Abstract
Background
The immune system plays a pivotal role in myocardial homeostasis and
response to injury. Interleukins‐4 and ‐13 are anti‐inflammatory type‐2
cytokines, signaling via the common interleukin‐13 receptor α1 chain
and the type‐2 interleukin‐4 receptor. The role of interleukin‐13
receptor α1 in the heart is unknown.
Methods and Results
We analyzed myocardial samples from human donors (n=136) and patients
with end‐stage heart failure (n=177). We found that the interleukin‐13
receptor α1 is present in the myocardium and, together with the
complementary type‐2 interleukin‐4 receptor chain Il4ra, is
significantly downregulated in the hearts of patients with heart
failure. Next, we showed that Il13ra1‐deficient mice develop severe
myocardial dysfunction and dyssynchrony compared to wild‐type mice
(left ventricular ejection fraction 29.7±9.9 versus 45.0±8.0; P=0.004,
left ventricular end‐diastolic diameter 4.2±0.2 versus 3.92±0.3;
P=0.03). A bioinformatic analysis of mouse hearts indicated that
interleukin‐13 receptor α1 regulates critical pathways in the heart
other than the immune system, such as extracellular matrix (normalized
enrichment score=1.90; false discovery rate q=0.005) and glucose
metabolism (normalized enrichment score=−2.36; false discovery rate
q=0). Deficiency of Il13ra1 was associated with reduced collagen
deposition under normal and pressure‐overload conditions.
Conclusions
The results of our studies in humans and mice indicate, for the first
time, a role of interleukin‐13 receptor α1 in myocardial homeostasis
and heart failure and suggests a new therapeutic target to treat heart
disease.
Keywords: cytokine, heart failure, receptor
Subject Categories: Growth Factors/Cytokines
Introduction
Activation of the immune system and release of proinflammatory
cytokines dictate the pathophysiology of acute and chronic myocardial
diseases.[62]1 Proinflammatory cytokines worsen adverse cardiac
remodeling and dysfunction by destructive effects on cardiomyocytes and
extracellular matrix.[63]2 Attempts to improve patient outcomes by
inhibition of pro‐inflammatory cytokines have failed and, in some
cases, have even led to exacerbation of heart failure (HF).[64]2,
[65]3, [66]4 Thus, there is a need to explore new immunomodulation
pathways to improve HF therapy.[67]5
The effects of anti‐inflammatory cytokines on the heart have been less
investigated. Interleukin (IL)‐4 and IL‐13 are T‐helper type‐2
anti‐inflammatory cytokines studied extensively for their involvement
in the pathogenesis of parasitic infection, asthma, and allergic
diseases.[68]6 However, their potential role in heart disease remains
controversial. Both activation and deficiency of IL‐13 and IL‐4 have
been linked to conflicting profibrotic[69]7, [70]8, [71]9 and
antifibrotic effects.[72]10, [73]11 Given these inconsistent results,
we aimed to determine the role of IL‐13 and IL‐4 signaling in the
heart. Both cytokines act via the common type‐2 interleukin‐4 receptor
(IL‐4R) composed of interleukin‐13 receptor α1 (IL‐13Rα1) and IL‐4Rα.
Hence, deficiency in IL‐4 could be compensated by activation of IL‐13
signaling via the common type‐2 IL‐4R, and vice versa. On the other
hand, specific deletion of the IL‐13Rα1 chain could prevent the effects
of compensatory activation and provide a unique opportunity to study
type‐2 IL‐4R signaling in the heart.[74]12
Here, we provide evidence for a previously unrecognized, protective,
regulatory role of IL‐13Rα1 and type‐2 IL‐4R signaling in myocardial
homeostasis, metabolism, and repair. A potential implication of our
study is the development of novel therapies for myocardial disease.
Methods
Human Heart Samples
Tissue samples were obtained from the left ventricular (LV) free wall
of 177 HF patients undergoing heart transplantation (94 ischemic and 77
idiopathic dilated cardiomyopathy, 1 valvular and 5 others) and from
136 unused donor hearts, enrolled in the MAGNet consortium
([75]http://www.med.upenn.edu/magnet/index.shtml). Gene expression
analysis was done as previously described.[76]13 All procedures
involving human tissue were approved by the Institutional Review Board
at the University of Pennsylvania and the Tel‐Hashomer Medical Center.
Mice
Il13ra1 ^−/− mice were generated as previously described.[77]14 In
these animals, disrupted Il13ra1 contains a lacZ reporter cassette used
for β‐galactosidase staining in mouse heart tissue. C57BL/6 wild‐type
(WT) mice were obtained from Harlan Laboratories (Rehovot, Israel). In
all experiments, age‐matched and sex‐matched mice were housed under
specific pathogen‐free conditions and maintained with 12‐hour light and
dark cycles, according to institutionally approved protocols of the
Animal Care Committee at the Tel‐Hashomer Medical Center, Tel‐Aviv
University.
Metabolic Studies
A glucose tolerance test was carried out in Il13ra1 ^−/− mice and their
control littermates, after 16 hours of overnight fasting and an
intraperitoneal injection of 2 g of glucose per kilogram body weight.
Blood glucose was measured on samples obtained by tail bleeding before
glucose administration and after 30, 60, 90, and 120 minutes, using a
FreeStyle Optium glucose meter (Abbott Diabetes Care, Alameda, CA). For
an insulin tolerance test, mice were fasted for 6 hours and injected
intraperitoneally with insulin (0.75 U kg^−1 body weight) (Eli Lilly,
Indianapolis, IN), and blood glucose levels were measured before and
15, 30, 60, 90, and 120 minutes after the injection. Body composition
analysis (fat mass) in mice was assessed by nuclear magnetic resonance
using a Bruker Mice Minispec NMR analyzer (Bruker Optics, Billerica,
MA).
Histological Analysis
To determine Il13ra1 gene expression in mouse heart, we used a
transgenic mouse that expresses a lacz‐interrupted Il13ra1 gene. Hearts
were harvested, cryosectioned into 5‐μm sections, and placed onto
slides. Sections were fixed with 0.125% glutaraldehyde, permeabilized
with 0.01% Na‐deoxycholate and 0.02% NP‐40. A signal was detected by
incubating with 1 mg/mL X‐gal at 37°C for 3 hours. Next, to visualize
cardiomyocytes in the X‐gal–stained heart sections, slides were
costained with antibodies against α‐cardiac actin (Santa Cruz
Biotechnology, Dallas, TX, catalog number sc‐58670).
