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Cardiovascular Research 2006 71(1):97-107; doi:10.1016/j.cardiores.2006.03.012
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Copyright © 2006, European Society of Cardiology

Dual level of interactions between calcineurin and PKC-{varepsilon} in cardiomyocyte stretch

Fanny Vincenta, Nicolas Duquesnesa, Christo Christovb, Thibaud Damyc, Jane-Lise Samuelc and Bertrand Crozatiera,*

aUnité INSERM U400, Créteil, France
bImaging plate-forme Institut Mondor de Médecine Moléculaire Créteil, France
cUnité INSERM U689CRCIL Paris, France

* Corresponding author. Present address: Unité INSERM U769 Faculté de Pharmacie 92296 Châtenay-Malabry, France. Tel.: +33 1 46 83 57 59; fax: +33 1 46 83 54 75. Email address: bertrand.crozatier{at}cep.u-psud.fr

Received 27 September 2005; revised 10 February 2006; accepted 14 March 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objectives Myocardial stretch activates a number of interconnected pathways including the protein kinase C (PKC) pathway that in turn activates mitogen activated protein kinases (MAPK), leading to gene expression stimulation and ventricular hypertrophy. A role of calcineurin has also been shown during hypertrophy. The goal of our study was to look for a possible interconnection between PKC and calcineurin in myocardial stretch.

Methods Neonatal rat cardiomyocytes were cultured for 5 days and a 15% stretch was applied. Expression of MAPK and PKC-{varepsilon} was evaluated by Western blot analysis. The specific role of PKC-{varepsilon} was evaluated by transfection of cardiomyocytes with a specific inhibitor peptide. Calcineurin and PKC-{varepsilon} complex formation and co-localization were evaluated by co-immunoprecipitation and by immunolocalization.

Results The PKC isoform involved in stretch-induced ERK and JNK activations was PKC-{varepsilon}. We show here that calcineurin is also found to be involved in the stretch response and that calcineurin and PKC-{varepsilon} co-operate at 2 levels during stretch. First, stretch-induced translocation of PKC-{varepsilon} from the cytosolic to the membrane fraction was inhibited by calcineurin inhibitors, indicating that calcineurin was necessary for PKC-{varepsilon} activation induced by stretch. A second level of interaction was the formation of a calcineurin–PKC-{varepsilon} complex, which increased during stretch. Immunofluorescent studies indicated that, after stretch, calcineurin and PKC-{varepsilon} were co-localized at the level of the perinuclear membrane. These results may have a major relevance in vivo since we also found similar PKC-{varepsilon}–calcineurin complexes in the phase of thoracic aortic stenosis in rats during which heart failure develops.

Conclusion Calcineurin appears to be necessary for stretch-induced PKC-{varepsilon} activation after which the phosphatase and the kinase are co-localized in a complex at the level of the perinuclear membrane where they may finely regulate the phosphorylation of their target proteins.

KEYWORDS MAP kinases; Protein kinase C; Signal transduction; Myocytes


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Cellular stretch is a major adaptational process in most organs and systems, particularly in the heart and vessels but also in skeletal muscles, bones, liver, kidneys, etc., with common mechanisms of cellular transduction [1]. In the heart, although various humoral factors such as catecholamines, vasoactive peptides, cytokines and growth factors contribute to the development of hypertrophy, myocardial stretch is the principal determinant of ventricular hypertrophy [2]. The hypertrophy process is an adaptational response to an increased work load. However, it is associated with an increased risk of mortality since it leads to heart failure, a major cause of death in developed countries [3]. Thus, the understanding of the molecular mechanisms leading to ventricular hypertrophy is the subject of intensive investigations.

