Important role of p38 MAP kinase/NF B signaling pathway in ... fileFeces-induced peritonitis (FIP) was employed as a model of sepsis. In vitro, cardiac myocytes were treated with plasma

Important role of p38 MAP kinase/NFκB signaling pathway in the sepsis-induced conversion of

cardiac myocytes to a proinflammatory phenotype

Min Yang 1, 3, Jun Wu 1, 3, Claudio Martin1, 2, Peter R. Kvietys1, 3, Tao Rui 1, 2, 3

1 Center for Critical Illness Research, Lawson Health Research Institute; 2 Critical Care Medicine,

London Health Science Centre; 3Department of Medical Biophysics, University of Western

Ontario, London, Ontario, Canada N6A 4G5

Running head: p38 MAP kinase and myocardial inflammation/dysfunction

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

Correspondence:

Tao Rui, M.D.

Center for Critical Illness Research

Lawson Health Research Institute

800 Commissioners Road E., VRL Rm A6-138

London, Ontario, CANADA N6A 4G5

Phone: (519) 685 8500 ext. 55075; FAX: (519) 685 8341; e-mail: [email protected]

Page 1 of 37

Copyright Information

Articles in PresS. Am J Physiol Heart Circ Physiol (December 14, 2007). doi:10.1152/ajpheart.01044.2007

Copyright © 2007 by the American Physiological Society.

mailto:[email protected]

1

Abstract

Septic plasma can convert murine cardiac myocytes to a proinflammatory phenotype. These

myocytes 1) have increased nuclear levels of NFκB, 2) release CXC chemokines and 3) promote

PMN transendothelial migration. The purpose of the present study was to evaluate the role of the

MAP kinases (p38 MAP kinase, ERK1/2, and 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 hrs after

either sham (saline) or FIP procedures. Myocyte supernatants were used for 1) detection of the

CXC chemokines (ELISA) and 2) assessment of their ability to promote PMN transendothelial

migration. In vivo, myocardial PMN accumulation was assessed by measuring 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 siRNA) 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 NFκB p65. Inhibition of p38 MAP kinase (SB202190) blocked both NFκB

p65 phosphorylation and NFκB 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 cardiomyocyte p38 MAP

kinase/NFκB signaling pathway plays an important role in the sepsis-induced conversion of

myocytes to a proinflammatory phenotype.

Key words: mice, chemokines, PMN transendothelial migration, myocardial contractility

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Introduction

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

PMN (5). In septic patients, one of the critical organs adversely affected in sepsis is the heart;

exhibiting histopathologic 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, NFκB.

The upstream signaling elements involved in NFκB activation in cardiac myocytes

conditioned with septic plasma remains unknown. However, others have shown that NFκB 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,

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3

which, if any, of these three MAP kinases is involved in the sepsis-induced activation/translocation

of NFκB 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/NFκB

signaling pathway is important to the development of a proinflammatory phenotype in cardiac

myocytes in vitro and myocardial inflammation and dysfunction in vivo.

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Materials and Methods

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 US 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 (i.p.) as

described previously (34). Sham mice were given 0.5 ml normal saline i.p. For the in vitro

studies, 6 hrs after FIP, the mice were anesthetized (Ketamine/Xylazine), exsanguinated (cardiac

puncture), and plasma obtained. Plasma from FIP (septic plasma) or sham (sham plasma) mice

was diluted 20% in M199 (containing 10% FCS) and used to condition cardiac myocytes as

previously described (22). For the in vivo studies, 6 hrs 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 previous 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 into cell culture plates (Corning). After 72 hrs 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

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microbead technique using a CD31 antibody capture approach was used to isolate the endothelial

cells. The endothelial cells (> 85 % purity; Dil-Ac-LDL) were cultured on cell culture inserts and

used for the PMN migration assays when confluent.

