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Am J Physiol Heart Circ Physiol 294: H994-H1001, 2008. First published December 14, 2007; doi:10.1152/ajpheart.01044.2007
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Important role of p38 MAP kinase/NF-{kappa}B signaling pathway in the sepsis-induced conversion of cardiac myocytes to a proinflammatory phenotype

Min Yang,1,3,* Jun Wu,1,3,* Claudio M. Martin,1,2 Peter R. Kvietys,1,3 and Tao Rui1,2,3

1Center for Critical Illness Research, Lawson Health Research Institute, 2Critical Care Medicine, London Health Science Center, and 3Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

Submitted 7 September 2007 ; accepted in final form 11 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Septic plasma can convert murine cardiac myocytes to a proinflammatory phenotype. These myocytes 1) have increased nuclear levels of nuclear factor-{kappa}B (NF-{kappa}B), 2) release CXC chemokines, and 3) promote polymorphonuclear neutrophil (PMN) transendothelial migration. The purpose of the present study was to evaluate the role of the mitogen-activated protein (MAP) kinases [p38 MAP kinase, extracellular signal-regulated kinase (ERK) 1/2, and c-Jun NH2-terminal kinase (JNK)] as upstream intracellular signaling components involved in this phenomenon. Feces-induced peritonitis (FIP) was employed as a model of sepsis. In vitro, cardiac myocytes were treated with plasma (20%) obtained 6 h after either sham (saline) or FIP procedures. Myocyte supernatants were used for 1) detection of the CXC chemokines (enzyme-linked immunosorbent assay) and 2) assessment of their ability to promote PMN transendothelial migration. In vivo, myocardial PMN accumulation was assessed by measuring myeloperoxidase (MPO) activity and function (dF/dt and heart work). Treatment of cardiac myocytes with septic plasma activated p38 MAP kinase and ERK1/2, but not JNK. Blockade approaches (inhibitors or small-interference RNA) indicated that only p38 MAP kinase played a role in the conversion of the myocytes to a proinflammatory phenotype. Time course studies indicated that phosphorylation of p38 MAP kinase preceded the phosphorylation of NF-{kappa}B p65. Inhibition of p38 MAP kinase (SB-202190) blocked both NF-{kappa}B p65 phosphorylation and NF-{kappa}B nuclear translocation. Confirmatory studies in vivo indicated that FIP resulted in an increase in myocardial MPO activity and dysfunction, events reversed by the inhibitor of p38 MAP kinase. Collectively, these data indicate that the cardiomyocyte p38 MAP kinase/NF-{kappa}B signaling pathway plays an important role in the sepsis-induced conversion of myocytes to a proinflammatory phenotype.

mice; chemokines; polymorphonuclear transendothelial migration; myocardial contractility; mitogen-activated protein kinase; nuclear factor-{kappa}B

SEPSIS IS A SYSTEMIC INFLAMMATORY response to severe infection that can lead to multiple organ failure (MOF) and, ultimately, death (1, 24, 32). A major contributing factor to the sepsis-induced MOF is believed to be tissue damage induced by invasion of various organs by circulating polymorphonuclear neutrophils (PMN) (5). In septic patients, one of the critical organs adversely affected in sepsis is the heart, which exhibits histopathological features of a classic acute inflammatory response, including PMN infiltration and cardiac dysfunction (9, 16). Studies in animal models indicate that activated PMN can directly injure cardiac myocytes (8, 30).

Neutrophil infiltration of the myocardium would be facilitated by the generation of a chemotactic gradient by resident interstitial cells. Immune cells (e.g., macrophages) are generally considered as major sources of the primary inflammatory mediators with chemotactic potential (26). We have recently provided evidence indicating that cardiac myocytes, per se, can play an important role in PMN infiltration of the heart (22). Exposure of isolated cardiac myocytes to plasma from septic animals converted them to a proinflammatory phenotype; these myocytes produced CXC chemokines, KC and LIX, and promoted PMN transendothelial migration. The conversion of cardiac myocytes to a proinflammatory phenotype was attributed to activation and nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B).

The upstream signaling elements involved in NF-{kappa}B activation in cardiac myocytes conditioned with septic plasma remain unknown. However, others have shown that NF-{kappa}B can serve as a target of mitogen-activated protein kinases (MAP kinases) (7, 45). There are three well-characterized subfamilies of MAP kinases: extracellular signal-regulated kinases (ERKs), the c-Jun NH2-terminal kinases (JNKs), and the p38 MAP kinase. All of these three MAP kinases have been implicated as cell signaling components involved in the generation of inflammatory mediators by a variety of cells (7, 13, 18, 19, 48). Thus the major objective of the present study was to identify which, if any, of these three MAP kinases is involved in the sepsis-induced activation/translocation of NF-{kappa}B in cardiomyocytes and the conversion of these myocytes to a proinflammatory phenotype.

Herein, we show for the first time that, in a septic milieu, the p38 MAP kinase/NF-{kappa}B signaling pathway is important to the development of a proinflammatory phenotype in cardiac myocytes in vitro and myocardial inflammation and dysfunction in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was approved by the University of Western Ontario Animal Care and Use Committee and conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of the Health (NIH Publication no. 85-23, revised 1996). C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) or cells derived from them were used for experiments.

