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Am J Physiol Heart Circ Physiol 276: H1655-H1663, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 5, H1655-H1663, May 1999

Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy

Klaus-Dieter Schlüter1, Andreas Simm2, Matthias Schäfer1, Gerhild Taimor1, and Hans Michael Piper1

1 Institut für Physiologie, Justus-Liebig-Universität, Giessen; and 2 Theodor-Boveri-Institut, Physiologische Chemie II, Universität Würzburg, Würzburg, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study investigated the role of early response kinase (ERK) and phosphatidylinositol 3 (PI 3)-kinase in ventricular cardiomyocytes from adult rat for the hypertrophic response to alpha -adrenoceptor stimulation. Parameters of the hypertrophic response were stimulation of protein synthesis and induction of creatine kinase BB. The alpha -adrenoceptor agonist phenylephrine (10 µmol/l) activated ERK2 and PI 3-kinase. The protein kinase C inhibitor bisindolylmaleimide (5 µmol/l) and the mitogen-activated protein kinase kinase inhibitor PD-98059 (10 µmol/l) but not the tyrosine kinase inhibitor genistein (100 µmol/l) blocked ERK2 activation. Inhibition of ERK2 activation abolished induction of creatine kinase BB by phenylephrine but not the increase in protein synthesis. The PI 3-kinase inhibitor wortmannin (100 nmol/l) blocked protein synthesis under alpha -adrenoceptor stimulation but did not interfere with ERK2 activation. Inhibition of the ERK2 pathway with PD-98059 did not affect PI 3-kinase activation. We conclude that ERK2- and PI 3-kinase-dependent pathways represent two mutually exclusive ways of signaling that lead to different aspects of the hypertrophic response to alpha -adrenoceptor stimulation.

tyrosine kinase; creatine kinase BB; protein synthesis; mitogen-activated protein kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIAC HYPERTROPHY is an important compensatory response of the heart to altered workload (15). Although this process is initially compensatory, hypertrophied myocardium may eventually become dysfunctional. Enlargement of cardiac mass is a well-established predictor of subsequent heart failure (13). Myocardial hypertrophy is characterized by an increased rate of protein synthesis and a reexpression of fetal-type proteins, e.g., creatine kinase B. The different intracellular signals involved in the induction of these cellular changes are only partly understood.

We have shown that the alpha -adrenoceptor agonist phenylephrine, but not the beta -adrenoceptor agonist isoprenaline, induces cardiac hypertrophy in newly isolated adult rat cardiomyocytes (22). Phenylephrine stimulates protein synthesis and induces the expression of the B-type isoform of creatine kinase (16, 22). The induction of creatine kinase BB activity under alpha -adrenoceptor stimulation is actinomycin D sensitive (22) and can therefore be taken as one example for alpha -adrenoceptor-mediated reexpression of a fetal-type protein. Phenylephrine thus mimics some typical aspects of myocardial hypertrophy in cardiomyocytes found in vivo. Isolated adult ventricular cardiomyocytes under alpha -adrenoceptor stimulation can therefore be used to study intracellular signaling leading to myocardial hypertrophy.

In neonatal cardiomyocytes, alpha -adrenoceptor stimulation by phenylephrine leads to an activation of genistein-sensitive tyrosine kinase(s), which in turn causes activation of mitogen-activated protein (MAP) kinase, also named early response kinase (ERK) (21, 26). The influence of ERK activation on protein synthesis in neonatal cardiomyocytes under alpha -adrenoceptor stimulation is still an open question. Some authors reported that ERK activation is not part of the intracellular signaling leading to an increase in protein synthesis (6, 25, 30), but others found that ERK activation is required for the increase in protein synthesis (9). Whether these results, obtained on neonatal cardiomyocytes, can be extrapolated to cells of the adult myocardium is unclear, inasmuch as these two cell types represent different stages of differentiation.

In adult cardiomyocytes the role of ERK activation in the growth response to alpha -adrenoceptor stimulation has not been addressed on the cellular level. This has been the aim of the present study, in which a well-characterized model of cultured ventricular cardiomyocytes from adult rats is used. In this model the cardiomyocytes are mechanically quiescent, and cells other than cardiomyocytes are not present. Intracellular signaling can therefore be studied independently of the contractile state of cardiomyocytes and of the influence of other cells. The following questions regarding the hypertrophic response to alpha -adrenoceptor stimulation were addressed with adult ventricular cardiomyocytes: 1) Is the ERK pathway activated? 2) Does protein kinase C (PKC) or a genistein-sensitive step represent upstream activators for ERK; i.e., does one or the other transduce the signal from adrenoceptor stimulation to ERK activation? 3) Is ERK activation required for the rise in protein synthesis, or, alternatively, is phosphatidylinositol 3 (PI 3)- kinase involved? 4) How does ERK activation relate to the induction of creatine kinase BB?


