Phosphorylation of Eukaryotic Translation Initiation Factor 2Bε by Glycogen Synthase Kinase-3β Regulates β-Adrenergic Cardiac Myocyte Hypertrophy

Construction of Vectors

The full-length rat eIF2Bε cDNA was obtained by reverse-transcription-PCR (Roche), using total RNA obtained from the rat brain, and subcloned into pcDNA3.1 (Invitrogen). Two eIF2Bε mutants were constructed using a mutagenesis kit (Stratagene). One mutant has a point mutation in the N-terminal region (eIF2Bε-F259L).¹⁶ This mutant has reduced intrinsic guanine nucleotide exchange function and acts as a dominant-negative.¹⁶ In another mutant, S535 was replaced with alanine (A) (eIF2Bε-S535A), which is the phosphorylation site of GSK-3β in rat eIF2Bε.

Adenoviral Vectors

Adenovirus harboring GSK-3β (AdGSK-3β) has been described.⁶ Adenovirus harboring LacZ (Ad-LacZ), eIF2Bε-wild-type (Ad-eIF2Bε-WT), eIF2Bε-F259L (Ad-eIF2Bε-F259L), and eIF2Bε-S535A (Ad-eIF2Bε-S535A) were constructed using AdenoX adenovirus construction kit (BD-Clontech).

GST-eIF2Bε Fusion Protein

cDNA encoding the amino acid residues 518 to 716 of eIF2Bε was generated by PCR and subcloned into pGEX-4T-3. Bacterially expressed fusion protein was purified using glutathione-Sepharose.

Primary Culture of Neonatal Rat Ventricular Myocytes

Primary cultures of ventricular cardiac myocytes were prepared from 1-day-old Crl:(WI)BR-Wistar rats (Charles River Laboratories) and purified using Percoll centrifugation.³ Culture media were changed to serum-free at 24 hours. Myocytes were cultured in a serum-free condition for 48 hours before experiments.

Phosphorylation of GST-eIF2Bε by GSK-3β

The cells were lysed with 1 mL of the lysis buffer, GSK-3β was immunoprecipitated with anti-mouse GSK-3β antibody (Transduction Labs), and the kinase reaction was performed using 1 μg of GST-eIF2Bε or GST-eIF2Bε-S535A, as described previously.⁶ Reaction mixtures were subjected to SDS-PAGE (12%). The gels were dried and subjected to autoradiography. Kinase reaction was also performed using whole-cell lysates or cold ATP followed by immunoblot analyses using anti-phospho S535 eIF2Bε antibody (Biosource International).

Immunoblot Analysis

For immunoblotting, cultured cells were lysed with the lysis buffer A, whereas tissue homogenates were prepared with the RIPA buffer (see the online data supplement available at http://circres.ahajournals.org).

Rat Model of Myocardial Infarction (MI)

Male Wistar rats (200 g) were used. Animals were anesthetized by intraperitoneal injection of ketamine (70 mg/kg) and xylazine (2 to 5 mg/kg). After orotracheal intubation, the thorax was opened left parasternally. The left anterior descending artery was ligated just below the left atrial appendage and the quality of the infarction was confirmed visually by the change of the color of the myocardium. The thorax was closed and the animals were extubated. At 24 hours, the animals were anesthetized, and the hearts arrested in diastole by injection of KCl. Immunoblot analyses were performed using tissue homogenates from the remote area. This study was approved by the Institutional Animal Care and Use Committee at New Jersey Medical School.

Myocyte Cell Surface Area and Sarcomeric Organization

Cells were fixed in PBS containing 4% paraformaldehyde. Immunostaining was performed using Texas Red phalloidin (Molecular Probes) and anti-α-sarcomeric actinin antibody (Sigma). Myocyte surface area was determined as described.³ Sarcomeric organization was classified as “nonorganized” when punctuated staining with random distribution was present, whereas “organized” indicated well-matured staining forming a clear striated pattern. Myocyte organization was determined to be “partially organized” when a less marked cytoskeletal organization was present with faint striated pattern.

