mm)
Cell diameter (mm)
Cell perimeter (mm)
Cell surface area (mm2)
WTSham
TGSham
WTTAC
TGTAC
6
5.8 + 0.3
5.5 + 0.4
160.4 + 8.1
172.7 + 7.6
1666.3 + 47.7
440.9 + 18.1
16.0 + 0.1
13.3 + 0.9
177.8 + 5.1
980.9 + 43.4
6
5.4 + 0.2
4.7 + 0.7
136.4 + 8.7þ
167.7 + 7.0
1563.4 + 43.0
422.8 + 21.8
15.7 + 0.2
12.3 + 0.1
174.3 + 2.8
885.6 + 10.2
5
9.9 + 0.7*
24.9 + 10.3*
218.0 + 10.6*
345.8 + 56.6*
1324.5 + 70.4*
333.0 + 13.6*
16.1 + 0.2
19.4 + 0.9*
207.7 + 3.2*
1428.6 + 42.6*
8
12.1 + 0.8*þ
47.0 + 8.5*þ
222.6 + 8.2*
404.9 + 39.0*
1213.6 + 66.2*
320.2 + 17.1*
15.7 + 0.1
20.0 + 0.6*
212.6 + 2.1*
1380.5 + 18.0*
Left atrial weight (AW), ventricular weight (VW), heart weight/body weight ratio (HW/BW).
*P , 0.05 vs. Sham.
þ
P , 0.05 vs. WT.
467
PP1 inhibitor-2 and heart failure
Figure 2 (A) Aortic cross-section area was determined 4 weeks after TAC
and Sham operation in WT (open square) and TG (closed square) mice. (B)
Maximum aortic pressure gradient (PGmax) was determined by use of
Doppler echocardiography across the stenosis. n ¼ 5–6; *P , 0.05 vs. Sham.
Figure 1 (A) Four-chamber view sections. (B) mRNA expression of ANF and
GAPDH. (C) Representative immunoblots of native and phosphorylated (p)
GSK3b. (D) Histological examination of ventricles on sections stained with
Sirius red. Arrowheads indicate focal fibrosis in TGTAC. (E) RT-PCR for analysis
of b-MHC mRNA in WT (open square) and TG (closed square) hearts. Signals
were normalized to cyclophilin A. n ¼ 5–7; *P , 0.05 vs. Sham; þP , 0.05
vs. WT.
TGSham (125 + 3 and 205 + 45%, respectively) vs. WTSham
(100 + 9 and 100 + 22%, respectively, n ¼ 6, P , 0.05).
TAC resulted in a comparable increase of both parameters
in TG (124 + 4 and 220 + 33%, respectively) compared
with WT (109 + 5 and 105 + 18%, respectively, n ¼ 12–14,
P , 0.05). TAC led to lung congestion in both groups likely
reflecting the onset of heart failure (Table 1). The histological analysis (Figure 1D) revealed a higher degree of fibrosis
in TGTAC (4.9 + 0.4 vs. 2.1 + 0.3% in TGSham) compared
with WTTAC (2.9 + 0.3 vs. 2.1 + 0.2% in WTSham, n ¼ 4–6,
P , 0.05). Interestingly, the increase in ventricular b-MHC
mRNA levels after TAC was more pronounced in TG
(Figure 1E). TAC was associated with higher mRNA levels
of collagen 1 and collagen 3 in TG and WT compared with
corresponding Sham-operated animals. However, there was
no difference between both groups (data not shown).
Study of ventricular sections indicated an unchanged percentage of TUNEL-positive nuclei in TG and WT mice after
TAC (0.31 + 0.03 vs. 0.24 + 0.02%, respectively, n ¼ 3–4,
P ¼ 0.1). Thus, atrial hypertrophy and increases in both
fibrosis and b-MHC content were the first indication of a
possibly detrimental effect of long-term PP1 inhibition by
I-2 in a TAC model.
3.2 Depressed contractility in TGTAC
The result of TAC was controlled 28 days after surgery by the
measurement of the aortic transverse area. As expected,
Doppler echocardiography revealed a similar reduction of
this parameter both in TGTAC and WTTAC compared with corresponding Sham mice (Figure 2A). The maximum pressure
gradient across the aortic stenosis was comparable
between both groups of TAC-operated mice (Figure 2B).
