INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BURN TRAUMA provokes cardiac injury and contractile dysfunction. Myocardial cellular disruption and hemodynamic alterations, including decreased cardiac output, shock, and left ventricular (LV) failure, have been documented in burned patients (24, 27, 31, 42). Deficits in myocardial contraction and relaxation have also been described in rats, guinea pigs, rabbits, and sheep (2, 7, 13-15, 17, 18, 23, 43). The contractile deficits are transient, appearing 2 h, resolving by 72 h after the burn, and occur despite fluid resuscitation to maintain adequate preload (13).

The molecular basis of burn-induced cardiac dysfunction is complex and incompletely understood. The delay between thermal injury and onset of impaired contractility suggests a multistep process, involving long- and short-range signaling and new gene expression. Intervention studies implicate proinflammatory molecules as major contributors to impaired contractility. Initial studies highlighted the role of oxygen-derived free radicals and leukocyte-derived products as mediators of contractile dysfunction (16-18). Additional investigations have underscored the importance of different proinflammatory mediators in this cardiac response. Tumor necrosis factor- (TNF-) inhibition prevents myocardial dysfunction (11), whereas intercellular adhesion molecule-1 blockade, protein kinase C inhibition, and pentoxifylline administration all reduce the degree of burn-triggered contractility impairment (15, 19, 38). Where each of these agents operates in the cascade of events that starts with tissue damage at the burn site and culminates in impaired relaxation and contraction of the heart needs to be established. Furthermore, the identity of burn-induced myocardial depressant signal(s) is still unknown.

The Toll/interleukin (IL)-1 signal transduction pathway mediates multiple steps in the host response to infection. This pathway operates in both the afferent (pathogen or injury sensing) and efferent (proinflammatory) arms of the innate response. It transduces signal from the Toll-like receptors (TLRs; of which there are at least 10) in the afferent limb (25, 34, 35) and from at least two different cytokine receptors (IL-1 and IL-18) in the efferent limb (5, 28). This pathway consists of the cytoplasmic Toll/IL-1 receptor homology (TIR) domain, the signaling domain common to the TLR and IL-1 family of receptors (MyD88), the IL-1 receptor-associated kinase (IRAK), and the TNF-associated factor 6 (TRAF6). After receptor activation, MyD88, an adapter protein essential for downstream signaling, moves to the receptor complex and provides a platform for IRAK binding (1, 3, 40). Within this complex, IRAK becomes phosphorylated and then dissociates from the receptor (5). The kinase then interacts with TRAF6, which has ubiquitin-conjugating functions and is essential for nuclear factor- B (NF-B) activation (6, 9). The signal is then distributed to multiple downstream signaling cascades, including NF-B, the stress-activated protein kinases (SAPK or Jun NH₂-terminal kinases or JNKs), and p38 mitogen-activated protein kinases (MAPKs), leading to a biological response such as cytokine production in macrophages or adhesion molecule expression in endothelial cells.

Its central role in the innate immune response to infection makes the Toll/IL-1 pathway a candidate mediator of the inflammatory response to burn injury. Specifically, we hypothesized that burn injury would activate Toll/IL-1 signaling and that interruption of signal transduction through this pathway would abrogate burn-induced myocardial dysfunction. To test this hypothesis, we compared signal transduction and contractile function in the hearts of wild-type (WT) and IRAK-deficient mice generated in our laboratory in response to burn injury.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents. Antisera used to immunoprecipitate JNK and p38 MAPK were gifts from Melanie Cobb (UT Southwestern, Dallas, TX). Antisera for blotting these kinases were purchased from Upstate Biotechnology (Lake Placid, NY) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Polyclonal antisera against IK (IKK)- and was purchased from Santa Cruz Biotechnology. Recombinant glutathione-S-transferase (GST)-coupled fusion proteins GST-IB-, GST-ATF2, and GST-cJun were kindly provided by Melanie Cobb. All other reagents, unless otherwise noted, were purchased from Sigma Chemical (St. Louis, MO).

