The purpose of the present study was to determine whether burn injury decreases myocardial protein synthesis and potential contributing mechanisms for this impairment. To address this aim, thermal injury was produced by a 40% total body surface area full-thickness scald burn in anesthetized rats, and the animals were studied 24 h later. Burn decreased the in vivo-determined rate of myocardial protein synthesis and translation efficiency by 25% but did not alter the protein synthetic rate in skeletal muscle. To identify potential mechanisms responsible for regulating mRNA translation in cardiac muscle, we examined several eukaryotic initiation factors (eIFs) and elongation factors (eEFs). Burn failed to alter eIF2B activity or the total amount or phosphorylation status of either eIF2α or eIF2Bϵ in heart. In contrast, hearts from burned rats demonstrated 1) an increased binding of the translational repressor 4E-BP1 with eIF4E, 2) a decreased amount of eIF4E associated with eIF4G, and 3) a decreased amount of the hyperphosphorylated γ-form of 4E-BP1. These changes in eIF4E availability were not seen in gastrocnemius muscle where burn injury did not decrease protein synthesis. Furthermore, constitutive phosphorylation of mTOR, S6K1, the ribosomal protein S6, and eIF4G were also decreased in hearts from burned rats. Burn did not appear to adversely affect elongation because there was no significant difference in the myocardial content of eEF1α or eEF2 or the phosphorylation state of eEF2. The above-mentioned burn-induced changes in mRNA translation were associated with an impairment of in vitro myocardial performance. Finally, 24 h postburn, the cardiac mRNA content of IL-1β, IL-6, and high-mobility group protein B1 (but not TNF-α) was increased. In summary, these data suggest that thermal injury specifically decreases cardiac protein synthesis in part by decreasing mRNA translation efficiency resulting from an impairment in translation initiation associated with alterations in eIF4E availability and S6K1 activity.
- eukaryotic initiation factors 2B and 4E
- high-mobility group protein B1
during the initial postburn period, cardiovascular complications are major contributors to mortality, particularly in those individuals with underlying pathology (44). Burn-induced alterations in cardiovascular function include tissue fluid sequestration, decreased circulating blood volume, reduced cardiac output, and an intrinsic defect in myocardial performance (1, 2, 5, 20, 37, 50). In general, the intrinsic contractile defects observed after a major burn [>30-40% total body surface area (TBSA)] affect both left and right ventricular function and contribute to therapeutic failure during the postburn period (1, 7, 20-23, 37, 38, 50). It is clear that hypovolemia per se is not the sole cause for the observed reduction in cardiac output and ventricular dysfunction because these defects are not fully corrected by aggressive fluid resuscitation (7, 22).
The physiological basis for the cardiac dysfunction is undoubtedly multifactorial, with increased circulating or tissue levels of a variety of mediators, including tumor necrosis factor (TNF)-α, reactive oxygen species, nitric oxide, calcium, and catecholamines (among others), being implicated in its pathogenesis (15, 20, 22, 23, 57). Regardless of the exact mechanism, a reduction in cardiac contractility is associated with derangements in myofibrillar architecture and a loss of the contractile proteins actin and myosin (24, 48). These data indicate that changes in cardiac myofibrillar proteins occur early after burn and may be caused by the inhibition of myocardial protein synthesis. The dynamic balance of myocardial protein content is dependent on both protein synthesis and protein degradation. The metabolism of contractile and other cardiac proteins is dynamic and rapid, with ∼50% of the myocardial protein turning over every 2-3 days (4). The current studies address the hypothesis that burn injury induces specific defects in the regulation of myocardial protein synthesis that are ultimately responsible for at least part of the observed cardiomyopathy. Although alterations in protein balance have been reported for skeletal muscle in response to burn (11), no information exists specifically related to burn-induced change in protein synthesis in the heart.
Alterations in cardiac protein synthesis may be caused by changes in either the efficiency of translation or the number of ribosomes (8, 46). The translation of mRNA into protein is often divided into three stages: initiation, elongation, and termination (8, 43). In sepsis and other catabolic conditions, the decreased protein synthesis in striated muscle is associated with defects in mRNA translation initiation, the first rate-determining phase of protein synthesis (26, 30-32, 47, 54). Translation initiation is regulated by a number of protein factors termed eukaryotic initiation factors (eIFs). One of these initiation factors, eIF2, mediates the first step in the initiation process that promotes the attachment of the initiator methionyl-tRNA (met-tRNAi) to the 40S ribosomal subunit to form the 43S preinitiation complex (43). A second critical point of translational regulation involves the binding of the 5′-end of cellular mRNA to the 43S preinitiation complex, a reaction mediated by the cap-binding protein complex eIF4F (45, 47). While the decreased muscle protein synthesis in other catabolic conditions has been generally linked to inhibition of peptide-chain initiation, the exact site of the molecular lesion often varies (8, 26, 30-32, 47). Furthermore, whether burn alters various eIFs in heart has not been previously reported.
