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Cardiovascular Research 74 (2007) 471 – 479
www.elsevier.com/locate/cardiores
Adiponectin accumulates in myocardial tissue that has been damaged by
ischemia-reperfusion injury via leakage from the vascular compartment
Rei Shibata a , Kaori Sato a , Masahiro Kumada b , Yasuhiro Izumiya a , Mina Sonoda b ,
Shinji Kihara b , Noriyuki Ouchi a , Kenneth Walsh a,⁎
a
Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany Street, W611, Boston, MA 02118, USA
b
Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, 2-2, Yamada-oka, Suita, Osaka, 565-0871, Japan
Received 11 October 2006; received in revised form 25 January 2007; accepted 2 February 2007
Available online 12 February 2007
Time for primary review 31 days
Abstract
Objectives: Adiponectin, a circulating adipocyte-derived hormone, exerts beneficial actions on hearts subjected to ischemia-reperfusion
injury. Adiponectin exists in plasma as three different oligomeric forms: trimer, hexamer and high molecular weight. This study investigated
the expression and myocardial accumulation of adiponectin in a murine model of ischemia-reperfusion injury.
Methods: Wild-type and adiponectin deficient mice were subjected to left anterior descending artery ligation followed by reperfusion.
Plasma adiponectin levels were analyzed by ELISA and adiponectin in heart was determined by immunohistochemical, Western blot and
real-time PCR analyses.
Results: Plasma adiponectin levels declined after myocardial ischemia-reperfusion injury due to reductions in high molecular weight and, to
a lesser extent, trimer and hexamer isoforms. Adiponectin protein was detected in injured but not sham-operated heart, and this was
accompanied by a negligible increase in adiponectin transcript in the myocardium. Systemic delivery of adiponectin to adiponectin knockout
(APN-KO) mice led to the accumulation of adiponectin in ischemia-reperfusion-injured, but not-uninjured hearts at levels comparable to
wild-type suggesting that cardiac expression of adiponectin does not appreciably contribute to its accumulation in the infarcted heart. The
serum half-life of adiponectin was 7.4 ± 0.3 h in ischemia-reperfusion and 9.7 ± 0.5 h in sham-operated mice (P N 0.05), whereas the half-life
of adiponectin in the damaged heart was 26.9 ± 2.2 h (P b 0.05).
Conclusions: These data show that adiponectin accumulates in the heart following ischemic damage primarily through leakage from the
vascular compartment, and that adiponectin has a longer half-life in damaged heart tissue than in plasma.
© 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Adiponectin; Ischemia-reperfusion; Myocardium; Infarction
1. Introduction
Adiponectin, also referred to as adipocyte complementrelated protein of 30 kDa (ACRP30) [1,2], is an adipocyte-
Abbreviations: AAR, area at risk; APN, adiponectin; CAD, coronary
artery disease; HMW, high molecular weight; IA, infarct area; I-R, ischemiareperfusion injury; KO, knockout; LAD, left anterior descending artery; LV,
left ventricular; WAT, white adipose tissue.
⁎ Corresponding author. Tel.: +1 617 414 2390; fax: +1 617 414 2391.
E-mail address: [email protected] (K. Walsh).
derived bioactive factor that is abundantly present in plasma.
In clinical studies, circulating adiponectin levels are downregulated in association with obesity-linked disorders
including hypertension, low grade inflammation and type 2
diabetes [3,4]. These obesity-linked disorders are implicated
in the severity and outcome of ischemic heart disease
including myocardial infarction [5,6]. Conversely, high
plasma adiponectin levels are associated with a lower risk
of CAD in some [7–10] but not all [11,12] studies. It is also
recognized that high plasma adiponectin levels are associated with a lower risk of myocardial infarction [13] and that
0008-6363/$ - see front matter © 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2007.02.010
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R. Shibata et al. / Cardiovascular Research 74 (2007) 471–479
adiponectin levels rapidly decline in men following acute
myocardial infarction [14].
Adiponectin exists in plasma as a trimer, hexamer or high
molecular weight (HMW) forms in humans and mice [15,16].
