Deficiency in Heat Shock Factor 1 (HSF-1) Expression Exacerbates Sepsis-induced Inflammation and Cardiac Dysfunction.

In the present study, we investigated whether absence of heat shock factor 1 (HSF-1) and inability to increase myocardial expression of heat shock proteins alter septic responses of inflammatory cytokines and myocardial contractility. HSF-1 knockout (hsf -/-) mice and wild type litter mates underwent a sterile (lipopolysaccharide; LPS) or infectious (Streptococcus pneumoniae or Klebsiella pneumoniae) septic challenge. Production of cytokines, TNF, IL-1ß, IL-6 and IL-10, in the blood and from cardiomyocytes was exaggerated in the hsf -/- mice compared to responses measured in wild type mice given an identical septic challenge. This enhanced compartmentalized myocardial inflammation was associated with significantly decreased cardiac contraction and diminished relaxation in the hsf -/- mice. However, lacking HSF-1 expression did not affect intracellular calcium and sodium responses in cardiomyocytes isolated from septic challenged mice, suggesting that ion loading was not a major or sustaining cause of the greater myocardial contractile defects in hsf -/- mice. In conclusion, our data indicated that HSF-1 and downstream heat shock proteins are essential components to support cardiac function in sepsis. Further studies are warranted to further define the precise mechanisms of HSF-1 mediated cardiac protection.

Molecular or pharmacologic inhibition of the CD14 signaling pathway protects against burn-related myocardial inflammation and dysfunction.

Signaling through toll-like receptor 4 (TLR4) plays an obligate role in burn-related myocardial dysfunction. We hypothesized that signaling through CD14, a cellular receptor for endotoxin that lacks a transmembrane domain but is coupled to TLR4, also plays a role in postburn myocardial inflammation and dysfunction. Burn covering 40% total body surface area (or sham burn for controls) was produced in wild-type (WT) and CD14 knockout (KO) as well as vehicle-treated and geldanamycin-treated WT mice (1 microg/g body weight) to inhibit CD14 signaling. Groups included (1) WT shams, (2) CD14 KO sham, (3) WT burns, (4) CD14 KO burns, (5) vehicle-treated WT shams, (6) geldanamycin-treated WT shams, (7) vehicle-treated WT burns, and (8) geldanamycin-treated WT burns. Twenty-four hours after burn, cardiac function (Langendorff) and cardiomyocyte secretion of inflammatory cytokines TNF-alpha, IL-1 beta, and IL-6 (in pg/mL; 5 x 10(4) myocytes) were studied in all groups. Relative to sham WT controls, burn trauma in increased cardiac myocyte secretion of inflammatory cytokines (TNF-alpha, IL-1 beta, and IL-6 rose from 59 +/- 10 to 171 +/- 8; 6 +/- 0.2 to 78 +/- 1; and 88 +/- 3 to 170 +/- 12 pg/mL, respectively; P < 0.05) and produced robust cardiac contractile dysfunction (left ventricular pressure and +dP/dt fell from 105 +/- 4 to 73 +/- 5 mmHg and 2,400 +/- 73 to 1,803 +/- 90 mmHg/s; P < 0.05). Inability to signal through the CD14/TLR4 pathway (induced by CD14/KO or inhibition of CD14 expression by administration of geldanamycin) attenuated TNF-alpha, IL-1 beta, and IL-6 production in response to burn injury and improved postburn myocardial contractile function. Our data suggest that signaling through the CD14 pathway plays an obligate role in cardiac inflammation/dysfunction which occurs after major burn injury.

Increasing percent burn is correlated with increasing inflammation in an adult rodent model.

Burn injury has been associated with systemic/compartmental inflammatory responses and myocardial dysfunction. We hypothesized that burn size correlates with the extent of cardiac inflammatory response/contractile dysfunction. Adult male Sprague-Dawley rats were divided to receive anesthesia, a 3-degree burn covering 20%, 30%, 40%, or 60% total body surface area (TBSA) plus fluid resuscitation (lactated Ringer, 4 mL/kg per percent burn); sham burn animals were included as controls. There were seven rats in each group. Rats were euthanized Twenty-four h postburn, and TNF-alpha, IL-1beta, and IL-6 were measured in the plasma and in supernatant from isolated cardiac myocytes by enzyme-linked immunosorbent assay. In addition, left ventricular function (Langendorff) was studied in vitro, and troponin levels were measured by enzyme-linked immunosorbent assay. There were progressive, statistically significant increases in plasma and myocyte inflammatory cytokine levels, as well as plasma troponin with increasing burn size. Similarly, left ventricular pressure (in millimeters of mercury) and +/-dP/dtmax (in millimeters of mercury per second) progressively fell with increasing burn size. However, myocardial contractile depression induced by 60% TBSA burn was similar to that produced by 40% TBSA burn. These data suggest that the degree of inflammatory response, cardiac tissue injury, and myocardial contractile depression were correlated directly with the percent TBSA burn. However, unlike inflammation and cardiac tissue damage, myocardial contractile depression reached a plateau, with maximal myocardial contraction and relaxation defects observed at 40% TBSA burn, which were not further aggravated by a larger (60%) burn.