Sepsis model. Feces induced peritonitis (FIP) was used to induce sepsis in mice. In brief, 0.5 ml of pooled fecal material (180 mg/ml normal saline) was given intraperitoneally as described previously (34). Sham mice were given 0.5 ml normal saline intraperitoneally. For the in vitro studies, 6 h after FIP, the mice were anesthetized (ketamine/xylazine) and exsanguinated (cardiac puncture), and plasma was obtained. Plasma from FIP (septic plasma) or sham (sham plasma) mice was diluted 20% in medium 199 (M199, containing 10% FCS) and used to condition cardiac myocytes as previously described (22). For the in vivo studies, 6 h after FIP, the hearts were harvested for biochemical assay of myeloperoxidase (MPO) activity or assessment of myocardial function in a Langendorff preparation.

Cells. Neonatal cardiac myocytes were isolated and cultured as previously described (22, 35, 38). Briefly, hearts were harvested, minced, and digested. After a washing step, the obtained cells were suspended in M199. Because myocytes adhere less avidly to plastic than other cell types (e.g., fibroblasts, endothelial cells), the myocytes were enriched by a preplating approach (to remove contaminating cells) before being seeded in cell culture plates (Corning). After 72 h in culture, the cells had formed a confluent monolayer consisting of 95% myocytes beating in synchrony and were used in experiments at this time.

Myocardial endothelial cells were isolated and cultured as previously described (22, 35, 36). Briefly, hearts were harvested, minced, and digested. After a washing step, a magnetic microbead technique using a CD31 antibody capture approach was used to isolate the endothelial cells. The endothelial cells (>85% purity; Dil-Ac-low density lipoprotein) were cultured on cell culture inserts and used for the PMN migration assays when confluent.

PMNs were isolated as previously described (35, 38). In brief, PMNs were isolated from the marrow of hindleg bones of adult mice and suspended in PBS. The cell suspension underwent Percoll gradient centrifugation. The PMNs were removed from the neutrophil-enriched fraction. This procedure yields 5–6 million white blood cells (WBCs), 95% of which are adult PMN as identified by acetic crystal violet staining. The PMN were used immediately in the migration assay.

In vitro experimental protocols. For measurement of intracellular signaling components (MAP kinases, NF-{kappa}B), the myocytes were incubated with either septic or sham plasma for different periods of time, washed with PBS, harvested, and used for Western blots and electrophoretic mobility shift assays (EMSAs). For assessment of cardiomyocyte conversion to a proinflammatory phenotype, the myocytes were conditioned for 4 h with septic or sham plasma, washed, and, additionally, incubated in M199 (FCS free) for another 1 h. Subsequently, the supernatants were collected for determination of KC and LIX levels and to assess their ability to promote PMN transendothelial migration.

PMN transendothelial migration. PMN transendothelial migration was assessed by using cell culture inserts as previously described (22, 35, 37). Briefly, cardiac endothelial cells were grown to confluence on fibronectin-coated cell culture inserts (3-µm-diameter pores). 51Cr-labeled PMN in M199 were added to the apical part of the endothelial monolayers (PMN: endothelial cell ratio of 10:1) and coincubated for 30 min with supernatants (from cardiac myocytes conditioned with sham or FIP plasma) introduced into the basal compartment. The percentage of the added PMNs that migrated from the apical to the basal aspect of the insert membrane was quantified.

Chemokine (KC and LIX) production. KC and LIX levels in supernatants from cardiac myocytes conditioned with plasma were determined by enzyme-linked immunosorbent assay (22). Briefly, supernatants were added to a 96-well enzyme immunoassay plate coated with capture antibodies (either rat anti-mouse KC or LIX monoclonal antibodies; R&D Systems). After addition of detection antibodies (biotinylated goat anti-mouse KC or LIX antibody, R&D system) and substrate 3,38,5,58-tetramethylbenzidine (TMB), color was developed by using an avidin-biotin complex peroxidase system (Sigma). Optical density was determined by a microreader (Bio-Rad) at 450 nm.

MAP kinase phosphorylation. ERK1/2, JNK, and p38 MAP kinase phosphorylation status in cardiac myocytes was determined by Western blot (37, 38). Plasma-conditioned myocytes were lysed. Cell protein (5 µg) was resolved on 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After being blocked, the membranes were blotted with either a relevant antibody against the phosphorylated MAP kinase or antibody against the total MAP kinase (Cell Signaling Technologies). Myocyte MAP kinase phosphorylation status was expressed as the ratio of phosphorylated to total MAP kinase.

Transfection of cardiac myocytes with small-interference RNA. Small-interference RNA (siRNA) specific for p38 MAP kinase (sc-29434) and transfection reagents (sc-29528) were purchased from Santa Cruz Biotechnology. The transfection of cardiac myocytes was carried out according to the manufacturer's instructions. Transfection efficiency was ~70% (Western blot), and the cardiac myocytes were used in experiments 48 h after the procedure.

Activation/translocation of NF-{kappa}B. Cardiac myocyte NF-{kappa}B activation and nuclei translocation was determined by Western blot and EMSA, respectively. Phosphorylation of p65, a subunit of NF-{kappa}B, was used as an indicator of NF-{kappa}B activation and assessed as described above. To assess NF-{kappa}B translocation, nuclear extracts were obtained from the cardiac myocytes for EMSA as previously described (38). A double-stranded oligonucleotide containing consensus binding sites for NF-{kappa}B (synthesized by Sigma) was labeled with [{gamma}-32P]ATP (Amersham) by using T4 polynucleotide kinase (MBI Fermentas). The sequence of the NF-{kappa}B oligonucleotide is 5'-AGGGACTTTCCGCTGGGGACTTTCC-3'. Labeled oligonucleotide (1 pmol) was incubated with 5 µg of nuclear protein in the presence or absence of a 50-fold excess of cold oligonucleotide for 30 min, and the reaction mixture was then loaded on native 5% polyacrylamide gel and electrophoresed at 250 volts in 0.5x Tris-borate-EDTA buffer. Dried gel was exposed to X-ray film (Kodak) for 16 h in cassettes with intensifying screens.

