Download Cardiomyocyte NF-kB p65 promotes adverse remodelling, apoptosis

Cardiovascular Research (2011) 89, 129–138
doi:10.1093/cvr/cvq274
Cardiomyocyte NF-kB p65 promotes adverse
remodelling, apoptosis, and endoplasmic
reticulum stress in heart failure
Tariq Hamid 1, Shang Z. Guo 1, Justin R. Kingery 1, Xilin Xiang 1, Buddhadeb Dawn 2,
and Sumanth D. Prabhu 1*
1
Department of Medicine, Louisville VAMC and Institute of Molecular Cardiology,University of Louisville, ACB, 3rd Floor, 550 South Jackson Street, Louisville, Louisville, KY 40202, USA;
and 2Division of Cardiovascular Diseases, University of Kansas Medical Center, Kansas City, KS 66160, USA
Received 11 May 2010; revised 30 July 2010; accepted 19 August 2010; online publish-ahead-of-print 25 August 2010
Time for primary review: 27 days
Aims
The role of nuclear factor (NF)-kB in heart failure (HF) is not well defined. We sought to determine whether
myocyte-localized NF-kB p65 activation in HF exacerbates post-infarction remodelling and promotes maladaptive
endoplasmic reticulum (ER) stress.
.....................................................................................................................................................................................
Methods
Non-transgenic (NTg) and transgenic (Tg) mice with myocyte-restricted overexpression of a phosphorylationand results
resistant inhibitor of kBa (IkBaS32A,S36A) underwent coronary ligation (to induce HF) or sham operation. Over 4
weeks, the remote myocardium of ligated hearts exhibited robust NF-kB activation that was almost exclusively
p65 beyond 24 h. Compared with sham at 4 weeks, NTg HF hearts were dilated and dysfunctional, and exhibited
hypertrophy, fibrosis, up-regulation of inflammatory cytokines, increased apoptosis, down-regulation of ER protein
chaperones, and up-regulation of the ER stress-activated pro-apoptotic factor CHOP. Compared with NTg HF,
Tg-IkBaS32A,S36A HF mice exhibited: (i) improved survival, chamber remodelling, systolic function, and pulmonary
congestion, (ii) markedly diminished NF-kB p65 activation, cytokine expression, and fibrosis, and (iii) a three-fold
reduction in apoptosis. Moreover, Tg-IkBaS32A,S36A HF hearts exhibited maintained expression of ER chaperones
and CHOP when compared with sham. In cardiomyocytes, NF-kB activation was required for ER stress-mediated
apoptosis, whereas abrogation of myocyte NF-kB shifted the ER stress response to one of adaptation and survival.
.....................................................................................................................................................................................
Conclusion
Persistent myocyte NF-kB p65 activation in HF exacerbates cardiac remodelling by imparting pro-inflammatory, profibrotic, and pro-apoptotic effects. p65 modulation of cell death in HF may occur in part from NF-kB-mediated transformation of the ER stress response from one of adaptation to one of apoptosis.
----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords
NF-kB † Heart failure † Cardiac remodelling † Apoptosis † ER stress
1. Introduction
The transcription factor nuclear factor (NF)-kB regulates genes that
coordinate stress, growth, and inflammatory responses.1,2 NF-kB
also influences cell survival and can induce either pro- or antiapoptotic genes depending on the cell type and stimulus.3 In the
endoplasmic reticulum (ER) stress response, for example, NF-kB
comprises an important signal for an alarm phase that ultimately
induces apoptosis in the face of prolonged ER stress,4 transforming
an initially compensatory mechanism into a maladaptive one. The
NF-kB family has five subunits—p65, RelB, c-Rel, p50, and p52—
that form homo- or heterodimers.1,2,5 Under resting conditions, inactive NF-kB dimers (classically p65/p50) are bound to inhibitor of kB
(IkB) in the cytoplasm, whereas on stimulation, IkB kinase (IKK)mediated IkB phosphorylation results in IkB ubiquitination and
nuclear translocation of NF-kB. NF-kB activation can also proceed
via an IkB-independent pathway releasing p50/RelB or p52/RelB.2,5
There are subunit-dependent differences in target gene specificity
related to the presence of transactivation domains (TADs).2,5 As
only p65, c-Rel, and RelB contain TADs, whereas p50 and p52 do
not, p50 and p52 homodimers can actually repress gene transcription.2,5 – 7 Hence, the ultimate transcriptional response engendered
* Corresponding author. Tel: +1 502 852 7959; fax: +1 502 852 7147, Email: [email protected]
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: [email protected].
130
is in part regulated by the constituent homo- or heterodimers
formed.
