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Cardiovascular Research 2007 75(1):118-128; doi:10.1016/j.cardiores.2007.03.003
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Copyright © 2007, European Society of Cardiology

The acute phase protein {alpha}2-macroglobulin induces rat ventricular cardiomyocyte hypertrophy via ERK1,2 and PI3-kinase/Akt pathways*

Manju Padmasekara, Rajender Nandigamaa, Maria Wartenbergb,c, Klaus-Dieter Schlütera and Heinrich Sauera,*

aDepartment of Physiology, Justus-Liebig-University Giessen, Aulweg 129, 35392 Giessen, Germany
bDepartment of Cell Biology, GKSS Research Institute Teltow, Germany
cClinic of Internal Medicine I, Friedrich Schiller University Jena, Germany

* Corresponding author. Tel.: +49 641 9947333; fax: +49 641 9947219. heinrich.sauer{at}physiologie.med.uni-giessen.de

Received 20 July 2006; revised 5 March 2007; accepted 7 March 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective {alpha}2-macroglobulin ({alpha}2M) is an acute phase protein released to the serum upon challenges such as cardiac hypertrophy and infarction. Here we report on the role of {alpha}2M in the induction of hypertrophic cell growth, contractile responsiveness of rat ventricular cardiomyocytes, and on the underlying extracellular regulated kinase 1,2 (ERK1,2) and phosphoinositide 3-kinase (PI3-kinase)/Akt pathways.

Methods Cell volume and cross-sectional areas were assessed as parameters of hypertrophic growth, and real time RT-PCR for the analysis of hypertrophy-related genes was performed. Protein synthesis was analyzed by 14C-phenylalanine incorporation. Activation of ERK1,2, PI3-kinase and Akt was assessed by immunohistochemical analysis of phosphorylated proteins. Contractile responsiveness was investigated by determination of cell shortening following electrical field stimulation. Intracellular calcium concentration [Ca2+]i was determined by fluo-3 microfluorometry.

Results Treatment of ventricular cardiomyocytes for 24 h with {alpha}2M significantly increased cell volume and protein synthesis as well as expression of hypertrophy-associated genes [brain natriuretic protein (BNP), β-myosin heavy chain (β-MHC), myosin light chain-2 (MLC-2), atrial natriuretic factor (ANF), and skeletal {alpha}-actin]. Comparable effects were achieved by treatment of cells with an antibody directed against the {alpha}2M-receptor LDL receptor-related protein-1 (LRP-1) and counteracted upon coincubation with receptor-associated protein (RAP), suggesting an involvement of {alpha}2M-LRP-1 signalling. Furthermore, {alpha}2M treatment increased sarcoplasmic reticulum Ca2+-ATPase (SERCA-2a) expression, diastolic and systolic [Ca2+]i, and contractile responsiveness after electrical stimulation. Shortly after {alpha}2M stimulation, activation of ERK1,2, Akt, and PI3-kinase pathways was observed. Consequently, {alpha}2M-induced protein synthesis was inhibited upon treatment with the ERK1,2 inhibitor UO126 as well as by LY294002 and wortmannin, which inhibit PI3-kinase, and by rapamycin, which inhibits mammalian target of rapamycin (mTOR) downstream of Akt.

Conclusions Our data show that {alpha}2M induces hypertrophic cell growth in rat ventricular cardiomyocytes via ERK1,2 and PI3-kinase/Akt and improves cardiac cell function.

KEYWORDS Cardiac hypertrophy; Acute phase proteins; {alpha}2-macroglobulin; SERCA


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Although it is generally accepted that {alpha}2M is the major endoprotease inhibitor in mammalian blood and is carrier of growth factors [1], not much is known about its biological function. In contrast to other protease inhibitors {alpha}2M selectively inhibits interactions between the targeted protease and macromolecular sites of action of the protease [2]. Protease binding to tetrameric human {alpha}2M involves a physical folding of the {alpha}2M molecule around the protease molecule, thereby creating a "trap" with the arms of the {alpha}2M polypeptides and forming a steric barrier that prevents contact between the protease and large substrates, active site-directed inhibitors, or antibodies. The conformational change of {alpha}2M results in exposure of a cryptic recognition site for the cell-surface clearance receptor LRP-1 [3]. Upon LRP-1 binding internalization of the activated complex of {alpha}2M, {alpha}2M*, is initiated by receptor-mediated endocytosis and transport to lysosomes for catabolism [4]. Recent studies have shown that binding of {alpha}2M* to cell surface receptors induces signalling pathways such as neuronal calcium signalling via N-methyl-D-aspartate receptors [5], activation of Akt/PDK signalling in macrophages [6], PI3-kinase-dependent activation of Akt and activation of ERK1,2 and p38 in prostate cancer cells [7]. However, it was suggested that a receptor distinct from LRP-1 may be responsible for the initiation of {alpha}2M*-mediated signalling pathways [8]. This receptor was identified as Grp78, the 78-kDa glucose-regulated protein that is a molecular chaperone of the Hsp70 family.

