Am J Physiol Heart Circ Physiol 291: H1900 –H1909, 2006.
First published April 28, 2006; doi:10.1152/ajpheart.00112.2006.
MyD88 and NOS2 are essential for Toll-like receptor 4-mediated survival
effect in cardiomyocytes
Xinsheng Zhu,1,* Huailong Zhao,1,* Amanda R. Graveline,2 Emmanuel S. Buys,2
Ulrich Schmidt,1 Kenneth D. Bloch,2,3 Anthony Rosenzweig,2,3 and Wei Chao1
1
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School,
Boston; 2Cardiovascular Research Center, Massachusetts General Hospital, Charlestown; and 3Division
of Cardiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Submitted 31 January 2006; accepted in final form 20 April 2006
signal transduction; cardiac; apoptosis; inducible nitric oxide synthase
represents a major cause of morbidity
and mortality in Western society. Because injured cardiac
tissues after a heart attack typically cannot regenerate, ischemic heart disease often leads to deterioration of myocardial
function and death. Therefore, it is critically important and
clinically relevant to understand the molecular mechanisms
governing ischemic myocardial injury and identify potential
targets for intervention.
Cardiomyocyte apoptosis, or programmed cell death, is an
important feature of ischemic myocardial injury. With the
ISCHEMIC HEART DISEASE
* X. Zhu and H. Zhao contributed equally to this study.
Address for reprint requests and other correspondence: W. Chao, Dept. of
Anesthesia & Critical Care, MGH, GRJ-4-462, 55 Fruit St., Boston, MA 02114
(e-mail: [email protected]).
H1900
employment of pharmacological inhibition of apoptosis,
transgenic cardiac expression of caspase, and genetic deletion of anti-apoptotic signaling pathway, previous studies
have demonstrated that cardiomyocyte apoptosis plays an important role in ischemic myocardial injury and cardiomyopathy
(19, 54, 56).
Innate immune signaling via Toll-like receptor 4 (TLR4) and
the downstream adaptor molecules MyD88 (38) and the serinethreonine kinase, interleukin-1 receptor-associated kinase
(IRAK-1) (21) plays an important role in host defense and
tissue inflammation (47). The heart expresses at least three
receptors involved in TLR signaling: CD14, TLR2, and TLR4
(12, 13, 30, 40). It is well documented that these receptors are
responsible for cardiac contractile dysfunction in various
pathological conditions such as endotoxemia (30, 40). In contrast, little is known about the role of innate immune signaling
in myocardial injury. A few recent studies have found that the
cardiac innate immune system is dynamically regulated in
response to ischemic injury and may play a role in cardiomyocyte survival. For example, myocardial TLR4 expression
is upregulated in response to myocardial ischemia and during
heart failure (13). Chao et al. (6) recently found that myocardial ischemia induces rapid activation of IRAK-1, a critical
component of TLR innate immune signaling (21) and that
adenovirus-mediated expression of IRAK-1 inhibited cardiomyocyte apoptosis in vitro. Moreover, administration of lipopolysaccaride (LPS), a TLR4 agonist, protects the heart
against ischemic injury (3, 39) and inhibits cardiomyocyte
apoptosis in vitro (6).
MyD88, originally isolated as one of the 12 myeloid differentiation primary response genes (34), is an adaptor protein
that plays an important role in TLR-interleukin-1 receptor
family signaling. Like TLR4-deficient mice (44), MyD88⫺/⫺
mice (25) lack the ability to respond to LPS, although MyD88independent pathways (e.g., TRIF-dependent IFN-␤ production) exist in TLR4 signaling (26). The role of MyD88 in cell
survival or death is not completely understood, and earlier
results have been conflicting (1, 23).