To determine IL‐13Rα1 presence in the human myocardium, a cardiac
tissue biopsy was obtained from the right atrium of a 70‐year‐old HF
patient during a coronary artery bypass graft surgery. The specimen was
fixed in formaldehyde 4%, paraffin embedded, and sectioned into 5‐mm
sections. The sections were immunostained with the primary antibodies
against IL‐13Rα1 (Abcam, Cambridge, MA, catalog number ab79277)
followed by incubation with peroxidase‐conjugated AffiniPure donkey
antirabbit (Jackson Immunoresearch Laboratories, West Grove, PA,
catalog number 711‐035‐152), according to the manufacturer's protocol.
For a negative control, the same samples and protocol were used, but
the primary antibody was omitted.
To analyze fibrosis and hypertrophy, hearts were harvested from
3‐month‐old Il13ra1 ^−/− and WT mice, washed with phosphate‐buffered
saline and then fixed in 4% paraformaldehyde overnight. Adjacent blocks
were embedded in paraffin, sectioned into 5‐μm slices, and stained with
Masson trichrome according to standard procedure. To quantify
perivascular fibrosis in comparably sized coronary arteries, we
photographed all arteries with a diameter of 50 to 80 μm in each slide
and analyzed collagen deposition by automated image analysis using
ImageJ software ([78]http://rsbweb.nih.gov/ij/).[79]15 To assess
cardiomyocyte hypertrophy and cardiac fibrosis in a pressure overload
model, hearts were harvested 3 weeks after transverse aortic
constriction (TAC). Wheat germ agglutinin staining was used to measure
cardiomyocyte diameter, and cardiac fibrotic area was evaluated after
Masson trichrome staining.
Pressure Overload Model in Mice
TAC was performed in 10‐week‐old Il13ra1 ^−/− and WT female mice.
Animals were anesthetized with 1% to 2% isoflurane in 100% oxygen
delivered through a volume‐cycled rodent respirator. Midline sternotomy
was performed, the aorta was exposed, and a 6.0 prolene suture was
placed around the aorta distal to the brachiocephalic artery. The
suture was tightened around a blunt 27‐gauge needle placed adjacent to
the aorta. The needle was then removed, and the chest and overlying
skin were closed with a 5‐0 absorbable suture. Mice were allowed to
recover from anesthesia under warm conditions. Mortality during and
immediately following the procedure was ≈10%.
Mouse Echocardiography
Transthoracic echocardiography and speckle‐tracking strain imaging were
performed with a mouse echocardiography system (Vevo 2100 Imaging
System; VisualSonics, Toronto, Ontario, Canada) equipped with a 22‐ to
55‐MHz linear‐array transducer (MS250 MicroScan Transducer,
VisualSonics, Toronto, Ontario, Canada).
Speckle‐tracking–based strain analysis was performed for strain
quantification in the radial axes. Echocardiographic parasternal
long‐axis images were acquired at a frame rate of 280 frames per
second. Three consecutive cardiac cycles were selected, and their
endocardium and epicardium borders were traced. Each LV image in long
axis was divided into 6 segments for regional speckle‐tracking–based
strain analysis: anterior base, anterior mid, anterior apex, posterior
apex, posterior mid, and posterior base. Peak strain data were recorded
from each segment for regional speckle‐tracking–based strain analysis.
Global strain of the LV was calculated as the averaged peak strain
obtained from all 6 segments.
Western Blotting
Proteins were extracted from the hearts of Il13ra1 ^−/− and WT mice or
H9C2 cells using a RIPA buffer (Sigma‐Aldrich, St. Louis, MO)
supplemented with Complete Mini, EDTA‐free, protease inhibitor cocktail
(Roche Diagnostics, Indianapolis, IN, catalog number: 11 836 170 001).
Following separation on an SDS‐PAGE, proteins were transferred to a
nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen
Corporation, Carlsbad, CA). Membranes were stained with a primary
antibody overnight at 4°C, washed, and incubated with the appropriate
secondary antibody for 45 to 60 minutes at room temperature. Specific
reactive bands were detected using the SuperSignal West Pico
Chemiluminescent Substrate (Thermo Scientific, Rockford, IL). The
antibodies used were anti–signal transducer and activator of the
transcription (STAT)3 (Cell Signaling Technology, Beverly, MA, catalog
number 9139), anti‐STAT6 (Cell Signaling Technology, Beverly, MA,
catalog number 5397), anti–phosphorylated STAT3 (Cell Signaling
Technology, Beverly, MA, catalog number 9145), anti–phosphorylated
STAT6 (Santa Cruz Biotechnology, Dallas, TX, catalog number
sc‐11762‐R), anti‐actin (Santa Cruz Biotechnology, Dallas, TX, catalog
number sc‐58670), and anti–α tubulin (Sigma‐Aldrich, Saint Louis, MO,
catalog number T9026).
IL‐13 Signaling in H9C2 Cardiomyoblast Cell Culture
H9C2 cells were cultured in DMEM medium (Biological Industries, Beit
Haemek, Israel) supplemented with 10% fetal bovine serum, 1%
penicillin: streptomycin (pen:strep) and 1% glutamine, at 37°C in a
humidified incubator with 5% CO[2]. Cells were seeded in 6‐well plates
at a concentration of 4×10^6 cells per well and treated with IL‐13
(10 ng/mL). Protein was extracted at 0, 2 minutes, 5 minutes,
15 minutes, 30 minutes, 1 hour, 6 hours, 24 hours, and 48 hours after
the addition of the cytokine, and western blot was performed for STAT6
and STAT3 signaling as described above.
Quantitative Real‐Time PCR in Mouse Heart
Mice were euthanized, and their hearts were harvested, washed in
phosphate‐buffered saline, and snap frozen in liquid nitrogen. RNA was
purified from snap‐frozen hearts with an RNeasy Mini Kit (Qiagen,
Valencia, CA) following the manufacturer's instructions. Reverse
transcription was performed using a High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, CA). Real‐time
quantitative polymerase chain reaction was performed using the
glyceraldehyde 3‐phosphate dehydrogenase (Gapdh) as a reference gene,
which showed stable levels of expression in WT and Il13ra1 ^−/− heart
samples. All reactions were run as triplicates. Reactions were
performed in a total volume of 10 μL containing cDNA equivalent to
50 ng of RNA from each sample. Real‐time quantitative polymerase chain
reaction was performed using an ABI StepOnePlus System (Applied
Biosystems, Foster City, CA). Primers were designed with PrimerBank
([80]http://pga.mgh.harvard.edu/primerbank/) or Primer‐BLAST
([81]http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The sequences of
all the primers and probes used for real‐time quantitative polymerase
chain reaction are listed in [82]Table.
Table 1.