Signalling cascades initiated in the heart by {alpha}2M have thus far not been investigated although recent studies have suggested a role for {alpha}2M in the development of cardiac hypertrophy [9]. The upregulation of hypertrophic response genes is an adaptive response towards increased work load to the cardiac muscle. During physiological hypertrophy increased expression of genes involved in the cell cycle, cell structure, intracellular signalling, protein synthesis and metabolism is observed, whereas during pathological hypertrophy genes involved in inflammation, wound healing, structural proteins, metabolism and survival are upregulated. Previously, a novel, high molecular weight (182 kDa) serum protein was identified in rats as a cardiac isoform of {alpha}2M [10], and it was suggested that cardiac {alpha}2M is involved in the development of cardiac hypertrophy in rats since injection of purified {alpha}2M into the tail vein of rats induced cardiac hypertrophy [9]. Although the 182-kDa cardiac isoform of {alpha}2M shared 100% sequence identity with hepatic {alpha}2M, it showed certain differences under denaturing conditions in isoelectric focusing and partial peptide mapping [9]. The same group recently demonstrated that the cardiac isoform of {alpha}2M is present and elevated in the hearts of diabetic patients with cardiac infarction, suggesting that {alpha}2M levels in plasma may be used as a biomarker for myocardial infarction in diabetic patients [11].

In the present study the signalling events involved in the induction of ventricular cardiac cell hypertrophy by {alpha}2M were investigated. Our data demonstrate that {alpha}2M induced cardiac hypertrophy in rat ventricular cardiomyocytes, which was mimicked by an antibody directed against LRP-1 and counteracted by RAP, thus indicating that LRP-1 signalling is critically involved in {alpha}2M-mediated signal transduction in cardiac cells. The signalling cascade involved activation of ERK1,2 as well as PI3-kinase/Akt signalling pathways and changes in SERCA-2a and Ca2+ signalling, which may be associated with induction of the hypertrophic cardiac phenotype and improvement of ventricular myocyte function.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Reagents
{alpha}2M isolated from human plasma was obtained from Sigma. Freshly isolated {alpha}2M from human plasma was a generous gift from Dr. S.V. Pizzo, Duke University Medical Center. The monoclonal mouse anti-LRP-1/{alpha}2M-R antibody was obtained from Research Diagnostics (Flanders, NJ). UO126, LY294002, wortmannin, and rapamycin were obtained from Calbiochem (Bad Soden, Germany). RAP protein was purchased from Research Diagnostics (Concord, MA).

2.2 Activation of {alpha}2M
Activation of {alpha}2M was performed as described previously [12]. Briefly, ammonium bicarbonate was added at a final concentration of 300 mM to {alpha}2M and incubated at room temperature overnight. The solution was subsequently dialyzed against endotoxin-free PBS.

2.3 Isolation procedure of rat ventricular cardiomyocytes
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Ventricular cardiomyocytes were obtained from 200–250 g male Wistar rats, suspended in basal culture medium and plated on 6-well culture plates that were pre-incubated with 4% serum in CCT medium for at least 2 h before plating. This facilitates the attachment of cardiomyocytes to the substratum. The basal culture (CCT) medium was modified medium199 including Earle's salts, 2 mM L-carnitine, 5 mM taurine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10 µM cytosine-D-arabinofuranoside (pH 7.4). After 2 h culture plates were washed with serum-free CCT medium containing 2% penicillin–streptomycin to remove dead cells and other debris. This results in cultures of approximately 90% quiescent, rod-shaped cells.

2.4 Incorporation of [14C]phenylalanine and changes in cellular protein
Incorporation of phenylalanine into cells was determined by exposing cultures to L-[14C]phenylalanine (0.1 µCi/ml) for 24 h and assessing the incorporation of radioactivity into acid-insoluble cell mass as reported earlier [13]. Nonradioactive phenylalanine (0.3 mM) was added to the medium to minimize variations in the specific activity of the precursor pool responsible for protein synthesis. These experiments were terminated after 24 h. Within this time period the incorporation of radioactivity from [14C]-phenylalanine into the acid-insoluble mass of the cell was linear, and the portion of cellular precursor radioactivity not incorporated into the acid-insoluble cell mass was small and identical under all investigated conditions. Experiments were terminated by removal of the supernatant medium from the cultures. Culture dishes were washed three times with ice-cold phosphate-buffered saline (PBS; composition in mM: 1.5 KH2PO4, 137 NaCl, 2.7 KCl, and 1.0 Na2HPO4, pH 7.4). Subsequently, ice-cold 10% (wt./vol.) trichloroacetic acid was added. After storage overnight at 4 °C, the acid was removed from the dishes. Radioactivity contained in this acid fraction was taken to represent the intracellular precursor pool. The dishes were then washed twice with ice-cold PBS. The remaining precipitate on the culture dishes was dissolved in 1 N NaOH–0.01% (wt./vol.) sodium dodecyl sulfate (SDS) by incubation for 2 h at 37 °C. In these samples protein content was determined, and the radioactivity was counted.

