INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE ADULT MYOCARDIUM has an intrinsic ability to respond to increased load with compensatory hypertrophic growth (4). The primary characteristic of hypertrophy is an overall increase in mass that results from accelerating the steady-state rate of protein synthesis in the cardiac muscle cell or cardiocyte (28). The initial mechanism for accelerating the rate of protein synthesis in response to increased load is via translational efficiency, defined as the rate of protein synthesis divided by RNA content (9, 27, 28). An increase in efficiency involves altering the activity of translational initiation factors (33). Our previous work (40) indicated that the active tension component of load in the adult myocardium increases the activity of translation initiation factor 4E (eIF-4E) as measured by its phosphorylation. eIF-4E functions as part of the eIF-4F complex consisting of three basic components: the cap-binding protein eIF-4E, which binds to the 7-methylguanosine (m⁷Gppp) cap of mRNA; eIF-4A, which functions as an ATP-dependent helicase to unwind mRNA secondary structure; and eIF-4G, which functions basically as a binding protein for coordinating the assembly of the eIF-4F complex (reviewed in Ref. 33). In the process of translational initiation, binding of eIF-4E to the m⁷Gppp cap of mRNA is required for the eIF-4F complex to perform its functions of unwinding mRNA secondary structure and facilitating binding of mRNA to the 40 S ribosomal subunit. Recent studies suggest that binding to capped mRNAs is more efficient when eIF-4E is assembled into the eIF-4F complex (7).

There are two primary mechanisms for regulating the specific activity of eIF-4E. The first mechanism is phosphorylation of eIF-4E on Ser-209, which increases its binding affinity for the m⁷Gppp caps of mRNA and stabilizes the eIF-4F complex (3, 12, 26). An increase in eIF-4E phosphorylation has been shown to occur in response to several types of anabolic stimuli including growth factors, hormones, and phorbol esters (29). The second mechanism is through 4E binding proteins (4E-BPs) 1, 2, and 3 (19, 34-36), which function as translational repressors by competing with eIF-4G for a common binding site on eIF-4E (6, 23). The activity of 4E-BPs is controlled by phosphorylation. The nonphosphorylated isoforms of 4E-BPs bind to eIF-4E with high affinity and prevent it from binding to eIF-4G to form the translationally active eIF-4F complex (23, 34). Conversely, the phosphorylation of 4E-BPs reduces the binding affinity for eIF-4E and thereby relieves translational repression because eIF-4E is able to bind to eIF-4G. Insulin and several other growth factors cause marked increases in 4E-BP phosphorylation and dissociation of 4E-BPs from eIF-4E, indicating the utility of this mechanism in several cell types for increasing eIF-4E activity in response to growth stimuli (5, 20, 21, 24).

In the adult cardiocyte, increased load in the form of electrically stimulated contraction resulted in a corresponding increase in eIF-4E phosphorylation (40). Phosphorylation of eIF-4E was dependent on active tension development because it did not occur when myofibrillar cross-bridge cycling and cardiocyte shortening were blocked during electrical stimulation with the chemical agent 2,3-butanedione monoxime (BDM). Prior studies (10) showed that the rate of cardiocyte protein synthesis was accelerated in response to electrically stimulated contraction by acutely increasing translational efficiency, and this effect was dependent on the generation of active tension. Taken together, these findings suggested that the active tension component of load utilizes eIF-4E phosphorylation as a downstream mechanism for increasing translational efficiency in the adult cardiocyte. In this study, we hypothesized that there is a direct correlation between eIF-4E phosphorylation and increased assembly of the translationally active eIF-4F complex. This hypothesis was tested by measuring the relative amount of eIF-4G associated with eIF-4E in contracting compared with quiescent cardiocytes. Furthermore, we tested whether changes in the activity of 4E-BPs have a role in regulating eIF-4F complex formation in response to electrically stimulated contraction. This was determined by measuring the phosphorylation state of 4E-BPs and the extent to which nonphosphorylated 4E-BPs are bound to the eIF-4E in contracting and quiescent cardiocytes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Electrical stimulation of cardiocyte contraction. Adult feline cardiocytes were isolated for primary cell culture as described previously (10). The cardiocytes were plated onto four-well culture trays (Nunc) that were coated with laminin at an initial plating density of 2 × 10⁵ cardiocytes per well. The dimensions of each well were 2.5 × 6.5 cm. After an overnight incubation, the cultures were maintained in serum-free medium as described by Volz et al. (38). The cardiocytes were stimulated to contract by delivering 130-V electrical pulses of alternating polarity through the culture medium via carbon electrodes using a custom-built electrical stimulator (13). The current between electrodes was 50-55 mA. Cardiocyte contraction was set at a frequency of 1 Hz and a pulse duration of 5 ms. Nonstimulated cardiocytes were quiescent and used as controls.

