Glucan phosphate attenuates cardiac dysfunction and inhibits cardiac MIF expression and apoptosis in...

Manuscript number: H-01264-2005R1

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Glucan Phosphate Attenuates Cardiac Dysfunction and Inhibits Cardiac MIF Expression

and Apoptosis in Septic Mice

Tuanzhu Ha1, Fang Hua1, Daniel Grant1, Yeling Xia1, Jing Ma1,

Xiang Gao2, Jim Kelley3, David L. Williams1, John Kalbfleisch4,

I. William Browder1, Race L. Kao1, and Chuanfu Li1*

Departments of Surgery1, Internal Medicine3, and Section of Medical Education4

East Tennessee State University, Johnson City, TN 37614

Animal Model Research Center2, Nanjing University, China, 210093

Running Head: Glucan phosphate prevents cardiac dysfunction in sepsis

Corresponding author:

Chuanfu Li, M.D. Department of Surgery East Tennessee State University Campus Box 70575 Johnson City, TN 37614-0575 Tel 423-439-6349 FAX 423-439-6259 Email Address: [email protected] Total words: 6,580

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Copyright Information

Articles in PresS. Am J Physiol Heart Circ Physiol (June 9, 2006). doi:10.1152/ajpheart.01264.2005

Copyright © 2006 by the American Physiological Society.


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Abstract

Myocardial dysfunction is a major consequence of septic shock and contributes to the

high mortality of sepsis. We have previously reported that glucan phosphate (GP) significantly

increased survival in a murine model of cecal ligation and puncture (CLP)-induced sepsis. In the

present study, we examined the effect of GP on cardiac dysfunction in CLP-induced septic mice.

GP was administered to ICR/HSD mice 1 hr before induction of CLP. Sham surgical operated

mice served as control. Cardiac function was significantly decreased six hrs after CLP-induced

sepsis compared with sham control. In contrast, GP administration prevented CLP-induced

cardiac dysfunction. Macrophage migration inhibitory factor (MIF) has been implicated as a

major factor in cardiomyocyte apoptosis and cardiac dysfunction during septic shock. CLP

increased myocardial MIF expression by 88.3% (p<0.05) and cardiomyocyte apoptosis by 7.8

fold (p<0.05) compared with sham control. GP administration, however, prevented CLP-

increased MIF expression, and decreased cardiomyocyte apoptosis by 51.2% (p<0.05) compared

with untreated CLP mice. GP also prevented sepsis-caused decreases in phospho-Akt, phospho-

GSK-3β and Bcl-2 levels in the myocardium of septic mice. These data suggest that GP

treatment attenuates cardiovascular dysfunction in fulminating sepsis. GP administration also

activates the PI3K/Akt pathway, decreases myocardial MIF expression and reduces

cardiomyocyte apoptosis.

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Introduction

Cardiovascular dysfunction is a major consequence of septic shock and contributes to the

high morbidity and mortality of sepsis (11; 12; 23). Current wisdom implies that following

severe injury or infectious challenge some patients respond by activating pro-inflammatory

signaling pathways and over-expressing inflammatory mediators that result in a systemic

inflammatory response that culminates in severe shock, multi-organ failure and death (34).

Despite extensive investigation, the cellular and molecular mechanisms that mediate myocardial

dysfunction during septic shock have remained elusive. Furthermore, developing effective

methods for preventing and/or treating sepsis-induced cardiovascular dysfunction has proven to

be difficult. A growing body of evidence suggests that there is a link between the innate immune

response and myocardial dysfunction in several important disease states including

ischemia/reperfusion injury (13), congestive heart failure (CHF) (21) and septic shock (12).

Glucan phosphate is a (1→3)-β-D-linked glucose ligand which has been reported to

modulate innate immunity and pro-inflammatory signaling in sepsis (46-49). We have reported

that glucan phosphate will significantly increase long-term survival (43), down regulate sepsis-

induced expression of Toll-like receptor 4 (TLR4) (44) and blunt tissue NFκB (43)and NF-IL6

(43) activation in a murine model of cecal ligation and puncture (CLP) induced polymicrobial

sepsis. Several groups have reported that TLR4 mediated NFκB activation contributes to

myocardial injury in response to ischemia/reperfusion (I/R) injury (26; 28; 35). We have

reported that glucan phosphate administration dramatically reduces myocardial damage in

response to I/R injury (28). The mechanisms of glucan-induced cardioprotection involve

decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKβ activity

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and decreased NFκB activity (28). In addition, glucan phosphate increased tyrosine

phosphorylation of the TLR4 transmembrane domain resulting in increased phosphoinositide-3-

kinase (PI3K)/Akt activity in the myocardium, which correlated with decreased cardiac myocyte

apoptosis following I/R (28). We have also shown that glucan phosphate increases long-term

survival in CLP sepsis via a PI3K/Akt dependent mechanism (45). Based on these data, we

hypothesized that glucan phosphate may exert a protective effect on cardiovascular function

during septic shock.

Macrophage migration inhibitory factor (MIF) is a neuropeptide and inflammatory

mediator which has been reported to play a critical role in sepsis-induced multiple organ failure

and immune homeostasis (8). Increased levels of circulating MIF have been observed in septic

animals and in patients with septic shock (6; 7). MIF is thought to play a role in host response to

endotoxin via modulation of TLR4 expression (37; 38). In support of this concept, neutralization

of MIF with specific antibody or through MIF gene deletion results in protection from lethal

endotoxemia and septic shock (5; 6). In addition, MIF has been implicated as an initiating factor

in myocardial inflammatory responses, cardiac myocyte apoptosis and cardiac dysfunction

during sepsis (11; 16). It is possible therefore, that modulation of MIF expression in the

myocardium could result in the improvement of cardiac dysfunction induced by septic shock. In

the present study, we evaluated left ventricular function in CLP-induced sepsis in the presence or

absence of glucan phosphate treatment. We observed that glucan phosphate administration

attenuated left ventricular dysfunction in CLP-induced sepsis. Glucan phosphate treatment also

inhibited myocardial MIF expression, activated PI3K/Akt, and decreased cardiac myocyte

apoptosis.

