Transcriptome Alteration in the Diabetic Heart by
Rosiglitazone: Implications for Cardiovascular Mortality
Kitchener D. Wilson1,3., Zongjin Li1., Roger Wagner2, Patrick Yue2, Phillip Tsao2, Gergana Nestorova4,
Mei Huang1, David L. Hirschberg4, Paul G. Yock2,3, Thomas Quertermous2, Joseph C. Wu1,2*
1Department of Radiology, Stanford University School of Medicine, Stanford, California, United States of America, 2Department of Medicine, Division of Cardiology,
Stanford University School of Medicine, Stanford, California, United States of America, 3Department of Bioengineering, Stanford University School of Medicine, Stanford,
California, United States of America, 4Human Immune Monitoring Center, Stanford University School of Medicine, Stanford, California, United States of America
Background: Recently, the type2 diabetesmedication, rosiglitazone, has come under scrutiny for possibly increasing the risk of
cardiac disease and death. To investigate the effects of rosiglitazone on the diabetic heart, we performed cardiactranscriptional
profiling and imaging studies of a murine model of type 2 diabetes, the C57BL/KLS-leprdb/leprdb(db/db) mouse.
Methods and Findings: We compared cardiac gene expression profiles from three groups: untreated db/db mice, db/db
mice after rosiglitazone treatment, and non-diabetic db/+ mice. Prior to sacrifice, we also performed cardiac magnetic
resonance (CMR) and echocardiography. As expected, overall the db/db gene expression signature was markedly different
from control, but to our surprise was not significantly reversed with rosiglitazone. In particular, we have uncovered a
number of rosiglitazone modulated genes and pathways that may play a role in the pathophysiology of the increase in
cardiac mortality as seen in several recent meta-analyses. Specifically, the cumulative upregulation of (1) a matrix
metalloproteinase gene that has previously been implicated in plaque rupture, (2) potassium channel genes involved in
membrane potential maintenance and action potential generation, and (3) sphingolipid and ceramide metabolism-related
genes, together give cause for concern over rosiglitazone’s safety. Lastly, in vivo imaging studies revealed minimal
differences between rosiglitazone-treated and untreated db/db mouse hearts, indicating that rosiglitazone’s effects on gene
expression in the heart do not immediately turn into detectable gross functional changes.
Conclusions: This study maps the genomic expression patterns in the hearts of the db/db murine model of diabetes and
illustrates the impact of rosiglitazone on these patterns. The db/db gene expression signature was markedly different from
control, and was not reversed with rosiglitazone. A smaller number of unique and interesting changes in gene expression
were noted with rosiglitazone treatment. Further study of these genes and molecular pathways will provide important
insights into the cardiac decompensation associated with both diabetes and rosiglitazone treatment.
Citation: Wilson KD, Li Z, Wagner R, Yue P, Tsao P, et al. (2008) Transcriptome Alteration in the Diabetic Heart by Rosiglitazone: Implications for Cardiovascular
Mortality. PLoS ONE 3(7): e2609. doi:10.1371/journal.pone.0002609
Editor: Steve E. Nissen, Cleveland Clinic Foundation, United States of America
Received April 4, 2008; Accepted June 4, 2008; Published July 9, 2008
Copyright: ? 2008 Wilson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding for this work was supplied in part by research grants from Burroughs Wellcome Foundation Career Award in Medical Scientist (JCW). KDW is a
recipient of a Stanford University Bio-X Graduate Student Fellowship and Society of Nuclear Medicine Student Fellowship. This study was designed by JCW in
collaboration with his academic colleagues. This investigation did not have separate industry funding. The funding sources had no role in the study design, data
collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final
responsibility for the decision to submit for publication.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Cardiovascular disease is the leading cause of morbidity and
mortality in patients with type 2 diabetes . While atherosclerotic
coronary artery disease is highly prevalent in many diabetic patients,
the occurrence of nonischemic cardiomyopathy (‘‘diabetic cardio-
myopathy’’) suggests that other processes such as microangiopathy,
metabolic factors, or myocardial fibrosis  might be involved.
Thiazolidinediones (TZDs) such as rosiglitazone are insulin
sensitizers that may also have beneficial properties for diabetic
cardiomyopathy. These agonists bind to a subfamily of nuclear
hormone receptors, the peroxisome proliferator-activated receptors
(PPARs, including a, b/d and c isoforms, of which the c isoform is
the most common target), and activate transcription factors that
modulate gene expression, ultimately leading to increased insulin
sensitivity in peripheral tissues through poorly defined pathways
[2,3]. However, a recent meta-analysis of clinical trial outcomes for
the PPARc agonist rosiglitazone found a significant increase in the
risk of myocardial infarction, and a borderline significant risk of
death from cardiovascular causes . The reasons for this increased
risk are unclear, and a better understanding of the effects of
rosiglitazone on the diabetic heart is urgently needed.
Despite the large number of studies on rosiglitazone and
diabetes, the transcriptional network changes by which PPARc
agonists and other TZDs promote insulin sensitivity are not well
characterized. A number of groups have studied gene expression
in the db/db mouse liver , as well as transcriptional changes
induced by TZDs in adipocytes, kidney, aorta, and pancreatic islet
PLoS ONE | www.plosone.org1 July 2008 | Volume 3 | Issue 7 | e2609
cells in various mouse models [6,7,8,9], but none has studied the
heart directly. Moreover, determining PPAR-agonist target tissues
is complicated by the fact that the three PPAR isoforms are not
distributed equally across all tissues. In adipose tissue, PPARc
predominates and promotes differentiation and lipid storage ,
resulting in suppression of lipolysis by insulin and reduction of
plasma free fatty acid concentrations. In the cardiovascular system,
PPARc is expressed in vascular smooth muscle and vascular
endothelium , but is believed to be in low abundance in
Animal models of type 2 diabetes present an optimal system for
studying the effects of rosiglitazone. A well-established murine
model is the C57BL/KLS-leprdb/leprdb(db/db) mouse that has a
mutation in the leptin receptor. Homozygous db/db mice become
obese by 3–4 wk of age and develop hyperglycemia at 4–8 wk.
