Left ventricular hypertrophy in mice with a cardiac-specific overexpression of interleukin-1.

Abstract

Recent studies have identified the importance of proinflammatory cytokines in the development of left ventricular (LV) hypertrophy. However, the precise role of interleukin-1 (IL-1), one of the major proinflammatory cytokines, in the myocardium is not fully understood. In this study, we investigated the pathophysiological consequences of cardiac expression of IL-1 in vivo. We generated mice with a cardiac-specific overexpression of human IL-1alpha. We then analyzed their heart morphology and functions. Histological and echocardiographic analyses revealed concentric LV hypertrophy with preserved LV systolic function in the mice. Our results suggest that myocardial expression of IL-1 is sufficient to cause LV hypertrophy.

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doi: 10.1152/ajpheart.00269.2005
291:H176-H183, 2006. First published 10 February 2006;
Am J Physiol Heart Circ Physiol
Isoda, Kohji Miyazaki and Fumitaka Ohsuzu
Kenichiro Nishikawa, Mikoto Yoshida, Masatoshi Kusuhara, Norio Ishigami, Kikuo
cardiac-specific overexpression of interleukin-1
Left ventricular hypertrophy in mice with a
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Left ventricular hypertrophy in mice with a cardiac-specific overexpression
of interleukin-1
Kenichiro Nishikawa, Mikoto Yoshida, Masatoshi Kusuhara,
Norio Ishigami, Kikuo Isoda, Kohji Miyazaki, and Fumitaka Ohsuzu
Internal Medicine-1, National Defense Medical College, Saitama, Japan
Submitted 18 March 2005; accepted in final form 30 January 2006
Nishikawa, Kenichiro, Mikoto Yoshida, Masatoshi Kusu-
hara, Norio Ishigami, Kikuo Isoda, Kohji Miyazaki, and Fumi-
taka Ohsuzu. Left ventricular hypertrophy in mice with a cardiac-
specific overexpression of interleukin-1. Am J Physiol Heart Circ
Physiol 291: H176–H183, 2006. First published February 10,
2006;doi:10.1152/ajpheart.00269.2005.—Recent
identified the importance of proinflammatory cytokines in the devel-
opment of left ventricular (LV) hypertrophy. However, the precise
role of interleukin-1 (IL-1), one of the major proinflammatory cyto-
kines, in the myocardium is not fully understood. In this study, we
investigated the pathophysiological consequences of cardiac expres-
sion of IL-1 in vivo. We generated mice with a cardiac-specific
overexpression of human IL-1?. We then analyzed their heart mor-
phology and functions. Histological and echocardiographic analyses
revealed concentric LV hypertrophy with preserved LV systolic
function in the mice. Our results suggest that myocardial expression of
IL-1 is sufficient to cause LV hypertrophy.
studieshave
myocardium; left ventricular systolic function; cardiovascular disease
INTERLEUKIN-1 (IL-1), one of the proinflammatory cytokines,
exerts a wide variety of effects, such as an immune response,
cell proliferation, and cell death, on many different cell types
(4). An elevated expression of IL-1 in hearts has been revealed
in a variety of cardiovascular diseases, including myocarditis
(22), myocardial infarction (18), and congestive heart failure
(13, 18, 22). IL-1 is also highly expressed in the hypertrophied
myocardium in vitro and in vivo (21).
We previously reported that mice with a ubiquitous overex-
pression of human IL-1? (hIL-1?), which were originally
generated as a mouse model of rheumatoid arthritis, unexpect-
edly showed prominent left ventricular (LV) hypertrophy (7).
The study did not, however, define whether the specific ex-
pression of IL-1? in the myocardium caused the LV hypertro-
phy, because the systemic expressions might induce the LV
hypertrophy. Therefore, to elucidate the role of myocardial
expression of IL-1? in the myocardium, we developed trans-
genic (Tg) mice with a constitutive overexpression of hIL-1?
restricted to cardiomyocytes under the control of ?-myosin
heavy chain (MHC) promoter.
The Tg mice exhibited concentric LV hypertrophy with a
preserved LV systolic function. Our data suggest that cardiac
expression of IL-1 thus is sufficient to cause LV hypertrophy.
