| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1Pediatrics, 2Molecular Biology, and 3Surgery, The University of Texas Southwestern Medical Center, Dallas, Texas 75390; and 4Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio 43210
Submitted 26 July 2001 ; accepted in final form 21 April 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
heart failure; signal transduction; contractile function; genetically altered mice
Three types of observations have linked myocardial dysfunction with
inflammation. First, acute infections and inflammatory diseases can
precipitate transient heart failure
(29–32,
39). Second, both microbial
and endogenous proinflammatory mediators, such as bacterial endotoxin
[lipopolysaccharide (LPS)], interleukin-1 (IL-1), and tumor necrosis factor
(TNF)-
directly depress myocardial contractility
(5,
9,
21,
35,
37,
38). Third, elevated
circulating concentrations of these molecules also occur in patients with
heart failure of any cause and are correlated with more severe myocardial
dysfunction (18,
27,
46).
Expression of the Toll-like receptor-4 (TLR4), the mammalian LPS sensor,
together with its intracellular signaling apparatus
(6,
7,
10,
13,
23,
33) in the heart, suggests a
mechanism whereby LPS could trigger cardiac dysfunction. In immune cells such
as macrophages, LPS signals through an intracellular pathway shared with the
type I IL-1 receptor (the Toll/IL-1 pathway). After activation of TLR4 or the
type 1 IL-1 receptor, MyD88 and IL-1 receptor associated kinase-1 (IRAK1) are
recruited to the activated receptor complex
(6,
24,
48). IRAK1 becomes
phosphorylated, dissociates from the receptor complex, and associates with TNF
receptor-associated factor 6
(6,
7). The signal is then
distributed to multiple downstream targets, including NF-
B, c-Jun
NH2-terminal kinase (JNK), and p38 MAPK. Deletion of MyD88,
IRAK1, or TNF receptor-associated factor 6 disrupts both IL-1 and LPS
signaling to distal pathways and disturbs normal physiological responses to
these proinflammatory mediators in innate immune cells
(1,
15,
16,
20,
25,
41,
44).
The presence of Toll/IL-1 signaling molecules in the heart and their upregulation in disease states such as congestive heart failure led to the hypothesis that the same pathway that initiates the inflammatory response to infection in immune cells also functions in the heart and mediates acute and chronic myocardial contractile dysfunction. Although previous studies (26, 45) have demonstrated a role for TLR4 in both LPS- and burn-induced myocardial dysfunction, a requirement for IRAK1 has only been shown in burn-triggered contractile impairment (43). Thus it is unknown whether TLR4 stimulation activates IRAK1 in the heart or whether IRAK1 function is necessary for contractile dysfunction after LPS administration. Studies reported here show that LPS activates IRAK1 in the heart. Moreover, IRAK1 inactivation alters cardiac signaling responses to LPS and protects mice from LPS-induced contractile function. Finally, IRAK1 deletion enhances survival in transgenic mice with myocarditis, cardiomyopathy, and lethal heart failure. Thus interference with Toll/IL-1 signaling, as occurs with IRAK1 inactivation, may represent a therapeutic strategy to ameliorate conditions associated with either acute or chronic cardiac contractile dysfunction.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
and IB- were purchased
from Santa Cruz Biotechnology and New England Biolabs, respectively.
Recombinant glutathione S-transferase-IB- and glutathione
S-transferase-c-Jun were provided by Melanie Cobb. Animals. All animals were used in compliance with the guidelines established by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center and performed in accordance with National Institutes of Health guidelines for the use of laboratory animals.
IRAK1-deficient and cardiac-specific TNF- overexpressor mice were
generated and genotyped as previously described
(4,
44). For generation of the
IRAK1 knockout (KO), the Irak1 gene was inactivated in murine
embryonic stem cells using targeted mutagenesis. Mutant stem cells were
injected into wild-type (WT) blastocysts and resulted in chimeric mice. Two
chimeric animals transmitted the mutant Irak1 gene through the
germline. Offspring from these founders were interbred to generate homozygous
IRAK1-deficient females and hemizygous IRAK1-deficient males (Irak1
is located on the X chromosome)
(44). These animals were on a
hybrid background (129Sv x C57BL/6). WT animals on a similar hybrid
background were used as controls for all experiments. Cardiac-specific
TNF--overexpressing mice were generated by microinjection of a
transgene encoding the murine TNF- sequence coupled to an
-myosin heavy chain promoter into the male pronucleus of fertilized
mouse eggs and implanted into pseudopregnant females. Offspring with the
integrated transgene became founders for this novel line of transgenic mice.