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Ventricular heart muscle cells were isolated from 200- to 250-g male Wistar rats, as previously described (17, 18). Isolated cells were suspended in FCS-free culture medium and plated at a density of 1.4 × 105 elongated cells/35-mm culture dish (type 3001, Falcon). The culture dishes had been preincubated overnight with 4% FCS in medium 199. The basic culture medium consisted of medium 199 with Earle's salts, 5 mmol/l creatine, 2 mmol/l L-carnitine, 5 mmol/l taurine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. To prevent growth of nonmyocytes, media were also supplemented with 10 µmol/l cytosine-D-arabinofuranoside.

Four hours after plating, cultures were washed twice with culture medium to remove round and nonattached cells and supplied with FCS-free experimental medium, in which cells were incubated for up to 24 h at 37°C. The experiments were carried out in basic culture medium (control), with additions of phenylephrine, isoprenaline, or phorbol 12-myristate 13-acetate (PMA) at concentrations indicated. Ascorbic acid (100 µmol/l) was added to all cultures as an antioxidant.

Incorporation of [14C]phenylalanine and changes in cellular protein and RNA mass. Incorporation of phenylalanine into cells was determined by exposing cultures to L-[14C]phenylalanine (0.1 µCi/ml) for 24 h and determining the incorporation of radioactivity into acid-insoluble cell mass, as described previously (16). Nonradioactive phenylalanine (0.3 mmol/l) was added to the medium to minimize variations in the specific activity of the precursor pool responsible for protein synthesis. In incorporation studies, experiments were terminated by removal of the supernatant medium from the cultures and washed three times with ice-cold PBS [composition (in mmol/l): 1.5 KH2PO4, 137 NaCl, 2.7 KCl, and 1.0 Na2HPO4, pH 7.4]. Subsequently, ice-cold 10% (wt/vol) TCA 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) SDS by incubation for 2 h at 37°C. In these samples, protein contents (4) and DNA contents (8) were determined, and the radioactivity was counted. RNA was determined from an aliquot of these samples after precipitation with an equal volume of 10% (wt/vol) perchloric acid in the remaining supernatant (14). The RNA content was also expressed relative to the DNA content of the samples.

Analysis of creatine kinase activities. Specific activity of the cytosolic creatine kinase was determined as described previously (22). Cultures were first washed twice with PBS. After addition of buffer A [composition (in mmol/l): 5 magnesium acetate, 0.4 EDTA, 2.5 dithiothreitol, 50 Tris · HCl, and 250 sucrose, pH 6.8] to the dishes, the cells were scrapped off, homogenized, and frozen until use at -14°C. For analysis, these samples were thawed, and the resulting suspension was sonicated and centrifuged at 12,000 g for 2 min. The supernatants were used for enzyme analysis. The activity of creatine kinase was determined according to Gerhardt (7) with standard ultraviolet methods.

The distribution of the cytosolic isozymes of creatine kinase, MM, MB, and BB, was analyzed according to Kaye et al. (11). The supernatants were applied to a 1-ml DEAE-cellulose column that had been equilibrated with buffer B [composition (in mmol/l): 20 NaCl, 5 magnesium acetate, 0.4 EDTA, and 100 Tris · HCl, pH 7.9]. The creatine kinase MM isozyme eluted directly with buffer B, creatine kinase MB with change of NaCl concentration to 40 mmol/l and pH to 6.4, and creatine kinase BB with change of NaCl concentration to 250 mmol/l and pH to 6.4.

Determination of p42 MAP kinase (ERK2). ERK2 was determined as described elsewhere in detail (24). Briefly, after stimulation, cells were lysed in lysis buffer [composition: 50 mmol/l Tris · Cl, pH 6.7, 2% (wt/vol) SDS, 2% (vol/vol) mercaptoethanol, and 1 mmol/l sodium orthovanadate]. Then nucleic acids were digested with benzonase (Merck, Darmstadt, Germany). After SDS-PAGE (100 µg protein/slot), proteins were transferred onto reinforced nitrocellulose by semidry blotting. The sheets were saturated with 2% (wt/vol) BSA and incubated for 2 h with rabbit polyclonal anti-rat p42 MAP kinase (0.2 µg/ml; Santa Cruz Biotechnology). After the sheets were washed, alkaline phosphatase-labeled sheep anti-rabbit IgG (50 mU/50 ml) was added for 2 h. Bands were visualized by alkaline phosphatase activity using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. For quantification the two bands on the blots were scanned densitometrically. The results were expressed as the ratio of the upper band (activated and phosphorylated ERK with retarded gel mobility) to the total amount of ERK determined on the Western blots.