METHODS
Generation of the Tg construct. A mature type of hIL-1? cDNA
(Immunex) was derived from a plasmid of the chicken ?-actin
promoter containing cytomegalovirus enhancer-hIL-1?. The plasmid
had been used for generation of mice with a ubiquitous overexpres-
sion of hIL-1? (17). The fragment containing hIL-1? cDNA was
excised by Bgl II/Xba I (770 bp). The terminal fragment was blunted
by the Klenow fragment (Takara Bio). The plasmid containing the
?-MHC promoter construct (pGL-?-MHC-SVpA) (11) was kindly
donated by Dr. Arthur M. Feldman. The fragment containing hIL-1?
cDNA was inserted into the plasmid that was digested by Hind III.
Finally, we generated the Tg construct pGL-?-MHC-hIL-1?-SVpA
(9.88 kbp). BamH I digestion produced a linear 6.96-kbp fragment
used for microinjection into mouse embryos (Fig. 1).
All studies were carried out according to the protocols approved by
the National Defense Medical College Board for Studies in Experi-
mental Animals.
Generation and identification of Tg mice. C57BL/6N mice were
used for the generation of Tg mice. The Tg mice were identified by
PCR with primers as follows: 5?-CAC ATA GAA GCC TAG CCC
AC-3? (forward) and 5?-TTC CAC CAC TGC TCC CAT TC-3?
(reverse). The PCR conditions were as follows: 30 cycles of denatur-
ation at 95°C for 10 s, annealing at 62°C for 15 s, and extension at
72°C for 90 s.
Quantitative PCR was performed to estimate the DNA copy num-
ber of transgene loci in each line. The primers specific for hIL-1? and
an internal normalizer gene, RNase P1, were used in multiplex PCR
on a PCR thermocycler (TaqMan 7700, Applied Biosystems, Foster
City, CA) following the manufacturer’s recommended conditions.
The following primers were used: hIL-1? [5?-TGT ATG TGA CTG
CCC AAG ATG AA-3? (forward) and 5?-CCT GTG ATG GTT TTG
GGT ATC TC-3? (reverse)] and RNase P1 [5?-CAC TTA CTG GTC
TGT GAG AAA TCC A-3? (forward) and 5?-TGC GTC TCC ACG
AGA TGC T-3? (reverse)]. The data are shown as the change in cycle
threshold (?Ct), which can be converted to the relative transgene
DNA copy number as follows: 2(??Ct).
Northern blot analysis. Total RNA was purified from quick-frozen
cardiac tissue specimens from 12-wk-old male mice using the RNeasy
protocol (Qiagen). Total RNA (5–10 ?g) was electrophoresed in 1.2%
agarose-formaldehyde gel, blotted onto nylon membranes (Roche
Molecular Biochemicals), and UV cross-linked. Hybridization was
performed overnight at 42°C using a digoxigenin-labeled probe gen-
erated with a digoxigenin DNA labeling kit (Roche Molecular Bio-
chemicals). The probe was a 660-bp Hind III/Hinc II fragment from
hIL-1? cDNA (Immunex). For visualization of digoxigenin-labeled
IL-1? cDNA, the membranes were washed and incubated with alka-
line phosphatase-labeled anti-digoxigenin Fab fragments and then in a
solution of alkaline phosphatase substrate (4-nitro blue tetrazolium
chloride and bromo-4-chloro-3-indolyl phosphate). The hybridization
signals were detected using a scanner (Epson). The mRNA levels
were qualified by 28S and 18S ribosomal RNA ethidium bromide
staining.
Determination of cytokine levels. The myocardial cytokine levels
were determined using a specific ELISA of hIL-1? (Japan Immunore-
Address for reprint requests and other correspondence: F. Ohsuzu, Internal
Medicine-1, National Defense Medical College, 3-2 Namiki Tokorozawa
Saitama, 359-0042, Japan.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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search Laboratories), mouse IL-1? (Biosource International), mouse
IL-1? and mouse TNF-? (Endogen), and mouse IL-6 (R & D
Systems). At 12 wk of age, male mice were killed with an overdose
of pentobarbital sodium (200 mg/kg ip), and the hearts were removed
and homogenized in ice-cold tissue protein-extraction reagent (T-PER,
Pierce Biotechnology) with protease inhibitors (Sigma-Aldrich). The
homogenized solutions were spun at 10,000 rpm for 20 min at 4°C.
The supernatants for the total protein level of 100 ?g/ml were applied
to the ELISA plate. The serum levels of hIL-1? were also analyzed
using a specific ELISA.
