PGC1a Plays a Critical Role in TWEAK–Induced Cardiac
Jianru Shi1., Bingbing Jiang1., Yiling Qiu1, Jian Guan1, Mohit Jain1, Xin Cao1, Michael Bauer1, Lihe Su2,
Linda C. Burkly3, Teresa C. Leone4, Daniel P. Kelly4, Ronglih Liao1*
1Cardiac Muscle Research Laboratory, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America,
2Protein Biochemistry Department, Biogen Idec, Inc, Cambridge, Massachusetts, United States of America, 3Immunology Department, Biogen Idec, Inc, Cambridge,
Massachusetts, United States of America, 4Cardiovascular pathobiology program, Sanford-Burnham Medical Research Institute at Lake Nona, Orlando, Florida, United
States of America
Background: Inflammatory cytokines play an important role in the pathogenesis of heart failure. We have recently found
the cytokine TWEAK (tumor necrosis factor (TNF)-like weak inducer of apoptosis), a member of the TNF superfamily, to be
increased in patients with cardiomyopathy and result in the development of heart failure when overexpressed in mice. The
molecular mechanisms underlying TWEAK-induced cardiac pathology, however, remain unknown.
Methodology and Critical Finding: Using mouse models of elevated circulating TWEAK levels, established through
intravenous injection of adenovirus expressing TWEAK or recombinant TWEAK protein, we find that TWEAK induces
a progressive dilated cardiomyopathy with impaired contractile function in mice. Moreover, TWEAK treatment is associated
with decreased expression of peroxisome proliferator-activated receptor gamma coactivator-1a (PGC1a) and genes required
for mitochondrial oxidative phosphorylation, which precede the onset of cardiac dysfunction. TWEAK-induced
downregulation of PGC1a requires expression of its cell surface receptor, fibroblast growth factor-inducible 14 (Fn14).
We further find that TWEAK downregulates PGC1a gene expression via the TNF receptor-associated factor 2 (TRAF2) and
NFkB signaling pathways. Maintaining PGC1a levels through adenoviral-mediated gene expression is sufficient to protect
against TWEAK-induced cardiomyocyte dysfunction.
Conclusion: Collectively, our data suggest that TWEAK induces cardiac dysfunction via downregulation of PGC1a, through
FN14-TRAF2-NFkB-dependent signaling. Selective targeting of the FN14-TRAF2-NFkB-dependent signaling pathway or
augmenting PGC1a levels may serve as novel therapeutic strategies for cardiomyopathy and heart failure.
Citation: Shi J, Jiang B, Qiu Y, Guan J, Jain M, et al. (2013) PGC1a Plays a Critical Role in TWEAK–Induced Cardiac Dysfunction. PLoS ONE 8(1): e54054. doi:10.1371/
Editor: Piero Anversa, Brigham and Women’s Hospital, United States of America
Received November 5, 2012; Accepted December 5, 2012; Published January 16, 2013
Copyright: ? 2013 Shi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in part, by National Institute of Health research grants HL099073 HL086967 and HL093147 (R.L.), R01HL058493,
R01HL101189, and R01DK045416 (D.P.K.) grants. M.J. is the recipient of NIH mentored award (K08 HL107451). J.S. and M.B. were supported by a postdoctoral
fellowship from American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: J.S., B.J., Y.Q., J.G., M.J., X.C., M.B., D.P.K. and R.L. have declared no competing interests for this work. L.S. and L.C.B. are employees of
Biogen-IDEC, and the authors confirm that this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Tumor necrosis factor (TNF)-like weak inducer of apoptosis
(TWEAK) is a recently identified member of the TNF superfamily
cytokines . Endogenously, TWEAK initially exists as a 249-
amino acid type II transmembrane homotrimer that is processed
rapidly into a soluble, circulating cytokine containing 156 amino
acids . TWEAK is a multifunctional cytokine involved in
regulating many biological processes including cell proliferation,
differentiation, apoptosis, migration, angiogenesis, and inflamma-
tion through binding to its cell surface signaling receptor, FGF-
inducible molecule 14 (Fn14) [3,4,5]. TWEAK and Fn14 are
typically expressed at relatively low levels in healthy conditions,
and their upregulation is linked with deleterious pathologic
conditions and disease states, such as renal damage, hypoxia/
reoxygenation, inflammatory diseases, and muscle dysfunction
[1,6,7,8]. Recently, we have identified an essential role for the
TWEAK-Fn14 axis in the development of dilated cardiomyopathy
. Circulating levels of TWEAK were found to be elevated in
patients with idiopathic dilated cardiomyopathy and overexpres-
sion of TWEAK resulted in structural remodeling and heart
failure in mice . Consistent with our findings, TWEAK levels
have also been found to be increased acutely in patients with
myocardial infarction and may predict short-term adverse events
In the present study, we find that TWEAK-induced cardiac
pathology in mice is associated with mitochondrial dysfunction.