2.5 Determination of cell contraction
Cell contractions were investigated as described previously [14]. Briefly, cells were allowed to contract at room temperature and analyzed using a cell-edge detection system. Cells were stimulated via two AgCl electrodes with biphasic electrical stimuli composed of two equal but opposite rectangular 50-V stimuli of 0.5 ms duration. Each cell was stimulated at 0.5, 1, 1.5 and 2 Hz for 1 min. Every 15 s the next five contractions were averaged. The mean of these four measurements at a given frequency was used to define the contractility of a given cell. Cell lengths were measured at a rate of 500 Hz via a line camera.

2.6 Imaging of [Ca2+]i in rat ventricular cardiomyocytes
Cells were loaded with fluo-3/AM dissolved in dimethylsulfoxide (DMSO) and supplemented with PluronicTM (Molecular Probes, Eugene, OR). The final concentration of fluo-3/AM in the cell culture medium was 10 µM. After a 20-min incubation cells were washed once in cell culture medium and transferred to an incubation chamber mounted to the stage of a Leica SP2 AOBS confocal laser scanning microscope (Leica, Bensheim, Germany) equipped with a 488-nm argon-ion laser for the excitation of fluo-3 fluorescence. Cell contractions were elicited by electrical stimulation of cardiac cells at 2 Hz. Relative changes in [Ca2+]i concentrations were presented as changes in fluo-3 fluorescence (F) in relation to basal fluo-3 fluorescence (F0).

2.7 Immunohistochemistry
Cardiomyocytes were treated with {alpha}2M for different time periods as indicated, and at the end of the reaction cardiomyocytes were fixed with 4% paraformaldehyde (PFA) for 20 min and then washed once with PBS. Subsequently, cardiomyocytes were permeabilized in ice-cold absolute methanol at –20 °C for 5 min. The myocytes were then washed with PBS supplemented with 0.01% Triton-X-100 (Sigma, Deisenhofen, Germany) to remove the methanol, and blocked against unspecific binding using 10% FCS in PBS for 1 h. They were then treated with appropriate primary antibodies overnight at 4 °C. The primary antibodies used were: polyclonal rabbit anti-phospho ERK1,2 (1:50), polyclonal rabbit anti-phospho JNK (1:50), polyclonal rabbit anti-phospho p38 (1:50), polyclonal rabbit anti-phospho Akt (1:50) (all from Cell Signalling Technologies, Frankfurt, Germany), mouse monoclonal anti-striated muscle {alpha}-actinin (1:400) (Sigma). After incubation with primary antibodies the cells were washed with 0.01% PBST for 3 times. Secondary antibody [anti-rabbit Cy5-labelled IgG H+L, anti-mouse FITC-labelled IgG H+L (both from Dianova, Hamburg, Germany)] was added and incubated for 1 h at room temperature. The cells were then mounted on slides with Fluoromount-G (Southern Biotech, Birmingham, Alabama). Immunofluorescence was recorded using a Leica SP2 AOBS confocal laser scanning microscope (Leica, Bensheim, Germany) equipped with a 633-nm He/Ne laser for the excitation of Cy5 and a 488-nm Argon-ion laser for the excitation of FITC.

2.8 Determination of cell size
Images of cardiomyocytes were taken using the transmission mode of the Leica confocal microscope. The length and width of the cardiomyocytes were determined using the Leica image analysis software. Cell volumes were calculated by the following algorithm: cell volume=(radius)2·{pi}·cell length, assuming a cylindrical cell shape.

2.9 Quantitative RT-PCR experiments
Total RNA from cardiomyocytes treated with {alpha}2M was prepared using the Trizol (Invitrogen) method followed by genomic DNA digestion using DNAse I (Invitrogen, Karlsruhe, Germany). Total RNA was determined by the OD260nm method. cDNA synthesis was performed using 1 µg of RNA in a total volume of 10 µl with MMLV RT (Invitrogen). Primer concentration for qPCR was 10 pM/20 µl. For each assayed gene, annealing temperature and the number of cycles resulting in linear amplification were tested. Primer sequences were as follows:

  1. HPRT
  2. Forward: 5'...CCAGCGTCGTGATTAGCGAT...3'
    Reverse: 5'...CAAGTCTTTCAGTCCTGTCC...3'
  3. ANF
  4. Forward: 5'...ATGGGCTCCTTCTCCATCAC...3'
    Reverse: 5'...TCTTCGGTACCGGAAGCTG...3'
  5. BNP
  6. Forward: 5'...GGGCTGTGACGGGCTGAGGTT...3'
    Reverse: 5'...AGTTTGTGCTGGAAGATAAGA...3'
  7. Skeletal {alpha}-actin
  8. Forward: 5'...TCAGGCGGTGCTGTCTCTCT...3'
    Reverse: 5'...TCCCCAGAATCCAACACGAT...3'
  9. β-MHC
  10. Forward: 5'...GCAGCTTATCAGGAAGGAATAC...3'
    Reverse: 5'...CTTGCGTACTCTGTCACTC...3'
  11. MLC-2
  12. Forward: 5'...TCACAATCATGGACCAGAACAGA...3'
    Reverse: 5'...TGATCATCTCATCGATCTCTTCGT...3'
  13. SERCA-2a
  14. Forward: 5'...CGAGTTGAACCTTCCCACAA...3'
    Reverse: 5'...AGGAGATGAGGTAGCCGATGAA...3'

2.10 Statistical analysis
Data are given as mean values±SEM, with n denoting the number of independent samples within one treatment group. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by adjusted t-tests with P values corrected by the Bonferroni method. A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Effects of {alpha}2M treatment on ventricular cardiac cell volume and protein synthesis
A cardiac isoform of {alpha}2M has previously been shown to induce cardiac hypertrophy in rats [9]. However, thus far no investigations on the effects of {alpha}2M on ventricular cardiac cells have been reported. To investigate whether {alpha}2M induced hypertrophic cell growth, rat ventricular cardiac cells were treated for 24 h with 40 µg/ml {alpha}2M, and cell volumes were calculated from transmission images recorded by confocal laser scanning microscopy. As shown in Fig. 1 {alpha}2M treatment significantly increased cell volume by approximately 30% (n=4). When ventricular cardiomyocytes were treated with the known hypertrophy-inducing {alpha}-adrenoceptor agonist phenylephrine a volume increase of approximately 22% was observed (n=4) (data not shown in Fig. 1).


Figure 1
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  		Fig. 1 Increase in cell volume of rat ventricular cardiomyocytes treated for 24 h with 40 µg/ml {alpha}2M. The images show representative transmission images of preparations of cardiomyocytes. The graph shows mean values±SEM of cardiomyocyte volumes of control cardiomyocytes and cells treated with {alpha}2M. Cell volumes were calculated from transmission images by computer-assisted image analysis. *P<0.05, significantly different from the untreated control.

 
Cell hypertrophy is associated with increased protein synthesis [13]. To investigate whether {alpha}2M increased protein synthesis in rat ventricular cardiomyocytes, 14Phe-incorporation was investigated after incubation of the cells for 24 h with either 40, 60, 100 or 120 µg/ml {alpha}2M (Fig. 2A, n=3). In parallel experiments cells were treated with an antibody directed against LRP-1 to investigate whether the hypertrophic effect was mediated through LRP-1-dependent signalling pathways (Fig. 2B, n=4). Treatment with {alpha}2M dose-dependently increased 14Phe-incorporation, indicating hypertrophy-associated protein synthesis. Interestingly, treatment with LRP-1 antibody either in the presence or absence of {alpha}2M resulted in comparable effects, which strongly suggests that {alpha}2M initiates LRP-1 signalling. To corroborate this finding, cardiomyocyte preparations were incubated with RAP, which binds to LRP and inhibits signal transduction by {alpha}2M. It was found that {alpha}2M increased protein synthesis to 126±2.5% as compared to the untreated control (set to 100%). Upon co-incubation with RAP, protein synthesis was significantly reduced to 107±2% (n=6). Incubation with RAP alone did not change the rate of protein synthesis (data not shown). Since commercial {alpha}2M preparations may contain impurities such as endotoxin, control experiments were performed with freshly isolated, endotoxin-free {alpha}2M (generous gift of Dr. S.V. Pizzo). Furthermore, purified preparations were incubated with ammonium bicarbonate, which is well known to activate {alpha}2M. With this purified and activated preparation, comparable protein synthesis was observed as that obtained with the commercial preparation used throughout the study (data not shown).


Figure 2
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  		Fig. 2 Effect of {alpha}2M and an antibody directed against the LRP-1/{alpha}2M-receptor on protein synthesis (14Phe-incorporation) in ventricular cardiomyocytes. (A) Concentration–response relationship of protein synthesis upon treatment of cells with different concentrations of {alpha}2M ranging from 40–120 µg/ml {alpha}2M. (B) Effects of {alpha}2M (40 µg/ml), {alpha}2M+LRP-1ab., and LRP-1ab. alone. Note that binding of the antibody to the LRP-1/{alpha}2M receptor results in stimulation of protein synthesis. *P<0.05, significantly different from the untreated control.