PMNs were isolated as previous described (35; 38). In brief, PMNs were isolated from the

marrow of hind leg 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, 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, NFκB), the myocytes were incubated with either septic or sham plasma for

different periods of time, washed with PBS, harvested and used for Westerns and EMSAs. For

assessment of cardiomyocytes conversion to a proinflammatory phenotype, the myocytes were

conditioned for 4 hrs, with septic or sham plasma, washed, and additionally incubation in M199

(FCS free) for another 1 hour. 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 co-incubated 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 ELISA (22). Briefly, supernatants were

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added into 96 well EIA plate coated with capture antibodies (either rat anti-mouse KC or LIX

monoclonal antibodies, R&D system). 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 ABC 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 blocking, the membranes were blotted with either a relevant antibody

against the phosphorylated MAP kinase or antibody against the total MAP kinase (Cell Signaling

Tech.). Myocyte MAP kinase phosphorylation status was expressed as the ratio of phosphorylated

to total MAP kinase.

Transfection of cardiac myocytes with siRNA. 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

manufacturer’s instructions. Transfection efficiency was approximately 70% (Western) and the

cardiac myocytes used in experiments 48 hrs after the procedure.

Activation/translocation of NFκB. Cardiac myocyte NFκB activation and nuclei

translocation was determined by Western blot and an electrophoretic mobility shift assay (EMSA),

respectively. Phosphorylation of p65, a subunit of NFκB, was used as an indicator of NFκB

activation and assessed as described above. To assess NFκB translocation, nuclear extracts were

obtained from the cardiac myocytes for EMSA as previously described(38). A double-stranded

oligonucleotide containing consensus binding sites for NFκB (synthesized by Sigma) was labeled

with γ-32P [ATP (Amersham)] by using T4 polynucleotide kinase (MBI Fermentas). The sequence

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of the NFκB oligonucleotide is 5’- AGGGACTTTCCGCTGGGGACTTTCC-3’. One picomole of

the labeled oligonucleotide 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

onto native 5% polyacrylamide gel, and electrophoresed at 250V in 0.5X Tris-borate EDTA buffer.

Dried gel was exposed to X-ray film (KODAK) for 16 hours in cassettes with intensifying screens.

Myocardial inflammation and dysfunction in vivo. As an index of PMN infiltration,

myeloperoxidase (MPO) activity in the myocardium was determined, as previously described (37).

Briefly, after euthanasia, hearts were excised, homogenized, and centrifuged. The pellet was re-

homogenized and sonicated for 10 s in 1 ml of 50 mM acetic acid (pH 6.0) containing 0.5%

CETOH detergent. Ten microliters of the samples were used in reactions for MPO activity

determined spectrophotometrically (650 nm) by measuring hydrogen peroxide dependent

oxidation of TMB.

Circulation PMN. White blood cell (WBC) count was measured on an LH750 Series

Beckman Coulter hematology analyzer (Beckman Coulter, Fullerton, CA, USA). 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 sample by the total WBC count.

A Langendorff heart preparation was used to evaluate heart function (28). In brief, after

euthanasia hearts were harvested and the aorta was retrograde attached to a Langendroff perfusion

system and perfused (2 ml/minute) with Krebs-Henseleit buffer (bubbled with 95% O2+5%CO2

gas mixture and maintained at 37 0C). 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).

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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.

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Results

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 LPS in a variety of cell types, including

cardiac myocytes (10; 15). As show in Figure 1, exposure of neonatal cardiac myocytes to septic

plasma activated both p38 MAP kinase and ERK1/2 as indicated by increase in the

phosphorylation status of p38 MAP kinase and ERK1/2. Septic plasma did not induce JNK

phosphorylation.

Pharmacologic inhibitors were used to identify which of the three MAP kinases play 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 hr with either a p38 MAP kinase inhibitor (SB202190), JNK inhibitor

(SP600125), or a MEK1/2 inhibitor which prevents ERK1/2 activation (U0126). As predicted

(22), supernatants collected from cardiac myocytes conditioned with plasma from FIP mice

promoted PMN transendothelial migration (Figure 2). The myocyte-induced PMN migration was

substantially diminished by the p38 MAP kinase inhibitor, while the inhibitors of JNK and

MEK1/2 were without effect (Figure 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 knock-down approach. As shown in Figure 2B,

transfection of cardiac myocytes with the siRNA targeting p38 MAP kinase reduced the ability of

the myocytes to promote PMN transendothelial migration.