Myocardial inflammation and dysfunction in vivo. As an index of PMN infiltration, MPO activity in the myocardium was determined, as previously described (37). Briefly, after death, hearts were excised, homogenized, and centrifuged. The pellet was rehomogenized and sonicated for 10 s in 1 ml of 50 mM acetic acid (pH 6.0) containing 0.5% hexadecyltrimethylammonium hydroxide. Samples (10 µl) were used in reactions for MPO activity determined spectrophotometrically (650 nm) by measuring hydrogen peroxide-dependent oxidation of TMB.

Circulation of PMN. WBC count was measured on an LH750 Series Beckman Coulter hematology analyzer (Beckman Coulter, Fullerton, CA). Blood films were made and stained with Wright-Giemsa. WBC differential was determined by an experienced clinical technologist blinded to experimental groups. The number of PMNs was calculated by multiplying the percent PMN in the sample by the total WBC count.

A Langendorff heart preparation was used to evaluate heart function (28). In brief, after death, hearts were harvested, and the aorta was retrograde attached to a Langendorff perfusion system and perfused (2 ml/min) with Krebs-Henseleit buffer (bubbled with 95% O2 + 5% CO2 gas mixture and maintained at 37°C). The apex of the left ventricle was attached to a light-weight rigid coupling rod by sutures. The rod was attached directly to a force transducer (FT-03) to record tension (g) and heart rate. Computer software (Powerlab Chart Software; AD Instruments) was used to determine heart work and the maximal first derivative of the force (dF/dt).

Statistical analysis. All of the values are presented as means ± SE. Statistical analysis was performed with the use of ANOVA and Student's t-test with Bonferroni correction for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of the MAP kinases involved in the conversion of cardiomyocytes to a proinflammatory phenotype by septic plasma. The three MAP kinases (ERK1/2, p38 MAP kinase, and JNK) can be activated by inflammatory cytokines or lipopolysaccharide (LPS) in a variety of cell types, including cardiac myocytes (10, 15). As show in Fig. 1, exposure of neonatal cardiac myocytes to septic plasma activated both p38 MAP kinase and ERK1/2 as indicated by an increase in the phosphorylation status of p38 MAP kinase and ERK1/2. Septic plasma did not induce JNK phosphorylation.


Figure 1
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  		Fig. 1. Effects of septic plasma on phosphorylation of mitogen-activated protein kinase (MAP kinase) in cardiac myocytes. Cardiac myocytes were treated with septic plasma. At 5–120 min after challenge, the cardiac myocytes were harvested for detection of MAP kinase phosphorylation by Western blot. As a control, either sham plasma (shown) or MEM (not shown) was used, neither of which had any effect on the phosphorylated or total levels of the MAP kinases. Treatment of cardiac myocytes with septic plasma induced phosphorylation (activation) of extracellular signal-regulated kinase (ERK) 1/2 and p38 MAP kinase, but not c-Jun NH2-terminal kinase (JNK). Data are representative of 2 experiments.

 
Pharmacologic inhibitors were used to identify which of the three MAP kinases plays a role in the PMN transendothelial migration induced by cardiac myocytes conditioned with septic plasma. The inhibitors (and their concentrations) were chosen based on existing literature supporting their specificity for blockade of the relevant kinases (4, 44, 46). The cardiac myocytes were pretreated for 1 h with either a p38 MAP kinase inhibitor (SB-202190), JNK inhibitor (SP-600125), or a mitogen/extracellular signal-regulated kinase (MEK) 1/2 inhibitor that prevents ERK1/2 activation (U-0126). As predicted (22), supernatants collected from cardiac myocytes conditioned with plasma from FIP mice promoted PMN transendothelial migration (Fig. 2). The myocyte-induced PMN migration was diminished substantially by the p38 MAP kinase inhibitor, whereas the inhibitors of JNK and MEK1/2 were without effect (Fig. 2A). These latter observations indicated that, of the three kinases, only p38 MAP kinase appeared to play a role in the myocyte-induced PMN migration. A role for p38 MAP kinase was confirmed using a knockdown approach. As shown in Fig. 2B, transfection of cardiac myocytes with the siRNA targeting p38 MAP kinase reduced the ability of the myocytes to promote PMN transendothelial migration.


Figure 2
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  		Fig. 2. Polymorphonuclear neutrophil (PMN) transendothelial migration induced by cardiac myocytes conditioned with septic plasma [feces-induced peritonitis (FIP)] is prevented by blockade of p38 MAP kinase. A: cardiac myocytes were pretreated with either U-0126 (20 µM; an inhibitor of ERK1/2 phosphorylation), SB-202190 (10 µM; a p38 MAP kinase inhibitor), or SP-600125 (10 µM; a JNK inhibitor) for 1 h. Subsequently, the cardiac myocytes were conditioned with either septic or sham plasma (4 h), washed, and incubated in medium 199 (M199) for another 1 h. Supernatants from the cardiac myocytes conditioned with FIP plasma increased PMN transendothelial migration compared with supernatants from myocytes conditioned with sham plasma. The PMN transendothelial migration was reduced by SB-202190, but not U-0126 or SP-600125. DMSO was the vehicle and was added to both sham and FIP groups (0.1% final concentration). B: cardiac myocytes were transfected with small-interference RNA (siRNA) targeting p38 MAP kinase 48 h before challenge with septic plasma and assessment of PMN migration (as described in Fig. 2A). The increase in PMN transendothelial migration induced by supernatants from cardiac myocytes treated with FIP plasma was reduced by the siRNA specific for p38 MAP kinase; the control RNA had no effect. P < 0.05 vs. sham (*) and vs. FIP (#); n = 4 experiments.