The purpose of the present study was to determine whether thermal injury acutely decreases myocardial protein synthesis and, if so, to determine whether this response was associated with defects in peptide-chain initiation, via alterations in eIF2 and/or eIF4F, or elongation. A burn-induced increase in myocardial synthesis of cytokines has been previously reported (21), and elevations in inflammatory cytokines have been reported to decrease translation initiation (32). Therefore, the myocardial mRNA content of several inflammatory cytokines, including interleukin (IL)-1β, IL-6, TNF-α, and the late-phase cytokine high-mobility group protein B1 (HMGB1, formerly HMG-1), was also determined.
METHODS AND MATERIALS
Animal preparation and experimental protocol. Adult specific pathogen-free male Sprague-Dawley rats (285-310 g; Charles River Breeding Laboratories, Cambridge, MA) were housed at a constant temperature, exposed to a 12:12-h light-dark cycle, and maintained on standard rodent chow and water ad libitum for at least 1 wk before experiments were performed. All experiments were approved by the Animal Care and Use Committee at the Pennsylvania State University College of Medicine and adhered to the National Institutes of Health guidelines for the use of experimental animals.
On the day of the experiment all rats were deeply anesthetized with an intramuscular injection of ketamine and xylazine (100 and 10 mg/kg, respectively), and body weight was determined. The hair on the dorsal and ventral surfaces of the animal was closely clipped, and animals were secured in an insulated template that exposed only the area of skin to be injured (18), as previously described (33). For the burn group, the surface of the skin exposed through the aperture in the template was immersed in 100°C water for 12 s on the dorsal surface and 7 s on the ventral surface. The template limits the burn area to a predetermined 40% total body surface area and the technique produces a full-thickness scald injury with complete destruction of the underlying neural tissue (21). Rats were immediately dried and resuscitated by the subcutaneous injection of lactated Ringer solution (10 ml). Sham-burn control rats were treated identically to those in the burn group, except they were immersed in 25°C water. All rats were injected subcutaneously with the analgesic buprenorphine (0.2 mg/kg) immediately after sham or burn injury. Rats were returned to individual cages, and food was withheld for the remainder of the experimental protocol. Burned rats displayed no discomfort or pain and moved freely within their cage. Tissues were sampled ∼24 h after the burn because previous studies indicated severe alterations in skeletal muscle protein balance, the insulin-like growth factor system, and cardiac function at this time point (11, 22, 33).
Protein synthesis and RNA content. The in vivo rate of protein synthesis was determined for various tissues using the flooding-dose technique, as described previously (30, 32, 52, 54). Animals were anesthetized with pentobarbital sodium, and a catheter was placed in the carotid artery. Rats were injected with l-[2,3,4,5,6-3H]phenylalanine (150 mM, 30 μCi/ml; 1 ml/100 g body wt) administered via the jugular vein. Thereafter, arterial blood samples were collected at 2, 6, and 10 min into heparinized syringes. The heart (ventricle only), gastrocnemius, liver, and kidney were rapidly excised and weighed. A portion of the gastrocnemius and heart from each animal was homogenized immediately for measurement of eIF2B activity and analysis of the eIF4E system, and the remaining tissue was frozen between aluminum clamps precooled in liquid nitrogen. The frozen tissues were powdered under liquid nitrogen using a mortar and pestle. Blood was centrifuged and plasma was collected. All tissue and plasma samples were stored at -70°C until analyzed. A portion of the powdered myocardium was used to estimate the rate of incorporation of [3H]phenylalanine into protein, exactly as described previously (30, 32, 52, 54).
Changes in the number of ribosomes or in the efficiency of mRNA translation may alter tissue protein synthesis (8, 46). Because ∼85% of the RNA is ribosomal RNA, changes in total RNA content reflect changes in the number of ribosomes. Total RNA content was measured from muscle homogenates, as previously described (30, 32, 52, 54). Translational efficiency was subsequently calculated by dividing the rate of protein synthesis for a particular tissue by the RNA content for that tissue.