Burn injury decreases myocardial Na-K-ATPase activity: role of PKC inhibition.

Cardiomyocyte sodium accumulation after burn injury precedes the development of myocardial contractile dysfunction. The present study examined the effects of burn injury on Na-K-ATPase activity in adult rat hearts after major burn injury and explored the hypothesis that burn-related changes in myocardial Na-K-ATPase activity are PKC dependent. A third-degree burn injury (or sham burn) was given over 40% total body surface area, and rats received lactated Ringer solution (4 ml.kg(-1).% burn(-1)). Subgroups of rats were killed 2, 4, or 24 h after burn (n = 6 rats/time period), hearts were homogenized, and Na-K-ATPase activity was determined from ouabain-sensitive phosphate generation from ATP by cardiac sarcolemmal vesicles. Additional groups of rats were studied at several times after burn to determine the time course of myocyte sodium loading and the time course of myocardial dysfunction. Additional groups of sham burn-injured and burn-injured rats were given calphostin, an inhibitor of PKC, and Na-K-ATPase activity, cell Na(+), and myocardial function were measured. Burn injury caused a progressive rise in cardiomyocyte Na(+), and myocardial Na-K-ATPase activity progressively decreased after burn, while PKC activity progressively rose. Administration of calphostin to inhibit PKC activity prevented both the burn-related decrease in myocardial Na-K-ATPase and the rise in intracellular Na(+) and improved postburn myocardial contractile performance. We conclude that burn-related inhibition of Na-K-ATPase likely contributes to the cardiomyocyte accumulation of intracellular Na(+). Since intracellular Na(+) is one determinant of electrical-mechanical recovery after insults such as burn injury, burn-related inhibition of Na-K-ATPase may be critical in postburn recovery of myocardial contractile function.

Bactericidal/permeability increasing protein attenuates the myocardial inflammation/dysfunction that occurs with burn complicated by subsequent infection.

Intubation and mechanical ventilation after burn contribute to pneumonia-related infection. Although postburn presence or absence of endotoxin has been described, inactivation of Toll-like receptor 4 signaling has been shown to improve postburn organ function, suggesting that LPS participates in burn-related susceptibility to infection. We hypothesized that bactericidal/permeability-increasing protein (rBPI) given postburn would attenuate myocardial inflammation/dysfunction associated with postburn septic challenge given 7 days postburn. Rats were given burn over 40% total body surface area, lactated Ringer 4 ml.kg(-1).% burn(-1); burns received either vehicle or rBPI, 1 mg.kg(-1).h(-1) for 48 h postburn. Postburn day 7, subgroups of burns and shams were given intratracheal Klebsiella pneumoniae, 4 x 10(6) CFU to produce burn complicated by sepsis; additional sham and burn subgroups received intratracheal vehicle to produce sham sepsis. Vehicle-treated groups: 1) sham burn + sham sepsis 2) sham burn + sepsis, 3) burn + sham sepsis, 4) burn + sepsis. rBPI-treated groups: 5) sham burn + sham sepsis, 6) sham burn + sepsis, 7) burn + sham sepsis, 8) burn + sepsis. Cardiomyocyte cytokine secretion and myocardial function were studied 24 h after septic challenge, postburn day 8. Pneumonia-related infection 8 days after vehicle-treated burn produced myocyte cytokine secretion (pg/ml), indicated by increased myocyte TNF-alpha, 549 +/- 46; IL-1beta, 50 +/- 8; IL-6, 286 +/- 3 levels compared with levels in sham myocytes (TNF-alpha, 88 +/- 11; IL-1beta, 7 +/- 1; IL-6, 74 +/- 10; P < 0.05). Contractile dysfunction was evident from lower left ventricular pressure +/-dP/dt values in this group compared with sham. rBPI attenuated myocyte cytokine responses to septic challenge and improved contractile function, suggesting that burn-related mobilization of microbial-like products contribute to postburn susceptibility to infection.