 
Our previous studies showed that the PMN transendothelial migration induced by cardiac myocytes conditioned with septic plasma was dependent on myocyte production of the chemokines KC and LIX (22). Thus we assessed whether p38 MAP kinase plays a role in chemokine production in our model. As expected, supernatants derived from cardiac myocytes conditioned with septic plasma had increased levels of KC and LIX. The increase in KC and LIX production was prevented by either pretreatment of the myocytes with an inhibitor of p38 MAP kinase, SB-202190, or transfection of the cells with siRNA targeting p38 MAP kinase (Fig. 3, A–D). The increase in KC and LIX production was not affected by inhibition of either ERK1/2 with U-0126 or JNK with SP-600125 (data not shown).


Figure 3
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  		Fig. 3. CXC chemokine (KC and LIX) production by cardiac myocytes conditioned with septic plasma (FIP) is prevented by blockade of p38 MAP kinase. Supernatants from cardiac myocytes conditioned with septic plasma contained increased levels of KC (A and C) and LIX (B and D) compared with those from the myocytes conditioned with sham plasma. The increase in KC and LIX production was reduced by either SB-202190 (10 µM) (A and B) or transfection of the cardiac myocytes with siRNA specific for p38 MAP kinase (C and D). Inhibition of mitogen/extracellular signal-regulated kinase (MEK) 1/2 with U-0126 (20 µM) or JNK with SP-600125 (10 µM) showed no effect (data not shown). For A and B, DMSO was used as the vehicle and was added to both sham and FIP groups (0.1% final concentration); for C and D, control siRNA had no effect on KC or LIX levels (data not shown). P < 0.05 vs. sham (*) and vs. FIP (#); n = 3 for A and B and n = 5 for C and D.

 
Taken together, these observations indicate that, of the three kinases evaluated, only ERK1/2 and p38 MAP kinase were activated in cardiac myocytes conditioned with septic plasma. Furthermore, only p38 MAP kinase appears to play a role in the conversion of cardiac myocytes to a proinflammatory phenotype (increased chemokine production and promotion of PMN transendothelial migration).

NF-{kappa}B activation/translocation in cardiomyocytes conditioned with septic plasma is dependent on p38 MAP kinase. The conversion of cardiac myocytes to a proinflammatory phenotype by septic plasma has been shown to be dependent on NF-{kappa}B translocation to the myocyte nucleus (22). To assess whether activation of p38 MAP kinase is prerequisite for NF-{kappa}B activation in the cardiac myocytes in our model, both phosphorylation of p38 MAP kinase and NF-{kappa}B p65 was evaluated. Within 2 min after exposure of the myocytes to septic plasma, there was an increase in p38 MAP kinase phosphorylation (Fig. 4A), whereas it required 5 min for phosphorylation of NF-{kappa}B p65 (Fig. 4B). These findings indicate that the phosphorylation of p38 MAP kinase precedes phosphorylation of the p65 subunit of NF-{kappa}B. To establish a causative link between phosphorylation of p38 MAP kinase and NF-{kappa}B p65, we used the p38 MAP kinase inhibitor SB-202190. As shown in Fig. 5A, the p38 MAP kinase inhibitor significantly diminished NF-{kappa}B p65 phosphorylation (Fig. 5A) and decreased nuclear levels of NF-{kappa}B (EMSA, Fig. 5B). Collectively, these results indicate that activation of p38 MAP kinase is an upstream event to NF-{kappa}B activation/translocation in cardiac myocytes challenged with septic plasma.


Figure 4
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  		Fig. 4. Phosphorylation (activation) of p38 MAP kinase occurs before phosphorylation of nuclear factor-{kappa}B (NF-{kappa}B) p65. Cardiac myocytes were exposed to septic (FIP) or sham plasma, and lysates were used for Western blot. A: septic plasma induced p38 MAP kinase activation at 2 min after exposure. B: septic plasma induced NF-{kappa}B p65 activation required at 5 min after exposure; n = 3.

 

Figure 5
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  		Fig. 5. Inhibition of p38 MAP kinase prevents NF-{kappa}B p65 phosphorylation (activation) and NF-{kappa}B nuclear translocation. Cardiac myocytes were pretreated with SB-202190 and challenged with either septic (FIP) or sham plasma as described in Fig. 2. Cardiac myocytes were assayed for NF-{kappa}B p65 phosphorylation by Western blot and nuclear translocation by electrophoretic mobility shift assay (EMSA). A: phosphorylation of NF-{kappa}B p65 was prevented by the p38 MAP kinase inhibitor. B: cardiac myocyte NF-{kappa}B nuclear translocation was induced by septic (FIP) plasma, an effect inhibited by the p38 MAP kinase inhibitor. Representative Western blot and EMSA are shown at top and densitometric analyses at bottom. DMSO was used as the vehicle and was added to both sham and FIP groups (0.1% final concentration). "comp," 50x cold oligonucleotide. P < 0.05 vs. sham (*) and vs. FIP (#); n = 3 for both A and B.

