Article

Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage

Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America.
PLoS ONE (Impact Factor: 3.23). 06/2009; 4(6):e5954. DOI: 10.1371/journal.pone.0005954
Source: PubMed
ABSTRACT
to assess the cardioprotective properties of a blueberry enriched diet (BD).
Reactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and vegetables.
Following 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability transition (t(MPT)) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD resulted in a 24% increase (p<0.001) of ROS indexed t(MPT). The remaining animals were subjected to a permanent ligation of the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD rats (p<0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the myocardial area at risk of BD compared to CD rats (p<0.01). In the subgroup of rats, after coronary ligation the original diet was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or accelerated rates of post MI cardiac remodeling and MI expansion.
A blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the potential to attenuate the development of post MI chronic heart failure.

Full-text

Available from: Edward L Spangler
Blueberry-Enriched Diet Protects Rat Heart from Ischemic
Damage
Ismayil Ahmet
1
, Edward Spangler
2
, Barbara Shukitt-Hale
3
, Magdalena Juhaszova
1
, Steven J. Sollott
1
,
James A. Joseph
3
, Donal d K. Ingram
2,4
, Mark Talan
1
*
1 Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America, 2 Laboratory of
Experimental Gerontology, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America, 3 USDA-
ARS, Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts, United States of America, 4 Nutritional Neuroscience and Aging Laboratory,
Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana, United States of America
Abstract
Objectives:
to assess the cardioprotective properties of a blueberry enriched diet (BD).
Background:
Reactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use
synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the
interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and
vegetables.
Methods and Results:
Following 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability
transition (t
MPT
) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD
resulted in a 24% increase (p,0.001) of ROS indexed t
MPT
. The remaining animals were subjected to a permanent ligation of
the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD
rats (p,0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the
myocardial area at risk of BD compared to CD rats (p,0.01). In the subgroup of rats, after coronary ligation the original diet
was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial
echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or
accelerated rates of post MI cardiac remodeling and MI expansion.
Conclusion:
A blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the
potential to attenuate the development of post MI chronic heart failure.
Citation: Ahmet I, Spangler E, Shukitt-Hale B, Juhaszova M, Sollott SJ, et al. (2009) Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage. PLoS
ONE 4(6): e5954. doi:10.1371/journal.pone.0005954
Editor: Alicia J. Kowaltowski, Instituto de Quı
´
mica, Universidade de Sa
˜
o Paulo, Brazil
Received March 3, 2009; Accepted May 20, 2009; Published June 18, 2009
This is an open-access article distributed under the terms of the Creative Comm ons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This work was supported by Intramural Research Program of the NIH, Natuonal Institute on Aging, and by USDA. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: talanm@grc.nia.nih.gov
Introduction
Reactive oxygen species (ROS) appear to play a major role in
ischemia-related myocardial injury [1–8]. ROS formation is triggered
by depletion of ATP and an overload of Ca
2+
[2,4]. In turn, ROS
trigger a specific mechanism leading to a change in mitochondrial
membrane permeability and eventually to collapse of mitochondrial
membrane potential [for review see 9]. It was originally believed that
ROS formation occurs primarily, or even exclusively, during
reperfusion, when oxygen interacts with the damaged mitochondrial
respiratory chain. However, it is now proven in experiments with
isolated cardiomyocytes [10] and in the intact hearts [11] that ROS
occurs during ischemia from residual O
2
.
The detrimental role of ROS in ischemic and ischemic-
reperfusion injury of the myocardium has naturally led to
increased interest in antioxidants as therapeutic agents [12].
Unfortunately, thus far, attempts to use synthetic antioxidants to
block or attenuate the detrimental effects of ROS have produced
mixed and mostly negative results [13–15], and increasingly
attention has been given to natural products [12].
Blueberries contain anthocyanins, polyphenols and flavonoids
and appear to have the highest antioxidant capacity among fruits
and vegetables [16]. Blueberry extract and blueberry enriched
diets have been shown to reduce age-related behavioral and
neuronal deficits in rodents [17–20]. Blueberry diets have also
inhibited inflammatory cytokines in rat glial cells [21]. Previous
research has also indicated that blueberry supplemented rats
showed less hippocampal cell loss following experimentally
induced stroke [22]. Additionally, it has been reported that
blueberry supplemented animals given intra-hippocampal injec-
tions of the neurotoxin, kainic acid, showed decreased hippocam-
pal cell loss, reduced cytokine activation, decreased microglial
activation and reduced cognitive deficits as compared to non-
supplemented kainic acid treated-rats [23,24]. Therefore, it seems
that blueberry supplementation appears to have a tissue-protective
effect.