Amount of eIF2 and eIF2B. The relative amounts and the phosphorylation state of the α-subunit of eIF2 (eIF2α) and the ϵ-subunit of eIF2B (eIF2Bϵ) in heart were estimated by protein immunoblot analysis, as described previously (30, 32, 53, 56). eIF2 and eIF2B were chosen because change in the content and/or activity of these initiation factors correlates with alterations in protein synthesis (43). eIF2 consists of three subunits of which the α-subunit appears important in regulating protein synthesis. Likewise, eIF2B is a multimeric protein consisting of five subunits, with the ϵ-subunit being the catalytic subunit. Previous studies have established that the expression of the ϵ-subunit is representative of the other subunits and the eIF2B holoenzyme. Tissues were homogenized, and samples were subjected to SDS-PAGE, exactly as previously described (30, 32, 53, 56). Immunoblotting for eIF2α was performed with a rabbit polyclonal antibody raised against the phosphorylated form of eIF2α (anti-Ser51) or an antibody recognizing both phosphorylated and unphosphorylated eIF2α (Cell Signaling Technologies, Beverly, MA). Similarly, immunoblotting for eIF2Bϵ was determined using either an affinitypurified anti-phosphopeptide antibody specific for eIF2Bϵ phosphorylated on Ser535 or a monoclonal anti-eIF2Bϵ antibody that recognizes both the phosphorylated and unphosphorylated form of the protein (Biosource International, Camarillo, CA). Antibodies were visualized using an enhanced chemiluminescence (ECL) procedure with the secondary antibody linked to horseradish peroxidase (Amersham). The blots were exposed to X-ray film in a cassette equipped with a DuPont Lightning Plus intensifying screen. Following development, the film was scanned (Microtek ScanMaker IV) and analyzed using NIH Image 1.6 software.
Determination of eIF2B activity. The guanine nucleotide exchange activity of eIF2B activity in heart was measured in postmitochondrial supernatants by the exchange of [3H]GDP bound to eIF2B for nonradioactively labeled GDP, as previously described (28, 30, 32).
Analysis of 4E-BP1·eIF4E and eIF4G·eIF4E complexes. The association of eIF4E with either 4E-BP1 or eIF4G was determined, as previously described (30-32, 52). Briefly, tissues were homogenized, and eIF4E as well as 4E-BP1·eIF4E and eIF4G·eIF4E complexes were immunoprecipitated from aliquots of 10,000 g supernatants using an anti-eIF4E monoclonal antibody. The antibody-antigen complex was collected by incubation with BioMag goat anti-mouse IgG beads (Perseptive Biosystems, Framingham, MA). The beads were collected by centrifugation, and the supernatants were subjected to electrophoresis either on a 7.5% polyacrylamide gel for analysis of eIF4G or on a 15% gel to quantify 4E-BP1 and eIF4E. Proteins were then electrophoretically transferred to nitrocellulose. The membranes were incubated with a mouse anti-human eIF4E antibody, a rabbit anti-rat 4E-BP1 antibody, or a rabbit anti-eIF4G antibody (Bethyl Laboratories, Montgomery, TX). The blots were developed using ECL, and autoradiographs were scanned and analyzed as described above.
Phosphorylation state of 4E-BP1, eIF4E, eIF4G, and mTOR. The phosphorylated and nonphosphorylated forms of eIF4E in tissue extracts were separated by isoelectric focusing (IEF) on a slab gel and analyzed by protein immunoblot analysis, as previously described (30-32, 52). The phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from tissue homogenates, and the various phosphorylated forms of 4E-BP1 were separated by SDS-PAGE and analyzed by protein immunoblotting as described above. In addition, other blots were incubated with either primary antibodies to total S6K1 (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated (Ser2448) and total mTOR (Bethyl Laboratories), or phosphorylated (Ser1108) and total eIF4G (Bethyl Laboratories). Autoradiographs were scanned and quantified as described above.
Analysis of elongation factors eEF1 and eEF2. Hearts were homogenized, and equal amounts of protein were subjected to SDS-PAGE, using Criterion Precast 10-20% Tris·HCl gradient gels (Bio-Rad Laboratories, Hercules, CA), followed by transfer of proteins to PVDF membranes as described previously (32, 55). Membranes were incubated with antibodies specific for eEF1A (Santa Cruz Antibodies, Santa Cruz, CA) or eEF2 (Dr. A. C. Nairn). The phosphorylation status of eEF2 in the tissue homogenate was analyzed by immunoblotting with an antibody that specifically recognized eEF2 phosphorylated on Thr56. The blots were developed using ECL, and autoradiographs were scanned and analyzed.