To generate transgenic KO double mutant mice, homozygous IRAK1-deficient
females were crossed to male TNF- overexpressors. Male transgenic
offspring were crossed to IRAK1 KO females and progeny with the TNF-
transgene were used in subsequent studies. Hybrid (C57Bl/6J x SJL) WT
mice also served as additional controls.
In vitro kinase reactions. At different times after LPS injection,
mice were euthanized, and the hearts were immediately removed and snap frozen
in liquid nitrogen. Longitudinal sections (25–40 mg) that included both
atrial and ventricular tissue were cut from frozen hearts, rinsed in ice-cold
phosphate-buffered saline to remove clotted blood, and minced into small
pieces using a number 11 scalpel blade. The tissue was then disrupted in a
Dounce homogenizer with 1 ml of either a HEPES lysis buffer [50 mM HEPES, pH
7.5; 150 mM NaCl; 1% Triton X-100; 10% glycerol, and 1 mM dithiothreitol
(DTT)] with protease (Complete Inhibitor, Roche; Indianapolis, IN) and
phosphatase inhibitors (20 mM NaF, 20 mM sodium glycerophosphate, and 0.5 mM
sodium orthovanadate) for all kinases except IRAK1, or a RIPA lysis buffer (50
mM Tris · HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.25% sodium
deoxycholate; 0.1% SDS; and 1 mM DTT) with the same protease and phosphatase
inhibitors. After a 30-min incubation on ice, lysates were cleared by
centrifugation at 20,000 g for 10 min at 4°C, and the supernatant
was transferred to a clean microcentrifuge tube. The total protein
concentration of the lysates was determined with the use of a protein assay
kit (Bio-Rad; Hercules, CA). Kinases were immunoprecipitated from 1 mg of
total cardiac protein in a minimum 250-µl volume with 1 to 5 µl of
antiserum on rocking platform overnight at 4°C. Protein A-conjugated
agarose (20 µl; Roche) was added and allowed to incubate for 2 h at
4°C. Precipitates were washed three times with lysis buffer and once with
incomplete kinase buffer composed of (in mM) 50 Tris · HCl (pH 7.4), 10
MgCl2, and 1 DTT. Beads were then resuspended in complete kinase
buffer composed of 50 mM Tris · HCl (pH 7.4), 10 mM MgCl2, 1
mM DTT, 50 µM "cold" ATP, and [
-32P]ATP (10
µCi/reaction) together with appropriate substrate (0.3 mg/ml) in a total
volume of 25 µl. The mixtures were incubated at 30°C for 30 min.
Reactions were stopped by adding an equal volume of 2x SDS-PAGE sample
buffer (200 mM Tris · HCl, 4% SDS, 0.04% bromophenol blue, and 20%
glycerol), followed by heating at 95°C for 5 min. Samples were
fractionated on a large-format 10% SDS-polyacrylamide gel and the proteins
were transferred to a polyvinylidene difluoride membrane (Immobilon-P,
Millipore; Bedford, MA). Membranes were air dried, exposed to phosphor storage
screens, and developed with the use of a phosphorimaging system (Molecular
Dynamics; Mountain View, CA). Fold catalytic activation was determined by
densitometric increases in signal above the 0-h time point.
Electromobility shift assays. A modified procedure based on the
method of Schreiber et al.
(34) was used. Longitudinal
heart sections (25 mg) were homogenized in ice-cold hypotonic lysis buffer
composed of (in mM) 10 Tris · HCl (pH 7.8), 5 MgCl2, 10 KCl,
0.3 EGTA, and 0.5 DTT and 0.3 M sucrose (protease inhibitors) and incubated on
ice for 15 min. After the addition of Nonidet P-40 (final concentration: 0.5%)
the mixture was vortexed and centrifuged to recover nuclei. The supernatant
was saved for IB immunoblots. Nuclear proteins were extracted from the
pellet that was composed of (in mM) 20 Tris · HCl (pH 7.8), 5
MgCl2, 320 KCl, 0.2 EGTA, and 0.5 DTT (plus protease inhibitors)
for 15 min on ice and centrifuged at 13,500 g for 15 min. Extracts
were stored at –80°C. Total protein concentration of nuclear
extracts was determined with a protein assay kit (Bio-Rad).