Three nitric oxide synthase (NOS) isoforms exist in the
heart: neuronal (NOS1), inducible (NOS2), and endothelial
(NOS3). With the utilization of genetically modified animal
models and pharmacological inhibitors, it has been demonstrated that all three NOSs play important roles in modulating
cardiac function and survival under various conditions (22, 50,
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Zhu, Xinsheng, Huailong Zhao, Amanda R. Graveline, Emmanuel S. Buys, Ulrich Schmidt, Kenneth D. Bloch, Anthony
Rosenzweig, and Wei Chao. MyD88 and NOS2 are essential for
Toll-like receptor 4-mediated survival effect in cardiomyocytes. Am J
Physiol Heart Circ Physiol 291: H1900 –H1909, 2006. First published
April 28, 2006; doi:10.1152/ajpheart.00112.2006.—Innate immune
system such as Toll-like receptor 4 (TLR4) represents the first line of
defense against infection. In addition to its pivotal role in host
immunity, recent studies have suggested that TLR4 may play a
broader role in mediating tissue inflammation and cell survival in
response to noninfectious injury. We and other investigators have
reported that cardiac TLR4 signaling is dynamically modulated in
ischemic myocardium and that activation of TLR4 confers a survival
benefit in the heart and in isolated cardiomyocytes. However, the
signaling pathways leading to these effects are not completely understood. Here, we investigate the role of MyD88, an adaptor protein of
TLR4 signaling, and inducible nitric oxide synthase (NOS2) in mediating TLR4-induced cardiomyocyte survival in an in vitro model of
apoptosis. Serum deprivation induced a significant increase in the
number of apoptotic cardiomyocytes as demonstrated by transferasemediated dUTP nick-end labeling (TUNEL) assay, nuclear morphology, DNA laddering, and DNA-histone ELISA. Lipopolysaccharide
(LPS), a TLR4 agonist, activated TLR4 signaling and led to significant reduction in apoptotic cardiomyocytes and improved cellular
function of surviving cardiomyocytes with enhanced Ca2⫹ transients
and cell shortening. We found that both TLR4 and MyD88 are
required for the LPS-induced beneficial effects as demonstrated by
improved survival and function in wild-type but not in TLR4⫺/⫺ or
MyD88⫺/⫺ cardiomyocytes. Moreover, genetic deletion or pharmacological inhibition of NOS2 abolished survival and functional rescue
of cardiomyocytes treated with LPS. Taken together, these data
suggest that TLR4 protects cardiomyocytes from stress-induced injury
through MyD88- and NOS2-dependent mechanisms.
MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
53). Myocardial expression of NOS2 is induced in response to
endotoxin-cytokine stimulation and to myocardial ischemia
(15). NOS2 may contribute to myocardial dysfunction during
endotoxemia (50), but its role in cardiac protection against
ischemic injury is controversial (11, 15, 32, 46).
In this study, we sought to determine the role of MyD88 and
NOS2 in modulating cardiomyocyte survival and function in
an in vitro model of apoptosis. Utilizing genetically modified
mice, we found that TLR4 mediates a cardiomyocyte survival
signal and that both MyD88 and NOS2 are critical components
of this signaling mechanism.
EXPERIMENTAL PROCEDURES
Animals
Neonatal Mouse Cardiomyocytes Isolation and Culture
Neonatal mouse cardiomyocytes were prepared from 1- to 3-dayold mice as described previously (52). More than 95% of cells were
cardiomyocytes as demonstrated by sarcomeric actin staining 4 days
after being plated. Neonatal cardiomyocytes were used for all apoptosis studies.
Adult Mouse Cardiomyocyte Isolation
Ventricular mouse cardiomyocytes were isolated as described previously (33) and used for the function studies: Ca2⫹ transients and cell
shortening.
Measurement of Cardiomyocyte Sarcomere Shortening and
Intracellular Calcium
Sarcomere shortening and intracellular calcium concentration transients were recorded simultaneously on an IonOptix system (Milton,
MA). Adult cardiomyocytes were incubated with membrane-permeable fluorescent indicator fura-2 AM (1 ␮M) (Molecular Probes) and
probenecid (0.5 mM). Cardiomyocytes were perfused with 1.2 mM
Ca2⫹ Tyrode solution and electrically paced at 2 or 1 Hz via platinum
wires. The Ca2⫹ transients and sarcomere shortening were analyzed
based on single cell averaged tracing. The final values were derived
from 20 –30 individual cells in each group and calculated for statistical analysis. Three to four mice from each group (mouse strain) were
employed to prepare cardiomyocytes for the functional studies.
In Vitro Model of Cardiomyocyte Apoptosis
Three to four days after being plated, neonatal cardiomyocytes
were washed three times and incubated in serum-free MEM containing 0.05% bovine serum albumin in an incubator containing 5%
CO2-95% air at 37°C. We have previously established that serum
deprivation induces a rapid activation of caspases (within 4 h) and
cardiomyocyte apoptosis (5, 6). In some studies, neonatal cardiomyocytes were exposed to hypoxic condition, which was created using
BD Bioscience GasPak EZ system.
Apoptosis Assays
DNA laddering. Neonatal cardiomyocytes (1 ⫻ 106) were lysed,
and DNA was extracted with phenol:chloroform. Genomic DNA
Fig. 1. Characterization of MyD88⫺/⫺ and Toll-like receptor-4 (TLR4)⫺/⫺ cardiomyocytes. A: expression of MyD88 in lung (L), heart (H), spleen (S) from adult
mice. Top, MyD88 Western blot (WB); bottom, ␤-actin and ␣-actinin WB. B: interleukin-1 receptor-associated (IRAK-1) kinase assay. Lipopolysaccharide (LPS)
concentration: 1,000 ng/ml. MBP, myclin basic protein. C: micrographs of adult cardiomyocytes, ⫻400. D: sarcomere length: measured and analyzed using the
IonOptix software (n ⫽ 30). WT, wild-type.
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All animal experiments were performed with the approval of the
Animal Care Committee of Massachusetts General Hospital.