The HMW form of adiponectin is lower in CAD patients,
whereas the HMW form increases during weight reduction in
obese subjects [16]. Several reports have shown that the HMW
form of adiponectin determines insulin sensitivity and glucose
tolerance [17–19].
Consistent with clinical observations, experimental studies
showed that adiponectin-knockout (APN-KO) mice exhibit
diet-induced insulin resistance, impaired endothelium-dependent vasodilation on atherogenic diets and impaired angiogenic response to ischemia [20,21]. Aortic constriction in
APN-KO mice results in enhanced concentric cardiac
hypertrophy and increased mortality [22,23], whereas adenovirus-mediated adiponectin expression attenuates cardiac
hypertrophy in APN-KO, wild-type (WT) and diabetic db/db
mice [22]. Adiponectin inhibits agonist-stimulated hypertrophy in cultured cardiac myocytes [22,24]. Recently, it was
demonstrated that APN-KO mice develop larger infarcts
following ischemia-reperfusion injury [25,26]. Conversely,
supplementation of adiponectin diminishes infarct size in
APN-KO and WT mice. Furthermore, adiponectin inhibits the
development of severe myocarditis in leptin-deficient obese
ob/ob mice [27]. These findings have led to the proposal that
the short-term administration of this factor may have clinical
utility in the treatment of patients suffering from acute
myocardial infarction [28].
Recently a number of studies have shown that adiponectin
transcript is upregulated in damaged tissues. For example,
adiponectin mRNA is detected in the liver of mice after carbon
tetrachloride-induced hepatic injury [29], and oxidative stress
upregulates adiponectin mRNA in skeletal muscle cells [30].
With regard to the heart, adiponectin protein is detected at
periphery of damaged myocytes in patients with myocardial
infarction and dilated cardiomyopathy [31,32], and this protein
is also detected in heart during the acute phase of virus-induced
myocarditis in mice [33]. Adiponectin transcript has been
detected in cultured cardiac myocytes [34] and in mouse
models of myocardial injury [33,35]. Collectively, these data
suggest that the endogenous production of this protein in
injured tissues may contribute to its protective actions, but this
has never been directly demonstrated. Therefore, a more
thorough understanding of the expression and pharmokinetic
properties of adiponectin is required to evaluate the therapeutic
potential of this protein for the treatment of heart disease.
In the present study, we examined the effect of myocardial
ischemia-reperfusion injury in mice on circulating levels of
adiponectin isoforms and assessed adiponectin expression
and accumulation in the heart. Our observations indicate that
despite a transient drop in plasma adiponectin following
ischemia-reperfusion, adiponectin accumulates in the heart
following injury, largely from leakage from the vascular
compartment, and that it has a longer half-life in damaged
heart tissue than in plasma.
2. Materials and methods
2.1. Materials
Bacterially-produced mouse adiponectin was prepared
as described previously [36]. The adenoviral vectors
expressing β-galactosidase (Ad-βgal) or murine adiponectin (Ad-APN) have been described previously [21].
Adiponectin antibody that recognizes globular domain of
mouse adiponectin, was purchased from R&D systems.
Collagen type III antibody was purchased from Rockland
Immunochemical. GAPDH antibody was purchased from
Biogenesis Inc.
2.2. Mouse model of myocardial ischemia-reperfusion
WT and APN-KO mice in a C57/BL6 background were
used for this study [25]. Study protocols conform to the
Guide for the Care and Use of Laboratory Animals published
by the US National Institutes of Health (Publication No. 8523, revised 1996). Mice at the ages of 10 to 12 weeks were
anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally). A left thoracotomy was performed, and the left
anterior descending artery (LAD) was visualized under a
microscope and ligated with 8–0 silk suture using snare
occluder. Area at risk and infarct size were determined as
described previously [25]. Mice were subjected to 30 min of
LAD ligation, followed by 1, 3, 6, 12, 24, 36, 48, 96 and
168 h of reperfusion. In some experiments, recombinant
adiponectin protein or PBS vehicle was injected into the
jugular vein of mice at 30 min before LAD ligation. In some
experiments, 2 × 108 plaque-forming units (pfu) of Ad-APN
or Ad-β-gal were injected into the jugular vein of APN-KO
mice 3 d prior to the ischemia-reperfusion injury. For
Western blot and real-time PCR analyses, Evans blue dye
was injected through the jugular vein for delineation of the
area at risk (AAR), and non-necrotic areas within the AAR
regions were collected under a dissecting microscope.