Double-stranded oligonucleotide corresponding to the consensus NF-B
binding site of the murine -light chain enhancer
(5'-AGTTGAGGGGACTTTCCCAGGC-3') was purchased from Amersham
Pharmacia Biotech (Piscataway, NJ). Oligonucleotide (3.5 pmol), T4
polynucleotide kinase (5 units) in 1x kinase buffer (Promega; Madison,
WI), and [-32P]ATP (30 µCi; DuPont-New England Nuclear;
Boston, MA) were incubated at 37°C for 60 min. The labeled probe was
separated from unbound ATP with the use of microcolumns (ProbeQuant G-50,
Amersham Pharmacia Biotech) and stored at –20°C. Nuclear proteins
(2.5 µg) were incubated with 500,000 counts/min of probe in the presence of
salmon sperm DNA (2 µg) in 1x gel shift buffer composed of (in mM) 20
HEPES, 50 KCl, 1 DTT, and 1 EDTA (pH 7.6) and 5% glycerol for 30 min at room
temperature. The mixtures were then separated on a nondenaturing 8%
polyacrylamide gel in 0.5x 25 mM Tris · HCl, 25 mM boric acid,
and 0.5 mM EDTA. The gel was dried and exposed to X-ray film (Kodak
BioMax).
Immunoblots. Samples containing cytosolic protein (50 µg) were
fractionated on a 12% SDS-polyacrylamide gel and then transferred onto a
polyvinylidene difluoride membrane (Immobilon-P, Millipore). Membranes were
washed in 1x Tris-buffered saline (TBS; 20 mM Tris · HCl and 140
mM NaCl, pH 7.5), blocked for 1 h at room temperature (1x TBS, 0.1%
Tween 20, and 5% nonfat dry milk), and then incubated with primary antibody
(rabbit polyclonal IB antibody, Santa Cruz Biotechnology) 1:1,000 in
dilution buffer (1x TBS, 0.1% Tween 20, and 5% nonfat dry milk)
overnight at 4°C. Membranes were washed four times in 1x TBS and
0.1% Tween 20, followed by incubation with peroxidase-labeled anti-rabbit IgG
(1:8,000 in dilution buffer, Santa Cruz) for1hat room temperature. Membranes
were washed four times at room temperature before the antigen-antibody
complexes were detected with enhanced chemiluminescence (Super Signal, Pierce;
Rockford, IL).
Langendorff-perfused hearts. To examine cardiac contractile
function, mice were divided into four experimental groups. Group 1
consisted of sham-treated WT mice (n = 5). Group 2 was
composed of sham-treated IRAK1 KO animals (n = 7). Group 3
was LPS-treated WT mice (n = 9), and group 4 was LPS-treated
IRAK1 KO animals (n = 10). Separate groups of alert mice from all
experimental groups were anti-coagulated with heparin sodium (100 units,
Elkins-Sinn, Cherry Hill, NJ) and euthanized by cervical dislocation. The
heart was rapidly removed and placed in ice-cold (4°C) Krebs-Henseleit
bicarbonate-buffered solution composed of (in mM) 118 NaCl, 4.7 KCl, 21
NaHCO3, 2.5 CaCl2, 1.2 MgSO4, 1.2
KH2PO4, and 11 glucose. All solutions were prepared each
day with demineralized, deionized water and bubbled with 95% O2-5%
CO2 (pH 7.4; PO2, 550 mmHg;
PCO2, 38 mmHg). Polyethylene-50 (PE-50) tubing
(Intramedic) was placed in the ascending aorta and connected via glass tubing
to a buffer-filled reservoir for perfusion of the coronary circulation at a
constant flow rate. Hearts were suspended in a temperature-controlled chamber
maintained at 38.6 ± 0.5°C, and the coronary arteries were perfused
by retrograde flow through the aortic stump cannula with the use of a constant
flow pump (model TIA, Ismatec, Cole Palmer; Vernon Hills, IL). Contractile
function was assessed by measuring intraventricular pressure with the PE-50
tubing threaded into the left ventricle (LV). LV pressure (LVP) was measured
with a Statham pressure transducer (model P23 ID, Gould Instruments; Oxnard,
CA) attached to the cannula, and the rates of LVP rise (+dP/dt) and
fall (–dP/dt) were obtained using an electronic differentiator
(model 7P20C, Grass Instruments, Quincy, MA), recorded (model 7DWL8P, Grass
Recording Instruments), and transferred to a Dell Pentium computer.