Determination of Cardiac Myocyte Hypertrophy

Total protein/DNA content and [³H]phenylalanine incorporation were determined as described.⁶

Quantitative PCR

Quantification of transcripts for atrial natriuretic factor (ANF) and α-myosin heavy chain (α-MHC) was performed by real-time PCR using GAPDH as a control.

Statistics

Data are given as mean±SEM. Statistical analyses were performed using ANOVA and the method of Tukey. Significance was accepted at the P<0.05 levels.

An expanded Materials and Methods section is provided in the online data supplement at http://circres.ahajournals.org.

Adenovirus Constructs and Anti-Phospho S535 eIF2Bε-Specific Antibody

We tested whether our adenovirus constructs cause expression of the proteins of interest in cardiac myocytes. Transduction of Ad-GSK-3β induced expression of GSK-3β (6-fold) versus untransduced myocytes at a multiplicity of infection (MOI) of 30 (Figure 1A) and increased the GSK-3β kinase activity as determined by immune complex kinase assays (Figure 1B). Transduction of Ad-eIF2Bε-WT, Ad-eIF2Bε-F259L, or Ad-eIF2Bε-S535A led to similar levels of overexpression of eIF2Bε proteins in cardiac myocytes (Figure 1C, bottom). We also tested the specificity of the anti-phospho S535 eIF2Bε antibody. Immunoblot analyses indicated that overexpression of eIF2Bε-WT and eIF2Bε-F259L increased S535 phosphorylation of eIF2Bε, whereas that of eIF2Bε-S535A failed to enhance phosphorylation of S535 (Figure 1C, top), confirming the specificity of the antibody detecting S535 phosphorylation.

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Figure 1. A, Immunoblot analyses with anti-GSK-3β antibody. Myocytes were transduced with Ad-GSK-3β at various MOI. B, Kinase activity of GSK-3β (dark columns) was determined by the immune complex kinase assay. White columns indicate activities in control immunoprecipitates. Mean of 6 samples in 3 experiments is shown. C, Immunoblot analyses with anti-phospho-S535 eIF2Bε antibody (top) and anti-eIF2Bε antibody (bottom). In A and C, experiments were conducted 3 times.

GSK-3β Phosphorylates eIF2Bε In Vitro and In Vivo in Cardiac Myocytes

We determined whether interventions known to change the total cellular activity of GSK-3β affect the extent of phosphorylation of eIF2Bε in vitro. In order to increase the total cellular activity of GSK-3β, myocytes were transduced with Ad-GSK-3β. To reduce the activity of GSK-3β, myocytes were treated with either isoproterenol (ISO, 10 μmol/L), an agonist for β-adrenergic receptors (β-ARs), for 10 minutes, or LiCl (10 mmol/L), a well-established inhibitor of GSK-3β, for 1 hour. First, kinase assays were performed using myocyte lysates and GST-eIF2Bε. Cell extracts prepared from GSK-3β overexpressing myocytes exhibited an enhanced kinase activity against GST-eIF2Bε by 2-fold. Transduction of Ad-LacZ did not significantly affect the basal kinase activity (data not shown). By contrast, extracts from ISO- or LiCl-treated myocytes exhibited reduced kinase activities toward GST-eIF2Bε by 49% and 51%, respectively (Figure 2A). Coomassie blue staining the SDS page gel with the duplicate samples indicated that protein at comparable levels was loaded (Figure 2A, bottom). These results suggest that cardiac myocyte extracts contain kinase activity toward GST-eIF2Bε and that GSK-3β is a predominant kinase phosphorylating GST-eIF2Bε in cardiac myocytes at baseline.