MyD88⫺/⫺ mice (kindly provided by Dr. Mason Freeman, Massachusetts General Hospital) were backcrossed six generations into the
C57BL/6J strain (25). TLR4⫺/⫺ (C57BL/10ScCr also referred to as
C57BL/10ScNJ, Stock No. 003752, with wild-type Il12rb2 allele),
TLR4⫹/⫹ (C57BL/10ScSn), and NOS2⫺/⫺ mice were purchased from
The Jackson Laboratory. NOS2⫺/⫺ mice were backcrossed for 10
generations into the C57BL/6J strain. Unless stated otherwise,
C57BL/6J mice were studied as the wild-type controls.
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MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
fragments of 1.5 ␮g were 32P-labeled by Klenow DNA polymerase
and subsequently separated by electrophoresis in 1.8% agarose gel (6).
Cell death ELISA. Apoptosis-induced histone-DNA fragments
were quantitated using ELISA (Roche).
TUNEL and Hoechst nuclear staining. Attached neonatal cardiomyocytes were fixed and permeabilized. Terminal deoxynucleotidyl
transferase was used to incorporate fluorescein-labeled dUTP into
3⬘-OH DNA ends generated by DNA fragmentation (Roche). Nuclei
were costained with Hoechst. Actin was stained with FITC-labeled
phalloidin (10 ␮g/ml). To quantitate apoptotic nuclei, 400 –900 nuclei
in each treatment group from ten ⫻400 magnification fields were
counted. The operator who counted nuclei was blinded to the experimental design.
Immunoblotting, Immunoprecipitation, and IRAK-1 Kinase Assay
For microscopic image of NOS2, neonatal cardiomyocyte cultures
(105 cells/chamber on 8-chamber slide) were treated with LPS. Cells
were fixed and permeabilized using a cytostaining kit from BD
PharMingen (San Diego, CA). NOS2 was stained with a monoclonal
antibody (1:100 dilution) (BD Bioscience, Clone 6) at 4°C overnight
and with Alexa Fluor 546-labeled anti-mouse IgG (1:2,000) for 1 h at
room temperature.
RNA Extraction and Quantitative RT-PCR Analysis
Total RNA was extracted from cultured cardiomyocytes using
TRIzol reagent, and cDNA was synthesized by reverse transcriptase
reaction. cDNA gene sequences corresponding to 18S ribosomal RNA
(rRNA) were amplified from cDNA by PCR and quantitated using an
ABI Prism 7000 (Applied Biosystems) with the forward primer
5⬘-TCATGTGGTGTTGAGGAAAGC-3⬘ and the reverse primer 5⬘TGGCGTGGATTCTGCATAATG-3⬘. NOS2 and NOS3 cDNA sequences were detected using Taqman primer sets supplied by Applied
Biosystems. Changes in relative gene expression normalized to 18S
rRNA levels were determined using the relative CT method.
Fig. 2. Role of TLR4 and MyD88 in the
anti-apoptotic effects of LPS-apoptotic nucleus analysis. A: micrographs of apoptotic
cardiomyocytes. Cells on 8-chamber slide
(105 cells/chamber) were treated in the presence (⫹Serum) or absence (serum deprivation, SD) of serum for 24 h and analyzed for
cell morphology (actin-phalloidin staining),
nuclear morphology (Hoechst), or in situ
DNA fragmentation (transferase-mediated
dUTP nick-end labeling, TUNEL). Dashed
arrow indicates a fragmented and positive
TUNEL nucleus and arrows indicate condensed nuclei and corresponding positive
TUNEL staining. Scale bar is shown on the
top left corner of each panel. B–D: effect of
LPS (1,000 ng/ml) on SD (24 h)-induced
apoptosis in WT, MyD88⫺/⫺, and TLR4⫺/⫺
cardiomyocytes. *P ⬍ 0.01, #P ⬍ 0.05 vs.
SD cardiomyocytes without LPS.
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These experiments were performed as described in detail previously (6). Briefly, cardiomyocytes were incubated in serum-free
medium for 30 min and treated with LPS. IRAK-1 immunoprecipitates were assayed for kinase activity using [␥-32P]ATP and myelin
basic protein as the substrates.
Immunocytochemistry
MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
Statistics
Unless stated otherwise, all data are expressed as means ⫾ SD of
at least three independent experiments and were analyzed with
ANOVA for statistical significance. The null hypothesis was rejected
for P ⬍ 0.05.
RESULTS
Characterization of TLR4⫺/⫺ and MyD88⫺/⫺
Cardiomyocytes
TLR4 and MyD88 are Essential for LPS-Induced Anti-Apoptotic Effect in Cardiomyocytes
Serum deprivation, an in vitro model of apoptosis, induced
significant apoptosis as demonstrated by transferase-mediated
dUTP nick-end labeling (TUNEL) staining, nuclear morphol-
ogy (condensation and fragmentation) (Fig. 2A), ELISA for
histone-DNA fragments (Fig. 3), and DNA laddering (Fig. 3).
