Post on 01-May-2023
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: Li@ETSU.EDU Total words: 6,580
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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.
Manuscript number: H-01264-2005R1
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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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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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26
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(B)
N S CLP N+G S+G CLP+G
End-
dias
tolic
Vol
ume
( μl)
0
40
50
60
#
*
N S CLP N+G S+G CLP+G
End-
syst
olic
Vol
ume
(μl)
0
20
40
60
N S CLP N+G S+G CLP+G
End
-sys
tolic
Pre
ssur
e (m
mH
g)
0
70
80
90
#
N S CLP N+G S+G CLP+G
End
-dia
stol
ic P
ress
ure
(mm
Hg)
0
1
2
3
4
5
N S CLP N+G S+G CLP+G
Car
diac
Out
put (
μl/m
in)
0
5000
10000
15000
20000
#
*
N S CLP N+G S+G CLP+G
Stro
ke W
ork
(mm
Hg*
ml)
0
1000
1500
2000
2500
3000
#
*
N S CLP N+G S+G CLP+G
dP/d
t max
(mm
Hg/
sec)
0
8000
10000
12000
#
*
N S CLP N+G S+G CLP+G
Ejec
tion
Frac
tion
(%)
0
50
60
70
80
#
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