Isolation of eIF-4E from cardiocytes. Cardiocytes were rinsed twice with ice-cold Hanks' balanced salt solution and scraped in lysis cell buffer (LCB) [20 mmol/l HEPES, pH 7.5, 100 mmol/l KCl, 0.2 mmol/l EDTA, 7 mmol/l -mercaptoethanol, 10% (vol/vol) glycerol, 0.5% (vol/vol) Triton X-100, 80 mmol/l 2-glycerophosphate, 50 mmol/l NaF, 0.2 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l benzamidine, 0.5 mmol/l sodium orthovanadate, 1 µmol/l okadaic acid, 1 µmol/l microcystin LR, 25 µg/ml leupeptin, 2 U/ml aprotinin, and 20 µg/ml chymostatin]. Two trays of cardiocytes were used for each experimental group. The material was homogenized by means of a Dounce homogenizer and centrifuged at 12,000 g for 10 min. Twenty microliters of washed 7-methyl-GTP-Sepharose 4B (m⁷GTP-Sepharose; Pharmacia) were added to the supernatant and incubated for 1 h at 4°C. The m⁷GTP-Sepharose was pelleted, washed three times with LCB buffer, resuspended in 50 µl of LCB buffer, and mixed with an equal volume of 2× loading buffer [100 mmol/l Tris, pH 6.8, 200 mmol/l dithiothreitol, 4% wt/vol SDS, 20% vol/vol glycerol, and 0.2% wt/vol bromphenol blue]. The samples were boiled for 5 min and subjected to SDS-PAGE using 15% polyacrylamide gels. Western blot analysis was carried out with eIF-4E, eIF-4G, or 4E-BP antibodies. The eIF-4G antiserum was provided by Dr. Simon Morley (31). Secondary antibody detection was done using the Renaissance chemiluminescence detection system (DuPont). The optical density of the signals was quantified by digital image analysis.

Immunoprecipitation of eIF-4E from cardiocytes. eIF-4E was immunoprecipitated from cardiocytes by the method of Kimball et al. with modifications (16). Cardiocytes were scraped in buffer A [20 mmol/l HEPES, pH 7.4, 100 mmol/l KCl, 2 mmol/l EGTA, 0.2 mmol/l EDTA, 1 mmol/l dithiothreitol, 50 mmol/l 2-glycerophosphate, 50 mmol/l NaF, 0.1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l benzamidine, 0.5 mmol/l sodium orthovanadate, and 0.5% (vol/vol) Triton X-100] and homogenized using a Dounce homogenizer. The homogenates were centrifuged at 12,000 g for 10 min, and eIF-4E was immunoprecipitated from the supernatant using a monoclonal eIF-4E antibody (16). The immune complexes were recovered using Biomag immunoglobulin G beads and subjected to Western blotting using 4E-BP and eIF-4E antibodies.

Isolation of 4E-BPs. Cardiocytes were scraped in LCB buffer, homogenized, and centrifuged as described in Isolation of eIF-4E from cardiocytes. The supernatant was boiled for 7 min, cooled at room temperature, and centrifuged at 12,000 g for 10 min. The supernatant was concentrated with the use of a Centricon 3 and subjected to SDS-PAGE using 17.5% polyacrylamide gels, followed by Western blotting with 4E-BP antibody. Secondary antibody detection was carried out using the Renaissance chemiluminescence detection system. The optical density of the signals was quantified by digital image analysis.

