Biomedical subjects

Jureta W Horton

Publications and source records attributed to Jureta W Horton.

At least 19 recordsLinked to original sources

PubMed2007

Role of interleukin-6 in cardiac inflammation and dysfunction after burn complicated by sepsis.

To examine the role of myocardial interleukin-6 (IL-6) in myocardial inflammation and dysfunction after burn complicated by sepsis, we performed 40% total body surface area contact burn followed by late (7 days) Streptococcus pneumoniae pneumonia sepsis in wild-type (WT) mice, IL-6 knockout (IL-6 KO) mice, and transgenic mice overexpressing IL-6 in the myocardium (TG). Twenty-four hours after sepsis was induced, isolated cardiomyocytes were harvested and cultured in vitro, and supernatant concentrations of IL-6 and tumor necrosis factor (TNF)-alpha were measured. Cardiomyocyte intracellular calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) concentrations were also determined. Separate mice in each group underwent in vivo global hemodynamic and cardiac function assessment by cannulation of the carotid artery and insertion of a left ventricular pressure volume conductance catheter. Hearts from these mice were collected for histopathological assessment of inflammatory response, fibrosis, and apoptosis. In the WT group, there was an increase in cardiomyocyte TNF-alpha, [Ca(2+)](i), and [Na(+)](i) after burn plus sepsis, along with cardiac contractile dysfunction, inflammation, and apoptosis. These changes were attenuated in the IL-6 KO group but accentuated in the TG group. We conclude myocardial IL-6 mediates cardiac inflammation and contractile dysfunction after burn plus sepsis.

Animals↗

PubMed2006

Sepsis and burn complicated by sepsis alter cardiac transporter expression.

UNLABELLED: Sepsis alone and burn complicated by sepsis produce significant cardiac dysfunction. Since calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism for cardiac abnormalities may be calcium dyshomeostasis. We hypothesized that sepsis and burn plus sepsis alter cardiac calcium transporter expression. Sprague-Dawley rats were divided into: (1) control, (2) sepsis (intratracheal S. Pneumoniae, 4x10(6) CFU), and (3) burn (40% TBSA) plus sepsis. Myocyte [Ca(2+)](i) and [Na(+)](i) were quantified with Fura-2 AM and SBFI, respectively. Western blot analysis of rat hearts used antibodies against the sarcoplasmic reticular Ca(2+) ATPase (SERCA), the L-type calcium channel, the Na(+)/Ca(2+) exchanger or the Na(+)/K(+) ATPase. RESULTS: Sepsis in the presence and absence of burn trauma increased [Ca(2+)](i) and [Na(+)](i). SERCA expression was decreased in the sepsis and burn plus sepsis groups while calcium channel expression was transiently increased in these sepsis groups. Na(+)/Ca(2+) exchanger expression exhibited a biphasic pattern of altered expression. Sepsis and burn plus sepsis reduced Na(+)/K(+) ATPase protein levels. These data suggest that altered transporter expression produce cardiomyocyte calcium and sodium loading and may contribute to sepsis-mediated cardiac contractile dysfunction.

Animals↗

PubMed2006

Endotoxin-induced cardiomyopathy and systemic inflammation in mice is prevented by aldose reductase inhibition.

BACKGROUND: Sepsis is a systemic inflammatory response syndrome characterized by excessive production of inflammatory cytokines and cardiovascular collapse. Postreceptor signaling events that lead to stress responses and cytokine production are sensitive to redox changes and products of lipid peroxidation. METHODS AND RESULTS: We tested the hypothesis that inflammatory signaling and cytokine generation during sepsis depend on the activity of the enzyme aldose reductase, which catalyzes the reduction of lipid peroxidation-derived aldehydes and their glutathione conjugates. The results of the present study show that pharmacological inhibition of aldose reductase by sorbinil or knockdown of the enzyme by small interfering RNA prevents the activation of nuclear factor-kappaB and the release of tumor necrosis factor-alpha from lipopolysaccharide-stimulated RAW264.7 or H9c2 cells. Increases in serum and cardiac cytokines in response to lipopolysaccharide challenge were suppressed by inhibition of aldose reductase. Treatment with sorbinil blunted the activation of protein kinase C, c-Jun NH2-terminal kinase, and p38, as well as phosphorylation of interleukin receptor-associated kinase, IkappaB-alpha, IkappaB kinase complex-alpha/beta, and phospholipase-gamma1 and -beta1. These changes were associated with decreased myocardial nuclear factor-kappaB and activating protein-1 activity, prostaglandin E2 production, induction of cyclooxygenase 2, and inducible nitric oxide synthase. Sorbinil treatment also induced functional recovery in myocardial fractional shortening in vivo and preserved contractile function of isolated perfused hearts. Inhibition of aldose reductase increased survival in mice injected with lethal doses of lipopolysaccharide. CONCLUSIONS: The present demonstration that aldose reductase mediates endotoxin-induced inflammation and cardiomyopathy suggests that inhibition of this enzyme may be useful to attenuate maladaptive host responses and to treat acute cardiovascular dysfunction associated with endotoxic shock.