We further identify peroxisome proliferator-activated receptor
gamma coactivator 1a (PGC1a), an essential regulator of
mitochondrial biogenesis and energy metabolism [11,12], to be
PLOS ONE | www.plosone.org1 January 2013 | Volume 8 | Issue 1 | e54054
downregulated in hearts from mice with increased circulating
TWEAK levels and in isolated cardiomyocytes exposed to
TWEAK. TWEAK-mediated downregulation of PGC1a, is found
to occur via an FN14-TRAF2-NFkB-dependent signaling pathway
and maintenance of PGC1a levels are shown to protect against
TWEAK-induced cardiac dysfunction. Selective targeting of the
FN14-TRAF2-NFkB-dependent signaling pathway or augmenting
PGC1a levels may serve as novel therapeutic strategies for
cardiomyopathy and heart failure.
Materials and Methods
C57BL/6J male mice (8 weeks old) were purchased from
Jackson Laboratory. Fn14 knockout mice (FN14 KO) and wild-
type (WT) counterparts have been previously reported and were
generated on the 129 strain background and backcrossed onto the
C57BL/6 strain, as described [13,14]. To study the in vivo role of
TWEAK, adenovirus expressing soluble murine TWEAK (Ad-
TWEAK) or control adenovirus expressing green fluorescent
protein (Ad-GFP) was delivered intravenously to mice through tail
vein injection at a dose of 1011viral particles per mouse as
described previously . In separate animals, recombinant Fc-
TWEAK (rTWEAK), a fusion protein of TWEAK with the
murine IgG2a Fc region, or an isotype-matched control IgG at
a dose of 200 mg per mouse was injected into mice intravenously
via tail vein injection. All animal procedures and handling were
carried out with the approval of Harvard Medical School, the
Institutional Animal Care and Use Committee (IACUC).
Non-invasive Transthoracic Echocardiography
Longitudinal cardiac function and chamber structure was
assessed in conscious mice through serial non-invasive trans-
thoracic echocardiography, using a Vevo2100 system (VisualSo-
nics) as previously described .
Cardiomyocyte Isolation and Culture
Adult rat ventricular cardiomyocytes were isolated from male
Wistar rats (Charles River Laboratories) using collagenase
perfusion and cell dissociation, as described previously .
Isolated cardiomyocytes were cultured in DMEM overnight prior
to incubation with 100 ng/ml of rTWEAK or control IgG at the
designated time. Similarly, adult ventricular cardiomyocytes were
isolated from WT and Fn14 KO mice using our established
protocols , and treated with IgG or rTWEAK as described
Cell Contractility Measurement
Adult rat cardiomyocytes were infected with adenovirus
expressing PGC1a (Ad-PGC1a) or Ad-GFP  for 24 hours
prior to treatment with 100 ng/ml IgG or rTWEAK for 48 hours.
Cardiomyocytes were then perfused with 1.2 mM Ca2+Tyrode’s
buffer at 37uC and field stimulated at a frequency of 5 Hz. Cell
shortening/relengthening was measured using video edge de-
tection (IonOptix, Milton, MA), as we have previously described
. Percent cell shortening (%CS) was calculated as the ratio of
the shortening length during systole over diastolic cell length.
Time to 90% relaxation defined as the time required from the
peak contraction to 90% relaxation.
TMRE (tetramethylrhodamine ethyl ester) Staining
Cardiomyocytes were cultured in petri-dishes (35 mm615 mm)
or 24-well plates overnight and subsequently treated with 100 ng/
ml IgG or rTWEAK for 24 hours, followed by incubation with
10 nM TMRE for 30 minutes. After washing with pre-warmed
PBS, culture dishes were placed in a LSM700 confocal microscopy
equipped with temperature-controlled chamber for live cell
imaging. TMRE fluorescence was assessed by excitation at
555 nm. On average, 5–7 pictures were taken from each dish.