Effects of burn injury on myocardial signaling and cytokine secretion: Possible role of PKC.

This study examined the effects of major burn injury on the cellular distribution of several PKC isoforms in adult rat hearts and examined the hypothesis that PKC plays a regulatory role in cardiomyocyte cytokine secretion. Burn trauma was given over 40% total body surface area in Sprague-Dawley rats. An in vitro model of burn injury included addition of burn serum, 10% by volume, to primary cardiomyocyte cultures (collagen perfusion). In vivo burn injury produced redistribution of PKCdelta, PKCepsilon, and PKCalpha from the cytosol (soluble) to the membrane (particulate) component of the myocardium. This activation of the PKC isoforms was evident 2 h after burn injury and progressively increased over 24 h postburn. Addition of burn serum to isolated myocytes produced similar PKC isoform redistribution from the soluble to the particulate compartment, promoted myocyte Ca2+ and Na+ loading, and promoted robust myocyte secretion of inflammatory cytokines similar to that reported after in vivo burn injury. Pretreating cardiomyocytes with either calphostin or PKCepsilon inhibitory peptide, a potent inhibitor of PKCepsilon, prevented burn serum-related redistribution of the PKCepsilon isoform and prevented burn serum-related cardiomyocyte secretion of TNF-alpha, IL-1beta, IL-6, and IL-10. These data suggest that the PKCepsilon isoform plays a pivotal role in myocardial inflammatory response to injury, altering cardiac function by modulating cardiomyocyte inflammatory cytokine response to injury.

The effects of early excision and grafting on myocardial inflammation and function after burn injury.

BACKGROUND: Sepsis is a frequent complication of burn injury despite absence of confirmed infection. Numerous investigators have proposed that the burn wound itself is a primary stimulus for postburn inflammation, and that early excision of the burn wound attenuates the hypermetabolic and inflammatory responses to burn injury. However, others have suggested that aggressive fluid resuscitation and correction of postburn fluid and electrolyte deficits should be the primary focus of intervention in the first 24 hours postburn. This present study determined whether excision and grafting of the burned wound within 30 minutes after injury abrogated myocardial inflammation and contractile defects that occur after burn injury. METHODS: In group 1, Sprague Dawley rats were given a third-degree burn over 20% total body surface area (TBSA), whereas rats in group 2 had burns over 30% TBSA and no wound excision. Rats in groups 3 and 4 had burn over 20% and 30% TBSA, respectively, followed by-wound excision and grafting (WE/G) within 30 minutes after completing burn injury. Group 5 included sham burn with no excision, whereas rats in groups 6 and 7 included shams that had either 20% or 30% normal skin excised and grafted to provide appropriate surgical controls. All rats received lactated Ringer's (4 mL/kg/% burn or percent wound excision). Twenty-four hours postburn, hearts were perfused (Langendorff) to assess ventricular function; myocytes were isolated to examine cytokine secretion and Ca2+/Na+ homeostasis. RESULTS: Burn in the absence of wound excision produced myocardial inflammation and contractile defects as indicted by a lower left ventricular pressure and lower rate of left ventricular pressure rise (+dP/dt) and fall (-dP/dt) response to maximal increases in preload or perfusate Ca2+ compared with responses measured in sham hearts. WE/G within 30 minutes after burn injury reduced myocyte secretion of proinflammatory cytokines and improved left ventricular pressure and +/-dP/dt responses to inotropic challenge. CONCLUSION: Cutaneous burn injury and the loss of the skin barrier function contribute, in part, to the myocardial inflammation which, in turn, contributes to myocardial contractile dysfunction that is characteristic of major burn injury.

Antioxidant vitamin therapy alters sepsis-related apoptotic myocardial activity and inflammatory responses.