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The objective of the current study was to assess the
cardioprotective properties of a blueberry enriched diet (BD) in
the isolated rat cardiomyocytes and in a rat model of myocardial
infarction induced by permanent ligation of a coronary artery.
Previously we have used this model to demonstrate the
cardioprotective effects of erythropoietin,
ˆ
2
-adrenergic receptors
agonists, and some dietary manipulations [25–27].
Methods
Experimental Design
One hundred and fourteen 2-mo old male Fischer-344 rats
(Charles River Laboratories Inc., Wilmington, MA) were housed
and studied in conformance with the NIH Guide for the Care and
Use of Laboratory Animals, Manual 3040–2 (1999), with
institutional Animal Care and Use Committee approval. They
were randomly divided into two diet groups: regular food (control
diet, CD) or blueberry-enriched diet (BD), as described previously
[28]. After 3 months on their respective ad libitum diets, 7 CD and
7 BD rats were randomly selected for cardiomyocytes isolation and
mitochondrial permeability transition experiments. The remaining
animals were subjected to baseline echocardiography followed by
a surgical intervention. Following previous protocol [26], 45 rats
from each diet group were subjected to a permanent ligation of the
left descending coronary artery, and 5 rats from each diet group
were sham operated. Among surviving animals, some were
sacrificed 24 hrs following surgery, and their hearts were harvested
for histological assessment (n = 8 and 9 for BD and CD groups,
respectively). Remaining rats were randomly divided into four
groups, 9 animals each. Two groups were continued on the same
diet they received prior to surgery, and two groups were switched
to the opposite diet (blueberry/blueberry, BB; control/control,
CC; blueberry/control, BC; and control/blueberry, CB). Addi-
tionally, two sham operated groups that had been maintained on
blueberry enriched and control diets were continued on the same
diet (BBs and CCs). All animals were subjected to repeated
echocardiography after 2, 6 and 10 weeks following MI induction.
After the last echo test they were euthanized, and their hearts were
harvested for histological assessment.
Diet
The diets were prepared by Harlan Teklad (Madison, WI) using
a reformulated NIH-31 diet by adding 20 g/kg lyophilized
blueberry (BD) or 20 g/kg dried corn (CD). To prepare the 2%
blueberry supplemented diet, blueberries were homogenized in
water, centrifuged, lyophilized and added to the NIH-31 rodent
chow. The amount of corn in the control diet was adjusted to
compensate for the added volume [see 28 for more information on
diet preparation and composition]. The two diets were isocaloric
within the error margin attributable to routine variations in the
nutritional value of the natural ingredients of the NIH-31 diet
[29]. The food consumption was determined by subtracting the
weight of feed remaining at the end of the week. Based on their
observed food consumption during the study, rats maintained on
the BD consumed an average of 394 mg/day of lyophilized
blueberries, roughly equivalent to 4.4 g/day of fresh blueberries.
Left Ventricular Myocytes Isolation for Mitochondrial
Permeability Transition Experiments
Single ventricular myocytes were isolated via a previously
described technique with minor modifications [30]. Briefly, rats
were anesthetized with sodium pentobarbital, and hearts were
rapidly excised and perfused with 40 ml of nominally Ca
2+
-free
bicarbonate buffer gassed with 95% O
2
-5%CO
2
at 37 uC. The
composition of buffer was the following (in mmol/L): NaCl 116.4,
KCl 5.4, MgSO
4
1.2, NaH
2
PO
4
1.2, glucose 5.6, and NaHCO
3
26.2, pH 7.4. Hearts were continuously perfused with bicarbonate
buffer containing 0.1% collagenase type B, 0.04 mg/ml protease
XVI, and 0.1% BSA type V for 4 min and 50
mmol/L Ca
2+
was
added. After 10 min perfusion, the left ventricle was minced and
incubated in bicarbonate buffer containing 100
mmol/L Ca
2+
for
10 min at 37uC. Myocytes were then resuspended in HEPES
buffer with gradually increasing Ca
2+
concentration up to
1 mmol/L and kept at room temperature until use. The
composition of the HEPES buffer was the following (in mmol/
L): NaCl 137, KCl 4.9, MgSO
4
1.2, NaH
2
PO
4
1.2, glucose 15,
HEPES 20, and CaCl
2
1.0 (adjusted pH to 7.4). Cardiac myocytes
viability was typically 70 to 80%.