Cardiomyocytes were hand-traced and quantified using SigmaPro
Membrane Protein Isolation
Membrane protein was isolated using a subcellular protein
fractionation kit (Thermo Scientific). Cardiomyocytes were de-
tached from culture dishes using a cell scraper and harvested into
ice-cold PBS. Cells were centrifuged at 500 6g for 5 minutes at
4uC, washed with ice-cold PBS, and centrifuged again at 500 6g
for 2 minutes. The cell pellet was suspended in cytoplasmic
extraction buffer containing protease inhibitors and incubated at
4uC for 10 minutes with gentle mixing. After centrifugation at 500
6g for 5 minutes, the separated supernatant contained the
cytoplasmic fraction. The remaining pellet was resuspended in
membrane extraction buffer containing protease inhibitors, in-
cubated at 4uC for 10 minutes with gentle mixing, and then
centrifuged at 3,000 6g for 5 minutes. The resulting supernatant
fraction provided the membrane proteins.
RNA Isolation and qRT-PCR
RNA was extracted using Trizol reagent (Invitrogen). Genomic
DNA was removed by using Turbo-DNA free kit (Ambion).
iScriptTM cDNA Synthesis Kit (Bio-Rad) was used for cDNA
synthesis and quantitative RT-PCR was performed using a CFX96
real-time PCR system (Bio-Rad). Primers used for qPCR include:
GATTCTCTTAC, reverse ATTAGGTCTGCCCTTTCTCC;
Western Blot Analysis
For protein isolation, heart tissues were homogenized or
cultured cardiomyocytes were harvested in cell lysis buffer (Cell
Signaling). Equal amounts of proteins were used for SDS/PAGE
and electrotransferred to a PVDF membrane (Millipore). The
membranes were treated with Odyssey Blocking buffer (Li-Cor) for
1 hour and incubated with appropriate primary antibodies
overnight at 4uC. After washing, blots were incubated with
corresponding secondary antibodies conjugated with IRDye
800CW or IRDye 680LT. Blots were then scanned and analyzed
using the Odyssey infrared scanner (Li-Cor).
Low glucose DMEM and Laminin were purchased from
Invitrogen. SC-514 and antibody against PGC1a were obtained
from Calbiochem. Antibodies against p-p65, p-IkBa and IkBa
were obtained from Cell Signaling Technology. Antibody against
GAPDH was obtained from R&D Systems. Ad-GFP and Ad-
TWEAK were provided by Biogen Idec Inc.
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org2 January 2013 | Volume 8 | Issue 1 | e54054
Figure 1. TWEAK induces cardiomyopathy and metabolic reprogramming in mouse hearts. (A) Fractional shortening (FS%, calculated as
the difference in chamber dimension between diastole and systole over the chamber dimension in diastole) and (B) LV ventricular diastolic chamber
dimension (LVID) were determined in mice by transthoracic echocardiography 1-day prior as well as 1, 2, and 3-week after intravenously delivery of
control, Ad-GFP or Ad-TWEAK. (C) Representative M-Mode echocardiographic images at one week and three weeks post Ad-GFP and Ad-TWEAK
injection in mice. Real time PCR analysis for expression of (D) PGCla and (E) OXPHOS genes in cardiac samples at 3-weeks post-Ad-GFP or Ad-TWEAK
injection. Real time PCR analysis for (F) PGCla and (G) OXPHOS genes in cardiac samples at 1 week (prior to the development of heart failure) post-Ad-
GFP or Ad-TWEAK injection. All real time PCR data were normalized to b-actin and presented relative to the Ad-GFP group. * p,0.05 vs. Ad-GFP, #
p,0.05 vs. Ad-TWEAK-injected mice at 1 week time point, N=5 for each group.
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org3 January 2013 | Volume 8 | Issue 1 | e54054
Figure 2. rTWEAK delivery results in cardiomyopathy and altered metabolic gene expression. (A) Fractional shortening (FS%) and (B) Left
ventricular diastolic chamber dimension (LVID), as determined by transthoracic echocardiography in mice one week after intravenous delivery of IgG
or rTWEAK. (C) Representative M-Mode echocardiographic images before and following IgG or rTWEAK injection in mice. Real time PCR analysis of
heart samples for expression of (D) PGCla and (E) OXPHOS genes, normalized to b-actin and presented relative to the IgG group. (F) Mitochondria
membrane potential in IgG and rTWEAK treated cardiomyocytes. The membrane potential was measured by TMRE staining. The fluorescent
intensities of TMRE were normalized to the total number of cardiomyocytes in the respective fields. * p,0.05 vs. IgG, N=3 for each group.