Construction of feline 4E-BP fusion protein. To produce an antibody against feline isoforms of 4E-BP, a histidine-tagged fusion protein was made with the use of a partial cDNA clone of 4E-BP1 obtained from a feline cardiocyte cDNA library. A cDNA clone encompassing nucleotides 197-385 of the rat 4E-BP1 sequence was produced using PCR (8). The open reading frame extended from Thr-49 to Ser-111. The cDNA was subcloned into a pQE-31 expression vector (Qiagen) to generate a 6×His-tagged fusion protein. After expression, the fusion protein was affinity purified with the Ni²⁺-NTA resin system (Qiagen) and used for production of polyclonal antibodies against feline 4E-BP.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To determine whether eIF-4F complex formation was increased by load in adult feline cardiocytes, we measured the relative amount of eIF-4G associated with eIF-4E in response to electrically stimulated contraction. eIF-4E was isolated from cardiocyte homogenates by binding to m⁷GTP-Sepharose, followed by Western blotting with both eIF-4G and eIF-4E antibodies. Figure 1A shows that the amount of eIF-4G bound to eIF-4E increased after 4 h of electrically stimulated contraction at 1 Hz compared with quiescent controls. To determine whether the increase in response to contraction was dependent on the generation of active tension, the cardiocytes were electrically stimulated in the presence of 7.5 mmol/l BDM, a chemical agent that inhibits the extent and velocity of sarcomere shortening by 70% and the extent of cardiocyte shortening by 90% (10). BDM has no significant effect on calcium transients during electrical stimulation, in effect uncoupling membrane depolarization and calcium transients from the generation of tension and shortening of the cardiocyte. The increase in binding of eIF-4G to eIF-4E was completely blocked by BDM, indicating that the increase in eIF-4F complex formation was linked to active tension development. Figure 1A also shows that the recovery of eIF-4E after purification with m⁷GTP-Sepharose was the same between treatment groups. Figure 1B contains summary data for five experiments, and these data show a positive correlation between the active tension component of load and eIF-4F complex formation.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effects of contraction on eIF-4F complex formation. A: representative Western blot from each treatment group. Con, quiescent control; BDM, 7.5 mmol/l 2,3-butanedione monoxime; Stim, electrically stimulated contraction at 1 Hz. B: summary data of 5 experiments. Ratio of eIF-4G to eIF-4E (eIF-4G/eIF-4E) for each experimental group was calculated as a percentage of quiescent control value. Values are means ± SE. * Significantly greater than control as determined by ANOVA followed by a Dunnett test (P < 0.05).

In the experiments represented in Fig. 2, the effects of other anabolic agents on eIF-4F complex formation were determined. Treatment with either insulin or phorbol ester over 4 h increased eIF-4F complex formation compared with controls. These increases were not blocked by treatment with BDM, indicating that increased eIF-4F complex formation was not dependent on active tension development. These findings mirrored previous results (40) showing that increased eIF-4E phosphorylation occurred in response to either insulin or phorbol ester treatment of adult cardiocytes and that both occurred independently of active tension development.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Effects of insulin or phorbol ester on eIF-4F complex formation. A: representative Western blot from each treatment group. Ins, 0.1 µmol/l insulin; BDM, 7.5 mmol/l BDM; PMA, 0.1 µmol/l phorbol 12-myristate 13-acetate. All treatments were carried out for 4 h. B: summary data of 4 experiments. eIF-4G/eIF-4E for each experimental group was calculated as a percentage of quiescent control value. Values are means ± SE. * Significantly greater than control as determined by ANOVA followed by a Dunnett test (P < 0.05).

To determine the role of 4E-BPs in regulating eIF-4F complex formation, a rabbit polyclonal antibody was produced against a fusion protein containing the feline form of 4E-BP1. The antibody detected two species of 4E-BP in quiescent cardiocytes (Fig. 3A). The slower-migrating species consisted of several isoforms in the range between 18 and 20 kDa, consistent with the mobility of 4E-BP1 (20). Quiescent cardiocytes contained predominantly the phosphorylated 4E-BP1 -isoform and a small amount of the highly phosphorylated 4E-BP1 -isoform. The 4E-BP1 -isoform was present at very low levels and was not detectable in cardiocyte homogenates. As a positive control, quiescent cardiocytes were treated with insulin to activate the signaling pathway leading to 4E-BP1 phosphorylation by a kinase referred to as mTOR (mammalian target of rapamycin) (2). Insulin caused a shift of virtually the entire pool of 4E-BP1- into the highly phosphorylated 4E-BP1 -isoform. The species of 4E-BP with faster mobility that were detected by the antibody had several isoforms in the range between 15 and 17 kDa. This protein, designated 4E-BP2, was similar to 4E-BP1 because the -isoform was not detectable in cell homogenates and insulin caused a shift into the highly phosphorylated -isoform. The Western blot in Fig. 3A (right) shows that the same results were obtained with the use of an antibody produced against recombinant 4E-BP1 provided by Lin and Lawrence (22), confirming the specificity of the feline 4E-BP antibody.