RNA extraction and Northern blotting. Total RNA was extracted from tissues with TRI Reagent (Molecular Research Center, Cincinnati, OH). RNA samples were resuspended in formamide and quantified by spectrophotometry. Riboprobes were synthesized from a rat template set (rCK-1) using an in vitro transcription kit (BD PharMingen, San Diego, CA). The labeled riboprobe was hybridized with 20 μg RNA using a ribonuclease protection assay (RPA) kit (BD PharMingen), as previously described (34). Protected RNAs were separated using a 5% acrylamide gel (19:1 acrylamide-bisacrylamide). Dried gels were exposed to a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA). The resulting data were quantified using ImageQuant software and normalized to rat ribosomal protein L32 mRNA. The ratio of each mRNA to L32 RNA was set at 1.0 arbitrary units (AU) for heart from control animals.
Northern blot analysis was performed to quantify the content of HMGB1 mRNA in cardiac tissue (34). Samples (25 μg) of total RNA were electrophoresed under denaturing conditions on a 1.1% agarose gel containing 6% formaldehyde. RNA was transferred to Nytran Supercharge membranes (Schleicher and Schuell, Keene, NH). After baking, blots were hybridized at 42°C in ULTRAhyb (Ambion, Austin, TX). Oligonucleotides for HMGB1 (5′-GCTGCTTGTCATCCGCAGCAGTGTTGTTCCACATCTCTCC-3′) were labeled by TdT (Promega, Madison, WI) tailing with [α-32P]dATP (Amersham, Arlington Heights, IL). A rat 18S oligonucleotide was used for normalization and was labeled in the same manner. All membranes were washed twice in 2× SSC/0.1% SDS at 42°C for 5 min and once in 0.2× SSC/0.1% SDS at 42°C for 15 min. Membranes were exposed to a phosphorimager screen, and the resultant data were calculated as described above for RPA.
In vivo hemodynamics and in vitro working heart preparation. In a separate experimental series, a catheter was surgically implanted in the carotid artery before burn injury so that mean arterial blood pressure (MABP) and heart rate (HR) could be assessed in conscious unrestrained rats before death (32). After determination of MABP and HR, rats were anesthetized with intravenously administered pentobarbital sodium, and in vitro myocardial performance was determined as described below. Blood pressure and HR were determined by connecting the arterial catheter to a pressure transducer (model P23, Gould Instruments; Oxnard, CA) attached to a polygraph (model 7D, Grass Instruments; Quincy, MA) (32).
To assess the in vitro mechanical function of hearts, rats were anesthetized with pentobarbital sodium, and hearts were removed from both burn and time-matched control animals. Both the aorta and the left atrial appendage were cannulated, and the latter was opened to a reservoir containing Krebs-Henseleit bicarbonate buffer gassed with 95% O2-5% CO2, pH 7.4 (37°C), and 5.5 mM glucose (39). Buffer pumped through the aorta perfused either the coronary arteries and was collected as it dripped off the heart or was pumped through the aortic outflow line. These two flows were measured and summed together for cardiac output. Aortic pressure was monitored with a transducer and polygraph in line with the aortic outflow cannula. Preload was varied by adjusting the height of the left atrial buffer reservoir above the heart, and afterload was primarily due to the resistance of a 23-gauge needle at the end of the aortic outflow line. After an initial 15-min perfusion period at a left atrial filling pressure (LAFP) of 15 cmH2O, a left ventricular function curve was generated by changing the LAFP to 10, 15, 20, 25, 30, and back to 15 cmH2O. At each preload, peak systolic pressure (PSP), HR, coronary flow, and aortic output were measured.
Myocardial high-energy phosphate concentrations and tissue water. In a separate group of rats the in vivo content of high-energy phosphate-containing compounds was determined. Control and burn rats were anesthetized with pentobarbital sodium, a tracheotomy was performed, and respiration was maintained with a Phipps and Byrd small animal respirator. The chest was opened, and the heart was frozen in situ with tongs precooled in liquid nitrogen. An aliquot of powdered tissue was extracted in cold PCA, neutralized, and used for the determination of ATP, ADP, AMP, creatine phosphate (CP), and creatine by standard fluorometric methods (35).
Another aliquot of powdered heart was weighed, dried overnight in an oven (60°C), and reweighed the next day to calculate the wet weight-to-dry weight ratio.
Statistics. Data were obtained from three separate experimental series each containing control and burn rats. For each study, rats were randomly assigned to either the experimental or control group. Experimental values are presented as means ± SE. The number of rats per group is indicated in the figure and table legends. Data were analyzed by unpaired Student's t-test to determine treatment effect (Instat, San Diego, CA). Statistical significance was set at P < 0.05.
