INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CARDIAC HYPERTROPHY is an adaptive process in the mature heart resulting from sustained high workload (18). Cardiac hypertrophy can also become maladaptive, and in this circumstance it is associated with sudden death. Agonists such as angiotensin II (ANG II), endothelin-1, and phenylephrine have been shown to mimic the hypertrophic response to increasing hemodynamic load (1, 31, 32). Treatment of cardiac myocytes in vitro with ANG II (23, 28, 29, 38) or phenylephrine (2, 4) results in measurable increases in protein synthesis within 48 h of treatment.

Protein translation can be divided into two phases: initiation and elongation (reviewed in Brown and Schreiber, Ref. 3). Translation initiation is highly regulated in cardiac myocytes. Agonists such as ANG II and phenylephrine appear to regulate translation initiation by increasing the activity of the serine-threonine kinase p70^S6 in a rapamycin-sensitive manner (2, 28). However, nothing is known about the regulation of elongation factors by these agents in cardiac myocytes. Two proteins, eukaryotic elongation factor-1 (eEF-1) and eEF-2, are essential components involved in extension of the polypeptide chain. eEF-2 is encoded by a single gene and is subject to several different types of posttranslational regulation. eEF-2 is the sole cellular target for the diphtheria toxin and is regulated by phosphorylation by a highly specific Ca²⁺/calmodulin-dependent kinase termed eEF2 kinase (reviewed in Nairn and Palfrey, Ref. 20). Phosphorylation by eEF2 kinase inhibits eEF-2 activity, a process that can be reversed by dephosphorylation with protein phosphatase 2A (PP2A) (21).

In the present study we examined the regulation of eEF-2 phosphorylation in cardiac myocytes following stimulation with ANG II. eEF2 is basally phosphorylated in cardiac myocytes, and ANG II treatment leads to a rapid dephosphorylation of eEF-2. This effect is mediated by the ANG II AT1 receptor and apparently involves a mechanism in which ANG II activates PP2A via the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways. Dephosphorylation of eEF-2 and the subsequent activation of protein elongation may contribute together with regulation of initiation to increased cardiac myocyte protein synthesis and cardiac hypertrophy.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cardiac myocyte isolation and culture. Neonatal cardiac myocytes were isolated from 1- to 3-day-old Sprague-Dawley rat hearts as previously described (11). Myocytes were initially plated in DMEM/F-12 supplemented with 5% horse serum, 2 g/l bovine serum albumin (fraction 5), 3 mmol/l pyruvic acid, 100 µmol/l ascorbic acid, 4 µg/ml transferrin, 0.7 ng/ml sodium selenium, 100 µmol/l 5-bromo-2-deoxyuridine, 50,000 units penicillin, and 50 mg streptomycin/500 ml. After the myocytes were allowed to attach for 24 h, the medium was changed to a defined serum-free DMEM/F-12 as above, except the horse serum was not included. This isolation procedure produced cultures containing >90% cardiac myocytes as confirmed by immunostaining with a monoclonal antibody against sarcomeric actin (MF20). Myocytes were used for studies after 72 h in serum-free culture.

Immunoblot analysis. After treatment, cardiac myocytes were washed twice with ice-cold Hank's-buffered saline (GIBCO) and scraped from the plate in 100 µl of lysis buffer (0.2% SDS, 4 mM sodium orthovanadate, 4 mM sodium molybdate, 1 µg/ml aprotinin, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, and 0.5 µg/ml DNAse) (Sigma). Soluble protein (50 µg) from each condition was separated by SDS-PAGE in a 10% gel and transferred overnight (Transblot, Bio-Rad) to nitrocellulose membranes (Bio-Rad). Immunodetection of total or phosphorylated eEF-2 utilized autoradiography with an ECL reagent (Amersham) (21). The autoradiographic phosphorylated and total eEF-2 signal from companion blots were compared using densitometry (Personal Densitometer, Molecular Dynamics; Sunnyvale, CA) and analyzed using ImageQuant software (Molecular Dynamics). The phosphorylated eEF-2 signal densities were normalized to the respective total eEF-2 signal to reflect the relative ratio of phosphorylated eEF-2 to total eEF-2. The reproducibility of this approach was confirmed in a minimum of three experiments per condition.