Confocal Microscopy and Determination of MPT-ROS
Threshold
Experiments were conducted as described previously [31],
employing a method to quantify the ROS-susceptibility for the
induction of MPT in individual mitochondria within cardiac
myocytes [32]. Briefly, isolated cardiac myocytes were exposed in
vitro to conditions that mimic oxidative stress by repetitive laser
scanning of a row of mitochondria in a myocyte loaded with the
membrane-potential-sensitive dye, tetramethylrhodamine methyl
ester (TMRM). This procedure results in incremental, additive
exposure of only the laser-exposed area to the photodynamic
production of ROS and consequent MPT induction. The
occurrence of MPT induction in a particular mitochondrion is
clearly identified by the instantaneous (,1 s) drop in its TMRM
fluirescence to background level signifying the immediate and
complete dissipation of mitochondrial membrane potential (DY)
causing the loss of its sequestered dye. Myocytes were loaded with
125 nM TMRM for at least 1 h at room temperature and imaged
with an LSM-510 inverted confocal microscope (Carl Zeiss Inc.,
Jena, Germany). Line scan images at 2 Hz were recorded from
mitochondria arrayed along individual myofibrils with excitation
at 568 nm and collecting emission at .560 nm, using a Zeiss
Plan-Apochromat 636/1.4 N.A. oil immersion objective, and the
confocal pinhole was set to obtain spatial resolutions of 0.4
mmin
the horizontal plane and 1
mm in the axial dimension. Images
were processed by MetaMorph software (Universal Imaging,
Downingtown, PA). The ROS threshold for MPT induction (t
MPT
)
was measured as the average time necessary to induce MPT in a
row consisting of ,25 mitochondria. Experiments were conducted
at 23uC. T
MPT
was measured in cardiomyocytes isolated from
hearts of rats exposed to CD or BD. The cardioprotective action of
insulin, which normally results in an enhancement of the MPT-
ROS threshold by ,35–40% [31], was used as a positive control
in the present experiments.
Coronary Artery Ligation
Rats were anesthetized with isoflorene (2% in Oxygen). The
surgical procedure was performed as previously described [25–27].
Briefly, in aseptic conditions a left thoracotomy was performed in
the fifth or sixth intercostal space. The rats were placed and
maintained on intermittent positive-pressure ventilation with 95%
02 and 5% CO2. A pericardiotomy was performed and the left
descending coronary artery was occluded by snaring and tying a
band of myocardium 2–3 mm to the left of the aorta and ligating it
with 5-0 silk sutures. Successful ligation was evident by blanching
of affected area of myocardium. After chest was surgically closed
the residual air was evacuated through a needle puncture. Animal
recovered from anesthesia in 10–15 minutes.
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Echocardiography
Echocardiography (Sonos 5500, a 12 MHz transducer) was
conducted under light anesthesia by sodium pentobarbital (30 mg/
kg, i.p) as previously described [25–27]. Briefly, parasternal long axis
views were obtained and recorded to ensure that the mitral and aortic
valves as well as the apex were visualized. Short axis views were
recorded at the mid-papillary muscle level. Endocardial area tracings
using the leading edge method were performed in 2D mode (short
and long axis views) from digital images captured on cineloop to
calculate end-diastolic and end-systolic left ventricular (LV) areas.
End-diastolic volume (EDV) and end-systolic volume (ESV) were
calculated by a modified Simpson’s method. Ejection fraction (EF)
was then derived as EF = (ED2ESV)/ED6100. MI size at the mid-
papillary muscle level was estimated from 2D short axis LV images at
end-diastole, and expressed as a percent of the LV endocardial
circumference. Infarct area was identified as a sharply demarcated
section of the LV free wall, which failed to thicken during systole. The
length of the akinetic part of the LV endocardial circumference was
measured from freeze-frame images at end-diastole. All measure-
ments were made by a single observer blind to the identity of the
tracings. All measurements were averaged over three to five
consecutive cardiac cycles. The reproducibility of measurements
was assessed in two sets of baseline measurements in ten randomly
selected rats, and the repeated measure variability did not exceed 5%.