Determination of PKC activity. The specific activity of PKC was determined in the membrane fraction of cardiomyocytes by the method of Chakravarthy et al. (5), which allows measurement of the activity of the enzyme in its native membrane-associated state, as described previously (23). Briefly, cardiomyocytes were incubated for the time indicated, rinsed with ice-cold PBS, and covered with a hypotonic lysis buffer [composition (in mmol/l): 1 NaHCO3, 5 MgCl2, and 0.1 phenylmethylsulfonyl fluoride (PMSF), pH 7.5]. The swollen cells were lysed by vigorous mixing, and the lysates were centrifuged at 4°C for 5 min at 1,000 g to sediment unlysed cells and the nuclei. The postnuclear supernatants were centrifuged at 4°C for 20 min at 35,000 g to sediment the cell membranes. The pelleted membranes were resuspended in assay buffer (composition: 50 mmol/l Tris · HCl, pH 7.5, 2 µmol/l CaCl2, 10 mmol/l MgCl2, 0.2 mmol/l PMSF, 2 mmol/l NaF, 0.2 mmol/l sodium pyrophosphate, and 0.2 mmol/l sodium vanadate). PKC activity in this fraction was measured using the specific substrate peptide PLSRTLSVAAKK (10). Membrane fractions phosphorylate this peptide with [gamma -32P]ATP over a 10-min period. The phosphorylated peptide was extracted from the reaction solution by a stepwise salt gradient on a DEAE-Sepharose column. The incorporated radioactivity was counted.

Determination of PI 3-kinase activation. PI 3-kinase activity was determined in immunoprecipitates, as described by Whitman et al. (28) with minor changes. Briefly, cardiomyocytes were washed twice with PBS, and the cells were lysed in lysis buffer [composition: 10% (vol/vol) glycerol, 1% (vol/vol) Nonidet P-40, and 1 mM PMSF]. After centrifugation (10 min at 10,000 g) the supernatant was used for immunoprecipitation with an antibody against the p85 alpha -subunit of bovine PI 3-kinase (IC Chemicals, Cologne, Germany), and the immunoprecipitates were sedimented with protein A-Sepharose. The pellets were washed with PBS, twice with buffer A (composition: 0.5 M LiCl, 0.1 M Tris, pH 7.4) and once with buffer B (composition: 10 mM Tris, pH 7.4, 100 mM NaCl, and 1 mM EDTA) and resuspended in 25 µl of buffer B. Phosphatidylinositol (1 mg/ml; Sigma Chemical, Deisenhofen, Germany) was dispersed by sonication in 5 mM HEPES buffer, pH 7.4, and 20 µl of this solution were added to the resuspended immunoprecipitates. After preincubation for 30 min at room temperature, the phosphorylation reaction was started by addition of 20 µCi of [gamma -32P]ATP in starting buffer containing 50 µM ATP and 5 mM MgCl2. The total volume in the reaction tubes was 50 µl. The reaction mixture was incubated for 20 min at 25°C and terminated by addition of 100 µl of 1 M HCl. Phospholipids were then extracted with 200 µl of CHCl3-MeOH (1:1). The organic phase was spotted onto a silica gel TLC plate pretreated with 1% (wt/vol) potassium oxalate. Phosphorylated products were separated by TLC in a CHCl3-MeOH-4 M Na4OH (9:7:2) developing solvent and visualized on a PhosphorImager (Molecular Dynamics). To quantify the activity of the immunoprecipitates, TLC plates were scanned densitometrically, and the amount of phosphorylated phosphatidylinositol was normalized to the spotted radioactivity on the plates, which varied between the reaction tubes.

Statistics. Values are means ± SE; n represents the number of culture preparations. Statistical comparisons were performed by one-way ANOVA, and the Bonferroni test was used for post hoc analysis (27). Differences with P < 0.05 were regarded as statistically significant.