Measurement of protein synthesis. Cardiac myocytes were cultured in serum-free DMEM/F12 supplemented with 2 µCi/ml of [³H]phenylalanine (NEN Life Sciences; Boston, MA). Okadaic acid (1-6 nM, Calbiochem), fostriecin (20-500 nM, Parke-Davis; Ann Arbor, MI), rapamycin (10-100 nM, Calbiochem), LY-29004 (1-20 nM, Calbiochem), losartan (10⁸-10⁵ M, Calbiochem), PD-123319 (10⁸-10⁵ M, RBI; Natick, MA), FK506 (50-200 ng/ml, Calbiochem), or PD-98059 (5-20 nM, Calbiochem) were added 30 min before the addition of ANG II (10⁷ M). Cultures were incubated for 48 h after which trichloroacetic acid-precipitable proteins were assayed for [³H]phenylalanine incorporation and DNA content as described by Sadoshima et al. (28). Results were performed in quadruplicate from three different myocyte isolations for each compound tested.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Angiotensin II regulates eEF-2 dephosphorylation. To examine ANG II-regulated phosphorylation of eEF-2, nonconfluent neonatal cardiac myocytes grown in serum-free media were treated with increasing concentrations of ANG II (10¹⁰-10⁷ M) for 30 min. Immunoblotting of cell lysates with a phospho-specific eEF-2 antibody demonstrated concentration-dependent dephosphorylation of eEF-2 with a maximum ANG II concentration of 10⁷ M (Fig. 1A). ANG II at 10⁷ M decreased the level of phosphorylated eEF-2 by ~3.5-fold. Treatment with ANG II (10⁷ M) also produced a rapid, time-dependent dephosphorylation of eEF-2 within 30 min of administration (Fig. 1B). By 30 min, the level of phosphorylated eEF-2 decreased approximately fourfold and persisted after 60 min of treatment. As shown in Fig. 1, A and B, the levels of total eEF2 were unaffected by any of the treatments used.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Angiotensin II (ANG II) concentration- and time-dependent dephosphorylation of eutaryotic elongation factor 2 (eEF-2). A: cardiac myocytes were treated with increasing concentrations of ANG II (1010-107 M) for 30 min, and 50 µg of soluble proteins were analyzed by SDS-PAGE and immunoblotting for phospho-specific (P-eEF-2) and total eEF-2 (T-eEF-2). B: cardiac myocytes were treated for 0-60 min with ANG II (107 M), and samples were also immunoblotted for phosphorylated and total eEF2. Numbers at the bottom of each pair of blots indicates the ratio of the signal for phosphorylated eEF-2 normalized to total eEF-2.

Angiotensin II stimulates eEF-2₁ dephosphorylation and protein synthesis via the AT₁ receptor. ANG II mediates its cardiovascular effects, including hypertrophy, through interaction with AT₁ and AT₂ receptors. To determine whether eEF-2 dephosphorylation is mediated by AT₁ or AT₂ receptors, isolated cardiac myocytes were pretreated with increasing concentrations of losartan (AT₁ inhibitor) or PD-123319 (AT₂ inhibitor) before stimulation with ANG II for 30 min. As shown in Fig. 2A, losartan blockade of the AT₁ receptor effectively inhibited eEF-2 dephosphorylation at a concentration of 10⁸ M, whereas inhibition of the AT₂ receptor with PD-123319 (Fig. 2B) up to a concentration of 10⁵ M did not block the effect of ANG II.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   ANG II stimulates dephosphorylation of eEF-2 via the AT1 receptor. Cardiac myocytes were treated with diluent (control), losartan (A, 108- 105 M), or PD-123319 (B, 108-105 M) for 30 min before stimulation with ANG II (107) for 30 min and immunoblotted for phosphorylated and total eEF-2. Numbers at the bottom of each pair of blots indicates the ratio of the signal for phosphorylated eEF-2 normalized to total eEF-2. C: [3H]phenylalanine incorporation at 48 h in response to ANG II, losartan + ANG II, or PD-123319 + ANG II. *P < 0.05, ANG II or PD-123319 groups vs. Control; **P < 0.05, losartan vs. ANG II groups by one-way ANOVA.