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Materials and Methods

Experimental animals: Age- and weight-matched male ICR/HSD mice were obtained

from Harlan Sprague Dawley (Indianapolis, IN). The mice were maintained in the Division of

Laboratory Animal Resources at East Tennessee State University (ETSU). The experiments

outlined in this manuscript conform with the Guide for the Care and Use of Laboratory Animals

published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

All aspects of the animal care and experimental protocols were approved by the ETSU

Committee on Animal Care.

Glucan phosphate: We selected glucan phosphate (GP) for this study because we have

previously demonstrated that GP will increase long-term survival in CLP sepsis (43-45) and it

decreases myocardial injury in response to I/R (28). Water soluble GP was prepared and

chemically characterized in our laboratory as previously described (22; 47).

CLP polymicrobial sepsis model. Cecal ligation and puncture was performed to induce

sepsis in mice as previously described (3; 42; 52). Briefly, the mice were anesthetized by

isoflurane inhalation and ventilated with room air using a rodent ventilator. A midline incision

was made on the anterior abdomen and the cecum was exposed and ligated with a 4-0 suture.

Two punctures were made through the cecum with an 18-gauge needle and feces were extruded

from the holes. The abdomen was then closed. Sham surgical operated mice served as the

surgical control group. Mice that were not subjected to surgery or anesthesia served as the

normal controls. For the treatment group, the animals were administered GP at 40 mg/kg body

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weight by intraperitoneal (i.p.) injection one hour prior to surgery. This dose of GP has been

shown to be effective in increasing survival of septic animals (43) and protecting the

myocardium from ischemia/reperfusion injury (28). There were 6 groups: normal control (N),

sham surgery control (S), CLP, N+ GP, S + GP, and CLP + GP with 4-8 mice in each group.

In separate experiments, a less severe model of CLP sepsis was employed in combination

with fluid resuscitation. Glucan phosphate (40 mg/kg) was administered to the experimental

mice one hr before surgical operation. CLP was performed as described above and a single

puncture was made through the cecum with a 20-gauge needle and feces were extruded from the

hole. After surgical operation, a single dose of resuscitative fluid (lactated Ringer’s solution, 50

ml/kg body weight) was immediately administered by subcutaneous injection. There were 6

groups which were the same as described above.

Experimental protocols: Mice were subjected to cecal ligation and puncture at time 0

and six hrs after CLP, cardiac function measurements were performed as described previously

(17; 20). To examine the effects of GP on the expression of MIF and cardiac myocyte apoptosis,

hearts were harvested and washed free of blood with ice-cold phosphate buffered saline (PBS). A

single heart tissue section (5 mm) was taken from each heart at the same anatomical locations,

immersion-fixed in 4% buffered paraformaldehyde, and embedded in paraffin for preparation of

tissue sections (17; 18; 36). The remaining heart tissue sections were immediately frozen in

liquid nitrogen and stored at -80 0C.

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In situ apoptosis assay: In situ cardiac myocyte apoptosis was examined by the TdT -

mediated dUTP nick end labeling (TUNEL) assay (Boehringer Mannheim, Indianapolis, IN) as

previously described (17; 18; 28). Sectioned heart tissue was embedded in paraffin. Three slides

from each block were evaluated for percentage of apoptotic cells using the TUNEL assay. Four

slide fields were randomly examined using a defined rectangular field area with 200 x

magnification. One hundred cells were counted in each field and apoptotic cardiac myocytes

were expressed as the percentage of total cells.

Immunohistochemistry: Immunohistochemistry was performed to examine Caspase-3

activity and MIF expression in heart sections using specific anti-capase-3 cleaved antibody (Cell

Signaling Technology) or anti-MIF antibody (17; 18) respectively as previously described (45).

Briefly, hearts from each group were harvested and one section was immersion-fixed in 4%

buffered paraformaldehyde, embedded in paraffin, cut at 5 µm, and stained with an antibody

directed against activated caspase-3 or MIF (17; 18). Three slides from each block were

evaluated with brightfield microscopy.

Western Blot: Cytoplasmic proteins were isolated from heart tissues and immunoblots

were performed as described previously (18; 27-30). Briefly, the cellular proteins were

separated by SDS-polyacrylamide gel electrophoresis and transferred onto Hybond ECL

membranes (Amersham Pharmacia, Piscataway, NJ). The ECL membranes were incubated with

appropriate primary antibody [anti-phospho-Akt, anti- phospho-GSK3β (anti-Ser9), anti-GSK-3β

(Cell Signaling Technology, Inc. Beverly, MA), anti-Akt, and anti-MIF (Santa Cruz

Biotechnology)] respectively, followed by incubation with peroxidase-conjugated second

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antibodies (Cell Signaling Technology, Inc.). The membranes were analyzed by the ECL system

(Amersham Pharmacia). The same membranes were stripped and re-probed with anti-GAPDH

(glyceraldehyde-3-phosphate dehydrogenase, Biodesign, Saco, Maine) as loading controls. The

signals were quantified by scanning densitometry and computer-assisted image analysis.