Western blot analysis. The protein samples were prepared from
heart tissue specimens from 12-wk-old male mice. Ventricular tissue
specimens were homogenized in ice-cold tissue protein-extraction
reagent containing protease inhibitors (Sigma-Aldrich). The extracts
of 50 ?g of protein were separated by SDS-PAGE, and the proteins
were electrophoretically transferred to polyvinylidene difluoride fil-
ters. The filters were washed and incubated with primary antibody:
rabbit polyclonal anti-nuclear factor (NF)-?B p65 antibody (Abcam),
anti-phosphorylated p65 antibody (Ser536) and anti-phosphorylated
p38 antibody (Cell Signaling Technology), and anti-p38 MAPK
antibody (Santa Cruz Biotechnology). The filters were washed and
then incubated with horseradish peroxidase-conjugated anti-rabbit
secondary antibody (Amersham Pharmacia Biotech). The signals were
visualized using the ECL Plus system (Amersham Pharmacia Bio-
tech). Densitometry was done using an image analysis software (Scion
Image, Scion) on a computer.
Histological analysis. At 12 wk of age the mice were killed with an
overdose pentobarbital sodium (200 mg/kg ip). The hearts were
removed and rinsed well in ice-cold normal saline and then fixed in
4% paraformaldehyde in PBS. The tissue blocks were embedded in
paraffin for sectioning (7 ?m). Tissue sections were stained with
hematoxylin-eosin or Masson’s trichrome. The regions in the left and
right ventricular free wall were used for the analysis. The myocyte
cross-sectional area was measured in the sections stained with Mas-
son’s trichrome as reported previously (5).
Myocardial ultrastructural analysis. An ultrastructural analysis of
morphological changes in Tg mice at 12 wk of age was performed
after the heart was fixed in conventional fixing solutions [4% (vol/vol)
paraformaldehyde and 1% (vol/vol) glutaraldehyde in PBS]. All
samples were processed after fixation and dehydration for embedding
in epoxy resin. Next, ultrathin sections from a midportion of the LV
free wall were stained with uranyl and lead acetate and then examined
by transmission electron microscopy (TEM; Nihon Electric).
Echocardiography. At 12 wk of age, male mice were anesthetized
intraperitoneally with a mixture of ketamine (100 mg/kg) and xylazine
(2.5 mg/kg), and their chests were shaved. Next, transthoracic echo-
cardiography was performed using a Hewlett-Packard Sonos 7500
ultrasound system equipped with a 15-MHz linear transducer (Phil-
ips). M-mode studies were analyzed as reported previously (7).
Measurement of blood pressure and heart rate. Arterial blood
pressure and heart rate were measured in conscious mice by the tail
cuff method.
Detection of gene expression. Fetal gene expression was detected
by purification of total RNA from cardiac ventricular tissue from
12-wk-old male mice with use of the RNeasy protocol (Qiagen).
First-strand cDNA was synthesized using RT with random hexamers
from 1 ?g of total RNA in a 20-?l reaction volume according to the
manufacturer’s protocol (Perkin-Elmer); then one-tenth of the result-
ing RT product was applied to 25 ?l of PCR solutions containing 1.5
mM Mg2?, 0.2 ?M primers, 0.2 mM dNTPs, and 2.5 U/100 ?l Taq
DNA polymerase (Perkin-Elmer). PCR conditions for the various
primers were as follows: 32 cycles of denaturation at 95°C for 15 s,
annealing at 58°C for 15 s, and extension at 72°C for 60 s for atrial
natriuretic factor [ANF, 5?-AAG AGG GCA GAT CTA TCG GA-3?
(forward) and 5?-TTG GCT TCC AGG CCA TAA TTG-3? (reverse)];
30 cycles of denaturation at 95°C for 15 s, annealing at 61°C for 15 s,
and extension at 72°C for 60 s for B-type natriuretic peptide [BNP,
5?-CAG CTC TTG AAG GAC CAA GG-3? (forward) and 5?-AAG
AGA CCC AGG CAG AGT CA-3? (reverse)]; 30 cycles of denatur-
ation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at
72°C for 60 s for ?-MHC [5?-AGA GGA GAA GGC CAA GAA
GG-3? (forward) and 5?-GCA GCC GCA TTA AGT TCT TC-3?