Histological Acquisition
At 24 hours after MI surgery, a subgroup of rats was
euthanized, and their hearts were excised. Using a 16G catheter,
3 ml of 5% Evans blue (Sigma) was rapidly injected into the aorta
to distinguish the perfused area (blue staining) from the under-
perfused area (no-staining). The atria and great vessels were
dissected away from the heart; the heart was cut transversely into
four slices from the base to apex. A section from mid-papillary
muscle level was immediately stored in liquid nitrogen for later
histological analysis. The other three samples were incubated at
37uC with 4% triphenyltetrazolium chloride (TTC, Sigma) for
30 min to distinguish the infarct area (unstained) from the area at
risk (AAR; brick red stained) in the under-perfused area. All
images were analyzed using NIH Image software. MI size was
expressed as a percent of the under-perfused area. Myocardial
sections (5
mm thick) were obtained from the frozen sample and
stained by Hematoxylene & Eosin (H&E Sigma) and TUNEL
(Apop Tag, Chemicon International, Temecula, CA). Inflamma-
tory cells (neutrophils and macrophages) were counted and
averaged from the five different fields of the AAR in H&E stained
sections in each heart. Apoptosis was assessed from TUNEL
stained sections of the AAR from 10 random fields of the AAR.
Care was taken to include only clearly identified myocyte nuclei.
Ten weeks after surgery, the remaining rats were euthanized
and the hearts were isolated and weighed. The heart weight to BW
ratio (HW/BW) was calculated. Myocardial sections (5
mm thick)
were obtained from the mid-papillary muscle level. MI size was
measured from Masson’s trichrome staining sections as the
average of infarct area of LV epicardial and endocardial length
divided by LV circumferences and expressed as percent of LV.
Cardiomyocyte density was averaged from 10 random fields.
Cardiomyocyte diameters were measured as a shortest axes
through a nucleus.
Statistical Analysis
All data are expressed as the mean 6 SEM. Echo-derived
indices were compared pairwise via two-way ANOVA for
repeated measurements. Histological and mitochondrial perme-
ability transition data were assessed by one-way ANOVAs. Group
differences at specific time-points were tested by Bonferroni = s
post-hoc test. (Prism v3.0, GraphPad Software Inc., San Diego,
CA). Statistical significance was accepted as P,0.05.
Results
Effects of BD on body weight and heart prior to MI
Prior to MI induction, the food consumption and body weight
gain did not differ between animals on BD or Control (regular)
diet (CD). Starting average body weight was 115 63 g in CD rats
and 112 64 g in BD rats, and after three months reached 351 65
and 348 65 g in CD and BD, respectively. Echo derived
morphometric and functional indices of the heart measured after
three months of BD or CD prior to MI induction also did not
differ between rats maintained on the two diets (Table 1).
Assessment of MPT-ROS Threshold
Figure 1 presents the cardioprotective effects of BD in isolated
cardiac myocytes as indexed by the ROS threshold for MPT
induction (t
MPT
). Compared to animals on CD, the t
MPT
was
increased in BD rats by 24% (p,0.001). This increase was
comparable to an increase induced by insulin, used as a positive
control (32%, p.0.05). Addition of insulin to cardiomyocytes from
BD rats did not produce any significant increase over and above
the increase provided by BD (35%, p.0.05).
Mortality
Three months of BD had no significant effect on mortality
related to ligation of a coronary artery. Perioperative mortality
(during the surgery and first 24 hrs after the surgery) was 36% and
40% for the CD and BD groups, respectively (p.0.05). During 10
weeks of observation following MI induction, there was no further
mortality, although one CD rat was lost during the echocardiog-
raphy due to complications of anesthesia.
Table 1. Echocardiographic indices in rats after 3 months of
control or blueberry-enriched diets.
Blueberry Diet Control Diet P
Body Weight (g) 347 6335364ns
Interventricular septum (mm) 1.06 60.02 1.02 60.02 ns
Posterior wall thickness (mm) 1.6 60.02 1.58 60.03 ns
Aortic Diameter (mm) 3.22 60.03 3.25 60.03 ns
Left Atrial Diameter (mm) 3.64 60.09 3.69 60.11 ns
LAD/AoD 1.13 60.03 1.14 60.03 ns
Heart Rate (beat/min) 354 6535865ns
Fractional Shortening (%) 53.3 60.6 52.7 60.7 ns
Left Ventricular Mass (g) 0.97 60.01 0.96 60.01 ns
End Systolic Volume (
mL) 105 6310663ns
End Diastolic Volume (
mL) 268 6527065ns
Stroke Volume (
mL) 162 6316464ns
Ejection Fraction (%) 60.6 60.7 60.7 60.8 ns
E (cm/sec) 73.9 60.9 75.4 61.2 ns
A (cm/sec) 52.1 61.4 53.0 61.2 ns
E/A 144 60.05 140 60.03 ns
Cardiac Index (
mL/g/min) 166 6 41636 5ns
doi:10.1371/journal.pone.0005954.t001
Cardioprotection by Blueberry
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Page 3
24 hrs following coronary ligation
Figure 2 illustrates the damage to the myocardium 24 hrs after
induction of MI in BD and CD animals. Areas at risk (AAR) did
not differ between hearts from CD and BD rats. However, the
average MI size, calculated either as fraction of AAR or as a
fraction of LV, was 22% smaller in BD than in CD rats (p,0.05).