Tissue protein synthesis. The in vivo rate of protein synthesis determined 24 h after thermal injury was decreased 25% in heart and increased 45% in liver (Fig. 1). In contrast, the protein synthetic rate was unaltered in response to burn in the predominantly fast-twitch gastrocnemius muscle and the kidney compared with tissues from control animals.
To determine whether a change in the number of ribosomes or the efficiency of mRNA translation was responsible for the burn-induced changes in heart and liver protein synthesis, the RNA content in each tissue was quantified. No significant burn-induced change in total RNA in either heart or liver was detected (Fig. 2, top). In contrast, the efficiency of translation, which provides an index of how rapidly the preexisting translational machinery is synthesizing protein, was decreased in heart and increased in liver in response to thermal injury (Fig. 2, bottom).
Content of eIF2 and eIF2B, and eIF2B activity. Alterations in the amount, availability, or activity of specific eIF proteins that regulate individual steps of the protein synthetic process might account for the impaired cardiac translational efficiency observed in burned rats. The binding of met-tRNAi to the 40S subunit to form the 43S preinitiation complex is mediated by eIF2. However, the relative content of the α-subunit of eIF2 was not altered in hearts from burned rats (Table 1). Furthermore, there was also no burn-induced change in the phosphorylation status of eIF2α, which is also known to impact the protein synthetic rate.
The ability of eIF2 to form a ternary complex can also be decreased via a reduction in eIF2B (43, 53). In the present study, there was no difference in the eIF2B activity measured in heart extracts prepared from burned and control rats (Table 1). Consistent with this observation is the lack of change in the relative amount of total and phosphorylated eIF2Bϵ (Table 1). Collectively, these data suggest that the burn-induced decrease in cardiac protein synthesis is not mediated by alterations in the eIF2/eIF2B system.
Availability of eIF4E. A second potential site for control of peptide-chain initiation involves regulating eIF4E availability (43, 45). Western blot analysis indicted there was no significant difference in the myocardial content of eIF4E between control and burned rats (Fig. 3). However, eIF4E can be sequestered into an inactive complex through binding of the translational repressor molecule 4E-BP1. Therefore, the ability of burn to modify the distribution of eIF4E in heart was subsequently determined. Figure 3 illustrates that the α- and β-forms of 4E-BP1 were detected in eIF4E immunoprecipitates of heart, and the content of the inactive eIF4E·4E-BP1 complex was increased more than twofold in hearts from burned rats (Fig. 3A). When eIF4E is bound to 4E-BP1 it is unable to interact with eIF4G to form the active eIF4E·eIF4G complex. Figure 3B illustrates that the amount of the eIF4E·eIF4G complex was reciprocally reduced by 53% in response to thermal injury compared with values from time-matched control animals.
Burn-induced changes in eIF4E distribution in the gastrocnemius were also assessed. In this striated muscle, there was no significant difference in the amount of inactive eIF4E·4E-BP1, active eIF4E·eIF4G, or the γ-isoform of 4E-BP1 between control and burned rats (data not shown). These data are consistent with the lack of a burn-induced change in protein synthesis in this fast-twitch skeletal muscle.
Phosphorylation of 4E-BP1, eIF4E, and eIF4G. One-dimensional SDS-PAGE allows the various phosphorylated forms of 4E-BP1 to be resolved into three bands (Fig. 3C, left). Hyperphosphorylation of 4E-BP1 decreases the association of the binding protein with eIF4E and increases translation. Figure 3C illustrates that the amount of 4E-BP1 in the hyperphosphorylated γ-form was decreased ∼40% in heart from burned rats. This change is consistent with the above-mentioned redistribution of eIF4E between 4E-BP1 and eIF4G.
The influence of burn on the phosphorylation state of eIF4E was also assessed because phosphorylation may increase the binding affinity of this protein for the 7-methylguanosine cap of mRNA and stabilizes the eIF4F complex (45). However, there was no detectable difference in myocardial eIF4E phosphorylation between burned and control rats (data not shown).
The interaction between eIF4E and eIF4G can also be regulated in part by the phosphorylation of eIF4G. In control rats there was constitutive phosphorylation of eIF4G in cardiac muscle, and thermal injury decreased the content of phosphorylated eIF4G by ∼35% (Fig. 4, top). This change did not result from a decreased content of total eIF4G in heart.