(reverse)]; 32 cycles of denaturation at 95°C for 15 s, annealing at
62°C for 15 s, and extension at 72°C for 60 s for ?-MHC [5?-cag ctc
ttg aag gac caa gg-3? (forward) and 5?-aag aga ccc agg cag agt ca-3?
(reverse)]; 30 cycles of denaturation at 95°C for 15 s, annealing at
61°C for 15 s, and extension at 72°C for 60 s for sarco/endoplasmic
reticulum Ca2?-ATPase [SERCA2a, 5?-AAG CTAT GGG AGT GGT
TGG TG-3? (forward) and 5?-ACA ACC AAG GGT CTC CAC
AG-3? (reverse)]; 30 cycles of denaturation at 95°C for 15 s, annealing
at 60°C for 15 s, and extension at 72°C for 60 s for ryanodine
Ca2?-release channels [CRC, 5?-AGG TGG TGC CGT ATC AGT
TC-3? (forward) and 5?-CAT CCA CAG CCT GGT AGG TT-3?
(reverse)]; and 30 cycles of denaturation at 95°C for 30 s, annealing
at 62°C for 15 s, and extension at 72°C for 120 s for GAPDH [5?-GGT
CCA GGG TTT CTT ACT CC-3? (forward) and 5?-AAG CCC ATC
ACC ATC TTC CA-3? (reverse)]. Densitometry was done using an
image analysis software (Scion Image) on a computer. mRNA levels
were corrected for the mRNA level of GAPDH and are expressed as
percentage of non-Tg mice.
Gene expressions of adhesion molecules were detected using a
quantitative PCR technique. RT was performed with TaqMan RT
reagent (Applied Biosystems) with random oligonucleotide primers.
The conditions were as follows: 25°C for 10 min, 48°C for 30 min,
and 95°C for 5 min. TaqMan probes and primer sets for intercellular
adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1
(VCAM-1), platelet-endothelial cell adhesion molecule 1 (PECAM-
1), and ?-actin mRNAs are commercially available and were pur-
chased from Applied Biosystems. Amplification was performed with
a sequence detection system (Prism 7700, Applied Biosystems) under
the following conditions: 50°C for 2 min, 95°C for 10 min, and 40
cycles at 95°C for 15 s and 60°C for 1 min. PCR results were analyzed
with Sequence Detector version 1.6 (Applied Biosystems). To correct
for variations in the total RNA content and unequal RT efficiency,
ICAM-1, VCAM-1, and PECAM-1 quantities were normalized to the
amount of ?-actin mRNA.
Statistical analysis. Values are means ? SE. Two-tailed Student’s
t-test and the ?2test were used for statistical analysis. P ? 0.05 was
considered to be statistically significant.
RESULTS
Generation of Tg mice. After three founder lines (Tg17,
Tg50, and Tg71) were obtained, quantitative PCR was used to
identify the homozygous members of each line. Mean ?Ct
values were ?4.2, ?3.0, and ?5.1 for Tg17, Tg50, and Tg71,
respectively. Because the most prominent increase in heart
weight-to-body weight ratio was observed in the Tg71 line,
Fig. 1. Transgene construct. Mouse ?-myosin heavy chain (?-MHC) promoter
was used as a cardiac-specific promoter to control expression of mature-type
human IL-1? (hIL-1?).
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male mice of the Tg71 line were used in this study. The Tg
mice show normal growth and normal gross appearance, and
the 1-yr survival rate of the Tg mice is similar to that of the
non-Tg mice (?95% for each group).
mRNA expressions of hIL-1?. A Northern blot analysis
demonstrated a high level of hIL-1? mRNA in the ventricular
tissue specimens of the Tg mice (Fig. 2A). Signals were either
weak or absent in other organs, such as the lung and spleen
(Fig. 2B).
Cytokine protein expressions. The hIL-1? levels of ventric-
ular tissue specimens in Tg mice were 73 pg/100 ?g protein,
on average, as determined by a specific ELISA (n ? 6 per
group; Table 1). However, the serum level of hIL-1? was too
low to be detected. There were no significant differences in
mouse IL-1? (too low to be detected in both groups), IL-1?