Chronic inflammation is a hallmark of heart failure (HF) and is a
predictor of overall prognosis.8 Failing myocardium exhibits augmentation of both pro-inflammatory cytokine expression9 and NF-kB activation.10,11 Circulating monocytes in HF are also pathologically
stimulated with augmented NF-kB activity.12 Despite the observation
that both myocardium and inflammatory cells in HF demonstrate
enhanced NF-kB, the pathophysiological role of NF-kB, whether it
is protective or detrimental, and the importance of cell-type specificity are not well defined. Studies of post-infarction left ventricular
(LV) remodelling in mice with targeted p50 deletion have yielded conflicting results. Two studies13,14 suggested that p50 exacerbates postinfarction remodelling and mortality, whereas another indicated the
opposite—that p50 is cardioprotective and alleviates remodelling.15
Importantly, none of these studies examined the degree of p50
subunit activation in wild-type (WT) HF or the importance of
myocyte (vs. non-myocyte) NF-kB. Accordingly, we tested the
hypothesis that chronic NF-kB p65 activation in myocytes is
pro-apoptotic and exacerbates post-infarction remodelling by using
mice with myocyte-restricted transdominant expression of a mutant
phosphorylation-resistant IkBa (IkBaS32A,S36A).16 IkBa is the
primary regulator of the p65/p50 heterodimer5 and masks the
nuclear localization sequence of p65;17 hence, IkBaS32A,S36A transgenic (Tg) mice are ideally suited for evaluating the effects of sustained
myocyte p65 activation.
2. Methods
All studies were performed in compliance with the NIH Guide for the
Care and Use of Laboratory Animals [DHHS publication (NIH) 85-23,
revised 1996]. Local approval was given by the University of Louisville
Institutional Animal Care and Use Committee (IACUC #08092).
Additional methodological details are also provided in the Supplementary
material online.
2.1 Mouse models
Male mice (10 – 22 weeks, 25 – 30 g) with myocyte-specific expression
(a-MyHC promoter) of a transdominant mutant human IkBa with serine
residues at positions 32 and 36 replaced by alanine (Tg-IkBaS32A,S36A)
were used. These mice have been characterized previously and do not
exhibit a baseline cardiac phenotype.16 The background strain is C57BL/
6; non-transgenic (NTg) littermates were used as controls.
2.2 Coronary ligation
Left coronary artery ligation or sham operation was performed in mice as
previously described18,19 and the mice were followed for 28 days after
surgery. The total mice used were: NTg, n ¼ 34; Tg-IkBaS32A,S36A, n ¼ 33.
2.3 Echocardiography
Mouse echocardiography was performed using a Philips Sonos 5500
machine and 15 MHz linear array transducer as described previously.18,19
2.4 Infarct size
Infarct size was measured 4 weeks after coronary ligation via morphological evaluation of freshly harvested LV tissue and image analysis. After
tissue harvest and dissection, the LV was quickly sectioned into five transverse slices and photographed with a digital camera. The epicardial circumference for the well-demarcated scar in each slice was determined
post hoc by videoplanimetry (NIH Image J) and summated for all slices,
T. Hamid et al.
and then normalized to total LV circumference measured in all slices.
Infarct (scar) size was expressed as a percentage of total LV.
2.5 Isolated cardiomyocyte studies and cell
transfection
H9c2 cells (ATCC) in serum-free DMEM media were seeded in 100 mm
tissue culture dishes and transfected for 24 h with the plasmid DNA
(5 mg/dish) using Transfectinw transfection reagent (BioRad) as described
previously.18 In some protocols, cells were also treated with either
recombinant mouse tumour necrosis factor (TNF) (20 ng/mL, BD Biosciences) or the ER stress inducer tunicamycin (TM, 10 mg/mL) for different time periods. Expression plasmids for NF-kB subunits p65 and p50
were purchased from Panomics. The expression plasmid for the
dominant-negative IKKb kinase mutant (KM) was a generous gift from
Dr Hiroyasu Nakano (Juntendo University School of Medicine, Japan).20
Calcium-tolerant adult mouse cardiomyocytes were isolated using modified Langendorff perfusion and collagenase digestion, and seeded in supplemented DMEM media as described previously.19,21
2.6 Western immunoblotting and
electrophoretic mobility shift assay
Non-infarcted tissue was used for molecular analyses. Total protein
extraction, SDS – PAGE western blotting, and immunodetection using
electro-chemiluminescence were performed as previously described 9,18,21
using commercially available antibodies. NF-kB subunits p65 and p50 were
also measured in nuclear extracts using a commercially available ELISA kit
(Panomics). NF-kB DNA-binding activity and subunit composition was
quantified by electrophoretic mobility shift assay (EMSA) and gel supershift as described previously.18
2.7 Quantitative real-time PCR
Total RNA extraction from LV tissue, cDNA synthesis, and quantitative
real-time PCR were performed as described previously.18 Relative levels
of mRNA transcripts for atrial natriuretic factor (ANF), connective
tissue growth factor (CTGF), TNF, interleukin (IL)-1b, and IL-6 were
determined using primer pairs previously detailed18 and normalized to
GAPDH expression using the DDCT comparative method.22
2.8 Histological analysis
H&E and Masson’s trichrome stains were used to determine cardiomyocyte cross-sectional area and myocardial fibrosis. Apoptosis was assessed
by terminal deoxytransferase-mediated dUTP nick-end labelling (TUNEL)
using the ApopTagw In Situ Detection Kit (Oncor) as described previously.23 Immunoreactivity or TUNEL positivity was quantified from at
least 20 random fields by light microscopy.