 
p38 MAP kinase plays a role in the sepsis-induced myocardial inflammation and dysfunction. Our results indicate that a signaling pathway involving p38 MAP kinase plays an important role in the conversion of cardiomyocytes to a proinflammatory phenotype in our in vitro model. Thus in vivo experiments were undertaken to assess whether p38 MAP kinase plays a role in the sepsis-induced myocardial inflammation and dysfunction. As shown in Fig. 6A, 6 h after induction of sepsis by FIP, myocardial MPO activity was increased. WBC analysis indicated that, 6 h after FIP, circulation PMNs significantly decreased (Table 1). The p38 MAP kinase inhibitor SB-202190 had a modest effect on this neutropenia (76% reduction with FIP + vehicle vs. 66% reduction with FIP + SB-202190). Thus the decrease in myocardial MPO activity was not related to changes in circulating PMN. The sepsis-induced increase in MPO activity was accompanied by decreases in myocardial contractility, heart rate, and heart work (Fig. 6, B–D). Both the increase in MPO activity and decrease in cardiac contractility, heart rate, and work were reversed by pretreatment of the mice with the p38 MAP kinase inhibitor SB-202190 (Fig. 6). Thus the in vivo experiments indicate that p38 MAP kinase plays an important role in the sepsis-induced myocardial inflammation and dysfunction.


Figure 6
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  		Fig. 6. p38 MAP kinase plays a role of in the sepsis-induced myocardial inflammation and dysfunction in vivo. A: myeloperoxidase (MPO) activity was increased in the hearts from FIP mice compared with those from sham mice. This increase in MPO activity was prevented by pretreatment of the mice with SB-202190 (2 mg/kg) 1 h before induction of the FIP. B–D: 6 h after the induction of the FIP, hearts were obtained for assessment of myocardial function (Langendorff). Hearts from FIP mice exhibited a decrease in myocardial contractility (dF/dt) (B), heart rate (C), and heart work (D). The myocardial function of the FIP hearts was improved when the mice were treated with SB-202190 before induction of the FIP. DMSO was used as the vehicle and was injected (ip) to both sham (5 µl DMSO in 0.5 ml saline) and FIP (5 µl DMSO in 0.5 ml fecal suspension) mice. P < 0.05 vs. sham (*) and vs. FIP (#); n = 4 for A and n = 5 for B–D.

 

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  		Table 1. White blood cell count and differential

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Myocardial dysfunction is a characteristic feature of sepsis (11, 33, 42, 50). The mechanisms involved in myocardial dysfunction in sepsis appear to be multifactorial (33, 42). One important event leading to myocardial dysfunction in sepsis is PMN infiltration in the myocardial interstitium (27, 43). PMN infiltration would be facilitated by the generation of a chemotactic gradient by resident cardiac cells. We have previously shown that exposure of cardiac myocytes to plasma from septic animals can convert the myocytes to a proinflammatory phenotype (22). Specifically, these myocytes 1) have increased nuclear levels of NF-{kappa}B, 2) produce the CXC chemokines KC and LIX, and 3) promote PMN transendothelial migration.

In the present study, using in vitro and in vivo approaches, we provide the following novel observations supporting a role for the p38 MAP kinase/NF-{kappa}B pathway in sepsis-induced myocardial inflammation and dysfunction. Herein, using a construct of the myocardial vascular-interstitial interface, we provide evidence that 1) both ERK1/2 and p38 MAP kinase are activated in cardiac myocytes challenged with septic plasma, 2) only p38 MAP kinase is involved in the conversion of the myocytes to a proinflammatory phenotype, 3) p38 MAP kinase activation is critical for the downstream activation/nuclear translocation of NF-{kappa}B, and 4) the p38 MAP kinase-induced phosphorylation of its subunit p65 is a prerequisite for NF-{kappa}B translocation to the nucleus. Finally, we extend the in vitro findings to the whole organ level by showing that p38 MAP kinase plays a role in sepsis-induced myocardial inflammation and cardiac dysfunction.

Septic plasma can be viewed as a pool of cytokines, chemokines, and other inflammatory mediators that have the potential to activate several intracellular signaling pathways in cardiac myocytes, some of which may lead to conversion of the cardiac myocytes to a proinflammatory phenotype (17). Previous studies(17, 22, 27) indicate that septic plasma can convert adult human and rat cardiomyocytes, as well as mouse neonatal myocytes, to a proinflammatory phenotype. The three MAP kinases (ERK1/2, JNKs, and p38 MAP kinase) can be both activated by inflammatory mediators and, in turn, generate inflammatory mediators (12, 31, 40, 47). In the present study, cardiac myocyte ERK1/2 and p38 MAP kinase, but not JNK, were phosphorylated (activated) by septic plasma (Fig. 1). Thus, although it has been reported that LPS can activate JNK in macrophages (48, 49), our results indicate that JNK is not activated in cardiomyocytes conditioned with septic plasma (Fig. 1).

Although both ERK1/2 and p38 MAP kinase were activated, blockade experiments indicated that ERK1/2 is not involved in the conversion of cardiomyocytes to a proinflammatory phenotype (Fig. 2). The exact role of ERK1/2 activation in cardiomyocytes challenged with septic plasma (Fig. 1) is not entirely clear. However, ERK1/2 has been implicated in the modulation of anti-apoptotic pathways in cardiomyocytes (20) and induction of adhesion molecules on endothelial cells (2). Irrespectively, it does not appear to play a role in the conversion of cardiomyocytes to a proinflammatory phenotype. Further studies are warranted to address the possible role(s) of ERK1/2 in cardiomyocytes under septic conditions.