(7.6 ? 0.7 and 8.1 ? 0.7 pg/100 ?g protein, respectively), and
TNF-? (1.1 ? 0.1 and 1.3 ? 0.1 pg/100 ?g protein, respec-
tively) in ventricular tissue specimens between the Tg and
non-Tg mice (n ? 6 per group; Table 1). The protein expres-
sion of IL-6, a downstream cytokine of IL-1, in the ventricular
tissue samples was greater in Tg than in non-Tg mice (20.6 ?
8.2 vs. 2.0 ? 1.1 pg/100 ?g protein, P ? 0.05), suggesting that
the Tg-derived hIL-1? was active as a proinflammatory cyto-
kine in mice.
Signal activation. IL-1 is known to activate the signaling
pathways of p38 MAPK as well as NF-?B (15). We thus
examined the signal activations of p38 MAPK and p65 NF-?B,
which are important intracellular signaling molecules in in-
flammation, to estimate whether transgene-derived hIL-1? is
biologically active as a proinflammatory cytokine in mice,
because the transgene was of human origin. The ratio of
phosphorylated p38 to p38 (Fig. 3A) and phosphorylated p65 to
p65 (Fig. 3B) was significantly higher (P ? 0.05) in Tg than in
non-Tg mice (1,350 ? 13 vs. 148 ? 4%, n ? 4 per group).
Heart morphology. Postmortem weight measurements dem-
onstrated a significant increase in heart weight-to-body weight
ratio in the Tg mice compared with the non-Tg mice at 6 and
12 wk of age (Table 2).
The tissue sections stained for hematoxylin-eosin revealed
an increase in the thickness of the ventricular free wall from the
Tg mice (Fig. 4A). A histological examination of hearts from
the Tg mice revealed a normal linear arrangement of myofi-
brils. No obvious myofibrillar disarray or prominent interstitial
infiltrating cells were observed in any of the sections from the
Tg mice (Fig. 4B).
The cross-sectional area of the ventricular myocytes mea-
sured on histological sections significantly increased by 20% in
the Tg mice compared with the non-Tg mice (280 ? 7 vs.
230 ? 6 ?m2, P ? 0.05; Fig. 5).
Heart ultrastructural analysis. An ultrastructural TEM study
revealed that mitochondria were rounded and greater in num-
ber and T tubules were enlarged in the Tg mice compared with
the non-Tg mice. The sarcomere structure of the Tg mice
appeared to be normal (Fig. 6).
Cardiac functions and hemodynamic parameters. An echo-
cardiographic analysis confirmed a decrease in LV end-dia-
stolic diameter in the Tg mice compared with the non-Tg mice
(3.18 ? 0.06 vs. 3.40 ? 0.08 mm), although the difference was
not significant (P ? 0.07). The end-diastolic LV posterior wall
thickness increased significantly in the Tg mice compared with
the non-Tg mice (0.92 ? 0.06 vs. 0.70 ? 0.05 mm, P ? 0.05),
indicating concentric LV hypertrophy in the Tg mice. The
percent fractional shortening of the Tg mice was comparable to
that of the non-Tg mice (45 ? 3 and 46 ? 4%, P ? NS). There
was no significant difference in heart rate and systolic blood
pressure between the non-Tg and Tg mice (Table 3).
mRNA expressions of hypertrophic markers and cell adhe-
sion molecules. Semiquantitative RT-PCR analyses demon-
strated an increase in ANF (175 ? 7%), BNP (190 ? 12%),
and ?-MHC (536 ? 40%) gene expressions and a decrease in
?-MHC (66 ? 4%), SERCA2a (80 ? 2%), and CRC (88 ?
3%) gene expressions in the Tg mice compared with the
non-Tg mice (n ? 4 per group; Fig. 7).
Fig. 2. Northern blot analysis for human
IL-1? (hIL-1?) mRNA. A: hIL-1? mRNA
expression in transgenic (Tg) and non-Tg
mice. B: selective hIL-1? mRNA expression
in tissues. Blots are representative of 3 sepa-
rate experiments. Arrow indicates hIL-1?
mRNA band.
Table 1. Cytokine levels of ventricular tissue
Non-Tg Tg
hIL-1?
mIL-1?
mIL-1?
mTNF-?
mIL-6
UD
UD
73.0?7.8*
UD
7.6?0.7
1.1?0.1
20.6?8.2*
8.1?0.7
1.3?0.1
2.0?1.1
Values are means ? SE expressed as pg/100 ng protein (n ? 6). Transgenic
(Tg) mice showed a significant increase in human (h) IL-1? and mouse (m)
IL-6 protein levels compared with non-Tg mice. UD, undetected. *P ? 0.05
vs. non-Tg.