Sequences of All Primers and Probes Used for Real‐Time Quantitative
Polymerase Chain Reaction
Gene Forward Reverse
Mmp12 CATGAAGCGTGAGGATGTAGAC TGGGCTAGTGTACCACCTTTG
Thbs1 GCAGCACACACAGAAGCATT CAATCAGCTCTCACCAGCAG
Stat6 CTCTGTGGGGCCTAATTTCCA CATCTGAACCGACCAGGAACT
Col3a1 CTGTAACATGGAAACTGGGGAAA CCATAGCTGAACTGAAAACCACC
Timp1 CCAGAACCGCAGTGAAGAGTT AAGCTGCAGGCACTGAGTG
Gapdh TCGTCCCGTAGACAAAATGG TTGAGGTCAATGAAGGGGTC
Tgfb1 TGACGTCACTGGAGTTGTACGG GGTTCATGTCATGGATGGTGC
Mmp9 GGACCCGAAGCGGACATTG CGTCGTCGAAATGGGCATCT
Col1a1 CGAAGGCAACAGTCGCTTCA GGTCTTGGTGGTTTTGTATTCGAT
Stat3a TaqMan probes
Stat3b TaqMan probes
[83]Open in a new tab
Plasma Cytokine Levels
To measure plasma pro‐ and anti‐inflammatory cytokine levels,
retro‐orbital bleeding was performed on 10‐week old Il13ra1 ^−/− and WT
mice under light isoflurane anesthesia. Whole blood was collected into
plasma collecting tubes (MiniCollect Tube, Greiner Bio‐One, Monroe, NC)
and placed on ice. Samples were then centrifuged for 10 minutes at
2000g. Cytokine plasma concentrations were measured using the
commercially available mouse Luminex Multiplex Platform (R&D Systems,
Minneapolis, MN) and enzyme‐linked immunosorbent assay (BioLegend, San
Diego, CA). All procedures where performed according to the
manufacturer's instructions.
Gene Arrays and Bioinformatic Analysis
To search for differentially regulated gene networks in the absence of
the Il13ra1 gene, we performed a comprehensive gene analysis by
hybridizing microarray chips with RNA probes prepared from mouse
Il13ra1 ^−/− and WT hearts. Briefly, total RNA was extracted from
hearts from 12‐week‐old Il13ra1 ^−/− and WT male mice, using EZ RNA
(Biological Industries, Beit Haemek, Israel) according to the
manufacturer's instructions. Total RNA was quantified by using a
spectrophotometer and confirmed by Qubit Fluorometric Quantitation
(Life Technologies, Grand Island, NY). Gene arrays were performed using
the Affymetrix Mouse Gene 2.0 ST Array (Affymetrix, Santa Clara, CA)
and were robust multi‐array average‐normalized.[84]16 Differentially
expressed genes were found by fitting linear models and computing
empirically moderated t‐statistics as implemented in the R/bioconductor
limma package[85]17 with a Benjamini‐Hochberg adjusted P‐value cutoff
of 5%.
Gene set enrichment analysis (GSEA) was based on software provided by
the Broad Institute of MIT and Harvard.[86]18 To retain statistical
power we limited the analysis to 171 gene sets that represented Kyoto
Encyclopedia of Genes and Genomes pathways. The sets were obtained from
the MSigDB v.4.0 database
([87]http://www.broadinstitute.org/gsea/msigdb/index.jsp) and included
all the gene sets in the CP: Kyoto Encyclopedia of Genes and Genomes
collection, which has between 15 and 500 genes (these cutoffs are the
default ones used by the Broad Institute's GSEA software). We retained
all the default parameters except that the null model was based on gene
set randomization to assess statistical significance, rather than on
the default phenotype randomization, in order to accommodate the small
sample size.
Constraint‐Based Modeling of Metabolism
A metabolic network consisting of m metabolites and n reactions can be
represented by an m×n stoichiometric matrix S, where m is the number of
metabolites, n is the number of reactions, and the entry S[ij]
represents the stoichiometric coefficient of metabolite i in reaction
j. A genome‐scale metabolic model imposes mass balance, directionality,
and flux capacity constraints on the space of possible fluxes in the
reactions of the metabolic network through a set of linear equations:
[MATH: S×V=0
:MATH]
(1)
[MATH: Vmin<V<Vmax :MATH]
(2)
V stands for the flux vector for all the reactions in the model. The
exchange of metabolites with the environment is represented as a set of
reactions, enabling a predefined set of metabolites to be either taken
up or secreted from the tissue. The steady‐state assumption represented
in Equation [88](1) constrains the production rate of each metabolite,
making it equal to its consumption rate. Enzymatic directionality and
flux capacity constraints define lower and upper bounds on the fluxes
and are embedded in Equation [89](2). Flux vectors satisfying these
conditions are referred to as feasible steady‐state flux distributions.
Integrative Metabolic Analysis Tool
We used a standard reconstruction of the human metabolic network
because it is thought to be close enough to the murine network but more
comprehensive than the available mouse models.[90]19 In each sample,
gene expression levels were discretized and classified according to the
following 3 levels: high (top 25%), low (bottom 25%), or moderate (the
remaining 50%). We then defined the metabolic state of the 2 conditions
of interest (WT versus Il13ra1‐deficient) by combining the samples for
each of the conditions. A gene was considered highly expressed if it
had been highly expressed in two‐thirds of the samples of the state,
and similarly for lowly expressed genes. The integrative metabolic
analysis tool (iMAT) analysis translates the metabolic state inferred
from gene expression into additional constraints in the metabolic
model. It then predicts a feasible solution space for the specific
condition by solving a mixed integer linear program that finds a
steady‐state flux distribution satisfying stoichiometric and
thermodynamic constraints while maximizing the number of reactions
whose activity is consistent with their expression.[91]20 To study the
metabolic phenotypes in each of the conditions, we constrained the iMAT
agreement level of gene expression and reaction activity to its maximum
in each of the conditions and then found the maximum and minimum
activity of each reaction subject to maintaining that agreement level
(ie, we conducted a flux variability analysis under the additional
constraint of the agreement level). We then conducted Monte‐Carlo
sampling to obtain 2000 flux vectors from the flux space defined by the
reaction activity limits.
Pathway Enrichment Analysis
We tested which functional metabolic pathways were enriched based on
the iMAT‐derived sampling analysis. First, based on the median, we
concluded whether a reaction was up‐ or downregulated. Second, we used
a hypergeometric statistical test (a binomial statistic representing
the likelihood of finding x out of K items in N drawings without
replacement from a group of M objects), to conclude regulation in the
pathway level. Finally, all pathways underwent a false discovery rate
multiple hypothesis correction test.