2.9 Statistical analysis
For two-group comparisons, we used the unpaired two-sample t-test. For
comparisons of more than two groups, we used one-way ANOVA if there
was one independent variable and two-way ANOVA if there were two
independent variables (e.g. genotype and ligation status). To adjust for
multiple comparisons, we performed a Bonferroni post-test. Pair-wise
comparisons were made between sham groups across genotypes, sham
vs. HF within each genotype, and HF groups across genotypes. A
P-value of ,0.05 was considered significant. Animal survival was evaluated
by the Kaplan– Meier method, and the log-rank test was used to compare
survival curves between NTg sham and HF, Tg sham and HF, and between
NTg HF and Tg HF. Continuous data are summarized as mean + SD.
NF-kB and heart failure
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Figure 1 p65 is the major NF-kB subunit chronically activated in the heart following myocardial infarction. (A) NF-kB DNA-binding activity and
subunit composition by EMSA and gel supershifts in nuclear extracts (NEs) from wild-type (WT) sham and HF hearts. p50(1) and p50(2) indicate
two different NF-kB p50 subunit-specific antibodies used in gel supershifts. Arrowhead, p65 supershift; arrow, RelB supershift. (B) NF-kB p65 and
p50 subunit-specific ELISA in NEs from WT sham and HF hearts 4 weeks after infarction. (C) Immunoblot analysis of the NF-kB subunits in cytosolic
(C) and nuclear (N) extracts from WT sham and HF hearts and corresponding densitometric values after normalizing with actin for cytosolic
expression and lamin A for nuclear expression. (D) NF-kB DNA-binding activity and subunit composition by EMSA, gel supershifts, and immunoblotting on NEs isolated from H9c2 cells transiently transfected with human NF-kB p50 in the presence or absence of TNF-a (20 ng/mL) for 30 min
(representative results from four independent experiments). (E) NF-kB p65 and p50 subunit-specific ELISA in NEs isolated from non-infarcted myocardium of WT animals at indicated times after coronary ligation. *P , 0.05 vs. sham; #P , 0.05 vs. naı̈ve; n ¼ 6 – 8/group unless otherwise indicated.
FP, free probe.
3. Results
3.1 NF-kB p65 is persistently activated in
the remodelling heart
WT C57BL/6 mice underwent coronary ligation or sham operation
and NF-kB activation was evaluated in remote myocardium 4 weeks
after surgery (Figure 1A–C ). EMSA revealed robust NF-kB activation
in failing hearts compared with sham. Gel supershift revealed that
p65 was the major subunit activated along with a minor amount of
RelB, but negligible p50, despite probing with two different p50
subunit antibodies (Figure 1A). The specificity of NF-kB DNA
binding was confirmed using cold competition with a 100-fold
unlabelled consensus sequence (see Supplementary material online,
Figure S1A). An alternative measurement for nuclear p65 and p50
using ELISA confirmed significant translocation of p65, but not p50,
in failing hearts over sham (Figure 1B). Moreover, immunoblotting
for NF-kB subunits in cytosolic and nuclear extracts revealed
augmented p65 (and RelB) nuclear translocation in failing hearts but
with diminished p50 translocation and no change in c-Rel (Figure 1C).
To exclude the possibility that the lack of p50 detection was related
to suboptimal antibody fidelity, H9c2 cells, an embryonic cardiomyoblast line, were transiently transfected with the expression plasmid
encoding human p50 in the presence or absence of TNF, and
EMSA/supershifts and immunoblotting of nuclear extracts were performed. p50 was readily detected in transfected cells using both
approaches (Figure 1D). Interestingly, p50 was not detectable by
either gel supershift or western immunoblotting in non-transfected
H9c2 cells even after TNF stimulation. In contrast, both p50 and
p65 were readily detected in HEK-293 cells upon TNF stimulation
(see Supplementary material online, Figure S1B). This indicates that
H9c2 cells may be used to selectively test p65-mediated responses.
We next performed a time course of p65 and p50 nuclear translocation in remote myocardium following infarction using ELISA
(Figure 1E). At 24 h following infarction, both p65 and p50
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Figure 2 Myocyte-specific overexpression of phosphorylation-resistant IkBaS32A,S36A attenuates NF-kB activation and improves post-infarction
remodelling. (A) Kaplan– Meier survival curves from NTg and Tg-IkBaS32A,S36A sham and HF mice. (B) NF-kB DNA-binding activity by EMSA in
nuclear extracts from NTg and Tg sham and HF hearts and corresponding densitometry (n ¼ 6 – 8/group). (C) Immunoblot analysis of the NF-kB
subunits in cytosolic (C) and nuclear (N) extracts from Tg sham and HF hearts. (D) Representative short-axis LV sections and M-mode echocardiograms from NTg and Tg sham and HF mice 4 weeks after operation. The white line indicates end-diastolic diameter. (E) Infarct size at 4 weeks in NTg
and Tg HF mice (n ¼ 10 – 16/group). *P , 0.05 vs. respective sham; #P , 0.05 vs. NTg HF.
translocation increased. However, at later time points, nuclear p65
levels increased further and remained persistently elevated, whereas
p50 levels returned to baseline.