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We further evaluated the mRNA expressions of cell adhe-
sion molecules, because no inflammatory infiltrates were mi-
croscopically detected in the Tg myocardium. The ICAM-1-
to-?-actin (4.73 ? 0.46 and 2.15 ? 0.32% for Tg and non-Tg,
respectively, P ? 0.05) and VCAM-1-to-?-actin (15.8 ? 2.0
and 8.3 ? 0.5% for Tg and non-Tg, respectively, P ? 0.05)
mRNA ratios were significantly elevated in the Tg myocar-
dium compared with the non-Tg myocardium (n ? 4 per
group). In contrast, the PECAM-1-to-?-actin mRNA ratio
(89.7 ? 3.9 and 88.8 ? 6.3% for Tg and non-Tg, respectively,
P ? NS) did not increase (n ? 4 per group).
DISCUSSION
In this study, we demonstrated concentric LV hypertrophy
with preserved LV systolic function in mice with a cardiac-
specific overexpression of mature-type hIL-1?.
An elevated expression of IL-1 in hearts has been revealed
in a variety of cardiovascular diseases, such as myocarditis
(22), myocardial ischemia and infarction (18), and congestive
heart failure (13, 18, 22). IL-1 is also implicated in the
pathogenesis of the hypertrophied myocardium in vitro and in
vivo (21). We found that mice with a ubiquitous overexpres-
sion of hIL-1?, which were originally generated as a mouse
model of rheumatoid arthritis, unexpectedly showed prominent
LV hypertrophy and congestive heart failure to death within 2
wk after birth. However, the findings we reported did not
address whether hIL-1? expressed in the myocardium caused
the LV remodeling, because hIL-1? was ubiquitously ex-
pressed in the Tg mice. Thus we generated and analyzed Tg
mice with a cardiac-specific overexpression of hIL-1? in this
study. The Tg mice showed an increased heart weight-to-body
weight ratio. A histological analysis revealed that the hyper-
trophy was the result of the increased size of the myocardium.
TEM observations revealed rounded mitochondria that were
increased in number. The overexpression of hIL-1? increased
mRNA expressions of ANF, BNP, and ?-MHC and decreased
mRNA expressions of ?-MHC, SERCA2a, and CRC in the
myocardium. An echocardiographic study revealed that the
end-diastolic LV wall was thicker in the Tg than in the non-Tg
mice. These findings are compatible with LV hypertrophy (9).
The responses in the Tg heart are further evidence that LV
hypertrophy is the result of hIL-1? overexpression, because
hypertrophy was observed without evidence of hemodynamic
overload.
Cardiac hypertrophy is defined as an enlargement of the
heart associated with an increase in cardiomyocyte cell volume
that occurs in response to diverse pathophysiological stimuli
such as hypertension, ischemic heart disease, valvular insuffi-
ciency, infectious agents, or mutations in sarcomeric genes
(14). Hypertrophic growth of the myocardium is thought to
Fig. 3. Western blot analysis. Representative
immunoblots of p38 MAPK (A), p65 NF-?B
(B), and phosphorylated (phospho) proteins
(A and B). Signal activations were assessed by
densitometry analysis of immunoblots. Ratios
of phosphorylated p38 to p38 and phosphor-
ylated p65 to p65 are expressed as percentage
of non-Tg values. Phosphorylation of p38
MAPK and p65 NF-?B was enhanced in Tg
mice compared with non-Tg mice. *P ? 0.05
vs. non-Tg.
Table 2. Heart and body weights
6 wk (n ? 10) 12 wk (n ? 20)
HW, mgBW, g HW/BW, mg/gHW, mgBW, g HW/BW, mg/g
Non-Tg
Tg
72.7?3.3
86.0?5.0
15.2?0.8
15.3?1.0
4.78?0.10
5.62?0.16*
102.5?4.2
123.4?5.7
22.3?0.8
22.3?1.4
4.60?0.08
5.53?0.13*
Values are means ? SE. Heart weight (HW)-to-body weight (BW) ratio was significantly higher in Tg than in non-Tg mice at 6 and 12 wk of age. *P ? 0.05
vs non-Tg.