During hypertrophy, the mitogen activated protein kinases (MAPK) ERK and JNK are activated through a number of pathways including protein kinase C [4]. The PKC family is composed of 12 different isoforms. The major PKC isoforms expressed in the heart are PKC-{alpha} and PKC-{varepsilon}. [5] PKC-{alpha} has been recently shown to be a major regulator of cardiac contractility with a propensity toward heart failure [6]. An important role of calcineurin, a calcium/calmodulin phosphatase, has also been shown in the hypertrophy process although conflicting results have been published [7]. Different involvements of calcineurin in the hypertrophy process may depend upon the model of hypertrophy [8]. These activation pathways and others have complex interactions. Such interactions were recently described between the calcineurin and the MAPK pathways [8].

During stretch, some of these pathways have been characterized: PKC, tyrosine kinases, integrins, etc. [9], activating the MAPKs [9,10] and leading to early genes stimulation and myocardial hypertrophy [11]. ERK activation was shown to involve PKC, in part through the effect of angiotensin II receptor stimulation by an autocrine/paracrine release of angiotensin II [10,12] or by a direct activation of these receptors without angiotensin II release [13].

Here we show, in cardiomyocytes, that calcineurin and protein kinase C epsilon (PKC-{varepsilon}) co-operate during stretch with complex interactions. Calcineurin is necessary for stretch-induced PKC-{varepsilon} translocation. Then, translocated PKC-{varepsilon} is associated with calcineurin in a signalling complex at the level of the perinuclear membrane.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
All investigations were performed in accordance with 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).

2.1 Cell culture and cardiomyocyte stretch
Hearts of 1–3 days old Wistar rats were dissociated in collagenase and pancreatin solution according to Iwaki et al. [14]. Myocytes were purified on a Percoll gradient (Amersham Biosciences, USA). Cells were plated on 6 wells flexible collagen1-coated plates (Flexcell International Corporation, USA) and kept for 72 h in serum containing medium. They were serum starved 48 h before the experiments. Cardiomyocytes were submitted to a static 15% of stretch during 15 min according to Dassouli et al. [15] in 3 of the 6 wells of each plate, the 3 other wells served as non-stretched controls. This time duration was chosen because preliminary experiments showed that it corresponded to the maximum MAPK activation. Stretch was performed either without drug or with a calcineurin inhibitor (FK506, 0.1 µM for 30 min) or with a PKC inhibitor (PMA, 1 µM for 24 h) or with the association of these drugs (in this case an angiotensin II receptor inhibitor telmisartan, 1 µM and a tyrosine kinase inhibitor genistein, 20 µM were added). In order to verify the viability of cultured cells, particularly after PMA treatment for 24 h, we performed cultures on transparent wells. The beating rate of the cultures was examined under a microscope and measured in spontaneous conditions and after isoproterenol treatment (1 µM for 5 min).

2.2 PKC isoform specific peptide transfection
Specific PKC-{varepsilon} isoform peptide and scrambled peptide (Santa Cruz Biotechnology, USA) were transfected after transient permeabilization of cardiac myocytes according to D Mochly-Rosen's laboratory [16].

2.3 PKC-{varepsilon} translocation
Translocation of PKC-{varepsilon} was evaluated by Western blot analysis of PKC-{varepsilon} isoform expression in cytosolic and particulate fractions obtained by differential centrifugation steps. Cell lysates obtained by pooling the cellular content of 3 wells were centrifuged at 14000 x g for 15 min (4 °C) in lysis buffer (Tris 12.5 mM, pH 7.4, EDTA 1 mM, EGTA 2.5 mM, NaF 100 mM and proteases inhibitors). Supernatants were kept (cytosolic fractions) and pellets were resuspended in lysis buffer containing 1% Triton X-100 (particulate fractions).

2.4 Immunoprecipitation of calcineurin and PKC-{varepsilon}
The content of 3 to 6 wells was pooled in order to reach an amount of about 300 µg of proteins. Immunoprecipitation with 5 µg of calcineurin antibody was performed as described [17] followed by Western blot analysis with PKC isoforms antibodies as described next. The reverse immunoprecipitation was performed using PKC-{varepsilon} antibody (2 µg) followed by calcineurin Western blot analysis. In this latter condition, the presence of total ERK or AKAP150 was investigated by Western blot analysis of the immunoprecipitate.