View larger version (60K): [in this window] [in a new window]   Fig. 3.   Specificity of feline eIF-4E binding protein (4E-BP) antibody. A: Western blot of total cell homogenates. Ins, 1 µmol/l insulin for 4 h. Left: blot employed antibody produced against a fusion protein of feline 4E-BP. Right: blot employed an antibody provided by Lin and Lawrence (22). 4E-BP1 and 4E-BP2, eIF-4E binding proteins 1 and 2, with shifts from - to -isoforms. B: Western blot using feline 4E-BP antibody for total homogenate, m7GTP-Sepharose-bound fraction, and unbound fraction. 1 and 2, 1 and 2, and 1 and 2 represent isoforms of 4E-BP1 and 4E-BP2, respectively. C: Western blot after immunoprecipitation with a monoclonal eIF-4E antibody. Left: blot shows feline 4E-BP antibody. Right: same blot reprobed with eIF-4E antibody. D: Western blot of either total homogenates or m7GTP-Sepharose-bound protein using 4E-BP antibody () or 4E-BP antibody preincubated for 1 h with 4E-BP fusion protein (+). Molecular weights of standards are indicated at left of each blot.

In experiments represented in Fig. 3B, the ability of 4E-BP isoforms to bind eIF-4E was determined. eIF-4E was purified from cardiocyte homogenates with m⁷GTP-Sepharose, followed by Western blotting. 4E-BP isoforms from either control or insulin-treated cardiocytes were compared in both the m⁷GTP-Sepharose-bound and unbound fractions. Also included on the blot were the total cell homogenates. A single 4E-BP1 isoform bound to m⁷GTP-Sepharose in the quiescent control, and it migrated faster than the 4E-BP1 -isoform. This represents the nonphosphorylated 4E-BP1 -isoform that has been shown to bind eIF-4E with high affinity. The phosphorylated 4E-BP1 - and -isoforms were recovered in the unbound fraction. Approximately one-half of the m⁷GTP-Sepharose-bound fraction and one-fifth of the total volume of the unbound fraction were run on the gel. These findings indicate that a large percentage of 4E-BP1 was phosphorylated in quiescent cardiocytes and interacted weakly, if at all, with eIF-4E to regulate its activity. Similar results were obtained for 4E-BP2. A faster-migrating -isoform was purified on m⁷GTP-Sepharose, indicating that it was bound tightly to eIF-4E. The - and -isoforms of this species were recovered in the unbound fraction and therefore did not bind to eIF-4E. Figure 3B also shows that the amount of the nonphosphorylated 4E-BP1 -isoform bound to m⁷GTP-Sepharose was markedly reduced in response to insulin treatment. The results obtained with the 4E-BP2 species were nearly identical.

In experiments represented in Fig. 3C, the binding of 4E-BP to eIF-4E was verified by immunoprecipitation using a monoclonal eIF-4E antibody (16). Only the -isoforms of 4E-BP bound to eIF-4E, a result identical to that obtained when eIF-4E was recovered using m⁷GTP-Sepharose (compare Fig. 3, B and C). Consistent with these results, there was a substantial reduction in the amount of 4E-BP that coimmunoprecipitated with eIF-4E when cardiocytes were treated with insulin.

Figure 3D shows data from a control experiment to further establish the specificity of the feline 4E-BP antibody. Western blots were loaded with either total cell homogenates or m⁷GTP-Sepharose-bound protein as indicated. The antibody reacted with some nonspecific proteins in total homogenates. However, the bands representing 4E-BPs were not detected when the antibody was preincubated with 4E-BP fusion protein, confirming the specificity of the feline antibody for 4E-BP isoforms in cardiocyte homogenates. Furthermore, the isoforms migrated at molecular weights consistent for 4E-BP1 and 4E-BP2.