Heart rate was increased to a similar level in TGTAC and
WTTAC (Table 2). The thickness of the intraventricular
septum was unchanged between both groups (Table 2). As
a main result of this study, we measured an increased
reduction of fractional shortening and ejection fraction in
TGTAC (Table 2). This is consistent with changes in the LV
end-diastolic and end-systolic diameters. Moreover, the
cardiac output was more profoundly depressed in TGTAC
(by 39%) compared with WTTAC (by 24%; Table 2). TAC
resulted in a decreased maximum pressure gradient across
the mitral valve in TG vs. WT mice (Table 2), suggesting
an impaired diastolic function. In vivo measurement of contractile parameters by LV catheterization revealed a 38%
lower LVPmax in TGTAC compared with WTTAC (Figure 3A).
The time to 90% relaxation was unchanged between
TAC-operated groups (Figure 3B). Moreover, we measured
a 49% lower dP/dtmax (Figure 3C) and a 46% lower dP/
dtmin (Figure 3D) in TGTAC vs. WTTAC. This decrease of contractile parameters in TGTAC was even more pronounced
when it was compared with basal values in the corresponding Sham group. Thus, the chronic inhibition of PP1 by overexpressed I-2 is deleterious for cardiac performance under
conditions of higher afterload.
3.3 Impaired cellular Ca21 handling in TGTAC
To test whether the depression of contractility in intact
TGTAC was paralleled by comparable changes at the cellular
level, we measured cell shortening and Ca2þ transients in
isolated myocytes. Under control conditions, cell shortening
(Figure 4A) was decreased by 43%, and time to 90%
re-lengthening (Figure 4B) was prolonged by 57% in TGTAC
compared with WTTAC. Application of increasing concentrations of Iso resulted in a blunted inotropic and lusitropic
response in myocytes of banded TG mice (Figure 4A and B,
respectively). However, at maximum concentrations of Iso
(1 mM), there were no differences between TG and WT myocytes (Figure 4A and B). Ca2þ transients were simultaneously
monitored in myocytes in the absence and presence of Iso
(Figure 4C). In the absence of Iso, the peak amplitude of
[Ca]i was reduced by 52% in TGTAC (Figure 4D). Both
potency and efficacy of Iso in regard to effects on D[Ca]i
were decreased in TGTAC vs. WTTAC (Figure 4D). Moreover,
we found a higher diastolic [Ca]i ratio in TGTAC at basal
and Iso-stimulated (0.1–1 mM) conditions (Figure 4E). The
time to 90% decay of [Ca]i was unchanged between both
groups at basal conditions (Figure 4F). However, the administration of 0.1 mM Iso was associated with a prolongation of
[Ca]i decay kinetics in TGTAC compared with WTTAC, whereas
468
S. Grote-Wessels et al.
Table 2 Echocardiographic and Doppler measurements
n
Heart rate (bpm)
LA (mm)
IVS (mm)
LVEDD (mm)
LVESD (mm)
FS (%)
EF Teichholz (%)
Card. output (mL/min)
MV PGmax (mmHg)
MV E-wave (cm/s)
WTSham
TGSham
WTTAC
TGTAC
6
418 + 11
2.1 + 0.1
1.0 + 0.1
3.4 + 0.2
2.2 + 0.1
35.7 + 2.6
65.9 + 3.5
21.4 + 1.2
2.0 + 0.2
71.5 + 3.5
5
410 + 5
2.1 + 0.1
0.9 + 0.0
3.5 + 0.1
2.3 + 0.1
32.3 + 5.0
60.2 + 7.0
21.3 + 1.1
1.7 + 0.2
65.7 + 3.4
5
477 + 19*
2.6 + 0.1*
1.1 + 0.1
3.7 + 0.2
2.9 + 0.2*
22.5 + 2.5*
45.7 + 4.0*
16.3 + 1.3*
3.3 + 0.4*
89.8 + 5.7*
6
508 + 11*
2.7 + 0.1*
1.1 + 0.1
4.1 + 0.1*
3.5 + 0.1*þ
14.7 + 1.7*þ
31.4 + 3.2*þ
12.9 + 0.5*þ
2.0 + 0.1þ
71.1 + 2.5þ
LA, left atrium; IVS, intraventricular septum; LVEDD, left ventricular end-diastolic diameter; LVESD, LV end-systolic diameter; FS, fractional shortening;
EF, ejection fraction; MV PGmax, maximum pressure gradient across the mitral valve.
