Short and Long Term Effects of (-)-Epicatechin on Myocardial Ischemia Reperfusion Injury

Katrina Go Yamazaki*, Diego Romero-Perez*, Maraliz Barraza-Hidalgo*, Michelle Cruz*, Maria Rivas*, Brenda Cortez-Gomez*, Guillermo Ceballos#, Francisco Villarreal*

*University of California, San Diego, Department of Medicine, San Diego, CA, #Escuela Superior de Medicina del Instituto Politécnico Nacional, Departamento de Farmacologia, Mexico

City, Mexico

Running head: Cardioprotection by epicatechin

Francisco Villarreal M.D. Ph.D. Professor of Medicine UCSD Cardiology 9500 Gilman Dr. 0613J BSB 4028 La Jolla, CA 92093 tel (858) 534-3630 lab (858) 534-1314 fax (858) 534-0522 email: fvillarr@ucsd.edu

Articles in PresS. Am J Physiol Heart Circ Physiol (June 20, 2008). doi:10.1152/ajpheart.00413.2008

Copyright © 2008 by the American Physiological Society.

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Abstract

Epidemiological studies have shown a correlation between flavonoid-rich diets and improved

cardiovascular prognosis. Cocoa contains large amounts of flavonoids, in particular flavanols

(mostly catechins and epicatechins). Flavonoids possess pleiotropic properties that may confer

protective effects to tissues during injury. We examined the ability of epicatechin to reduce short

and long-term ischemia reperfusion (IR) myocardial injury. Epicatechin (1mg/kg/day) pre-

treatment (Tx) was administered daily via oral gavage to male rats for 2 or 10 days. Controls

received water. Ischemia was induced via a 45 min coronary occlusion. Reperfusion was

allowed until 48 h or 3 weeks while Tx continued. We measured infarct (MI) size (%),

hemodynamics, myeloperoxidase (MPO) activity, tissue oxidative stress, and matrix

metalloproteinase-9 (MMP-9) activity in 48 h groups. Cardiac morphometry was also evaluated

in 3 week groups. With 2 days of Tx, no reductions in MI size occurred. After 10 days, a

significant ~50% reduction in MI size occurred. Epicatechin rats demonstrated no significant

changes in hemodynamics. Tissue oxidative stress was significantly reduced in the epicatechin

group vs. controls. MMP-9 activity demonstrated limited increases in the infarct region with

epicatechin. By 3 weeks, a significant 32% reduction in infarct size was observed with Tx,

accompanied with sustained hemodynamics and preserved chamber morphometry. In

conclusion, epicatechin Tx confers cardioprotection in the setting of IR injury. The effects are

independent of changes in hemodynamics, are sustained over time, and are accompanied by

reduced levels of indicators of tissue injury. Results warrant the evaluation of cocoa flavanols as

possible therapeutic agents to limit ischemic injury.

Keywords: myocardial infarction, catechin, epigallocatechin, cocoa, chocolate

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Introduction

Known risk factors for the development of cardiovascular disease (CVD) include hypertension, a

poor diet, obesity, lack of physical activity, smoking and diabetes. Diet has been established as

one of the most important lifestyle factors that can strongly influence the incidence of CVD.

Polyphenols are widely distributed in plants and are known as flavonoids (30). Flavonoids can

be divided into 4 classes, flavones, flavanols, flavonols and anthocyanins (34). All 4 classes

have in common phenolic rings with variations in the number and arrangement of the hydroxyl

groups as well as the nature and extent of alkylation and/or glycosylation of these groups (34).

Flavonoids have been long recognized for their free radical scavenging activity (34).

Epidemiological evidence indicates a negative correlation between consumption of flavonoid

rich foods or beverages and incidence of cardiovascular disease (CVD) (20, 23, 25, 26). Wine

has garnered attention because its polyphenol concentration is relatively high and may be partly

responsible for the prolonged longevity associated with the “French Paradox” (2, 13). However,

cocoa powder contains by far the largest amount of flavonoids, in particular the subtype known

as flavanols (up to 10% flavanols by weight) (11). Main flavanols in cocoa are catechins and

epicatechins present in mono- or multimeric forms (11, 26). Interest in the beneficial effects of

cocoa derived flavonols emerged from observations of Kuna Indians living off Panama. Kuna

islanders have a very low incidence of CVD in particular, hypertension. The low incidence of

CVD is related to environmental factors and not genetic since protection is lost when Kuna