 
3.2 Increase in cardiac gene expression upon treatment of ventricular cardiomyocytes with {alpha}2M
Pathological myocardial hypertrophy is characterized by an increase in cardiomyocyte protein and the expression of a gene profile reminiscent of early embryonic development. To investigate whether {alpha}2M treatment of ventricular cardiac myocytes initiated cardiac gene expression, ANF, β-MHC, MLC-2 and skeletal {alpha}-actin mRNA expression was investigated by real-time RT-PCR. Treatment with {alpha}2M significantly increased the expression of the cardiac genes ANF, β-MHC, MLC-2 and skeletal {alpha}-actin (Fig. 3, n=4). Furthermore, an increase in BNP expression to approximately 700% of the untreated control was observed (not shown). Incubation with an anti-LRP-1 antibody increased cardiac gene expression comparably to that observed with {alpha}2M (see Fig. 3), thus suggesting that indeed LRP-1-mediated signalling pathways were activated by {alpha}2M treatment.


Figure 3
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  		Fig. 3 Increase in cardiac gene expression upon treatment of rat ventricular cardiomyocytes with either {alpha}2M or an antibody directed against the LRP-1/{alpha}2M receptor. The cells were treated for 24 h with either {alpha}2M or the anti-LRP-1 antibody. Subsequently, the expression of the cardiac genes ANP, β-MHC, MLC-2 and {alpha}-actin was assessed by real-time RT-PCR. *P<0.05, significantly different to the untreated control.

 
3.3 Effects of {alpha}2M on ventricular cell shortening
It is generally accepted that the early hypertrophic response of the heart to pressure overload is an adaptive process that acts to preserve heart function. However, long-lasting myocardial hypertrophy is transformed into a non-adaptive type of hypertrophy that finally results in heart failure [15]. It should therefore be assumed that treatment of cardiac myocytes with hypertrophy-inducing agents ameliorates myocyte function. To investigate contractile function of ventricular cardiomyocytes in the presence of {alpha}2M, cell shortening following electrical stimulation with frequencies of 0.5, 1.0, 1.5 and 2.0 Hz was investigated directly after addition of {alpha}2M to the incubation medium or 24 h thereafter (Fig. 4). Treatment with {alpha}2M visibly increased cell shortening of cardiomyocytes as early as 5 min after addition of the compound to the incubation medium, although no statistical difference was achieved (data not shown, n=4). A more pronounced effect of the compound on cell shortening was observed after 24-h incubation with {alpha}2M, which attained statistical significance at a stimulation frequency of 2 Hz (see Fig. 4, n=3, 78 cells). Since the PI3-kinase/Akt/mTOR pathway has been shown to be involved in cardiac cell hypertrophy [16] and Akt increases calcium entry into cardiac myocytes [17,18], we investigated whether inhibition of this pathway by the mTOR antagonist rapamycin (100 nM) would affect cardiac cell function. We observed that rapamycin alone increased the contractility of cardiomyocytes. A non-significant reduction of contractility was observed for the combination of rapamycin and {alpha}2M (data not shown). The data of these experiments are in line with previous observations of Schoffstall et al. [19], who demonstrated that there was little or no effect of rapamycin on maximum calcium-activated isometric force, whereas calcium sensitivity was increased at some rapamycin concentrations in rabbit skeletal and cardiac muscle and rat cardiac muscle.


Figure 4
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  		Fig. 4 Effects of {alpha}2M treatment on contractile function of rat ventricular cardiomyocytes. Cells were treated with {alpha}2M (40 µg/ml) for 24 h. Subsequently, quiescent cells were electrically stimulated at frequencies ranging from 0.5 Hz to 2 Hz. Cell shortening was assessed by video-assisted cell edge detection and presented as % of cell shortening (dL) of the original cell length (L). *P<0.05, significantly different from the untreated control.

 
3.4 Effects of {alpha}2M on resting calcium, the amplitude of calcium spikes, and SERCA-2a expression
The improvement of cardiac contractility following treatment with {alpha}2M should be associated with increased diastolic as well as systolic calcium levels. Indeed, addition of {alpha}2M increased basal [Ca2+]i approximately twofold, whereas a 1.5-fold increase in the peak amplitude of [Ca2+]i spikes after electrical stimulation was observed (Fig. 5A–C). Furthermore, treatment of rat ventricular cardiomyocytes with either {alpha}2M or anti-LRP-1 antibody increased SERCA-2a expression to 400±6% and 300±22% of the untreated control (set to 100%) (Fig. 6, n=5, 50 cells). When ventricular cells were treated with phenylephrine a 270±22% increase in SERCA-2a expression was observed.