Serum insulin levels increase as early as 10–14 days, peak at 6–
8 wk, then decrease afterward. These mice continue to be
hyperinsulinemic throughout life and ultimately develop cardio-
myopathy (contractile dysfunction), as evidenced from metabolic
experiments using cultured db/db cardiomyocytes , echocar-
diographic studies , isolated working heart preparations ,
and direct in situ ventricular pressure measurements . Recent
studies by our lab and others have delineated the longitudinal
structural and metabolic cardiomyopathic changes in the db/db
mouse using cardiac magnetic resonance (CMR) and Fluorine-18–
2-fluoro-2-deoxy-d-glucose ([18F]FDG) PET scanning .
To address the many questions concerning the molecular and
functional effects of rosiglitazone on the diabetic heart, we used
CMR imaging and echocardiagraphic studies to assess the
development of cardiomyopathy in untreated and rosiglitazone-
treated db/db mice, as well as normal db/+ mice. These studies
were followed by cardiac gene expression profiling of the diabetic
and control phenotypes to understand the underlying molecular
changes in heart tissue. A comparison of the cardiac transcrip-
tomes of rosiglitazone-treated db/db mice with untreated db/db and
normal db/+ mice thus provides insights into the effects of
rosiglitazone on the heart, either through direct or indirect
mechanisms, as well as clues to its potential toxicity.
Animal and sample tissue preparation
Homozygous db/db mice (Jackson Laboratories, Bar Harbor,
ME) were maintained on a normal chow diet and housed in a
room with a 12:12-h light-dark cycle and an ambient temperature
of 22uC. For the treatment group, homozygous db/db mice were
maintained on 5 mg/kg/day (approximately 0.225 mg/mouse/
day) rosiglitazone (GlaxoSmithKline, London, UK) that was
mixed with normal chow. Unless otherwise stated, heterozygous
db/+ littermates were used as control animals. All protocols were
approved by the Administrative Panel on Laboratory Animal Care
at Stanford University and were carried out in accordance with
the guidelines of the American Association for Accreditation of
Laboratory Animal Care. At the end of the study, animals were
euthanized with a lethal dose of isoflurane. Immediately after
death, wet heart weight (HW) and body weight (BW) were
measured. Whole hearts, pancreas, and liver were harvested and
preserved in TRIzol reagent (Invitrogen, San Diego, CA) for
subsequent mRNA isolation, and a subset of hearts were fixed in
10% formalin for histological evaluation.
Insulin tolerance testing
Insulin tolerance testing was performed on mice after a 6-h fast.
At the time of testing, a bolus of human regular insulin (Eli Lilly,
Indianapolis, IN; 0.75 IU/kg) was injected intraperitoneally. In
blood derived from a tail nick, glucose levels were then determined
with a FreeStyle blood glucose monitoring system (Abbott
Laboratories, Abbott Park, IL) at baseline and 30, 60, and
120 min after injection.
Insulin Enzyme-Linked ImmunoSorbent Assay (ELISA)
After a 6-h fast, roughly 200 ml of blood was obtained from each
animal via retroorbital bleeding. Samples were placed in 500- ml
tubes containing EDTA (Becton-Dickinson, Franklin Lakes, NJ)
and centrifuged at 4uC and 13,200 rpm for 10 min. Approxi-
mately 50–75 ml of supernatant (serum) was then collected for
further processing. Serum insulin concentrations were measured
with a Mercodia mouse insulin enzyme immunoassay kit (Alpco
Diagnostics, Salem, NH).
Nonesterified fatty acids (NEFA)
Plasma nonesterified fatty acid concentrations were measured
using a HR series NEFA-HR(2) colorimetric assay kit (Wako
Chemicals USA, Richmond, VA).
Left ventricular functional analysis with echocardiogram
Echocardiography was performed by a blinded investigator (ZL)
using the Siemens-Acuson Sequioa C512 system equipped with a
multi-frequency (8–14 MHz) 15L8 transducer. Analysis of M-
mode images was performed using the Siemens built-in software.
Left ventricular end diastolic diameter (EDD) and end-systolic
diameter (ESD) were measured and used to calculate fractional
shortening (FS) by the following formula: FS=[EDD-ESD]/EDD.
LV volume at end diastolic (EDV) and end-systole (ESV) were
calculated by the bullet method as follows: EDV=0.856C-
SA(d)6L(d), ESV=0.856CSA(s)6L(s), where CSA(d) and (s) are
endocardial area in end-diastole and end-systole, respectively,
obtained from short-axis view at the level of the papillary muscles.
L(d) and L(s) are the LV length (apex to mid-mitral annulus plane)
in end-diastole and end-systole, respectively, obtained from the
parasternal long-axis view. LV ejection fraction (EF%) was
calculated as: EF%=(EDV2ESV)6100/EDV.