2.5 Western blot analysis of ERK, JNK, PKC isoforms, calcineurin and AKAP150
Expressions of ERK and JNK in cells lysates, calcineurin or PKC isoforms in immunoprecipitates and PKC-{varepsilon} in cytosolic and particulate fractions were analysed by Western blot analysis. For the evaluation of each protein, 20 µg of proteins were deposited on each lane except for particulate fractions for which 40 µg proteins were used. After blockade of non-specific sites with Tween Tris Buffer Saline 5% milk, membranes were probed with antibodies raised against ERK and JNK non-phosphorylated and phosphorylated forms (Santa Cruz Biotechnology, USA). PKC-{alpha}, -{delta}, -{varepsilon} or -{zeta} isoforms or calcineurin or AKAP150 expression was evaluated using specific polyclonal antibodies (Santa Cruz Biotechnology, USA). After additional incubation with a secondary antibody labelled with horseradish peroxidase (Santa Cruz Biotechnology, USA), signals were detected by ECL (Amersham Biosciences).

2.6 Calcineurin phosphatase activity assay
Calcineurin phosphatase activity was determined using a colorimetic assay kit (Calbiochem). The assay was done on 12 µg of proteins with or without EGTA. The difference in free phosphate released from reactions in assay buffer or in EGTA buffer indicates specific calcineurin activity.

2.7 Immunocytochemistry of PKC-{varepsilon} and calcineurin
Cardiomyocytes were fixed in control conditions and after 15 min stretch with 2% paraformaldehyde/triton X100 0.1% and incubated with mouse anti-calcineurin (1/50) and rabbit anti-PKC-{varepsilon} (1/100) primary antibodies. PKC-{varepsilon} primary antibody was detected by FITC-conjugated rabbit antibody. Calcineurin bound primary antibody was detected by a biotinylated secondary anti-mouse antibody (Vector Laboratories, Burlingame, CA, USA), followed by an incubation with an avidin–peroxidase complex (Vector Laboratories, Burlingame, CA, USA) and TSA cyanine 3 (Perkin ElmerTM, Boston, USA), all used according to the manufacturer's instructions.

2.8 Microscopy and image processing
Images of double-labeled cardiomyocytes were acquired on a motorized stage Axioplan 2 microscope (objective) coupled to an AxioCam CCD using the Multidimensionnal acquisition module of AxioVision 4.2 (all from Zeiss, Germany). In order to avoid cross-talk between green (FITC) and red (Cy3) channels appropriate camera gain and offset values were chosen to avoid hypersaturation of pixels, and emission signals were detected through narrow bandpass filters. During processing, the linear intensity distribution histograms of the acquired images were uniformly rescaled to exclude 1{per thousand} of both histogram ends' pixels using the Best Fit option of AxioVision 4.0 (Zeiss). Finally, to further eliminate cross-talk, two-channel images were processed in the Wide-field Multichannel Unmixing module of AxioVision 4.2.

2.9 Semi-quantitative assessment of signal subcellular localization
In order to semi-quantitatively validate visual inspection of cells immunostained for PKC-{varepsilon}, subcellular signal intensity localization of 42 control cells and 43 stretched cells was performed. The localization of the most intense (intensity above the 66th percentile of all brightness values of the image of the cell) was determined using the Multisegmentation module of the KS400 3.0 image analysis software (Zeiss) and two thresholds were determined in the grey value histogram: one including all lightness values of the cells permitting to segment the cell from background and a second one delimiting 33% of the brightest pixels (light grey region). According to subcellular distribution, all 85 cells could be divided into three patterns: (i) a predominantly cytoplasmic localization with inconstant minimal perinuclear extension; (ii) a perinuclear rim embracing no more than 60% of the nuclear circumference, and (iii) a perinuclear corona embracing 80–100% of the nuclear circumference.