Aldehyde Reductase↗

PubMed2006

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.

Animals↗

PubMed2006

Cardiac mitochondrial damage and loss of ROS defense after burn injury: the beneficial effects of antioxidant therapy.

Mechanisms of burn-related cardiac dysfunction may involve defects in mitochondria. This study determined 1) whether burn injury alters myocardial mitochondrial integrity and function; and 2) whether an antioxidant vitamin therapy prevented changes in cardiac mitochondrial function after burn. Sprague-Dawley rats were given a 3 degrees burn over 40% total body surface area and fluid resuscitated. Antioxidant vitamins or vehicle were given to sham and burn rats. Mitochondrial and cytosolic fractions were prepared from heart tissues at several times postburn. In mitochondria, lipid peroxidation was measured to assess oxidative stress, mitochondrial outer membrane damage and cytochrome-c translocation were determined to estimate mitochondrial integrity, and activities of SOD and glutathione peroxidase were examined to evaluate mitochondrial antioxidant defense. Cardiac function was measured by Langendorff model in sham and burn rats given either vitamins or vehicle. Twenty-four hours postburn, mitochondrial outer membrane damage was progressively increased to approximately 50%, and cytosolic cytochrome-c gradually accumulated to approximately three times more than that measured in shams, indicating impaired mitochondrial integrity. Maximal decrease of mitochondrial SOD activity occurred 8 h postburn ( approximately 63.5% of shams), whereas maximal decrease in glutathione peroxidase activity persisted 2-24 h postburn ( approximately 60% of shams). In burn animals, lipid peroxidation in cardiac mitochondria increased 30-50%, suggesting burn-induced oxidative stress. Antioxidant vitamin therapy prevented burn-related loss of membrane integrity and antioxidant defense in myocardial mitochondria and prevented cardiac dysfunction. These data suggest that burn-mediated mitochondrial dysfunction and loss of reactive oxygen species defense may play a role in postburn cardiac dysfunction.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

Echocardiography assessment of myocardial function after burn injury.

INTRODUCTION: Echocardiography provides noninvasive and clinically relevant assessment of left ventricle (LV) function in injury and disease. In this present study, we hypothesized that application of transthoracic echocardiography in awake mice would provide in vivo assessment of myocardial performance that correlates with in vitro assessment of LV function using isolated perfused hearts (Langendorff). METHODS: Burn over 40% of the total body surface area (or sham burn) was given in C57/BL6 mice 22 to 24 g; lactated Ringer fluid resuscitation was given intraperitoneally. Transthoracic echocardiography was performed at baseline and at designated intervals over 7 days postburn. Subgroups of mice were killed at intervals and hearts perfused to assess LV pressure and +/-dP/dt max responses to inotropic challenge (n = 8/group per time). RESULTS: Burn produced myocardial depression, evidenced by a fall in fractional shortening from 69% +/- 3% at baseline to 50% +/- 2% (12 h postburn), 58% +/- 3% (24 h), 59% +/- 2% (48 h), and 62% +/- 2% (72 h) (P < 0.05). Burn-related changes in in vivo LV performance were paralleled by in vitro evidence of myocardial contractile depression; LV pressure progressively fell from 97 +/- 2 mmHg at baseline to 84 +/- 7 mmHg (12 h), 63 +/- 2 (24 h), 79 +/- 4 mmHg (48 h postburn) (P < 0.05). Analysis of variance and multiple comparison procedure. Myocardial recovery occurred by day 8 postburn. CONCLUSIONS: Echocardiography provides measures of cardiac function in a conscious animal, which correlate with contractile depression measured in vitro using Langendorff preparations. Echocardiography is a valuable tool for noninvasive assessment of postburn myocardial function.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

Innate immunity SNPs are associated with risk for severe sepsis after burn injury.