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preserve pump function, although prolongation of the hyper-
trophic state is a leading predictor for the development of
sudden death and heart failure (6, 12). In our study, the
cardiac-specific hIL-1?-overexpressing mice showed LV hy-
pertrophy but a preserved systolic function and a normal 1-yr
survival rate. However, we revealed that the pathological
significance of the LV hypertrophy observed in this study was
an increase in ANF, BNP, and ?-MHC gene expression and a
decrease in ?-MHC, SERCA2a, and CRC gene expression in
the myocardium. Therefore, these results suggest that the
hypertrophy may be a consequence of compensatory mecha-
nisms. The result is different from those observed in the
ubiquitous hIL-1?-overexpressing mice, which demonstrated
congestive heart failure to death within 2 wk after birth (7). We
speculate that the phenotypic difference most likely is due to
the different regulation of the hIL-1? expression by each
promoter as well as the distinct degrees of hIL-1? expression.
The gene expression controlled under the ?-MHC promoter
can affect the myocardium only after birth, whereas the ex-
pression under the CAG promoter can affect the myocardium
even before birth (20). The IL-1 transgene regulated under the
CAG promoter acts on fetal myocardial cells, which maintain
the capacity to divide. On the other hand, the IL-1 transgene
under the ?-MHC promoter acts on mature myocardial cells,
which have no capacity to divide. This could lead to an
increase in myocyte number and volume and cause severe
ventricular hypertrophy and congestive heart failure in CAG-
IL-1 Tg mice. In addition, because of a ubiquitous overexpres-
sion of IL-1 in CAG-IL-1 Tg mice, mononuclear cells and
macrophages overexpressing IL-1 appeared to gain a tissue-
destructive phenotype (17), which was likely to cause robust
inflammatory infiltrations in the hearts and, subsequently, an
aggravation of myocardial injury.
The effects of TNF-?, which is also a proinflammatory
cytokine, on the myocardium have been well investigated. A
recent report demonstrated that a chronic overexpression of
TNF-? (TNF-? Tg) in mice induced LV hypertrophy, eventual
LV dilation, and congestive heart failure (8). In contrast, in our
Fig. 4. Heart morphology. A: representative
hematoxylin-and-eosin
slices of cardiac ventricles. Heart mass was
increased in Tg mice. Arrows indicate thick-
ness of left ventricular free wall. Scale bar,
1.0 mm. B: representative hematoxylin-and-
eosin- and Masson’s trichrome-stained sec-
tions from adult heart of non-Tg and Tg mice.
Sections revealed a normal linear arrange-
ment of myofibrils in Tg mice. Scale bars, 30
?m.
(HE)-stained thin
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Fig. 5. Cross-sectional area of ventricular cardio-
myocytes (n ? 50 per mouse) was evaluated on
Masson’s trichrome-stained sections of hearts in
non-Tg and Tg mice (n ? 4 per group). Cross-
sectional area was increased in Tg mice. Scale bars,
20 ?m.
Fig. 6. Heart ultrastructural analysis. High-
magnification images of representative slices
from a compact layer from left ventricle il-
lustrate mitochondria (M) and T tubule (ar-
rowhead) alterations in Tg heart. Z, Z bands;
S, sarcomere. Scale bars, 2 ?m.
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mice, a persistent overexpression of IL-1 in the heart induced
ventricular hypertrophy, but not ventricular dilatation or con-
gestive heart failure. These phenotypic differences might be
explained by the cytokines expressed in the heart. The TNF-?
Tg mice showed a significant increase in the myocardial
TNF-? and IL-1? levels (8). Kadokami et al. (8) reported that,
in TNF-? Tg mice treated with a TNF-?-neutralizing antibody,
LV systolic function was improved to the same level as in the
non-Tg mice; however, these TNF-? Tg mice still showed a
high expression of myocardial IL-1? without any attenuation
of development of LV hypertrophy. In our study, IL-1 Tg mice
showed a significant increase in the myocardial level of hIL-
1?, but not TNF-? (7).
There has also been considerable interest in the relative roles
of IL-1? and IL-1? in cardiac hypertrophy. Both isoforms have
been demonstrated to be involved in cardiac hypertrophy (1,
10), and mRNA expressions of IL-1? in hypertrophied myo-
cardium were reported to be higher than those of IL-1? (1, 10).