Materials. Falcon tissue culture dishes were obtained from Becton-Dickinson (Heidelberg, Germany). Boehringer Mannheim (Mannheim, Germany) was the source for glutamine-free medium 199, FCS, and bisindolylmaleimide (BIM). Cytosine-D-arabinofuranoside, L-carnitine, creatine, taurine, l-phenylephrine hydrochloride, dl-isoproterenol hydrochloride, and PMA were obtained from Sigma Chemical. All other chemicals were of analytic grade.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of ERK2. Activation of ERK2 in adult ventricular cardiomyocytes under alpha -adrenoceptor stimulation was determined on Western blots of whole cell protein samples by retarded gel mobility of the activated (phosphorylated) compared with the nonactivated (nonphosphorylated) form of ERK2 (Fig. 1A). For quantification, ERK2 activation was expressed as the ratio of the activated form of ERK2 to the total amount of ERK2 (Fig. 1B). Time course experiments showed that alpha -adrenoceptor stimulation led to an activation of ERK2 (Fig. 1A). Activation was significant within 5 min and was maximal after 15 min (Fig. 1B). At this time, ~60% of blotted ERK2 was found in the activated form. After 60 min, however, ERK2 activation could no longer be seen (Fig. 1, A and B). In contrast, ERK2 was not activated within the same time span in the presence of the beta -adrenoceptor agonist isoprenaline (Fig. 1, A and B).



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  		Fig. 1.   Stimulation of mitogen-activated protein (MAP) kinase (MAPK) in adult rat cardiomyocytes. Phenylephrine (PE, 10 µmol/l) or isoprenaline (Iso, 1 µmol/l) was added to cultures as indicated. Whole cell extracts of cultures were loaded on 12% SDS polyacrylamide gels and blotted on nitrocellulose. Blot was incubated with a monoclonal p42 MAPK (ERK2) antibody. Activation of ERK2 is indicated by appearance of a double band. A: representative example. B: ERK2 activation as ratio of activated (MAPK*) to total MAPK (MAPKtotal). Solid bars represent activation in presence of PE; open bars represent stimulation in presence of Iso. Values are means ± SE for 3 experiments. * P < 0.05 vs. 0 min.

Role of PKC activation in ERK2 activation. A direct activator of PKC, PMA (100 nmol/l), also activated ERK2 within 15 min. At 15 min after addition of PMA, 76 ± 11% of total ERK2 was found in the activated form (P < 0.05 vs. untreated control cultures, n = 3). Activation of ERK2 in the presence of PMA indicated that ERK2 can be activated via PKC. It was therefore investigated whether the specific activity of PKC was increased in the membranous fraction of cardiomyocytes under the same conditions in which ERK2 was activated. Within 15 min, phenylephrine caused a 2.4-fold increase and PMA a 3.1-fold increase of membranous PKC activity (Fig. 2). Isoprenaline, which did not activate ERK2, did not activate membranous PKC (Fig. 2).


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  		Fig. 2.   Stimulation of protein kinase C (PKC) in membrane fraction of cardiomyocytes by exposure of cells to PE (10 µmol/l), phorbol 12-myristate 13-acetate (PMA, 100 nmol/l), or Iso (1 µmol/l) for 15 min. Values are means ± SE for 3 experiments expressed as percentage of basal activity. Basal activity was 120 ± 17 pmol of phosphate incorporated · 10 min-1 · 10 µg membrane protein-1. * P < 0.05 vs. control.

The role of PKC in the alpha -adrenoceptor-mediated activation of ERK2 in adult ventricular cardiomyocytes was further investigated in experiments in which the activation of PKC was inhibited by BIM. BIM at 5 µmol/l abolished the activation of ERK2 in the presence of phenylephrine (Fig. 3). At this concentration, BIM also suppressed the hypertrophic response to phenylephrine. Stimulation of protein synthesis and induction of creatine kinase BB by phenylephrine were reduced to insignificant levels (Fig. 4). BIM alone did not change basal [14C]phenylalanine incorporation [4.3 ± 0.4 × 10-2 vs. 4.1 ± 0.3 × 10-2 dpm/µg DNA, not significant (NS), n = 4] or basal creatine kinase BB activity (0.08 ± 0.03 vs. 0.07 ± 0.01 U/mg protein, NS, n = 4). In summary, the effects of phenylephrine on ERK2 activation, stimulation of protein synthesis, and induction of creatine kinase BB are dependent on the activation of PKC.


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  		Fig. 3.   Stimulation of ERK2 in adult rat cardiomyocytes. Cultures were analyzed under control conditions and after 15 min in presence of PE or PE + bisindolylmaleimide (BIM) at concentrations indicated. ERK2 activation was analyzed and quantified as described in Fig. 1 legend. Values are means ± SE for 3 experiments. * P < 0.05 vs. control.



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  		Fig. 4.   Incorporation of [14C]phenylalanine (14C-Phe) into cardiomyocyte cultures (left) and stimulation of creatine kinase BB (CK-BB) activity (right) by exposure of cardiomyocytes to PE (10 µmol/l) or PE + BIM (5 µmol/l) for 24 h. Mean basal 14C-Phe incorporation was 4.1 ± 0.3 × 10-2 dpm/µg DNA, and mean basal CK-BB activity was 0.07 ± 0.01 U/mg protein. Values are means ± SE for 4 experiments. * P < 0.05 vs. control.