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Figure 2. A through C, Cardiac myocytes were stimulated with or without ISO (10 μmol/L, 10 minutes) and LiCl (10 mmol/L, 1 hour). A, top, Total cell lysates were subjected to kinase assays, using GST-eIF2Bε as a substrate. Bottom, Coomassie blue staining of the gel with the duplicate samples. B through E, Myocyte extracts were subjected to immunoprecipitation using anti-GSK-3β antibody. Immune complex kinase assays were performed using GST-eIF2Bε as a substrate. Phosphorylation of GST-eIF2Bε is shown. In C through E, Ab(−) indicates that immunoprecipitation was performed using control IgG. B, All myocytes were transduced with either Ad-LacZ (30 MOI) or Ad-GSK-3β (30 MOI). Stimulation with ISO (10 μmol/L, 10 minutes) and LiCl (10 mmol/L, 1 hour) reduced, whereas overexpression of GSK-3β increased kinase activities against GST-eIF2Bε. C, Myocytes were stimulated with ISO (10 μmol/L) for 1 or 2 hours. D, To verify that S535 is phosphorylated by GSK-3β, GST-eIF2Bε-S535A was used as a substrate. E, Kinase assays were performed, using cold ATP followed by immunoblotting against phospho-S535 eIF2Bε. In B, C, and E, the value of untreated samples was expressed as 1.0. In D and E, only background levels of phosphorylation were observed in samples immunoprecipitated with control IgG [Ab(−)]. In A and D, the results shown are representative of 3 experiments. In B, C, and E, the graphs show the mean of 5, 4, and 6 experiments, respectively. In all graphs, *P<0.05 vs Control.

In order to further confirm that GSK-3β in the myocyte extract has kinase activities toward GST-eIF2Bε, experiments were repeated using immune complex kinase assays. GSK-3β immunoprecipitated from control myocytes phosphorylated GST-eIF2Bε, and its activity was enhanced in Ad-GSK-3β-transduced myocytes but reduced in ISO- or LiCl-treated myocytes (Figure 2B). The activity of GSK-3β was not affected by transduction of Ad-LacZ (not shown). Longer treatment with ISO also significantly reduced the GST-eIF2Bε kinase activity (Figure 2C). Immunoprecipitates with control antibody [indicated as Ab(−)] did not show kinase activity toward GST-eIF2Bε (Figure 2C), confirming the specificity of the immune complex kinase assays.

In order to test if serine 535 is subjected to phosphorylation, additional immune complex GSK-3β kinase assays were conducted using the same cell extracts and GST-eIF2Bε-S535A. Only weak background phosphorylation was observed when GST-eIF2Bε-S535A was used as a substrate compared with when GST-eIF2Bε wild-type (WT) was used, confirming that GSK-3β predominantly phosphorylates S535 of GST-eIF2Bε (Figure 2D). Kinase reaction was also performed using cold ATP, and immunoblotting was performed using the anti-phospho-S535 eIF2Bε antibody. S535 phosphorylation of eIF2Bε was reduced in ISO-treated myocytes, whereas it was enhanced in GSK-3β-transduced myocytes (Figure 2E). These results suggest that GSK-3β phosphorylates S535 of GST-eIF2Bε.

In order to further test whether S535 of endogenous eIF2Bε is phosphorylated and whether S535 phosphorylation of eIF2Bε correlates with cellular activities of GSK3β, immunoblot analyses were performed using anti-phospho-S535 eIF2Bε antibody. Although phosphorylation of eIF2Bε at S535 was significantly stimulated by overexpression of GSK-3β in cardiac myocytes (Figure 3A), it was significantly decreased by LiCl or ISO treatment, as well as by a specific GSK-3β inhibitor [GSK-3β inhibitor I (20 μmol/L); Figure 3B]. Decreases in phosphorylated S535 eIF2Bε were observed with both 1 and 10 μmol/L of ISO at 10 minutes and 1 hour (Figure 3C). These interventions did not significantly affect the total amount of eIF2Bε, as determined using nonphosphospecific anti-eIF2Bε antibody. ISO-induced decreases in S535 phosphorylation of eIF2Bε was inhibited in the presence of propranolol, an antagonist for β-ARs, but not by prazosin, an antagonist for α₁-adrenergic receptors (Figure 3D), suggesting that decreases in S535 eIF2Bε phosphorylation induced by ISO at doses of up to 10 μmol/L are predominantly mediated by β-ARs. Endothelin-1 (100 nmol/L), a Gαq-agonist, also transiently reduced phosphorylation of S535 eIF2Bε (Figure 3E).