3.2 Myocyte NF-kB abrogation improves
post-infarction survival and alleviates LV
remodelling
Echocardiography revealed no baseline differences in LV structure or
systolic function between NTg and Tg-IkBaS32A,S36A mice (data not
shown). The Kaplan –Meier survival curves (Figure 2A) revealed significantly increased mortality for NTg HF mice over sham at 28-day postinfarction but only a non-significant trend (P ¼ 0.088) towards
increased mortality in Tg-IkBaS32A,S36A HF mice compared with
sham. Tg-IkBaS32A,S36A HF mice exhibited improved survival in comparison to NTg HF. At baseline, there was a four-fold increase in
IkBa protein expression in Tg-IkBaS32A,S36A hearts when compared
with NTg hearts (see Supplementary material online, Figure S2A). In
NTg HF hearts, IkBa levels were markedly diminished compared
with NTg sham, consistent with augmented NF-kB activation (see
Supplementary material online, Figure S2B). IkBa protein levels,
while still relatively high, also decreased in Tg-IkBaS32A,S36A failing
hearts when compared with Tg-IkBaS32A,S36A sham. EMSA of
nuclear extracts from failing hearts revealed robust activation of
NF-kB in NTg HF vs. sham (Figure 2B). In Tg-IkBaS32A,S36A mice,
although NF-kB was also activated in HF, such activation was markedly reduced when compared with NTg. Immunoblotting of cytosolic
and nuclear extracts revealed that as in WT HF, p65 was the primary
subunit activated in Tg-IkBaS32A,S36A HF hearts with negligible p50
(Figure 2C). Presumably, this reflects non-myocyte (interstitial and
inflammatory cell) NF-kB and non-canonical, IkBa-independent
activation.
Figure 2D depicts representative short-axis LV sections and
M-mode echocardiograms and Table 1 presents the group echocardiographic and gravimetric data. There was LV dilatation (increased
LV end-diastolic volume and LV end-systolic volume) and systolic dysfunction (reduced LV ejection fraction and Vcf ) in both NTg and
Tg-IkBaS32A,S36A HF mice. However, compared with NTg HF, LV dilatation and dysfunction were attenuated in Tg-IkBaS32A,S36A HF. LV,
right ventricle (RV), and lung weight normalized to tibia length (TL)
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NF-kB and heart failure
Table 1 Echocardiographic and gravimetric data from
NTg and Tg-IkBaS32A,S36A mice
Tg-IkBaS32A,S36A
NTg
these cytokines was markedly up-regulated in NTg HF hearts over
sham (Figure 4). In contrast, Tg-IkBaS32A,S36A HF hearts exhibited
either diminished (TNF) or unchanged (IL-1b and IL-6) cytokine
expression compared with sham.
.............................. ...............................
Sham
HF
Sham
HF
522 + 29
521 + 50
531 + 38
510 + 38
....................................................................................
HR (b.p.m.)
LVEDD (mm)
LVESD (mm)
4.0 + 0.4
2.0 + 0.2
5.1 + 0.6*
4.2 + 0.5*
3.7 + 0.4
1.9 + 0.2
4.7 + 0.6*
3.1 + 0.8*,#
FS (%)
49 + 3
18 + 4*
48 + 5
34 + 11*,#
LVEDV (mL)
LVESV (mL)
46 + 21
16 + 9
82 + 24*
56 + 21*
34 + 15
10 + 5
64 + 24*,#
30 + 17*,#
LVEF (%)
66 + 7
37 + 12*
70 + 9
55 + 12*,#
AWT (mm)
Vcf (circ/s)
0.66 + 0.09 0.88 + 0.19* 0.70 + 0.04 0.73 + 0.17
10.4 + 1.7
3.9 + 0.8*
9.0 + 1.0
7.0 + 2.2*,#
LV weight/TL
(mg/mm)
3.7 + 0.2
4.8 + 0.9*
3.7 + 0.4
4.8 + 1.0*
RV weight /TL
(mg/mm)
1.0 + 0.1
1.6 + 0.8*
1.0 + 0.2
1.4 + 0.6*
Lung weight/TL
(mg/mm)
5.9 + 0.57
9.3 + 2.8*
6.0 + 0.7
6.9 + 1.5#
HF, heart failure; HR, heart rate; LV, left ventricular; EDD, end-diastolic diameter; EDV,
end-diastolic volume; ESD, end-systolic diameter; ESV, end-systolic volume; FS,
fractional shortening; EF, ejection fraction; AWT, anterior wall thickness at
end-diastole; Vcf, velocity of circumferential fibre shortening; TL, tibia length; RV, right
ventricle. n ¼ 7–12/group.
