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Cardiovascular Research Advance Access first published online on May 3, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn113
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

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
2 Institut für Pathologie und Neuropathologie, Universitätsklinikum Essen, Germany
3 Institut für experimentelle und klinische Pharmakologie, Medical Center Hamburg-Eppendorf, Germany
4 Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster, Germany
5 Institut für Anatomie and IZKF, Universitätsklinikum Münster, Germany
6 Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Martin-Luther-Universität Halle-Wittenberg, Germany
7 Klinik für Anästhesiologie und Intensivmedizin, Medical University Hannover, Germany

* Corresponding author. Institut für Pharmakologie und Toxikologie, Westfälische Wilhelms-Universität, Domagkstr. 12, 48149 Münster, Germany. Tel: +49 251 8355510; fax: +49 251 8355501.E-mail address: kirchhef{at}uni-muenster.de

Received 12 June 2007; revised 15 April 2008; accepted 28 April 2008

Time for primary review: 47 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
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 β-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 Ca2+ 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.

KEYWORDS Hypertrophy; Heart failure; Protein phosphatase; Transgenic mice; Pressure overload; Ca2+


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
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 β-adrenergic receptors (β-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 cAMP-dependent signalling pathway leads to an activation of the PKA. This increases cardiac output as a result of a higher phospholamban (PLB)Ser16 phosphorylation and Ca2+ uptake into the sarcoplasmic reticulum (SR), and leads to enhanced cytosolic Ca2+ transients.3 The increased protein kinase A (PKA)-activated phosphorylation of the ryanodine receptor (RyR), the troponin inhibitor (TnI), and the L-type Ca2+ 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 β-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 β-AR by β-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 Ca2+ 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 β-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 Ca2+ 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 Ca2+ handling, and does not represent a therapeutic option in the treatment of heart failure.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Experimental animals
We used a TG mouse lineage overexpressing ~40-fold a COOH-terminally 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 (=tribromoethanol) 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 µm 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 {alpha}-32P-labelled dCTP (Amersham Biosciences, Piscataway, NJ, USA).

2.5 Western blot analysis
Fifty milligrams of frozen individual ventricles was homogenized at 4°C 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 µg 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), PP1{alpha}c, PP2A, GSK3β, phospho-GSK3β, 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, β-myosin heavy chain (MHC), brain natriuretic peptide (BNP), {alpha}-skeletal actin, collagen 1, and collagen 3. All transcripts were normalized to mouse cyclophilin A.

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 µL of 50 mM Tris/HCl (pH 7.4), and 10 µL 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 30°C. 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 30°C, the reaction was stopped on ice by adding 20 µL of 50% TCA and 30 µL 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 –80°C. 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 Ca2+ 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 23°C and intracellular Ca2+ 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 β-adrenergic stimulation was tested by application of isoproterenol (Iso, 0.1–1 µM).

2.11 Detection of apoptosis
Tyrode-perfused heart tissue was fixed in formalin, embedded in paraffin, and cut into 5 µm 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
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
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 = 7) and WTTAC (13.5 ± 2.9, n = 5) compared with TGSham (2.1 ± 0.5, n = 5) and WTSham (1.3 ± 0.4, n = 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 {alpha}-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 GSK3β in TGSham (125 ± 3 and 205 ± 45%, respectively) vs. WTSham (100 ± 9 and 100 ± 22%, respectively, n = 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 = 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 = 4–6, P < 0.05). Interestingly, the increase in ventricular β-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 = 3–4, P = 0.1). Thus, atrial hypertrophy and increases in both fibrosis and β-MHC content were the first indication of a possibly detrimental effect of long-term PP1 inhibition by I-2 in a TAC model.


Figure 1
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  		Figure 1 (A) Four-chamber view sections. (B) mRNA expression of ANF and GAPDH. (C) Representative immunoblots of native and phosphorylated (p) GSK3β. (D) Histological examination of ventricles on sections stained with Sirius red. Arrowheads indicate focal fibrosis in TGTAC. (E) RT-PCR for analysis of β-MHC mRNA in WT (open square) and TG (closed square) hearts. Signals were normalized to cyclophilin A. n = 5–7; *P < 0.05 vs. Sham; +P < 0.05 vs. WT.

 


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  		Table 1 Gravimetrical and morphometric analysis of myocardium and inner organs

 
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.


Figure 2
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  		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 = 5–6; *P < 0.05 vs. Sham.

 


Figure 3
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  		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 = 4–5; *P < 0.05 vs. Sham; +P < 0.05 vs. WT.

 


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  		Table 2 Echocardiographic and Doppler measurements

 
3.3 Impaired cellular Ca2+ 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 Ca2+ 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 µM), there were no differences between TG and WT myocytes (Figure 4A and B). Ca2+ 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 {Delta}[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 µM) 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 µM Iso was associated with a prolongation of [Ca]i decay kinetics in TGTAC compared with WTTAC, whereas the application of maximum Iso resulted in a comparable decay (Figure 4F). Thus, overexpression of I-2 is associated with an impaired cellular Ca2+ handling under conditions of sustained afterload stress. Moreover, the potency of Iso stimulation in respect to its contractile response was decreased in TGTAC.


Figure 4
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  		Figure 4 Effects of TAC on parameters of cellular contraction and Ca2+ 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 Ca2+ 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 µM Iso in TGTAC vs. WTTAC. n = 13–23; +P < 0.05 vs. WT.

 
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 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 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 = 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 = 4). It is possible—albeit not confirmed by 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
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  		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 PP1{alpha}/PP2A and the endogenous mouse and transgenic human I-2 are shown. (C) Summarized data of PP1{alpha} and PP2A protein levels. n = 4–7; *P < 0.05 vs. Sham; +P < 0.05 vs. WT.

 


Figure 6
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  		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 = 4–7; *P < 0.05 vs. Sham.

 
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 Ca2+ 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).


Figure 7
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  		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 = 4–7; *P < 0.05 vs. Sham.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
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 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 Ca2+ handling, leading to a lower peak amplitude of [Ca]i and an inability to normalize cytosolic [Ca]i in diastole. The alterations in Ca2+ 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 cardiac-specific 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 Ca2+ 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 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 adenoviral-mediated 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 GSK3β 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 β-AR. In line with this, heart-directed overexpression of the β2-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 β1-AR triggered interstitial matrix remodelling and fibrosis.36 Consistently, in banded TG hearts, we found an increased content of β-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 β-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 Ca2+ handling. Interestingly, the inotropic response after application of β-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 β-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 Ca2+ handling and an attenuated inotropic effect of β-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.


   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).

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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