Statistical Analysis
Statistical analysis was performed with the R statistical package,
version 3.2.2 (R Foundation for Statistical Computing, Vienna,
Austria), for human cardiac gene expression data, and GraphPad Prism
version 6.00 for Windows (GraphPad Software, San Diego, CA) for all the
mouse experiments. All values are expressed as mean±SEM. Kruskal‐Wallis
rank sum test was applied to compare the expression of genes in human
unused donor hearts with different groups of HF. Differences between
means of 2 groups were compared by Student t test or Mann‐Whitney test,
where data were not normally distributed. One‐way ANOVA with Bonferroni
correction was used to compare radial strain and strain rate between
different heart segments. Two‐way repeated‐measures ANOVA with
Bonferroni correction was used to test whether measurements of weight,
glucose tolerance test, insulin tolerance test, and LV function and
structure after TAC operations varied over time among the experimental
groups. Differences were considered significant at a P<0.05.
Results
Expression of Il13ra1 in the Human Heart
First, we aimed to determine whether IL‐13Rα1 is present in the human
heart. We obtained cardiac tissue samples from the right atrium of a
patient with HF and found a robust staining for IL‐13Rα1 (Figure [92]1A
through [93]1C). Next, we analyzed gene expression of tissue from the
hearts of patients with end‐stage HF (n=177) and unused donor hearts
(controls, n=136), obtained by the MAGNet consortium
([94]www.med.upenn.edu/magnet). Remarkably, the expression of the
chains comprising type‐2 IL‐4R, Il13ra1 and Il4ra were downregulated in
the failing hearts of patients with ischemic and dilated
cardiomyopathy, compared with controls (Figure [95]1D and [96]1E). In
contrast, the expression of the unspecific Il2rg subunit, common to
type‐1 IL‐4R and other cytokine receptors, was upregulated in the
failing hearts (Figure [97]1F). The expression of Il13 and Il13ra2 did
not differ between donor and failing hearts, but Il4 was downregulated
in the failing hearts (Table [98]S1). Notably, we found a trend for
increased expression of the Il13 gene in a subset of donor hearts with
a history of diabetes mellitus (Figure [99]1G; Table [100]S2). These
results suggest that signaling via IL‐13Rα1 and the type‐2 IL‐4R could
be implicated in the pathobiology of HF.
Figure 1.
Figure 1
[101]Open in a new tab
Type‐2 interleukin (IL)‐4R signaling is differentially expressed
between human failing and donor hearts. A, Staining for IL‐13Rα1 in a
cardiac tissue biopsy obtained from the right atrium of a 70‐year‐old
heart failure patient during a coronary artery bypass graft surgery
(×100). B, IL‐13Rα1 is present on cardiomyocytes in the human
myocardium (×400). C, Same cardiac tissue sample excluding the primary
antibody (negative control, ×100). D, Reduced Il13ra1 expression in
failing hearts (n=177) compared with unused donor hearts (n=136)
(P=3.94×10^–17, Kruskal‐Wallis test). E, Reduced Il4ra expression in
failing hearts (n=177) compared with unused donor hearts (n=136)
(P=7.89×10^–13, Kruskal‐Wallis test). F, Overexpression of Il2rg in
failing hearts compared to unused donor hearts (n=136) (P=0.004,
Kruskal‐Wallis test). G, Il13 is upregulated in donor hearts (n=136)
with a history of diabetes mellitus (P=0.097, Mann‐Whitney test). CM
indicates cardiomyocyte; LVEF, left ventricular ejection fraction; V,
blood vessel.
Role of IL‐13Rα1 in Cardiac Structure and Function
To further explore the role of IL‐13Rα1 in the heart, we studied hearts
of IL‐13Rα1 whole‐body knockout mice (Il13ra1 ^−/−). These mice harbor
a functional deletion of type‐2 IL‐4R but have an intact type‐1 IL‐4R
and therefore provide an opportunity to distinguish between the roles
of type‐1 and type‐2 IL‐4R in the myocardium.[102]14 We first aimed to
determine whether IL‐13Rα1 is expressed in mouse hearts. Because the
construction of Il13ra1 ^−/− mice included an in‐frame insertion of a
lacZ reporter gene,[103]14 we used β‐galactosidase activity as a
biomarker of Il13ra1 expression. Double staining for both cardiac actin
and β‐galactosidase activity revealed that Il13ra1 is expressed by
cardiomyocytes in normal mouse myocardium (Figure [104]2A and [105]2B,
Figure [106]S1).
Figure 2.
Figure 2
[107]Open in a new tab
Il13ra1 is expressed in mouse hearts and plays an important role in
cardiac function and structure. A, Staining for β‐galactosidase
activity for detection of lacZ reporter in the hearts of Il13ra1 ^−/−
mice reveals myocardial and blood vessel expression of Il13ra1 (×400).
B, Double staining for cardiac actin and β‐galactosidase activity
demonstrates Il13ra1 expression in striated cardiomyocytes (×400).
Echocardiography assessment of cardiac structure and function of 10‐
and 22‐week‐old Il13ra1 ^−/− and WT male mice (Student t test). C,
Decreased diastolic posterior wall thickness, in 22‐week‐old Il13ra1
^−/− mice. D, No difference in diastolic anterior wall thickness
between study groups. E, Increased LVEDD in 22‐week‐old Il13ra1 ^−/−
mice. F, Systolic dysfunction in Il13ra1 ^−/− mice at 10 and 22 weeks
of age. G, Reduced radial strain in Il13ra1 ^−/− mice, with the main
difference involving the posterior segments. H, Reduced radial strain
rate in Il13ra1 ^−/− mice (*P<0.05 and **P<0.01, 1‐way ANOVA). I,
Representative regional strain analysis of LV movement during cardiac
cycle demonstrates marked desynchrony in contractility of Il13ra1 ^−/−
mouse heart compared with WT. AW;d indicates anterior wall diastole;
CM, cardiomyocyte; LV, left ventricle; LVEDD, left ventricular
end‐diastolic diameter; LVEF, left ventricular ejection fraction; MyC,
myocardial; PW;d, posterior wall diastole; V, blood vessel; WT, wild
type.