*P , 0.05 vs. respective sham.
#
P , 0.05 vs. NTg HF.
all significantly increased in NTg HF hearts over sham, indicating
biventricular hypertrophy and pulmonary congestion (secondary to
elevated LV filling pressure) (Table 1). LV/TL and RV/TL were
similar between Tg-IkBaS32A,S36A HF and NTg HF mice. However,
lung/TL was significantly decreased in Tg-IkBaS32A,S36A HF mice, indicating less pulmonary congestion and lower filling pressures. Notably,
infarct size at 4 weeks was equivalent in NTG and Tg-IkBaS32A,S36A HF
mice (Figure 2E), suggesting that these changes did not result from
different degrees of injury but rather to subsequent differences in
remodelling.
3.3 Myocyte NF-kB abrogation prevents
pro-inflammatory cytokine expression
and fibrosis without impacting
hypertrophy in HF
Consistent with the gravimetric data, both NTg and Tg-IkBaS32A,S36A
HF hearts exhibited increased ANF gene expression and myocyte
diameter when compared with sham, consistent with cardiac hypertrophy (Figure 3A). However, there were no differences in these parameters between NTg and Tg HF. Collagen deposition in both
remote and border zone myocardium was significantly increased in
the failing heart (Figure 3B). The degree of fibrosis was markedly attenuated, however, in Tg-IkBaS32A,S36A HF when compared with NTg
HF, and not different from Tg-IkBaS32A,S36A sham. Moreover, cardiac
gene expression of CTGF, a pro-fibrotic matrix-associated protein,
was increased five-fold in NTg HF over sham, but such up-regulation
was completely prevented in Tg-IkBaS32A,S36A HF. As NF-kB is a
potent inducer of inflammation, we next examined gene expression
of the pro-inflammatory cytokines TNF, IL-1b, and IL-6. Each of
3.4 Myocyte NF-kB abrogation decreases
apoptosis and promotes adaptive ER stress
responses in HF
We have previously demonstrated that sustained p65 overexpression
is pro-apoptotic in H9c2 cardiomyocytes, and that, along with NF-kB,
other mediators of the alarm phase of ER stress—p38 mitogenactivated protein kinase (MAPK) and c-Jun N-terminal kinase
(JNK)—are activated in failing hearts.18 Hence, we evaluated the
effects of myocyte NF-kB abrogation on apoptosis and ER stress.
As shown in Figure 5A, TUNEL-positive nuclei were markedly
increased in NTg HF hearts over NTg sham but significantly attenuated in Tg-IkBaS32A,S36A HF, which in turn was not significantly augmented over Tg-IkBaS32A,S36A sham.
The adaptive phase of ER stress is characterized by increased
expression of the ER chaperone proteins Grp78, Grp94, and calreticulin that help stabilize protein folding intermediates. In the face of
excessive ER stress, however, alarm phase mediators (e.g. NF-kB,
p38 MAPK, and JNK) are activated that ultimately up-regulate the
pro-apoptotic transcription factor CHOP (GADD153) and proteolytically process caspase-12 to induce cell death.4 Figure 5B shows
protein expression of Grp78, Grp94, and CHOP in NTg and
Tg-IkBaS32A,S36A sham and HF hearts. When compared with sham,
NTg HF hearts exhibited significantly reduced expression of Grp78
and Grp94 and robust up-regulation of CHOP demonstrating that
in established HF, there is a preponderance of alarm phase ER
stress responses associated with the augmented apoptosis. In contrast, Tg-IkBaS32A,S36A HF hearts maintained the expression of
Grp78 and Grp94 and exhibited no significant change in CHOP
when compared with sham, indicating preservation of adaptive ER
stress responses.
3.5 NF-kB activation is required for ER
stress-mediated apoptosis in
cardiomyocytes
As shown in Figure 6A, prolonged (≥8– 24 h) stimulation of H9c2 cardiomyocytes with the classical pharmacological ER stress inducer TM
increased phosphorylation and degradation of IkBa and NF-kB
nuclear translocation. This was associated with phosphorylation of
PERK and JNK1/2, up-regulation of Grp78, Grp94, and CHOP, and
increased cleavage of PARP and caspase-3, all consistent with activation of ER stress and ER stress-mediated apoptosis (Figure 6B). To
determine the role of NF-kB in ER stress-mediated cell death, the
experiments were repeated after transfection and overexpression of
a dominant-negative (DN) IKKb mutant. In separate validation
studies, DN IKKb was highly effective in blocking NF-kB activation
in response to TNF stimulation (see Supplementary material online,
Figure S3). As seen in Figure 6B, DN IKKb overexpression prevented
IkBa degradation but had no effect on expression of Grp78, Grp94,
and CHOP or the phosphorylation of PERK. However, there was suppression of JNK1/2 phosphorylation and ER stress-mediated apoptosis
with considerably less cleavage of PARP and caspase-3. The significance of JNK1/2 phosphorylation is shown in Figure 6C—pharmacological JNK inhibition with SP600125 during ER stress diminished
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T. Hamid et al.