*P , 0.05 vs. Sham.
þ
P , 0.05 vs. WT.
Figure 3 Haemodynamic parameters were obtained by LV catheterization
under basal conditions in WT (open square) and TG (closed square) mice.
(A) Maximum LV pressure. (B) Time to 90% relaxation. (C ) Maximum rate of
LV pressure development (dP/dtmax). (D) Minimum rate of LV pressure
decline (dP/dtmin). n ¼ 4–5; *P , 0.05 vs. Sham; þP , 0.05 vs. WT.
the application of maximum Iso resulted in a comparable
decay (Figure 4F). Thus, overexpression of I-2 is associated
with an impaired cellular Ca2þ handling under conditions
of sustained afterload stress. Moreover, the potency of Iso
stimulation in respect to its contractile response was
decreased in TGTAC.
3.4 Reduced protein phosphatase 1 activity
in TGTAC
To study the effects of TAC on the interaction between overexpressed I-2 and PP1, we measured the PP activity in ventricular homogenates. TGSham exhibited a 33% reduction of
total PP activity compared with WTSham (Figure 5A). The
total PP activity was still decreased by 16% in TG vs. WT
hearts after TAC (Figure 5A). To determine the relative contribution of PP1 and PP2A to the total PP activity, we added
Figure 4 Effects of TAC on parameters of cellular contraction and Ca2þ transients were measured in response to increasing doses of Iso in WT (open circle)
and TG (closed circle). (A) Cell shortening. (B) Blunted acceleration of relaxation in TGTAC. (C) Representative Ca2þ transient recordings at 0.5 Hz under
basal and Iso-stimulated conditions. (D) Peak amplitude of [Ca]i. (E) Diastolic
[Ca]i ratio. (F) Time to 90% decay of [Ca]i was increased after application of
0.1 mM Iso in TGTAC vs. WTTAC. n ¼ 13–23; þP , 0.05 vs. WT.
3 nM okadaic acid (Figure 5A). This concentration completely inhibits PP2A activity, as shown previously.25 In the presence of okadaic acid, we observed a similar pattern of PP
activity as in the absence of okadaic acid, suggesting that
the decrease of total PP activity in pressure-overloaded TG
hearts is due to a reduced activity of PP1. TAC did not
469
PP1 inhibitor-2 and heart failure
Figure 6 (A) RT-PCR analysis of I-1 mRNA expression in WT (open square)
and TG (closed square) hearts normalized to the endogenous marker
protein cyclophilin A. (B) TCA extracts from heart homogenates were subjected to SDS–PAGE and immunoblot analysis. Homogenate from neonatal
rat cardiomyocytes, infected with a rat I-1 adenovirus, was used as a
control (Ad I-1). Pre-incubation with a pre-immune serum prevented the
detection of I-1 (Ad I-1*). n ¼ 4–7; *P , 0.05 vs. Sham.
immunoblotting—that I-1 protein is increased by banding
along with the higher I-1 mRNA levels. However, I-1 seems
to be of minor relevance considering that the PP1 activity
was not changed in WTTAC vs. WTSham and was not inhibited
to a higher degree in TGTAC vs. TGSham.
Figure 5 (A) Homogenates were prepared from WT (open square) and TG
(closed square) ventricles and total protein phosphatase activity was
assayed using 32P-labelled phosphorylase a as substrate (left panel). PP1
activity was determined in the presence of 3 nM OA (middle panel). PP2A
activity was calculated as the difference between total PP and PP1 activity
(right panel). (B) Homogenates were also subjected to immunoblot analysis.
Blots were incubated with specific antibodies and representative autoradiograms of the catalytic subunits of PP1a/PP2A and the endogenous mouse
and transgenic human I-2 are shown. (C ) Summarized data of PP1a and
PP2A protein levels. n ¼ 4–7; *P , 0.05 vs. Sham; þP , 0.05 vs. WT.
affect PP2A activity in TG or WT hearts (Figure 5A). To test
whether the changes in PP activity in TGTAC were
accompanied by similar alterations at the protein level,
we measured the content of PP1 by immunoblotting
(Figure 5B). The PP1 protein levels were unchanged
between Sham animals (Figure 5C). Pressure overload
resulted in an increase in the protein level of the catalytic
subunit of PP1 in TG compared with WT hearts. The
protein level of the catalytic subunit of PP2A was not different between all groups studied (Figure 5B and C). To exclude
compensatory changes in the protein level of I-2, we performed immunoblotting with antibodies recognizing I-2
either of human or murine origin (Figure 5B). The resulting
protein level of the overexpressed truncated human I-2
gene remained constant after TAC in TG hearts (n ¼ 5–7,
data not shown). Furthermore, the protein level of the
endogenous mouse full-length I-2 was unchanged between
all groups studied (data not shown). I-1 expression was
measured at the mRNA level in banded TG and WT mice.