Our previous studies showed that the PMN transendothelial migration induced by cardiac

myocytes conditioned with septic plasma was dependent on myocyte production of the

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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, SB202190, or transfection of the cells with siRNA targeting p38 MAP kinase (Figure 3 A-

D). The increase in KC and LIX production was not affected by inhibition of either ERK1/2 with

U0126 or JNK with SP600125 (data not shown).

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.

Further, 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).

NFκB 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 NFκB translocation to the

myocyte nucleus (22). To assess whether activation of p38 MAP kinase is prerequisite for NFκB

activation in the cardiac myocytes in our model, both phosphorylation of p38 MAP kinase and

NFκB p65 were evaluated. Within 2 min. after exposure of the myocytes to septic plasma there

was an increase in p38 MAP kinase phosphorylation (Figure 4A); whereas, it required 5 min for

phosphorylation of NFκB p65 (Figure 4B). These findings indicate that the phosphorylation of

p38 MAP kinase precedes phosphorylation of the p65 subunit of NFκB. To establish a causative

link between phosphorylation of p38 MAP kinase and NFκB p65, we used the p38 MAP kinase

inhibitor, SB202190. As shown in Figure 5A, the p38 MAP kinase inhibitor significantly

diminished NFκB p65 phosphorylation (Figure 5A) and decreased nuclear levels of NFκB (EMSA,

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Figure 5B). Collectively, these results indicate that activation of p38 MAP kinase is an upstream

event to NFκB activation/translocation in cardiac myocytes challenged with septic plasma.

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 Figure 6A, six hrs

after induction of sepsis by FIP, myocardial MPO activity was increased. WBC analysis indicated

that 6 hrs after FIP, circulation PMNs significantly decreased (Table 1). The p38 MAP kinase

inhibitor, SB202190 had a modest effect on this neutropenia (76% reduction with FIP + vehicle vs

66% reduction with FIP + SB202190). 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 (Figure 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, SB202190 (Figure 6).

Thus, the in vivo experiments indicate that p38 MAP kinase plays an important role in the sepsis-

induced myocardial inflammation and dysfunction.

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Discussion

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 into 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 NFκB, 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/NFκB 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 NFκB, and 4) the p38 MAP kinase-

induced phosphorylation of its subunit p65 is a prerequisite for NFκB 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 which have the potential to activate several intracellular signalling 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 pro-inflammatory phenotype.

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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 (Figure 1). Thus, although it has been reported that LPS can activate

JNK in macrophage(48; 49), our results indicate that JNK is not activated in cardiomyocytes

conditioned with septic plasma (Figure 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 (Figures 2). The exact role of ERK1/2 activation in cardiomyocytes challenged with

septic plasma (Figure 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 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. Pharmacologic inhibition of p38

MAP kinase or knock down of p38 MAP kinase attenuated the myocyte 1) production of the

chemokines (LIX and KC) and 2) induced PMN transendothelial migration (Figures 2 and 3).

These findings are in general agreement with previous studies implicating p38 MAP kinase in

myocyte production of proinflammatory cytokines (TNF and IL-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 NFκB activation and nuclear translocation occurs is through

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the phosphorylation of IκB by IΚK (6) and subsequent degradation of IκB by the 26S proteasome.

The loss of IκB unmasks the nuclear localization sequence on NFκB, 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 NFκB-mediated gene transcription by phosphorylating the p65

subunit of NFκB. However, it is not entirely clear whether p65 phosphorylation by p38 MAP

kinase is important in 1) NFκB 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 NFκB in cardiac

myocytes challenged with septic plasma (Figures 4 and 5).

Our in vitro studies indicate that the p38 MAP kinase/NFκB 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 (Figure 6A) and dysfunction (Figure 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 NFκB 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 NFκB activation (Figure 5 A) and translocation to nuclei (Figure 5 B). This

observation indicates that p38 MAP kinase is pivotal to activation NFκB. However, inhibition of

either p38 MAP kinase (Figure 2 and 3) or NFκB(22) only partially decreased myocyte chemokine

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production and PMN transendothelial migration. Thus, it appears likely that other signaling

pathway(s) may be involved in the conversion of cardiac myocytes to 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 NFκB 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/NFκB signaling pathway may provide a

therapeutic regimen to alleviate sepsis-induced inflammation and dysfunction in the heart and,

potentially, other organs as well.