Herein, the blockade experiments indicated that p38 MAP kinase is involved in the conversion of cardiomyocytes to a proinflammatory phenotype. Pharmacological inhibition of p38 MAP kinase or knock down of p38 MAP kinase attenuated the myocyte production of the chemokines (LIX and KC) and induced PMN transendothelial migration (Figs. 2 and 3). These findings are in general agreement with previous studies implicating p38 MAP kinase in myocyte production of proinflammatory cytokines (tumor necrosis factor and interleukin-1) by LPS (19, 29). However, to our knowledge, this is the first report showing that p38 MAP kinase activation is a prerequisite for cardiac myocyte conversion to a proinflammatory phenotype capable of generating chemokines and promoting PMN migration.

The classic pathway by which NF-{kappa}B activation and nuclear translocation occurs is through the phosphorylation of inhibitory factor {kappa}B (I{kappa}B) by I{kappa}B kinase (6) and subsequent degradation of I{kappa}B by the 26S proteasome. The loss of I{kappa}B unmasks the nuclear localization sequence on NF-{kappa}B, thereby allowing it to translocation to the nucleus and initiate the transcription of relevant genes (3). The p38 MAP kinase appears to play a role in NF-{kappa}B-mediated gene transcription by phosphorylating the p65 subunit of NF-{kappa}B. However, it is not entirely clear whether p65 phosphorylation by p38 MAP kinase is important in 1) NF-{kappa}B translocation to the nucleus or 2) the actual process of transcription once bound to nuclear DNA, or both (14, 25, 41). Herein, we provide evidence that p65 phosphorylation by p38 MAP kinase is critical for nuclear translocation of NF-{kappa}B in cardiac myocytes challenged with septic plasma (Figs. 4 and 5).

Our in vitro studies indicate that the p38 MAP kinase/NF-{kappa}B signaling pathway is important in the conversion of cardiomyocytes to a proinflammatory phenotype in sepsis. The results of our in vivo studies support this contention and provide evidence to indicate that this proinflammatory phenotype may play a role in myocardial dysfunction. Mice rendered septic exhibited myocardial inflammation (Fig. 6A) and dysfunction (Fig. 6, B–D). These findings are consistent with observations in septic patients indicating 1) myocardial PMN infiltration (autopsy analyses) (9) and 2) myocardial dysfunction (hemodynamic analyses) (16). In the present study, we show for the first time that the sepsis-induced inflammation and dysfunction can be prevented by an inhibitor of p38 MAP kinase.

Transcription factor NF-{kappa}B can be activated by multiple upstream signaling components, including p38 MAP kinase (21, 23, 39). In the present study, inhibition of p38 MAP kinase completely prevented NF-{kappa}B activation (Fig. 5A) and translocation to nuclei (Fig. 5B). This observation indicates that p38 MAP kinase is pivotal to activation of NF-{kappa}B. However, inhibition of either p38 MAP kinase (Figs. 2 and 3) or NF-{kappa}B (22) only partially decreased myocyte chemokine production and PMN transendothelial migration. Thus it appears likely that other signaling pathway(s) may be involved in the conversion of cardiac myocytes to the proinflammatory phenotype in sepsis.

In summary, studies using isolated cardiac myocytes conditioned with septic plasma indicate that, of the three MAP kinases targeted (ERK1/2, JNK, and p38 MAP kinase), only p38 MAP kinase is involved in the activation/translocation of NF-{kappa}B in cardiac myocytes and their conversion to a proinflammatory phenotype. Our in vivo studies indicate that p38 MAP kinase plays a role in sepsis-induced myocardial inflammation and cardiac dysfunction. Collectively, our findings suggest that targeting the p38 MAP kinase/NF-{kappa}B signaling pathway may provide a therapeutic regimen to alleviate sepsis-induced inflammation and dysfunction in the heart and, potentially, other organs as well.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by an operating grant from the Canadian Institutes of Health Research to T. Rui (MOP 81303) and P. R. Kvietys (MOP 13668).