The amount of I-1 mRNA was enhanced in banded compared
with Sham groups (Figure 6A). However, there was no difference between TGTAC and WTTAC (Figure 6A). In Sham hearts,
the content of I-1 was also tested by immunoblotting
(Figure 6B). Here, we found an unchanged I-1 protein level
(2.5 + 0.7 arbitrary units in TGSham and 2.4 + 0.8 in
WTSham, n ¼ 4). It is possible—albeit not confirmed by
3.5 Reduced increase in phospholamban
phosphorylation in TGTAC
The level of cardiac regulatory SR proteins was measured by
immunoblotting (Figure 7A). The level of the main SR Ca2þ
storage protein, calsequestrin, was unchanged between all
groups studied (Figure 7B). Thus, the protein level of calsequestrin was used as a loading control in all immunoblotting
experiments. The protein levels of SERCA2a and PLB were
similar under TAC and Sham conditions between TG and
WT hearts (Figure 7B). The RyR was reduced to a comparable level in TGTAC and WTTAC (Figure 7B). In addition, we
tested the phosphorylation state of PLB and the RyR
(Figure 7A). The phosphorylation state of PLBSer16 tended
to be enhanced in TGSham compared with WTSham, but the
difference did not reach statistical significance
(Figure 7C). TAC resulted in a higher phosphorylation state
of PLBSer16 in both groups. However, this increase was less
pronounced in TG (by 56%) compared with WT hearts (by
249%, P , 0.05). In contrast, the phosphorylation state of
PLBThr17 (Figure 7C) was unchanged between all groups.
Finally, the phosphorylation state of RyRSer2809 exhibited a
similar reduction in TGTAC and WTTAC (Figure 7A) as observed
for the total RyR protein (Figure 7B). In other words, a
change in RyR phosphorylation was not different between
TG and WT hearts in response to TAC (data not shown).
4. Discussion
One of the major characteristics of the failing heart is an
altered balance between protein kinase and phosphatase
activities in favour of dephosphorylation. Several studies
detected a higher protein level and/or activity of PP1 in
human end-stage heart failure and in animal models of
heart hypertrophy or failure.11,12 Thus, it was hypothesized
470
Figure 7 Immunological detection of SR proteins in homogenates of WT
(open square) and TG (closed square) hearts. Blots were probed with specific
antibodies as described in Section 2. (A) Representative autoradiograms of
PLB, calsequestrin (CSQ), SERCA2a, the RyR (left panel) and of phosphorylated SR proteins (right panel). (B) Averaged data of protein analysis. (C )
Statistical evaluation of phosphorylated regulatory proteins. WTSham is set
to 100%. For quantification, phosphorylated proteins were related to the
native forms. n ¼ 4–7; *P , 0.05 vs. Sham.
that inhibition of PP1 activity may represent a therapeutic
option in reducing the progression of contractile
dysfunction.
Unexpectedly, we found that overexpression of a constitutively active form of I-2 did not attenuate heart failure
development in a model of pressure overload. This conclusion is based on the systolic and diastolic dysfunction
that was observed in the intact animal and at the cellular
level in TGTAC. The worsening of cardiac function was associated with an impaired Ca2þ handling, leading to a lower
peak amplitude of [Ca]i and an inability to normalize cytosolic [Ca]i in diastole. The alterations in Ca2þ signalling
are due, at least in part, to a reduced stimulation of phosphorylation of PLBSer16. It is conceivable that the higher proportion of PP1 protein in TGTAC compared with TGSham mice
contributes to the lower stimulation of PLBSer16 phosphorylation, albeit total PP1 activity was decreased in TG vs. WT.