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Acknowledgments

This work was supported by an operating grant from Canadian Institutes of Health Research to TR

(MOP81303) and PRK (MOP 13668). The authors would like to thank Ms Leslie Gray-Statchuk

for assistance with circulating WBC counts.

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Figure legends

Figure 1. Effects of septic plasma on phosphorylation of MAP kinases 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 ERK1/2 and p38 MAP kinase, but not

JNK. Representative of 2 experiments.

Figure 2. PMN transendothelial migration induced by cardiac myocytes conditioned with septic

plasma (FIP) is prevented by blockade of p38 MAP kinase. A, Cardiac myocytes were pretreated

with either U0126 (20 µM; an inhibitor of ERK1/2 phosphorylation), SB202190 (10 µM; a p38

MAP kinase inhibitor), or SP600125 (10 µM; a JNK inhibitor) for 1 hr. Subsequently, the cardiac

myocytes were conditioned with either septic or sham plasma (4 hrs), washed, and incubated in

M199 for another 1 hr. Supernatants from the cardiac myocytes conditioned with FIP plasma

increased PMN transendothelial migration as compared to supernatants from myocytes

conditioned with sham plasma. The PMN transendothelial migration was reduced by the

SB202190, but not U0126 or SP600125. DMSO was as the vehicle and 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 hrs prior to challenge with septic plasma

and assessment of PMN migration (as described in Figure 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; # p<0.05 vs FIP, n=4.

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Figure 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 level of KC (A, C) and LIX (B, D) as

compared with those from the myocytes conditioned with sham plasma. The increase in KC and

LIX production was reduced by either SB202190 (10 µM) (A, B) or transfection of the cardiac

myocytes with siRNA specific for p38 MAP kinase (C, D). Inhibition MEK1/2 with U0126 (20

µM or JNK with SP600125 (10 µM) shown 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,

D, control siRNA had no effect on KC or LIX levels (data not shown). * p<0.05 vs sham, #p<0.05

vs FIP, n=3 for A and B, n=5 for C and D.

Figure 4. Phosphorylation (activation) of p38 MAP kinase occurs prior to phosphorylation of

NFκB 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 NFκB p65 activation required at 5 min after exposure. n=3.

Figure 5. Inhibition of p38 MAP kinase prevents NFκB p65 phosphorylation (activation) and

NFκB nuclear translocation. Cardiac myocytes were pretreated with SB202190 and challenged

with either septic (FIP) or sham plasma as described in Figure 2. Cardiac myocytes were assayed

for NFκB p65 phosphorylation by Western blot and nuclear translocation by EMSA. A, The

phosphorylation of NFκB p65 was prevented by the p38 MAP kinase inhibitor. B, Cardiac

myocyte NFκB nuclear translocation was induced by septic (FIP) plasma; an effect inhibited by

the p38 MAP kinase inhibitor. Representative Western blot and EMSA are shown above and

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densitometric analyses below. DMSO was used as the vehicle and added to both sham and FIP

groups (0.1% final concentration). “comp”; 50 X cold oligonucleotide *p<0.05 vs sham, #p<0.05

vs FIP, n=3 for both A and B.

Figure 6. p38 MAP kinase plays a role of in the sepsis-induced myocardial inflammation and

dysfunction in vivo. A, MPO activity was increased in the hearts from FIP mice as compared to

those from sham mice. This increase in MPO activity was prevented by pretreatment of the mice

with SB202190 (2 mg/kg) 1 hr prior to induction of the FIP. B – D, Six hours after the induction

of the FIP, hearts were obtained for assessment of myocardial function (Langendroff). 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 the SB202190 prior to induction of the FIP. DMSO was used as the vehicle and was

injected (i.p.) 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, #p<0.05 vs FIP, n=4 for A and n=5 for B-D.

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Figure 1. Effects of septic plasma on phosphorylation of MAP kinases 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 ERK1/2 and p38 MAP kinase, but not JNK. Representative of 2 experiments.