In experiments represented in Fig. 4, changes in the 4E-BP1 isoform pattern were measured in response to electrically stimulated contraction to determine whether load increases 4E-BP activity. 4E-BP1- was the predominant isoform in quiescent controls, but there was a small amount of the -isoform. The -isoform was barely detectable in total homogenates. Electrically stimulated contraction for 4 h shifted a portion of the 4E-BP1- pool into 4E-BP1 -isoform. The same results were obtained with 4E-BP2, suggesting that both species of 4E-BP were phosphorylated by the same kinase pathway. The shift into the -isoform was blocked when rapamycin was added. As a positive control, cardiocytes were treated for 4 h with insulin to shift both species 4E-BP into the highly phosphorylated -isoform. This shift was blocked in the presence of rapamycin.

View larger version (71K): [in this window] [in a new window]   Fig. 4.   Effects of insulin (A) or electrically stimulated contraction (B) on 4E-BP isoform shifts in adult feline cardiocytes. Western blots were performed using total cell homogenates. Rapa, 100 ng/ml rapamycin; Ins, 1 µmol/l insulin. All treatments were carried out for 4 h.

These experiments were done three times, and the optical densities of the - and -isoforms for each species of 4E-BP were quantified by digital image analysis. The -isoform comprised 92 ± 4% (mean ± SE) of the 4E-BP1 pool in quiescent controls. The percentage of 4E-BP1- was reduced to 64 ± 5% in electrically stimulated cardiocytes, indicative of a significant shift into the highly phosphorylated -isoform as shown in Fig. 4 (P < 0.05 vs. control). In all three experiments, insulin treatment shifted the entire pool of 4E-BP1- into the -isoform (P < 0.005 vs. control). Rapamycin blocked the shift into the highly phosphorylated -isoform in response to either insulin or electrically stimulated contraction. The results obtained for 4E-BP2 were essentially identical. The percentage of 4E-BP2- was 94 ± 6% in quiescent controls. Contraction significantly reduced 4E-BP2- to 63 ± 11% (P < 0.05 vs. control), whereas insulin caused a 100% shift into 4E-BP2- in all three experiments (P < 0.005 vs. control). The shift into 4E-BP2- was also blocked by rapamycin treatment.

The recovery of the -isoforms of 4E-BP1 and 4E-BP2 on m⁷GTP-Sepharose indicated that a small pool of 4E-BP was bound tightly to eIF-4E. We therefore examined the effects of either insulin or electrically stimulated contraction on the binding of both -isoforms of 4E-BP to eIF-4E. Figure 5 shows that neither 4E-BP1- nor 4E-BP2- bound to eIF-4E after insulin treatment. However, the binding to eIF-4E was restored when rapamycin was added simultaneously with insulin. Rapamycin treatment alone had no effect on 4E-BP binding to eIF-4E. Figure 5 further shows that electrically stimulated contraction did not produce a decrease in the amount of either 4E-BP1- or 4E-BP2- bound to eIF-4E compared with quiescent controls. This experiment was repeated three times, and the same results were obtained.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Effects of insulin or electrically stimulated contraction on 4E-BP binding to eIF-4E. eIF-4E was purified on m7GTP-Sepharose and used for Western blotting with 4E-BP and eIF-4E antibodies. Ins, 1 µmol/l insulin; Rapa, 100 ng/ml rapamycin. All treatments were carried out for 4 h. Similar results were obtained in 2 other experiments.