Phosphorylation of S6K1 and the ribosomal protein S6. S6K1 is activated by undergoing multiple serine-threonine phosphorylation events. The most slowly migrating forms of this protein represent the heavily phosphorylated and, thus, highly active form of the kinase (14). There was a basal level of S6K1 phosphorylation in hearts from control rats. In response to burn, the mobility of the bands increased, indicating a relative dephosphorylation and inactivation of S6K1 (Fig. 5, top).
The phosphorylation status of the ribosomal protein S6, a physiologically relevant S6K1 substrate, was also determined. Figure 5, middle and bottom, illustrates that there was a constitutive phosphorylation of S6 in hearts from control rats, and the relative content of phosphorylated S6 was decreased 55% in hearts after thermal injury.
mTOR. The mammalian target of rapamycin (mTOR) is a proline-directed serine-threonine protein kinase and is believed to be a common intermediate in the phosphorylation of both 4E-BP1 and S6K1. Although thermal injury did not alter the cellular content of total mTOR protein, there was an ∼60% decrease in the phosphorylation of mTOR (Ser2448) in hearts from burned rats (Fig. 6).
eEF-1 and -2. Compared with values from time-matched control rats, thermal injury did not significantly alter the content of eEF1α (control = 100 ± 11 AU vs. burn = 114 ± 9 AU), eEF2 (control = 100 ± 12 AU vs. burn = 98 ± 12 AU), or the amount of phosphorylated eEF2 (control = 100 ± 9 AU vs. burn = 107 ± 9 AU) in heart.
Myocardial cytokine mRNA content. Elevations in circulating and tissue levels of inflammatory cytokines can impair muscle protein synthesis (32). Therefore, the effect of thermal injury on the steady-state mRNA content for IL-1β, IL-6, TNF-α, and HMGB1 was determined. Hearts from burned rats demonstrated an ∼2-fold increase in the mRNA content for IL-1β and HMGB1 as well as more than a 10-fold increase in the IL-6 mRNA content 24 h after injury (Fig. 7). In contrast, there was no significant change in the expression of TNF-α at the time point examined.
Myocardial high-energy phosphate content and tissue water. Protein synthesis consumes a large fraction of cellular energy, and an “energy defect” represents one possible mechanism to account for the reduced translation efficiency after burn. However, there was no significant difference in the cardiac concentrations of ATP, ADP, AMP, CP or creatine between control and burned rats (Table 2). Therefore, it is doubtful that a reduction in high-energy phosphates was responsible for the reduced rates of myocardial protein synthesis and translational efficiency observed rats after thermal injury.
Thermal injury did not significantly alter cardiac muscle water content. The dry weight-to-wet weight ratio for heart was not different between burned rats (14.5 ± 0.2%) and time-matched control animals (14.7 ± 0.2%).
In vivo hemodynamics and in vitro myocardial function. Twenty-four hours after burn injury, there was no significant difference in the MABP between control and burn rats (control = 117 ± 11 mmHg vs. burn = 112 ± 7 mmHg). However, burned rats did demonstrate a marked tachycardia (control = 345 ± 38 beats/min vs. burn = 441 ± 59 beats/min; P < 0.05).
Hearts removed from burned rats and perfused in vitro in the working mode demonstrated a consistent decrease in both PSP and CO over the range of LAFPs (Fig. 8); however, only the decrement in CO achieved statistical significance. The product, CO × PSP, which provides an estimate of myocardial work, was also decreased in hearts from burned rats (∼40-50%) compared with values from control rats.
In the present study, thermal injury decreased the rate of ventricular protein synthesis. This decrease appears relatively selective because no change was seen in skeletal muscle or kidney, and the rate of hepatic protein synthesis was increased in response to burn. The cellular content of ribosomes may be rate limiting for protein synthesis under some conditions (8, 46). However, the abundance of ribosomes, as estimated by the total RNA content, was not altered in heart. Therefore, these data indicate that the burn-induced decrease in myocardial protein synthesis results primarily from an impairment in the efficiency of polypeptide synthesis and not from a diminished ribosomal content. While the above-mentioned changes represent an early response to thermal injury, the possibility that a decreased ribosome content may modulate myocardial protein synthesis at later times after burn cannot be excluded.