TUNEL(+) cardiomyocytes in the AAR are presented in
Figure 3. Among CD rats, 9% of cardiomyocytes in the AAR
were stained positively for apoptosis (8248 per 10
5
cardiomyocytes,
as shown at Fig. 3), while among BD rats the number of
TUNEL(+) cardiomyocytes was reduced to 2% of the total
number of cardiomyocytes (p,0.05).
The density of inflammatory cells in the AAR is illustrated in
Figure 4. In the AAR of BD rats, there were 40% less
inflammatory cells (neutrophiles and macrophages) than in the
AAR of CD rats (p,0.05).
To summarize, 24-hrs following coronary ligation, the BD
rats had smaller MI and significantly reduced necro-apoptosis and
inflammation in the myocardium surrounding MI than CD rats.
LV remod eling and function during 10 weeks following
MI
The CD and BD rats that survived surgery and were not
selected for the acute, 24-hr study were randomly divided into four
groups (n = 9 at each group): CD rats that continued on the
control diet (CC), BD rats that continued on blueberry enriched
diet (BB), BD rats that were switched after surgery from blueberry-
enriched to the control diet (BC), and CD rats that were switched
from the control to the blueberry-enriched diet (CB). Two groups
of sham operated animals were maintained on the same diet as
before surgery (BBs and CCs).
Figure 5 illustrates the results of Echo measured progression of
LV volumes and MI expansion and functional decline in these
four groups from baseline (pre-MI, 0 wks) to 10 weeks following
MI induction. The MI size in the original BD groups (BB and BC)
Figure 1. Cellular mechanism of cardioprotection. Blueberry-
enriched diet reduces the MPT susceptibility to ROS (t
MPT
) in cardiac
myocytes. The t
MPT
was measured in cells isolated from the hearts of
rats, fed for 3 months a control (CD) or blueberry-enriched (BD) diet,
before or after exposure to insulin 30 nM (as the positive control) for
20 min. * - p,0.01 vs Control.
doi:10.1371/journal.pone.0005954.g001
Figure 2. MI 24 hrs following permanent coronary ligation. On the left are samples of the hearts of BD and CD rats subjected to TTC staining:
white - necrotic area; red - area at risk (AAR); blue - viable, well-perfused myocardium. On the right is the graph comparing the AAR expressed as a
percent of LV and MI size expressed as a percent of LV or AAR in BD and CD rats. *- p,0.01 vs. CD.
doi:10.1371/journal.pone.0005954.g002
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Figure 3. Apoptosis in the myocardial AAR 24-hrs following permanent coronary ligation. TUNEL staining. Number of TUNEL positive
cardiomyocyte nuclei are normalized for 10
5
cardiomyocytes. *- p,0.01 vs CD.
doi:10.1371/journal.pone.0005954.g003
Figure 4. Inflammation in the myocardial AAR 24-hrs following permanent coronary ligation. Hematoxylene & Eosin staining. *- p,0.01 vs CD.
doi:10.1371/journal.pone.0005954.g004
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was significantly smaller than in CD groups (CC and CB) at each
time-point. When BB and CC groups were compared with each
other, the ANOVA revealed significant group and time effects, as
well as a significant time x group interaction, indicating that in CC
group the MI size expanded at a faster rate than in the BB group.
However, when BB rats were compared with CB and CC with BC
groups, no statistically significant time x group interactions were
found. Comparison of Aswitched@ groups (CC vs CB and BB vs
BC) showed a trend to slow or to accelerate the MI expansion
depending on addition or removal of blueberry, respectively;
however the ANOVA derived group x time interaction
approached significance (p,0.07) only for the comparison
between BB and BC groups, i.e., removal of blueberry from the
diet after MI induction accelerated the MI expansion.
The rate of EDV expansion was similar among all groups.