Experimental animals. All animals were used in compliance with the guidelines established by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center and performed in accordance with National Institutes of Health guidelines for the use of laboratory animals. IRAK-deficient mice were generated as previously described (37). Briefly, the Irak gene was inactivated in murine embryonic stem cells using targeted mutagenesis. Mutant stem cells were injected into WT blastocysts and resulted in chimeric mice. Two chimeric animals transmitted the mutant Irak gene through the germline. Offspring from these founders were interbred to generate homozygous IRAK-deficient females and hemizygous IRAK-deficient males [Irak is located on the X chromosome (37)]. These animals were on a hybrid background (129Sv × C57BL/6). WT animals on an identical hybrid background were used as controls for all experiments.

Burn procedure. Mice were deeply anesthetized (methoxyflurane), and their sides and back were closely clipped and then carefully shaved from the base of the tail to the base of the neck. Animals were then assigned to sham burn or burn groups. In those animals designated for burn trauma, a cutaneous burn injury was produced over 40% of the total body surface area by applying brass probes (2 × 3 cm with a 3-mm thickness) heated to 100°C in boiling water to the animals' side and back for 5 s. Animals designated for sham burn group received identical regimens of anesthesia and handling but no burn injury was given. After burn trauma was completed, the mice were given lactated Ringer fluid resuscitation (4 mg/kg per %burned surface area) intraperitoneally. All animals received analgesic (0.05 mg/kg im buprenorphine) every 8 h after burn trauma (41). Animals were monitored closely for the first 8 h after burn trauma to determine adequate recovery from the anesthesia, animal responsiveness to external stimuli, the absence of pain, and the ability to consume food and water.

In vitro kinase reactions. At different times following burn injury, mice were euthanized, and the heart from each mouse was immediately removed and snap-frozen in liquid nitrogen. Twenty five- to forty-milligram sections were removed from the frozen hearts, rinsed in ice-cold phosphate-buffered saline (PBS) to remove clotted blood, and minced into small pieces using a no. 11 scalpel blade. The tissue was then disrupted in a Dounce homogenizer with 1 ml of HEPES lysis buffer [50 mM HEPES, pH 7.5; 150 mM NaCl; 1% Triton X-100; 10% glycerol, and 1 mM dithiothreitol (DTT)] with protease (Roche Complete inhibitor; Indianapolis, IN) and phosphatase inhibitors (20 mM NaF; 20 mM sodium glycerophosphate; 0.5 mM sodium orthovanadate). After a 30-min incubation period on ice, lysates were cleared by centrifugation at 20,000 g for 10 min at 4°C, and the supernatant was transferred to a clean microcentrifuge tube. Total protein concentration of lysates was determined using a kit (Bio-Rad; Hercules, CA). Kinases were immunoprecipitated from 1 mg of total cardiac protein in a minimum 250-µl volume with 1-5 µl of antiserum on a rocking platform overnight at 4°C. Protein A-conjugated agarose (20 µl; Roche Biochemicals; Indianapolis, IN) was added and allowed to incubate for 2 h at 4°C. Precipitates were washed three times with lysis buffer and once with incomplete kinase buffer (50 mM Tris · HCl, pH 7.4; 10 mM MgCl₂, and 1 mM DTT). Beads were then resuspended in complete kinase buffer (50 mM Tris · HCl, pH 7.4; 10 mM MgCl₂, 1 mM DTT, and 50 µM "cold" ATP) and [³²P]ATP (10 µCi/reaction) together with the appropriate substrate (0.3 mg/ml) in a total volume of 25 µl. The mixtures were incubated at 30°C for 30 min. Reactions were stopped by adding an equal volume of 2× SDS-PAGE sample buffer (200 mM Tris · HCl, 4% SDS, 0.04% bromophenol blue, and 20% glycerol) followed by heating at 95°C for 5 min. Samples were fractionated using SDS-PAGE, and the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore; Bedford, MA). Membranes were air-dried, exposed to phosphor storage screens, and developed using a phosphorimaging system (Molecular Dynamics; Mountain View, CA).