As shown in Fig. 2A, apoptotic cardiomyocytes were characterized by cell shrinking (actin staining), nuclear fragmentation
and condensation (Hoechst staining), and positive TUNEL.
The serum deprivation-induced apoptosis was time dependent
with 1.5 ⫾ 0.9% at 0 min and up to 11.4 ⫾ 3.5% at 48 h. LPS
at the concentrations ranging from 10 to 2,500 ng/ml, when
added at the time of serum deprivation, led to a significant
reduction in the number of apoptotic cells (data not shown).
LPS failed to exhibit anti-apoptotic effect when added 3– 6 h
after serum deprivation started as demonstrated by DNA laddering (data not shown). LPS exhibited similar anti-apoptotic
effect in neonatal murine cardiomyocytes exposed to transient
hypoxia (Fig. 4). To elucidate the role of TLR4 and MyD88 in
mediating the survival effect, we studied TLR4⫺/⫺ and
MyD88⫺/⫺ cardiomyocytes. The data indicated that serum
deprivation induced a comparable level of cardiomyocyte apoptosis among wild-type, TLR4⫺/⫺, and MyD88⫺/⫺ cardiomyocytes (Figs. 2 and 3). The apoptotic rates were 9.7 ⫾ 2.7% (n ⫽
3), 9.6 ⫾ 1.3% (n ⫽ 5), and 8.6 ⫾ 3.2% (n ⫽ 4), respectively,
for wild-type, TLR4⫺/⫺, and MyD88⫺/⫺ cardiomyocytes. The
anti-apoptotic effects of LPS (1,000 ng/ml) observed in wildtype cardiomyocytes, however, were abolished in TLR4⫺/⫺
and MyD88⫺/⫺ cardiomyocytes as demonstrated by apoptotic
nuclei counting (Fig. 2, C and D), DNA fragmentation ELISA
Fig. 3. Role of TLR4 and MyD88 in LPS-induced
survival effect: cell death ELISA and DNA laddering. A: cell death ELISA. Cells were incubated in
serum-free MEM containing 0.05% BSA for 8 h.
Results were normalized against the control cells
incubated in the presence of serum. n ⫽ 4, each
performed in triplicates. *P ⬍ 0.01 vs. cells in the
presence of serum; **P ⬍ 0.05 vs. cells exposed to
SD but without LPS, 1,000 ng/ml; #P ⬍ 0.05 vs.
cells in the presence of serum. B–D: DNA laddering, LPS (1,000 ng/ml) inhibits SD-induced DNA
fragmentation in WT but not in MyD88⫺/⫺ or
TLR4⫺/⫺ cardiomyocytes. B: C57BL/6J WT cardiomyocytes. Similar results were obtained with
C57BL/10ScSn (TLR4⫹/⫹) cells (data not shown).
C: TLR4⫺/⫺. D: MyD88⫺/⫺. LPS (1,000 ng/ml) or
IGF-1 (100 nM) was added at the time of SD.
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MyD88 expression was confirmed by PCR (data not shown)
and Western blots (Fig. 1A). MyD88 immunoreactivity was
detected in the lung, heart, and spleen of wild-type but not
MyD88⫺/⫺ mice. MyD88 protein levels were decreased in
MyD88⫹/⫺ mice. LPS induced IRAK-1 activation in wild-type
cardiomyocytes but not in MyD88⫺/⫺ or TLR4⫺/⫺ cardiomyocytes (Fig. 1B). However, cardiomyocytes isolated from adult
wild-type, TLR4⫺/⫺, and MyD88⫺/⫺ mice shared similar morphology and striation patterns and had similar sarcomere length
(Fig. 1, C and D).
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MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
NOS2 is Critical for TLR4-Mediated Survival and Function
Recovery After Serum Deprivation
Fig. 4. LPS inhibits cardiomyocyte apoptosis in a model of transient hypoxia.
Cells on 8-chamber slide (105 cells/chamber) were incubated without or with
LPS (1,000 ng/ml) in a hypoxic chamber (BD GasPak EZ system) at 37°C for
6 h and then in an incubator containing 5% CO2-95% air for 18 h. Cells were
washed, fixed, and permeabilized. Apoptotic nuclei were labeled with TUNEL
staining. All nuclei were costained with Hoechst. A: microscopic images of
nuclei. B: quantitative data of apoptotic nuclei. n ⫽ 4, *P ⬍ 0.01.