Cardiac magnetic resonance imaging
To prepare for scanning, induction of anesthesia was accom-
plished with 2% isoflurane and 1 l/min oxygen. Respiratory rate
was monitored and used to manually calibrate the maintenance
dose of isoflurane at 1.25–1.5%. Platinum needle ECG leads were
inserted subcutaneously in the right and left anterior chest wall.
Respiration was monitored with a pneumatic pillow sensor
positioned along the abdomen. Body temperature was maintained
at 36–37uC by a flow of heated air thermostatically controlled by a
rectal temperature probe. Heart rate (HR), respiratory rate, and
body temperature were recorded every 4 min during image
acquisition. Magnetic resonance images were acquired by a
blinded investigator (PY) with a 4.7-T magnet (Bruker BioSpin,
Fremont, CA) controlled by a Varian Inova Console (Varian, Palo
Alto, CA), using a transmit-receive quadrature volume coil with an
inner diameter of 3.5 cm. For particularly obese animals, a larger
coil with an inner diameter of 6 cm was utilized. Image acquisition
was gated to the ECG R-wave (Small Animal Instruments, Stony
Brook, NY). Coronal and axial scout images were used to position
a two-dimensional imaging plane along the short axis of the left
ventricular (LV) cavity. Gated gradient echo sequences were then
used to acquire sequential short-axis slices spaced 1 mm apart
from apex to base. For each sequence, 12 cine frames
encompassing one cardiac cycle were obtained at each slice level
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(TR)=100–140 ms, echo time (TE)=2.8–3.5 ms, number of
repeats (NEX)=8, field of view (FOV)=30630 mm, ma-
trix=1286128, flip angle=60u. For each short-axis slice,
planimetry measurements of LV myocardial area were conducted
off-line by tracing the epicardial and endocardial borders at end
systole and end diastole with MRVision software (MRVision,
Winchester, MA). For these purposes the papillary muscles were
considered part of the LV cavity. Anteroseptal and posterior wall
thickness measurements were performed on a midventricular slice
at the level of the papillary muscles at end diastole. LV mass
(LVM) was derived from the sum of the differences between the
end-diastolic epicardial and endocardial areas from apex to base,
adjusted for the specific gravity of myocardial tissue (1.055 g/ml).
LV end-diastolic (LVEDV) and end-systolic (LVESV) volumes
were calculated as the sum of the endocardial areas of each slice
from the apex to the LV outflow tract at end diastole and end
systole, respectively. LV ejection fraction (LVEF) was calculated as
(LVEDV2LVESV)/LVEDV. Cardiac output (CO) was calculat-
ed as (LVEDV2LVESV)/(HR).
the followingsequence parameters: acquisitiontime
RNA isolation and quality control
Total RNA was isolated separately from five heart samples for
each condition using TRIzol reagent (Invitrogen, San Diego, CA)
according to the manufacturer’s instructions for a total of 15 distinct
RNA samples. Total RNA was purified using RNeasy columns
(Qiagen, Chatsworth, CA). RNA concentration was measured by
spectrophotometry, and RNA integrity assessed with an Agilent
Nano Chips according to the manufacturer’s instructions. RNA was
judged as suitable for array hybridization only if samples exhibited
intact bands corresponding to 18S and 28S ribosomal RNA subunits
and had a RNA Integrity Number (RIN) greater than six. Universal
Reference RNAs for mouse (Stratagene, La Jolla, CA) were
purchased as microarray reference controls.
Labeling reaction and hybridization
Using Low RNA Input Fluorescent Linear Amplification Kits
(Agilent Technologies, Santa Clara, CA, USA), cDNA was reverse
transcribed from each RNA sample, and cRNA was then
transcribed and fluorescently labeled from each cDNA sample.
The fifteen mouse heart tissue cRNA samples were labeled with
Cy5 and the Universal Mouse reference cRNA was labeled with
Cy3, yielding a total of 5 biological replicates per condition. The
resulting cRNA was purified using an RNeasy kit (Qiagen,
Valencia, CA, USA) followed by quantification of the cRNA by
spectroscopy using an ND-1000 spectrophotometer (NanoDrop
Technologies, Wilmington, DE, USA). 825 ng Cy3- and Cy5-
labeled and amplified cRNA was mixed and fragmented according
to the Agilent technology protocol. cRNA was hybridized to
4644K whole human genome microarray slides from Agilent (Part
G4112F) according to the manufacturer’s instructions. The
hybridization was carried in a rotating hybridization chamber in
the dark at 65uC for 17 h.
Washing, scanning, and feature extraction
Slides were washed with Gene Expression Wash Buffer 1 and 2
(Agilent Technologies, Santa Clara, CA) followed by Acetonitrile.
A final wash in Stabilization and Drying Solution was performed
to prevent Cy-5 degradation by ozone. The array was scanned
using an Agilent G2505B DNA microarray scanner under
extended Dynamic range. The image files were extracted using
the Agilent Feature Extraction software version 9.5.1 applying
LOWESS background subtraction and dye-normalization. Fur-
ther analyses were performed with BRB ArrayTools Version 3.4
(National Cancer Institute) and TIGR MeV software (http://
Real-time quantitative PCR for confirmation of microarray
RNA to cDNA conversion was performed with a SuperScript
First-Strand cDNA synthesis kit (Invitrogen). The cDNA was then
used as a template in a TaqMan real-time PCR assay with the ABI
Prism 7700 Sequence Detection System (Applied Biosystems, Foster
City, CA). All samples were run in triplicates. Specialized premade
gene expression assay reagents for potassium channel, subfamily K,
member 1 (Kcnk1; catalog no. Mm00434624_m1), matrix metallo-
proteinase 3 (Mmp3; Mm00440295_m1), beta-1,4-N-acetyl-galac-
tosaminyl transferase 1 (B4galnt1; Mm00484653_m1), carboxyl
ester lipase (Cel; Mm00486975_m1), and N-acylsphingosine
amidohydrolase 2 (Asah2; Mm00479659_m1), purchased from
Applied Biosystems, were used for these experiments. Threshold
cycles were placed in the logarithmic portion of the amplification
curve, and each sample was referenced to 18S RNA (part
no. 4319413E) amplification to control for the total amount of
RNA. Fold differences between samples (relative quantification)
were calculated with the delta-delta method [S1/S2=22(T1–T2)],
where S1 and S2 represent samples 1 and 2 and T1 and T2 denote
the threshold cycles for S1 and S2.