Immunoblots of precipitated kinases. Kinases were immunoprecipitated as above and washed six times with lysis buffer with inhibitors. After addition of SDS loading dye and heating at 95°C for 5 min, samples were fractionated on a 10% denaturing polyacrylamide gel and transferred to a PVDF membrane. Membranes were then blocked with PBS with 0.05% Tween-20 (PBS-T)-4% bovine serum albumin (BSA) solution overnight. Membranes were then incubated with primary antibody (diluted 1:5,000 for anti-JNK, and 1:3,000 for anti-p38) in PBS-T/4% BSA for 1 h. Membranes were washed and secondary antibodies were added (diluted 1:30,000 in PBS-T/BSA) for 30 min before detection using an enhanced chemiluminescence protocol (Amersham Pharmacia Biotech; Piscataway, NJ).

Langendorff-perfused hearts. To examine cardiac contractile function, separate groups of awake mice from all experimental groups [sham-treated WT (n = 5), sham-treated IRAK knockout (KO) (n = 8), burned WT (n = 5), or burned IRAK KO (n = 6)] were anticoagulated with heparin sodium (100 units, Elkins-Sinn; Cherry Hill, NJ) and euthanized by cervical dislocation. The heart was rapidly removed and placed in ice-cold (4°C) Krebs-Henseleit bicarbonate-buffered solution (in mM: 118 NaCl, 4.7 KCl, 21 NaHCO₃, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, and 11 glucose). All solutions were prepared each day with demineralized, deionized water and bubbled with 95% O₂-5% CO₂ (pH, 7.4; PO₂, 550 mmHg; PCO₂, 38 mmHg). Polyethylene (PE)-50 Intramedic tubing was placed in the ascending aorta and connected via glass tubing to a buffer-filled reservoir for perfusion of the coronary circulation at a constant flow rate. Hearts were suspended in a temperature-controlled chamber maintained at 38.6 ± 0.5°C, and the coronary arteries were perfused by retrograde flow through the aortic stump cannula using a constant flow pump (model TIA, Ismatec, Cole Palmer; Vernon Hills, IL). Contractile function was assessed by measuring intraventricular pressure with Intramedic tubing (PE-50) threaded into the left ventricle. LV pressure (LVP) was measured with a Statham pressure transducer (model P23 ID, Gould Instruments; Oxnard, CA) attached to the cannula, and the rates of LVP rise (+dP/dt) and fall (dP/dt) were obtained using an electronic differentiator (model 7P20C, Grass Instruments; Quincy, MA), recorded (model 7DWL8P, Grass Recording Instruments), and transferred to a Dell Pentium computer. Starling relationships were determined by plotting LV systolic pressure and ±dP/dt_max values against increases in coronary flow or perfusate Ca²⁺ concentration. Because the heart rate varied, hearts were paced through an electrode attached to the right atrium (4.8-5.0 amps for 1-ms duration, Grass Stimulator).