Indians migrate to mainland (21, 29). Kuna islanders drink large amounts of cocoa rich in

flavanols vs. mainland migrants. Studies relate the low indices of CVD to the consumption of

cocoa beverages and not other factors such as fruit or protein, physical activity or alcohol

intake. In addition to their free radical scavenging activity, there are other clues as to the

mechanisms that may explain cocoa effects. Consumption of flavanol rich cocoa leads to

vasodilation which can be reversed by the use of the NO synthesis inhibitors (12, 18). Other

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effects of flavanoids/flavanols include the inhibition of platelet adhesion, low-density lipoprotein

(LDL) oxidation, inflammation, reactive oxygen species generation, eicosanoid synthesis and

insulin resistance (for reviews see, (10, 33). Recent reports link the effects of cocoa to

epicatechin. The ingestion of epicatechin in humans can reproduce the hypotensive, antioxidant

and insulin sensitizing effects of cocoa (35).

In spite of the identification of many candidate compounds, which have the potential to

reduce infarct size, none have demonstrated the capacity to retain the effect over the long-term

(3, 24, 45). Most pharmacological agents studied to date have only managed to postpone cell

death. On the basis of the reported pleiotropic actions of flavanols, it is reasonable to propose

that on the short-term, these agents may ameliorate tissue injury secondary to ischemia

reperfusion (IR) and that in the long-term their continued actions may lead to improved

outcomes. We hypothesize that the administration of epicatechin to rats leads to a sustained

reduction in infarct size and that this phenomena is secondary to a suppression of mediators of

reperfusion injury.

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Materials and Methods

Animal Groups and Epicatechin treatment

Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 250-300 g (~8 weeks of

age) were used. All procedures were approved by the Institutional Animal Care and Use

Committee and conform to published NIH guidelines for animal research. Epicatechin (1

mg/kg/day; Sigma-Aldrich, St. Louis, MO) or vehicle (water) was administered by oral gavage

once/day beginning either 2 or 10 days before thoracotomy and continuing until the time of the

terminal study (48 h or 3 weeks). This dose was noted in humans to reproduce the vascular (i.e.

hypotensive) effects of dark chocolate (35). Groups of normal (i.e. no surgery was performed in

these groups) animals were treated with water (n=6) or epicatechin (n=6) for 10 d and were

used solely to evaluate the effects of epicatechin on baseline hemodynamics. For 48 h studies,

groups included IR (n=21), IR + 2 d epicatechin (n=7) and IR + 10 d epicatechin (n=15) and

were used to measure infarct size. Subgroups of 10 d treatment (n=8 per each) were used to

measure hemodynamics. For 48 h biochemical determinations subgroups included IR (n=8) and

IR + 10 d epicatechin (n=7). The 48 h timepoint was selected to be able to clearly distinguish

regions of necrotic tissue from viable myocardium. For 3 week studies, groups included sham

(n=5), sham + 10 d epicatechin (n=4), IR (n=7) and IR + 10 d epicatechin (n=8). Sham animals

underwent a thoracotomy but were not subjected to IR.

Ischemia/ Reperfusion Surgery

Animals were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10

mg/kg), intubated, and positive-pressure ventilated. A left thoracotomy was then performed. In

IR animals, the left anterior descending coronary artery was ligated for 45 min, released and the

suture left in place as a point of reference. The chest was closed in layers and animals allowed

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to recover. Successful occlusion and reperfusion was verified by visual inspection of left

ventricle (LV) color.

Hemodynamics

For hemodynamic measurements, animals were anesthetized with 5% isoflurane and

maintained with 1-2% isoflurane. The right carotid artery was exposed via a neck dissection.

Carotid and LV pressures were acquired using a micromanometer (2 French, 140 cm; Millar

instruments Inc. Houston, TX) introduced via the carotid artery. Hemodynamics were digitally

recorded for subsequent analysis using WINDAQ software (version 2.15, DATAQ Instruments

Inc).

Terminal Studies

Hearts were excised and weighed. In 48 h hearts, the area at risk (AAR) was determined by the

reocclusion of the snare and infusion of trypan blue into the cannulated aorta. Hearts was

sectioned into five 2 mm rings and stained using triphenyltetrazolium chloride. Computer

assisted image analysis was used using blinded operators. Results are expressed as infarct

area (IA) as a function of the area at risk (AAR). For 3 week studies, hearts were processed as

above and sectioned to identify the infarct (i.e. scar) area. The images of unfixed, stained rings

were also used to measure internal and external chamber diameters, and anterior and septal

wall thicknesses.