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Figure 3. Immunoblot analyses with anti-phospho-S535 eIF2Bε-specific antibody (top) or anti-total (nonphosphospecific) eIF2Bε antibody (bottom). A, Overexpression of GSK-3β increased, whereas addition of LiCl (10 mmol/L, 1 hour) or ISO (10 μmol/L, 10 minutes) reduced phosphorylation of eIF2Bε. B, Cardiac myocytes were treated with GSK-3β inhibitor I (20 μmol/L) for 1 hour. C, Myocytes were stimulated with ISO (1 or 10 μmol/L) for either 10 minutes or 1 hour. D, Myocytes were stimulated with ISO (10 μmol/L) for 10 minutes in the presence of propranolol (10 μmol/L) or prazosin (10 μmol/L). E, Myocytes were stimulated with endothelin-1 (100 nmol/L) for indicated durations. In C and E, the result shown is representative of 3 experiments. In A, B, and D, the graphs show the mean of 7, 3, and 3 experiments, respectively. In all graphs, *P<0.05 vs Control.

We examined whether phosphorylation of S535 eIF2Bε is also reduced in an in vivo model of cardiac hypertrophy. To this end, we used the rat model of MI, where significant levels of hypertrophy in the remote area can be observed within 24 to 48 hours.^17,18 Tissues obtained from the remote area 24 hours after MI exhibited significantly lower levels of S535 eIF2Bε phosphorylation than those from sham animals (Figure 4).

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Figure 4. Adult male rats were subjected to coronary artery ligation (n=8) or sham operation (n=3). Left ventricular (LV) tissue homogenates from the remote region harvested at 24 hours after MI and those from comparable LV area in sham-operated rats were subjected to immunoblot analyses with anti-phospho-S535 eIF2Bε and total eIF2Bε antibody. *P<0.01 vs Control.

eIF2Bε Plays an Essential Role in Mediating Cardiac Hypertrophy, Although Its Ability to Mediate Cardiac Hypertrophy Negatively Correlates With the Level of S535 Phosphorylation

To elucidate the role of eIF2Bε in cardiac myocyte hypertrophy, we examined the effect of procedures that either increase or decrease S535 phosphorylation of eIF2Bε on changes in myocyte size. Treatment of myocytes with ISO or LiCl, which reduces S535 phosphorylation of eIF2Bε, increased myocyte size, whereas overexpression of GSK-3β, which increases S535 phosphorylation of eIF2Bε, reduced myocyte size at baseline and partially prevented ISO-induced increases in myocyte size (Figure 5A). Treatment with GSK-3β-inhibitor I increased cell size to a similar extent as LiCl, whereas addition of LiCl to ISO did not further increase myocyte size (Figure 5A). Propranolol completely abolished ISO-induced myocyte size, whereas prazosin did not prevent it, confirming that the hypertrophic effects of ISO are mediated primarily by β-ARs.