   ACKNOWLEDGMENTS
 
We thank Leslie Gray-Statchuk for assistance with circulating white blood cell counts.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Rui, Center for Critical Illness Research, Lawson Health Research Institute, 800 Commissioners Road E., VRL Rm A6-138, London, Ontario, Canada N6A 4G5 (e-mail: trui{at}uwo.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* M. Yang and J. Wu contributed equally to the paper. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet 365: 63–78, 2005.[CrossRef][Web of Science][Medline]
  2. Bai YP, Liu YH, Chen J, Song T, You Y, Tang ZY, Li YJ, Zhang GG. Rosiglitazone attenuates NF-kappaB-dependent ICAM-1 and TNF-alpha production caused by homocysteine via inhibiting ERK1/2/p38MAPK activation. Biochem Biophys Res Commun 360: 20–26, 2007.[CrossRef][Web of Science][Medline]
  3. Baldwin AS Jr. Series introduction: the transcription factor NF-kappaB and human disease. J Clin Invest 107: 3–6, 2001.[CrossRef][Web of Science][Medline]
  4. Bein K, Odell-Fiddler ET, Drinane M. Role of TGF-beta1 and JNK signaling in capillary tube patterning. Am J Physiol Cell Physiol 287: C1012–C1022, 2004.[Abstract/Free Full Text]
  5. Brown KA, Brain SD, Pearson JD, Edgeworth JD, Lewis SM, Treacher DF. Neutrophils in development of multiple organ failure in sepsis. Lancet 368: 157–169, 2006.[CrossRef][Web of Science][Medline]
  6. Brown M, McGuinness M, Wright T, Ren X, Wang Y, Boivin GP, Hahn H, Feldman AM, Jones WK. Cardiac-specific blockade of NF-kappaB in cardiac pathophysiology: differences between acute and chronic stimuli in vivo. Am J Physiol Heart Circ Physiol 289: H466–H476, 2005.[Abstract/Free Full Text]
  7. Cowan KJ, Storey KB. Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress. J Exp Biol 206: 1107–1115, 2003.[Abstract/Free Full Text]
  8. Davani EY, Boyd JH, Dorscheid DR, Wang Y, Meredith A, Chau E, Singhera GK, Walley KR. Cardiac ICAM-1 mediates leukocyte-dependent decreased ventricular contractility in endotoxemic mice. Cardiovasc Res 72: 134–142, 2006.[Abstract/Free Full Text]
  9. Fernandes Junior CJ, Iervolino M, Neves RA, Sampaio EL, Knobel E. Interstitial myocarditis in sepsis. Am J Cardiol 74: 958, 1994.[CrossRef][Web of Science][Medline]
  10. Fernandes A, Falcao AS, Silva RF, Brito MA, Brites D. MAPKs are key players in mediating cytokine release and cell death induced by unconjugated bilirubin in cultured rat cortical astrocytes. Eur J Neurosci 25: 1058–1068, 2007.[CrossRef][Web of Science][Medline]
  11. Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 348: 138–150, 2003.[Free Full Text]
  12. Issa R, Xie S, Khorasani N, Sukkar M, Adcock IM, Lee KY, Chung KF. Corticosteroid inhibition of growth-related oncogene protein-{alpha} via mitogen-activated kinase phosphatase-1 in airway smooth muscle cells. J Immunol 178: 7366–7375, 2007.[Abstract/Free Full Text]
  13. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298: 1911–1912, 2002.[Abstract/Free Full Text]
  14. Kim HJ, Lee HS, Chong YH, Kang JL. p38 Mitogen-activated protein kinase up-regulates LPS-induced NF-kappaB activation in the development of lung injury and RAW 264.7 macrophages. Toxicology 225: 36–47, 2006.[Web of Science][Medline]
  15. Klintman D, Li X, Santen S, Schramm R, Jeppsson B, Thorlacius H. p38 mitogen-activated protein kinase-dependent chemokine production, leukocyte recruitment, and hepatocellular apoptosis in endotoxemic liver injury. Ann Surg 242: 830–838, 2005.[CrossRef][Web of Science][Medline]
  16. Kumar A, Haery C, Parrillo JE. Myocardial dysfunction in septic shock. Crit Care Clin 16: 251–287, 2000.[CrossRef][Web of Science][Medline]
  17. Kumar A, Kumar A, Michael P, Brabant D, Parissenti AM, Ramana CV, Xu X, Parrillo JE. Human serum from patients with septic shock activates transcription factors STAT1, IRF1, and NF-kappaB and induces apoptosis in human cardiac myocytes. J Biol Chem 280: 42619–42626, 2005.[Abstract/Free Full Text]
  18. Kumar S, Boehm J, Lee JC. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2: 717–726, 2003.[CrossRef][Web of Science][Medline]
  19. Li M, Georgakopoulos D, Lu G, Hester L, Kass DA, Hasday J, Wang Y. p38 MAP kinase mediates inflammatory cytokine induction in cardiomyocytes and extracellular matrix remodeling in heart. Circulation 111: 2494–2502, 2005.[Abstract/Free Full Text]
  20. Liu Q, Hofmann PA. Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes. Am J Physiol Heart Circ Physiol 286: H2204–H2212, 2004.[Abstract/Free Full Text]
  21. Liu SF, Malik AB. NF-kappa B activation as a pathological mechanism of septic shock and inflammation. Am J Physiol Lung Cell Mol Physiol 290: L622–L645, 2006.[Abstract/Free Full Text]
  22. Madorin WS, Rui T, Sugimoto N, Handa O, Cepinskas G, Kvietys PR. Cardiac myocytes activated by septic plasma promote neutrophil transendothelial migration: role of platelet-activating factor and the chemokines LIX and KC. Circ Res 94: 944–951, 2004.[Abstract/Free Full Text]
  23. Madrid LV, Mayo MW, Reuther JY, Baldwin AS Jr. Akt stimulates the transactivation potential of the RelA/p65 Subunit of NF-kappa B through utilization of the Ikappa B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 276: 18934–18940, 2001.[Abstract/Free Full Text]
  24. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 348: 1546–1554, 2003.[Abstract/Free Full Text]
  25. Mitra S, Abraham E. Participation of superoxide in neutrophil activation and cytokine production. Biochim Biophys Acta 1762: 732–741, 2006.[Medline]