Figure 3 Divergent effects of myocyte-specific NF-kB inhibition on hypertrophic and fibrotic responses in the failing heart. (A) Representative
haematoxylin – eosin histomicrographs and quantitation of myocyte cross-sectional area, and ANF gene expression (quantitative RT – PCR analysis,
normalized to GAPDH expression) from NTg and Tg-IkBaS32A,S36A sham and HF hearts. (B) Masson’s trichrome stains and quantitation of interstitial
fibrosis (combined border and remote zone), and CTGF expression in remodelled myocardium by real-time PCR. *P , 0.05 vs. respective sham;
#
P , 0.01 vs. NTg HF. n ¼ 4 –6/group. Scale bar ¼ 15 mm.
Figure 4 Effect of myocyte-specific NF-kB inhibition on pro-inflammatory cytokine expression in the failing heart. Normalized gene expression of
TNF, IL-6, and IL-1b by quantitative RT – PCR analysis in NTg and Tg-IkBaS32A,S36A sham and HF hearts (n ¼ 4 – 5/group). *P , 0.05 vs. respective
sham; #P , 0.01 vs. NTg HF.
IkBa degradation and CHOP up-regulation and similarly attenuated
PARP and caspase-3 cleavage.
We next assessed ER stress-mediated cell death in adult NTg and
Tg-IkBaS32A,S36A cardiomyocytes. As seen in the phase contrast
images and quantitation in Figure 6D, Tg-IkBaS32A,S36A myocytes exhibited improved survival in response to 24 h of TM exposure when
compared with NTg myocytes. As seen in Figure 6E, prolonged TM
stimulation of NTg myocytes induced IkBa degradation (indicative
of NF-kB activation), up-regulation of the ER chaperones Grp78,
Grp94, and calreticulin, and induction of CHOP and apoptosis
suggested by a decrease in procaspase-12 (indicating increased proteolytic processing) and increased cleavage of PARP. In contrast,
akin to the HF studies, Tg-IkBaS32A,S36A myocytes exhibited
augmented ER chaperones but with marked suppression of CHOP,
procaspase-12 processing, and PARP cleavage indicating abrogation
of ER stress-dependent apoptosis.
4. Discussion
We have demonstrated that chronic NF-kB activity in the murine
failing heart is primarily related to p65 and not p50, and that sustained
myocyte-localized p65 activation reduces survival and promotes postinfarction LV remodelling, imparting pro-fibrotic, pro-inflammatory,
and pro-apoptotic effects. We have also shown that NF-kB is
required for ER stress-mediated apoptosis in cardiomyocytes and
that abrogation of myocyte NF-kB p65 in isolated cardiomyocytes
NF-kB and heart failure
135
Figure 5 Myocyte-specific NF-kB abrogation attenuates apoptosis and exacerbates maladaptive ER stress in the failing heart. (A) Representative
TUNEL stain histomicrograph depicting an apoptotic nucleus (arrow) from a non-transgenic (NTg) HF heart and the corresponding quantitation
from NTg and Tg-IkBaS32A,S36A sham and HF hearts. Scale bar ¼ 15 mm. (B) Immunoblot analysis of ER stress proteins in NTg and Tg sham and
HF hearts and corresponding densitometry. *P , 0.05 vs. respective sham; #P , 0.01 vs. NTg HF; n ¼ 4 – 6/group.
or in the failing heart in vivo shifts the ER stress response to one of
adaptation rather than apoptosis. The results establish multifaceted
roles for myocyte NF-kB in general, and the p65 subunit in particular,
related to inflammation, ER stress, and apoptosis that ultimately
exacerbate cardiac remodelling and dysfunction in HF.
Failing hearts exhibit chronic activation of NF-kB and up-regulation
of NF-kB-responsive genes.9 – 11 Although classically considered a
pro-survival transcription factor, studies suggest that NF-kB serves
as a control point that can induce either survival or death depending
on the cell type and nature of the stimulus.3 Indeed, NF-kB can
up-regulate both anti-apoptotic genes (TRAF1/2, cIAP-1/2, A1/Bfl-1,
Bcl-XL, and cFLIP) and pro-apoptotic genes (Fas, FasL, DR4, DR5,
DR6, TRAIL, and p53).3,24 – 26 Whether NF-kB confers primarily detrimental or beneficial effects in chronic HF is controversial.26 Using
p50 null mice, two groups reported that p50 deficiency improved survival and ameliorated post-infarction remodelling, fibrosis, hypertrophy, and dysfunction,13,14 but did not attenuate inflammatory
cytokine expression.14 In contrast, a third study using magnetic resonance imaging reported the opposite—that p50 deletion aggravated LV
remodelling, dysfunction, hypertrophy, and fibrosis and increased
inflammation.15 Although the underlying reasons for this disparity
are unclear, none of these studies determined the status of NF-kB
subunit activation in chronically remodelled myocardium (and hence
the biological importance of p50 activity) in WT HF. Also, unclear
is the relative importance of myocyte vs. non-myocyte (e.g. inflammatory cell) responses as somatic p50 null mice were used. Cell typespecific effects may be of importance in HF, given that adoptive transfer mouse models with selective p50 deficiency in the bone marrow
(and not in cardiomyocytes) exhibit cardioprotection during acute
ischaemia/reperfusion.27
In contrast to our results in post-infarction remodelling, two recent
studies suggest that cardiomyocyte-localized NF-kB plays a protective
role in pressure-overload hypertrophy in that cardiomyocyte-specific
deletion of either IKKb or the NF-kB essential modulator (NEMO
and IKKg) exacerbates apoptosis, hypertrophy, fibrosis, and the development of HF after transverse aortic constriction.28,29 These results
may relate to differences in experimental model and serve to underscore the stimulus- and disease-specific effects of NF-kB. However,
upstream IKKs also activate NF-kB-independent signalling pathways,30
many of which (e.g. p53 and FOXO3A) can also potentially influence
remodelling. Moreover, in these studies of IKK ablation, the relative
makeup of the NF-kB subunits was not quantified, and consequently,
the role of subunit-specific responses in these effects remains unclear.