Hemodynamic measurements: Mice were anesthetized with isoflurane inhalation and

ventilated with room air using a rodent ventilator. A microconductance pressure catheter (Millar

Instruments Inc., Houston, TX) was positioned in the left ventricle (LV) via the right carotid

artery for continuous registration of LV pressure-volume loops (17; 20) using the PowerLab

system (AD Instruments, Inc., Colorado Springs, CO). A cuvette calibration method was used to

convert the conductance voltage into volume units by filling nonconductive cuvettes of known

diameter with heparin treated mouse blood. Parallel conductance from surrounding structures

was determined by intravenous (external jugular vein) injection of a small bolus (15 μl) of

hypertonic saline (15% NaCl). All measurements were performed while ventilation was turned

off momentarily. Indices of systolic and diastolic cardiac performance were derived from LV

pressure-volume data obtained at steady state. Cardiac output, ejection fraction, stroke volume

and stroke work were chosen as indices of cardiac function.

Statistical analysis: Figures present group mean levels and the corresponding error of the

mean (sem). Analysis of variance (ANOVA) and the Kruskal-Wallis(KW) procedure were used

to assess difference between the 6 group means and 6 group medians (KW). Specific

comparisons of interest (S vs CLP, CLP vs CLP+G) were judged by the least significant

difference test and the t-test (when ANOVA was significant) and by the Mann-Whitney U-test

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when a normal distribution was not indicated (using residuals and the Anderson-Darling test).

Probability levels of 0.05 and smaller are used for reporting in the Figures.

Results

Glucan phosphate prevented cardiac dysfunction in septic mice. In vivo cardiac function

was measured 6 hrs after the mice were subjected to CLP using the Millar pressure-volume

conductance system. As shown in Figure 1(A), the levels of end-systolic volume were

significantly reduced in CLP-induced septic shock mice without fluid resuscitation. CLP-induced

septic shock also resulted in significant suppression of cardiac function as evidenced by

reduction of stroke work by 47.7%, and cardiac output by 41.1%, respectively, compared with

sham control. In addition, end-systolic pressure (mmHg) was decreased by 15.1%, dp/dt max

(mmHg/sec) by 25.7%, and ejection fraction by 11.7% in the CLP group compared with sham

control. Following glucan phosphate administration, end-systolic volume and end-diastolic

volume were maintained at normal levels. Glucan phosphate treatment prevented sepsis-induced

cardiac dysfunction. When compared to the CLP group, glucan phosphate administration

increased cardiac output by 53.8%, ejection fraction by 11.7%, dp/dt max (mmHg/sec) by

49.8%, and stroke work by 58.6%, respectively. Cardiac function in glucan treated CLP mice

was not significantly different from normal or sham control animals.

Figure 1(B) shows that fluid resuscitation immediately following surgery maintained

circulating blood volume in CLP-induced septic mice as evidenced by the levels of end-systolic

volume which were not reduced compared with sham control. However, CLP-induced sepsis

with fluid resuscitation still resulted in cardiac dysfunction which showed a similar pattern when

compared to CLP-induced septic mice without fluid resuscitation (Fig. 1A). It was also noted

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that the effect of glucan was similar in the presence or absence of fluid resuscitation.

Glucan phosphate prevented increased MIF expression in the myocardium of CLP-

induced septic mice. Neutralization of MIF has been shown to reverse endotoxin-induced

myocardial dysfunction in an experimental rat model (11). To examine the effect of glucan

phosphate on the expression of MIF in the myocardium of septic mice, we analyzed the

expression of MIF in the hearts by immunoblot and immunohistochemistry. As shown in Figure

2A, the levels of MIF in the myocardium were significantly increased by 88.3% in CLP mice

compared with sham control (0.98 ± 0.12 vs 0.52 ± 0.10). In glucan phosphate treated mice, the

levels of MIF in the myocardium were not significantly different from normal or sham controls

(Figure 2A). Immunohistochemical examination showed increased expression of MIF in cardiac

myocytes of CLP mice (Figure 2B). Glucan phosphate treatment prevented the increased MIF

expression in cardiac myocytes from CLP mice (Figure 2B).

Glucan phosphate inhibited sepsis-induced cardiac myocyte apoptosis. Cardiac myocyte

apoptosis plays a major role in cardiac dysfunction (24; 25; 41). Therefore, we examined the

effect of glucan phosphate administration on cardiac myocyte apoptosis in septic mice using the

TUNEL assay. Figure 3A shows that cardiac myocyte apoptosis was significantly increased

(7.8 fold) in the myocardium of septic mice compared with sham control (39.63 ± 2.04% vs 4.49

± 0.80%). The percentage of apoptotic cells in glucan phosphate treated CLP mice was also

significantly increased compared to the controls, however, the increase was significantly less

than in the CLP mice. Activation of caspase-3 is an established marker for apoptotic cells. As

shown in Figure 3B, caspase-3 activity was increased in the myocardium of septic mice as

evidenced by immunohistochemistry with specific anti-cleaved caspase-3 antibody when

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compared with sham control. We observed decreased caspase-3 activity in the myocardium of

glucan phosphate treated CLP septic mice compared with the untreated CLP mice (Figure 3B).