2.3. Adiponectin level and oligomeric state in plasma
Total mouse adiponectin levels were determined with
adiponectin ELISA kits (Otsuka Pharmaceutical Co Ltd.).
For this measurement blood (20 μl) was collected from tail
vein at the time of ischemia-reperfusion surgery and at 0, 1,
3, 6, 12, 24, 36, 48, 96 and 168 h after reperfusion. To
determine the oligomeric form of adiponectin, blood samples
were collected by heart puncture from mice on postoperative
24 h. The oligomeric state of adiponectin was analyzed by
gel filtration chromatography and ELISA on fractions of
eluted proteins as described previously [16].
2.4. Immunohistochemical analysis
Mice were sacrificed and Left Ventricular (LV) tissue
was obtained at 0, 3, 12, 24, 48, 96 and 168 h after
R. Shibata et al. / Cardiovascular Research 74 (2007) 471–479
ischemia-reperfusion injury. Tissue samples were embedded in OCT compound (Sakura Finetech USA Inc) and
frozen in liquid nitrogen. Tissue sections (7 μm in
thickness) were incubated with goat polyclonal antiadiponectin antibody or control IgG antibody (Santa Cruz
Biotechnology). In some experiments, double fluorescence
staining was performed on frozen heart sections. Sections
were stained with rabbit polyclonal anti-collagen type III
antibody followed by the treatment with rhodamineconjugated secondary antibody to detect collagen type
III, and subsequently with goat polyclonal anti-adiponectin
antibody followed by the treatment with FITC-conjugated
secondary antibody to detect adiponectin. The signals were
detected and analyzed using a fluorescence microscope
(Nikon Diaphot).
2.5. Western blot analysis
Heart tissue samples (non-necrotic AAR and sham)
obtained at 0, 3, 12, 24, 48, 96 and 168 h after surgery and
subcutaneous white adipose tissue (WAT) from control
mice were homogenized in lysis buffer. Proteins (30 μg)
were separated with denaturing SDS-PAGE. Following
transfer to membranes, immunoblot analysis was performed with the indicated antibodies. This was followed by
incubation with secondary antibody conjugated with HRP.
2.6. Determination of adiponectin mRNA
Adiponectin mRNA level in myocardium and WAT was
quantified by real-time PCR. Total RNA from heart (nonnecrotic AAR and sham) and WAT were prepared with the
use of a Qiagen kit. The cDNA was produced using oligo-dT
primer (ThermoScript RT-PCR Systems, Invitrogen). PCR
was performed on iCycler iQ Real-Time PCR Detection
System (BIO-RAD) using SYBR Green 1 as a double
standard DNA specific dye (Applied Biosystems) [3]. In
some experiments, total RNA were isolated from heart and
liver of Ad-APN-treated APN-KO mice at 24 h after
ischemia-reperfusion or sham-operated APN-KO mice.
PCR was performed using Taq DNA Polymerase (Takara).
Primers were: 5′-AGGTTGGATGGCAGGC-3′ and 5′GTCTCACCCTTAGGACCAAGAA-3′ for mouse adiponectin and 5′-TCACCACCATGGAGAAGGC-3′ and 5′GCTAAGCAGTTGGTGGTGCA-3′ for mouse GAPDH.
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3. Results
3.1. Plasma adiponectin levels decline after ischemiareperfusion injury
We assessed the time-dependent changes of plasma
adiponectin levels in mice following myocardial ischemiareperfusion injury. For these experiments, the LAD was
ligated for 30 min followed by 24 h of reperfusion in C57/
BL6 mice (WT). Under the conditions of these assays, this
injury led to a 30.5% ± 2.4% (P b 0.05) infarct area (IA)
relative to AAR at 24 h in a parallel set of 5 WT mice
(Table 1). Circulating adiponectin levels, determined from
sequential tail vein bleeds (n = 5 mice), declined by 35%
(P b 0.01) at 24 h after ischemia-reperfusion (Fig. 1A).