However, the statistical evaluation of ESV expansion showed a
significant time x group interaction between BB and CC groups,
indicating that in CC group the ESV expanded at an accelerated
rate compared to BB. However, the time x group interaction was
not significant when the BB group was compared with CB and CC
was compared with BC groups. Comparison of switched groups
showed the tendency of removal or addition of blueberry to the
diet to affect the rate of ESV expansion, but the time x group
interaction did not reach statistical significance.
Two weeks after MI induction, the EF was significantly higher in
BD groups (BB and BC) than in CD groups (CC and CB). In
subsequent tests (at 6 and 10 weeks), the EF remained higher in BB
rats compared to both CD groups. ANOVA revealed a significant
time x group interaction between BB and CC groups, an indication
of the difference in the rate of EF decline; however, the interaction
was not statistically significant when CC was compared with the BC
condition and BB was compared with the CB condition. The
interactions between Aswitched@ groups (CC vs CB and BB vs BC)
were also not statistically significant. Nevertheless, the differences
between BC and CB groups that were statistically significant at the
second week disappeared in the subsequent tests due to elevation of
EF in CB and its reduction in BC.
Figure 6 represents the progression of echo-derived indices of
posterior wall thickness (PWth/EDV) and LV mass. All MI
animals had significantly thinner PWth normalized for EDV than
sham operated animals, however the thinning of the posterior wall
was attenuated in BB group compared to CC rats at all time-
points. In animals fed blueberry after MI induction (BB and CB),
the PWth progressively reduced (p,0.05 for group x time
interaction), while in CC and BC groups the PWth did not
change or even increased. LV mass was higher in animals fed
blueberry-enriched diet following MI (BB and CB) than in the CC
and BC conditions.
To summarize, comparison of experimental groups, those for
which the diet was continued after MI induction with those, for
which the diet was switched to the opposite condition, suggests
that blueberry-enriched diet might attenuate the post MI cardiac
remodeling and MI expansion even when started after MI
induction.
Figure 5. LV remodeling and MI expansion. Echo derived EDV, ESV, EF, and MI size during 10 weeks of post MI observation. Data on sham
operated groups (BBs and CCs) is shown for refernce and is not included in statistical comparison. *- p,0.05 vs. CC; # -p,0.05 vs CB.
doi:10.1371/journal.pone.0005954.g005
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Figure 7 represents the results of histological assessment of the
hearts harvested after 10 weeks of post MI observation.
Histologically measured MI size was highly correlated (r
2
= 0.77)
with MI size obtained from the last (10 weeks) Echo test. Ten
weeks following coronary ligation, the average MI size in both BD
groups was significantly smaller than in CD groups (24%
difference, p,0.05). The HW/BW ratio was significantly higher
in BB group than in both CC and BC. Cardiomyocyte density was
reduced in all post-MI animals, but was significantly better
preserved among groups fed the blueberry-enriched diet following
MI (BB and CB) than in control diet groups (CC and BC). The
measurements of single cardiomyocyte diameters did not reveal
any significant differences among groups.
Discussion
The epidemic explosion in the incidence of chronic heart failure
(CHF) in Western society resulted in great human suffering and
Figure 6. LV hypertrophy. Echo derived measurements of posterior wall thickness normalized for EDV (Pwth/EDV) and LV mass during 10 weeks of
post MI observation. Data on sham operated groups (BBs and CCs) is shown for refernce and is not included in statistical comparison. * - p,0.05 vs
CC; { -p,0.05 vs BC.
doi:10.1371/journal.pone.0005954.g006
Figure 7. Histological assessment 10 weeks following coronary ligation. From left to right MI size, heart weight normalized for body weight
(HW/BW), cardiomyocyte density, and average cardiomyocyte diameter. Data on sham operated groups (BBs and CCs) is shown for refernce and is not
included in statistical comparison. * - p ,0.05 vs CC; # -p,0.05 vs CB; { -p,0.05 vs BC.
doi:10.1371/journal.pone.0005954.g007
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has produced tremendous burden on health care and social
services. Since the majority of the CHF develops consequently to
MI, the most effective CHF prevention is the prevention of MI
from occurrence or minimization of myocardial damage after MI
occurrence. The former is targeted through changing life style and
diet, leading to reduction of cholesterol and other risk factors,
strategies whose arsenal was recently expanded by using
pharmacological tools such as statins [33–35]. The latter is
achieved by rapid and vigorous revascularization and early
pharmacological therapy. However, a large fraction of heart
attacks is not associated with increased level of cholesterol and its
etiology, and thus means of prevention, remain unclear.