This study examined the effects of antioxidant vitamins on several aspects of sepsis-related myocardial signaling cascades. Sprague-Dawley rats were divided into four groups: group 1, vehicle-treated shams; group 2, sham-operated rats given antioxidant vitamins (vitamin C, 24 mg/kg; vitamin E, 20 U/kg; vitamin A, 417 U/kg; and zinc, 3.7 ng/kg) by oral gavage in 0.5 ml water twice daily for 3 days and no septic challenge (vitamin-treated, sham-operated rats); group 3, intratracheal delivery of Streptococcus pneumoniae, 4 x 10(6) colony forming units in a volume of 0.3 ml phosphate buffer solution; group 4, S. pneumonia challenge as described for group 3 plus antioxidant vitamins (as described for group 2). Hearts collected 24 h after septic challenge were used to examine several aspects of cell signaling and ventricular function. As a result, when compared with sham-operated rats, sepsis in the absence of antioxidant therapy promoted NF-kappaB activation, increased mitochondrial cytochrome c release, increased myocyte cytokine secretion, increased caspase activation, and impaired left ventricular function. Antioxidant vitamin therapy plus septic challenge prevented NF-kappaB activation, reduced mitochondrial cytochrome c release, decreased caspase activity, abrogated cardiomyocyte secretion of inflammatory cytokines, and improved myocardial contractile function. In conclusion, antioxidant vitamin therapy abrogated myocardial inflammatory cytokine signaling and attenuated sepsis-related contractile dysfunction, suggesting that antioxidant vitamin therapy may be a potential approach to treat injury and disease states characterized by myocardial dysfunction.

Hypertonic saline dextran after burn injury decreases inflammatory cytokine responses to subsequent pneumonia-related sepsis.

The present study examined the hypothesis that hypertonic saline dextran (HSD), given after an initial insult, attenuates exaggerated inflammation that occurs with a second insult. Adult rats (n = 15 per group) were divided into groups 1 (sham burn), 2 [40% total body surface area burn + 4 ml/kg isotonic saline (IS) + 4 ml.kg(-1).% burn(-1) lactated Ringer solution (LR)], and 3 (burn + 4 ml/kg HSD + LR), all studied 24 h after burns. Groups 4 (sham burn), 5 (burn + IS + LR), and 6 (burns + HSD + LR) received intratracheal (IT) vehicle 7 days after burns; groups 7 (burn + IS + LR) and 8 (burn + HSD + LR) received IT Streptococcus pneumoniae (4 x 10(6) colony-forming units) 7 days after burn. Groups 4-8 were studied 8 days after burn and 24 h after IT septic challenge. When compared with sham burn, contractile defects occurred 24 h after burn in IS-treated but not HSD-treated burns. Cardiac inflammatory responses (pg/ml TNF-alpha) were evident with IS (170 +/- 10) but not HSD (45 +/- 5) treatment vs. sham treatment (80 +/- 15). Pneumonia-related sepsis 8 days after IS-treated burns (group 7) exacerbated TNF-alpha responses/contractile dysfunction vs. IS-treated burns in the absence of sepsis (P < 0.05). Sepsis that occurred after HSD-treated burns (group 8) had less myocyte TNF-alpha secretion/better contractile function than IS-treated burns given septic challenge (group 7, P < 0.05). We conclude that an initial burn injury exacerbates myocardial inflammation/dysfunction occurring with a second insult; giving HSD after the initial insult attenuates myocardial inflammation/dysfunction associated with a second hit, suggesting that HSD reduces postinjury risk for infectious complications.

Cardiac myocyte accumulation of calcium in burn injury: cause or effect of myocardial contractile dysfunction.

Myocardial calcium accumulation and myocardial injury occur after burn trauma. However, whether altered calcium dyshomeostasis occurs as a result of myocardial injury/dysfunction or whether altered calcium handling initiates myocardial injury and contractile abnormalities remains unclear. In addition, the specific mechanisms by which burn injury promotes calcium entry into cardiac myocytes, specifically L-type channels and the sodium-calcium exchanger, remain unclear. This study addressed the hypothesis that burn trauma promotes cardiomyocyte calcium accumulation, in part, via reverse mode function of the sodium/calcium exchanger and via L-type channels. Myocardial calcium accumulation, in turn, alters performance. Burn trauma (40% TBSA and sham burn for controls) was accomplished in Sprague-Dawley rats. Burns received fluid resuscitation (lactated Ringer's at 4 ml/kg/% burn). Hearts were harvested at several time points after burn injury (2, 4, 8, 12, 24, 48, 72 hours, and 8 days after burn) and were perfused with collagenase/bovine serum albumin-containing buffer to produce enzymatic digestion. Myocytes were then resuspended in MEM buffer, loaded with 2 mug/ml Fura 2AM for 45 minutes or 2 microg of sodium-binding benzofurzan isophthalate for 2 hours at room temperature in the dark. Cells were washed to remove extracellular dye and placed on a glass slide on the stage of a Nikon inverted microscope interfaced with Grooney optics. A computer-controlled filter changer allowed alternation between 340/380 excitation wavelengths; fluorescence was measured at 510 nm. Cardiac function (Langendorff) was measured in parallel groups at each time period (n = 6-7 hearts/time point). Cardiomyocyte accumulation of sodium occurred before alterations in myocyte calcium levels, and sodium/calcium dyshomeostasis preceded cardiac contraction deficits. Interventions that altered calcium flux through L-type channels (amlodipine) or sodium/calcium exchange (amiloride) attenuated burn-related myocyte calcium accumulation and improved contractile function. Our finding that myocyte sodium loading precedes myocyte calcium accumulation suggests a role for the reverse mode function of the sodium/calcium exchanger in burn trauma.