Plasma adiponectin returned to pre-injury levels 7 days after
surgery. To corroborate these findings, plasma adiponectin
levels at 24 h after sham-operation or ischemia-reperfusion
were determined in a third set of WT mice (n = 12 for each
condition). At this time point, plasma adiponectin levels
were 29% lower (P b 0.01) in mice after ischemia-reperfusion compared with those in sham-operated mice (Table 1).
The distribution of adiponectin oligomers in plasma at
24 h after ischemia-reperfusion or sham-operation was
analyzed by ELISA on column elution fractions following
gel filtration chromatography (n = 4 mice per condition).
Representative elution profiles of plasma adiponectin from
individual WT mice after sham-operation or ischemiareperfusion are shown in Fig. 2a. All oligomeric forms of
adiponectin were significantly decreased after ischemiareperfusion injury, although the HMW form showed the
greatest decrease (Fig. 2b). The level of HMW declined by
33% (P b 0.01), whereas hexamer and trimer forms declined
by 13% (P b 0.05) and 12% (P b 0.05), respectively.
3.2. Expression and accumulation of adiponectin in injured
heart
The presence of adiponectin in the heart was assessed by
immunohistochemistry, immunoblot and real-time polymerase chain reaction assays. Representative photographs of
2.7. Statistical analysis
Data are presented as mean ± SD. The mean value was
compared between 2 groups using an unpaired t test. The
comparison among more than 3 groups was performed by
analysis of variance (ANOVA) with Scheffe's Ftest.
Comparison of adiponectin half-life between different
experimental groups was performed by a repeated measure
ANOVA. A value of P b 0.05 was accepted as statistically
significant.
Fig. 1. Decreased plasma adiponectin levels following ischemia-reperfusion
injury. Plots of time course of plasma adiponectin levels from the same series
of individual mice following ischemia-reperfusion injury (n = 5). ⁎P b 0.05,
⁎⁎P b 0.01 vs. WT mice before surgery.
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R. Shibata et al. / Cardiovascular Research 74 (2007) 471–479
Table 1
Infarct size after ischemia-reperfusion
WT
APN-KO
APN-KO + APN
% AAR/LV
% IA/AAR
% IA/LV
51.9 ± 6.2
50.3 ± 4.1
49.6 ± 1.7
30.5 ± 6.2
50.1 ± 4.8⁎⁎
31.9 ± 3.9⁎⁎
15.5 ± 1.5
28.1 ± 2.2⁎⁎
16.8 ± 2.7⁎⁎
Infarct size was determined 24 h after ischemia-reperfusion. Recombinant
adiponectin protein (APN) was injected into jugular vein of APN-KO mice
at 30 min before LAD ligation. ⁎⁎P b 0.01 APN-KO + APN versus APN-KO
and APN-KO versus WT.
myocardium tissue stained with anti-adiponectin antibodies
are shown in Fig. 3a. Adiponectin protein was readily
detected in the myocardium of the AAR at 24 h after
ischemia-reperfusion in WT mice, whereas little or no
adiponectin could be detected in hearts of WT mice that had
undergone sham surgery. No signal was detected in
histological sections of hearts from APN-KO mice (data
not shown). Western blotting analysis detected adiponectin
protein in heart after ischemia-reperfusion, whereas little or
no expression of adiponectin could be detected in shamoperated WT mice (Fig. 3b). The level of adiponectin protein
in injured heart was approximately 30% of the level found in
subcutaneous white adipose tissues (WAT). Adiponectin
mRNA in the myocardial non-necrotic AAR and WAT was
quantified by real time PCR (Fig. 3c). Ischemia-reperfusion
Fig. 3. The expression of adiponectin in the ischemic area of WT mouse
heart. a) Representative immunostaining of adiponectin from WT heart
sections at 24 h after sham-operated or ischemia-reperfusion injury
(magnification, ×400; bar indicates 50 μm) (n = 5 in each group). b)
Expression of adiponectin protein in heart tissues from WT mice at 24 h after
sham-operation or ischemia-reperfusion injury and in white adipose tissue
(WAT) by Western blot analysis. Relative adiponectin levels were quantified
using the NIH image. Immunoblots were normalized to GAPDH (⁎⁎P b 0.01
vs. sham-operated WT, mice n = 3–5). c) Adiponectin mRNA levels in WT
mouse heart at 24 h after sham-operation or ischemia-reperfusion. WAT was
used as a positive control for adiponectin expression. Results are expressed
adiponectin mRNA levels relative to GAPDH mRNA levels. ⁎P b 0.05,
⁎⁎P b 0.01 vs. sham-operated WT mice (n = 3–6).