Figure 5
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  		Fig. 5 Effects of {alpha}2M treatment on systolic and diastolic [Ca2+]i in rat ventricular cardiomyocytes. Cells were treated with 40 µg/ml {alpha}2M for 24 h. Subsequently, cells were loaded with fluo-3/AM and electrically stimulated intracellular Ca2+ responses were recorded by confocal laser scanning microscopy. (A, B) representative traces of Ca2+ responses in contracting cardiomyocytes under control conditions (A) and in the presence of {alpha}2M (B). (C) Amplitudes of resting (diastolic) and peak (systolic) [Ca2+]i in ventricular cardiomyocytes which remained either untreated (filled bars) or were treated with {alpha}2M (open bars). *P<0.05, significantly different from the untreated control.

 

Figure 6
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  		Fig. 6 Increase in SERCA-2a mRNA expression upon treatment of rat ventricular cardiomyocytes with either {alpha}2M or an antibody directed against the LRP-1/{alpha}2M receptor. Cells were treated for 24 h. Subsequently mRNA expression was evaluated by real-time RT-PCR. *P<0.05, significantly different from the untreated control.

 
3.5 Effects of {alpha}2M on MAPK, PI3-kinase and Akt activation
Treatment of macrophages [20] and prostate cancer cells [21] with {alpha}2M has been previously shown to activate the mitogen-activated protein kinases ERK1,2, JNK and p38. Signalling events elicited by {alpha}2M in cardiac cells, however, have not yet been investigated. To investigate MAPK activation, ventricular cardiac cells were labelled with antibodies directed against the phosphorylated, active forms of the MAPKs. It was shown that {alpha}2M transiently activated ERK1,2 with maximum values observed after 5 min (Fig. 7, n=3). This was significantly inhibited in the presence of the MEK inhibitor UO126 (data not shown). In contrast, no significant activation of JNK or p38 was observed (see Fig. 7, n=3). Furthermore, {alpha}2M treatment of ventricular cardiomyocytes activated p85, the regulatory unit of PI3-kinase with maximum values observed between 15 and 30 min after stimulation. Akt, which is downstream of PI3-kinase, was also activated with maximum activity seen between 5 and 15 min after incubation with {alpha}2M (Fig. 8A, B, n=3).


Figure 7
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  		Fig. 7 Effects of {alpha}2M treatment on the activation of ERK1,2, JNK and p38. Rat ventricular cardiomyocytes were treated with 40 µg/ml {alpha}2M and fixed after incubation times as indicated. Subsequently, they were stained with antibodies directed against the active (phosphorylated) forms of the proteins. The state of activation was evaluated by confocal laser scanning microscopy and computer-assisted image analysis. *P<0.05, significantly different from the untreated control.

 

Figure 8
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  		Fig. 8 Stimulation of PI3-kinase (A) and Akt (B) activation upon treatment with {alpha}2M. Rat ventricular cardiomyocytes were treated with 40 µg/ml {alpha}2M and fixed after incubation times as indicated. Subsequently, they were stained with antibodies directed against the active (phosphorylated) forms of the proteins. The state of activation was evaluated by confocal laser scanning microscopy and computer-assisted image analysis. *P<0.05, significantly different from the untreated control.

 
3.6 Effects of inhibition of ERK1,2 and PI3-kinase/Akt signalling on {alpha}2M-induced protein synthesis
The data of the present study demonstrate that {alpha}2M treatment of ventricular cardiac cells induces cell hypertrophy and protein synthesis and activates ERK1,2 as well as PI3-kinase/Akt signalling cascades. To investigate whether activation of signalling cascades is related to the hypertrophic response, {alpha}2M-induced 14Phe incorporation was investigated in the presence of the mitogen activated protein kinase kinase (MEK) inhibitor UO126 (10 µM) (Fig. 9A, n=3), the PI3-kinase inhibitors LY294002 (50 µM) (Fig. 9B, n=4) and wortmannin (100 nM) (Fig. 9B, n=4), and rapamycin (100 nM), which inhibits mTOR signalling pathway downstream of Akt (Fig. 9C, n=4). As shown in Fig. 9A–C, all applied inhibitors totally abolished the increase in protein synthesis observed after treatment of cells with {alpha}2M, thus indicating that {alpha}2M-induced ERK1,2 and PI3-kinase/Akt signalling is involved in the hypertrophic response.