Next, we sought to define the role of IL‐13Rα1 in the structure and
function of the heart. Using a small animal echocardiography, we found
a significant systolic dysfunction in Il13ra1 ^−/− male mice at the age
of 10 weeks, accompanied by mild LV dilatation and posterior wall
thinning at 22 weeks (Figure [108]2C through [109]2F; Tables [110]S3
and [111]S4). These findings were supported by a speckle‐tracking
strain analysis, a more sensitive method for assessing global and
region‐specific myocardial contractility.[112]21 Radial strain and
strain rate, parameters of myocardial contractility, were significantly
reduced in Il13ra1 ^−/− mice compared with controls (Figure [113]2G and
[114]2H). Furthermore, speckle‐tracking echocardiography revealed
marked desynchronization in contractility of different segments of the
LV (Figure [115]2I), a characteristic of advanced HF. Finally, to
exclude proinflammatory cytokines as a cause of cardiac dysfunction in
the mutant mice, we measured plasma cytokines in WT and Il13ra1 ^−/−
mice. Interestingly, the plasma levels of tumor necrosis factor‐α, a
biomarker of inflammation, which has myogenic and antifibrotic
properties,[116]22 were lower in Il13ra1 ^−/− compared with WT mice.
Levels of other cytokines were similar (Figure [117]S2). Furthermore,
myocardial dysfunction in Il13ra1 ^−/− was exclusive to male but not
female mice (Figure [118]S3). These findings indicate that Il13ra1
deficiency is associated with significant myocardial dysfunction.
STAT3 and STAT6 Mediate Myocardial IL‐13Rα1 Signaling in the Myocardium
Because of the sex‐specific effects of Il13ra1 deficiency on mouse
phenotype, we focused our further studies on male mice. Cytokines and
their receptors exert their transcriptional modifications via
activation of the STAT family of genes. Particularly, STAT3 and STAT6
are implicated in IL‐13/IL‐4 signaling in several cell lines.[119]23,
[120]24 Significantly, our human heart data indicated that Il13ra1 is
correlated with Stat3 and Stat6 gene expression (Figure [121]3A through
[122]3D). To confirm these findings, we analyzed gene expression and
proteins from hearts of WT and Il13ra1 ^−/− mice. Indeed, Stat3a,
Stat3b, and Stat6 gene expression were downregulated in Il13ra1 ^−/−
male mice (Figure [123]3E and [124]3F). Western blot analysis of heart
lysates indicated fewer total and phosphorylated STAT3 and STAT6
proteins in the hearts of Il13ra1 ^−/− than in WT mice
(Figure [125]3G).
Figure 3.
Figure 3
[126]Open in a new tab
Interleukin (IL)‐13Rα1 regulates STAT3 and STAT6 signaling in human and
mouse hearts. Spearman rank correlation between Il13ra1 gene expression
in failing human heart (n=177 samples) and (A) Stat3 expression. B,
Stat6 expression. Spearman rank correlation between Il13ra1 gene
expression in human donor hearts (n=136 samples) and (C) Stat3
expression. D, Stat6 expression. Real‐time quantitative polymerase
chain reaction from hearts of wild‐type and Il13ra1 ^−/−. E, Reduction
in Stat6 in mutant hearts by 41%. F, Reduction in Stat3a and Stat3b
expression by 33% and 27% (Student t test; each experiment was
performed in triplicate). G, western blot demonstrates a reduction in
total and phosphorylated STAT3 and STAT6 proteins in Il13ra1 ^−/− mouse
hearts. H9C2 rat cardiomyoblasts were cultured in 6‐well plates at a
concentration of 4×10^6 cells per well and treated with IL‐13
(10 ng/mL). Protein was extracted at consecutive time points, and
western blot was performed for STAT6 and STAT3 signaling. H, IL‐13
caused STAT6 phosphorylation, which peaked 15 minutes after treatment.
IL‐13 increased total STAT3 α and β proteins, and caused a bimodal
phosphorylation of STAT3 α and β with an early (5 minutes) and late
(6 hours) activation. P indicates phosphorylated; T, total.
To confirm these findings and localize them to cardiomyocytes, we
stimulated cultured rat cardiomyoblasts (H9C2 cell line) with IL‐13
cytokine (10 ng/mL) and demonstrated an increase in STAT6 and STAT3α
and β phosphorylation and total STAT3α and β protein (Figure [127]3H).
Our findings suggest that STAT3 and STAT6 mediate IL‐13Rα1 signaling in
the myocardium. These results are important because STAT3 and to a
lesser extent STAT6 play an important role in cardiac homeostasis,
modulating cell‐to‐cell signaling among the different components of the
myocardium and regulating myocardial repair.[128]25
Pathway Analysis of IL‐13Rα1‐Dependent Gene Regulation in the Mouse Heart
To gain further insight into the mechanism underlying LV dysfunction in
Il13ra1 ^−/− mice, we performed Affymetrix gene array from RNA purified
from hearts of WT and Il13ra1 ^−/− mice. A total of 549 genes were
differentially expressed in Il13ra1 ^−/− compared with WT hearts
(empirical Bayes moderated t test; BH‐adjusted P<0.05). Immune function
and cell adhesion genes were significantly enriched with GO terms (data
not shown). To understand the causes underlying the Il13ra1 ^−/−
phenotype, we conducted GSEA of the microarray data. Briefly, GSEA
tests whether the definition of gene sets, based on external biological
knowledge (eg, known pathways), is collectively up‐ or downregulated
with respect to a given phenotype.[129]18 Here, we tested the
differential expression of 171 gene sets based on Kyoto Encyclopedia of
Genes and Genomes pathways and found that 55 pathways were
differentially expressed between Il13ra1 ^−/− and WT mouse hearts
(false discovery rate q<0.05). Of these, 53 pathways were significantly
downregulated in Il13ra1‐deficient mice hearts, and 2 were
significantly upregulated (Figure [130]4A). As expected, many of the
downregulated gene sets in the Il13ra1‐deficient hearts were related to
inflammation and immune response. However, we found downregulated
pathways that were previously unknown to be controlled by IL‐13Rα1 in
the heart. These new pathways included extracellular matrix (ECM)
receptor interaction, cell cycle, lysosome, focal adhesion, and
apoptosis. The 2 pathways that were significantly enriched in the
mutant hearts were maturity‐onset diabetes mellitus of the young and
neuroactive ligand‐receptor interaction (Figure [131]4B). Thus, our
bioinformatic analysis suggests that IL‐13Rα1 regulates important
pathways in the heart other than the immune system, such as ECM and
glucose metabolism.
Figure 4.
Figure 4
[132]Open in a new tab
Cardiac gene‐array analysis of biological pathways targeted by
interleukin (IL)‐13Rα1 in the heart. A, GSEA reveals altered biological
pathways and processes based on predefined KEGG gene sets. Distribution
of all gene sets based on NES and FDR q‐values corresponding to these
scores. B, Data show enriched KEGG gene sets upregulated in Il13ra1
^−/− mice hearts (green) and 20 most enriched KEGG gene sets
downregulated in Il13ra1 ^−/− mouse hearts (red). FDR indicates false
discovery rate; GSEA, gene set enrichment analysis; KEGG, Kyoto
Encyclopedia of Genes and Genomes; NES, normalized enrichment score.