Fig. 2. Adiponectin isoforms are decreased after ischemia-reperfusion injury.
a) Representative elution profiles of adiponectin in plasma from WT mice
after sham-operation (closed circle) or ischemia-reperfusion injury (open
circle). Plasma was collected at 24 h after sham-operation or ischemiareperfusion. Plasma was fractionated by gel filtration chromatography, and
the adiponectin levels in each fraction were determined by ELISA. b)
Percent changes of each form of adiponectin between sham-operation and
ischemia-reperfusion surgery (⁎P b 0.05, ⁎⁎P b 0.01, n = 4 in each group).
injury led to a small, but reproducible, increase in
adiponectin mRNA levels in the heart (1.5-fold). However
adiponectin mRNA levels in heart under these conditions are
more than 33,000-fold lower than in WAT.
To examine whether adiponectin accumulates in the heart
as the result of leakage from the vascular compartment,
R. Shibata et al. / Cardiovascular Research 74 (2007) 471–479
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Fig. 4. Accumulation and kinetics of exogenous adiponectin in the heart after acute ischemic injury. APN-KO mice were subjected to ischemia-reperfusion injury
at 30 min after injection of exogenous adiponectin. a) Immunostaining of adiponectin at 3 and 96 h in myocardial ischemic area of APN-KO mice after injection
of exogenous bacterially-produced adiponectin (magnification, ×400; bar indicates 50 μm) (n = 3 in each group). b) Double fluorescence staining of adiponectin
(green) and collagen type III (red) in myocardial ischemic region of APN-KO mice at 3 h after injection of exogenous adiponectin. Co-localization is indicated by
yellow in the merged images (magnification, ×400; bar indicates 50 μm). c) Detection of exogenous adiponectin in ischemic heart tissues at 0, 3, 12, 24, 48, 96
and 168 h in APN-KO mice after injection of adiponectin by Western blot analysis. Relative adiponectin levels were quantified using the NIH image.
Immunoblots were normalized to GAPDH. (n = 3 in each group). d) Plasma adiponectin levels at 0.5, 3, 12, 24, 36, 48 and 96 h in APN-KO mice in response to
ischemia-reperfusion (I-R, open circle) or sham (closed circle) treatment after injection of exogenous adiponectin. Plasma adiponectin levels were determined by
ELISA (n = 3 in each group).
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R. Shibata et al. / Cardiovascular Research 74 (2007) 471–479
Fig. 5. Accumulation of adiponectin protein in damaged heart tissue. a)
Adiponectin mRNA in heart and liver from sham-operated APN-KO mice or
APN-KO mice treated with Ad-APN at 24 h after ischemia-reperfusion
injury. Adiponectin mRNA expression was determined by PCR. b)
Expression of adiponectin protein in heart tissues from WT mice and
APN-KO mice treated with Ad-APN or Ad-βgal at 24 h after shamoperation or ischemia-reperfusion injury. Relative adiponectin levels were
quantified from Western blot analyses using the NIH image (n = 3–5).
Immunoblots were normalized to GAPDH. ⁎⁎P b 0.01 relative to shamoperated WT mice.