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org4 January 2013 | Volume 8 | Issue 1 | e54054
All data are expressed as mean 6 SEM. Student’s t test and
ANOVA analyses were used for statistical comparison between
two groups and multiple groups, respectively. A P value ,0.05 was
considered to be significant.
TWEAK Induces Cardiomyopathy with Mitochondrial
Damage and Decreased Expression of PGC1a
To elucidate the underlying mechanisms by which TWEAK
induces cardiac dysfunction, we utilized our established mouse
model of TWEAK induced cardiomyopathy. Increased circulating
level of TWEAK was achieved via adenovirus-mediated gene
delivery as we described previously . Consistent with our
previous observations, mice receiving Ad-TWEAK developed
a gradual decrease in contractile function and enlarged ventricular
chamber dimensions in vivo, measured by serial non-invasive
transthoracic echocardiography (Figures 1A–1C). Derangements
in myocardial substrate utilization have been shown to result in
cardiac dysfunction [19,20,21]. We, therefore, measured the level
of PGC1a, a transcriptional co-activator that is essential for
mitochondrial biogenesis and expression of genes involved in
cardiac metabolism, including oxidative phosphorylation (OX-
PHOS) genes such as Cyt C, Cox2, Atp5o, Ndufb5 and Sdha.
PGC1a expression was significantly downregulated in Ad-
TWEAK-injected mice in which cardiac dysfunction was readily
apparent (3 week time-point) (Figure 1D), and in agreement with
the down-regulation of PGC1a, cardiac expression of multiple
OXPHOS genes was also significantly decreased (Figures 1E).
Importantly, we found that the downregulation of PGC1a and
OXPHOS gene expression occurred as early as 1-week after Ad-
Figure 3. Fn14 is required for TWEAK-mediated down-regulation of PGC1a expression and TRAF2 membrane translocation.
Cardiomyocytes isolated from WT and Fn14 KO mice were treated with 100 ng/ml IgG or rTWEAK, and then harvested for determination of PGC1a
expression and TRAF2 membrane translocation. (A) Real time PCR analysis for PGCla expression post IgG and rTWEAK treatment, normalized to b-
actin and presented relative to the IgG treated WT group. (B) Real time PCR analysis for gene expression profiling of TRAF family proteins, expressed
as the mRNA ratio of TRAF/b-actin. (C) Immunoblot analysis for TRAF2 membrane translocation after IgG and rTWEAK treatment for 10 min. Lower
panel shows Ponceau S staining of PVDF membranes that was used for evaluation of protein loading amount. (D) Bar-graph shows the relative levels
of TRAF2 by densitomitric analysis with IgG treated WT group as 1-fold. * p,0.05 vs. IgG treatment in WT group, # p,0.05 vs. rTWEAK treatment in
WT group. (E) Silencing of TRAF2 using shRNA (sh-TRAF2) prevents TWEAK-induced downregulation of PGC1a. * p,0.05 vs. IgG in sh-Scramble group,
and # p,0.05 vs. rTWEAK in sh-Scramble group.
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org5January 2013 | Volume 8 | Issue 1 | e54054
TWEAK injection (Figures 1F and 1G), which preceded the
development of cardiac dysfunction. These results suggest that the
decreased PGC1a expression may play a causal role in mediating
TWEAK-induced cardiac dysfunction.
To exclude the potential effects of adenoviral delivery and gene
expression in the Ad-TWEAK mouse model, we developed a novel
mouse model with tail vein delivery of recombinant TWEAK
(rTWEAK). As shown in Figures 2A–2C, delivery of rTWEAK
resulted in the development of cardiac dysfunction with a signif-
icant decrease in contractile function and ventricular dilation after
1-week of delivery. This degree of dysfunction is comparable to
that in Ad-TWEAK-injected mice at 3-weeks. Furthermore,
similar to Ad-TWEAK-injected mice, rTWEAK administration
significantly downregulated the cardiac expression of PGC1a and
multiple OXPHOS genes (Figures 2D and 2E). Using TMRE,
a mitochondrial membrane potential sensitive fluorescence dye,
we observe a significantly reduction in TMRE fluorescence signal
in cardiomyocytes exposed to rTWEAK as compared to IgG
(Figure 2F), indicating that mitochondrial membrane potential was
reduced by direct exposure of cells to TWEAK.
Fn14 is Necessary for TWEAK-mediated Changes in
PGC1a Expression and TRAF2 Translocation
We have previously identified Fn14 as a critical receptor that
mediates TWEAK-induced cardiac dilatation and dysfunction .