Glucan phosphate prevented the decrease in phospho-Akt and phospho-GSK-3β in the

myocardium of septic mice. Activation of the PI3K/Akt signaling pathway has been shown to

prevent cardiac myocyte apoptosis (15; 51). We have demonstrated that glucan phosphate

increases PI3K/Akt activity in ischemic rat hearts and that the increase in PI3K/Akt activation

correlates with decreased myocardial apoptosis (28). We have previously shown that glucan

phosphate increased long-term survival in CLP sepsis via a PI3K/Akt dependent mechanism

(45). To examine the effect of glucan phosphate on the activation of PI3K/Akt in the

myocardium of septic mice, we examined the levels of phospho-Akt. Figure 4A shows that

the levels of the phospho-Akt were reduced in the myocardium of CLP mice compared with

sham controls (p≤0.086). In contrast, glucan phosphate treatment prevented the sepsis-induced

decrease in myocardial phospho-Akt levels (Figure 4A). The levels of phospho-Akt in the

myocardium of glucan phosphate treated CLP mice were significantly higher than in the CLP

group and not significantly different from sham controls. GSK-3β is a downstream target of

the PI3K/Akt pathway (32). As shown in Figure 4B the levels of phospho-GSK-3β (Ser-9) in

the myocardium were significantly reduced (64.7%) in CLP septic mice when compared with

sham controls. In contrast, the levels of phospho-GSK-3β in the myocardium of glucan

phosphate treated CLP septic mice was significantly higher than in the CLP mice and not

significantly different from sham or normal controls (Figure 4B).

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Glucan phosphate prevented the decrease in Bcl-2 levels in the myocardium of septic

mice. Bcl-2 is an important molecule for cell survival and anti-apoptosis, therefore, we

examined the effect of glucan phosphate administration on the expression of Bcl-2 in the

myocardium following CLP. Figure 5 shows that Bcl-2 levels in the myocardium of CLP-

induced septic mice were significantly decreased by 46.5% compared with sham control. In

contrast, administration of glucan phosphate prevented the decrease in the levels of Bcl-2

observed in untreated CLP mice. Levels of Bcl-2 in the glucan phosphate treated CLP mice were

not significantly different from sham controls.

Discussion

An important finding in the present study is that glucan phosphate administration

attenuated left ventricular cardiac dysfunction in CLP sepsis. Glucan phosphate attenuation of

cardiac dysfunction positively correlated with increased PI3K/Akt activity, decreased MIF

expression and reduction of cardiac myocyte apoptosis in septic mice. These results suggest that

activation of PI3K/Akt, inhibition of MIF expression, and reduced cardiac myocyte apoptosis in

the myocardium by glucan phosphate could explain, in part, the mechanisms of improved cardiac

function in sepsis.

The septic shock model induced by CLP in the present study is a hypodynamic sepsis

model which is characterized by reduced levels of end-systolic volume and cardiac output. The

hypovolemia during sepsis is usually caused by vasodilatation due to inflammatory cytokines,

resulting in maldistribution of blood flow and myocardial depression. Adequate fluid

resuscitation, therefore, is one of the keystones in the management of septic shock. In the

present study, we have observed that following fluid resuscitation, the levels of end-systolic

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volume in CLP animals were maintained at the control levels, indicating that fluid resuscitation

significantly improved circulating blood volume. However, cardiac output in CLP mice was still

significantly decreased compared with sham control, suggesting that CLP-induced septic shock

results in significant myocardial suppression independent of fluid status. Tao et al (39) have

shown that cardiac function was significantly reduced in CLP mice with fluid resuscitation.

Albuszies et al reported that a combination of fluid resuscitation and norepinephrine resulted in

significantly increased cardiac output in CLP-induced septic mice (1). Collectively, these data

suggest that prevention of cardiac dysfunction could be an important strategy in management of

septic shock.

Clinical and experimental studies have shown that myocardial dysfunction is an early and

fatal complication of septic shock (11; 12; 23; 39) and that the TLR4 mediated NFκB activation

signaling pathway could be an early molecular event leading to cardiac dysfunction during septic

shock (33; 40). We have previously shown that glucan phosphate significantly increased

survival in CLP mice (43) and the mechanisms involved down regulating the expression of

TLR4 and blunting NFκB activation in the lung, liver, and spleen (44). Therefore, we postulated

that glucan phosphate administration could also improve myocardial function in the septic mice.

To evaluate our hypothesis, we examined cardiac function in CLP-induced sepsis with or without

glucan phosphate treatment. We observed that the cardiac function was significantly depressed

in untreated CLP mice. In glucan phosphate treated CLP mice, however, cardiac function was

maintained at control levels. We have previously shown that glucan phosphate administration

significantly blunted NFκB activation both in septic mice (43) and in ischemic hearts (28).

NFκB is a critical transcription factor in TLR mediated signaling pathways and plays a critical

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role in regulation of the expression of a number of genes, including inflammatory cytokines such

as TNFα and IL-1β, which have been shown to suppress cardiac function synergistically during

sepsis (10). Unfortunately, anti-TNFα or anti-IL-1β therapy did not result in increased survival

in patients with septic shock (12). Furthermore, we have reported that glucan treatment in CLP

mice did not result in significant changes in serum cytokine levels, even though survival

outcome was increased (45). Therefore, it is likely that the improved cardiac function observed

in septic animals treated with glucan phosphate is mediated by mechanisms that are independent

of inflammatory cytokine expression.