APN-KO mice were subjected to ischemia-reperfusion
injury 30 min after the intravenous injection of 1.0 μg/g
recombinant adiponectin protein. Parallel experiments were
performed on APN-KO mice that received vehicle (PBS). In
the absence of exogenous adiponectin, IA/AAR × 100% was
50.1 ± 4.8% in APN-KO mice after 24 h of reperfusion
(Table 1), whereas adiponectin protein administration led to
a 37% decrease in infarct size at 24 h after injury, consistent
with our previous findings [25]. At 3, 12, 24, 48, 96 and
168 h after injection of adiponectin protein, mice were killed
and adiponectin accumulation in heart was assessed by
immunohistochemistry (Fig. 4a). There was no detectable
adiponectin prior to surgery in histological sections from
hearts of APN-KO mice that had received adiponectin
protein 3 h prior. In contrast, adiponectin protein was readily
observed in the vasculature and myocardium of ischemic
heart. This pattern of adiponectin distribution appeared
similar to that seen in WT hearts subjected to ischemiareperfusion injury (Fig. 3a). The immunoreactivity appeared
maximal at 3 h after injection of adiponectin and gradually
declined at 12, 24, 48 and 96 h (Fig. 4a and data not shown).
Adiponectin protein expression was not detected in hearts at
168 h after intravenous injection of protein.
To examine whether adiponectin co-localizes with
extracellular matrix in the injured myocardium, dual
immunofluorescence staining was performed on adiponectin
and collagen type III, a major component of the myocardial
collagen matrix [37]. Hearts from APN-KO mice were
harvested 3 h after the administration of adiponectin protein
(2.5 h after the initiation of ischemia-reperfusion injury).
Histological sections were co-stained for adiponectin (green)
and collagen type III (red). Merged images revealed that
these proteins co-localized (yellow) in the vasculature and
myocardium of ischemic heart (Fig. 4b).
The changes of adiponectin protein levels, assessed by
semi-quantitative Western immunoblot analysis, paralleled
the changes in immunoreactivity following ischemia-reperfusion injury (Fig. 4c). The highest levels of adiponectin
protein were detected 3 h after the injection of adiponectin
and levels declined at 12, 24, 48 and 96 h. No adiponectin
could be detected at 168 h. No adiponectin was detected 3 h
after adiponectin delivery in sham surgery hearts. Using
these data, the calculated half-life of exogenous adiponectin
in the myocardial AAR was 26.9 ± 2.2 h.
We also examined the clearance of intravenously injected
adiponectin in APN-KO mice. Recombinant adiponectin
1.0 μg/g was delivered intravenously through the jugular
vein 30 min prior to ischemia or sham surgery (n = 3). At
different time points blood was sequentially obtained from
tail vein and serum adiponectin levels were determined by
ELISA (Fig. 4d). The calculated half-life of adiponectin in
plasma was similar under both these conditions (7.4 ± 0.3 h in
ischemia-reperfusion-treated mice and 9.7 ± 0.5 h in shamoperated mice, P N 0.05). However, the half-life of adiponectin in the plasma was significantly lower than in the
myocardial AAR (P b 0.05).
Finally, we compared adiponectin levels in WT mice and
in APN-KO mice that received 2 × 108 pfu of Ad-APN or
Ad-βgal via the jugular vein 3 d prior to myocardial
Table 2
Plasma adiponectin levels
Mouse genotype
I-R
Adenovirus
Adiponectin (μg/ml)
WT
WT
APN-KO
APN-KO
APN-KO
APN-KO
−
+
−
+
−
+
−
−
Ad-βgal
Ad-βgal
Ad-APN
Ad-APN
13.9 ± 1.3
10.1 ± 1.4
b0.05
b0.05
10.0 ± 1.7
8.4 ± 1.2
Measurements were made at 24 h after surgery. Adenoviral vectors were
delivered intravenously via the jugular vein 3 days prior to surgery.
Adiponectin levels are reported as mean ± standard error.
R. Shibata et al. / Cardiovascular Research 74 (2007) 471–479
ischemia-reperfusion injury (30 min to 24 h, respectively) or
sham-operation. This mode of delivery leads to the
transduction of liver (Fig. 5a), and no adiponectin could be
detected in hearts of sham-operated APN-KO mice that had
received Ad-APN (Fig. 5b). In contrast, a low level of
adiponectin could be detected in hearts from sham-operated
WT mice, and this could represent the endogenous production of adiponectin by cardiac tissues. Following
ischemia-reperfusion injury, similar levels of adiponectin
protein were detected by Western immunoblot in the ischemic regions of the myocardium between WT mice and the
APN-KO mice that had been treated with Ad-APN (Fig. 5b).