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Figure 5. Cardiac myocytes were transduced with Ad-GSK-3β, Ad-eIF2Bε, Ad-eIF2Bε-S535A, Ad-eIF2Bε-F259L, or Ad-LacZ (30 MOI). Forty-eight hours after transduction, myocytes were treated with or without LiCl (10 mmol/L), GSK-3β-inhibitor I (20 μmol/L), or ISO (10 μmol/L) for an additional 48 hours. In some experiments, myocytes were stimulated with ISO (10 μmol/L) in the presence of propranolol (10 μmol/L) or prazosin (10 μmol/L). Mean cell size of unstimulated myocytes was expressed as 100. A, Inhibition of GSK-3β by ISO or LiCl (48 hours) increased myocyte size. Overexpression of GSK-3β reduced myocyte size and partially prevented ISO-induced increases in myocyte size. B, Effects of overexpression of eIF2Bε (wild-type and mutants) on myocyte size. Overexpression of eIF2Bε-S535A induced significant myocyte hypertrophy. WT-eIF2Bε alone moderately increased myocyte size, whereas addition of ISO or LiCl to Ad-WT-eIF2Bε-transduced cells enhanced myocyte hypertrophy as much as overexpression of eIF2Bε-S535A. Overexpression of eIF2Bε-F259L reduced myocyte size and partially inhibited ISO-induced increases in myocyte size. *P<0.05 vs Control; #P<0.05 vs eIF2Bε-WT transduction. C, Overexpression of eIF2Bε-S535A abolished negative regulation of hypertrophy by GSK-3β. Myocytes were transduced with either Ad-LacZ alone, Ad-GSK-3β plus Ad-LacZ, or Ad-GSK-3β plus Ad-eIF2Bε-S535A. In all cases, the total amount of the adenovirus transduced was 60 MOI. Forty-eight hours after transduction, myocytes were stimulated with or without ISO (10 μmol/L) for 48 hours. Data shown in A through C were obtained at least from 6 experiments for each stimulus. Myocyte size was measured from at least 50 myocytes per stimulus in each experiment.

In order to further dissect the role of eIF2Bε and its phosphorylation, we examined the effect of wild-type or mutant eIF2Bε overexpression on cardiac hypertrophy at baseline and in response to GSK-3β inhibition. Overexpression of eIF2Bε-WT alone modestly increased myocyte size (+31±11%), whereas inhibition of GSK-3β in eIF2Bε-overexpressing myocytes by ISO (+84±33% versus eIF2Bε-WT only; P<0.05) or LiCl (+73±22% versus eIF2Bε-WT only; P<0.05) markedly enhanced the effect of eIF2Bε (Figures 5B and 6⇓A). Myocytes overexpressing eIF2Bε-S535A, which cannot be phosphorylated by GSK-3β, induced increases in myocyte size (+107±35%) as strongly as ISO (10 μmol/L) (+95±25%) (Figures 5B and 6⇓A). Thus, the ability of eIF2Bε to mediate cardiac hypertrophy positively correlated with the amount of S535-unphosphorylated eIF2Bε. Overexpression of eIF2Bε-S535A in combination with ISO treatment (48 hours) did not cause an additive effect on the increase in CS (+12±19%, P=NS versus eIF2Bε-S535A overexpression only; Figure 5B). By contrast, overexpression of a dominant-negative form of eIF2Bε (eIF2Bε-F259L) significantly reduced the myocyte size at baseline and in response to ISO (Figures 5B and 6⇓A), suggesting that eIF2Bε plays a critical role in mediating both baseline cardiac myocyte growth as well as β-adrenergic cardiac hypertrophy. Inhibitory effects of GSK-3β on baseline as well as ISO-induced cardiac hypertrophy were abolished in the presence of eIF2Bε-S535A (Figure 5C), suggesting that S535 phosphorylation of eIF2Bε plays an important role in mediating the inhibitory effects of GSK-3β on cardiac hypertrophy.

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Figure 6. A, Representative immunostaining of myocytes with Texas Red phalloidin. Myocytes with the following treatment are shown: Control (Cont), Ad-eIF2Bε-S535A transduction, ISO (10 μmol/L, 48 hours), LiCl (10 mmol/L, 48 hours), Ad-eIF2Bε-F259L transduction, eIF2Bε-WT transduction, and Ad-eIF2Bε-F259L transduction plus ISO (10 μmol/L, 48 hours). B, Greater magnifications depicting the highlighted areas from the panels in A to evaluate sarcomeric organization. Experiments were conducted 5 times. C, Representative immunostaining of myocytes with α-sarcomeric actinin. Texas Red-conjugated anti-mouse IgG antibody was used as secondary antibody. Myocytes with the following treatment are shown: Control (Cont), ISO (10 μmol/L, 48 hours), Ad-eIF2Bε-S535A transduction, and Ad-eIF2Bε-F259L transduction plus ISO (10 μmol/L, 48 hours). Slides shown are representative of 11 experiments in A and B and 6 experiments in C.