Recent studies have shown that MIF is expressed in the myocardium (11; 16) and that

MIF neutralization by anti-MIF antibody reversed endotoxin or burn injury-induced cardiac

dysfunction (11; 50). Anti-MIF treatment also protected TNFα knockout mice, which were

sensitive to CLP and succumbed quickly to uncontrolled infection from lethal peritonitis induced

by CLP (11). The septic TNFα knockout mice were protected even if the treatment was started 8

hrs after the onset of bacterial peritonitis (11). In the present study, we observed an inverse

relationship between cardiac function and myocardial MIF levels in sepsis. Specifically, cardiac

function was significantly depressed, while myocardial MIF expression was significantly

increased in the CLP mice. In contrast, glucan phosphate treated septic animals showed normal

cardiac function and myocardial MIF levels that were equivalent to the untreated controls. These

data suggest that glucan phosphate preserved cardiac function in septic mice while preventing up

regulation of MIF expression in the myocardium. The mechanism(s) by which glucan phosphate

prevented myocardial MIF expression are unclear. Recent studies suggest that IL-1β-induced

MIF synthesis by human endometrial stromal cells is mediated via NFκB activation, since

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blockade of NFκB translocation into the nucleus significantly inhibited MIF secretion (9). MIF

also regulates TLR4 expression (37; 38). Activation of the TLR4 signaling pathway leads to

NFκB activation (37; 38). In addition, MIF-deficient macrophages were found to be

hyporesponsive to LPS stimulation due to down regulation of TLR4 expression (37; 38). In our

previous studies we have reported that glucan phosphate blunted TLR4 up regulation and

inhibited NFκB activation in CLP sepsis. Therefore, it is possible that the effect of glucan

phosphate on MIF expression in the myocardium may involve modulation of sepsis-induced

TLR4 and NFκB signaling.

Cardiac myocyte apoptosis plays an important role in cardiac dysfunction (28).

Numerous studies have shown that apoptosis plays a significant role in the morbidity and

mortality associated with sepsis (4). By way of example, prevention of apoptosis with caspase

inhibitors significantly improved survival in murine CLP-induced sepsis (10; 24). Support for

this concept can also be found in the work of Bommhardt et al (4). These investigators reported

that mice that constitutively over express active Akt in their lymphocytes showed decreased

lymphocyte apoptosis, a TH1 cytokine propensity, and a marked improvement in survival

outcome in response to CLP sepsis (4). We have previously shown that CLP-induced sepsis

significantly increased apoptosis in the lung and spleen (45). In the present study we observed

that cardiac myocyte apoptosis was significantly increased in septic mice. Glucan phosphate

administration significantly reduced cardiac myocyte apoptosis and decreased caspase-3 activity

in the myocardium of the septic mice. In addition, glucan phosphate prevented the decrease in

expression of Bcl-2 in the myocardium in septic mice. The results were consistent with our

previous observation that glucan phosphate significantly decreased splenocyte apoptosis and

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caspase-3 activity in CLP-induced septic mice (45). Recent studies suggested that death

receptor-mediated apoptotic signaling contributes to septic shock-induced apoptosis (41). For

example, caspase-8 activity was significantly increased in the myocardium of LPS-induced

cardiac dysfunction (24) and in vivo delivery of caspase-8 or Fas siRNA improved the survival

of septic mice (41). Interestingly, stimulation of TLRs can result in apoptosis by triggering pro-

apoptotic signaling (2; 19; 31) and blocking TLR signaling by transfection of dominant negative

MyD88 or dominant negative FADD (dnFADD) reduced cell death (19). These observations

suggest that death receptor-mediated signaling is involved in TLR mediated apoptosis. We have

observed that over expression of TLR2 and TLR4 contributed to apoptosis (14) and that glucan

phosphate administration significantly reduced I/R mediated cardiac myocyte apoptosis through

modulation of the TLR4 mediated signaling pathway (28). Glucan phosphate administration also

reduced the expression of TLR4 in the tissues of CLP mice (44). Thus, we speculate that glucan

phosphate treatment reduces cardiac myocyte apoptosis by modulating TLR4 mediated apoptotic

signaling pathways in the myocardium of septic mice.

Activation of the PI3K/Akt signaling pathway has been shown to prevent apoptosis and

promote cell survival (15; 51). We have reported that inhibition of PI3K/Akt by wortmannin

significantly increased apoptosis and resulted in a change in the distribution of splenocyte

apoptotic profiles in CLP sepsis (45). We have also shown that glucan phosphate mediated

protection in CLP sepsis (45) and myocardial I/R injury (28) through a PI3K/Akt dependent

mechanism. In the present study we observed that glucan phosphate prevented the decrease in

myocardial phospho-Akt levels in response to sepsis. Glucan treatment also resulted in increased

myocardial phosphorylation of GSK-3β. Phosphorylation of Akt at ser473 activates the enzyme,

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while phosphorylation of GSK-3β at Ser-9 results in its inactivity (32). The data showed that

glucan treatment activates myocardial Akt and inactivates myocardial GSK-3β. These changes

in Akt/GSK-3β activity correlate with decreased myocardial apoptosis and improved cardiac

function in CLP sepsis (45).

In summary, glucan phosphate administration attenuated cardiac dysfunction in CLP

sepsis. The mechanisms by which glucan phosphate attenuated cardiac function include;

activation of Akt, inhibition of MIF expression, and reduction of cardiac myocyte apoptosis.

The present study also indicates that increased expression of MIF and cardiac myocyte apoptosis

in the myocardium could contribute to the depression of cardiac function in CLP-induced sepsis.

Future studies will determine whether specific blocking of MIF expression will prevent septic

shock induced cardiac dysfunction and whether treatment with glucan phosphate after sepsis has

been initiated will prevent cardiac dysfunction. In addition, studies determining the molecular

mechanisms by which glucan phosphate exerts its cardio-protective effect will be pursued.

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ACKNOWLEDGEMENTS

This work was supported in part by NIH RO1 HL071837, AHA-0051480B and AHA-

0255038B to CL. This work was also supported in part by NIH GM53552, NIH AI45829 and

NIH AT00501 to DLW; ETSU Research Development Committee grant to TH; and National

Gongguan Project of China (NGGPOC) 2001BA710B to XG.

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Reference List

1. Albuszies G, Radermacher P, Vogt J, Wachter U, Weber S, Schoaff M, Georgieff M and Barth E. Effect of increased cardiac output on hepatic and intestinal microcirculatory blood flow, oxygenation, and metabolism in hyperdynamic murine septic shock. Crit Care Med 33: 2332-2338, 2005.