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ANG II-dependent dephosphorylation of eEF-2 and stimulation of protein synthesis requires active PP2A. The rapid dephosphorylation of eEF-2 that takes place with ANG II treatment must require the activity of a phosphatase. We explored whether inhibition of PP2A (21, 25-27) with the specific inhibitors, okadaic acid (IC₅₀ for PP2A 0.1-1 nM; 10-20 nM for PP1) (5) or fostriecin (IC₅₀ for PP2A 3.2 nM; 131 µM for PP1) (35), could block ANG II-mediated dephosphorylation of eEF-2. As shown, okadaic acid (Fig. 3A) and fostriecin (Fig. 3B) blocked, in a dose-dependent manner, the dephosphorylation of eEF-2 caused by ANG II. This occurred at a concentration of okadaic acid and fostriecin specific for PP2A (1 and 100 nM, respectively). In contrast, treatment of myocytes with FK506 (50-200 ng/ml), a specific inhibitor of the related serine-threonine phosphatase, protein phosphatase 2B (PP2B, calcineurin) (17), had no effect on ANG II-stimulated dephosphorylation of eEF-2 (Fig. 3C).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   ANG II-dependent dephosphorylation of eEF-2 requires active protein phosphatase 2A (PP2A). Cardiac myocytes were treated with the PP2A inhibitors okadaic acid (OA, 1-6 nM) (A) or fostriecin (20-200 nM) (B) or the PP2B inhibitor FK506 (50-200 ng/ml) (C) for 30 min before stimulation with ANG II (107 M) for 30 min. Soluble proteins were immunoblotted for phosphorylated and total eEF-2. Numbers at the bottom of each pair of blots indicates the ratio of the signal for phosphorylated eEF-2 normalized to total eEF-2. D: [3H]phenylalanine incorporation at 48 h in response to ANG II + okadaic acid, fostriecin, or FK506. *P < 0.05 vs. control; **P < 0.05 vs. ANG II.

ANG II signals through the MAPK cascade to stimulate dephosphorylation of eEF-2 and activation of protein synthesis. ANG II stimulation of cardiac myocytes is known to rapidly activate the MAPK cascade leading to phosphorylation of MAPK (extracellularly regulated kinase 1/2, ERK1/2) (28, 29, 38). The time sequence of activation of MAPK in cardiac myocytes was determined by treating with ANG II for 0-30 min and immunoblotting for phosphorylated and total MAPK. As shown in Fig. 4A, ANG II maximally activated MAPK between 5 and 20 min with levels of phosphorylated MAPK decreasing by 30 min. The mitogen-activated protein or ERK kinase (MEK1/2) inhibitor PD-98059 (4, 7, 12) was found to effectively block ANG II-dependent activation of MAPK at a concentration of ~5 nM (Fig. 4B). Similarly, PD-98059 blocked dephosphorylation of eEF-2 in a dose-dependent manner with a half-maximum inhibitory concentration of 5 nM (Fig. 4C). As shown in Table 1, PD-98059 (5-20 nM) significantly inhibited basal protein synthesis in a dose-dependent manner. However, the ANG II-dependent increase in protein synthesis was blocked at all concentrations of PD-98059 used.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   Effect of mitogen-activated protein or extracellularly regulated kinase kinase (MEK1/2) inhibition on ANG II-stimulated dephosphorylation of eEF-2. Cardiac myocytes were treated with ANG II (107 M) for 1-30 min (A) or water (control) or the MEK1/2 inhibitor PD-98059 (PD, 1-20 nM) for 30 min before stimulation with ANG II (107 M) for 30 min (B and C). Soluble proteins were immunoblotted for phosphorylated (P-MAPK) and total mitogen-activated protein kinase (T-MAPK) (A and B) or phosphorylated (P-eEF2) and total (T-eEF2) eEF-2 (C). In C, numbers at the bottom of each pair of blots indicates the ratio of the signal for phosphorylated eEF-2 normalized to total eEF-2.