2.10 Aortic stenosis
After anaesthesia with intraperitoneal xylazine (50 mg/kg) and ketamine (100 mg/kg), ascending aortic stenosis was performed in 25 day-old Wistars female rats (Iffa Credo, France), by placing a partially occluded Weck hemoclip (Atrauclip, Pliling®) on the ascending aorta as described in [18]. Additional age-matched animals underwent sham operation to serve as controls (sham group). Animals were studied 2 and 20 weeks after operation.

Before sacrifice, rats were anaesthetised with intraperitoneal Xylasine (50 mg/kg) and Ketamine (100 mg/kg) and left hemodynamics parameters were measured. Briefly using a pressure head connected to a needle, left hemodynamics parameters were measured by transparietal way. Measured parameters included left systolic pressure, left end-diastolic ventricular pressure. The hearts were dissected, trimmed free of large vessels, weighed and frozen in isopentane precooled with liquid nitrogen and stored at – 80 °C.

2.11 Data calculations. Statistical analysis
The intensity of the bands of Western blots was quantified by scanning densitometry with NIH image 1.34 software. In order to get an estimation of the PKC-{varepsilon} amount present in particulate or cytosolic fraction, the densitometric values of the Western blots were divided by 20 and 40 in cytosolic and particulate fractions respectively since the respective amount of protein in each lane was 20 and 40 µg. This leads to an estimation of the PKC-{varepsilon} content per µg of protein of each fraction. It was supposed that the total amount of proteins of particulate and cytosolic fraction was not modified during a 15 min stretch so that it was possible to compare the amount of proteins in each fraction in different conditions and to calculate a translocation index as particulate/total contents with total=cytosolic+particulate.

Values were expressed as means±1 s.e.m. They were compared with ANOVA followed by a Newman–Keuls test when multiple comparisons were performed. When only 2 conditions were compared, a student t-test was used.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Stretch-induced ERK and JNK activations are dependent upon calcineurin and PKC
We first verified that stretch-induced ERK and JNK activation in cardiomyocytes. A 15 min stretch induced an increase of phosphorylated forms of ERK 1 and ERK 2 without change in total ERK isoform expression (Fig. 1a). Treatment with an inhibitor of calcineurin, FK506 (0.1 µM 30 min) blocked stretch-induced ERK phosphorylation by 50% (Fig. 1a and c). Pretreatment with PMA for 24 h blocked by more than 50% ERK activation (Fig. 1a and c) and the effects of these 2 drugs were not additive since their association did not induce a larger decrease of ERK phosphorylations than PMA or FK506 alone (Fig. 1a and c).


Figure 1
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  		Fig. 1 Typical ERK (a) and JNK (b) immunoblots (upper traces: phosphorylated forms, lower traces: total isoforms) with a calcineurin inhibitor (FK506) or with a PKC inhibitor (PMA for 24 h) or with the association of these drugs. Corresponding means±s.e.m of 7 experiments (N=4 for drug association) expressed as a % increase in ERK (c) or JNK (d) phosphorylations above the corresponding non-stretched control without treatment. Stretched values with treatment were compared to the value obtained without treatment (first column of each panel). *p<0.05.

 
We performed the same experiment for JNK activation. Stretch induced a significant increase in JNK1 and 2 phosphorylations without change in total JNK isoform expression (Fig. 1b). Similarly to ERK, inhibition of calcineurin by FK506 and inhibition of PKC by PMA 24 h blocked stretch-induced JNK phosphorylations by more than 50% with no additional effect of these 2 drugs (Fig. 1b and d). To eliminate some non-specific effect of FK506, we used another calcineurin inhibitor, cyclosporin A (0.5 µM, 30 min). Cyclosporin A led to similar results (data not shown).

Cell viability was verified by the evaluation of the beating rate of cell cultures in 2 different cell isolations. The spontaneous beating rate was 112.6+/ – 15.7 beats/min in basal conditions and it was not significantly different 24 h after incubation with PMA (100.0+/ – 11.1 beats/min; 7 and 11 wells respectively). Isoproterenol treatment (1 µM for 5 min) induced similar significant increases (both ps<0.05) in the beating rate by 49.7% in control cells and 41.3% in cells treated with PMA to 167.4+/ – 17.6 and 141.3+/ – 11.0 beats/min respectively.