The lack of a burn-induced change in skeletal muscle protein synthesis is consistent with previous in vivo and in vitro studies examining protein metabolism that indicate the primary determinant of the burn-induced negative nitrogen balance is an accelerated rate of protein degradation (17, 41, 62). However, this response contrasts markedly with the consistent decrease in skeletal muscle protein synthesis observed in other catabolic conditions, such as sepsis and inflammation (30-32, 47, 52). The reason for this difference is not readily apparent given that many of the same inflammatory mediators are comparably increased in these different conditions. The increased hepatic protein synthesis observed after thermal injury is consistent with the accelerated rate of synthesis and secretion of acute phase proteins (13, 19). Moreover, the increased hepatic protein synthesis observed in burned rats appears to result from a corresponding increase in translational efficiency.
The inhibition of cardiac muscle protein synthesis and translational efficiency may result from a burn-induced block in either peptide-chain initiation and/or elongation-termination. Although translational controls operate most frequently during the initiation phase, elongation has been demonstrated as a control point in the translation of specific mRNAs (36). Both eEF1 and eEF2 are critical for catalyzing the sequential addition of amino acid residues to the carboxy-terminal end of the nascent peptide. A reduction in the tissue content of elongation factors has been causally linked to diminished rates of protein synthesis during certain types of catabolic stress (36, 55, 56). However, there was no detectable alteration in the content of these two critical eEFs or the extent of eEF2 phosphorylation in hearts from burned rats. These results provide supportive evidence that the initiation of polypeptide chains, not their elongation, is the primary site of the burn-induced defect in myocardial protein synthesis.
Two of the steps involved in translation initiation are important regulatory points in the overall control of in vivo protein synthesis. The first step controlling initiation is the binding of met-tRNAi to the 40S ribosomal subunit to form the 43S preinitiation complex, a step required for all initiation events. This reaction is mediated by eIF2 and is regulated by the activity of another initiation factor, eIF2B. Both anabolic (60) and catabolic stimuli (27, 40, 53) have been reported to regulate mRNA translation via changes in the eIF2/eIF2B system. However, burn injury did not produce a detectable change in either eIF2 or eIF2B, and therefore this system is an unlikely mediator of the decreased protein synthetic rate. This finding is consistent with our previous studies in TNF-infused or LPS-treated rats where the decreased cardiac protein synthesis was also independent of changes in the eIF2/eIF2B system (30, 32).
The second important control point involves recruitment of the 43S preinitiation complex to the mRNA, which is mediated by the eIF4F complex (43, 46). This complex is heterotrimeric, being composed of eIF-4E, -4G and -4A. In muscle, eIF4E is the least abundant of the eIF4F subunits and under many conditions is considered to be a limiting factor in eIF4F complex assembly (10). eIF4E binds to the mRNA cap structure present at the 5′-end of all nuclear transcribed mRNAs to form an eIF4E·mRNA complex. This complex then binds to the scaffold protein eIF4G and the RNA helicase eIF4A to form the active eIF4F complex, thereby allowing translation to proceed. While the total amount of eIF4E in hearts from burned rats was not altered, there was a marked redistribution of this protein as evidenced by the decreased amount of the active eIF4E·eIF4G complex and corresponding increase in the amount of the inactive eIF4E·4E-BP1 complex. The binding of eIF4E to eIF4G is controlled in part by a family of cap-dependent translational repressors, referred to as eIF4E-binding proteins. The most prominent member of this family in muscle is 4E-BP1. This binding protein acts as a molecular antagonist by completing with eIF4G for a common binding site on eIF4E (16). The hyperphosphorylation of 4E-BP1 liberates it from eIF4E and thereby facilitates binding of eIF4E with eIF4G. In the present study, the burn-induced decrease in the phosphorylation of 4E-BP1 was associated with the redistribution of eIF4E. The triumvirate of decreased 4E-BP1 phosphorylation, redistribution of eIF4E to the inactive complex, and impaired protein synthesis has been observed in cultured cardiomyocytes and heart under other conditions (32, 51). In contradistinction, there was no alteration in eIF4F complex assembly or 4E-BP1 phosphorylation in skeletal muscle after burn, and this lack of change is internally consistent with the unaltered rate of protein synthesis in this tissue.