OBJECTIVE: To analyze allelic association with clinical outcome in a cohort of burn patients. PATIENTS: Two hundred twenty-eight individuals with burns > or =15% total body surface area without significant non-burn related trauma who survived >48 hours post-admission were enrolled. One hundred fifty-nine of these patients were analyzed previously. METHODS: Candidate polymorphisms within interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), cellular differentiation marker 14 (CD14) and toll-like receptor 4 (TLR4) were evaluated by logistic regression analysis for association with increased risk for severe sepsis (sepsis plus organ dysfunction or shock). RESULTS: After adjustment for age, burn size, ethnicity, gender and inhalation injury, alleles at TNF-alpha (308G, p=0.013), TLR4 (+896G, p=0.027), IL-6 (174C, p=0.040) and CD14 (159C, p=0.047) were significantly associated with an increased risk for severe sepsis. CONCLUSIONS: Carriage of variant alleles at immune response genes were associated with increased risk for severe sepsis after burn injury.

Adult↗

PubMed2005

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.

Animals↗

PubMed2005

Sodium/hydrogen exchange activity in sepsis and in sepsis complicated by previous injury: 31P and 23Na NMR study.

OBJECTIVE: Sepsis or septic shock occurs frequently in sick and injured patients and is associated with a significant mortality. Myocardial contractile dysfunction has been proposed to be a major determinant of sepsis-related mortality. This study was directed to examine the role of Na/H exchange activity in myocardial defects after sepsis or after sepsis complicated by a previous burn injury. DESIGN: Laboratory study. SETTING: University research laboratory. SUBJECTS: Sprague-Dawley rats (300-350 g, males). INTERVENTIONS: Cardiac function, cellular Na and Ca, myocardial pH, and high-energy phosphates were examined in perfused hearts harvested after sepsis alone (intratracheal Streptococcus pneumoniae, 0.4 mL of 1 x 10 CFU/mL), after sepsis complicated by previous burn injury (40% total body surface area), and after amiloride (a selective inhibitor of Na/H exchange) treatment of either sepsis alone or sepsis plus burn. MEASUREMENTS AND RESULTS: The ratio of Na signal from the intracellular compartment (Nai) compared with an external standard (monitored by Na-NMR spectroscopy, TmDOTP shift reagent) increased by 70% in sepsis alone and by 41% in sepsis complicated by previous burn injury compared with shams. Cardiac adenosine triphosphate and intracellular pH (P nuclear magnetic resonance spectroscopy) were unchanged by sepsis or sepsis plus burn. Left ventricular pressure and maximal change in pressure over time were reduced after sepsis or after sepsis plus burn injury. Amiloride treatment in either sepsis or sepsis complicated by a previous burn injury prevented myocardial Na and Ca accumulation, attenuated sepsis-related lactic acidosis, and improved left ventricular function. CONCLUSION: Our results suggest that sepsis-related cardiac dysfunction is mediated, in part, by Na/H exchange activity, and inhibition of Na/H exchange activity improves cardiac function after sepsis alone or sepsis complicated by a previous injury.

Amiloride↗

PubMed2005

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.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Nitric oxide donors alter cardiomyocyte cytokine secretion and cardiac function.