Under these conditions, plasma levels of adiponectin were
10.1 ± 1.4 μg/ml in WT and 8.4 ± 1.2 μg/ml in APN-KO mice
that received Ad-APN at 24 h after reperfusion (Table 2).
These data suggest that leakage from the vascular compartment is the predominant source of adiponectin in the
damaged myocardium.
4. Discussion
Recently it has been shown that adiponectin exerts
cardioprotective activities in models of pathological hypertrophy [22], viral myocarditis [27] and ischemia-reperfusion
injury [25,26]. In the present study, we analyzed adiponectin
levels in plasma and heart tissue before and after myocardial
ischemia-reperfusion injury to elucidate aspects of the
pharmacokinetics of adiponectin therapy for cardiac disorders. Total plasma adiponectin levels were found to decline
transiently in mice following myocardial ischemia-reperfusion injury. At the 24 h timepoint decline was attributed to
reductions in HMW and, to lesser extents, hexamer and
trimer isoforms of adiponectin. Relatively abundant adiponectin protein could be detected in the ischemic area of
hearts but the level of adiponectin protein in non-injured
hearts was lower by a factor of 3. In experiments involving
systemic delivery of adiponectin protein to APN-KO mice,
adiponectin could be detected in hearts following ischemiareperfusion injury, but not in non-injured hearts. These data
suggest that myocardial damage is required for the
appreciable accumulation of adiponectin protein in heart.
Previously, it has been shown that adiponectin transcript
is almost exclusively expressed by adipose tissue [2].
Recently, however, it was reported that adiponectin transcript
is synthesized by mouse cardiomyocytes [34], and is
upregulated in virus-induced damaged myocardium
[33,35]. Consistent with these latter reports, we find a 1.5fold elevation of adiponectin mRNA in heart following
ischemia-reperfusion injury. However, quantitative RT-PCR
analysis reveals that adiponectin transcript levels in injured
heart are lower by a factor of 33,000, and by a factor of
49,000 in non-injured heart, compared to that in adipose
tissue. In contrast, the level of adiponectin protein in injured
heart is within a factor of 3 of that in adipose tissue,
suggesting that endogenous production of adiponectin is
unlikely to be a major source of this protein in injured heart.
477
To test directly whether leakage from the vascular compartment is a major source of this protein in damaged heart, an
adenoviral vector expressing adiponectin was delivered to
APN-KO mice prior to ischemia-reperfusion injury. Systemic delivery results in adiponectin expression predominantly from liver, and no expression could be detected in
non-injured heart in our experiments. Following systemic
delivery, adenoviral vectors are reported to interact with
blood proteins that direct their binding to receptors on the
hepatic cell surface [38]. The rescue of APN-KO mice with
the systemic delivery of Ad-APN restored plasma adiponectin levels to 80% the level found in WT mice.
Furthermore, following ischemia-reperfusion injury, myocardial adiponectin levels were within 87% of that in WT
heart (Fig. 5, no statistically significant difference between
WT and APN-KO treated with Ad-APN). Collectively, these
data suggest that adiponectin protein accumulates in the
heart following injury largely as a result of leakage from the
vascular compartment.
Consistent with the notion that adiponectin accumulates in
damaged heart tissue due to breaches in the endothelial barrier,
adiponectin protein can be detected in the subendothelial space
at the early phase of catheter-injured vessels but not in the
intact artery [39]. Adiponectin protein is also detected in the
endothelium and subendothelial space of injured human
vessels identified by thrombus attachment, but not in vessels
with an intact endothelium [40]. Solid-phase binding assays
have shown that adiponectin can bind to collagen type I, III and
V, presumably through interactions with its collagen-like
domain [39]. Collagens type I and III are major components of
the cardiac extracellular matrix, and cardiac fibroblasts and
endothelial cells produce collagens type I and III after
myocardial infarction [37]. In this regard, adiponectin protein
has been detected at the periphery of damaged myocytes in
patients with myocardial infarction and dilated cardiomyopathy [31,32]. In the present study, it was shown that
adiponectin protein was systemically delivered in APN-KO
mice, and adiponectin co-localized with myocardial collagen
type III following ischemia-reperfusion injury in APN-KO
mice that were treated with recombinant adiponectin.