In experiments represented in Fig. 6, we tested whether increased eIF-4F complex formation was associated with phosphorylation of the 4E-BP1 or 4E-BP2 -isoform. Both insulin and electrically stimulated contraction significantly increased the ratio of eIF-4G to eIF-4E (eIF-4G/eIF-4E) in the absence of rapamycin, consistent with the results in Figs. 1 and 2. Rapamycin had different effects on the ability of either insulin or electrically stimulated contraction to increase eIF-4F complex formation. The increase in eIF-4G/eIF-4E in response to insulin was not affected by inhibiting 4E-BP phosphorylation with rapamycin. In contrast, eIF-4G/eIF-4E was significantly lower when cardiocytes were electrically stimulated to contract in the presence of rapamycin. This effect on eIF-4F complex formation occurred even though electrically stimulated cardiocytes had the same amount of the 4E-BP -isoforms bound to eIF-4E in the presence or absence of rapamycin. These data suggest that the inhibitory effects of rapamycin on contraction-induced eIF-4F complex formation occur via a mechanism other than 4E-BP phosphorylation. One possible mechanism is via FK506-binding protein (FKBP-12), which is tightly coupled with the ryanodine receptor/calcium-release channel in the cardiocyte and regulates its activity (42). Rapamycin binds to FKBP-12 and blocks FKBP-ryanodine receptor interactions (17). To test the effects of rapamycin on contractility, the extent and velocity of sarcomere shortening were measured in freshly isolated, nonadherent cardiocytes (Fig. 7). The cardiocytes were incubated for 2 h in medium containing rapamycin and then electrically stimulated to contract at 0.25 Hz. Sarcomere motion was recorded in individual cardiocytes after 20 contractions using a laser-diffraction method (14). The extent of sarcomere shortening was significantly decreased by 41%, and the velocity of sarcomere shortening was decreased by 51%. These data suggest that rapamycin affected eIF-4F complex formation by decreasing contractility rather than blocking the phosphorylation of 4E-BP.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Effects of rapamycin on eIF-4F complex formation. A: representative Western blot from each treatment group. Ins, 1 µmol/l insulin; Rapa, 100 ng/ml rapamycin. All treatments were carried out for 4 h. Source of minor band that migrates faster than eIF-4G is unknown, but it may represent some degradation of eIF-4G on gel. B: summary data of 6 experiments. eIF-4G/eIF-4E for each experimental group was calculated as a percentage of quiescent control value. Values are means ± SE. * Significantly greater than control as determined by ANOVA followed by a Dunnett test (P < 0.05).

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Effects of rapamycin on contractile function. Freshly isolated cardiocytes were incubated in medium containing 1 µg/ml rapamycin for 2 h. Cardiocytes were electrically stimulated to contract at 0.25 Hz, and 6-8 recordings (average) were taken for each cardiocyte. Values are means ± SE (n = 5 cells). * P < 0.0001 vs. control as determined by unpaired t-test.

To confirm that increases in eIF-4G/eIF-4E reflected significant shifts in the fraction of the total eIF-4G pool bound to eIF-4E, the percentage of eIF-4G in the m⁷GTP-Sepharose-bound fraction was measured (Fig. 8). Cardiocyte homogenates were incubated with m⁷GTP-Sepharose to bind eIF-4E, and the unbound material was retained after the beads were pelleted by centrifugation. After the m⁷GTP-Sepharose was washed, the bound fractions were eluted with 100 µM m⁷GTP and adjusted to the same volume as the unbound fractions. Equivalent volumes were used for Western blotting with eIF-4G and eIF-4E antibodies. As shown by the summary data in Fig. 8B, 34% of the eIF-4G pool in the quiescent control was recovered by m⁷GTP-Sepharose and was therefore bound to eIF-4E. This amount was unchanged in quiescent cardiocytes treated for 4 h with rapamycin. Insulin and electrically stimulated contraction increased the percentage of eIF-4G in the m⁷GTP-Sepharose-bound fraction to 74 and 57%, respectively. Rapamycin did not significantly affect the percentage of eIF-4G bound to eIF-4E in insulin-treated cardiocytes, but it did partially block the shift in electrically stimulated cardiocytes (41%). These data are consistent with the eIF-4G/eIF-4E data (see Fig. 6). Figure 8A also shows that eIF-4E was recovered in the m⁷GTP-Sepharose-bound fraction but was not detected on Western blots of the unbound fractions. These findings suggest that eIF-4G competes for a limited pool of eIF-4E in adult cardiocytes.