Statistical analysis. Separate analysis of cardiac functions were conducted for each of LVP, +dP/dt, and dP/dt as a function of treatment group (factor 1) and either coronary flow rate or calcium level (factor 2). First, a repeated-measures analysis of variance was performed. In all instances, the factor 1-2 interaction was significant at the 0.05 level. A simple effects analysis, employing the Student-Newman-Keuls procedure, was then conducted at each level of factor 2 to discern differences among the treatment groups. The sum of the within-group and within-animal sums of squares was divided by their total degrees of freedom to provide an estimate of mean square error. This was divided by the harmonic mean of the treatment group sizes in obtaining the standard error of the mean. Satterthwaite's approximation was employed to obtain the degrees of freedom for the Studentized range critical values at the 0.05 level of significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Altered intracellular signaling in hearts of burned IRAK-deficient mice. We hypothesized that IRAK is involved in transducing the burn-induced signal in the myocardium. To test this hypothesis, we examined kinase cascade activation in WT and IRAK-deficient hearts from animals subjected to burn injury. Specifically, we assayed for activation of IKK-, JNK, and p38 MAPK using immunoprecipitation in vitro kinase reactions. We employed this assay because it directly measures catalytic activity of isolated kinases, thereby permitting assessment of both duration and intensity of signaling activity. Indirect assays, such as immunoblots against phosphorylated kinases, indicate that the kinase could be active but can neither distinguish between catalytically active and inactive phosphorylated forms and give no indication of the degree of kinase activity in the isolated extracts. We selected these signaling cascades for two reasons. First, they operate directly downstream of IRAK in both TLR and IL-1 receptor family signaling in cells such as macrophages and fibroblasts (21, 34), so we would anticipate impaired or altered signaling to these targets if burn injury signals through IRAK. Second, all three pathways regulate TNF- production in innate immune cells. NF-B activation is required for TNF- transcription following lipopolysaccharide (LPS) treatment of macrophages (30, 32), whereas the JNK-SAPK and p38 MAPK pathways regulate TNF mRNA translation (22, 33). TNF- has been implicated as a potent endogenous myocardial depressant substance produced in response to burns and other injuries (11, 26).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Impaired burn-triggered IB kinase- (IKK-) activity in interleukin-1 receptor-associated kinase (IRAK)-deficient mouse hearts. Hearts were harvested from wild-type (WT) and IRAK knockout (KO) mice at the indicated times following a 40% total body surface area (TBSA) full thickness burns. Heart tissue was homogenized in lysis buffer, and IK- was immunoprecipitated from the extracts. Precipitates were incubated in the presence of bacterially expressed recombinant IB- and radioactive ATP for 30 min, and the reaction mix was fractionated on a 10% SDS-polyacrylamide, transferred to a polyvinylidene difluoride membrane, and exposed to a phosphorimaging screen. IK- undergoes rapid activation in WT hearts within 30 min of burn injury. This is sustained up to 60 min but declines to baseline levels by 2 h. KO hearts, in contrast, show no significant activation of IKK- above baseline at any time point following burn injury. The experiment shown is one of three representative trials. IP, immunoprecipitation; GST, glutathione-S-transferase.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Shortened Jun NH2-terminal kinase (JNK) activation in IRAK-deficient mouse hearts. Mice were burned, and hearts were harvested as before. After lysis, JNK was immunoprecipitated from the lysates as described. A: immunoprecipitates were incubated in an in vitro kinase assay using c-Jun as a substrate, fractionated, and transferred as before. JNK from the hearts of WT burned animals exhibits increased activity starting 30 min after burn injury and continues through 2 h with some decline in activity by 4 h. IRAK-deficient hearts show a rapid upregulation by 30 min that is sustained up to 1 h but has returned to baseline by 2 h. B: to verify that equal amounts of JNK immunoprecipitated from lysates, parallel precipitations were performed, fractionated using SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Immunoblots of precipitated JNK were then performed as described. Bands shown represent the two major isoforms of JNK in mice (46 kDa and 55 kDa). Experiments shown are one of three representative trials. WB, Western blot.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Attenuated p38 activity in hearts from burned IRAK KO mice. Thermal injury, organ harvest, and lysate preparation was conducted as for previous in vitro kinase assays. p38 was immunoprecipitated from heart extracts using a rabbit polyclonal antiserum raised against recombinant protein. A: precipitated proteins were then used in in vitro kinase assays with a recombinant fragment of the transcription factor ATF2 as a substrate. Reactions were fractionated, transferred, and exposed to phosphorstorage screens as before. As seen in the previous kinase assays, within 30 min after burn injury, p38 undergoes catalytic activation, which begins to decline at 1 h. KO hearts, in contrast, show less pronounced activation above baseline at 30 min. Both WT and KO hearts exhibit a second smaller increase in catalytic activity at 2 h following burn injury, but this is not a consistent finding on repeated trials. B: immunoblot of immunoprecipitated p38 shows equivalent amounts of immunoprecipitated protein from all hearts.