Therefore, dietary or behavioral manipulations, which could
increase the myocardial resistance to ischemic damage, are
extremely valuable. Thus far, only exercise has been shown
experimentally to increase the resistance of the myocardium to
ischemic damage [36–38]. We have recently reported, in
experiments on rats, that dietary regimen using intermittent,
every-other-day fasting significantly increased the myocardial
tolerance to ischemic damage [27]. In the present study we have
shown for the first time that a regular dietary supplement of
blueberry extract attenuates coronary ligation-induced myocardial
damage.
Three months of blueberry-enriched diet did not affect food
intake or body weight of rats. It also did not have any effect on
morphometric or functional echocardiographic indices of the
heart. However, experiments with single cardiomyocytes isolated
from the LV of BD and CD rats showed significantly increased
threshold of ROS-susceptibility for the induction of mitochondrial
permeability transition with the blueberry diet. In other words, at
the mitochondrial level, the tolerance of cardiomyocytes to
oxidative stress was increased with this dietary supplement.
The results of our in vivo experiments showed that in the
myocardium of rats maintained on a blueberry-enriched diet,
24 hrs after coronary ligation the number of cardiomyocytes in the
area at risk stained positively for apoptosis was 4.5 fold reduced
compared to that in rats on the control diet; the number of
inflammatory cells was reduced almost in half; and MI size
resulting from a coronary ligation was 22% smaller. A subset of
rats, in which LV remodeling was followed after induction of MI
via serial echocardiography, also showed a reduction in original
myocardial damage (two weeks after surgery) in BD rats which was
naturally translated into significant attenuation of post MI LV
remodeling: 10 weeks after coronary ligation rats maintained on
blueberry-enriched diet had 14% smaller EDV, 26% smaller ESV,
and 82% higher EF than rats fed a control diet. Moreover, 10
weeks after coronary ligation the resulting MI in rats maintained
on blueberry-enriched diet prior to and post MI was 31% smaller
than in rats maintained on control diet. The positive effect of a
blueberry enriched diet post-MI was also confirmed by the
assessment of cardiomyocyte density in the myocardium. The
reduction of cardiomyocyte density observed 10 weeks after
induction of MI in rats maintained on control diet (CC) and
reflecting usual loss of cell associated with chronic heart failure
[39] was significantly attenuated among BB and CB groups, but
was not found in BC group.
This study was designed as a proof of concept and was not
intended to analyze the signaling pathways responsible for
reduction of necro-apoptosis and inflammation in the myocardium
after MI in animals on blueberry-enriched diet. The one thing that
could be stated with certainty on the basis of our finding is that this
signaling was associated with increased mitochondrial permeabil-
ity transition threshold. Most probably, based on our previous
extensive research, the effect was mediated through a number of
possible kinases (31), however the ROS scavenging mechanism
also cannot be excluded (32). Some inferences can also be made on
the basis of previous studies. Fruits and vegetable rich in
polyphenolics are known to delay or reverse the deleterious effects
of aging on neurocommunication and behavior [see 40 for
review]. Blueberry extract was shown to significantly inhibit the
lipopolysacharide-induced inflammatory response in brain mi-
croglia. This effect was due to down-regulation of iNOS mRNA
and suppression of iNOS proteins, i.e., blueberry extract may
inhibit one of the primary steps in the inflammatory stress pathway
[41]. In the same model (BV-2 mouse microglial cells), blueberry
extract treatment also inhibited COX-2 mRNA and protein
expression [41], which is known to be associated with proin-
flammatory stimuli [42,43], and the proinflammatory cytokines
IL-1b and TNF-a [41]. Extrapolating from these findings, it is
reasonable to assume that in the present study similar oxidative/
inflammatory stress signals may be operational. Much more
difficult is to explain the reduced necro-apoptosis in our model.
Blueberry-enriched diet is known to have some anti-cancer
properties [44,45] and one of the anti-cancer mechanisms is the
activation of apoptosis [46].
An additional promising direction in understanding the tissue-
protective properties of blueberry might be in exploring the
possible effects of BB treatment on glucose metabolism and,
specifically, its insulin-like properties [47,48]. Tissue-protective
properties of insulin are expanded well beyond its control of
hyperglycemia. It suppresses the production of TNF-a, IL-6, and
other pro-inflammatory cytokines and enhances the synthesis of
anti-inflammatory cytokines, IL-4 and IL-10 [for review see 49].