Role of ERK2 activation in the hypertrophic response. Whether one or both of the PKC-dependent parameters of hypertrophic responses, stimulation of protein synthesis or induction of creatine kinase BB, depend on ERK activation was investigated. For this purpose, PD-98059, a novel MAP kinase kinase (MEK) inhibitor, was used. PD-98059 inhibited ERK2 activation dose dependently; at 10 µmol/l it abolished the activation of ERK2 by phenylephrine (Fig. 5). In the presence of PD-98059 (10 µmol/l), induction of creatine kinase BB caused by phenylephrine was also abolished (Fig. 6), but stimulation of protein synthesis was not altered significantly. In the absence of phenylephrine, PD-98059 did not influence basal [14C]phenylalanine incorporation (3.9 ± 0.2 × 10-2 vs. 4.1 ± 0.3 × 10-2 dpm/µg DNA, NS, n = 4) or basal creatine kinase BB activity (0.06 ± 0.02 vs. 0.07 ± 0.01 U/mg protein). The results thus show that induction of creatine kinase BB, but not stimulation of protein synthesis, depends on ERK2 activation in the presence of phenylephrine.


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  		Fig. 5.   Stimulation of ERK2 in adult rat cardiomyocytes. Cultures were analyzed under control conditions and after 15 min in presence of PE or PE + PD-98059 (PD). ERK2 activation was analyzed and quantified as described in Fig. 1 legend. Values are means ± SE for 3 experiments. * P < 0.05 vs. control.



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  		Fig. 6.   Incorporation of 14C-Phe into cardiomyocyte cultures (left) and stimulation of CK-BB activity (right) by exposure of cardiomyocytes to PE (10 µmol/l) or PE + PD (10 µmol/l) for 24 h. See Fig. 4 legend for details. Values are means ± SE for 4 experiments. * P < 0.05 vs. control.

Role of a genistein-sensitive step in the hypertrophic response under alpha -adrenoceptor stimulation. Whether a genistein-sensitive step, indicating the involvement of genistein-sensitive tyrosine kinase(s), is involved in the hypertrophic response to phenylephrine was evaluated. Cardiomyocytes were stimulated with phenylephrine in the presence or absence of genistein (100 µmol/l). Genistein reduced the induction of creatine kinase BB but did not affect the increase in [14C]phenylalanine incorporation by phenylephrine (Fig. 7). Under control conditions, genistein did not influence [14C]phenylalanine incorporation (4.0 ± 0.4 × 10-2 vs. 4.1 ± 0.3 × 10-2 dpm/µg DNA, NS, n = 4) or creatine kinase BB activity (0.07 ± 0.03 vs. 0.07 ± 0.01 U/mg protein, NS, n = 4). The effect of genistein on the alpha -adrenoceptor-mediated induction of creatine kinase BB was dose dependent, with an EC50 of 13 µmol/l. Genistein had an effect similar to PD-98059, the MEK inhibitor, on the two investigated parameters of the hypertrophic response to phenylephrine, i.e., stimulation of protein synthesis and induction of creatine kinase BB. Therefore, whether genistein inhibits ERK2 activation was analyzed.


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  		Fig. 7.   Incorporation of 14C-Phe into cardiomyocyte cultures (left) and stimulation of CK-BB activity (right) by exposure of cardiomyocytes to PE (10 µmol/l) or PE + genistein (Gen, 100 µmol/l) for 24 h. See Fig. 4 legend for details. Values are means ± SE for 4 experiments. * P < 0.05 vs. control.

Activation of ERK2 in the presence of phenylephrine or PMA was unaltered when genistein was applied (Fig. 8). These results indicated that a genistein-sensitive step in the signaling pathway, leading from alpha -adrenoceptor stimulation via activation of PKC and ERK2 to induction of creatine kinase BB, is downstream of ERK2 or in series with the ERK2 pathway.


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  		Fig. 8.   Stimulation of ERK2 in adult rat cardiomyocytes. Cultures were analyzed under control conditions (C) and after 15 min in presence of PE (10 µmol/l), PE + Gen (100 µmol/l), PMA (100 nmol/l), or PMA + Gen. MAPK activity was analyzed and quantified as described in Fig. 1 legend. Values are means ± SE for 3 experiments. * P < 0.05 vs. control.