To determine whether Fn14 is also necessary for TWEAK-
induced downregulation of PGC1a expression, PGC1a gene
expression in response to TWEAK treatment was examined in
cultured cardiomyocytes isolated from adult WT and Fn14
knockout mice. Loss of Fn14 completely abolished TWEAK-
mediated downregulation of PGC1a (Figure 3A).
Activation of Fn14 may result in membrane translocation of
TRAF (tumor necrosis factor receptor associated factor) leading to
activation of downstream signaling pathways . Gene expres-
sion profiling of TRAF family proteins revealed that among all six
TRAF family genes examined, TRAF2 is the most abundant in
adult cardiomyocytes (Figure 3B). Subcellular fractionation of
cardiomyocytes after rTWEAK or IgG treatment for 10 minutes
showed that TWEAK increased membrane translocation of
TRAF2 proteins, in an Fn14 dependent manner (Figure 3C and
3D). Importantly, silencing TRAF2 expression by shRNA
targeting TRAF2 (sh-TRAF2) in cardiomyocytes partially reversed
PGC1a expression following TWEAK treatment, while scramble
shRNA (sh-Scramble) showed no effect (Figure 3E).
Activation of NFkB Mediates TWEAK-induced
Downregulation of PGC1a Expression
Treatment of cardiomyocytes with rTWEAK, but not IgG,
further induced downstream mediators, including IkBa phosphor-
ylation, degradation, and resynthesis, accompanied by the
phosphorylation of NFkB p65, which occurred as early as 10
minutes and persisted for several hours (Figure 4A). As shown in
Figure 4B, inhibition of NFkB activation via a selective IkB kinase-
b inhibitor, SC-514, completely abolished TWEAK regulation of
PGC1a expression, indicating a requirement for NFkB activation
in the downregulation of PGC1a.
Maintaining PGC1a Prevents TWEAK-induced
Cardiomyocyte Contractile Dysfunction
To test our hypothesis that the downregulation of PGC1a plays
a causal role in TWEAK-induced cardiac dysfunction, adenoviral-
mediated PGC1a expression was used in cultured cardiomyocytes
for 24 hours prior to treatment with IgG or rTWEAK. Upon
rTWEAK treatment, PGC1a expression was downregulated in
Ad-GFP-infected cells but maintained at normal levels in Ad-
PGC1a-infected cells (Figure 5A). rTWEAK significantly impaired
cardiomyocyte contractility as revealed by reduced percent cell
shortening (%CS) (Figures 5B and 5C) and prolonged time of cell
relaxation (Figure 5D). Strikingly, maintaining PGC1a expression
prevented rTWEAK impaired cell contractility (Figures 5B–5D).
These data support the notion that downregulation of PGC1a
plays a critical role in mediating TWEAK-induced cardiomyocyte
In the present study, we reveal that the cytokine TWEAK
downregulates PGC1a and mitochondrial OXPHOS gene
expression in cardiomyocytes, which contributes to TWEAK-
induced cardiac dysfunction. Moreover, we find that TWEAK
regulates PGC1a expression via Fn14/TRAF2/NFkB-dependent
signaling pathways (Figure 6).
PGC1a is a transcriptional coactivator that is preferentially
expressed in tissues with high-energy demand and greater
Figure 4. Activation of NFkB mediates TWEAK-induced down-
regulation of PGC1a. (A) Immunoblots of phospho-p65, phospho-
IkBa, total-IkBa and GAPDH in isolated cardiomyocytes incubated with
100 ng/ml IgG or rTWEAK at designated time points. (B) Inhibition of
NFkB activation with SC-514 (25 mM) abolished TWEAK-mediated
downregulation of PGC1a expression. * p,0.05 vs. IgG and # p,0.05
vs. rTWEAK in the absence of SC-514.
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org6 January 2013 | Volume 8 | Issue 1 | e54054
mitochondrial abundance, including the heart [23,24]. PGC1a
directly coactivates the transcription factors PPARs and estrogen-
related receptors (ERRs) and regulates mitochondrial fatty acid b-
oxidation as well as expression of genes related to electron
transport chain and oxidative phosphorylation . PGC1a also
coactivates nuclear respiratory factor (NRF) and the expression of
mitochondrial transcription factor A (Tfam), which are essential
for mitochondrial DNA replication and transcription .
Expression levels of PGC1a are intricately linked to the
maintenance of the cardiac structure and function. Genetic
ablation or overexpression of PGC1a has been demonstrated to
result in cardiac dysfunction [18,26,27], thereby suggesting that
PGC1a is required for maintenance of proper heart function.