Tumor necrosis factor-alpha-induced caspase activation mediates endotoxin-related cardiac dysfunction.

OBJECTIVE: Sepsis-induced cardiac dysfunction is a serious clinical syndrome characterized by hypotension, decreased systemic vascular resistance, and elevated cardiac index. Although cytokines such as tumor necrosis factor (TNF)-alpha have been shown to play a significant role early in this response, the downstream effects of TNF-alpha signaling on cardiac function, specifically its relationship to apoptosis, have not been fully elucidated. DESIGN: Previous studies from our laboratory have identified endotoxin-induced apoptosis in cardiac cells in vitro. To further determine the role of lipopolysaccharide-induced apoptosis in vivo, mice were injected intraperitoneally with lipopolysaccharide (4 mg/kg), and cardiac apoptosis was detected and inhibited using a broad-spectrum caspase inhibitor. SETTING: University research laboratory. SUBJECTS: Adult male wild-type (B6:129PF1/J) and TNF receptor 1/receptor 2 (TNFR-1/2) knockout mice (B6;129S-Tnfrsf1aTnfrsf1b). INTERVENTIONS: We sought to determine the dependence of cardiac apoptosis on TNF-alpha signaling and determine the physiologic role of caspase activation on lipopolysaccharide-induced cardiac dysfunction. MEASUREMENTS AND MAIN RESULTS: Cardiac apoptosis was determined at baseline and at 2, 4, 8, and 24 hrs by detection of capase-3 and -8 activity, cytoplasmic levels of Bax/Bcl-2, cleaved caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL) staining of histologic sections in wild-type and TNFR-1/2 knockout mice. To determine the role of caspase activation in lipopolysaccharide-induced cardiac dysfunction, a broad-spectrum caspase inhibitor Z-Val-Ala-Asp (ome)-FMK (sad) was given, and cardiac function was determined in isolated beating hearts (Langendorff preparation). Our experiments determined that caspase-3-dependent apoptosis was active in cardiac tissue by 2 hrs and that this activation was completely mediated by TNFR-1/2. The Bax/Bcl-2 ratios supported the finding and time course of apoptosis, whereas TUNEL staining of cardiac tissue sections identified sporadic apoptotic ventricular cells. The administration of zVAD significantly inhibited myocardial caspase-3 activity and preserved cardiac physiologic function (Langendorff preparation). CONCLUSIONS: Endotoxin induces a TNF-alpha-dependent apoptotic cascade in the myocardium, which contributes to the development of cardiac dysfunction.

Macrophage migration inhibitory factor mediates late cardiac dysfunction after burn injury.

We have recently demonstrated that macrophage migration inhibitory factor (MIF) is a myocardial depressant protein and that MIF mediates late, prolonged cardiac dysfunction after endotoxin challenge in mice. Because many factors, including endotoxin, have been implicated in the pathogenesis of cardiac dysfunction after burn injury, we tested the hypothesis that MIF might also be the mediator of prolonged cardiac dysfunction in this model. At 4 h after 40% total body surface area burn in anesthetized mice, serum MIF levels increased significantly compared with baseline (2.2-fold). This increase was accompanied by a significant decrease in cardiac tissue MIF levels (2.1-fold decrease compared with controls). This pattern was consistent with MIF release from preformed cytoplasmic stores in the heart and other organs. To determine whether MIF mediates cardiac dysfunction after burn injury, mice were pretreated with anti-MIF neutralizing monoclonal antibodies or isotype control antibodies. Beginning 4 h after burn injury (and continuing through 48 h), burned mice demonstrated a significantly depressed left ventricular shortening fraction of 38.6 +/- 1.8%, compared with the normal controls (56.0 +/- 2.6%). Mice treated with anti-MIF displayed an initial depression of cardiac function similar to nontreated animals but then showed rapid restoration of cardiac function with complete recovery by 24 h, which persisted for the duration of the protocol. This study is the first to demonstrate that MIF mediates late, prolonged cardiac dysfunction after burn injury and suggests that MIF blockade should be considered a therapeutic target for the treatment of burn injury.