Figure 9
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  		Fig. 9 Effects of the MEK inhibitor UO126 (A), the PI3-kinase inhibitors LY294002 and wortmannin (B), and the mTOR inhibitor rapamycin (C) on protein synthesis in rat ventricular cardiomyocytes. The cells were treated with the respective substances for 24 h. Protein synthesis was evaluated by determination of 14Phe-incorporation into synthesized proteins. *P<0.05, significantly different as indicated.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The acute phase response is a nonspecific inflammatory reaction of the host that occurs shortly after any tissue injury. The response includes changes in the concentration of plasma proteins called acute phase proteins, some of which decrease in concentration (negative acute phase proteins), such as albumin or transferrin, and others of which increase in concentration (positive acute phase proteins), such as C-reactive protein, serum amyloid A, haptoglobin, {alpha}-1-acid glycoprotein, ceruloplasmin and {alpha}2M. Most positive acute phase proteins are glycoproteins synthesized mainly by hepatocytes upon stimulation by proinflammatory cytokines and released into the bloodstream. Within the heart acute phase proteins have been thus far mainly considered to be factors of cardiovascular risk, whereas their biological function has been widely neglected [22]. Previous studies have demonstrated that a cardiac isoform of the acute phase protein {alpha}2M induces cardiac hypertrophy in vivo [9,10], suggesting that upon pressure overload expression of {alpha}2M is increased within the heart and exerts autocrine or paracrine hypertrophic effects on cardiac cells. Moreover, {alpha}2M was effective when administered through the tail vein of rats, suggesting that the compound gains access to cardiac cells through the coronary vasculature [23]. The data of the present study used {alpha}2M isolated from human plasma to study the hypertrophic effects of this compound. It was shown that a hypertrophic response was elicited by {alpha}2M in ventricular rat cardiomyocytes that was comparable in its extent to the hypertrophic response observed by treatment of cells with the {alpha}-adrenergic agonist phenylephrine. From the data of our study we cannot conclude that the hypertrophic response of ventricular cardiomyocytes as well as the underlying signalling cascade were solely initiated by the native {alpha}2M protein. On the contrary, our experiments showed that {alpha}2M which was activated by the ammonium bicarbonate method stimulated protein synthesis to nearly the same extent as native protein. From this it could be concluded that the effects of {alpha}2M on cardiomyocytes were indeed mediated by the activated form of {alpha}2M which was presumably activated by proteinases released from the cells into the cell culture medium.

In the present study concentrations of 40–120 µg/ml of {alpha}2M were used. From the literature it is known that in vivo plasma concentrations of {alpha}2M in humans attain 2–4 mg/ml [24]. Okubo demonstrated that the level of {alpha}2M in sera from normal male rats is 32±4 µg/ml [25]. During acute inflammation {alpha}2M levels can increase to 2 mg/ml in rats [26]. Schreiber et al. reported an increase in the {alpha}2M concentration during inflammation in rats from 14 µg/ml to 4.5 mg/ml [27]. However, it should be noted that the concentration of {alpha}2M* that is circulating is difficult to determine, since in vivo {alpha}2M* is taken up by liver cells within minutes [28].

The hypertrophic response towards {alpha}2M was apparently mediated through specific signalling through its receptor LRP-1, since a comparable stimulation of hypertrophic protein synthesis and cardiac gene expression was achieved by LRP-1 receptor activation using an antibody directed against LRP-1, and the LRP receptor antagonist RAP abolished {alpha}2M-induced protein synthesis. These findings exclude that {alpha}2M induces ventricular cell hypertrophy by acting as a carrier for growth factors and cytokines, e.g. TGF-β1, TGF-β2 and PDGF BB, which has been previously reported [29]. To our knowledge, nothing is so far known about LRP-1 expression and function within the heart. Recent studies suggest that LRP-1 exerts signalling functions that are related to cell migration, proliferation and vascular permeability [30]. Furthermore LRP-1 has been recently shown to regulate the composition of the extracellular matrix in mouse embryonic fibroblasts [31]. The data of the present study demonstrate that besides increasing the cell size of cardiomyocytes, {alpha}2M treatment enhanced contractile function and raised resting [Ca2+]i as well as the mean amplitudes of systolic [Ca2+]i. The changes in [Ca2+]i may be associated to the increase in SERCA expression, resulting in an increased Ca2+ load within the sarcoplasmic reticulum and consequently higher peak [Ca2+]i during systolic contraction. Several studies have shown that increased SR Ca2+-ATPase expression improves Ca2+ cycling and myocardial function [32]. In this respect it has been recently shown that SERCA expression both at the mRNA and protein level is upregulated by preload in rabbit multicellular muscle strips and is associated with improved myocardial performance, thus underlining the functional relevance of the preload-induced expression changes [33].