(Fig. 3A), and by DNA laddering (Fig. 3, C and D). To
ascertain whether or not other intracellular surviving pathways
were still intact in the TLR4⫺/⫺ and MyD88⫺/⫺ cardiomyocytes, we treated the cells with IGF-1, a known survival growth
factor that elicits its anti-apoptotic effect via phosphatidylinositol 3⬘-kinase and Akt (10, 55). As shown in Figs. 2 and 3,
insulin-like growth factor (IGF-1) maintained its anti-apoptotic
effect in both TLR4⫺/⫺ and MyD88⫺/⫺ cardiomyocytes. Of
note, in LPS-treated MyD88⫺/⫺ cells, apoptosis rate is 8.9 ⫾
2.2% (Fig. 2D), slightly higher than that in LPS-treated
TLR4⫺/⫺ cells (7.2 ⫾ 1.3%) but statistically insignificant (P ⫽
0.22, n ⫽ 4).
LPS Improves Ca2⫹ Transients and Cell Shortening After
Serum Deprivation: Role of TLR4 and MyD88
To assess the function of those surviving cardiomyocytes
after serum deprivation and the impact of LPS treatment, we
measured Ca2⫹ handling and cell shortening of isolated adult
mouse cardiomyocytes. Cardiomyocyte function was significantly inhibited after 3 h of serum deprivation with ⬃26%
reduction in maximal velocity (Vdep) and 23% in amplitude
(⌬F/F0) of Ca2⫹ transients compared with cells incubated in
serum-containing medium (data not shown). LPS, added at the
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To examine the role of NOS2 in the TLR4-induced beneficial
effects by LPS, we utilized the selective NOS2 inhibitor N6-(1iminoethyl)-L-lysine dihydrochloride (L-NIL) and cardiomyocytes
from mice congenitally deficient for NOS2 (31). We first tested
the TLR4 dependency of the cardiac NOS2 induction in response
to LPS administrated intraperitoneally. Eight hours after systemic
administration of LPS, there was an increase in NOS2 protein in
the heart from wild-type mice but not from that of TLR4⫺/⫺ or
NOS⫺/⫺ mice (Fig. 6A,b). Moreover, to demonstrate the specific
phenotypic effects of the gene knockout in these mice, we also
tested the IRAK-1 kinase activity in response to LPS. As anticipated, the IRAK-1 activation is severely impaired in TLR4deficient mice but remained largely intact in NOS2⫺/⫺ mice (Fig.
6A,a), suggesting that the upstream signaling of NOS2⫺/⫺ mice is
intact. Similarly, in cultured wild-type neonatal cardiomyocytes,
LPS induced a rapid induction of NOS2 mRNA, but not NOS3,
with a 55-fold increase at 4 h and 186-fold at 8 h (Fig. 6B). The
NOS2 protein expression was also increased in neonatal cardiomyocytes in response to LPS as demonstrated by immunocytochemistry and immunoblotting (Fig. 6, C and D).
As shown in Fig. 7A, serum deprivation for 24 h led to a
fourfold increase in apoptotic cells (2.3 ⫾ 0.6% of the control
vs. 9.6 ⫾ 1.7% of serum deprivation). LPS inhibited the serum
deprivation-induced apoptosis in control cells (9.6 ⫾ 1.7% in
serum deprivation cells vs. 4.0 ⫾ 0.8% in serum deprivation
cells treated with LPS, P ⬍ 0.01). However, in the presence of
L-NIL, the LPS-induced survival benefit was blocked (7.8 ⫾
1.8% of cardiomyocytes exposed to serum deprivation ⫹
L-NIL vs. 5.7 ⫾ 1.7% of cells exposed to LPS ⫹ serum
deprivation ⫹ L-NIL, P ⬎ 0.1, n ⫽ 4). L-NIL had no significant
effect on the level of apoptotic cells in serum or on the level of
apoptosis induced by serum deprivation. IGF-1-induced survival effect was not affected by L-NIL (Fig. 7A). Moreover,
TLR4 activation failed to inhibit serum deprivation-induced
apoptosis in the NOS2⫺/⫺ cardiomyocytes as demonstrated by
DNA laddering (Fig. 7B), by apoptotic nuclei counting (Fig.
7C), and by histone-DNA fragment ELISA (Fig. 7D). In
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time of serum deprivation, significantly improved the function
with a 27% increase in both Vdep and ⌬F/F0 and a 21%
increase in maximal returning velocity to baseline (Vret) (Fig. 5
and Table 1, group II vs. group I). LPS also led to a 50%
increase in the Vdep of sarcomere shortening, a 58% increase in
sarcomere shortening, and a 64% increase in Vret (Fig. 5 and
Table 2, group II vs. group I). It is noteworthy that LPS also
improved calcium handling and contractile function of cardiomyocytes in the presence of serum (data not shown). To test
whether TLR4 and MyD88 mediate the function improvement
induced by LPS in the serum deprivation model, we studied
adult TLR4⫺/⫺ and MyD88⫺/⫺ cardiomyocytes. As shown in
Fig. 5 and Tables 1 and 2, compared with wild-type cardiomyocytes, TLR4⫺/⫺ and MyD88⫺/⫺ cardiomyocytes showed
similar response to electric pacing. Importantly, unlike wildtype cardiomyocytes, neither TLR4⫺/⫺ nor MyD88⫺/⫺ cardiomyocytes responded to LPS treatment with no detectable
difference in all Ca2⫹ transient and cell shortening parameters
between LPS-treated and the control cells (Tables 1 and 2,
group IV vs. group III and group VI vs. group V).