Contractile function was determined by plotting LV systolic pressure and
±dP/dtmax values against increases in coronary
flow, achieved by increasing the flow rate of a pump calibrated to deliver
between 1 and 4 ml/min into the cannulated aortic stump, or perfusate
Ca2+ concentration. After removal, cardioplegia,
cannulation, and perfusion, ex vivo denervated hearts typically beat between
280 and 310 beats/min. If the rhythm was too slow, too fast, or irregular, the
hearts were captured at their intrinsic rates and then paced at 
300
beats/min through an electrode attached to the right atrium (4.8–5.0 A
for 1-ms duration, Grass Stimulator) for the duration of the contractile
function studies. There is no difference between groups with regard to the
requirements for pacing.
Histology. Hearts were fixed by immersion in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 6 µm. Sections were stained with hematoxylin and eosin for routine examination and with Masson's trichrome to reveal collagen. Coded samples were scored by a single veterinary pathologist (D. F. Kusewitt) located at another institution for severity of myocarditis on a scale of 0–3 (0, no myocarditis; 1, mild myocarditis; 2, moderate myocarditis; 3, severe myocarditis). The reader was blinded to the animals' genotypes. Severity scores were based on the extent of myocardial involvement and the degree of interstitial cellularity and fibrosis in affected areas. Less than 25% involvement of the heart was scored as mild myocarditis; in cases of moderate myocarditis, 25–50% of the heart was affected; in cases of severe myocarditis, >50% of the heart was involved.
Statistics. Separate analyses of cardiac functions were conducted
for each of LVP, +dP/dt, and –dP/dt as a function of
treatment group (factor 1: WT sham vs. LPS, KO sham vs. LPS, sham WT
vs. KO) and either coronary flow rate or calcium level (factor 2). A
two-way analysis of variance was performed. In those instances in which the
factor 1–2 interaction was significant at the 0.05
level, Bonferroni-corrected post tests were then conducted at each level of
factor 2 (coronary flow rate or perfusate calcium) to discern
differences among the treatment groups. Survival curves of TNF-
transgenic and transgenic/IRAK1-deficient mice were compared using a
Kaplan-Meier survival estimate.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
B
activation (14). The responses
of hearts from WT and IRAK1-deficient mice
(44) were then compared.
Animals were injected with LPS (1 mg/kg ip), their hearts were removed, and
NF-B activation was examined at various times. Catalytic activation of
IKK-, the kinase that phosphorylates IB- and targets it
for degradation, was assayed first. As shown in
Fig. 1B, IKK-
catalytic activity increased rapidly in WT hearts, reached 15-fold activation
after 30 min, and then declined, returning to baseline by 3 h. In contrast,
IKK- activity in IRAK1-deficient hearts was diminished and delayed
compared with WT, with peak activation sevenfold above baseline. Similar
results were obtained in two other trials. Thus IRAK1 governs both the
kinetics and peak activity of the principle kinases controlling NF-B
activation in the heart, an effect similar to that seen in LPS-treated
macrophages (41). This finding
is also reflected by the delayed and incomplete LPS-induced IB-
degradation (Fig. 1C)
and reduced NF-B DNA binding activity in IRAK1-deficient compared with
WT hearts (Fig. 1D).
Together, these findings demonstrate that LPS-mediated NF-B activation
in the heart operates through an IRAK1-dependent pathway.
|
The contribution of IRAK1 to JNK signaling in the heart was also examined.
JNK activity controls LPS-induced TNF- production in macrophages
(12,
40). This cytokine is produced
in cardiac myocytes after LPS stimulation and causes myocardial contractile
dysfunction (4,
5,
38). LPS treatment of WT mice
triggered a 28-fold increase in cardiac JNK activity in the experiment shown
in Fig. 1E. Peak
activation that occurred 15 min after injection returned to basal levels by 60
min. IRAK1-deficient hearts, on the other hand, exhibited a severely
attenuated and delayed response to LPS injection
(Fig. 1E),
demonstrating that IRAK1 is required for LPS-induced JNK activation in heart
tissue.