3.2 Inhibition of {varepsilon} isoform of PKC
We next studied the involvement of specific isoform of PKC by using a specific inhibitor peptide developed by D. Mochly-Rosen's laboratory [16]. This peptide ({varepsilon}-inh) mimics the specific receptor for activated protein kinase C (RACK) for PKC-{varepsilon}. PKC-{varepsilon} inhibitor peptide blocked stretch-induced activations of ERK and JNK but the scramble peptide did not (Fig. 2).


Figure 2
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  		Fig. 2 Examples of the effect of a PKC-{varepsilon} inhibitor peptide ({varepsilon}-inh) treatment compared with a control scramble peptide (scram) on stretch-induced ERK (a) and JNK (b) activations. Means of 4 experiments (pERK in panel c) or 3 experiments (pJNK in panel d). *p<0.05 with non-stretched. {dagger}p<0.05 with stretch with scramble peptide.

 
3.3 Activation of PKC-{varepsilon} by stretch
Activation of PKC-{varepsilon} was studied by translocation from cytosolic to particulate fractions. These fractions were obtained by differential centrifugation as described in Methods. Stretch induced a significant increase in PKC-{varepsilon} content in particulate fraction (Fig. 3a and b). PMA 24 h totally down-regulated PKC-{varepsilon} expression in cytosolic and particulate fractions as expected (Fig. 3). Surprisingly, cyclosporin A blocked PKC-{varepsilon} translocation showing that calcineurin is necessary for PKC-{varepsilon} translocation (Fig. 3).


Figure 3
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  		Fig. 3 a) PKC-{varepsilon} expression of cytosolic and particulate fractions prepared from cell lysates were compared during control and stretch without treatment or with PMA 24 h or cyclosporin A (CyA, 0.5 µM). b) and c) means of 5 experiments measuring PKC-{varepsilon} content in particulate (b) and cytosolic (c) fractions. Values are expressed in densitometric values divided by 40 and 20 in particulate and cytosolic fraction respectively to get an estimate of PKC-{varepsilon} content in these fraction/µg of corresponding proteins. *p<0.05 with the non-stretched control. d) translocation index as measured by the ratio between PKC-{varepsilon} in particulate and in total fractions.

 
3.4 Calcineurin and PKC-{varepsilon} form a complex during stretch in cardiomyocytes from neonatal rat hearts
We performed an immunoprecipitation of calcineurin in cell lysates from non-stretched and stretched cardiomyocytes and we looked for proteins that co-precipitated with calcineurin. We found, in the immunoprecipitate, the presence of PKC-{varepsilon} that was increased by stretch (Fig. 4a). We did not find the other isoforms of PKC ({alpha}, {delta} and {zeta}, Fig. 4b). To confirm this result, we performed reverse experiments (immunoprecipitation of PKC-{varepsilon} and calcineurin Western blot, Fig. 4c) and control experiments with immunoprecipitations followed by Western blotting with the same antibody (Fig. 4d). We found a weak interaction between calcineurin and PKC-{varepsilon} in control cardiomyocytes that was markedly increased by stretch (Fig. 4c).


Figure 4
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  		Fig. 4 Immunoprecipitation with 5 µg of calcineurin antibody was performed as described [18] followed by Western blot analysis with PKC isoforms antibodies. The reverse immunoprecipitation was performed using PKC-{varepsilon} antibody (2 µg) followed by calcineurin Western blot analysis. a) Immunoblots (IB) of PKC-{varepsilon}, isoform after calcineurin immunoprecipitation (IP) with the corresponding graphs of the means±s.e.m of 4 experiments. *p<0.05 non-stretched versus stretched. b) PKC-{alpha}, PKC-{delta}, PKC-{zeta} immunoblots of calcineurin IP and of cell lysates showing the absence of IP of these PKC isoforms. c) Typical example of a immunoprecipitation of PKC-{varepsilon} followed by calcineurin immunoblot (left) and mean±s.e.m of 4 experiments (right). *p<0.05. d) Control of the immunoprecipitation: calcineurin IB after calcineurin IP and PKC-{varepsilon} IB after PKC-{varepsilon} IP. Specific bands at the expected molecular weights are shown and similar in control and in stretch.