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MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
contrast, IGF-1 maintained its anti-apoptotic effect in the
NOS2⫺/⫺ cardiomyocytes (Fig. 7, C and D). We next examined whether NOS2 also plays a role in TLR4-mediated functional improvement observed in wild-type cardiomyocytes. As
shown in Fig. 5 and Tables 1 and 2 (group VIII vs. group VII),
LPS failed to improve Ca2⫹ transients and cell shortening
under serum-free conditions in NOS2⫺/⫺ cardiomyocytes.
DISCUSSION
Previous studies have found that LPS, a TLR4 agonist,
confers a cardioprotective effect in both isolated hearts and
cultured cardiomyocytes (3, 6, 39). However, the signaling
pathways leading to the cardiac protection are not completely
understood. In the present study, we explored the role of TLR4
Table 1. Analyzed Ca2⫹ transient parameters in WT, TLR4⫺/⫺, MyD88⫺/⫺, and NOS2⫺/⫺ cardiomyocytes
WT
Group I
Group II
TLR4⫺/⫺
Group III
Group IV
MyD88⫺/⫺
Group V
Group VI
NOS⫺/⫺
Group VII
Group VIII
Serum
LPS
Vdep, U/s
⌬F/F0, %
Vret, U/s
tau, s
⫺
⫺
⫺
⫹
26.8⫾12.1
34.2⫾12.2*
21.9⫾7.7
27.9⫾7.7*
⫺3.3⫾1.0
⫺4.0⫾1.6*
0.098⫾0.043
0.076⫾0.013
⫺
⫺
⫺
⫹
57.2⫾27.1
46.5⫾15.7
31.4⫾12.1
25.6⫾12.2
⫺7.2⫾7.4
⫺5.9⫾3.4
0.111⫾0.054
0.119⫾0.074
⫺
⫺
⫺
⫹
26.1⫾14.4
30.8⫾15.7
22.5⫾10.4
24.9⫾10.9
⫺4.4⫾3.4
⫺3.9⫾3.5
0.095⫾0.025
0.108⫾0.038
⫺
⫺
⫺
⫹
32.3⫾16.7
34.9⫾18.4
28.9⫾13.1
29.6⫾13.5
⫺3.1⫾1.8
⫺2.9⫾2.0
0.132⫾0.039
0.134⫾0.044
Values are means ⫾ SD. LPS, lipopolysaccharide; WT, wild-type; TLR4, Toll-like receptor-4; NOS, nitric oxide synthase; Vdep, maximal velocity departing
from baseline; ⌬F/F0, amplitude change (%) of Ca2⫹ transients; Vret, maximal velocity returning to baseline; tau, time constant, fit single exponential to baseline.
Data in each group were recorded from 20 to 30 single adult cardiomyocytes and analyzed. At least three mice were used in each group to prepare isolated
cardiomyocytes. *P ⬍ 0.05 compared with control without LPS treatment within each pair of groups, e.g., group II vs. group I or group IV vs. group III.
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Fig. 5. LPS improves Ca2⫹ transients and cardiomyocyte shortening after SD. Critical role of TLR4, MyD88,
and inducible nitric oxide synthase (NOS2). Representative tracing of Ca2⫹ flux and cell shortening. Adult
mouse cardiomyocytes were freshly prepared and incubated in serum-free MEM for 3 h in the presence or
absence of LPS (1,000 ng/ml). A: WT cardiomyocytes.
Top, 3 original tracing and averaged tracing of Ca2⫹
transients; bottom, corresponding 3 original tracing and
averaged tracing of sarcomere shortening. Cardiomyocytes were paced at 2 Hz. B: TLR4⫺/⫺, MyD88⫺/⫺, and
NOS2⫺/⫺ cardiomyocytes. Top, averaged tracing of
Ca2⫹ transients; bottom, corresponding averaged tracing of sarcomere shortening. Cardiomyocytes were
paced at 1 Hz. Accumulated data recorded from 20 to 30
adult cardiomyocytes are shown in Tables 1 and 2.