Specifically, cardioprotective effects of insulin were demonstrated
clinically [50] and in experimental models [51]. Multiple
mechanisms of cardioprotection were proposed: coronary dilata-
tion, anti-inflammatory and anti-apoptotic; however, the most
fundamental to cardioprotection remained insulin = s ability to
stabilize the mitochondrial permeability transition (52). Thus, it is
conceivable that increased myocardial tolerance to hypoxia after
prolonged feeding with a blueberry-enriched diet, as well as
elevation of mitochondrial permeability transition threshold shown
in our study, are due to up-regulation of insulin sensitivity.
Therefore, the Apreventive@ part of our experiment clearly
demonstrated that blueberry-enriched diet increased tolerance to
ischemic damage of myocardium in a rat model of a permanent
coronary ligation. The Atreatment@ part of the study, however,
while less obvious, is also very promising. The switching of the diet
immediately after coronary ligation to its opposite, i.e., blueberry-
enriched to control and control to blueberry-enriched, revealed a
tendency to affect the post MI progression of LV remodeling by
accelerating or attenuating it respectively. This outcome indicates
the possibility of adding blueberry supplementation to the
therapeutic arsenal which should be evaluated further in
experimental models of CHF.
In summary, in experiments examining rats maintained on a
blueberry enriched diet, we found increased myocardial tolerance
to ischemic damage. At a cellular level, the blueberry diet
increased cardiomyocyte survival by elevating the mitochondrial
permeability transition ROS threshold. In in vivo experiments this
diet reduced the size of myocardial infarction induced by a
permanent coronary ligation by attenuating necro-apoptosis and
inflammation in the area at risk. The beneficial effects of the
blueberry diet were extended after coronary ligation by continuing
the attenuation of the post-MI LV remodeling and MI expansion.
The only non-pharmacological intervention capable to produce
similar cardioprotective effect so far was the intermittent fasting
(27). To the best of our knowledge, this is the first demonstration of
Cardioprotection by Blueberry
PLoS ONE | www.plosone.org 8 June 2009 | Volume 4 | Issue 6 | e5954
Page 8
the effectiveness of a readily available natural product in
acceptable quantity to significantly limit myocardial damage
resulted from induced ischemia.
Author Contributions
Conceived and designed the experiments: IA BSH SJS JJ DKI MT.
Performed the experiments: IA ES MJ. Analyzed the data: IA ES MJ SJS
DKI MT. Contributed reagents/materials/analysis tools: BSH. Wrote the
paper: IA BSH MJ SJS JJ DKI MT.
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  • Source
    • "Our results suggest that this BE may have an antioxidant effect in hypercholesterolemic rats because of the presence of polyphenols that are preferentially incorporated into membrane lipid bilayers and act as hydrogen donors, trapping free radicals, inhibiting the formation of lipid radicals (Soobrattee et al., 2005). The antioxidant effects of blueberries have also been reported by studies using cellular and animal models of oxidative stress (Ahmet et al., 2009; Sellappan et al., 2002 ). Oxidative modifications in proteins, lipids and DNA are considered to be among the molecular mechanisms leading to endothelial dysfunction and atherosclerosis (Aydin et al., 2009). "
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    • "Owing to richness in antioxidant, it protected against reactive oxygen species (ROS) -induced ischemic injuries. Development of post-myocardial infarction and chronic heart failure was notably blunted [16]. The nutritional effect of V. angustifolium consumption was assessed on the markers of oxidative stress, inflammation and endothelial function in 18 male volunteers with risk factors for cardiovascular disease. "
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    • "The data showed that both grape skin and flesh were equally cardioprotective as evidenced by improved recovery from ischemia, smaller infarct size and reduced MDA content in the heart as compared to the control group [220]. Blueberry supplementation in rats that underwent coronary ligation induced myocardial infarction showed a 22% improvement in survival rates as well as reduction in the expansion of myocardial infarcts [221,222]. Mechanistically, this might be achieved by stimulating the synthesis of NOS via the Akt pathway, as was the case in chick embryonic cardiomyocytes stimulated with grape seed extract, thereby protecting from I/R induced cell death [223]. "
    [Show abstract] [Hide abstract] ABSTRACT: Numerous studies have demonstrated the importance of naturally occurring dietary polyphenols in promoting cardiovascular health and emphasized the significant role these compounds play in limiting the effects of cellular aging. Polyphenols such as resveratrol, epigallocatechin gallate (EGCG), and curcumin have been acknowledged for having beneficial effects on cardiovascular health, while some have also been shown to be protective in aging. This review highlights the literature surrounding this topic on the prominently studied and documented polyphenols as pertaining to cardiovascular health and aging.
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