 
We measured the activity of calcineurin in order to examine whether PKC-{varepsilon} present in the complex could modify calcineurin activity. The results show an absence of significant change in calcineurin activity induced by stretch (1.89+/ – 0.48 nmol/mg proteins/min in control conditions and 1.35+/ – 0.34 nmol/mg proteins/min; NS).

3.5 Calcineurin–PKC-{varepsilon} complex formation in adult rat hearts subjected to thoracic aortic stenosis
To test the relevance of these findings in vivo, we used a model of thoracic rat aortic stenosis. As shown in Fig. 5a in typical examples of ventricular slices of hearts arrested in diastole, aortic stenosis produced a marked ventricular dilatation (30+/ – 2% increase in end-diastolic diameter as measured in vivo by echocardiography) and a dramatic cardiac hypertrophy with 100% hypertrophy 4 months after the insult (Fig. 5b). Rats with an aortic stenosis presented a marked increase in end-diastolic pressure and systolic pressure (Fig. 5c). Immunoprecipitation of PKC-{varepsilon} was performed on heart lysates followed by Western blot analysis of calcineurin. Some co-immunoprecipitation was found in sham-operated rats. Four months after the stenosis, when signs of heart failure were present, co-immunoprecipitation was dramatically increased (Fig. 5d and e). This result shows that calcineurin–PKC-{varepsilon} complex formation is stimulated when the heart is dilated in the final phase of heart failure.


Figure 5
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  		Fig. 5 a) Examples of left ventricular slices of hearts arrested in diastole by KCl injection obtained from sham-operated rats and from rats subjected to aortic stenosis. b) Left ventricular weight of sham-operated rats and rats subjected to thoracic aortic stenosis. c) Left ventricular (LV) end-diastolic (EDP) and systolic (SP) pressures in these animals. d) Representative example of co-immunoprecipitation experiments and corresponding means (e). Immunoprecipitation was performed similarly as in Fig. 4 in adult rat hearts without (sham) or with an aortic stenosis for 4 months. *p<0.01 between sham-operated rats and rats with aortic stenosis.

 
3.6 Cellular localization of the calcineurin–PKC-{varepsilon} complex
We then studied the localization of calcineurin and PKC-{varepsilon} by fluorescent microscopy. In resting conditions, calcineurin and PKC-{varepsilon} were found mostly in the cytosol with some variable weak staining in the perinuclear region (Fig. 6a). Stretch induced a weakening of PKC-{varepsilon} cytoplasmic staining and its mobilization towards the perinuclear region where it appeared as circular and intense and co-localized with calcineurin (Fig. 6a, overlay). Different patterns of PKC-{varepsilon} localization are shown in Fig. 6b. Control cardiomyocytes presented cytosolic or segmental localizations whereas stretched cardiomyocytes mostly presented annular localizations (Fig. 6c).


Figure 6
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  		Fig. 6 a) Immunolocalizations of PKC-{varepsilon}, calcineurin and overlay images during control conditions and after stretch. The co-localization of the enzymes in the perinuclear membrane during stretch is shown by the yellow color of the perinuclear contour. b) Representative pictures of recalculated images of 3 types: (i) a predominantly cytoplasmic localization with inconstant minimal perinuclear extension; (ii) a perinuclear rim embracing no more than 60% of the nuclear circumference, and (iii) a perinuclear corona embracing 80–100% of the nuclear circumference. c) Histogram showing the calculated cellular distribution of PKC-{varepsilon} distributions in control and stretch. The statistical difference was calculated using a {chi}2 test. d) Immunolocalizations of PKC-{varepsilon}, {alpha}-actinin and overlay images during control conditions and after stretch.