This is supported by the fact that PP1 activity accounts for
90% of the PLB phosphatase activity30 and that cardiacspecific overexpression of the PP1 catalytic subunit in TG
mice was associated with an exclusive dephosphorylation
of PLBSer16.13 An altered phosphorylation state of both
PLBThr17 and the RyR was not detected in TGTAC and, therefore, cannot contribute to the impaired Ca2þ transients.
In a recent study, transcoronary delivery of a recombinant
adenovirus and of an adeno-associated virus encoding
full-length I-2 improved LV fractional shortening, reduced
S. Grote-Wessels et al.
the chamber size, and extended the survival time of
cardiomyopathic hamsters.31 This was associated with a
lower protein level and activity of PP1 and an enhanced phosphorylation of PLBSer16. Consistently, short-term adenoviralmediated expression of a truncated, constitutively active
I-1, which cannot be activated by a PKA-dependent phosphorylation,7 completely restored cardiac function and partially reversed remodelling in rats with pre-existing heart
failure due to pressure overload.16 This was associated with
a reduced PP1 activity. In the present study, we used a truncated form of I-2 that is equally potent as full-length I-2, but
that is not regulated by a GSK3-dependent phosphorylation.19,32 Normally, phosphorylation shifts I-2 from an
active to an inactive form that restores PP1 activity. This
I-2 phosphorylation may represent an important adaptive
mechanism in heart failure since LV hypertrophy and
cardiac failure were paralleled by a higher phosphorylation
and protein level of I-2 in a rat model with chronic renal
hypertension.33 Here, the higher protein level and degree
of phosphorylation of GSK3b in both Sham- and TAC-operated
TG mice may represent a compensatory mechanism to overcome the frustrating activation of I-2. As shown by Zhang
et al., 34 the reduced PP1 activity likely contributes to an
increased phosphorylation of GSK3 in TG. Thus, we speculate
that phosphorylation of I-2 is required in stressed myocardium
to resist haemodynamic failure and that the long-term inhibition of PP1 evokes similar detrimental effects on cardiac
structure and function as observed after long-term stimulation of b-AR. In line with this, heart-directed overexpression of the b2-AR gene was associated with contractile
dysfunction, atrial enlargement, and focal fibrosis after
aortic stenosis.35 In addition, sympathetic overactivation in
mice with overexpression of b1-AR triggered interstitial
matrix remodelling and fibrosis.36 Consistently, in banded
TG hearts, we found an increased content of b-MHC which
is the predominant marker of fibrosis in the hypertrophic
mouse heart.37
The hypothesis of deleterious cardiac effects of a long-term
PP1 inhibition, similar to long-term stimulation of b-AR, is also
supported by the fact that we found a reduced potency of Iso in
isolated myocytes of banded TG mice. This effect was
accompanied by similar changes in the cellular Ca2þ handling.
Interestingly, the inotropic response after application of
b-adrenergic agonists was unchanged between non-operated
TG and WT mice,17 suggesting a specific contribution of
pressure overload in TG mice. A reduction of sensitivity and/
or the maximum inotropic effect after stimulation by
b-adrenergic agonists is a common feature of cardiac hypertrophy and end-stage heart failure.38
In summary, here we provide a chain of evidence demonstrating that pressure overload in TG mice with overexpression of a constitutively active form of I-2 leads to reduced
PP1 activity, development of fibrosis, and contractile
failure. These effects were associated with an impaired
Ca2þ handling and an attenuated inotropic effect of
b-adrenergic stimulation. We conclude that a chronic inhibition of PP1 by I-2 is not a therapeutic option in the treatment of heart failure. PP1 may have even a beneficial
regulatory role in the stressed myocardium. Indeed,
increased PP1 activity protected from ischaemia–reperfusion injury and contractile failure.39 Further studies are
necessary to clarify the regulation of PP1 under conditions
of chronic cardiac stress.
471
PP1 inhibitor-2 and heart failure
Acknowledgements
We thank N. Hinsenhofen, M. Schulik, N. Nordsiek, and L. Fortmüller
(IZKF-ZPG4a) for excellent technical assistance. Grant support:
IZKF-The1-04/68 and SFB656-Z2 (Theilmeier), SFB656-C3 (Fabritz),
DFG-Bo1263/9-1 (Boknik), DFG-MU 1376/10-3 (Müller), and
DFG-FOR-604 and EUGene Heart (El-Armouche).
19.
20.
Conflict of interest: none declared.
21.
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