ANG II signals through LY-294002- but not rapamycin-sensitive pathways to stimulate dephosphorylation of eEF-2. In cardiac myocytes, the actions of ANG II (28) and phenylephrine (2) appear to involve the mammalian target rapamycin (mTOR)/p70^S6K signaling pathway. Both LY-294002 (2, 23, 24), an inhibitor of PI3K, and rapamycin (2, 28), an inhibitor of mTOR, both block ANG II-dependent stimulation of protein synthesis in cardiac myocytes. As shown in Fig. 5A, LY-294002 treatment resulted in a dose-dependent inhibition of ANG II-dependent dephosphorylation of eEF-2. LY-294002 at a concentration of 20 nM completely inhibited ANG II-dependent dephosphorylation of eEF-2 with a half-maximal effect at ~10 nM. Unlike LY-294002, rapamycin, at a concentration up to 100 nM, did not block ANG II-dependent dephosphorylation of eEF-2 (Fig. 5B). Rapamycin at concentrations up to 50 nM has been shown to block ANG II-stimulated protein synthesis (2, 28). As shown in Table 1, LY-294002 (1-20 nM) and rapamycin (10-100 nM) both significantly suppressed basal protein synthesis in cardiac myocytes in a dose-dependent manner. LY-294002 and rapamycin also inhibited ANG II-induced protein synthesis at all concentrations examined.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Effect of phosphoinositol 3-kinase (PI3K) and mammalian target rapamycin (mTOR) inhibition on ANG II-stimulated dephosphorylation of eEF-2. Cardiac myocytes were treated with water (control), the PI3K inhibitor LY-294002 (1-20 nM) (A), or the mTOR inhibitor rapamycin (10-100 nM) (B) for 30 min before stimulation with ANG II (107 M) for 30 min. Soluble proteins were immunoblotted for phosphorylated and total eEF-2. Numbers at the bottom of each pair of blots indicates the ratio of the signal for phosphorylated eEF-2 normalized to total eEF-2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Increased protein synthesis is the cardinal feature of cardiac hypertrophy. Protein synthesis is tightly regulated in cells by various molecules that interact with the translational machinery of the ribosome. ANG II has been clearly shown to produce cardiac hypertrophy in vitro and to increase protein synthesis in vivo, although the exact mechanisms regulating this process in the heart have not been completely identified. ANG II has been found to regulate translation initiation but to date the regulation of translation elongation by ANG II has not been investigated.

Translation elongation is a tightly regulated process controlled by eEF-2, the activity of which is related to its phosphorylation status. Phosphorylation of eEF-2 by eEF-2 kinase results in complete inhibition of polypeptide synthesis, and this can be reversed by the dephosphorylation of the factor by PP2A (21). In the present study, treatment with the PP1/PP2A inhibitors okadaic acid and fostriecin both blocked ANG II-stimulated dephosphorylation of eEF-2; however, treatment with the PP2B inhibitor FK506 had no effect. On the basis of the dose responses for inhibition of PP1 and PP2A for okadaic acid and fostriecin and the cell permeability of these two inhibitors, these results support the idea that PP2A is involved in the dephosphorylation of eEF-2 that is modulated by ANG II. Treatment with okadaic acid and fostriecin blocked both basal and ANG II-stimulated protein synthesis. Together, these results suggest that ANG II treatment leads to activation of PP2A and the dephosphorylation of eEF-2 and that this contributes to the increased protein synthesis observed in cardiac myocytes. Basal protein synthesis was also inhibited by both okadaic acid and fostriecin, indicating a role for PP2A in regulation of protein translation possibly via an increase in eEF-2 phosphorylation. Although it is likely that the regulation of eEF-2 phosphorylation occurs via a direct effect of PP2A on eEF-2, it is possible that PP2A also may play some role in the regulation of eEF-2 kinase activity. Notably, a previous study in cultured adult cardiac myocytes has indicated that insulin appears to regulate both eEF-2 phosphorylation and eEF-2 kinase activity (36). In the present study, FK506 had no effect on basal or the ANG II-dependent increase in protein synthesis. The role of PP2B inhibitors such as FK506 and cyclosporin in mediating cardiac hypertrophy is controversial (6, 16, 19, 39) but likely involves the inactivation of genomic pathways linked to cardiac hypertrophy. It is clear from the present study, however, that at least in response to ANG II, a FK506-sensitive pathway is not linked to the regulation of eEF2 dephosphorylation.