Similar data were obtained for measurement of the total myocyte protein content (data not shown). Using [³H] phenylalanine incorporation, we confirmed that GSK-3β significantly inhibited both basal and ISO-induced increases in the rate of protein synthesis (Figure 7A). Expression of eIF2Bε-S535A was sufficient to stimulate the rate of protein synthesis (Figure 7A), whereas increases in the rate of protein synthesis were significantly attenuated in the presence of eIF2Bε-F259L (Figure 7B), consistent with the results of the cell size analyses.

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Figure 7. Measurement of protein synthesis by [³H]phenylalanine incorporation. Cardiac myocytes were transduced with Ad-GSK-3β, Ad-eIF2Bε, Ad-eIF2Bε-S535A, Ad-eIF2Bε-F259L, or Ad-LacZ (30 MOI). Forty-eight hours after transduction, myocytes were incubated with 5 μCi/mL of [³H]phenylalanine. ISO stimulation was performed for 24 hours, whereas myocytes were incubated with [³H]phenylalanine for the last 12 hours. [³H]phenylalanine incorporation in unstimulated myocytes without adenovirus transduction was designated as 1. Each column represents mean of 4 wells. Similar results were obtained from 3 other experiments using different myocyte preparations.

eIF2Bε Partially Mediates Actin Organization, but Does Not Regulate Expression of ANF or αMHC in Response to β-AR Stimulation

We examined whether transduction of eIF2Bε-S535A influences other key features of cardiac hypertrophy, such as sarcomeric organization and expression of ANF in cardiac myocytes. Although expression of eIF2Bε-S535A did not change the proportion of myocytes with highly organized actin, it increased myocytes with modest levels of actin organization (Figures 6B and 8⇓A). Similar results were obtained when myocytes were treated with LiCl (Figures 6B and 8⇓A), which increases the levels of S535-unphosphorylated eIF2Bε. We also examined whether inhibition of endogenous eIF2Bε function affects these cardiac phenotypes in response to β-AR stimulation. Expression of eIF2Bε-F259L significantly blocked ISO-induced increases in myocytes with well-organized actin (Figures 6B and 8⇓A). In order to further evaluate the role of S535 eIF2Bε dephosphorylation in sarcomeric organization, α-sarcomeric actinin staining was conducted (Figure 6C). ISO caused a clear striated pattern of α-sarcomeric actinin staining, indicating induction of well-organized sarcomere. Expression of eIF2Bε-S535A modestly increased α-sarcomeric actinin positive staining (in both fibrous and punctuated patterns) without striated patterns, suggesting that eIF2Bε-S535A partially induces formation of premyofibril-like structures,¹⁹ but it is not sufficient to cause mature sarcomere. eIF2Bε-F259L significantly, but not completely, inhibited ISO-induced increases in myocytes with well-organized sarcomere. These results suggest that eIF2Bε is partially required for ISO-induced sarcomere organization.

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Figure 8. A, Semiquantitative analyses of sarcomeric organization. eIF2Bε-S535A and LiCl (10 mmol/L, 48 hours) induced modest levels of actin organization. eIF2Bε-F259L partially blocked ISO-induced actin organization. Eight to 12 different slides obtained from 4 experiments were analyzed for each condition (n=116 to 225 myocytes for each condition). *P<0.05 vs Ad-LacZ; #P<0.05 vs Ad-LacZ+Iso. B, Cardiac myocytes were transduced with Ad-GSK-3β, Ad-eIF2Bε, Ad-eIF2Bε-S535A, Ad-eIF2Bε-F259L, or Ad-LacZ (30 MOI). Forty-eight hours after transduction, myocytes were treated with or without LiCl (10 mmol/L) or ISO (10 μmol/L) for an additional 24 hours. Total RNA was subjected to RT-PCR. Level of ANF expression was normalized by that of GAPDH and was expressed as 1 in control virus-transduced myocytes. Graph shows the mean of 6 individual experiments. *P<0.05 vs Control. C, Cardiac myocytes were transduced with Ad-eIF2Bε-S535A, Ad-eIF2Bε-F259L, or Ad-LacZ (30 MOI). Forty-eight hours after transduction, myocytes were treated with or without ISO (10 μmol/L) for an additional 24 hours. Immunostaining with anti-ANF antibody and anti-sarcomeric myosin antibody (MF20) was performed. Representative microscope pictures of the double staining are shown. Experiments were conducted 3 times.