Reduced ECM Deposition in the Hearts of Il13ra1 ^−/− Mice
The GSEA of microarray data suggests that IL‐13Rα1 regulates cardiac
ECM (Figure [133]5A), which is important for LV structural integrity,
provides a scaffold for myocardial cells, and regulates myocardial
function. Furthermore, the ECM components control communication among
myocardial cells and are critical for myocardial repair and
regeneration. Indeed, loss of ECM leads to disruption of LV structure,
cardiomyocyte slippage, adverse cardiac dilatation, and HF.[134]26 On
the other hand, uncontrolled accumulation of ECM, ie, fibrosis,
facilitates myocardial stiffness and results in diastolic dysfunction
and HF.[135]27 Staining WT and Il13ra1 ^−/− hearts revealed a
significant decrease in perivascular collagen in the hearts of Il13ra1
^−/− mice (Figure [136]5B through [137]5D), which was consistent with
reduced expression of myocardial collagens (collagens I, III) and
thrombospondin‐1 (Figure [138]5E through [139]5G). Together, our
results indicate that IL‐13Rα1 regulates cardiac ECM, and that Il13ra1
deficiency is associated with reduced myocardial ECM.
Figure 5.
Figure 5
[140]Open in a new tab
Interleukin (IL)‐13Rα1 regulates cardiac extracellular matrix
deposition under homeostasis. A, GSEA of KEGG ECM receptor interaction
pathway is enriched with genes that are downregulated in Il13ra1 ^−/−
(n=3) compared to WT (n=4) hearts. The bars represent genes included in
the pathway, which are sorted by their differential expression from the
most down‐regulated in Il13ra1 ^−/− (left) to the most up‐regulated
ones (right). The green curve corresponds to the GSEA enrichment score.
B, Representative perivascular collagen deposition in WT heart (Masson
trichrome). C, Representative perivascular collagen deposition in an
Il13ra1 ^−/− heart (Masson trichrome). D, Reduced perivascular collagen
deposition in Il13ra1 ^−/− hearts compared to WT (Student t test). E,
Real‐time quantitative polymerase chain reaction from hearts of WT and
Il13ra1 ^−/−. Reduced Col3a1 expression in Il13ra1 ^−/− hearts. F,
Reduced Col1a1 expression in Il13ra1 ^−/− hearts. G, Reduced Thbs1
expression in Il13ra1 ^−/− hearts (Student t test; each experiment was
performed in triplicate). H, Schematic outline of the TAC model
protocol performed. I, Reduced posterior wall hypertrophy 3 weeks after
TAC in Il13ra1 ^−/− females as assessed by echocardiography (*P<0.05
and **P<0.01, 2‐way ANOVA). ECM indicates extracellular matrix; FDR,
false discovery rate; GSEA, gene set enrichment analysis; KEGG, Kyoto
Encyclopedia of Genes and Genomes; NES, normalized enrichment score;
TAC, transverse aortic constriction; WT, wild type.
Next, we sought to determine the role of IL‐13Rα1 in cardiac fibrosis.
Pressure overload exposes the myocardium to significant stress, which
is characterized by cardiomyocyte hypertrophy, fibrosis, apoptosis, and
adverse LV remodeling.[141]28 To simulate the effect of pressure
overload, we induced TAC in 10‐week‐old female Il13ra1‐deficient and WT
mice (Figure [142]5H). The selection of female mice was based on our
finding that Il13ra1 ^−/− leads to spontaneous LV dysfunction in male
but not in female mice. Three weeks after TAC, Il13ra1 ^−/− female mice
developed less posterior wall hypertrophy compared with WT controls
(Figure [143]5I), with no difference in cardiac systolic function
(Table [144]S5). Significantly, staining the hearts for collagen
deposition revealed that the Il13ra1 ^−/− myocardium was resistant to
fibrosis (Figure [145]6A and [146]6B). Moreover, Il13ra1 ^−/− displayed
slightly less cardiomyocyte hypertrophy (Figure [147]6C and [148]6D),
suggesting that the main protective effect against cardiac hypertrophy
was mediated by inhibition of ECM deposition. These structural
differences in the Il13ra1 ^−/− hearts were accompanied by a reduction
in the expression of genes regulating fibrosis and hypertrophy,
including transforming growth factor β (Tgfb), matrix metalloproteinase
(Mmp) 12, tissue inhibitor of metalloproteinase (Timp) 1 and Mmp9
(Figure [149]6E through [150]6H). Consistent with these findings in
mice, we found a positive correlation between Il13ra1 and Tgfb and
Timp1 expression in failing human hearts (Figure [151]S4). Overall, our
results indicate that Il13ra1 deficiency in female mice reduces
fibrosis and hypertrophy during pressure overload.
Figure 6.
Figure 6
[152]Open in a new tab
Interleukin (IL)‐13Rα1 regulates cardiac fibrosis under pathological
conditions. A, Representative images of cardiac fibrosis in WT and
Il13ra1 ^−/− hearts 3 weeks after TAC. B, Reduced cardiac fibrotic area
in Il13ra1 ^−/− compared to WT hearts 3 weeks after TAC (Mann‐Whitney
rank sum test). C, Representative images of cardiomyocyte hypertrophy
in WT and Il13ra1 ^−/− hearts 3 weeks after TAC (WGA). D, Reduced
cardiomyocyte hypertrophy in Il13ra1 ^−/− compared to WT hearts 3 weeks
after TAC (Student t test). Reduced gene expression of key cardiac
remodeling‐associated genes in Il13ra1 ^−/− compared to WT hearts after
TAC. E, Tgfb. F, Mmp12. G, Timp1. H, Mmp9 (Student t test; each
experiment was performed in triplicate). TAC indicates transverse
aortic constriction; WGA, wheat germ agglutinin; WT, wild type.
Il13ra1 Deficiency Leads to Metabolic Abnormalities
Both our human gene expression analysis and the enrichment of
Maturity‐onset diabetes mellitus of the young in GSEA of Il13ra1 ^−/−
mice hearts, suggest a possible link between IL‐13Rα1 signaling and
diabetic hearts. Indeed, Il13ra1 ^−/− mice displayed several systemic
metabolic abnormalities including increased weight and fat gain, as
well as mild abnormalities in glucose metabolism (Figure [153]S5). To
determine the effect of IL‐13Rα1 on cardiac metabolism, we analyzed the
data obtained from Il13ra1 ^−/− and WT mice cardiac gene arrays using
genome scale metabolic modeling, which is a constraint‐based
computational approach that has been widely used to study human
metabolism in health and in disease.[154]29 We utilized iMAT, which
integrates gene expression levels measured under different conditions
to predict the most likely distribution of metabolic enzyme fluxes.