2. Aliprantis AO, Yang R-B, Weiss DS, Godowski P and Zychlinsky A. The apoptotic signaling pathway activated by Toll-like receptor-2. The EMBO Journal 19: 3325-3336, 2000.

3. Baker CC, Chaudry IH, Gaines HO and Baue AE. Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model. Surgery 94: 331-335, 1983.

4. Bommhardt U, Chang KC, Swanson PE, Wagner TH, Tinsley KW, Karl IE and Hotchkiss RS. Akt decreases lymphocyte apoptosis and improves survival in sepsis. J Immunol 172: 7583-7591, 2004.

5. Bozza M, Satoskar AR, Lin GS, Lu B, Humbles AA, Gerard C and David JR. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J Exp Med 189: 341-346, 1999.

6. Calandra T, Echtenacher B, Le Roy D, Pugin J, Metz CN, Hultner L, Heumann D, Mannel D, Bucala R and Glauser MP. Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nature Med 6: 164-170, 2000.

7. Calandra T, Froidevaux C, Martin C and Roger T. Macrophage Migration Inhibitory Factor and Host Innate Immune Defenses against Bacterial Sepsis. JID 187: S385-S390, 2003.

8. Calandra T and Roger T. Macrophage Migration Inhibitory Factor: A Regulator of Innate Immunity. Nature Reviews Immunology 3: 791-800, 2003.

9. Cao W-G, Morin M, Metz C, Maheux R and Akoum A. Stimulation of Macrophage Migration Inhibitory Factor Expression in Endometrial Stromal Cells by Interleukin 1, beta Involving the Nuclear Transcription Factor NFkB. Biology of Reproduction 73: 565-570, 2005.

10. Carlson DL, Willis MS, White DJ, Horton JW and Giroir BP. Tumor necrosis factor-α-induced caspase activation mediates endotoxin-related cardiac dysfunction. Crit Care Med 33: 1021-1028, 2005.

11. Chagnon F, Metz CN, Bucala R and Lesur O. Endotoxin-Induced Myocardial Dysfunction Effects of Macrophage Migration Inhibitory Factor Neutralization. Circ Res 96: 1095-1102, 2005.

of 31



20

12. Court O, Kumar A, Parrillo JE and Kumar A. Clinical review: Myocardial depression in sepsis and septic shock. Crit Care 6: 500-508, 2002.

13. Entman, M. L., Michael, L., Rossen, R. D., Dreyer, W. J., Anderson, D. C., Taylor, A. A., and Smith, C. W. Inflammation in the course of early myocradial ischemia. FASEB J 5, 2529-2537. 1991.

14. Fan W, Ha T, Li Y, Ozment-Skelton T, Williams DL, Kelley J, Browder IW and Li

C. Overexpression of TLR2 and TLR4 susceptibility to serum deprivation-induced apoptosis in CHO cells. Biochem Biophys Res Comm 337: 840-848, 2005.

15. Fujio Y, Nguyen T, Wencker D, Kitsis RN and Walsh K. Akt Promotes Survival of Cardiomyocytes In Vitro and Protects Against Ischemia-Reperfusion Injury in Mouse Heart. Circulation 101: 660-667, 2000.

16. Garner LB, Willis MS, Carlson DL, DiMaio JM, White MD, White DJ, Adams IGA, Horton JW and Giroir BP. Macrophage migration inhibitory factor is a cardiac-derived myocardial depressant factor. AJP - Heart 285: 2500-2509, 2003.

17. Ha T, Hua F, Li Y, Ma J, Gao X, Kelley J, Zhao A, Haddad GE, Williams DL, Browder IW, Kao RL and Li C. Blockade of MyD88 Attenuates Cardiac Hypertrophy and Decreases Cardiac Myocyte Apoptosis in Pressure Overload Induced Cardiac Hypertrophy in vivo. Am J Physiol Heart Circ Physiol 290: H985-H994, 2006.

18. Ha T, Li Y, Hua F, Ma J, Gao X, Kelley J, Zhao A, Haddad GE, Williams DL, Browder IW, Kao RL and Li C. Reduced cardiac hypertrophy in toll-like receptor 4-deficient mice following pressure overload. Cardiovasc Res 68: 224-234, 2005.

19. Into T, Liura K, Yasuda M, Kataoka H, Inoue N, Hasebe A, Takeda K, Akira S and Shibata KI. Stimulation of human Toll-like reeptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappaB activation. Cell Microbiol 6: 187-199, 2004.

20. Kao RL, Zhang F, Yiang Z-J, Gao X and Li C. Cellular Cardiomyoplasty Using Autologous Satellite Cells: from Experimental to Clinical Study. Basic Appl Myol 13: 23-28, 2003.

21. Katz SD, Rao R and Berman JW. Pathophysiological correlates of increased serum tumor necrosis factor in patients with congestive heart failure. Circulation 90: 12-16, 1994.

22. Kim YT, Kim E, Cheong C, Williams DL, Kim CW and Lim ST. Structural Characterization of beta-D-(1-3, 1-6) Glucans using NMR Spectroscopy. Carbohyd Res 328: 331-341, 2000.

23. Krishnagopalan S, Kumar A, Parrillo JE and Kumar A. Myocardial dysfunction in the patient with sepsis. Curr Opin Crit Care 8: 376-388, 2002.

of 31



21

24. Lancel S, Joulin O, Favory R, Goossens JF, Kluza J, Chopin C, Formstecher P, Marchetti P and Neviere R. Ventricular Myocyte Caspases Are Directly Responsible for Endotoxin-Induced Cardiac Dysfunction. Circulation 111: 2596-2604, 2005.