The phosphorylation and activation of the serine/threonine protein kinase S6K1 is also an important element of the protein synthetic signaling pathway (14) because it phosphorylates a number of proteins, including the small ribosomal subunit protein S6. The phosphorylation of S6K1 and S6 are associated with a stimulation of muscle protein synthesis and accelerated rates of mRNA translation initiation (9). Phosphorylation of S6 enhances translation of a subset of mRNAs containing 5′-terminal tracts of oligopyrimidines (e.g., 5′-TOP mRNAs), including those that encode for ribosomal proteins and elongation factors (25). In the present study, thermal injury decreased the extent of both S6K1 and S6 phosphorylation in heart. Additionally, the recruitment of the translational machinery to the 5′-end of mRNA can be enhanced by eIF4G phosphorylation, which was decreased in burned rats. Hence, in addition to the decreased formation of eIF4F, the dephosphorylation and inactivation of S6K1 and eIF4G may also limit myocardial protein synthesis in response to burn. The serine/threonine kinase mTOR is believed to represent a common upstream intermediate important in the phosphorylation of both 4E-BP1 and S6K1 (14, 47). Therefore, impaired mTOR activity might account for the dual defects in the phosphorylation of 4E-BP1 and S6K1 as well as the subsequent inhibition of myocardial mRNA translation in response to thermal injury. This conclusion is consistent with the observed decrease in mTOR phosphorylation in hearts from burned rats.
The above-mentioned impairment in cardiac protein synthesis and mRNA translation was temporally associated with a marked dysfunction in mechanical function. The burn-induced impairment in intrinsic myocardial performance has been reported previously (1, 7, 20-23) and is consistent with known deficits in cardiac contraction and relaxation (15, 20-23). Our data and that of others (61) indicate the impairment in cardiac function and protein synthesis is not related to a deficit in high-energy phosphates. The etiology of the contractile defect has been extensively investigated, and a large number of putative mediators have been implicated. Several lines of evidence suggest that the multifunctional cytokine TNF-α has maladaptive effects in the heart. First, TNF-α depresses myocardial protein synthesis and myocardial function (6, 32). Second, cardiomyocytes isolated from burned rats secrete more TNF-α under basal conditions and in response to endotoxin than myocytes from control rats (21). Finally, agents that neutralize TNF-α action prevent the burn-induced decrement in myocardial function (15). The present study demonstrates that TNF-α mRNA content was not upregulated in hearts 24 h after induction of burn. This lack of change was not entirely unexpected given the known transient nature of the increase in TNF-α mRNA and protein induced by other catabolic insults (34). However, cardiac TNF-α mRNA content was also examined at the 4-h postburn injury and found not to be elevated (unpublished observations). In contrast, there was a marked increase in gene expression for IL-6 and a modest increase in IL-1β mRNA at 24 h after thermal injury. The secretion of both cytokines has been previously demonstrated to be increased in cardiomyocytes isolated from burned rats (21). Furthermore, the cardiac mRNA content of HMGB1, a representative late-phase inflammatory cytokine (42, 58), was also modestly increased after thermal injury. Although HMGB1 is an evolutionarily conserved protein that is ubiquitously expressed, data regarding alterations in the tissue mRNA expression of this mediator in response to traumatic injury are sparse. In this regard, thermal injury is known to increase HMGB1 mRNA in liver and lung between 24 and 72 h (12), and a nonlethal dose of endotoxin has been reported to increase HMGB1 mRNA in skeletal and cardiac muscle (34). For the cytokines examined, we cannot exclude the possibility that a portion of the cardiac mRNA content was contributed by residual blood cells present within the tissue or vasculature at the time tissue was freeze-clamped. The overall physiological relevance of the burn-induced increase in cytokine mRNA in hearts remains to be determined.
In summary, the results of the present investigation provide evidence that thermal injury selectively inhibits cardiac protein synthesis in rats and that this defect is caused by a diminished translational efficiency rather than a reduction in the number of ribosomes. Furthermore, the impairment in mRNA translation could not be ascribed to changes in either the eIF2/eIF2B system or alterations in elongation. Instead the burn-induced decrease in myocardial protein synthesis was associated with a decreased phosphorylation of 4E-BP1 that led to a redistribution of eIF4E from the active eIF4E·eIF4G complex to the inactive eIF4E·4E-BP1 complex, and a decreased phosphorylation of S6K1, eIF4G, and mTOR. These data suggest that the burn-induced cardiomyopathy results from an inhibition of translation initiation secondary to marked changes in eIF4E availability and S6K1 phosphorylation and that these changes may contribute to the observed defect in intrinsic myocardial performance.
This work was supported in part by National Institutes of Health Grants HL-66443 and GM-39277.
We thank D. Wu, G. Nystrom, and N. Deshpande for excellent technical assistance. We also sincerely thank all of those individuals that have provided various reagents: Dr. M. J. Birnbaum, University of Pennsylvania, for antibody to phosphorylated S6; Drs. Leonard S. Jefferson and Scot R. Kimball Pennsylvania State College of Medicine, for the eIF4E antibody and [3H]GDP; and Angus C. Nairn, Rockefeller University, for the eEF2 antibody.
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