Expression of eIF2Bε-S535A did not affect mRNA expression of ANF at baseline (Figure 8B). Inhibition of endogenous eIF2Bε by eIF2Bε-F259L also failed to inhibit ISO-induced increases in ANF mRNA expression (Figure 8B). Stimulation of cardiac myocyte with ISO (10 μmol/L for 48 hours) increased typical perinuclear staining of ANF. By contrast, eIF2Bε-S535A failed to induce the perinuclear staining of ANF and eIF2Bε-F259L did not inhibit ISO-induced increases in the ANF staining (Figure 8C). Expression of eIF2Bε-S535A also did not significantly change expression of αMHC gene, a sarcomeric protein (online Figure 1, in the online data supplement at http://circres.ahajournals.org). These results are consistent with the notion that inhibition of GSK-3β and subsequent dephosphorylation of eIF2Bε at S535 induces a modest degree of actin reorganization, but does not affect ANF or αMHC expression. Furthermore, activity of eIF2Bε is required for full sarcomeric reorganization but not for ANF expression in response to β-AR stimulation.

eIF2Bε Is a Regulator of Protein Translation Initiation in Cardiac Myocytes

Protein synthesis is a complex process involving three essential steps: initiation, elongation, and termination.²⁰ One of the critical steps controlling initiation of protein translation is the binding of eukaryotic translation initiation factor 2 (eIF2) to the activated initiator tRNA (met-tRNAmet) and subsequent formation of a ternary complex that binds to the 40S ribosomal subunit. This process requires activities of eIF2Bε in order to stimulate the GDP/GTP exchange reaction of eIF2. GSK-3β phosphorylates human eIF2Bε at S540 (S535 in the rat sequence) and inactivates it.^15,21 Thus, eIF2Bε is an attractive candidate molecule mediating regulation of cardiac hypertrophy by GSK-3β. Our results indicate that GSK-3β is the predominant kinase regulating the phosphorylation status of S535 eIF2Bε in cardiac myocytes, keeping S535 eIF2Bε phosphorylated at baseline, and that overexpression of eIF2Bε S535A is sufficient to stimulate cardiac hypertrophy. Furthermore, expression of eIF2Bε-F259L significantly inhibited β-adrenergic hypertrophy, suggesting that regulation of eIF2Bε is an essential step for β-adrenergic cardiac hypertrophy. Taken together, our results strongly suggest that inhibition of protein translation initiation through phosphorylation of S535 eIF2Bε plays an important role in mediating antihypertrophic effects of GSK-3β, and that its reversal by β-AR stimulation in part mediates increases in protein synthesis in cardiac myocytes. It should be noted that ISO caused a greater increase in cell size than LiCl or GSK-3β inhibitor I. Thus, additional mechanisms are also involved in β-adrenergic hypertrophy. Although the fact that ISO failed to significantly enhance the effect of eIF2Bε-S535A on hypertrophy at the first view contradicts from this notion, it may be explained by the possibility that the capacity of myocyte to undergo hypertrophy is almost saturated when eIF2Bε-S535A is overexpressed in our experimental conditions.