IRAK inactivation partially protects against burn-induced cardiac contractile dysfunction. To determine whether differences in intracellular signaling were associated with altered cardiac function, we compared the contractile responses of WT and IRAK-deficient mice to burn injury. Animals underwent a 40% total body surface area burn, received fluid resuscitation, and were euthanized 24 h after injury, a time associated with maximal burn-induced cardiac contractile defects (41). The hearts were removed, and contractile function was assessed ex vivo using modified Langendorff isolated perfusion preparations. WT and IRAK-deficient hearts from sham-treated animals exhibit similar systolic and diastolic responses to increased coronary flow (Fig. 4) and perfusate calcium concentration (Fig. 5). As seen in Fig. 4A, hearts from WT burned mice had marked decreases in systolic performance, as determined by depressed maximal LV developed pressure as well as a reduced rate of maximal pressure generation (+dP/dt_max) compared with hearts from sham-treated animals. These same hearts also showed impaired diastolic function, with decreased ventricular relaxation rates (dP/dt_max) compared with unburned WT animals (Fig. 4A). These deficits were evident in responses to either increasing coronary flow rate (Fig. 4A) or increasing calcium ion concentration in the coronary perfusate (Fig. 5A).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   IRAK inactivation protects against burn-induced cardiac contractile function: responsiveness to increased coronary flow. WT and IRAK-deficient mice were subjected to thermal injury as described. Twenty-four hours after burns, contractile function was assessed using a modified Langendorff isolated perfusion preparation. A: WT hearts show marked impairment in maximal left ventricular (LV) pressure generation (LVP; left), maximal rate of intraventricular pressure generation (+dP/dtmax; middle), and maximal rate of relaxation (dP/dtmax; right) with increases in coronary flow rate following burn injury. B: IRAK-KO mice, in contrast, demonstrate less pronounced decreases in contractile function, either systolic or diastolic, following burn injury.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   IRAK inactivation protects against burn-induced cardiac contractile function: responsiveness to increased calcium ion concentration. WT and IRAK-deficient mice were subjected to thermal injury as described. Twenty-four hours after burns, contractile function was assessed using a modified Langendorff isolated perfusion preparation. A: WT hearts exhibit pronounced depression in maximal LVP, +dP/dtmax, and dP/dtmax with increases in calcium concentration following burn injury. B: IRAK-KO mice, in contrast, show less marked decreases in contractile function, both systolic and diastolic, following burn injury. There were no differences in baseline contractile function between sham-treated WT and KO mice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Results from the foregoing studies demonstrate that IRAK contributes to burn-induced myocardial contractile dysfunction. Because the Toll/IL-1 signaling pathway participates in both injury-sensing (afferent) and proinflammatory (efferent) responses, the site of IRAK function could be at either step. The timing of downstream pathway activation (IKK-b, JNK, and p38) within 30 min in WT hearts suggests that burn injury first engages this signaling cascade in the afferent limb of the innate immune response. Thus, it is likely that a burn injury signal first activates a TLR. Identification of burn injury-sensing TLR(s) may require some time because there are 10 known TLRs [(10, 29, 36) and Bruce Beutler, personal communication]. These studies are currently underway in this laboratory. TLR activation leads in turn to the production of multiple cytokines. Of these, TNF- and IL-1 have been implicated as endogenous myocardial depressant substances (4, 11). Attenuated signaling downstream of TLRs in IRAK-deficient mice leads to impaired TNF- production (34), therefore, protection against cardiac dysfunction seen in burn-injured IRAK KO mice may result from a diminished TNF- response. Moreover, the absence of IRAK function downstream of the IL-1 receptor in the efferent response may also protect IRAK-deficient mice from the cardiac depressant effects of this cytokine. Finally, IRAK catalytic activity has been implicated in TNF--mediated NF-B activation (39). Because prevention of NF-B activation blocks LPS-triggered cardiac contractile dysfunction (8, 12), loss of all IRAK function, including catalytic function, could further attenuate burn-induced myocardial dysfunction in IRAK-deficient mice by partially blocking TNF--dependent NF-B activity.

Because it may act in series, IRAK may exert a greater cumulative effect in burn-induced myocardial dysfunction than molecules operating at a single step in this response. This could make IRAK an attractive therapeutic target for the development of inhibitors to prevent myocardial depression after large burns. Presently, however, it is unclear whether IRAK catalytic activity (the most viable target for drug screening efforts) is required for the cardiac response to burn injury. Protection of IRAK-deficient mice from myocardial dysfunction merely indicates that the molecule participates in the response. Further studies, such as those using mice with a kinase-defective form of IRAK (so-called IRAK knockins), will be necessary to determine whether catalytic activity contributes to burn-triggered myocardial dysfunction.