25. Lancel S, Petillot P, Favory R, Stebach N, Lahorte C, Danze PM, Vallet B, Marchetti P and Neviere R. Expression of apoptosis regulatory factors during myocardial dysfunction in endotoxemic rats. Crit Care Med 33: 492-496, 2005.

26. Langdale LA, Wilson L, Jurkovich GJ and Liggitt HD. Effects of immunomodulation with interferon-gamma on hepatic ischemia-reperfusion injury. Shock 11: 356-361, 1999.

27. Li C, Browder W and Kao RL. Early activation of transcription factor NF-κB during ischemia in perfused rat heart. Am J Physiol 276: H543-H552, 1999.

28. Li C, Ha T, Kelley J, Gao X, Qiu Y, Kao RL, Browder W and Williams DL. Modulating Toll-like receptor mediated signaling by (1-->3)-β-D-glucan rapidly induces cardioprotection. Cardiovascular Research 61: 538-547, 2004.

29. Li C, Ha T, Liu L, Browder W and Kao RL. Adenosine prevents activation of transcription factor NF-kappa B and enhances activator protein-1 binding activity in ischemic rat heart. Surgery 127: 161-169, 2000.

30. Li C, Kao RL, Ha T, Kelley J, Browder IW and Williams DL. Early Activation of IKKβ during in vivo myocardial ischemia. Am J Physiol Heart Circ Physiol 280: H1264-H1271, 2001.

31. Lopez M, Sly LM, Luu Y, Young D, Cooper H and Reiner NE. The 19-kDa Mycobacterium tuberculosis Protein Induces Macrophage Apoptosis Through Toll-Like Receptor-2. J Immunol 170: 2409-2416, 2003.

32. Martin M, Rehani K, Jope RS and Michalek SM. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nature Immunology 6: 777-784, 2005.

33. Nemoto S, Vallejo JG, Kneufermann P, Misra A, Defreitas G, Carabello BA and Mann DL. Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart. Am J Physiol Heart Circ Physiol 282: H2316-H2323, 2002.

34. Oberholzer A, Oberholzer C and Moldawer LL. Sepsis Syndromes: Understanding the Role of Innate and Acquired Immunity. Shock 16: 83-96, 2001.

35. Oyama J, Blais JrC, Liu X, Pu M, Kobzik L, Kelly RA and Bourcier T. Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice. Circulation 109: 784-789, 2004.

36. Ribeiro OG, Maria DA, Adribuch S, Pechberty S, Cabrera WHK, Morisset J, Ibanez OM and Seman M. Convergent alteration of granulopoiesis, chemotactic activity,

of 31



22

and neutrophil apoptosis during mouse selectin for high acute inflammatory response. J Leukoc Biol 74: 497-506, 2003.

37. Roger T, David J, Glauser MP and Calandra T. MIF regulates innate immune responses through modulation of Toll-like receptor 4. Nature 414: 920-924, 2001.

38. Roger T, Froidevaux D, Martin C and Calandra T. Macrophage migration inhibitory factor (MIF) regulates host responses to endotoxin through modulation of Toll-like receptor 4 (TLR4). J Endotoxin Res 9: 119-123, 2003.

39. Tao W, Enoh VT, Lin CY, Johnston WE, Li P and Sherwood ER. Cardiovascular dysfunction caused by cecal ligation and puncture is attenuated in CD8 knockout mice treated with anti-asialoGM1. Am J Physiol Regul Integr Comp Physiol 289: R478-R485, 2005.

40. Tavener SA, Long EM, Robbins SM, McRae KM, Remmen HV and Kubes P. Immune Cell Toll-Like Receptor 4 Is Required for Cardiac Myocyte Impairment During Endotoxemia. Circ Res 95: 700-707, 2004.

41. Wesche-Soldato DE, Chung C-S, Lomas-Neira J, Doughty LA, Gregory SH and Ayala A. In vivo delivery of caspase-8 or Fas siRNA improves the survival of septic mice. Blood 106: 2295-2301, 2005.

42. Williams DL, Ha T, Li C, Kalbfleisch JH and Ferguson DAJr. Early Activation of Hepatic NFkB and NF-IL6 in Polymicrobial Sepsis Correlates with Bacteremia, Cytokine Expression and Mortality. Ann Surg 230: 95-104, 1999.

43. Williams DL, Ha T, Li C, Kalbfleisch JH, Laffan JJ and Ferguson DA. Inhibiting early activation of tissue nuclear factor-κB and nuclear factor interleukin 6 with (1-->3)-β-D-glucan increases long-term survival in polymicrobial sepsis. Surgery 126: 54-65, 1999.

44. Williams DL, Ha T, Li C, Kalbfleisch JH, Schweitzer J, Vogt W and Browder IW. Modulation of tissue Toll-like receptor 2 and 4 during the early phases of polymicrobial sepsis correlates with mortality. Crit Care Med 31: 1808-1818, 2003.

45. Williams DL, Li C, Ha T, Ozment-Skelton T, Kalbfleisch JH, Preiszner J, Brooks L, Breuel K and Schweitzer JB. Modulation of the Phosphoinositide 3-Kinase Pathway Alters Innate Resistance to Polymicrobial Sepsis. J Immunol 172: 449-456, 2004.

46. Williams DL, Lowman DW and Ensley HE. Introduction to the Chemistry and Immunobiology of β-Glucans. In: Toxicology of 1->3-Beta-Glucans. Glucans as a Marker for Fungal Exposure, edited by Young SH and Castranova V. New York: Taylor & Francis, 2004, p. 1-34.