Bone marrow-derived cells contribute to contractile dysfunction in endotoxic shock.

How infection precipitates depressed contractility is incompletely understood but may involve the immune, nervous, and endocrine systems as well as the heart itself. In this study, we examined the role of Toll-like receptor 4 (TLR4) in LPS-induced myocardial contractile depression. Eighteen hours following endotoxin challenge, we compared contractile responses in hearts from wild-type (WT) and TLR4-deficient mice using modified Langendorff preparations. Unlike hearts from WT mice, TLR4-deficient hearts did not reveal significant contractile dysfunction following LPS administration, as measured by decreased responses in maximal left ventricular pressure, +dP/dtmax, and -dP/dtmax in ex vivo Langendorff preparations. These findings indicate a requirement for TLR4 in LPS-induced contractile depression. To determine the contribution of bone marrow-derived TLR4 function to LPS-induced myocardial dysfunction, we generated TLR4 chimeras using adoptive transfer between histocompatible mouse strains: either TLR4-deficient mice with TLR4+/+ bone marrow-derived cells or TLR4+/+ animals lacking TLR4 in their hematopoietic cells. We then compared the contractile responses of engrafted animals after LPS challenges. Engraftment of TLR4-deficient mice with WT marrow restored sensitivity to the myocardial depressant effects of LPS in TLR4-deficient hearts (P < 0.05). Inactivation of bone marrow-derived TLR4 function, via transplantation of WT mice with TLR4-/- marrow, however, did not protect against the depressant effect of endotoxin. These findings indicate that bone marrow-derived TLR4 activity is sufficient to confer sensitivity to mice lacking TLR4 in all other tissues. However, because inactivation of marrow-derived TLR4 function alone does not protect against endotoxin-triggered contractile dysfunction, TLR4 function in other tissues may also contribute to this response.

Effects of burn serum on myocardial inflammation and function.

Large cutaneous burns are clearly recognized to produce acute myocardial contractile dysfunction. This study used a model of burn serum challenge in either primary cardiomyocyte cultures or isolated perfused hearts to examine several aspects of burn-serum-related contractile dysfunction as well as myocardial inflammatory responses. Despite the absence of detectable LPS in burn serum, pretreating isolated cells or perfused hearts with recombinant bactericidal permeability-increasing protein (rBPI21) prevented both the inflammatory cytokine cascade and the cardiac contractile dysfunction induced by burn serum treatment of myocytes or ventricular muscle preparations. Our finding that anti-TNF strategies applied to isolated myocytes or hearts before burn serum challenge prevented myocardial inflammation and contractile dysfunction suggested that LPS or LPS-like factors may require the action of second messengers such as TNF-alpha and IL-1beta to mediate LPS-related myocardial depressant effects. Our finding that experimental approaches neutralizing circulating LPS provided cardioprotection suggested that bacterial endotoxin or LPS-like molecules contribute, in part, to burn-related myocardial contractile dysfunction.

Selective decontamination of the digestive tract attenuated the myocardial inflammation and dysfunction that occur with burn injury.

This study examined the effects of oral antibiotics to selectively decontaminate the digestive tract (SDD) on postburn myocardial signaling, inflammation, and function. We hypothesized that antibiotic therapy to eliminate pathogens from the gastrointestinal (GI) tract would reduce myocardial inflammatory responses and improve postburn myocardial performance. Sprague-Dawley rats received polymyxin E (15 mg), tobramycin (6 mg), and 5-flucytosin (100 mg) by oral gavage twice daily for 3 days preburn and 24 h postburn. Experimental groups included 1) sham burn given vehicle (3 ml water), 2) sham plus SDD, 3) burn over 40% total body surface area (TBSA) plus SDD, and 4) burn over 40% TBSA given vehicle. All burns received lactated Ringer solution (4 mg.kg(-1).%burn(-1)); myocardial signaling (PKCepsilon/p38 MAPK/NF-kappaB) was studied 2, 4, and 24 h postburn; and cytokine secretion (systemic and myocyte secreted cytokines, ELISA) and cardiac function were examined 24 h postburn. Vehicle-treated burn injury increased myocardial PKCepsilon/p38 MAPK expression, promoted NF-kappaB nuclear translocation, promoted TNF-alpha, IL-1beta, IL-6, and IL-10 secretion, and impaired myocardial function. SDD attenuated burn-related proinflammatory myocardial signaling, cytokine secretion, and myocardial contractile defects. Our data suggest that burn-related loss of GI barrier function and translocation of microbial products serve as upstream mediators of postburn myocardial inflammatory signaling and dysfunction.