  26. Moser B, Wolf M, Walz A, Loetscher P. Chemokines: multiple levels of leukocyte migration control. Trends Immunol 25: 75–84, 2004.[CrossRef][Web of Science][Medline]
  27. Neviere RR, Cepinskas G, Madorin WS, Hoque N, Karmazyn M, Sibbald WJ, Kvietys PR. LPS pretreatment ameliorates peritonitis-induced myocardial inflammation and dysfunction: role of myocytes. Am J Physiol Heart Circ Physiol 277: H885–H892, 1999.[Abstract/Free Full Text]
  28. Peng T, Lu X, Feng Q. Pivotal role of gp91phox-containing NADH oxidase in lipopolysaccharide-induced tumor necrosis factor-alpha expression and myocardial depression. Circulation 111: 1637–1644, 2005.[Abstract/Free Full Text]
  29. Peng T, Lu X, Lei M, Moe GW, Feng Q. Inhibition of p38 MAPK decreases myocardial TNF-alpha expression and improves myocardial function and survival in endotoxemia. Cardiovasc Res 59: 893–900, 2003.[Abstract/Free Full Text]
  30. Poon BY, Ward CA, Cooper CB, Giles WR, Burns AR, Kubes P. Alpha(4)-integrin mediates neutrophil-induced free radical injury to cardiac myocytes. J Cell Biol 152: 857–866, 2001.[Abstract/Free Full Text]
  31. Ratthe C, Pelletier M, Chiasson S, Girard D. Molecular mechanisms involved in interleukin-4-induced human neutrophils: expression and regulation of suppressor of cytokine signaling. J Leukoc Biol 81: 1287–1296, 2007.[Abstract/Free Full Text]
  32. Riedemann NC, Guo RF, Ward PA. The enigma of sepsis. J Clin Invest 112: 460–467, 2003.[CrossRef][Web of Science][Medline]
  33. Rudiger A, Singer M. Mechanisms of sepsis-induced cardiac dysfunction. Crit Care Med 35: 1599–1608, 2007.[CrossRef][Web of Science][Medline]
  34. Rui T, Stetsenko I, Martin CM, Kvietys PR. Conversion of the cardiac myocytes to a proinflammatory phenotype in sepsis: role of p38 MAP kinase (Abstract). FASEB J 20: A646, 2006.[Free Full Text]
  35. Rui T, Cepinskas G, Feng Q, Ho YS, Kvietys PR. Cardiac myocytes exposed to anoxia-reoxygenation promote neutrophil transendothelial migration. Am J Physiol Heart Circ Physiol 281: H440–H447, 2001.[Abstract/Free Full Text]
  36. Rui T, Cepinskas G, Feng Q, Kvietys PR. Delayed preconditioning in cardiac myocytes with respect to development of a proinflammatory phenotype: role of SOD and NOS. Cardiovasc Res 59: 901–911, 2003.[Abstract/Free Full Text]
  37. Rui T, Feng Q, Lei M, Peng T, Zhang J, Xu M, Dale AE, Xenocostas A, Kvietys PR. Erythropoietin prevents the acute myocardial inflammatory response induced by ischemia/reperfusion via induction of AP-1. Cardiovasc Res 65: 719–727, 2005.[Abstract/Free Full Text]
  38. Rui T, Kvietys PR. NFkappaB and AP-1 differentially contribute to the induction of Mn-SOD and eNOS during the development of oxidant tolerance. FASEB J 19: 1908–1910, 2005.[Abstract/Free Full Text]
  39. Saccani S, Pantano S, Natoli G. p38-Dependent marking of inflammatory genes for increased NF-kappa B recruitment. Nat Immunol 3: 69–75, 2002.[CrossRef][Web of Science][Medline]
  40. Seybold J, Thomas D, Witzenrath M, Boral S, Hocke AC, Burger A, Hatzelmann A, Tenor H, Schudt C, Krull M, Schutte H, Hippenstiel S, Suttorp N. Tumor necrosis factor-alpha-dependent expression of phosphodiesterase 2: role in endothelial hyperpermeability. Blood 105: 3569–3576, 2005.[Abstract/Free Full Text]
  41. Song YJ, Jen KY, Soni V, Kieff E, Cahir-McFarland E. IL-1 receptor-associated kinase 1 is critical for latent membrane protein 1-induced p65/RelA serine 536 phosphorylation and NF-kappaB activation. Proc Natl Acad Sci USA 103: 2689–2694, 2006.[Abstract/Free Full Text]
  42. Supinski GS, Callahan LA. Polyethylene glycol-superoxide dismutase prevents endotoxin-induced cardiac dysfunction. Am J Respir Crit Care Med 173: 1240–1247, 2006.[Abstract/Free Full Text]
  43. Tavener SA, Kubes P. Cellular and molecular mechanisms underlying LPS-associated myocyte impairment. Am J Physiol Heart Circ Physiol 290: H800–H806, 2006.[Abstract/Free Full Text]
  44. Yagi R, McBurney D, Horton WE Jr. Bcl-2 positively regulates Sox9-dependent chondrocyte gene expression by suppressing the MEK-ERK1/2 signaling pathway. J Biol Chem 280: 30517–30525, 2005.[Abstract/Free Full Text]
  45. Yang SH, Sharrocks AD, Whitmarsh AJ. Transcriptional regulation by the MAP kinase signaling cascades. Gene 320: 3–21, 2003.[CrossRef][Web of Science][Medline]
  46. Yellaturu CR, Rao GN. A requirement for calcium-independent phospholipase A2 in thrombin-induced arachidonic acid release and growth in vascular smooth muscle cells. J Biol Chem 278: 43831–43837, 2003.[Abstract/Free Full Text]
  47. Zarubin T, Han J. Activation and signaling of the p38 MAP kinase pathway. Cell Res 15: 11–18, 2005.[CrossRef][Web of Science][Medline]
  48. Zhao Q, Shepherd EG, Manson ME, Nelin LD, Sorokin A, Liu Y. The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38. J Biol Chem 280: 8101–8108, 2005.[Abstract/Free Full Text]
  49. Zhao Q, Wang X, Nelin LD, Yao Y, Matta R, Manson ME, Baliga RS, Meng X, Smith CV, Bauer JA, Chang CH, Liu Y. MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock. J Exp Med 203: 131–140, 2006.[Abstract/Free Full Text]
  50. Zhu X, Bagchi A, Zhao H, Kirschning CJ, Hajjar RJ, Chao W, Hellman J, Schmidt U. Toll-like receptor 2 activation by bacterial peptidoglycan-associated lipoprotein activates cardiomyocyte inflammation and contractile dysfunction. Crit Care Med 35: 886–892, 2007.[CrossRef][Web of Science][Medline]



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