IRAK1 mediates LPS-induced myocardial contractile dysfunction. Sepsis causes myocardial contractile dysfunction (30, 32), and administration of endotoxin to both humans and experimental animals mimics the myocardial depression of sepsis (30, 39). To determine whether differences in intracellular signaling were also associated with altered myocardial contractile function, the contractile responses of hearts from LPS-treated WT and IRAK1-deficient mice were compared. Animals were injected with either LPS (1 mg/kg ip) or an equal volume of normal saline. Eighteen hours later, the hearts were removed and contractile function was assessed ex vivo with the use of modified Langendorff-isolated perfusion preparations. There was no difference in baseline contractile function between hearts from WT or IRAK1-deficient mice. Both exhibited similar systolic maximal developed LVP and rate of pressure generation (+dP/dtmax) and diastolic rate of relaxation (–dP/dtmax) responses to increasing coronary flow rates (Fig. 2) and perfusate calcium concentrations (Fig. 3A). As expected, WT LPS-treated hearts exhibited significant myocardial contractile dysfunction compared with sham-treated WT hearts. This impaired function affects both systole and diastole and persists at increasing coronary flow rates (Fig. 2) and extracellular calcium concentrations (Fig. 3). In marked contrast, IRAK1-deficient hearts are protected against LPS-induced contractile dysfunction. Hearts from LPS-treated IRAK1-deficient animals showed no significant decreases in maximal developed LVP, +dP/dtmax, or –dP/dtmax in response to increasing coronary flow rates (Fig. 2). Similarly, hearts from IRAK1-deficient mice challenged with LPS were also resistant to the myocardial depressant effects of LPS, as determined by their systolic and diastolic responses to increasing calcium concentrations (Fig. 3). Thus IRAK1 inactivation blocks acute LPS-triggered cardiac contractile failure, implicating this kinase as a transducer of the acute myocardial depressant effects of endotoxin.
|
|
IRAK1 inactivation prolongs survival in mice that overexpress
TNF- in the heart. Although IRAK1 is crucial to acute
LPS-induced myocardial depression, its role in conditions characterized by
chronically impaired contractility is unknown. To determine whether IRAK1
contributes to chronic contractile dysfunction as well, IRAK1 was inactivated
in transgenic mice with a lethal form of heart failure. IRAK1-deficient mice
were crossed with transgenic mice that overexpress TNF- in the heart.
These TNF- transgenic animals develop myocarditis, dilated
cardiomyopathy, heart failure, and experience 100% early lethality
(4). Survival of the transgenic
animals and the TNF- transgenic/IRAK1 KO double mutants was compared.
Figure 4A depicts the
survival curves of the two strains of animals. Elimination of IRAK1 function
markedly enhanced the 150-day survival of TNF- transgenic mice. All of
the transgenic animals with functional IRAK1 succumbed during the test period,
whereas 70% of those lacking the kinase were still alive after 150 days.
Therefore, IRAK1 inactivation protects mice against this form of lethal heart
failure.
|
Because heart failure in the transgenic mice results from cardiac
overexpression of the proinflammatory cytokine TNF-, and IRAK1 also
mediates injury sensing and inflammation at several different steps in the
innate immune response, it is possible that decreased mortality was due to a
diminished inflammatory response in transgenic/KO hearts. On average, hearts
from 75-day-old WT mice and IRAK1 KO mice appeared normal in size and
consistency. In contrast, hearts from the TNF- overexpressors were
markedly dilated, thin walled, and pale. Most hearts from double mutants
(TNF- transgenic/IRAK1 KO) appeared normal, but occasionally (<10%)
a transgenic/KO animal would have a dilated heart similar to the transgenic
animals. These rare animals were also clinically ill. Hearts from WT
(n = 4), TNF- transgenic (n = 7), IRAK1 KO
(n = 7), and TNF- transgenic/KO (n = 4) mice were
removed for histopathological examination by a veterinary pathologist who was
blinded to the animals' genotypes. Hearts were classified as having no, mild,
moderate, or severe myocarditis (Table
1). WT and IRAK1-deficient hearts lacked evident cardiac lesions
(Fig. 4B). Hearts from
TNF- transgenic mice exhibited diffuse interstitial cellularity, with
mononuclear inflammatory cells and fibroblasts, as well as interstitial
fibrosis in both atria and ventricles, as previously described
(4). Cardiac myocytes from
these transgenic animals showed degenerative changes, including cytoplasmic
vacuolization and hyalinization, nuclear enlargement, vesiculation, and the
development of Anitschkow-like nuclear changes
(Fig. 4B). TNF-
transgenic/IRAK1 KO hearts were histologically indistinguishable from
TNF- transgenic hearts and exhibited either moderate or severe
myocardial lesions (Fig.