 
3.7 Presence of other proteins in the complex
The presence of 2 other proteins (ERK and AKAP150) in the PKC-{varepsilon} complex was investigated by Western blot of the immunoprecipitate. As shown in Fig. 7, ERK was present in the complex in the control state. It was also present after stretch but was not increased. In contrast, AKAP150 was not found in the complex, neither in control conditions nor after stretch.


Figure 7
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  		Fig. 7 Representative examples of Western blots of ERK (left panels) and AKAP150 (right panels) in PKC-{varepsilon} immunoprecipitates (IP). ERK 1 and 2 were present in similar amounts in control conditions and after stretch. In contrast, AKAP150 was not present in the immunoprecipitates in any conditions. The cell lysates (L) were added on the same gels in order to verify the precise localization of the AKAP bands.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Both ERK and JNK are known to be activated in response to stretch in new born rat cardiomyocytes [9,10]. The precise mechanism of stretch-induced MAPK activation is not completely known. ERK activation was shown to involve PKC, in part through the effect of released angiotensin II [10] but the role of PKC in JNK activation is less clear. Our results confirm the increased expression of ERK and JNK phosphorylated forms after a 15 min stretch without change in total isoforms (Fig. 1). Our data also show that, similarly with ERK, JNK activation by stretch was reduced by more than 50% by PKC down-regulation induced by PMA, indicating a similar PKC pathway in both ERK and JNK activations in this model.

4.1 PKC-{varepsilon} and calcineurin involvement in MAPK activation during stretch
The-{varepsilon} isoform of PKC was suspected to be involved since a PKC-{varepsilon} translocation has been shown during left ventricular dilatation of the adult heart [19]. However, its direct role has not been evaluated in ERK and JNK activations except, very recently, the demonstration of PKC-{varepsilon} involvement in ERK activation during preconditioning [20]. The activation of PKC-{varepsilon} during stretch was evidenced by its translocation from cytosolic to particulate fractions (Fig. 3). The effect of PKC-{varepsilon} on ERK and JNK activations during stretch was confirmed by manipulating the interaction of the PKC with its specific anchoring protein, termed receptor for the activated C kinases (RACK) [21] (Fig. 2).

The other pathway that was found to be involved in MAPK activation was the calcineurin pathway. Calcineurin, a calcium/calmodulin phosphatase, has been reported to play a role in cardiomyocyte hypertrophy. It is known to act by dephosphorylation of the transcription factor NFAT, enabling it to translocate to the nucleus [7]. Several studies showed that calcineurin may be involved in MAPK activation in models in which intracellular calcium is increased (isoproterenol [22] or angiotensin II [21] treatments). The treatment of cardiomyocytes with FK506, a calcineurin inhibitor, blocked by more 50% of both ERK and JNK isoforms (Fig. 1). This is partly in agreement with the data of De Windt et al. who showed in hearts of mice overexpressing calcineurin that cyclosporin treatment inhibited JNK but not ERK overactivation observed in this model [23]. The effect of calcineurin blockade was in the same order of magnitude as that induced by PMA 24 h. Furthermore, the effects of these 2 drugs on stretch-induced MAPK activation were not additive since their association did not induce a larger decrease of ERK or JNK phosphorylation than PMA or FK506 alone (Fig. 1). This strongly suggested a co-operativity between these enzymes.

4.2 Co-operativity of calcineurin with PKC-{varepsilon}
The activation of PKC-{varepsilon} during stretch was also demonstrated by its translocation from cytosolic to particulate fractions. The inhibition of PKC-{varepsilon} translocation by calcineurin blockade was surprising but a similar co-operativity between PKC-{varepsilon} and calcineurin was found in which calcineurin is necessary for PKC-{varepsilon} activation during angiotensin infusion [24]. The mechanism was unknown. A dephosphorylation by calcineurin of a protein to which PKC-{varepsilon} is bound may be a mechanism but it is presently purely hypothetical.