Interestingly, we observed a reduced expression of PGC1a and
OXPHOS genes in response to TWEAK treatment. Importantly,
the downregulation of these genes occurred while heart function
was still maintained and prior to the development of functional
decline, suggesting a temporal association between suppression of
PGC1a expression by TWEAK and the development of cardiac
dysfunction. In contrast, we have previously found that genetic
overexpression of TWEAK does not result in increased cardiac
apoptosis, unlike with other cytokines, prior to the development of
contractile dysfunction and heart failure . The causal relation-
ship between PGC1a downregulation and cardiac dysfunction is
supported by our data demonstrating that maintenance of PGC1a
expression in cardiomyocytes protected against TWEAK-induced
cardiac dysfunction. Notably, high degree of chronic overexpres-
sion of PGC1a has been associated with the development of
cardiac dysfunction  and therefore, adenoviral gene expression
approaches used in this study aimed to maintain PGC1a in
TWEAK-treated cells at control cell levels.
We have previously found that TWEAK-induced cardiac
dysfunction requires expression of Fn14 . Fn14 has a short
cytoplasmic tail that contains a TRAF consensus-binding motif
. The recruitment of TRAFs to the TRAF binding motif leads
to activation of signaling cascades and regulates a variety of
cellular functions including survival and death, among other
functions . To date, there are six known structurally related
members in TRAF family, of which TRAF1, TRAF2, TRAF3 and
TRAF5 are able to bind with Fn14 . In adult cardiomyocyte,
our data have identified TRAF2 as the most abundant TRAF
member. TWEAK stimulation was further found to be capable of
initiating membrane translocation of TRAF2 in an Fn14-de-
pendent manner. Interestingly, knockdown of TRAF2 with
shRNA is sufficient to prevent TWEAK-induced downregulation
of PGC1a expression, indicating that TRAF2 is the principal
proximal signaling member involved in this event.
Figure 5. Maintenance of PGC1a levels protects against TWEAK-induced contractile dysfunction in isolated cardiomyocytes. Isolated
cardiomyocytes were infected with adenovirus (MOI of 10) expressing GFP or PGC1a for 24 hours prior to treatment with IgG or rTWEAK for 48 hours.
(A) Western blot analysis of PGC1a expression, normalized with GAPDH. N=3 independent biological replicates group. (B) Isolated cardiomyocyte
function (%CS) was determined using edge detection method. (C) Representative tracings of single cell shortening at indicated conditions. (D) Time
to 90% relaxation in isolated cardiomyocytes. Cellular function was assessed in three independent biological replicates and data from 8–12 cells was
averaged as N=1 for a given experiment. * p,0.05 vs. IgG and # p,0.05 vs. rTWEAK in Ad-GFP group.
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org7 January 2013 | Volume 8 | Issue 1 | e54054
A universal characteristic of TWEAK signaling through Fn14 is
the activation of NFkB signaling pathway with modulation of
numerous downstream target genes [30,31,32]. NFkB p65 is
present in the cytoplasm and is associated with the repressor
protein IkBa. Proinflammatory stimuli such as interleukin-1, TNF,
and lipopolysaccharides can activate IkB kinase-b that induces
IkBa phosphorylation and proteosome-mediated degradation,
which results in NFkB phosphorylation, nuclear translocation,
and binding to DNA . In this study, we demonstrate that
TWEAK induces IkBa phosphorylation accompanied by IkBa
degradation and resynthesis, as well as NFkB p65 phosphorylation
in cardiomyocytes, consistent with previous reports [31,34].
Interestingly, a selective IkB kinase-b inhibitor, SC-514, prevents
the downregulation of PGC1a by TWEAK, indicating that
TWEAK repression of PGC1a expression requires activation of
NFkB signaling pathway. This is consistent with the observation
that other cytokines, such as TNFa, suppress PGC1a expression
through activation of NFkB . It has also been reported that
lipopolysaccharide suppresses PGC-1a gene expression at the
transcriptional level, and blocking the toll-like receptor-4/NFkB
axis prevented the downregulation of PGC-1 coactivator expres-
sion by LPS. However, how activation of NFkB suppresses
PGC1a gene expression is unclear and remains under investiga-
In summary, our data suggest that Fn14-TRAF2-NFkB-de-
pendent suppression of PGC1a expression plays a crucial role in
TWEAK-induced cardiac dysfunction. Modulation of PGC1a
expression or antagonism of Fn14-TRAF2-NFkB may serve as
candidate therapeutic targets in preventing TWEAK-induced
The authors wish to thank Mr. Soeun Ngoy at Brigham and Women’s
Hospital Cardiovascular Physiology Core for his assistance in animal work.