GSK-3β Regulates S535 Phosphorylation of eIF2Bε in Cardiac Myocytes

eIF2Bε is phosphorylated by various kinases, including GSK-3β and casein kinases 1 and 2 (CK1 and CK2).²² Whereas GSK-3β has been shown to be a predominant kinase regulating phosphorylation of S535 of eIF2Bε, which renders eIF2Bε inactive, CK1 and CK2 stimulate the activity of eIF2Bε through phosphorylation at sites distinct from S535.²² Our results using anti-phospho-S535-specific antibody indicate that phosphorylation of S535 eIF2Bε is significantly reduced by GSK-3β inhibitors, including LiCl and GSK-3β-inhibitor I. This result clearly indicates that GSK-3β is a major kinase regulating S535 phosphorylation of endogenous eIF2Bε in cardiac myocytes. It has been shown that insulin stimulation, which presumably inhibits the kinase activity of GSK-3β, fails to change eIF2Bε phosphorylation in the heart.²³ Although this result seems contradictory to our observation, changes in phosphorylation of S535 were not specifically determined in that study.²³ It is possible that changes in S535 phosphorylation were masked by phosphorylation of other sites by CK1 and CK2 in this previous report.

A recent study showed that reduced phosphorylation of eIF2Bε by a GSK-3β inhibitor was not sufficient to increase activity of eIF2Bε in CHO cells.²⁴ In this case, concomitant stimulation of eIF2Bε by other kinases may be required to increase the activity of eIF2Bε. At present, we do not exclude the role of other eIF2Bε kinases, which presumably phosphorylate sites other than S535 and activate eIF2Bε, in cardiac myocyte protein synthesis. It should be noted, however, that overexpression of eIF2Bε-WT only modestly increased myocyte size and protein synthesis probably because some of the overexpressed eIF2Bε is rendered inactive by phosphorylation through GSK-3β (Figure 1C). In fact, our results indicate that phosphorylation of S535 is increased in myocytes overexpressing eIF2Bε. This result suggests that basal phosphorylation of eIF2Bε by other kinases alone, even if it is present, may not overcome the effect of S535 phosphorylation by GSK-3β. This notion is consistent with the observation that overexpression of eIF2Bε-S535A more strongly promotes myocyte growth than eIF2Bε-WT. Thus, in conjunction with the aforementioned results showing that GSK-3β is the predominant kinase phosphorylating S535 eIF2Bε, our results are consistent with the notion that S535 phosphorylation of eIF2Bε by GSK-3β critically regulates cardiac protein synthesis.

eIF2Bε Differentially Regulates Various Hypertrophic Phenotypes in Response to β-Adrenergic Receptor Stimulation

Although an increase in protein synthesis is one of the most important features of cardiac hypertrophy, it is also accompanied by other phenotypic changes, including activation of the fetal gene program and cytoskeletal reorganization. Our results suggest that eIF2Bε differentially regulates these other aspects of hypertrophy. Overexpression of eIF2Bε-S535A strongly increased protein synthesis, protein content, and myocyte size, but it did not significantly induce ANF expression. Also, inhibition of eIF2Bε failed to inhibit ISO-induced upregulation of ANF. This suggests that induction of ANF expression by ISO is mediated through mechanisms distinct from regulation of protein synthesis. In this regard, we have previously shown that phosphorylation and inhibition of GATA4 plays an important role in mediating inhibition of ANF expression by GSK-3β.⁶

The effect of eIF2Bε on cytoskeletal organization has not been reported previously. Overexpression of eIF2Bε-S535A partially induced actin and sarcomere reorganization in cardiac myocytes and dominant-negative eIF2Bε reduced the proportion of myocytes with well-organized actin and sarcomere in ISO stimulated myocytes. These results suggest that dephosphorylation of S535 eIF2Bε may contribute to ISO-induced cytoskeletal reorganization, and that activity of eIF2Bε is required for full sarcomeric reorganization on β-AR stimulation. These findings are consistent with the notion that each phenotype in β-adrenergic cardiac hypertrophy is mediated by distinct mechanisms. The predominant function of eIF2Bε in β-adrenergic hypertrophy is regulation of cell size through protein synthesis, whereas eIF2Bε also contributes to cytoskeletal reorganization, but not ANF expression.