Histologic analysis of cardiac tissue
At the time of death, a select group of hearts were fixed in 10%
formalin, cut at the midventricular level, and embedded in paraffin
blocks. The blocks were then sectioned into short-axis slices, which
were subsequently stained with hematoxylin and eoxin according to
standard protocols. High-power fields magnified to 406 from the
midportion of the LV free wall were photographed.
For all tests, one-way ANOVA was performed. All P values
,0.05 were considered significant. For microarray analysis, the
Significance Analysis of Microarrays (SAM) algorithm was used to
identify genes with statistically significant differences in expression
among the conditions. SAM is a statistically rigorous test that
incorporates a FDR calculation to correct for multiple testing
errors. Gene Ontology group overrepresentation analysis was
performed using Fisher’s exact test with FDR correction through
High Throughput GoMiner software.
Insulin resistance in rosiglitazone-treated, untreated and
We analyzed a number of metabolic parameters to confirm
insulin resistance in our db/db murine model of diabetes before
proceeding with microarrays. Insulin resistance is strongly
associated with obesity, and one mechanism may be the
generation of metabolic messengers such as free fatty acids by
adipose tissue that inhibit insulin action on muscle [18,19]. We
measured plasma non-esterified fatty acid (NEFA) and insulin
levels to follow insulin resistance in our three groups of mice.
Figure 1a shows plasma NEFA levels in the three groups of mice.
Compared to untreated db/db mice, NEFA levels in the
rosiglitazone-treated db/db mice decreased dramatically. Fasting
insulin levels (Figure 1b) and insulin tolerance testing (Figure 1c)
further confirmed the improved insulin sensitivity with rosiglita-
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Body and heart morphometric analysis
In addition to the obesity associated with type 2 diabetes,
rosiglitazone and other TZDs are widely known to cause further
weight gain due to fluid retention and altered adipogenesis .
Mean body weights of both treated and untreated db/db mice were
markedly higher than db/+ controls at baseline (Figure 1d).
Furthermore, rosiglitazone-treated mice had generally higher
mean body weights when compared to untreated mice, as
expected. The heart/body weight ratio was significantly higher
in the db/+ group when compared to the treated and untreated
Figure 1. Insulin resistance and mean body/heart weights of rosiglitazone-treated, untreated, and control mice. (A) Plasma NEFA
levels at 0, 1, and 3 months in untreated db/db mice (n=10), rosiglitazone-treated db/db mice (n=10), and db/+ mice (n=10). NEFA levels were
higher in both the treated and untreated db/db groups at baseline when compared to db/+ control mice. Compared to untreated db/db mice, NEFA
levels in the rosiglitazone-treated db/db group decreased significantly during the first month (960.9 vs. 1501.0 mmol/L, P,0.05), and decreased
further by the third month to near db/+ control levels. (B) Fasting insulin levels in the three groups at 0, 1, and 3 months. At baseline, both treated
and untreated db/db groups had increased levels of insulin compared to db/+ control. Insulin levels dramatically increased in untreated db/db mice at
1 month, but progressively decreased in rosiglitazone-treated mice. (C) Insulin tolerance testing at 3 months. Serum glucose levels after insulin
injection were significantly higher in untreated db/db mice when compared to db/+ mice; the rosiglitazone-treated db/db group had moderately
elevated glucose levels after insulin administration. These results corroborate the NEFA and insulin studies that demonstrate improved insulin
sensitivity with rosiglitazone treatment. (D) Mean body weights of the three groups of mice at 0, 1, and 3 months. Weights of both treated and
untreated db/db mice were markedly higher than db/+ controls at baseline. Furthermore, rosiglitazone-treated mice had generally higher mean body
weights when compared to untreated mice. (E) Mean heart/body weight ratios at sacrifice (4 months after treatment initiation). No significant
difference between treated and untreated groups (3.27 vs. 2.58, P=0.13), though there does appear to be a downward trend in the data. Heart/body
weight ratio was significantly higher in the db/+ group compared to both db/db groups. Values are mean6SEM. *P,0.05 vs. age-matched db/+;
#P,0.05 vs. age-matched untreated db/db.
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db/db groups (Figure 1e). However, histological analysis did not
reveal obvious differences at the microscopic level (Figure 2).