Contributed to data interpretation and scientific discussion: LCB DPK MJ.
Conceived and designed the experiments: JS BJ MJ RL. Performed the
experiments: JS JG MB YQ XC. Analyzed the data: JS BJ. Contributed
reagents/materials/analysis tools: LS LCB TCL DPK. Wrote the paper: JS
BJ MJ RL.
Figure 6. Graphical illustration summarizing the proposed mechanism underlying TWEAK-induced cardiac dysfunction. Normal
PGC1 levels maintain OXPHOS gene expression and mitochodrial function to support cardiomyocyte contractility (left panel). TWEAK downregulates
PGC1 levels through FN14-TRAF2-NFkB-dependent signaling, leading to impaired OXPHOS gene expression and mitochondrial dysfunction, and
reduced cardiomyocyte contractility (right panel).
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org8 January 2013 | Volume 8 | Issue 1 | e54054
1. Burkly LC, Michaelson JS, Zheng TS (2011) TWEAK/Fn14 pathway: an
immunological switch for shaping tissue responses. Immunol Rev 244: 99–114.
2. Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, et al. (1997)
TWEAK, a new secreted ligand in the tumor necrosis factor family that weakly
induces apoptosis. J Biol Chem 272: 32401–32410.
3. Campbell S, Burkly LC, Gao HX, Berman JW, Su L, et al. (2006)
Proinflammatory effects of TWEAK/Fn14 interactions in glomerular mesangial
cells. J Immunol 176: 1889–1898.
4. Burkly LC, Michaelson JS, Hahm K, Jakubowski A, Zheng TS (2007)
TWEAKing tissue remodeling by a multifunctional cytokine: role of
TWEAK/Fn14 pathway in health and disease. Cytokine 40: 1–16.
5. Winkles JA (2008) The TWEAK-Fn14 cytokine-receptor axis: discovery, biology
and therapeutic targeting. Nat Rev Drug Discov 7: 411–425.
6. Weinberg JM (2011) TWEAK-Fn14 as a mediator of acute kidney injury.
Kidney Int 79: 151–153.
7. Yepes M, Winkles JA (2006) Inhibition of TWEAK activity as a new treatment
for inflammatory and degenerative diseases. Drug News Perspect 19: 589–595.
8. Kumar A, Bhatnagar S, Paul PK (2012) TWEAK and TRAF6 regulate skeletal
muscle atrophy. Curr Opin Clin Nutr Metab Care 15: 233–239.
9. Jain M, Jakubowski A, Cui L, Shi J, Su L, et al. (2009) A novel role for tumor
necrosis factor-like weak inducer of apoptosis (TWEAK) in the development of
cardiac dysfunction and failure. Circulation 119: 2058–2068.
10. Chorianopoulos E, Jarr K, Steen H, Giannitsis E, Frey N, et al. (2012) Soluble
TWEAK is markedly upregulated in patients with ST-elevation myocardial
infarction and related to an adverse short-term outcome. Atherosclerosis 211:
11. Finck BN, Kelly DP (2007) Peroxisome proliferator-activated receptor gamma
coactivator-1 (PGC-1) regulatory cascade in cardiac physiology and disease.
Circulation 115: 2540–2548.
12. Rowe GC, Jiang A, Arany Z (2010) PGC-1 coactivators in cardiac development
and disease. Circ Res 107: 825–838.
13. Girgenrath M, Weng S, Kostek CA, Browning B, Wang M, et al. (2006)
TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor
cells and skeletal muscle regeneration. EMBO J 25: 5826–5839.
14. Jakubowski A, Ambrose C, Parr M, Lincecum JM, Wang MZ, et al. (2005)
TWEAK induces liver progenitor cell proliferation. J Clin Invest 115: 2330–
15. Bauer M, Cheng S, Jain M, Ngoy S, Theodoropoulos C, et al. (2011)
Echocardiographic speckle-tracking based strain imaging for rapid cardiovascu-
lar phenotyping in mice. Circ Res 108: 908–916.
16. Shi J, Guan J, Jiang B, Brenner DA, Del Monte F, et al. (2010) Amyloidogenic
light chains induce cardiomyocyte contractile dysfunction and apoptosis via
a non-canonical p38alpha MAPK pathway. Proc Natl Acad Sci U S A 107:
17. Liao R, Jain M (2007) Isolation, culture, and functional analysis of adult mouse
cardiomyocytes. Methods Mol Med 139: 251–262.