Protective role of heat stress in burn trauma.

OBJECTIVE: This study was designed to determine whether cutaneous burn injury up-regulated expression of myocardial heat shock protein (HSP)70 and to determine a potential cardioprotective role of inducible HSP70 (iHSP70) in postburn myocardial contractile function. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Adult Hartley guinea pigs. INTERVENTIONS: The first set of studies determined whether heat stress (increasing body temperature to 42 degrees C for 20 mins) in adult Hartley guinea pigs would increase expression of myocardial iHSP70. MEASUREMENTS AND MAIN RESULTS: Our model of heat stress increased expression of inducible HSP in the myocardium (Western blot), and this response persisted 1, 2, 4, and 24 hrs after the initial heat stress. We then determined whether burn trauma over 40% total body surface area (TBSA) increased myocardial expression of iHSP70. Time-matched sham and burned guinea pigs were killed 1, 2, 4, 12, 18, or 24 hrs postburn, and hearts were used either to examine myocardial iHSP70 expression by Western blot or to determine myocardial contractile function (Langendorff). Burn trauma produced a two-fold increase in myocardial iHSP70 that was evident as early as 1 hr postburn and persisted 24 hrs postburn; increased iHSP70 expression occurred despite only a modest and transient increase in body temperature after burn trauma. We then determined whether heat shock stress before burn trauma provided a protective or detrimental effect on cardiac function. Body temperature was increased to 42 degrees C for 20 mins, animals were allowed to recover, and body temperature returned to baseline; burn trauma was then produced (40% TBSA) either 1, 2, 4, or 24 hrs after the initial heat stress. Myocardial contraction and relaxation deficits were evident after burn trauma alone; however, heat stress 1 hr before burn trauma improved left ventricular developed pressure and positive or negative maximum change in pressure in time and shifted left ventricular function curves upward and leftward from those calculated for burn in the absence of heat stress, indicating improved ventricular performance. Increasing the time between the initial heat stress and burn injury decreased the cardioprotective effects of heat stress. Thus, organ protection was evident only when the time period between the initial heat stress and the second insult was brief (1 hr). CONCLUSIONS: Our finding that the amount of myocardial iHSP70 remained constantly elevated after heat stress while the cardio-protective effect afforded by a prior heat stress declined with time suggested that the initial heat stress evoked several compensatory/adaptive mechanisms that may include modulation of autonomic nervous system responses, changes in metabolic function, modulation of pro/anti-inflammatory cytokine responses, and heat stress-related alterations in antioxidant capacity.

Gender-related differences in myocardial inflammatory and contractile responses to major burn trauma.

Gender-related differences in immune responses to hemorrhage and sepsis have been described. However, most trauma studies continue to limit experimental models to males to avoid the variable responses associated with hormonal fluctuation in proestrus/estrus females. In the present study, male and female (either diestrus or proestrus/estrus) Sprague-Dawley rats (250-325 g) were given a third-degree scald burn over 40% total body surface area and fluid resuscitated (4 ml/kg per %burn of lactated Ringer solution); sham burn males and diestrus as well as sham burn proestrus/estrus female rats were included to provide controls. Twenty-four hours postburn, hearts were either perfused to examine mechanical function (Langendorff, n = 8 to 9 hearts/group) or to prepare cardiomyocytes (collagenase digestion, n = 4 to 5 hearts/group). Left ventricular developed pressure and the positive and negative first derivative of left ventricular pressure responses to increases in preload were significantly lower in burned males compared with responses measured in either burned proestrus/estrus or burned diestrus females; burn trauma increased cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide to a lesser extent in proestrus/estrus females than levels secreted by either diestrus females or males. Similarly, myocytes from proestrus/estrus females accumulated significantly less sodium/calcium compared with values measured in males (P < 0.05). Our data confirm gender-related differences in myocardial function and myocardial inflammatory responses to burn injury.