47. Williams DL, McNamee RB, Jones EL, Pretus HA, Ensley HE, Browder IW and Di Luzio NR. A method for the solubilization of a (1-3)-β-D-glucan isolated from Saccharomyces cerevisiae. Carbohyd Res 219: 203-213, 1991.

of 31



23

48. Williams DL, Pretus HA, McNamee RB, Jones EL, Ensley HE, Browder IW and Di Luzio NR. Development, physicochemical characterization and preclinical efficacy evaluation of a water soluble glucan sulfate derived from Saccharomyces cerevisiae. Immunopharmacol 22: 139-156, 1991.

49. Williams DL, Rice PJ, Herre J, Willment JA, Taylor PR, Gordon S and Brown GD. Recognition of fungal glucans by pattern recognition receptors. Recent Devel Carbohydrate Res 1: 49-66, 2003.

50. Willis MS, Carlson DL, DiMaio JM, White MD, White DJ, Adams IGA, Horton JW and Giroir BP. Macrophage migration inhibitory factor mediates late cardiac dysfunction after burn injury. AJP-Heart 288: 795-804, 2005.

51. Wu W, Lee W-L, Wu YY, Chen D, Liu T-J, Jang A, Sharma PM and Wang PH. Expression of Constitutively Active Phosphatidylinositol 3-Kinase Inhibits Activation of Caspase 3 and Apoptosis of Cardiac Muscle Cells. J Biol Chem 275: 40113-40119, 2000.

52. Yang S, Chung CS, Ayala A, Chaudry IH and Wang P. Differential alterations in cardiovascular responses during the progression of polymicrobial sepsis in the mouse. Shock 17: 55-60, 2002.

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FIGURE LEGENDS

Fig. 1. Glucan phosphate administration prevented left ventricle (LV) dysfunction in CLP-

induced septic mice. Glucan phosphate was administrated to mice by i.p. injection one hr before

the mice were subjected to CLP. Surgical operated mice served as sham control. (A) The

experimental mice did not receive fluid resuscitation. (B) The experimental mice were given

fluid resuscitation. Six hrs after CLP, left ventricle hemodynamic parameters were examined.

There were 4-8 mice in each group. * P<0.05 compared to age-matched respective sham control;

# P<0.05 compared to the CLP group. N = Normal; S = Sham; CLP = cecal ligation and

puncture; G = glucan phosphate.

Fig. 2. Glucan phosphate administration prevented increased expression of myocardial MIF in

septic mice. Glucan phosphate was administrated to mice by i.p. injection one hr before the mice

were subjected to CLP. Six hrs after CLP, hearts were harvested. Cellular proteins were isolated

from the hearts and the expression of MIF was examined by Western blot with specific

antibodies (A). Immunohistochemistry was performed for examination of MIF with specific

antibody in heart tissue sections embedded in paraffin (B). There were 4-8 mice in each group. *

P<0.05 compared to age-matched respective sham control; # P<0.05 compared with the CLP

group. N = Normal; S = Sham; CLP = cecal ligation and puncture; G = glucan phosphate.

Fig. 3. Glucan phosphate administration decreased cardiac myocyte apoptosis in CLP-induced

septic mice. Glucan phosphate was administered to mice by i.p. injection one hr before the mice

were subjected to CLP. Surgical operated mice served as sham control. Six hrs after CLP, the

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hearts were harvested and sectioned for the TUNEL assay of apoptosis (A) and

immunohistochemistry of caspase-3 activity with anti-cleaved caspase-3 antibody (B). In the

TUNEL assay, the blue color shows the nucleus of each cell and the dark brown color indicates

positive cardiac myocyte apoptosis which is marked by red arrows. In immunohistochemistry

examination, caspase-3 activity in the cardiac myocytes is indicated by brown color staining.

There were 4 mice in each group. * P<0.01 compared with age-matched respective sham control.

# P< 0.05 compared with the CLP group. S = Sham; CLP = cecal ligation and puncture; G =

glucan phosphate.

Fig. 4. Glucan phosphate administration prevented decreases in the phosphorylation of Akt and

GSK-3β in the myocardium of CLP-induced septic mice. Glucan phosphate was administrated

to mice by i.p. injection one hr before the mice were subjected to CLP. Surgical operated mice

served as sham control. Six hrs after CLP, hearts were harvested and the cellular proteins were

isolated for examination of phosphorylation of Akt (A) and phosphorylation of GSK-3β (Ser-9)

(B) by Western blot with specific antibodies. There were 4-8 mice in each group. * P<0.05

compared to age-matched respective sham control; # P<0.05 compared with the CLP group. N =

Normal; S = Sham; CLP = cecal ligation and puncture; G = glucan phosphate.

Fig. 5. Glucan phosphate administration prevented decreased levels of Bcl2 in the myocardium

of CLP-induced septic mice. Glucan phosphate was administrated to mice by i.p. injection one hr

before the mice were subjected to CLP. Surgical operated mice served as sham control. Six hrs

after CLP, hearts were harvested and the cellular proteins were isolated for examination of Bcl2

by Western blot with specific antibody. There were 4-8 mice in each group. * P<0.05 compared

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to age-matched respective sham control; # P<0.05 compared with the CLP group. N = Normal;

S = Sham; CLP = cecal ligation and puncture; G = glucan phosphate.

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Fig. 1(A)

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Fig. 1(B)

N S CLP N+G S+G CLP+G

End-

dias

tolic

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ume

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0

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-dia

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(mm

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Fig. 2(B)

Fig. 2(A)

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Fig. 3(A)

Fig. 3(B)

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Fig. 4(B)

Fig. 5

Fig. 4(A)

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