The coupling of the AT₁ receptor to upstream signal transduction pathways leading to eEF-2 dephosphorylation remains to be established. We specifically examined the importance of the MAPK/ERK signaling pathway in this process. In the present study and in others (28, 29), ERK1/2 is activated within 5 min of ANG II stimulation, and in the current study the peak of MAPK phosphorylation is observed at 10 min. In comparison, the ANG II-induced dephosphorylation of eEF-2 begins after 5 min and reaches a maximal level after 30 min. This suggests that activation of the MAPK cascade may lie upstream of the signaling pathway involved in the dephosphorylation of eEF-2. The MAPK/ERK cascade is likely an important component in the regulation of myocyte hypertrophy because PD-98059 has also been shown to prevent the increase in myofibrillar organization stimulated by endothelin-1 or phenylephrine (4). Consistent with this notion, the present study demonstrates that treatment with PD-98059 not only blocks ANG II-dependent dephosphorylation of eEF-2 but also inhibits ANG II-stimulated and basal protein synthesis. The mechanism of ANG II activation of MAPK in cardiac myocytes is unclear; however, in vascular smooth muscle cells ANG II stimulates MAPK activation by transactivation of the EGF receptor by a Ca²⁺-dependent mechanism (10). There is also evidence from neuronal cells and PC12 pheochromocytoma cells, which exclusively express the AT₂ receptor, that the AT₂ receptor suppresses MAPK by activation of a phosphatase 1 (14, 15). The importance of this balance of MAPK activation/inactivation in cardiac myocytes at different ages in response to ANG II remains to be established. The signaling pathway(s) downstream MAPK that result in dephosphorylation of eEF-2 are not known but clearly involve PP2A. MAPK is regulated by PP2A (37); however, there is no evidence that MAPK regulates PP2A.

PI3K are membrane enzymes that phosphorylate the hydroxyl group at position 3 on the inositol ring of phosphatidylinositol 4-phosphate in response to stimulation of tyrosine kinase or G protein-coupled receptors (33). PI3K are also activated in cardiac myocytes in response to ANG II (23). Inhibition of a LY-294002-sensitive pathway in myocytes blocked ANG II-stimulated protein synthesis (23). In the present study, LY-294002 also blocked ANG II-stimulated protein synthesis in addition to inhibiting ANG II-dependent dephosphorylation of eEF-2. These findings suggest that PI3K activation by ANG II is likely to be part of the upstream pathway(s) that regulate eEF-2 dephosphorylation. The precise mechanism(s) involved in linking PI3K to dephosphorylation of eEF-2 are not known but likely involve MAPK (22) and/or PP2A. In vascular smooth muscle cells, ANG II-dependent stimulation of PI3K is directly linked to activation of Akt (protein kinase B) (10). Because PP2A and Akt can interact (13), it will be of interest to identify whether PI3K, Akt, and PP2A are all linked in cardiac myocytes to the regulation of eEF2 dephosphorylation.

The macrolide antibiotic rapamycin is also an inhibitor of protein synthesis in cardiac myocytes acting downstream of PI3K (2, 28), blocking activation of p70^S6K and subsequent translation initiation (8). As shown in the present study, rapamycin unlike the PI3K inhibitor LY-294002 does not block the ANG II-stimulated dephosphorylation of eEF-2. Both, however, block ANG II-stimulated protein synthesis. This suggests a model where ANG II-dependent activation of PI3K via the AT₁ receptor results in the stimulation of both the initiation and elongation phases of protein synthesis by activation of MAPK and PP2A. This regulation of both translation initiation and elongation by upstream signaling pathways is likely to be a conserved process because MAPK has also been shown to regulate the activity of the initiation control protein eIF4E (24) and in this report the elongation protein eEF-2. Clearly multiple signaling pathways are likely to be involved in the ANG II-induced regulation of cardiac hypertrophy to balance the phosphorylation/dephosphorylation of key translation initiation and elongation control proteins.