OBJECTS: The mechanisms by which nitric oxide produces beneficial/detrimental effects on physiologic function are unclear. In this study, we hypothesized that nitric oxide promotes cyclic guanosine monophosphate (cGMP) formation, which, in turn, promotes cardiomyocyte secretion of inflammatory cytokines as well as accumulation of intracellular Na+/Ca2+; these factors contribute to altered cardiac contractile function. DESIGN: Laboratory study. SETTING: Medical Center. SUBJECTS: Adult Sprague Dawley rats weighing 325-350 g. INTERVENTIONS: Cardiomyocytes were prepared by collagenase perfusion of rat hearts; cells were plated (5 x 10(4) cells/microtiter well) and challenged with either vehicle or nitric oxide donor (S-nitroso-N-acetyl-penicillamine [SNAP] or PAPA NONOATE, 3-[2-Hydroxy-2-nitroso-1-propythdrazinol]-1-propanamine], NOC-15 [PAPA-NO], 0.3 or 1.0 mM of each nitric oxide donor) in the presence/absence of methylene blue (10 microM/L to inhibit cGMP). After 3 hrs, supernatants were collected to measure nitrite/nitrate (nitric oxide), cytokines (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6), and cGMP levels; cells were then loaded with a fluorescent indicator (Fura-2AM or sodium-binding benzofurzan isophthalate) to measure myocyte Ca2+ or Na+, respectively. Parallel experiments included the addition of nitric oxide donor (0.3 or 1.0 mM SNAP or PAPA-NO) to perfused hearts in presence or absence of the methylene blue to examine cGMP-mediated effects on myocardial contraction-relaxation, while other experiments determined a) potential lipopolysaccharide contamination of myocyte preparations; and b) whether a cGMP analogue recapitulated the effects of nitric oxide donors on cytokine secretion. MEASUREMENTS AND MAIN RESULTS: Nitric oxide donors produced a dose-dependent increase in cGMP levels in myocyte supernatants as well as an increase in myocyte cytokine secretion, increased myocyte loading of Na+/Ca2+, and produced myocardial contractile dysfunction. Addition of the cGMP analog, 8-bromo-cGMP, recapitulated the effects of nitric oxide donors on myocyte cytokine secretion. Nitric oxide donor-related effects were ablated by pretreatment of myocytes or isolated hearts with methylene blue. Treatment of myocytes with recombinant bactericidal/permeability-increasing protein to scavenge lipopolysaccharide confirmed that cytokine responses to nitric oxide donors were not related to lipopolysaccharide contamination of myocyte preparations. CONCLUSIONS: We suggest that nitric oxide synthesis in injury and disease promotes cGMP formation, which, in turn, modulates cardiac contraction/relaxation by a) altering cardiomyocyte secretion of inflammatory cytokines and b) altering myocyte handling of Na+/Ca2+.

Animals↗

PubMed2005

Rho-associated kinase modulates myocardial inflammatory cytokine responses.

Rho, a Ser-Thr kinase identified as a member of the RAS GTPase super family, is highly expressed in the heart, and has been implicated in the development of heart failure. GTPase Rho is located downstream of Gq, and Rho and the associated kinase (Rho kinase) regulate myofibril organization, apoptosis, and myofibrillar sensitivity to calcium. Myocardial injury and dysfunction occur after major burn injury, and this phenomenon has been linked to cardiac myocyte synthesis and the secretion of proinflammatory cytokines. Whether Rho-associated kinase modulates any aspect of cardiomyocyte synthesis of inflammatory mediators, contributing to myocardial dysfunction, has not been studied and was the focus of this study. Hearts were collected at several times postburn to determine if an acute injury such as thermal trauma altered myocardial Rho kinase expression. In addition, cardiomyocytes were isolated (collagenase digestion) from adult control Sprague Dawley rats, plated (5 x 10 cells/microtiter well), incubated with medium alone or in the presence of burn serum (collected 24 h after burn over 40% total body surface area in rats) in a CO2 incubator at 37 degrees C in the presence/absence of specific Rho-kinase inhibitors (HA1077, 10 microM or Y27632, 10 microM). After 18 h, supernatants were collected to measure secreted cytokines (enzyme-linked immunoabsorbant assay), cells were loaded with Fura-2AM (2 microg) or sodium-binding benzofuran isophthalate (2 microg) for 45 min at 37 degrees C, and fluorescence was measured with an InCyt IM2 fluorescence imaging system to measure myocyte calcium and sodium. In parallel studies, cells were examined to determine if burn serum challenge increased Rho kinase in this cell population. In vivo burn injury or in vitro burn serum challenge of isolated myocytes increased Rho-kinase expression and promoted cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 6, and increased cardiomyocyte calcium and sodium levels compared with values measured when myocytes were incubated in medium alone (P < 0.05). Pretreating cardiomyocytes with Rho-kinase inhibitor (HA1077 or Y27632) prevented burn serum-related upregulation of Rho-kinase and attenuated the associated inflammatory cytokine responses, and attenuated myocyte calcium and sodium loading. Our data suggest that the Rho-kinase pathway is one potential upstream regulator of cardiac inflammatory response to burn injury.