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MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
Table 2. Analyzed sarcomere shortening parameters in WT, TLR4⫺/⫺, MyD88⫺/⫺, and NOS2⫺/⫺ cardiomyocytes
WT
Group I
Group II
TLR4⫺/⫺
Group III
Group IV
MyD88⫺/⫺
Group V
Group VI
NOS2⫺/⫺
Group VII
Group VIII
Serum
LPS
Sarcomere
Length, ␮m
Vdep, ␮m/s
Sarcomere
Shortening, %
Vret, ␮m/s
tau, s
⫺
⫺
⫺
⫹
1.82⫾0.04
1.82⫾0.07
⫺0.96⫾0.44
⫺1.44⫾0.66*
1.30⫾0.68
2.06⫾0.86*
0.50⫾0.31
0.82⫾0.49*
0.098⫾0.043
0.076⫾0.013
⫺
⫺
⫺
⫹
1.81⫾0.04
1.79⫾0.05
⫺2.88⫾1.48
⫺2.54⫾1.40
3.82⫾1.78
3.60⫾1.97
1.90⫾1.43
1.66⫾1.13
0.047⫾0.026
0.051⫾0.032
⫺
⫺
⫺
⫹
1.81⫾0.05
1.81⫾0.06
⫺2.53⫾1.20
⫺2.44⫾1.48
3.71⫾1.53
3.33⫾1.70
1.68⫾1.07
1.42⫾0.96
0.134⫾0.145
0.117⫾0.119
⫺
⫺
⫺
⫹
1.78⫾0.08
1.77⫾0.05
⫺2.01⫾1.40
⫺2.09⫾1.24
3.50⫾1.95
3.98⫾2.21
1.34⫾1.27
1.21⫾0.93
0.087⫾0.073
0.069⫾0.035
and its downstream molecules MyD88 and NOS2 in mediating
cardiomyocyte survival and function in an in vitro model of
apoptosis. Using genetically modified mice, we demonstrate
that cardiomyocyte TLR4 mediates a direct anti-apoptotic
signal and preserves cardiomyocyte function through both
MyD88- and NOS2-dependent mechanisms.
The heart expresses at least three pattern recognition receptors for a group of microorganisms with pathogen-associated
Fig. 6. LPS induces NOS2 induction. A: LPS induces cardiac IRAK-1 activation and NOS2 induction in adult mice. Mice were injected intraperitoneally with
LPS (50 mg/kg) (L) or PBS (P). Eight hours later, the hearts were removed and the myocardial tissues analyzed for IRAK-1 kinase activity (a) and for NOS2
protein expression (b). a: IRAK-1 kinase assay. Myocardial tissues were immunoprecipitated for IRAK-1. The IRAK-1 immunoprecipitates were then devided
and assayed for IRAK-1 kinase activity (top) or Western blot (bottom). b: NOS2 immunoblotting. Myocardial proteins (150 ␮g) were loaded in each lane,
separated in 4 –20% gradient SDS-PAGE, and blotted for NOS2. Actin was blotted to ascertain equal sample loading. B: NOS gene expression. Neonatal
cardiomyocytes were treated with LPS (1,000 ng/ml). Relative level of NOS transcripts was measured by quantitative RT-PCR. n ⫽ 3– 4. *P ⬍ 0.05. C:
immunocytochemistry of NOS2 in cultured mouse neonatal cardiomyocytes. Cardiomyocytes were incubated without (a) or with (b) LPS (1,000 ng/ml) for 24 h.
Cells were then fixed, permeabilized, and immunostained with monoclonal anti-NOS2 antibody (1:100). Nuclei were costained with Hoechst shown in blue. D:
NOS2 protein expression in cultured neonatal cardiomyocytes. Cells were incubated without (lane 1) or with (lane 2) LPS (500 ng/ml) for 24 h. Cells were lysed
and NOS2 protein immunoblotted. Cell lysates of LPS/IFN␥-treated macrophages (MØ) (BD Bioscience) were used as the positive control for NOS2 with a
molecular mass of 130 kDa (lane 3). Actin was blotted to ascertain equal loading of cardiomyocyte samples (lanes 1 and 2).
AJP-Heart Circ Physiol • VOL
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Values are means ⫾ SD. The data in each group were recorded from 20 to 30 single adult cardiomyocytes and analyzed. At least three mice were used in
each group to prepare isolated cardiomyocytes. *P ⬍ 0.05 compared with the control without LPS treatment within each pair of groups.
MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
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molecular patterns, including CD14, TLR4, and TLR2 (12, 13,
30, 40). The role of pattern recognition receptors in cell death
or survival is not completely understood. Frantz et al. (12)
reported that blocking of TLR2 signaling by using a specific
antibody increases hydrogen peroxide-induced cell death in
vitro, suggesting an anti-apoptotic role for TLR2 in cardiomyocytes (12). In a noninfectious lung injury model, a recent
study has demonstrated a proinflammatory and prosurvival role
for both TLR2 and TLR4 (23). The study found that extracellular matrix hyaluronan, produced in response to lung injury,
acts on both TLR2 and TLR4 to stimulate inflammatory cytokine production and to confer an anti-apoptotic effect in lung
epithelial cells in vivo (23). Consistent with this report, our
study in TLR4-deficient cardiomyocytes demonstrates that
TLR4 mediates a direct survival benefit against serum deprivation-induced apoptosis. Different from the current study, a
previous report found that TLR4-deficient mice exhibit reduced tissue inflammation and myocardial infarction after
ischemia-reperfusion, suggesting a role of TLR4 in mediating
myocardial tissue inflammation and injury (43). The reason for
the difference is unclear. We hypothesize that the systemic
deficiency of TLR4 could have confounded the in vivo model.