Left ventricular functional analysis with echocardiogram
Previous groups have observed a significant decrease in left
ventricular ejection fraction (LVEF) and fractional shortening (FS)
in db/db mice when compared with db/+ controls [14,21]. In one
study, the FS was reduced by as much as 16% at 12 weeks of age
. However, more accurate CMR studies of cardiac contrac-
tility have not confirmed the dramatic differences seen in the
echocardiographic studies, observing only an approximately 2%
reduction in LVEF at 22 weeks of age . Furthermore, clinical
echocardiographic studies of type 2 diabetic patients treated with
rosiglitazone found no significant changes in LVEF after 52 weeks
of treatment . To investigate cardiac contractility and verify
these previous findings, we assessed cardiac contractility in the
three mouse groups using echocardiography (Figure 3). The
mean LVEF and FS levels of the untreated db/db group were not
significantly different from those in the db/+ group, though there
does appear to be a downward trend in contractility in the db/db
group. Further, we found no significant LVEF or FS differences
between the rosiglitazone-treated db/db group and untreated db/db
group, confirming the results of the clinical study that found no
evidence of rosiglitazone-induced changes in cardiac contractility.
Cardiac magnetic resonance imaging
Using cardiac magnetic resonance (CMR) scanning, our group
has previously shown progressive cardiomyopathic changes in db/
db mice when compared to db/+ controls . Left ventricular
mass (LVM), interventricular septal thickness (IVST) and posterior
wall thickness (PWT) were significantly increased in db/db mice,
while LVEF was only marginally decreased. For the CMR
imaging in this new study (Figure 4), we included rosiglitazone-
treated db/db mice in addition to the untreated db/db and db/+
groups, and scanned them at 8 weeks after treatment initiation to
look for cardiomyopathic changes. CMR scans revealed signifi-
Figure 2. High-power light microscopy slides of myocardial tissue from rosiglitazone-treated and untreated db/db mice, and db/+
controls. Individual cardiomyocytes from all three groups appear qualitatively similar based on morphology and size, though the untreated db/db
cardiomyocytes exhibit very mild thickening. No increases in inflammatory cells, cell death, fibrosis, or other processes were observed in the two db/
db groups compared to db/+ controls.
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cant, albeit subtle, cardiomyopathic changes in untreated db/db
mice compared to db/+ controls (Figure 4). However, rosiglita-
zone treatment resulted in no significant improvements in cardiac
contractility, which confirmed our echocardiographic studies.
Gene expression analysis
Whole-heart RNA from five mice from each of the three groups
after four months with or without treatment was used for microarray
analysis. Overall, there were significant transcriptional differences
between the db/+ and both db/db groups, while differences between
rosiglitazone-treated and untreated db/db groups were much less
dramatic. Figure 5a shows a 3-dimensional scaling plot based on
principal component analysis of the expression data, which
graphically demonstrates that rosiglitazone did not restore db/db
transcriptomes to the normal db/+ state.
Genes that exhibited significant differential expression across
the three groups were identified using the SAM statistical
algorithm (selected genes are listed in Tables 1, 2; full gene txt
files are included as Supplemental Gene Lists S1, S2).
Interesting genes that were significantly upregulated in the
untreated db/db group compared to control include PPARc
(Pparg, the receptor for rosiglitazone), FK506 binding proteins
(Fkbp5 and Fkbp10), several potassium channel proteins (Kcnk1
and Kcnd3), two matrix metalloproteinases (Mmp3 and Mmp8), a
cyclin-dependent kinase inhibitor (Cdkn1), and myostatin (Gdf8)
(Table 1). Specific downregulated genes of interest include the
anti-apoptotic gene B-cell lymphoma–leukemia 2 (Bcl2), several
tumor necrosis factor-alpha (TNFa) genes and transforming
growth factor (TGFb) genes, vascular endothelial growth factor
(Vegfc), adiponectin (Adipoq), and apelin (Apln). We confirmed
the microarray results of 4 selected genes with real-time PCR
(Figures 5b, 5c). When we compared the db/db rosiglitazone
treated group to control, we found that upregulated genes include
the previously-mentioned Mmp3 and Kcnk1 transcripts, as well as
a potassium channel-interacting gene (Kcnip4), ryanodine recep-
tor (Ryr1), Myosin IIIB (Myo3b), Patched homolog 1, and apelin
While identifying individual genes that are differentially
regulated in each of the conditions is informative, it is also useful
to ascertain which cellular processes are up- or down- regulated;
therefore, we performed the Gene Ontology pathway overrepresen-
tation analysis using Fisher’s exact test. Lipid and protein
metabolism, fatty acid beta-oxidation, cell death, apoptosis,
peroxisome organization, and biogenesis were significantly upregu-
lated in untreated db/db mice when compared to control db/+ mice
(Supplemental Tables S1, S2, S3, S4). Among major pathways
thatweresignificantlydownregulated are cellproliferationandcycle,
immune response, blood vessel and vasculature development, and
anti-apoptosis. Interestingly, comparing the rosiglitazone-treated db/
db mice to control mice, we did not see a reversal of the pathways
that were upregulated in the untreated db/db group. The additional
pathways that were significantly upregulated in the rosiglitazone-
treated db/db hearts, however, included secretion,exocytosis,protein
and intracellular transport, cellular protein metabolism, and
Few studies have studied the global cardiac gene expression
changes resulting from rosiglitazone treatment. Understanding
these molecular changes will help elucidate the potential
mechanism(s) for the increased cardiac disease observed in diabetic
patients treated with this drug. Our microarray results demon-
Figure 3. Left ventricular functional analysis with echocardiogram. The mean left ventricular ejection fraction (LVEF) and fractional
shortening (FS) of the untreated db/db group (72.462.3% and 36.261.7%, respectively) were not significantly different from the db/+ group
(76.964.3%, P=0.11 and 39.763.6%, P=0.11, respectively), though there appears to be a downward trend in contractility between these two groups.