Role of PI 3-kinase activation on alpha -adrenoceptor-mediated hypertrophic growth. The previous results indicated that PKC activation, but not ERK2 activation, is involved in the stimulation of protein synthesis by phenylephrine. It was then investigated whether the PI 3-kinase pathway takes part in the signaling pathway leading to hypertrophy. Wortmannin, an inhibitor of PI 3-kinase, dose dependently attenuated the stimulation of protein synthesis under the influence of phenylephrine; it abolished this response at 100 nmol/l (Fig. 9). In the absence of phenylephrine, wortmannin did not change basal [14C]phenylalanine incorporation (4.6 ± 0.4 × 10-2 vs. 4.4 ± 0.5 × 10-2 dpm/µg DNA, NS, n = 4). Similar results were also obtained with LY-294002 (100 µmol/l), a chemically distinct PI 3-kinase inhibitor. It also reduced [14C]phenylalanine incorporation from 57 ± 8% above untreated controls to 6 ± 5% (P < 0.05 vs. without LY-294002, n = 4).


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  		Fig. 9.   Incorporation of 14C-Phe into cardiomyocyte cultures. Experiments were performed for 24 h under serum-free conditions (control) or in presence of PE or PE + wortmannin (Wort). Values are means ± SE for 4 experiments. * P < 0.05 vs. control.

It was finally confirmed that alpha -adrenoceptor stimulation leads to a PKC-dependent activation of PI 3-kinase. Phenylephrine activated PI 3-kinase, and this activation was abolished in the presence of wortmannin (Fig. 10) and in the presence of BIM, an inhibitor of PKC. In contrast, the presence of the MEK inhibitor PD-98059 had no effect (Fig. 10). To show more directly the PKC dependency of PI 3-kinase activation, it was further investigated whether direct stimulation of PKC by PMA activates PI 3-kinase. This was indeed the case, and this response was also attenuated by BIM (Fig. 11). These results showed that activation of PI 3-kinase follows activation of PKC but is independent of the activation of ERK2. In a reciprocal experiment the effect of the PI 3-kinase inhibitor wortmannin on ERK2 activation was tested (Fig. 12). It was found that wortmannin did not influence ERK2 activation elicited by phenylephrine or PMA (Fig. 11). In addition, wortmannin did not attenuate the induction of creatine kinase BB, which normally follows ERK2 activation. Phenylephrine induced creatine kinase BB activity from 0.07 ± 0.01 to 0.13 ± 0.03 mU/mg protein in the absence of wortmannin (P < 0.05, n = 4 experiments) and to 0.12 ± 0.01 mU/mg protein in the presence of wortmannin (NS vs. phenylephrine, n = 4).


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  		Fig. 10.   Phosphatidylinositol 3 (PI 3)-kinase activation in adult rat cardiomyocytes. Cultures were analyzed under control conditions (C) and after 15 min in presence of PE (10 µmol/l), PE + BIM (5 µmol/l), PE + Wort (100 nmol/l), or PE + PD (100 µmol/l). PI 3-kinase activity was analyzed and quantified as explained in MATERIALS AND METHODS. Values (means ± SE for 3 experiments) represent phosphorylation of phosphatidylinositol of immunoprecipitates of 85-kDa subunit of PI 3-kinase. * P < 0.05 vs. control.



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  		Fig. 11.   PI 3-kinase activation in adult rat cardiomyocytes. Cultures were analyzed under control conditions (C) and after 15 min in presence of PE (10 µmol/l), PE + BIM (5 µmol/l), PMA (100 nmol/l), or PMA + BIM. PI 3-kinase activity was analyzed and quantified as explained in MATERIALS AND METHODS. Values (means ± SE for 3 experiments) represent phosphorylation of phosphatidylinositol of immunoprecipitates of 85-kDa subunit of PI 3-kinase. * P < 0.05 vs. control; + P < 0.05 vs. BIM-treated cultures.



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  		Fig. 12.   Stimulation of ERK2 in adult rat cardiomyocytes. Cultures were analyzed under control conditions (C) and after 15 min in presence of PE (10 µmol/l), PE + Wort (100 nmol/l), PMA (100 nmol/l), or PMA + Wort. ERK2 activation was analyzed and quantified as described in Fig. 1 legend. Values are means ± SE for 3 experiments. * P < 0.05 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study investigated the role of ERK in the hypertrophic response to alpha -adrenoceptor stimulation in ventricular cardiomyocytes from adult rat. Parameters of the hypertrophic response were induction of creatine kinase BB, representing induction of fetal-type proteins, and stimulation of protein synthesis, indicating myocyte growth. The main findings of this study are that, in adult rat ventricular cardiomyocytes, 1) alpha -adrenoceptor stimulation leads to a transient activation of ERK2, 2) ERK2 activation requires activation of PKC, but not of a genistein-sensitive step, 3) ERK activation is required for induction of the fetal-type isoform of creatine kinase, creatine kinase BB, but not for the stimulation of protein synthesis, and 4) the stimulation of protein synthesis requires activation of PI 3-kinase, but induction of creatine kinase BB does not.