Importantly, iMAT can be used to predict not only the activity of a
certain metabolic reaction and its direction but also (within a genome
scale metabolic context) all posttranscriptional properties not
accessible by gene expression alone.[155]20 Pathway enrichment analysis
revealed a significant downregulation of genes related to metabolic
reactions associated with glycolysis, the tricarboxylic acid cycle, and
upregulation of genes of the pyruvate metabolism pathway in the hearts
of Il13ra1‐deficient mice. More specifically, we found an increase in
the core reactions of pyruvate metabolism involved in the production of
advanced glycosylation end products such as methylglyoxal,
lactoylglutathione, and lactaldehyde (Figure [156]7A through [157]7C;
Tables [158]S6 through [159]S8). These advanced glycosylation end
products are increased in the plasma and tissues of diabetic patients
and are believed to contribute to micro‐ and macrovascular
complications by promoting cellular apoptosis, inflammation, and ECM
crosslinking and expression.[160]30 Thus, our results indicate that
Il13ra1 deficiency dysregulates glucose metabolism in the heart.
Figure 7.
Figure 7
[161]Open in a new tab
Interleukin (IL)‐13Rα1 in myocardial metabolism. A, Schematic
description of the input used to simulate cardiac Il13ra1 ^−/−
metabolism using GSMM. High‐throughput data from cardiac tissue of
wild‐type (n=4) and Il13ra1 ^−/− (n=3) mice, and a GSMM were used as
inputs. The iMAT algorithm integrates the gene‐expression data with a
genome scale model in order to find a feasible solution space for
metabolic flux distribution, thus enabling description of
posttranscriptional modifications not shown by gene expression alone.
B, Pathway enrichment analysis over the set of common reactions
upregulated or downregulated in Il13ra1 ^−/− cardiac metabolism,
computed via hypergeometric test and corrected for multiple hypotheses
using a false discovery rate <0.05. C, Guided by changes in
gene‐expression iMAT presents an aberrant energy metabolism in
Il13ra1‐deficient hearts, consisting of a decrease in glycolysis and
tricarboxylic acid cycle, with the upregulation of pyruvate. GSMM,
genome scale metabolic modeling; iMAT, integrative metabolic analysis
tool; TCA cycle, tricarboxylic acid cycle. Glycolytic metabolites: GLC,
glucose; G6P, glucose 6‐phosphate; F6P, fructose 6‐phosphate; FDP,
fructose 1,6‐bisphosphate; DHAP, dihydroxyacetone phosphate; G3P,
glyceraldehyde 3‐phosphate; 1,3BPG, 1,3 bisphosphoglycerate; 3PG,
3‐phosphoglycerate; 2PG, 2‐phosphoglycerate; PEP, phosphoenolpyruvate;
PYR, pyruvate. TCA cycle metabolites: CIT, citrate; ICIT, isocitrate;
AKG, α‐ketoglutarate; SUCCOA, succinyl‐CoA; SUC, succinate, FUM,
fumarate; MAL, malate; OAA, oxaloacetate. Advance glycosylation end
products: MTHGXL, methylglyoxal; LACG, lactoylglutathione; LALD,
lactaldehyde.
Discussion
Our study provides new evidence that IL‐13Rα1 signaling plays an
essential regulatory role in myocardial homeostasis under physiological
and pathological conditions. We demonstrated the pivotal role of
IL‐13Rα1 in the heart by detailed bioinformatic analysis of myocardial
samples from patients with and without HF and correlated them with
Il13ra1‐deficient mice. We demonstrated that IL‐13Rα1 is linked to
pathways associated with ECM deposition and glucose metabolism. Il13ra1
and Il4ra genes are downregulated in the hearts of human patients with
end‐stage HF. Il13ra1 deficiency in male mice causes marked LV
dysfunction, which is associated with impaired deposition of myocardial
ECM and abnormal glucose metabolism. We showed that IL‐13Rα1 regulates
downstream activation of STAT3 and STAT6 in cardiomyocytes and that its
deficiency attenuates cardiac fibrosis in TAC‐operated female mice.
Together, our results suggest that the immune system, by IL‐13Rα1
signaling, plays a key role in cardiac homeostasis and failure in
humans and mice.
The role of IL‐4 and IL‐13 signaling in myocardial disease has been
controversial. IL‐4 and IL‐13 have been shown to exert powerful
profibrotic effects within the heart, liver, intestines, and
lungs[162]7, [163]8, [164]9 and have been implicated in various chronic
fibrotic diseases.[165]31 In contrast, Il13‐deficient male mice display
increased cardiac fibrosis in models of myocarditis and myocardial
infarction.[166]10, [167]11 Based on our findings, it may now be
possible to resolve these seemingly contradictory findings. Because
IL‐4 and IL‐13 have overlapping signaling via type‐2 IL‐4R, deletion of
1 cytokine may lead to increased signaling by the other cytokine and to
a paradoxical increase in fibrosis and tissue damage. Indeed,
Il13‐deficient mice with myocarditis had increased levels of
IL‐4.[168]10 On the other hand, the Il13ra1‐deficient mouse, which we
used in our experiments, does not respond to either IL‐4 or IL‐13 via
the type‐2 IL‐4R. Thus, we were able to demonstrate that type‐2 IL‐4R
signaling plays a pivotal protective role in the myocardium.
Interestingly, Il13ra1 deficiency in female mice reduces fibrosis
during pressure overload. The significance of this observation is
unclear. Although fibrosis and scar formation are essential responses
to acute myocardial injury, uncontrolled fibrosis may lead to adverse
cardiac remodeling and HF.[169]27
Our results also support the paradigm of STAT3 as a key mediator of
myocardial structure and function regulating cardiac ECM deposition and
hypertrophy. Similar to our findings in Il13ra1‐deficient mice, earlier
studies have shown that STAT3 has sex‐specific effects in the
myocardium. Male but not female cardiomyocyte‐restricted
STAT3‐deficient mice develop impaired cardiac function, ventricular
remodeling and dilatation with advancing age.[170]32, [171]33 Moreover,
cardiomyocyte STAT3 inhibition resulted in decreased collagen synthesis
in cultured cardiac fibroblasts and attenuated pressure
overload–induced cardiac fibrosis and hypertrophy.[172]34 Overall,
STAT3 seems to be a key mediator of IL‐13Rα1 signaling in the heart.