Our results demonstrate that after the initial 24 h during which
both p65 and p50 translocate to the nucleus, NF-kB activity in noninfarcted murine myocardium is almost entirely p65. Given the
primacy of nuclear p65 in chronically failing myocardium, and as
there is known subunit specificity in the ensuing transcriptional
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T. Hamid et al.
Figure 6 NF-kB activation mediates ER stress-induced apoptosis in cardiomyocytes. (A) H9c2 cardiomyocytes were treated with vehicle (V) or TM
(10 mg/mL) for the indicated times and NF-kB activation was evaluated by immunoblotting for total and phosphorylated IkBa and DNA-binding by
EMSA. (B) H9c2 cells were transiently transfected either with 5 mg of empty plasmid as a control or IKKb KM expression vector for 24 h before
treating with TM (10 mg/mL) for the indicated times and cell lysates were prepared. Total lysates (25 – 30 mg) were then immunoblotted for ER
stress markers and mediators of apoptosis. (C) H9c2 cardiomyocytes were treated as in (A) in the presence and absence of the JNK inhibitor
SP600125 (20 mM) and total cell lysates (25 – 30 mg) were immunoblotted for ER stress and apoptotic markers. (D) Equal numbers of adult
mouse cardiomyocytes isolated from naı̈ve NTg and Tg-IkBaS32A,S36A mice were treated either with vehicle (DMSO, V) or TM (10 mg/mL) for
24 h. Shown are the representative bright field images before and after treatment and quantitation of remaining rod-shaped myocytes at 24 h. (E)
Adult mouse cardiomyocytes were isolated from naı̈ve NTg and Tg hearts and plated on collagen coated dishes for 6 h before treating with TM
(10 mg/mL) for the indicated times. Total lysates were then immunoblotted for ER stress markers and mediators of apoptosis. Results in each
panel are representative of three to five independent experiments.
responses,2,5 p65-specific gene targets are likely to have more important roles in cardiac remodelling. Indeed, whereas p65 generally
induces gene transactivation, the p50 subunit is considered to
inhibit rather than stimulate transcriptional activity in part because
of an absence of TADs.2,5 – 7 Moreover, LPS stimulation of p502/2
mouse embryonic fibroblasts markedly increases nuclear p65
expression.15 Hence, the divergent experimental results with p50
ablation discussed above may be related to unrecognized differences
in the degree of compensatory p65 activation and/or up-regulation in
mice with p50 deletion. In our study, we specifically targeted
myocyte-localized p65 via transdominant expression of
phosphorylation-resistant IkBa, which masks the nuclear localization
sequence of p65.17
In our model, chronic NF-kB p65 activation in myocytes aggravated
LV remodelling, dilatation, and dysfunction following myocardial
infarction. Consistent with its central role in triggering inflammation,
myocyte p65 activation was an absolute requirement for the
up-regulation of the inflammatory cytokines TNF, IL-1b, and IL-6 in
the failing heart. Indeed, Tg-IkBaS32A,S36A HF mice actually exhibited
reduced cytokine expression in comparison to sham; the myocyte
localization of these effects is consistent with failing myocytes
serving as robust sources of inflammatory cytokines. Moreover, suppression of myocardial cytokine expression was associated with amelioration of interstitial fibrosis, consistent with the known pro-fibrotic
effects of pro-inflammatory mediators.8
Although LV chamber size was significantly smaller in
Tg-IkBaS32A,S36A HF mice when compared with NTg HF mice, the
abrogation of myocyte NF-kB in these mice did not impact the
degree of cardiac hypertrophy, as assessed by complementary
approaches of gravimetry, ANF expression, and myocyte size. This
observation is somewhat surprising given that in neonatal cardiomyocytes NF-kB activation is required for G-protein-coupled receptor
137
NF-kB and heart failure
agonist-induced hypertrophy,31 but serves to highlight important
differences between responses in isolated cells vs. in vivo pathology.