The lack of TLR4 in extracardiac sources such as macrophages, lymphocytes, and neutrophils could have direct impact
on the myocardial inflammation and injury after ischemia-reperfusion. Recent studies have supported the notion that the
extracardiac source of TLR4 plays an important role in mediating
cardiac injury and dysfunction under certain pathological conditions such as endotoxemia (49). Therefore, direct contribution of
AJP-Heart Circ Physiol • VOL
cardiac TLR4 activation to myocardial inflammation and injury
after transient ischemia in vivo remains to be determined.
The present study also provides evidence that LPS elicits a
direct function preserving, rather than depressing, effect in
cardiomyocytes and that TLR4, MyD88, and NOS2 play a
critical role in mediating the functional rescue of surviving
cardiomyocytes after serum deprivation. It is unclear whether
the functional recovery is the result of improved cell survival
or vice versa. It is possible that the function-preserving benefit
of TLR4 activation may not be the results of improved cell
survival but rather contribute to cardiomyocyte survival. It has
been reported that improved calcium handling (e.g., by overexpressing sarcoplasmic reticulum Ca2⫹-ATPase pump) decreases ventricular arrhythmias and may contribute to reduced
myocardial infarction after ischemia-reperfusion (7).
To dissect the TLR4-mediated signaling pathways that are
responsible for LPS-induced survival and function improvement in isolated cardiomyocytes, we utilized mice genetically
deficient for MyD88 (25). These mice lack the ability to
respond to LPS as measured by B cell proliferation and
cytokine production. However, MyD88-independent pathways
exist in TLR4 signaling (26). We found that TLR4 activation
failed to protect the MyD88⫺/⫺ cardiomyocytes against serum
deprivation-induced apoptosis. To determine whether or not
other survival pathways are intact in MyD88⫺/⫺ cells, IGF-1
was used as a control to block apoptosis and protects cardiomyocytes (4). The survival benefit of IGF-1 is dependent on
activation of phosphatidylinositol 3⬘-kinase and Akt (PKB)
(10, 55). These data suggest that genetic deletion of MyD88
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Fig. 7. NOS2 deletion or inhibition abolishes LPSinduced anti-apoptotic effect. A: effect of N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) on LPS-induced
anti-apoptotic effect. Cardiomyocytes were treated with
or without 0.5 mmol/l L-NIL for 30 min in regular
medium. Cells were subsequently washed and incubated
in either serum-containing or serum-free medium for
24 h. A second dose of L-NIL was added at the time of
SD as indicated. As indicated, some cultures also received LPS (1,000 ng/ml) or IGF-1 (10⫺7 M). n ⫽ 4.
*P ⬍ 0.01 vs. SD without L-NIL; **P ⬍ 0.05 vs. SD
cells with L-NIL; #P ⬎ 0.1 vs. SD cells with L-NIL.
B–D: LPS failed to inhibit SD-induced apoptosis in
NOS2⫺/⫺ cardiomyocytes. B: DNA laddering from
three experiments with similar results. C: apoptotic
nuclei. n ⫽ 5. #P ⬎ 0.05 vs. SD, *P ⬍ 0.01 vs. SD. D:
cell-death ELISA. Each data point represents mean ⫾
SD of triplicate samples. Three separate experiments
were performed with similar results.
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MYD88 AND NOS2 MEDIATE TLR4-INDUCED MYOCYTE SURVIVAL
AJP-Heart Circ Physiol • VOL
mals, the physiological significance of the present findings
needs to be confirmed in animal models.
In summary, in addition to its role in host immunity, recent
studies have suggested that TLR4 signaling may play a broader
role in mediating tissue inflammation and repair by recognizing
various endogenous mediators (23, 41, 51). Some of these
endogenous mediators are produced in response to myocardial
ischemia (29, 37) and have potent anti-ischemic and anti-apoptotic effects (28, 42). The current studies present clear evidence
that activation of TLR4 signaling via MyD88 and NOS2
confers a survival benefit in cardiomyocytes and suggest a
potentially important role for TLR4 signaling as an intrinsic
cardiac protective mechanism. Identification and characterization of this survival pathway may provide novel targets for
intervention for ischemic myocardial injury.
GRANTS
This work was supported in part by grants from the National Heart, Lung,
and Blood Institute (HL-04336 to W. Chao, HL-070896 to K. D. Bloch, and
HL-073363 to A. Rosenzweig) and from the American Heart Association (to
X. Zhu and E. S. Buys).
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