Although we can reliably assay kinase cascade activation in the hearts of burned animals, the signal-to-noise ratio is low. This probably represents a dosing phenomenon. In previous studies of LPS-triggered signaling in the heart, we used a single pharmacological dose of LPS (1 mg/kg ip) and were able to delineate clearly the onset and termination of intracellular signaling in the heart (34). Pathway activation following burn injury is less intense and also more prolonged, suggesting a less potent (or lower dose) signal released more gradually.

The most important finding from these studies, however, is that IRAK inactivation partially blocks burn-induced contractile dysfunction. This finding carries two fundamental consequences. First, it further implicates Toll/IL-1 signaling in the regulation of physiological function outside the immune system: cardiac contractility. Our previous studies have already suggested that IRAK mediates LPS-induced contractile dysfunction and rescues mice from a lethal form of heart failure (34). Findings described here expand IRAK function to a third model of cardiac dysfunction, one more closely related to a clinical setting.

Incomplete protection against myocardial dysfunction afforded by IRAK inactivation also implies that the burn-induced myocardial depression is a complex phenomenon, operating through more than one pathway. This resistance is less pronounced than that seen following LPS challenge (34). There are at least two reasons why IRAK deletion offers a partial protective effect. First, the burn injury is severe, and it may generate several signals that lead to contractile failure. A subset of these signals may activate receptors that in turn activate IRAK (e.g., one or more TLRs), whereas others may engage receptors that signal via unrelated pathways. Alternatively, burn injury signals may activate receptors upstream of both IRAK-dependent and IRAK-independent pathways. This second notion has been supported in recent analyses of TLR4 signaling (20) and may pertain to burn injury as well. Furthermore, neither possibility is exclusive of the other and both could occur in vivo.

Two alternative models of how Toll/IL-1 signaling affects contractility can be envisioned. First, this signaling system could operate in the myocyte to effect myocyte contractile dysfunction (a cell-autonomous model). Conversely, nonmyocyte cell types could sense the burn injury signal (e.g., monocytes, cardiac fibroblasts, and endocardial or endothelial cells) and produce secondary signals (e.g., cytokines such as TNF- and IL-1) that directly depress myocyte contractility (a cell nonautonomous model). The first model has not been scrutinized, and although the second model has been examined, definitive support for it is still lacking. Furthermore, how burn injury is sensed and results in impaired contractility may share features of both models.

Finally, these studies highlight the complexity of the phenomenon of burn-induced myocardial depression. The signaling assays detect a burn-induced signal within 30 min after injury. The differential pattern of pathway activation in WT and IRAK-deficient hearts and partially spared contractility of hearts from burned animals lacking IRAK indicate that one or more factors produced in response to burns signal through IRAK and the Toll/IL-1 pathway but provide only a partial explanation for the cardiac response to burn injury. Furthermore, use of a single genetic mutant in these studies constrains the conclusions to be drawn about IRAK function in this complex response. Whereas the findings point to a role for IRAK, delineation of the exact contribution will require additional studies. It is unclear, for example, how chronic IRAK inactivation affects the cardiac response to burn injury. IRAK deficiency from birth could underestimate the impact of this protein on burn-induced cardiac depression because of the engagement of multiple mechanisms to compensate for its absence. Alternatively, the opposite scenario, one in which IRAK inactivation exaggerates the role of the molecule in myocardial dysfunction, could also pertain. Similar difficulties arise with the use of pharmacological tools to interrupt signaling through a pathway: in vivo preburn or postburn administration of an agent thought to be specific for some aspect of the pathway may exert multiple effects, both in the heart and throughout the rest of the body. Systematic application of multiple experimental approaches, including genetic (using additional IRAK alleles, as well as other pathway mutants and transgenics), pharmacological (with known receptor and kinase inhibitors, as well as ligand chelators), and hybrid (delivery of genetically engineered peptides and antisense technology) should help clarify the exact role of IRAK in the myocardial response to severe burns.