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Figure 2. PMN transendothelial migration induced by cardiac myocytes conditioned with septic plasma (FIP) is prevented by blockade of p38 MAP kinase. A, Cardiac myocytes were pretreated with either U0126 (20 M; an inhibitor of ERK1/2 phosphorylation),

SB202190 (10 M; a p38 MAP kinase inhibitor), or SP600125 (10 M; a JNK inhibitor) for 1 hr. Subsequently, the cardiac myocytes were conditioned with either septic or sham plasma (4 hrs), washed, and incubated in M199 for another 1 hr. Supernatants from the cardiac myocytes conditioned with FIP plasma increased PMN transendothelial migration

as compared to supernatants from myocytes conditioned with sham plasma. The PMN transendothelial migration was reduced by the SB202190, but not U0126 or SP600125.

DMSO was as the vehicle and 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 hrs prior to challenge with septic plasma and

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assessment of PMN migration (as described in Figure 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; # p<0.05 vs FIP, n=4. 209x296mm (300 x 300 DPI)

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Figure 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 level of KC (A, C) and LIX (B, D) as compared with those from the myocytes conditioned with sham plasma. The increase in KC and LIX production was reduced by either SB202190 (10 M) (A, B) or transfection of the cardiac myocytes with siRNA specific for p38 MAP kinase (C, D).

Inhibition MEK1/2 with U0126 (20 M or JNK with SP600125 (10 M) shown 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, D, control siRNA had no effect on KC or LIX levels (data not shown). * p<0.05 vs sham, #p<0.05 vs FIP, n=3 for A and B,

n=5 for C and D. 296x209mm (300 x 300 DPI)

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Figure 4. Phosphorylation (activation) of p38 MAP kinase occurs prior to phosphorylation of NF 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 B p65 activation required at 5 min

after exposure. n=3.

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Figure 5. Inhibition of p38 MAP kinase prevents NF B p65 phosphorylation (activation) and NF B nuclear translocation. Cardiac myocytes were pretreated with SB202190 and

challenged with either septic (FIP) or sham plasma as described in Figure 2. Cardiac myocytes were assayed for NF B p65 phosphorylation by Western blot and nuclear

translocation by EMSA. A, The phosphorylation of NF B p65 was prevented by the p38 MAP kinase inhibitor. B, Cardiac myocyte NF 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 above and densitometric analyses below. DMSO was used as the vehicle and added to both sham and FIP groups (0.1% final concentration). ¡°comp¡±; 50 X cold oligonucleotide *p<0.05 vs sham, #p<0.05 vs FIP, n=3 for both A

and B.

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Figure 6. p38 MAP kinase plays a role of in the sepsis-induced myocardial inflammation and dysfunction in vivo. A, MPO activity was increased in the hearts from FIP mice as compared to those from sham mice. This increase in MPO activity was prevented by

pretreatment of the mice with SB202190 (2 mg/kg) 1 hr prior to induction of the FIP. B ¨C D, Six hours after the induction of the FIP, hearts were obtained for assessment of

myocardial function (Langendroff). 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 the SB202190

prior to induction of the FIP. DMSO was used as the vehicle and was injected (i.p.) to both sham (5 l DMSO in 0.5 ml saline) and FIP ( 5 l DMSO in 0.5 ml fecal suspension)

mice. *P<0.05 vs sham, #p<0.05 vs FIP, n=4 for A and n=5 for B-D. 209x296mm (300 x 300 DPI)

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Values are means SE (n=6 mice/group). WBC, white blood cell; PMN, polymorphonuclear neutrophil; LY, lymphocyte; MO, monocyte. DMSO was used as the

vehicle and was injected (i.p.) to both sham (5 l DMSO in 0.5 ml saline) and FIP ( 5 lDMSO in 0.5 ml fecal suspension) mice. # p< 0.05, *p<0.01, vs corresponding

sham+vehicle group. 254x190mm (96 x 96 DPI)

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Documents

Important role of p38 MAP kinase/NF B signaling pathway in ... fileFeces-induced peritonitis (FIP) was employed as a model of sepsis. In vitro, cardiac myocytes were treated with plasma