Although the relative pathogenic contributions of IL-1? and
IL-1? to myocardial hypertrophy remain to be fully elucidated,
the roles of IL-1? in cardiac hypertrophy have been well
investigated. The expression of IL-1? increased in hypertro-
phied hearts with pressure overload in vivo (21) and in hyper-
trophied myocardium in vitro (13). In addition, in vitro exper-
iments have revealed that IL-1? can induce cardiomyocyte
hypertrophy (19, 23). The two isoforms bind to the same
receptor, IL-1 type 1 receptor, to elicit the biological effects of
IL-1 (4). However, the precursor form of IL-1?, in contrast to
IL-1?, is known to act as a membrane-associated IL-1 (4). We
could not detect hIL-1? protein in the serum, suggesting that
membrane-associated IL-1 on the cardiomyocytes contributes
to cardiac hypertrophy through cell-to-cell interaction. Further-
more, by histological evaluation, we recognized a lack of
inflammatory infiltrates in the Tg myocardium. Thus we mea-
sured the mRNA expressions of cell adhesion molecules.
ICAM-1 and VCAM-1 were found to induce the firm adhesion of
inflammatory cells on the vascular surface, whereas PECAM-1
plays a critical role in the extravasation of leukocytes into
underlying tissue as previously reported (2). In our study, we
demonstrated an increase in ICAM-1 and VACM-1, but not
PECAM-1, gene expression in the Tg myocardium. The
chronic cardiac-specific overexpression of IL-1? did not aug-
ment PECAM expression in the myocardium, which might
result in a lack of leukocyte extravasation into the myocardial
tissue.
Niki et al. (17) reported that the bone marrow macrophages
derived from hIL-1?-overexpressing mice stimulated prolifer-
ation of murine cells and that the mitogenic activity was
suppressed by the neutralizing antibody against hIL-1?. In our
study, to confirm the transactivation of murine myocardium by
hIL-1?, we demonstrated an increase in IL-6 protein levels,
phosphorylated p38 MAPK, and phosphorylated p65 (Ser536)
NF-?B in the Tg hearts. IL-6, a downstream cytokine activated
by IL-1, was reported to be a cardioprotective and hypertrophic
signal via gp130. Thus gp130 signals as an effector might be
involved in the pathogenesis of the phenotypes observed in this
study. Both p38 MAPK and NF-?B have been traditionally
implicated as pivotal intracellular mediators of the inflamma-
tory responses induced by IL-1. p38 MAPK is also associated
with myocardial hypertrophy. However, it remains to be fully
elucidated whether p38 MAPK itself plays a role in the onset
and development of myocardial hypertrophy in mammalian
adult hearts (3, 16, 24).
Table 3. Cardiac functions and hemodynamic parameters
LVEDd, mm
LVESd, mm
FS, %PWT, mm HR, beats/minSBP, mmHg
Non-Tg
Tg
3.40?0.08
3.18?0.06
1.83?0.10
1.74?0.09
46?4
45?3
0.70?0.05
0.92?0.06*
556?34
544?30
98?12
101?14
Values are means ? SE (n ? 6). Echocardiographic analysis revealed concentric left ventricular (LV) hypertrophy and preserved LV systolic functions in Tg
mice. No significant difference in heart rate (HR) and systolic blood pressure (SBP) was observed between non-Tg and Tg mice. LVEDd and LVESd, LV
end-diastolic and end-systolic diameter; PWT, posterior systolic wall thickness; FS, fractional shortening. *P ? 0.05 vs. non-Tg.
Fig. 7. Gene expressions analyzed using
semiquantitative RT-PCR. RT-PCR bands
are representative of 4 separate experiments.
RT-PCR analyses demonstrated an increase
in atrial natriuretic factor (ANF), B-type na-
triuretic protein (BNP), and ?-myosin heavy
chain (?-MHC) gene expressions and a de-
crease in ?-myosin heavy chain (?-MHC),
sarco/endoplasmic reticulum Ca2?-ATPase
type 2 (SERCA2a), and CRC gene expres-
sions in Tg mice compared with non-Tg mice
(n ? 4 per group).
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In summary, we generated mice with a cardiac-specific
overexpression of hIL-1?. The Tg mice showed concentric LV
hypertrophy with a preserved LV systolic function. Our find-
ings suggest that the cardiac expression of IL-1 may, therefore,
cause LV hypertrophy in vivo.
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