Animals↗

PubMed2005

Murine in vivo myocardial contractile dysfunction after burn injury is exacerbated by pneumonia sepsis.

We evaluated hemodynamic and cardiac contractile dysfunction in a murine model of 40% contact burn complicated by Streptococcus pneumoniae (1 x 10(5) CFU) sepsis. Male, 9- to 10-week-old C57/BL6 mice were divided into the following groups: sham burn, sham sepsis; 24 h after burn alone; 24 h after sepsis alone; 7 days after burn alone; and 7 days after burn followed by pneumonia sepsis. Hemodynamic and cardiac contractile function was assessed with carotid artery cannulation and left ventricular pressure-volume analysis. At 24 h after burn, there were significant decreases in all load-insensitive contractility variables including the end-systolic pressure volume relationship, preload-recruitable stroke work, and maximum elastance, but there were no changes in global hemodynamics. Twenty-four hours after sepsis, there was similar cardiac contractile dysfunction, along with a decrease in cardiac output, but mean arterial pressure was maintained with an increase in systemic vascular resistance. Late burn (7 days) was associated with a recovery of all contractility variables except the end-systolic pressure volume relationship. However, sepsis induced during the late burn period was associated with a significant decrease in heart rate and cardiac output, but mean arterial pressure was still maintained with increased systemic vascular resistance. With burn complicated by sepsis, all cardiac contractility variables showed profound contractile dysfunction. Our data indicate that burn complicated by sepsis is associated with more pronounced cardiac contractile dysfunction than burn alone or sepsis alone.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

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.

Animals↗

PubMed2004

Role of cytosolic vs. mitochondrial Ca2+ accumulation in burn injury-related myocardial inflammation and function.

This study was designed to examine the role of mitochondrial Ca2+ homeostasis in burn-related myocardial inflammation. We hypothesized that mitochondrial Ca2+ is a primary modulator of cardiomyocyte TNF-alpha, IL-1beta, and IL-6 responses to injury and infection. Ventricular myocytes were prepared by Langendorff perfusion of hearts from adult rats subjected to sham burn or burn injury over 40% of total body surface area to produce enzymatic (collagenase) digestion. Isolated cardiomyocytes were suspended in MEM, cell number was determined, and aliquots of myocytes from each experimental group were loaded with fura 2-AM (2 microg/ml) for 1) 45 min at room temperature to measure total cellular Ca2+, 2) 45 min at 30 degrees C followed by incubation at 37 degrees C for 2 h to eliminate cytosolic fluorescence, and 3) 20 min at 37 degrees C in MnCl2 (200 microM)-containing buffer to quench cytosolic fura 2-AM signal. In vitro studies included preparation of myocytes from control hearts and challenge of myocytes with LPS or burn serum (BS), which have been shown to increase cytosolic Ca2+. Additional aliquots of myocytes were challenged with LPS or BS with or without a selective inhibitor of mitochondrial Ca2+, ruthenium red (RR). All cells were examined on a stage-inverted microscope that was interfaced with the InCyt Im2 fluorescence imaging system. Heat treatment or MnCl2 challenge eliminated myocyte cytosolic fluorescence, whereas cells maintained at room temperature retained 95% of their initial fluorescence. Compared with Ca2+ levels measured in sham myocytes, burn trauma increased cytosolic Ca2+ from 90 +/- 3 to 293 +/- 6 nM (P < 0.05) and mitochondrial Ca2+ from 24 +/- 1 to 75 +/- 2 nM (P < 0.05). LPS (25 microg/5 x 10(4) cells) or BS (10% by volume) challenge for 18 h increased cardiomyocyte cytosolic and mitochondrial Ca2+ and promoted myocyte secretion of TNF-alpha, IL-1beta, and IL-6. RR pretreatment decreased LPS- and BS-related rise in mitochondrial Ca2+ and cytokine secretion but had no effect on cytosolic Ca2+. BS challenge in perfused control hearts impaired myocardial contraction/relaxation, and RR pretreatment of hearts prevented BS-related myocardial contractile dysfunction. Our data suggest that a rise in mitochondrial Ca2+ is one modulator of myocardial inflammation and dysfunction in injury states such as sepsis and burn trauma.

Animals↗