In our study, adiponectin levels in WT mice significantly
declined 35% after ischemia-reperfusion, which is comparable to the 29% decrease found in human after myocardial
infarction [14]. The accumulation of adiponectin in damaged
regions of the heart could contribute to the decline of
circulating adiponectin levels following acute cardiac injury.
In addition, tumor necrosis factor (TNF)-α and interleukin
(IL)-6 are known to suppress adiponectin transcript expression in adipose tissue [4], and serum levels of TNF-α and IL6 rapidly increase after ischemia-reperfusion injury in the
heart [41,42]. Thus, the upregulation inflammatory cytokines
following acute myocardial injury may contribute to the
decline in endogenous adiponectin levels in WT mice
through a reduction in its rate of synthesis in adipose tissue.
Adiponectin transcript is upregulated in the injured heart
in response to ischemia-reperfusion or virus injury [33,35].
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R. Shibata et al. / Cardiovascular Research 74 (2007) 471–479
Furthermore, adiponectin mRNA levels in skeletal muscle
and liver are increased under conditions of inflammation or
oxidative stress [29,30,43]. In contrast, adiponectin is
negatively regulated by pro-inflammatory cytokines or
oxidative stress in adipose tissue [4,44]. Collectively, these
data suggest that the upregulation of adiponectin by stress in
non-adipose tissues may confer localized protection to
injured tissue at a time when its expression by adipose
tissues decline leading to a reduction in circulating levels of
adiponectin. However, the mechanisms that contribute to the
differential regulation of adiponectin between adipose and
non-adipose tissues are unknown.
Combs et al. have reported that the half-life of
recombinant murine adiponectin is 6 h in FVB mice [45],
which is compatible with our data using APN-KO mice
after injection of bacterially-produced adiponectin protein
(9.7 ± 0.5 h). More recently, Peake et al. reported that
human adiponectin had a half-life of 14.3 h when injected
into rabbits [46]. Furthermore, they reported that the HMW
and trimer forms had half-lives of 13.0 and 17.5 h,
respectively. Currently, the cardioprotective role of each
oligomeric form of adiponectin is unknown, and future
studies will be required to clarify the specific actions of
each adiponectin oligomer on the injured heart. Of note, it
is shown here that adiponectin has a longer half-life in
damaged myocardial tissue, where it is bound to collagen
matrix, than in the plasma. It is conceivable that the
different oligomeric forms of adiponectin have different
affinities for matrix and, thus, would exhibit different halflives in injured tissues.
In summary, this work shows that myocardial ischemiareperfusion injury leads to a reduction in serum levels of
adiponectin in mice. Low levels of adiponectin protein are
detected in uninjured heart, but relatively high levels of
adiponectin are detected in the heart following acute injury.
While ischemia-reperfusion injury results in a small upregulation of adiponectin transcripts in the heart, myocardial levels of
this protein are similar between WT mice and Ad-APN-treated
APN-KO mice that synthesize adiponectin from non-myocardial tissues. Collectively, these data indicate that adiponectin
accumulates in damaged heart primarily as the result of
leakage from the vascular compartment. These findings are
consistent with the notion that adiponectin enters damaged
myocardial tissue, and participates in the wound healing
process by binding to matrix.
Acknowledgments
This work was supported by the National Institutes of
Health grants HL66957, HL77774, AR40197 and AG15052
to K. Walsh and Grant-in-Aid for Scientific Research on
Priority Areas to S. Kihara. R. Shibata was supported by a
grant from the American Heart Association Postdoctoral
Fellowship Award, Northeast Affiliate. N. Ouchi was
supported by an American Heart Association Scientist
Development Grant, Northeast Affiliate.
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