Further, there were no significant LVEF or FS differences between the rosiglitazone-treated db/db group (74.363.9% and 37.863.1%, respectively)
and untreated db/db group (P=0.43 and 0.40, respectively). Values are mean6SEM.
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strate that rosiglitazone does not reverse many of the significant
gene expression and pathway changes that occur in untreated
diabetic hearts, such as apoptosis and lipid metabolism. Further-
more, our microarray analysis indicates that rosiglitazone did not
upregulate important insulin-regulated glucose transporters such as
Glut4 in the heart, a finding that has been reported previously in
unique genes which appear to be differentially regulated between
rosiglitazone-treated and untreated animals that may give mecha-
nistic insights into rosiglitazone’s effects on the diabetic heart.
We were initially puzzled to find that PPARc expression was
upregulated in the hearts of untreated diabetic mice, as it has been
reported to be low in cardiomyocytes [12,24,25]. However, several
studies have shown PPARc expression to be important to cardiac
metabolism. One study found that rosiglitazone-treated Zucker
fatty rat hearts had improved glucose uptake as well as improved
contractility compared to lean rat hearts . Another study
found that mice lacking PPARc in cardiomyocytes exhibited mild
cardiac hypertrophy . These findings may reflect an indirect
effect of rosiligitazone on the heart, as demonstrated by a study in
which metabolic changes in PPARc-treated db/db hearts were
thought to be secondary to changes in the supply of exogenous
substrates . It appears that cardiac PPARc expression may
play an important though poorly understood role in cardiac
physiology, and it is possible that modulation of PPARc signaling
may affect the response to cardiac ischemia.
Matrix metalloproteinases are known to play a critical role in
atherosclerosis and cardiovascular tissue remodeling, mediating
the balance between matrix accumulation and degradation .
We found that rosiglitazone caused significant upregulation of
Mmp3, which encodes the matrix metalloproteinase stromelysin.
Mmp3 is typically expressed by macrophages within the
atherosclerotic plaques found in coronary artery disease ,
and has been shown to promote plaque rupture, myocardial
infarction and aneurysm . It is therefore possible that the in vivo
upregulation of Mmp3 by rosiglitazone will promote plaque
rupture, leading to increased rates of myocardial infarction, as well
as increasing tissue remodeling after ischemia.
In both untreated and rosiglitazone-treated db/db mice, we found
several potassium channel-related genes that were highly upregu-
lated, including Kcnip4 and Kcnk1. These findings support the
hypothesis that cardiomyocytes from db/db hearts exhibit electro-
physiological alterations such as attenuated outward K+currents
[21,30]. In general, glycolytic ATP production is important for the
Figure 4. Cardiac magnetic resonance imaging. CMR imaging at 8 weeks after treatment initiation of rosiglitazone-treated db/db mice (n=4),
untreated db/db mice (n=4) and db/+ groups (n=4). Left ventricular mass (LVM), interventricular septal thickness (IVST) and posterior wall thickness
(PWT) were significantly increased in both treated and untreated db/db mice relative to db/+ controls, while LVEF was decreased (though not
significantly in the rosiglitazone-treated group). No significant differences in LVM, IVST, PWT, or LVEF between rosiglitazone-treated and untreated
db/db mice were detected (P=0.82, 0.66, 0.14, and 0.88, respectively). Values are mean6SEM. *P,0.05 vs. age-matched db/+.
Rosiglitazone and the Heart
PLoS ONE | www.plosone.org7 July 2008 | Volume 3 | Issue 7 | e2609
normal function of cardiac membrane ion channels and pumps, and
inhibition of glycolysis causes altered intracellular ion concentrations
that may affect cardiac action potentials. Specifically, Kcnip4
modulates A-type potassium channels and has been shown to
increase expression of cardiac A-type Kv-4 channels . Kcnk1
encodes a two-pore potassium channel, TWIK-1, which plays a
major role in setting the resting membrane potential in many cell
types . Although itis unclear to what degree cardiacarrhythmias
might have played a role in the mortality shown in the meta-analysis
of rosiglitazone trials , the changes we observed in cardiomyocyte
potassium channel expression raise some concerns.
Rosiglitazone treatment also upregulated four genes related to
sphingolipid metabolism: B4galnt1, Cel, Asah2, and St3gal5. Found
in eukaryotic plasma membranes, sphingolipids have emerged as a
class of lipid mediators believed to be important for endogenous
modulation of cardiovascular function [33,34]. Generation of
sphingolipid metabolites such as ceramide, sphingosine, and
sphingosine-1-phosphate can activate smooth muscle cell prolifera-
tion, endothelial cell differentiation, apoptosis and cell death,
migration, vasoconstriction, and dilation. Specifically, ceramide is
an important mediator of lipotoxicity in the heart, and accumulation
of this metabolite has been associated with cardiomyocyte apoptosis
. Recent evidence indicates that the TZD pioglitazone causes
increased de novo ceramide synthesis in rat hearts , although
another group has reported decreased ceramide accumulation and
subsequent reduction in apoptosis . Regardless of the specific
mechanism involved, our microarray data suggest a significant role
for sphingolipid metabolism in the rosiglitazone-treated diabetic
heart which we are actively investigating further.