In the present study the hypertrophic response elicited by {alpha}2M was preceded by the activation of distinct intracellular signalling cascades, i.e. the transient activation of ERK1,2, PI3-kinase and Akt. Consequently, pretreatment with specific inhibitors of the signalling cascades under investigation abolished the stimulation of protein synthesis observed after {alpha}2M treatment. ERK1,2 activation upon treatment of cardiac cells with hypertrophic stimuli has been demonstrated in numerous studies [34] and has been previously shown to be associated with angiotensin 2 — but not phenylephrine-mediated cardiac cell hypertrophy. An activation of the ERK1,2 signalling cascade has been furthermore shown in Smad 4-deficient cardiomyocytes, which developed cell hypertrophy and reexpression of fetal genes [35]. Besides activation of ERK1,2 upon treatment of ventricular cardiomyocytes with {alpha}2M an activation of Akt and PI3-kinase was observed. The role of the PI3-kinase/Akt pathway in cardiac hypertrophy is well established. It was demonstrated that targeted overexpression of constitutively active PI3-kinase in the heart increased organ size while expression of a dominant-negative mutant decreased it [16]. The hypertrophic response following PI3-kinase activation is apparently associated with Akt downstream of PI3-kinase, since transgenic expression of Akt in the heart led to increased size of the heart and cardiomyocytes [36,37]. Akt activation furthermore enhanced left ventricular (LV) function, which was apparently due to an increase in L-type Ca2+ current density and – comparable to the results of the present study – SERCA-2a protein levels [17]. Interestingly, an increased contractile function and SERCA-2a activity was likewise demonstrated by nuclear targeting of Akt. However, in this study the amplitude of [Ca2+]i transients and L-type Ca2+ current density was not different between the transgenic and the control animals, and no hypertropic cell growth was observed [18]. Whether Akt activation in hypertrophic cardiac cells is associated with fetal gene expression remains controversial. From the literature it is apparent that ANF is also activated in an Akt-dependent way [38]. Barac et al. recently reported that Fas-mediated hypertrophy which resulted in increased ANP and sarcomeric actin expression, was abolished upon inhibition of the PI3-kinase/Akt pathway [39]. Hiraoka et al. reported that Akt/PKB activation is indispensable for ANP expression and stimulation of protein synthesis, but not for sarcomeric reorganization [40]. Conversely, Shiojima et al. reported that adenovirus-mediated overexpression of myristoylated Akt1 in cultured, neonatal rat ventricular myocytes promotes hypertrophy in the absence of ANF expression or reorganization of the actin cytoskeleton [41]. The conflicting data regarding Akt-dependent gene expression may arise from different times of Akt activation used in various studies. Schiekofer et al. [42] recently discussed that physiological hypertrophy occurs by short-term activation of Akt, which induces hypertrophy in the absence of fetal gene expression. However, chronic expression of constitutively activated Akt1 in the heart replicates biochemical and histological aspects of heart failure, including induction in the expression of fetal genes.

In the present study cardiac cell hypertrophy-associated protein synthesis was abolished in the presence of rapamycin, which inhibits mTOR. mTOR is a 289-kDa evolutionarily conserved serine/threonine kinase which can phosphorylate and thereby inactivate the eukaryotic translation initiation factor 4E-BP1, leading to increased protein translation. mTOR also phosphorylates and activates p70S6K, which is a short isoform of the ribosome S6 kinase (S6K) 1. S6K1 and S6K2 are key regulators of cell growth through control of protein translation [30]. It has been conclusively demonstrated that the PI3K/Akt/mTOR signalling pathway plays an important role in the development of cardiac hypertrophy, since inhibition of mTOR signalling with rapamycin regressed established cardiac hypertrophy induced by pressure overload [43,44]. Furthermore, it has been recently shown that the toll-like receptor-4-mediated pathway and PI3-kinase/Akt/mTOR signalling are involved in the development of cardiac hypertrophy in vivo [45].

Although acute phase proteins including {alpha}2M [46] have been established as markers of cardiac disease and dysfunction, nearly nothing is known about their function in adaptive responses to cardiac insults like cardiac hypertrophy or cardiac infarction. Systemic administration of rapamycin has been used successfully for the treatment of transplant rejection in clinical practice, and it has therefore been suggested to be a useful therapeutic means to suppress cardiac hypertrophy in patients [44]. Besides possible physiological roles for acute phase proteins in cardiac hypertrophy and infarction, C-reactive protein and {alpha}2M have been shown to be related to the severity of coronary atherosclerosis [47]. This may be treatable by rapamycin, since it has been shown to attenuate atherosclerosis progression in animal models [48] as well as neointima formation in coronary stents [49]. Assuming that during acute phase protein-induced cardiac hypertrophy as well as atherosclerosis an over-activation of the PI3-kinase/Akt/mTOR pathway occurs, interference with this signalling pathway may be clinically exploitable for the treatment of both, hypertrophic and coronary heart disease.

Time for primary review 41 days


   Acknowledgement
 
This work was supported by the Jürgen Manchot Foundation, Düsseldorf, Germany and by the Graduate College 534 of the German Research Foundation.


   Notes
 
* Gerd Heusch (Universitätsklinikum Essen, Essen, Germany) served as Guest Editor for this article. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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