Macrophage migration inhibitory factor is a cardiac-derived myocardial depressant factor.

Macrophage migration inhibitory factor (MIF) is a pluripotent proinflammatory cytokine that is ubiquitously expressed in organs, including the heart. However, no specific role for MIF in modulating cardiac performance has yet been described. Therefore, we examined cardiac MIF expression in mice after LPS challenge (4 mg/kg) and tested the hypothesis that MIF is a mediator of LPS-induced cardiac dysfunction. Western blots of whole heart lysates, as well as immunohistochemistry, documented constitutive MIF protein expression in the heart. Cardiac MIF protein levels significantly decreased after LPS challenge, reaching a nadir at 12 h, and then returned to baseline by 24 h. This pattern was consistent with MIF release from cytoplasmic stores after endotoxin challenge. After release of protein, MIF mRNA levels increased 24-48 h postchallenge. To determine the functional consequences of MIF release, we treated LPS-challenged mice with anti-MIF neutralizing antibodies or isotype control antibodies. Anti-MIF-treated animals had significantly improved cardiac function, as evidenced by a significant improvement in left ventricular (LV) fractional shortening percentage at 8, 12, 24, and 48 h after endotoxin challenge. In support of these findings, perfusion of isolated beating mouse hearts (Langendorff preparation) with recombinant MIF (20 ng/ml) led to a significant decrease in both systolic and diastolic performance [LV pressure (LVP), positive and negative first derivative of LVP with respect to time, and rate of LVP rise at developed pressure of 40 mmHg]. This study demonstrates that MIF mediates LPS-induced cardiac dysfunction and suggests that MIF should be considered a pharmacological target for the treatment of cardiac dysfunction in sepsis and potentially other cardiac diseases.

IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction.

Myocardial contractile dysfunction accompanies both systemic and cardiac insults. Septic shock and burn trauma can lead to reversible contractile deficits, whereas ischemia and direct inflammation of the heart can precipitate transient or permanent impairments in contractility. Many of the insults that trigger contractile dysfunction also activate the innate immune system. Activation of the innate immune response to infection is coordinated by the conserved Toll/interleukin-1 (IL-1) signal transduction pathway. Interestingly, components of this pathway are also expressed in normal and failing hearts, although their function is unknown. The hypotheses that Toll/IL-1 signaling occurs in the heart and that intact pathway function is required for contractile dysfunction after different insults were tested. Results from these experiments demonstrate that lipopolysaccharides (LPS) activate Toll/IL-1 signaling and IL-1 receptor-associated kinase-1 (IRAK1), a critical pathway intermediate in the heart, indicating that the function of this pathway is not limited to immune system tissues. Moreover, hearts lacking IRAK1 exhibit impaired LPS-triggered downstream signal transduction. Hearts from IRAK1-deficient mice also resist acute LPS-induced contractile dysfunction. Finally, IRAK1 inactivation enhances survival of transgenic mice that develop severe myocarditis and lethal heart failure. Thus the Toll/IL-1 pathway is active in myocardial tissue and interference with pathway function, through IRAK1 inactivation, may represent a novel strategy to protect against cardiac contractile dysfunction.

I kappa B overexpression in cardiomyocytes prevents NF-kappa B translocation and provides cardioprotection in trauma.

This study examined the effects of either IkappaBalpha overexpression (transgenic mice) or N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) administration (proteosome inhibitor in wild-type mice) on cardiomyocyte secretion of tumor necrosis factor-alpha (TNF-alpha) and on cardiac performance after burn trauma. Transgenic mice were divided into four experimental groups. IkappaBalpha overexpressing mice were given a third-degree scald burn over 40% of the total body surface area or wild-type littermates were given either a scald or sham burn to provide appropriate controls. Pharmacological studies included ALLN (20 mg/kg) administration in either burned wild-type mice or wild-type shams. Burn trauma in wild-type mice promoted nuclear factor-kappaB (NF-kappaB) nuclear translocation, cardiomyocyte secretion of TNF-alpha, and impaired cardiac performance. IkappaBalpha overexpression or ALLN treatment of burn trauma prevented NF-kappaB activation in cardiac tissue, prevented cardiomyocyte secretion of TNF-alpha, and ablated burn-mediated cardiac contractile dysfunction. These data suggest that NF-kappaB activation and inflammatory cytokine secretion play a significant role in postburn myocardial abnormalities.