We previously showed that alpha -adrenoceptor stimulation induces protein synthesis and creatine kinase BB in adult ventricular cardiomyocytes. Both effects depend on the activation of PKC (2, 22). These results are confirmed in the present study, in which signal transduction of the hypertrophic response to alpha -adrenergic stimulation is analyzed. Using the same cell preparation used in the earlier study, we found that alpha -adrenoceptor stimulation causes activation of ERK2. This activation of ERK2 depends on PKC activation, because 1) it is coincident in time with PKC activation, 2) it can be inhibited by BIM, a PKC inhibitor, in a dose-dependent way, and 3) it can be provoked not only by alpha -adrenoceptor stimulation but also by use of PMA, a direct activator of PKC. In this last aspect, our study confirms the results of Lazou et al. (12), who reported that PMA activates ERK in adult myocardium and isolated adult rat ventricular cardiomyocytes. In that study, norepinephrine was found to activate ERK in whole myocardium (12), but whether the activated ERK was located in cardiomyocytes or other cells remained unproven.

beta -Adrenoceptor stimulation with isoprenaline has no effect on ERK2 activity, nor does it activate PKC or alter any of the investigated parameters of hypertrophy (16, 22) in these mechanically quiescent adult cardiomyocytes. Our findings are in apparent contrast to reports that in neonatal cardiomyocytes (3) or the beating myocardium of adult hearts (12) beta -adrenoceptor stimulation activates ERK. Neonatal cardiomyocytes, however, represent a different stage of cardiac differentiation, and in beating myocardium beta -adrenoceptor effects might be mediated through nonmuscular cell types or changes in the mechanical load. The present study is the only one demonstrating on the cellular level for the adult type of cardiomyocytes that when mechanical effects are excluded, beta -adrenoceptor stimulation does not cause ERK2 activation.

In this study, whether ERK2 activation is involved in either of the investigated outcomes of phenylephrine-mediated PKC activation, i.e., stimulation of protein synthesis or induction of creatine kinase BB, was tested. Activation of ERK2 was inhibited by the specific MEK inhibitor PD-98059 in a dose-dependent manner. In the presence of PD-98059, induction of creatine kinase BB by phenylephrine was abolished. This shows that ERK2 activation is required for the induction of creatine kinase BB under alpha -adrenoceptor stimulation. Whether ERK2 activation under alpha -adrenoceptor stimulation is also required for the transcriptional activation of other fetal-type genes, e.g., atrial natriuretic peptide (ANP), was not investigated in our study. Recent studies on neonatal cardiomyocytes studying the expression of ANP indicated, however, that activation of MEK is not involved in the intracellular signaling leading to reexpression of ANP (19). Phenylephrine augmented protein synthesis in adult cardiomyocytes in the presence of PD-98059. This demonstrated that this growth effect of alpha -adrenoceptor stimulation is independent of ERK2 activation.

We further found that stimulation of protein synthesis in the presence of phenylephrine is mediated through activation of PI 3-kinase. This causal relationship is documented by the observation that 1) PI 3-kinase is indeed activated in the presence of phenylephrine in a PKC-dependent way and 2) protein synthesis is inhibited in a dose-dependent manner by PI 3-kinase inhibitors wortmannin and LY-294002. Wortmannin, however, does not influence ERK2 activation in the presence of phenylephrine. Conversely, the MEK inhibitor PD-98059 does not affect phenylephrine-induced PI 3-kinase activation. From these results, it is concluded that, downstream of PKC activation, alpha -adrenoceptor stimulation activates two mutually independent pathways of signal transduction: one mediated by ERK2 activation, which leads to induction of creatine kinase BB, and another that is mediated by PI 3-kinase activation, which causes stimulation of protein synthesis. Our conclusions might be limited to the pharmacological profiles of the inhibitors used in this study, i.e., PD-98059 and wortmannin. PD-98059 is a highly specific inhibitor of MEK and has been used in several studies to discriminate between the ERK pathway and other intracellular steps. Wortmannin, however, has side effects on MEK, myosin light-chain kinase, and PI 4-kinase activation, but at much higher concentrations than used in this study. Here we demonstrate that wortmannin does not interfere with ERK2 activation at 100 nmol/l, although it completely abolishes PI 3-kinase activation. Thus the use of the inhibitors may justify the conclusions.