The downregulation of the hormone adiponectin in the diabetic
state has been well established in adipocytes and serum , but it
was not until 2005 that Pineiro et al. showed evidence that
cardiomyocytes also synthesize adiponectin . Confirming a
recent report by Natarajan et al. , our results demonstrate a
reduction of adiponectin in the diabetic heart, though we did not
observe restoration of its expression after rosiglitazone treatment. In
contrast, the signaling peptide apelin, a recently discovered regulator
of cardiac and vascular function , was downregulated in the
Figure 5. Multidimensional scaling plot of the expression data and real-time PCR analysis. (A) 3-dimensional scaling plot provides a
graphical representation of high-dimensional expression data in low dimensions. Each point within a ‘‘cloud’’ represents a single microarray, and the
similarity within a set of microarrays is indicated by their physical proximity to one other. As evidenced from the figure, each group (db/+ control,
untreated db/db, and rosiglitazone-treated db/db) clusters into a distinct grouping, indicating that each group has a similar transcriptome that can be
distinguished from the two other groups. Taqman real-time PCR of four selected genes normalized to 18s shows (B) significant upregulation of Kcnk1
in untreated db/db vs. db/+ mice and (C) significant upregulation of all four genes in rosiglitazone-treated vs. untreated db/db mice. These data
confirm the altered regulatory patterns of these four genes in the microarray data. Values are mean6SEM. *P,0.05 vs. control.
Rosiglitazone and the Heart
PLoS ONE | www.plosone.org8 July 2008 | Volume 3 | Issue 7 | e2609
untreated db/db group but was restored to normal levels after
rosiglitazone treatment. Apelin is expressed in the endothelium of
heart, kidney, and lung, and its receptor (APJ) is expressed by
myocardial cells and some vascular smooth muscle cells. Apelin has
inotropic effects on cardiac contractility and has been shown to
decrease systemic vascular resistance, and is therefore likely
beneficial during heart failure. In fact, apelin signaling may augment
inhibition of the renin-angiotensin system , which is known to
exacerbate heart failure when left unchecked. Though not well
understood, the altered expression of apelin we observed in the
diabetic heart in response to rosiglitazone may present a novel
pathway for the study of diabetic cardiomyopathy.
In conclusion, this study has mapped the genomic expression
patterns in the hearts of the db/db murine model of diabetes and the
effects of rosiglitazone on these patterns. Overall, as we expected, the
db/db gene expression signature was markedly different from control,
but to our initial surprise was not significantly reversed with
rosiglitazone. In fact, many of the transcriptional changes induced
rosiglitazone. In particular, we have highlighted a number of
Table 1. Untreated db/db vs. db/+ control mice.
Symbol RefSeq IDUGCluster Name Score(d)
Kcnk1NM_008430Mm.10800 Potassium channel, subfamily K, member 17.82 26.03
Gdf8 NM_010834- growth differentiation factor 8 (Gdf8)5.12 15.93
Egfbp2 NM_010115- epidermal growth factor binding protein type B (Egfbp2) 4.77 9.47
Fkbp5 NM_010220Mm.276405 FK506 binding protein 59.766.11
Mmp8NM_008611Mm.16415 Matrix metallopeptidase 85.225.93
Edn3NM_007903Mm.9478Endothelin 36.59 5.72
Cdkn1a NM_007669Mm.195663 Cyclin-dependent kinase inhibitor 1A (P21) 5.133.74
Kcnj4 NM_008427 Mm.140760 Potassium inwardly-rectifying channel, subfamily J, member 45.06 2.68
Map3k6 NM_016693 Mm.36640 Mitogen-activated protein kinase kinase kinase 69.39 2.54
Map3k15 BC031147Mm.386889 Mitogen-activated protein kinase kinase kinase 155.88 2.10
Fkbp10NM_010221Mm.3894 FK506 binding protein 105.212.09
Mmp3 NM_010809Mm.4993 Matrix metallopeptidase 34.301.90
Kcnd3 NM_019931 Mm.44530Potassium voltage-gated channel, Shal-related family, member 35.93 1.77
Ryr3 XM_619795Mm.436657 Ryanodine receptor 35.45 1.74
PpargNM_011146 Mm.3020 Peroxisome proliferator activated receptor gamma5.511.42
Tnfrsf22NM_023680Mm.261384Tumor necrosis factor receptor superfamily, member 22 4.061.40
Aifm2NM_153779 Mm.286309Apoptosis-inducing factor, mitochondrion-associated 2 5.621.39
Il4ra NM_001008700Mm.233802|Mm.441865 interleukin 4 receptor, alpha (Il4ra)4.961.37
Sod1BC057074 Mm.431677 superoxide dismutase 1, soluble (cDNA clone IMAGE:5697222), partial cds.4.02 1.34
Adipor2 NM_197985 Mm.291826 Adiponectin receptor 24.421.22
Myo9a AK029836Mm.249545 Myosin IXa
Casp2NM_007610Mm.3921|Mm.433648caspase 2 (Casp2)
VegfcNM_009506Mm.1402Vascular endothelial growth factor C
Il13ra1NM_133990Mm.24208Interleukin 13 receptor, alpha 1
Gja1NM_010288Mm.378921Gap junction membrane channel protein alpha 1
Il13ra1NM_133990Mm.24208Interleukin 13 receptor, alpha 1
Tnfaip8NM_134131Mm.27740Tumor necrosis factor, alpha-induced protein 8
Adrb2NM_007420Mm.5598Adrenergic receptor, beta 2
Tnfaip8l1NM_025566 Mm.2312Tumor necrosis factor, alpha-induced protein 8-like 1
Il16NM_010551Mm.10137 Interleukin 16
Bcl2NM_009741257460|Mm.462969 B-cell leukemia/lymphoma 2 (Bcl2), transcript variant 1
Tnfrsf13cNM_028075Mm.240047 Tumor necrosis factor receptor superfamily, member 13c
AdipoqNM_009605 Mm.3969Adiponectin, C1Q and collagen domain containing
Selected genes that were differentially expressed across untreated db/db mice and db/+ controls. All of the genes presented in the tables have statistically significant
expression changes. Listed are the most important parameters, including the standard identifications of the gene, a statistical measure of significance (‘‘Score(d)’’), and
magnitude of the change (‘‘Fold Change’’).