The major new finding of this study is that PKC-{varepsilon} and calcineurin have another interaction with the formation of a novel protein–protein complex. Functional proteomic studies showed that PKC-{varepsilon} is associated with a number of proteins such as Src or ERK during cardiac preconditioning [25] but calcineurin was not found in the complexes as it was here in myocyte stretch.

It had been found in other systems or organs that calcineurin and PKC-{varepsilon} may be associated with the anchoring protein AKAP [26]. The absence of PKC-{varepsilon} binding to AKAP after stretch (Fig. 7) was not surprising since Takahashi et al. showed that PKC-{varepsilon} and calcineurin are associated with AKAP in an inactive hypophosphorylated form [27] and not and in active form. In addition, overlay experiments suggest that calcineurin and PKC-{varepsilon} can directly interact with each other in a complex (results not shown).

The immunolocalization pattern we found in which PKC-{varepsilon} is mobilized towards the perinuclear area and co-localized with calcineurin during stretch is not typical of PKC-{varepsilon} in cardiomyocytes. After TGFβ treatment, PKC-{varepsilon} was found at the cell periphery at the contact regions between adjacent cells [28]. In contrast, our laboratory showed a cross-striated staining in normal rabbit hearts [29]. The same cross-striation was observed in PMA or norepinephrine treated rat cells [30]. We thus looked for a localization of PKC-{varepsilon} in sarcomeres [28,29] by an immunostaining by {alpha}-actinin (Fig. 6d) that showed a clear sarcomere localization but PKC-{varepsilon} appeared as diffuse in the cytoplasm without any co-localization both in control conditions and after stretch during which PKC-{varepsilon} appeared mostly in the perinuclear region. This strongly suggests a localization in the Golgi apparatus, in line with the localization in the Golgi shown by Csukai et al. [31]. In addition, in another study [28], in aFGF-treated myocytes, PKC-{varepsilon} localized to the perinuclear region of 90% of the cells giving annular staining similarly to what we found in stretched cardiomyocytes. These data show that subcellular localization of PKC depends on the particular stimulus involved. The localization in the perinuclear region during stretch strongly suggests that both enzymes act in concert in gene activation.

This novel signaling complex is a new example of the association of a phosphatase with a kinase. This complex may act at the level of the target proteins as found in the brain in which an anchored type 1 protein phosphatase (PP1) limited NMDA channel activity in the basal state whereas PKA activation overcame constitutive PP1 activity [32]. Another possibility is a modification of the activity of the phosphatase by the associated kinase as shown in Jurkat T cells in which a protein serine–threonine kinase (calmodulin kinase) was regulated by a tightly associated serine–threonine phosphatase (PP2A) [33]. The interactions between the kinase and the phosphatase we show here is a new type of interaction since we found that the enzymes co-operate at 2 levels. In a first step, calcineurin is necessary for PKC-{varepsilon} stretch-induced translocation to the membranes. Then PKC-{varepsilon} and calcineurin form a complex at the level of the perinuclear membrane where it may be a fine regulator of the degree of phosphorylation of its targets: MAPK studied here and possibly other target proteins that are activated during the development of the hypertrophy process.

Until now, PKC-{varepsilon} and calcineurin were known as major independent key factors in the development of cardiac hypertrophy. Our data show that they interact in concert in the hypertrophy process. The dual level of interaction we show here adds another complexity in the direct and indirect interactions between calcineurin–NFAT and ERK signalling pathways recently demonstrated [8]. In this study, an interaction between ERK 1/2 and calcineurin was found. Here we also found ERK in the PKC-{varepsilon}–calcineurin complex, adding a complexity to the interactions between these proteins. These results are probably of major importance from a clinical point of view since we show that these results mostly obtained during stretch of cardiomyocytes are also found in the late phase of the hypertrophy process during which stretch is present. Thus, any intervention of one of these enzymes may interfere with the activity of the other and thus open new therapeutic perspectives.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported in part by a grant of the Fondation de France. FV was the recipient of grants from AREMCAR and GRRC.


   Notes
 
Time for primary review 29 days


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

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