Given the absence of changes in myocyte hypertrophy, an alternate
mechanism for the marked differences in LV remodelling between
Tg-IkBaS32A,S36A HF and NTg HF may be attributed to differences in
the rates of apoptosis and cardiac cell loss, together with attendant
changes in replacement fibrosis. Indeed, Tg-IkBaS32A,S36A HF hearts
exhibited a three-fold reduction in the apoptotic rate, suggesting
that p65 activation has a substantial impact on apoptosis. These
results are consistent with our prior work, demonstrating that
NF-kB activation in H9c2 cardiomyocytes is pro-apoptotic in a TNF
receptor (R)1-dependent manner.18 In that study, we also showed
that TNFR1 signalling aggravated, whereas TNFR2 signalling ameliorated, post-infarction remodelling, apoptosis, inflammation, and
NF-kB activation in vivo. Taken together with our current results,
differential modulation of myocyte NF-kB activity likely contributes
to the divergent effects of the two TNF receptors in HF.
One mechanism by which NF-kB can influence cell death is through
the ER stress response, which is known to be activated in HF.32 Cellular stress (e.g. altered redox state) results in the accumulation of
unfolded proteins in the ER and the release of ER chaperone proteins
Grp78, Grp94, and calreticulin.4 The release of ER chaperones initially
triggers adaptive responses to restore homeostasis and reduce ER
protein load including activation of the ER transmembrane proteins
PERK, Ire1, and ATF6, suppression of global mRNA translation, and
selective up-regulation of ER chaperones and proteins involved in
the retrograde transport of misfolded proteins from the ER to the
cytosol. However, in the face of excessive ER stress, an alarm phase
is initiated by the activation of several kinases including p38MAPK,
JNK, and IKK, which subsequently results in NF-kB nuclear translocation. The end-outcome of alarm gene activation is cell death secondary to the induction of pro-apoptotic transcription factors such as
CHOP, and, in rodents, the proteolytic cleavage of caspase-12.4,33
Our results confirm that in H9c2 cardiomyocytes, a cell type that
does not express p50 (Figure 1), TM-mediated ER stress induces
PERK phosphorylation and the up-regulation of ER chaperones, but
that prolonged exposure phosphorylates JNK1/2, up-regulates
CHOP, and causes apoptosis.
In the remote myocardium of NTg failing hearts, 4 weeks after
infarction, there was down-regulation of the ER chaperones Grp78
and Grp94, but very obvious and robust expression of CHOP accompanying the increased apoptosis, indicating preponderance of the
alarm phase of ER stress. However, attenuation of NF-kB p65 activation in Tg-IkBaS32A,S36A failing hearts was accompanied by persistent
expression of Grp78 and Grp94 (suggesting greater ER protein folding
capacity) and no change in CHOP, suggesting that NF-kB serves as a
switch to convert the ER stress response from one of adaptation to
alarm in chronic HF. Indeed, in TM-treated H9c2 cells, NF-kB inhibition with IKKb KM overexpression prevented both JNK1/2 phosphorylation and apoptosis. Moreover, Tg-IkBaS32A,S36A adult
cardiomyocytes were resistant to TM-induced ER stress-related apoptosis, exhibiting maintenance of ER chaperones, suppression of
CHOP, and attenuation of procaspase-12 processing, consistent
with the in vivo results in Tg-IkBaS32A,S36A HF hearts. These results
support a model in which p65 activation is the primary driver of detrimental ER stress responses and ER stress-mediated apoptosis in HF.
Notably, these results are consistent with a recent study in rat insulinoma cells, demonstrating that NF-kB inhibition prevents
thapsigargin-induced ER stress-mediated cell death but not nitric
oxide-mediated apoptosis.34 Taken together, our results establish
that a complex interplay between cytokine-mediated inflammatory
responses, ER stress, and apoptosis underlies the detrimental effects
of myocyte NF-kB activation in the failing heart.
In summary, NF-kB activation in the murine failing heart is primarily
the p65 subunit, with negligible p50. Chronic augmentation of
myocyte-localized p65 activity is detrimental in post-infarction HF,
increases mortality, and aggravates pathological remodelling and dysfunction with pro-inflammatory, pro-fibrotic, and pro-apoptotic
effects. The modulation of cell death in the failing heart by NF-kB
p65 occurs at least in part by its regulation of the alarm phase of
the ER stress response, as it is required for ER stress-mediated cardiomyocyte apoptosis. Indeed, in failing myocytes, NF-kB p65 activity
may serve as a nodal point in the transformation of the ER stress
response from one of adaptation to one of cell death. Hence, targeted
blockade of p65 in the heart may be a useful therapeutic strategy to
maintain homeostatic responses to ER stress and ameliorate myocyte
loss in the remodelling heart.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Conflict of interest: none declared.
Funding
This work was supported by a VA Merit Award (S.D.P.), NIH grants
HL-78825 and HL-99014 (S.D.P.), and an AHA SDG award 0835456N
(T.H.).
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