4B). Thus, on the basis of histological evaluation, loss
of IRAK1 function does not attenuate cardiac inflammation in mice
overexpressing TNF- in cardiomyocytes.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
B and JNK activity, displaying
diminished amplitude and timing of downstream signaling required for
TNF- production in other cells
(40). These findings are
consistent with previously published reports. Giroir and colleagues
(11) first demonstrated
LPS-driven cardiac TNF-, whereas Frantz and colleagues
(10) have shown TLR4
expression in normal myocytes and failing hearts, and Baumgarten et al.
(3) have implicated TLR4 in
this cardiac-specific TNF- response. Activation of cardiac NF-B
by LPS has also been reported previously
(3,
8,
14,
36) with the observation that
NF-B inhibition protects against LPS-induced myocardial contractile
dysfunction (8,
14,
36). The present study
demonstrates a requirement for the Toll/IL-1 signaling intermediate IRAK1 in
the cardiac signaling response to LPS. Moreover, LPS-induced JNK catalytic
activation in the heart and the effect of IRAK1 inactivation on cardiac JNK
signaling also represents a novel finding. In fact, WT and IRAK1-deficient
hearts exhibit endotoxin-triggered signaling profiles that resemble those seen
in macrophages (41), which
also showed attenuated and delayed NF-B and MAPK responses. The
macrophage studies, however, were conducted in vitro, so there may be
important differences in signal transduction between these two tissues. Thus
the myocardium possesses an LPS-sensing apparatus, and IRAK1 regulates the
cardiac signaling response to endotoxin. The results from these studies also implicate IRAK1 in acute LPS-induced contractile dysfunction. WT LPS-treated hearts exhibited predictable and reproducible impaired contractility compared with sham-treated hearts, whereas the response of LPS-challenged IRAK1-deficient hearts was no different from sham-treated hearts lacking IRAK1. Thus IRAK1 function is required for the development of LPS-induced contractile depression.
These findings reinforce the importance of Toll/IL-1 signaling in acute myocardial contractile dysfunction by demonstrating its requirement in a second model of cardiac depression. Recent work in our laboratory (43) has demonstrated that IRAK1 transduces a signal that leads to myocardial dysfunction after large body surface area burns (35–40% total body surface area). Additional studies indicated that TLR4 was a principal sensor for burn-induced myocardial dysfunction because mice harboring inactivating TLR4 mutations failed to develop cardiac depression after injury (45). Furthermore, contractile dysfunction after burn injury could be prevented by administrations of the LPS inhibitor recombinant bactericidal/permeability-increasing protein (rBPI21) (45). Together, the results from the burn studies suggested that myocardial depression might be due to LPS translocated from the gut of injured animals, although other possible mechanisms could not be excluded. Regardless of whether LPS is responsible for myocardial depression in burn injury, the findings presented in the current experiments document a clear role for IRAK1 in contractile dysfunction after acute exogenous endotoxin administration.
In addition to its effect on acute LPS-mediated contractile dysfunction,
IRAK1 may also be involved in chronically impaired contractility. IRAK1
inactivation enhances survival in animals with congestive heart failure due to
cardiac-specific TNF- overexpression. How IRAK1 deletion exerts this
protection is unknown. Histological analysis suggests that IRAK1 deletion does
not directly affect the degree of inflammation seen in transgenic hearts.
Double mutant animals still synthesize TNF- in the heart and still
exhibit an inflammatory infiltrate similar to transgenic mice with IRAK1, but
nonetheless live longer. Interestingly, contractile function in transgenic
animals at 50 days of life, the time when some of these animals begin to
die is significantly impaired compared with double mutant animals of the same
age (data not shown), but this additional observation fails to reveal a
mechanism linking IRAK1 activation to worsening heart failure.