View larger version (49K): [in this window] [in a new window]   Fig. 8.   Binding of eIF-4G to eIF-4E in cardiocyte homogenates. A: representative Western blot from each treatment group. Rapa, 100 ng/ml rapamycin; Ins, 0.1 µmol/l insulin. All treatments were carried out for 4 h. There was no eIF-4E detected on Western blots of unbound fraction. B: summary data of 3 experiments. Amount of eIF-4G in bound fraction was calculated as a percentage of total amount of eIF-4G (bound + unbound). Values are means ± SE. * Significantly greater than control as determined by ANOVA followed by a Dunnett test (P < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our previous work indicated that eIF-4E phosphorylation is a specific anabolic end point for increasing translational initiation in response to mechanical loading of the adult cardiocyte (40). This conclusion was based on studies showing that phosphorylation of eIF-4E increases the binding affinity for the m⁷Gppp cap present on virtually all mRNA molecules (26). Two lines of evidence in the present study indicate that eIF-4F complex formation is linked to eIF-4E phosphorylation in the adult cardiocyte. First, eIF-4F complex formation increased in response to several known anabolic stimuli for adult cardiocytes, all of which caused a corresponding increase in eIF-4E phosphorylation (40). Furthermore, the magnitude of the increase that occurred in response to each individual anabolic stimulus was similar to the extent of eIF-4E phosphorylation. Second, the increase in eIF-4F complex formation produced in response to electrically stimulated contraction was dependent on the generation of active tension development. Phosphorylation of eIF-4E was also dependent on active tension development. Thus, as a result of eIF-4E phosphorylation, translational efficiency can be increased via a mechanism whereby more eIF-4F complex is positioned to unwind mRNA secondary structure and to bind mRNA to the 40 S ribosomal subunit. It is also possible that an increase in eIF-4F complex formation may trigger hypertrophic growth by selective translation of mRNAs encoding proteins that are critically involved in regulating growth, because many of these mRNAs require the helicase activity of the eIF-4F complex to melt excessive secondary structure in the 5'-untranslated region (24).

By increasing translational efficiency, the rate of protein synthesis in the adult cardiocyte can be accelerated without increasing transcription or altering gene expression. Increased efficiency accelerated the rate of protein synthesis when adult cardiocytes in vitro were subjected to acute increases in load in the form of either electrically stimulated contraction to generate active tension or stretch on a deformable membrane to increase passive strain (10, 11, 15). A similar responsiveness to load has been observed in vivo; rapid increases in translational efficiency occurred in response to pressure overload of adult myocardium (9, 25, 32). The positive correlation between load and eIF-4F complex formation provides a mechanism for increasing translational efficiency in the cardiocyte because the eIF-4F complex controls a rate-limiting step for peptide chain initiation, namely, the binding of the 43 S preinitiation complex to mRNA. Precisely how load triggers an increase in the eIF-4F complex has not been determined, but there are two leading possibilities. The first possibility is that the load-dependent increase in eIF-4E phosphorylation enhances cap binding and subsequently promotes the assembly of the eIF-4F complex on mRNA. This mechanism is dependent on eIF-4E binding to the mRNA cap as a free subunit before the assembly of the eIF-4F complex (33). However, this possibility is unlikely because a recent study has shown that the binding of eIF-4E to the cap structure in vitro requires that eIF-4E is bound to eIF-4G, providing strong evidence that prior assembly of the eIF-4F complex is required for cap recognition and binding (7). The second possibility is that increased load somehow stabilizes the eIF-4F complex in parallel with eIF-4E phosphorylation. Approximately 85% of eIF-4E found in the eIF-4F complex is phosphorylated, and the eIF-4F complex is stabilized by eIF-4E phosphorylation (3, 18). Furthermore, studies have shown that the reactions governing the phosphorylation and dephosphorylation of eIF-4E in vitro are more efficient when eIF-4E is part of the eIF-4F complex (37).

Another mechanism for regulating eIF-4F complex formation is via the activity of 4E-BPs, which compete with eIF-4G for a common binding site on eIF-4E (6, 23). We found that >90% of both the 4E-BP1 and 4E-BP2 pools in quiescent cardiocytes consisted of the -isoform and that the phosphorylated - and -isoforms of 4E-BP did not bind to eIF-4E. The -isoforms were not detectable in total homogenates of adult cardiocytes. Thus the -isoforms apparently comprise a very small percentage of the total 4E-BP pool, consistent with findings reported for the 4E-BP1 -isoform in smooth muscle cells (5). The -isoforms of each 4E-BP were detected on purification of eIF-4E with m⁷GTP-Sepharose, and data shown in Fig. 3B confirms that they migrated faster than the -isoform of 4E-BPs.