Based on our findings and previous reports,[173]6, [174]35, [175]36 we
suggest a dual role for IL‐13Rα1 in both myocardial homeostasis and
repair. IL‐13Rα1 signaling is critical for cardiac ECM integrity under
physiological conditions, but continuous, uncontrolled stimulation of
IL‐13Rα1/STAT3 signaling during chronic cardiac stress is maladaptive
and may induce excessive ECM accumulation, cardiac fibrosis and HF. Our
findings are in line with previous reports, suggesting that IL‐13
signaling has healing, reparative effects on tissues, such as skeletal
muscle and the lungs.[176]37, [177]38
In the present report, we show that IL‐13Rα1–deficient mice exhibited
mild metabolic abnormalities and altered myocardial energy metabolism
consistent with the diabetic heart. However, these mice are protected
from myocardial fibrosis, which is often associated with diabetic
cardiomyopathy. The effect of immune stimuli on metabolic pathways has
recently been recognized.[178]39 For example, stimulation of
macrophages with IL‐4 can induce oxidative phosphorylation and M2
polarization, whereas activation of cells through pattern recognition
receptors such as Toll‐like receptor 4 (TLR4) induces HIF1α expression
and promotes glycolysis, and M1 polarization.[179]39 Inflammatory
macrophages (M1) use glycolysis, the tricarboxylic acid cycle, the
pentose phosphate pathway, fatty acid synthesis, and amino acid
metabolism to proliferate and to support the production of inflammatory
cytokines.[180]39 M2 macrophages, which exhibit a more
anti‐inflammatory phenotype, use the tricarboxylic acid cycle, fatty
acid oxidation and arginine flux into the arginase pathway.[181]39 Our
results are complementary to recent publications showing that IL‐13
plays an important role in glucose metabolism in skeletal muscle cells
and the liver. Jiang et al showed that IL‐13 increases glucose
oxidation in skeletal muscle myotubes from diabetic and nondiabetic
patients.[182]40 IL‐13 deficiency in mice leads to increased weight
gain, hyperglycemia, and hepatic insulin resistance due to the
dysregulation of the IL‐13Rα1/STAT3 axis in hepatocytes.[183]41
Together, our findings could be relevant for the development of new
therapies for diabetic cardiomyopathy.
We are aware of several limitations in our work. First, we analyzed
human cardiac biopsies, which contained various myocardial cells such
as cardiomyocytes, fibroblasts, and endothelial cells. Thus, our
findings cannot be solely attributed to a deficiency of the receptor in
cardiomyocytes. It is possible that loss of Il13ra1 in fibroblasts also
plays a role in the development of cardiac dysfunction. Further studies
using cell‐specific Il13ra1 deletion are required to localize our
results to a specific cell type. Second, to describe the effect of
IL‐13Rα1 on cardiac metabolic pathways, we used a constraint‐based
model, a widely used computational approach for studying metabolism on
a genome scale, which has been implicated in various tissues and
conditions.[184]20, [185]42, [186]43 A validation at the protein level
could strengthen our new findings. Third, to generate Il13ra1 ^−/−
mice, the Il13ra1 gene was replaced by a cassette, which consists of a
β‐galactosidase enzyme gene and a neomycin resistance gene.[187]14 The
construct deletes amino acids 15 824 through 22 414 of IL‐13R1
contained in exons 2 to 4 of the gene. Although replacement of the
endogenous gene at a given locus can theoretically alter
transcriptional patterns in any transgenic mouse model, this unwanted
effect has not been described in previous publications using the
Il13ra1‐deletion/lacz reporter mouse model.[188]38, [189]44, [190]45,
[191]46
In summary, our human data, together with results observed from mouse
studies, provide new insights into the pleiotropic roles of IL‐13Rα1 in
the heart during normal conditions and stress. A deeper understanding
of the role of IL‐13Rα1 in heart disease may ultimately pave the way
for the development of effective treatment for adverse heart remodeling
and failure.
Sources of Funding
This project was partially supported by a grant from the Israeli
National Nanotechnology Initiative and Helmsley Charitable Trust for a
focal technology area on Nanomedicines for Personalized Theranostics
(Leor and Blum).
Disclosures
None.
Supporting information
Table S1. Differential Expression Analysis of IL‐13/IL‐4 Cytokines and
Receptor Subunits in Failing (n=177) and Donor Human Hearts (n=136)
Obtained From the MAGNet Consortium
Table S2. Differential Expression Analysis of IL‐13/IL‐4 Cytokines and
Receptor Subunits in Donor Hearts With a History of Diabetes Obtained
From the MAGNet Consortium
Table S3. Echocardiography Assessment of Cardiac Structure and Function
of 10‐Week‐Old Il13ra1‐Deficient and Wild‐Type Male Mice
Table S4. Echocardiography Assessment of Cardiac Structure and Function
of 22‐Week‐Old Il13ra1‐Deficient and Wild‐Type Male Mice
Table S5. Echocardiography Assessment of Cardiac Structure and Function
of Il13ra1‐Deficient and Wild‐Type Female Mice After Transverse Aortic
Constriction
Table S6. Glycolysis Cycle Reactions Change in Il‐13ra1 ^−/− Hearts
Compared to Wild‐Type Controls, According to Integrative Metabolic
Analysis Tool Increased Flux: “↑”, Decreased Flux: “↓”
Table S7. Tricarboxylic Acid Cycle Reactions Change in Il‐13ra1 ^−/−
Hearts Compared to Wild‐Type Controls, According to Integrative
Metabolic Analysis Tool Increased Flux: “↑”, Decreased Flux: “↓”
Table S8. Pyruvate Reactions Change in Il‐13ra1 ^−/− Hearts Compared to
Wild‐Type Controls, According to Integrative Metabolic Analysis Tool
Increased Flux: “↑”, Decreased Flux: “↓”
Figure S1. Staining for β‐galactosidase activity for detection of lacZ
reporter in the hearts of Il13ra1 ^−/− mice, showing that the Il13ra1
gene is expressed in all parts of mice myocardium with no visible
differences among base, middle, and apex sections.
Figure S2. Reduced plasma tumor necrosis factor in Il13ra1‐deficient
mice.
Figure S3. No difference in cardiac structure and function between
10‐week‐old Il13ra1 ^−/− and wild‐type female mice as assessed by
echocardiography.
Figure S4. Spearman rank correlation between Il13ra1 gene expression
and cardiac remodeling‐associated genes in human failing hearts (n=177)
obtained from the MAGNet consortium.
Figure S5. Il13ra1 ^−/− mice display metabolic abnormalities.
[192]Click here for additional data file.^ (462.7KB, pdf)
Acknowledgments