Two general possibilities could help explain this result. First, IRAK1 may
be required for normal TNF- signaling. Most studies to date have failed
to uncover a role for IRAK1 in TNF- signaling, although one report has
suggested that IRAK1 potentiates IL-1 signaling to NF-B
(47). According to this view,
loss of IRAK1 function would attenuate cardiac responses to TNF-,
thereby lessening the degree of contractile dysfunction in transgenic hearts
lacking IRAK1. Alternatively, IRAK1 may promote lethality by transducing
other, TNF--independent signals in progressive heart failure.
Importantly, patients with severe (New York Hospital Association class III and
IV) congestive heart failure and edema exhibit elevated serum LPS and
proinflammatory cytokine concentrations
(18,
27), which may be both markers
for worsening cardiac function and stimuli contributing to further
deterioration in contractility. Normally, the presence of endotoxin would be
expected to worsen contractile function, if the host is insensitive to this
compound; however, as occurs in the double mutant mice, myocardial function
might be spared. Therefore, a loss of IRAK1 function through genetic deletion
may abrogate three major proximal signals that trigger cardiac dysfunction.
Direct myocardial depressant actions of LPS and IL-1 are blocked, and
LPS-induced TNF- production by noncardiac cells is also impaired, such
that the heart is exposed to less circulating TNF-. Loss or diminution
of these three negative inotropic inputs may slow the deterioration of
contractile function in double mutant mice and thereby prolong survival.
Determining the precise role for IRAK1 in lethality is this model of heart
failure; however, it will require further study.
Although they identify IRAK1 as an important participant in LPS-induced
myocardial dysfunction, these studies cannot pinpoint the site of IRAK1 action
in this response. Myocardial protection is correlated with altered myocardial
signaling responses to LPS, but it is impossible to discern whether this
protection stems from disrupted signaling in the heart or some other
tissue(s). LPS responsiveness is presumably disrupted in all IRAK1-deficient
tissues. Thus these studies cannot determine whether the myocardial protection
seen in KO mice is due to the lack of IRAK1 in the heart of its absence from
some other cell type. How, then, does LPS cause myocyte contractile
dysfunction? Two models, both testable, can be envisioned. In the first, LPS
sensing and impaired contractility both occur in the cardiac myocyte (a
cell-autonomous model). Conversely, nonmyocyte cell types either in the heart
itself or elsewhere (e.g., fibroblasts, macrophages, monocytes, endocardial or
endothelial cells, or hepatocytes) could sense LPS and produce secondary
signals (e.g., cytokines such as TNF- or IL-1 or other molecules) that
directly depress myocardial contractility (a cell nonautonomous model).
Furthermore, the models are not mutually exclusive.
Regardless of the site of IRAK1 action, the data presented here suggest that a signaling pathway considered part of the innate immune system can regulate a nonimmune physiological response. IRAK1 inactivation blocks the usual negative effect of LPS, a bacterial product with well-described immunostimulatory properties, on cardiac contractility. Most studies of Toll/IL-1 signaling have focused on the immune consequences of pathway stimulation. This report, however, represents one of the earliest attempts to link pathway activation to physiological alterations outside the immune system. These findings have two important implications. First, demonstration that IRAK1 regulates LPS-induced contractile depression highlights the interrelatedness of different systems in integrated biological responses to infection. Second, it may force reconsideration of what properly constitutes innate immunity, usually viewed as a form of host defense mediated by bone marrow-derived cells and specialized molecules to aid in antibacterial responses, thrombosis, and inflammation.
Why, for example, does the heart, rarely a direct target of infection, have a functional Toll/IL-1 pathway? Could Toll/IL-1 signaling enact a broader injury-sensing role not limited to the detection of infection, with physiological consequences extending beyond the immune system? Recent reports seem to support this possibility. Several groups have implicated endogenous molecules, such as highly conserved heat shock proteins (28), the breakdown products of the extracellular matrix (42), and products released from necrotic cells (19) as activators of Toll/IL-1 signaling. It is therefore possible that certain Toll-like receptors on neighboring viable heart cells actually sense direct cardiac injury resulting from ischemia or trauma, when necrotic myocytes release their contents? Subsequent pathway activation could trigger responses in uninjured cells that might protect them from injury, lead to the characteristic postinjury inflammatory infiltrate, or precipitate postinjury dysfunction, to name a few possible responses. Thus the Toll/IL-1 pathway may represent a ubiquitous, generic injury sensor that serves to alert the host to damage to one of its parts and initiate the containment and healing responses.