For neonatal cardiomyocytes it has been suggested that genistein-sensitive tyrosine kinases are involved in alpha -adrenoceptor-mediated hypertrophic effects (6). This genistein-sensitive step was localized upstream of ERK2. In the adult cardiomyocytes investigated here, induction of the fetal-type creatine kinase BB includes a genistein-sensitive step. The analysis reveals, however, that this genistein-sensitive step is not upstream of ERK2, because ERK2 activation in the presence of phenylephrine was not influenced by genistein. Inasmuch as ERK2 activation is known to involve a tyrosine phosphorylation, this finding indicates that this tyrosine phosphorylation is not mediated by a genistein-sensitive kinase. This is consistent with results of others showing that ERK2 activation is insensitive to genistein, e.g., ERK activation in rat liver macrophages by PMA (1) or ERK activation in neonatal cardiomyocytes under mechanical stretch (29). The genistein-sensitive step, which is necessary to induce creatine kinase BB, is located downstream of ERK or in series with ERK. The kind of genistein-sensitive step involved in the inhibitory effect of genistein on creatine kinase induction was not investigated further. One might speculate that activation of p90rsk, which is activated by ERK, requires a genistein-sensitive phosphorylation (p90rsk is indeed tyrosine phosphorylated). In this case, the genistein-sensitive step would be located downstream of ERK activation. Alternatively, induction of creatine kinase BB may require the activation of genistein-sensitive transcription factors, e.g., signal transducer and activator of transcription and other ERK2-dependent transcription factors. Taken together, the comparison between the published data on rat neonatal cardiomyocytes (6) and data of the present study regarding the intracellular signaling leading to ERK2 activation under alpha -adrenoceptor stimulation revealed differences in the signaling of hypertrophic stimuli between the neonatal- and adult-type cardiomyocytes.

The intracellular signaling by which alpha -adrenoceptor stimulation evokes a stimulation of protein synthesis is genistein insensitive. This signaling requires an activation of PI 3-kinase, which is known to need a tyrosine phosphorylation for full activation. As in the case of ERK2 activation, our results indicated that this tyrosine phosphorylation is not mediated through a genistein-sensitive kinase. These observations are consistent with the reports by other authors that PI 3-kinase activity is not influenced by genistein (20).

Our study suggests that the hypertrophic effects of alpha -adrenoceptor stimulation are caused by PKC activation. The conclusion might be limited to the PKC inhibitors used, but this is unlikely. In our study we used the selective, cell-permeable PKC inhibitor BIM, which is structurally similar to the less selective inhibitor staurosporine. BIM is known to inhibit PKC (inhibition constant = 10 nmol/l) and, with much less potency, protein kinase A (inhibition constant = 2 µmol/l). In previous studies we used two other PKC inhibitors, staurosporine and calphostine, with essentially the same results obtained here with BIM with respect to the hypertrophic response of cardiomyocytes (2, 22, 23). These other inhibitors are less specific than BIM for PKC, but the identity of effects indicates that they act by a common mechanism, i.e., PKC inhibition. Of course, we cannot rule out that BIM interferes with protein kinases other than PKC and protein kinase A. Such an effect, however, is unknown on any cell type. Finally, our conclusion that ERK activation caused by phenylephrine is mediated through PKC activation is not based only on the inhibitory potential of BIM. It is based on the following findings: 1) phenylephrine activates PKC before MEK activation (Fig. 2), 2) direct stimulation of PKC by PMA stimulates ERK2, and 3) BIM inhibits the ERK2 activation by alpha -adrenoceptor stimulation (Fig. 3). In summary, the data provided by this study and the previous studies from our group seem to justify our conclusions.

In summary, our study shows that alpha -adrenoceptor stimulation of adult cardiomyocytes leads to an activation of PKC and subsequently of the ERK2 pathway. This is required for the induction of the fetal-type creatine kinase BB, a characteristic feature of alpha -adrenoceptor-mediated myocardial hypertrophy. Independent of ERK2 activation, alpha -adrenoceptor stimulation also causes the activation of the PI 3-kinase-dependent pathway. This is also dependent on an activation of PKC but mediates the stimulatory action on protein synthesis.


    ACKNOWLEDGEMENTS

This work was supported by Deutsche Forschungsgemeinschaft Pi 162/11-1 and SFB 353 Project C2. This work was part of the thesis by M. Schäfer.


    FOOTNOTES

Address for reprint requests and other correspondence: K.-D. Schlüter, Physiologisches Institut, Justus-Liebig-Universität, D-35392 Giessen, Germany (E-mail: Klaus-Dieter.Schlueter{at}physiologie.med.uni-giessen.de).

Received 3 November 1997; accepted in final form 13 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 276(5):H1655-H1663
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