Rosiglitazone and the Heart
PLoS ONE | www.plosone.org9 July 2008 | Volume 3 | Issue 7 | e2609
rosiglitazone modulated genes and pathways that may play a role in
the pathophysiology of the increase in cardiac mortality seen in the
meta-analysis by Nissen et al. The cumulative upregulation of (1) a
matrix metalloproteinase gene that has previously been implicated in
plaque rupture, (2) potassium channel genes involved in membrane
potential maintenance and action potential generation, and (3)
concern over rosiglitazone’s safety. Interestingly, a second meta-
same negative cardiovascular effects as rosiglitazone . We believe
likely also be found with pioglitazone treatment, though with some
 and hepatocytes  that found similar, but not identical,
expression profiles after treatment with pioglitazone, rosiglitazone,
that govern diabetic cardiovascular function and disease.
zone-treated db/db mice.
Found at: doi:10.1371/journal.pone.0002609.s001 (0.03 MBRTF)
Upregulated Gene Ontology pathways with rosiglita-
rosiglitazone-treated db/db mice.
Found at: doi:10.1371/journal.pone.0002609.s002 (0.02 MBRTF)
Downregulated Gene Ontology pathways with
Found at: doi:10.1371/journal.pone.0002609.s003 (0.04 MBRTF)
Upregulated Gene Ontology pathways in untreated
Found at: doi:10.1371/journal.pone.0002609.s004 (0.05 MBRTF)
Downregulated Gene Ontology pathways in untreated
Gene List S1
vs. db/+ analysis
Found at: doi:10.1371/journal.pone.0002609.s005 (0.15 MB
txt file of microarray results from db/db+rosigli-
tazone vs db/db analysis.
Found at: doi:10.1371/journal.pone.0002609.s006 (0.04 MB
txt file of microarray results from untreated db/db
Gene List S2
Conceived and designed the experiments: PT JW. Performed the
experiments: KW ZL PY GN MH. Analyzed the data: TQ RW KW
JW ZL PY PGY. Contributed reagents/materials/analysis tools: TQ PT
PY GN DH. Wrote the paper: RW KW JW. Other: The Human Immune
Monitoring Center at Stanford University facility played a role in the
design of the experiments and discussion of the results. The Human
Immune Monitoring Center at Stanford University reviewed the final draft
of the paper. Director of the Human Immune Monitoring Center at
Stanford University that supervised a technician that conducted all the
Agilent microarray experiments and data analysis: DH.
Table 2. Rosiglitazone-treated vs. untreated db/db mice.
Symbol RefSeq IDUGCluster NameScore(d)
Kcnip4NM_030265 Mm.160172Kv channel interacting protein 44.46 4.76
Ptch1 NM_008957Mm.228798 Patched homolog 16.673.32
Ryr1 NM_009109Mm.439745 Ryanodine receptor 1, skeletal muscle 5.273.17
Kcnk1 NM_008430Mm.10800 Potassium channel, subfamily K, member 16.38 2.84
Myo3b AK033795 Mm.99648|Mm.458853 adult male epididymis cDNA, RIKEN full-length enriched library,
Mmp3 NM_010809 Mm.4993Matrix metallopeptidase 34.702.03
Tnfsf18NM_183391 Mm.276823 Tumor necrosis factor (ligand) superfamily, member 183.82 1.86
Apln NM_013912 Mm.29262Apelin3.52 1.80
Anxa13 NM_027211 Mm.237985 Annexin A134.04 1.64
St3gal5NM_011375 Mm.38248ST3 beta-galactoside alpha-2,3-sialyltransferase 55.56 1.62
Col22a1XM_193814 Mm.322500 Collagen, type XXII, alpha 13.651.45
Col4a1 NM_009931 Mm.738Procollagen, type IV, alpha 1 4.981.26
Kras NM_021284 Mm.383182 V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog4.40 1.23
B4galnt1 BC022180 Mm.386762 Beta-1,4-N-acetyl-galactosaminyl transferase 17.83 1.44
CelNM_009885 Mm.236017 Carboxyl ester lipase6.31 1.38
PparbpNM_013634 Mm.12926Peroxisome proliferator activated receptor binding protein 3.68 1.29
Tnfrsf10bNM_020275Mm.193430 Tumor necrosis factor receptor superfamily, member 10b3.54 1.27
Asah2 NM_018830Mm.104900N-acylsphingosine amidohydrolase 2 4.381.21
Myh9NM_022410 Mm.29677Myosin, heavy polypeptide 9, non-muscle4.031.19
Bdkrb1NM_007539Mm.377078 Bradykinin receptor, beta 1
Hsf1NM_008296Mm.347444 Heat shock factor 1
Col23a1 AK162470Mm.154093|Mm.39236712 days embryo embryonic body between diaphragm region and neck
cDNA, RIKEN full-length enriched library, clone:9430076L03
Selected genes that were differentially expressed across rosiglitazone-treated and untreated db/db mice.
Rosiglitazone and the Heart
PLoS ONE | www.plosone.org10July 2008 | Volume 3 | Issue 7 | e2609
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PLoS ONE | www.plosone.org11 July 2008 | Volume 3 | Issue 7 | e2609