Finally, these studies raise the possibility that IRAK1 or other members of the Toll/IL-1 signaling pathway may be targets for the therapy of cardiac contractile dysfunction. Pinpointing how IRAK1 exacerbates contractile dysfunction and limits survival represent critical first steps in developing such a strategy. The protection that genetic inactivation of IRAK1 affords mice with either acute or chronic contractile dysfunction, however, provides a theoretical basis for the development of new therapies for congestive heart failure. It may someday be possible, for example, to prolong survival in patients with end-stage heart failure through pharmacological IRAK1 inhibition.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
Present address of S. B. Haudek: Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
B prevents endotoxin-induced
myocardial dysfunction. Am J Physiol Heart Circ
Physiol 280:
H962–H968, 2001. B/Toll-like receptor pathway in induction of
inflammatory and tissue-repair gene expression by necrotic cells. J
Immunol 166:
7128–7135, 2001. in
conscious dogs. J Clin Invest
90: 389–398,
1992.[Web of Science][Medline]
and tumour necrosis
factor-. Br J Pharmacol
114: 27–34,
1995.[Web of Science][Medline]
B- protein promote
tolerance to endotoxin. Am J Physiol Heart Circ
Physiol 275:
H1084–H1091, 1998.) translation: glucocorticoids inhibit
TNF-alpha translation by blocking JNK/SAPK. Mol Cell
Biol 17:
6274–6282, 1997.This article has been cited by other articles:
|
|
 |
|
  U. Maitra, N. Singh, L. Gan, L. Ringwood, and L. Li IRAK-1 Contributes to Lipopolysaccharide-induced Reactive Oxygen Species Generation in Macrophages by Inducing NOX-1 Transcription and Rac1 Activation and Suppressing the Expression of Antioxidative Enzymes J. Biol. Chem., December 18, 2009; 284(51): 35403 - 35411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Maitra, J. S. Parks, and L. Li An Innate Immunity Signaling Process Suppresses Macrophage ABCA1 Expression through IRAK-1-Mediated Downregulation of Retinoic Acid Receptor {alpha} and NFATc2 Mol. Cell. Biol., November 15, 2009; 29(22): 5989 - 5997. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. RAMARAJ and V. L. SORRELL Peripartum cardiomyopathy: Causes, diagnosis, and treatment Cleveland Clinic Journal of Medicine, May 1, 2009; 76(5): 289 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lockett, M. G. Goebl, and M. A. Harrington Transient membrane recruitment of IRAK-1 in response to LPS and IL-1{beta} requires TNF R1 Am J Physiol Cell Physiol, August 1, 2008; 295(2): C313 - C323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Luo, H.-J. Kwon, S. Montano, M. Georgiadis, M. G. Goebl, and M. A. Harrington Phosphorylation of SIMPL modulates RelA-associated NF-{kappa}B-dependent transcription Am J Physiol Cell Physiol, March 1, 2007; 292(3): C1013 - C1023. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Subla, S. A. Khan, D. Behl, and S. G. Peters Sepsis and Myocardial Depression in a Young Woman Mayo Clin. Proc., June 1, 2005; 80(6): 810 - 814. [Abstract] [PDF]
|
|
|
|
 |
|
 M. S. Willis, D. L. Carlson, J. M. DiMaio, M. D. White, D. J. White, G. A. Adams IV, J. W. Horton, and B. P. Giroir Macrophage migration inhibitory factor mediates late cardiac dysfunction after burn injury Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H795 - H804. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Knuefermann, Y. Sakata, J. S. Baker, C.-H. Huang, K. Sekiguchi, H. S. Hardarson, O. Takeuchi, S. Akira, and J. G. Vallejo Toll-Like Receptor 2 Mediates Staphylococcus aureus-Induced Myocardial Dysfunction and Cytokine Production in the Heart Circulation, December 14, 2004; 110(24): 3693 - 3698. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Tavener, E. M. Long, S. M. Robbins, K. M. McRae, H. Van Remmen, and P. Kubes Immune Cell Toll-Like Receptor 4 Is Required for Cardiac Myocyte Impairment During Endotoxemia Circ. Res., October 1, 2004; 95(7): 700 - 707. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P < 0.05.