Essentially all of the -isoforms of 4E-BP from cardiocyte homogenates were bound to eIF-4E as indicated by recovery on m⁷GTP-Sepharose. In contrast, the phosphorylated -isoforms were not recovered even though they constituted such a large percentage of the total 4E-BP pool. These results are consistent with previous findings showing that the nonphosphorylated -isoform of 4E-BP1 binds tightly to eIF-4E, the binding of the -isoform to eIF-4E is much weaker, and the -isoform does not bind to eIF-4E at all (1, 19, 21, 34). Thus, under steady-state conditions, a large portion of the 4E-BP pool in the adult cardiocyte does not appear to be interacting with eIF-4E to sequester it and limit eIF-4F complex formation. This fact may be an important determinant for the ability to regulate eIF-4E phosphorylation in response to an anabolic stimulus such as mechanical load. For example, it has been shown that phosphorylation of eIF-4E in vitro by some isoforms of protein kinase C is inhibited when eIF-4E is bound to 4E-BP (41). Because the adult cardiocyte maintains a relatively high percentage of 4E-BP-, an isoform that interacts weakly with eIF-4E, phosphorylation of eIF-4E can occur in response to a growth stimulus without increasing 4E-BP phosphorylation.

The results in Figs. 5 and 6 suggest that eIF-4F complex formation can increase independently of 4E-BP phosphorylation in the adult cardiocyte. This conclusion is based on two observations. First, insulin caused a complete shift of each 4E-BP into its highly phosphorylated -isoform and a corresponding decrease in the binding of 4E-BP -isoforms to eIF-4E. In comparison, electrically stimulated contraction had a smaller effect on 4E-BP phosphorylation. Despite these differences in 4E-BP phosphorylation, electrically stimulated contraction increased eIF-4F complex formation to the same extent as insulin. Second, insulin increased eIF-4F complex formation even when the dissociation of 4E-BP- from eIF-4E was prevented by treatment with rapamycin. These data suggest that 4E-BPs were not limiting the binding of eIF-4G to eIF-4E. This observation is not unique to the adult cardiocyte because rapamycin treatment to block 4E-BP phosphorylation in 3T3 fibroblasts did not prevent an acute increase in eIF-4F complex formation produced by serum stimulation and did not significantly affect translational initiation or protein synthesis (30). Given that rapamycin did not block eIF-4F complex formation in insulin-treated cardiocytes and that rapamycin treatment alone did not increase the amount of 4E-BP bound to eIF-4E, these findings suggest that eIF-4E might be sequestered in the eIF-4F complex. The findings of a recent study (39) using adult rat cardiocytes are consistent with eIF-4E sequestration: acute treatment with rapamycin did not increase the amount of 4E-BP1 bound to eIF-4E. Other studies indicate that eIF-4E could be sequestered by relatively slow exchange from the eIF-4F complex or by stabilization of the eIF-4F complex by eIF-4E phosphorylation (3, 30). The stability of the eIF-4F complex is underscored by studies showing that prolonged exposure to rapamycin (>20 h) decreased eIF-4F complex formation by 40% and protein synthesis by 40-50%, whereas the effects of rapamycin on inhibition of 4E-BP phosphorylation were maximal by 1 h (1, 30).

Our data suggest that the inhibitory effects of rapamycin on eIF-4F complex formation occurred by decreasing contractility rather than blocking the phosphorylation of 4E-BP. This conclusion is based on several observations. First, the amount of 4E-BP -isoforms associated with eIF-4E remained the same when cardiocytes were electrically stimulated to contract in the presence or absence of rapamycin. Second, the velocity and extent of sarcomere shortening were significantly depressed by 51 and 41%, respectively, when cardiocytes were electrically stimulated to contract in the presence of rapamycin. Similar findings were observed in a skinned skeletal muscle preparation in which 1 µmol/l rapamycin decreased force generation by >50% after just eight contractions (17). The decrease in contractility may be the result of rapamycin binding to FKBP-12, which causes a loss of depolarization-induced calcium release in cardiocytes by inhibiting the closure of the ryanodine receptor/calcium-release channel (42). Third, the ability of rapamycin to inhibit eIF-4F complex formation in electrically stimulated cardiocytes mirrored the results obtained with BDM, a chemical agent that decreases contractility by 70% as measured by the velocity and extent of sarcomere shortening (10). Neither rapamycin nor BDM blocked the ability of insulin to increase eIF-4F complex formation in quiescent cardiocytes, suggesting that inhibitory effects of these agents occur as a result of preventing active tension generation during contraction.