18. Lehman JJ, Barger PM, Kovacs A, Saffitz JE, Medeiros DM, et al. (2000)
Peroxisome proliferator-activated receptor gamma coactivator-1 promotes
cardiac mitochondrial biogenesis. J Clin Invest 106: 847–856.
19. Horowitz JD, Chirkov YY, Kennedy JA, Sverdlov AL (2010) Modulation of
myocardial metabolism: an emerging therapeutic principle. Curr Opin Cardiol
20. Stanley WC, Recchia FA, Lopaschuk GD (2005) Myocardial substrate
metabolism in the normal and failing heart. Physiol Rev 85: 1093–1129.
21. Schilling J, Kelly DP (2011) The PGC-1 cascade as a therapeutic target for heart
failure. J Mol Cell Cardiol 51: 578–583.
22. Brown SA, Richards CM, Hanscom HN, Feng SL, Winkles JA (2003) The Fn14
cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3
and 5 and mediates nuclear factor-kappaB activation. Biochem J 371: 395–403.
23. Liang H, Ward WF (2006) PGC-1alpha: a key regulator of energy metabolism.
Adv Physiol Educ 30: 145–151.
24. Finck BN, Kelly DP (2006) PGC-1 coactivators: inducible regulators of energy
metabolism in health and disease. J Clin Invest 116: 615–622.
25. Puigserver P, Spiegelman BM (2003) Peroxisome proliferator-activated receptor-
gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and
metabolic regulator. Endocr Rev 24: 78–90.
26. Arany Z, He H, Lin J, Hoyer K, Handschin C, et al. (2005) Transcriptional
coactivator PGC-1 alpha controls the energy state and contractile function of
cardiac muscle. Cell Metab 1: 259–271.
27. Russell LK, Mansfield CM, Lehman JJ, Kovacs A, Courtois M, et al. (2004)
Cardiac-specific induction of the transcriptional coactivator peroxisome
proliferator-activated receptor gamma coactivator-1alpha promotes mitochon-
drial biogenesis and reversible cardiomyopathy in a developmental stage-
dependent manner. Circ Res 94: 525–533.
28. Wiley SR, Winkles JA (2003) TWEAK, a member of the TNF superfamily, is
a multifunctional cytokine that binds the TweakR/Fn14 receptor. Cytokine
Growth Factor Rev 14: 241–249.
29. Arch RH, Gedrich RW, Thompson CB (1998) Tumor necrosis factor receptor-
associated factors (TRAFs)–a family of adapter proteins that regulates life and
death. Genes Dev 12: 2821–2830.
30. Enwere EK, Holbrook J, Lejmi-Mrad R, Vineham J, Timusk K, et al. (2012)
TWEAK and cIAP1 Regulate Myoblast Fusion Through the Noncanonical NF-
kappaB Signaling Pathway. Sci Signal 5: ra75.
31. Moreno JA, Izquierdo MC, Sanchez-Nino MD, Suarez-Alvarez B, Lopez-
Larrea C, et al. (2011) The inflammatory cytokines TWEAK and TNFalpha
reduce renal klotho expression through NFkappaB. J Am Soc Nephrol 22: 1315–
32. Roos C, Wicovsky A, Muller N, Salzmann S, Rosenthal T, et al. (2010) Soluble
and transmembrane TNF-like weak inducer of apoptosis differentially activate
the classical and noncanonical NF-kappa B pathway. J Immunol 185: 1593–
33. Shih VF, Tsui R, Caldwell A, Hoffmann A (2011) A single NFkappaB system for
both canonical and non-canonical signaling. Cell Res 21: 86–102.
34. Brown SA, Ghosh A, Winkles JA (2010) Full-length, membrane-anchored
TWEAK can function as a juxtacrine signaling molecule and activate the NF-
kappaB pathway. J Biol Chem 285: 17432–17441.
35. Alvarez-Guardia D, Palomer X, Coll T, Davidson MM, Chan TO, et al. (2010)
The p65 subunit of NF-kappaB binds to PGC-1alpha, linking inflammation and
metabolic disturbances in cardiac cells. Cardiovasc Res 87: 449–458.
PGC1a and TWEAK-Induced Heart Failure
PLOS ONE | www.plosone.org9 January 2013 | Volume 8 | Issue 1 | e54054