Tissue collection

The 48 h excised hearts were perfused with cold saline to remove blood. LV free wall was

separated, and divided into two parts, the infarct region and the border zone. The right ventricle

was also taken to be used as the remote or non-ischemic region.

Gelatin zymography

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Heart samples ( 50 mg) were homogenized in 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.2 mM

EDTA, 25% glycerol, 100 µg/ml phenylmethylsulfonyl fluoride, and 0.2 kallikrein inhibitory

units/ml aprotinin. Samples (10 µg of protein) were analyzed by SDS-PAGE as described (14).

An internal control (human MMP-2/MMP-9, Chemicon, Temecula, CA) was loaded to normalize

between gels. Bands of gelatinolytic activity were digitally quantified (Kodak 1D, Eastman

Kodak, Rochester, NY).

Myeloperoxidase (MPO) Assay

The MPO assay was performed as previously described with modification (14). Tissue samples

were homogenized in MPO lysis buffer (50 mmol/L KH2PO4 pH 6.0, 0.5%

hexadecyltrimethylammonium bromide) and incubated on ice for 30 min. Following

centrifugation, the supernatants were reacted with 0.4 mmol/L tetramethylbenzidine (Sigma)

and 0.006% H2O2 in 50 mmol/L phosphate at pH 6.0. Absorbance was monitored and MPO

activity expressed as relative units/min.

Glutathione (GSH/GSSG) Assay

Tissue samples were homogenized in ice cold homogenization buffer (154mM KCl, 5mM

diethylenetriaminepentaacetic acid (DPTA), and 0.1 M potassium phosphate, pH 6.8). After

centrifugation, an aliquot was removed for protein determination using the bicinchoninic acid

method (Pierce Chemical Co., Rockford, IL). Immediately after an aliquot was taken, one

volume of cold acid buffer (40mM HCl, 10mM DPTA, 20mM ascorbic acid, and 10%

trichloroacetic acid (TCA) was added to one volume of homogenate. The suspension was

centrifuged and the resulting supernatant solution was centrifuged through a 0.45-µm

microcentrifuge filter (Millipore Corp., Bedford, MA). Reduced and oxidized glutathione, GSH

and GSSG respectively, levels were determined as previously described by Senft et al., using

the fluorophore o-phthalaldehyde (OPA) (37).

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Statistical Analysis

Results are expressed as mean ± S.E.M. Comparisons between means were analyzed, as

appropriate, by student’s t-tests or one-way ANOVA followed by Bonferroni t-test. A value of

p<0.05 was considered statistically significant.

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Results

Hemodynamics

Hemodynamic parameters were measured in normal, and normal + 10 d epicatechin rats.

Epicatechin treatment only raised heart rate vs. normal. Values recorded 48 h after IR (table 1)

demonstrate no significant changes in either heart rate, LV end-diastolic/peak systolic pressure,

or mean aortic pressure between IR groups. In 3 week studies (table 2), hemodynamic

parameters were recorded in sham and IR animals and results were comparable between

untreated vs. treated groups.

Infarct size and morphometry

For the 2 day pre-treatment study, the AAR was 50±4% and 56±2% and were statistically not

different between the IR and IR + 2 d epicatechin groups respectively. The IR group and the IR

+ 2 d epicatechin group also had comparable (p>0.05) infarct areas (infarct area/area at risk) of

41±8% and 34±4% respectively. In the 10 day pre-treatment study (figure 1), infarct area was

52 ± 2% vs. 26 ±3% (p<0.0001). There was no difference in the area at risk, 47±3% and 49±2%,

between the two groups. Figure 2 summarizes the 3 week results. The IR animals infarct area

was 50±6% vs. 34±6% (p<0.05). The two groups had similar area at risks (48±4% and 45±4%).

As shown in table 2, IR animals treated with epicatechin demonstrate comparable post IR

morphometric changes to those of 3 week untreated IR animals. Anterior wall vs. septal wall

thicknesses yielded a statistical difference only in the vehicle treated IR group. Changes in

infarct size and morphometry appear independent from altered hemodynamics since

epicatechin did not modify these in a manner that would explain the observed results. No

differences in survival rates were noted between any groups.

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MPO Activity

We assessed the capacity of epicatechin to alter inflammatory cell infiltration in the myocardium

by measuring MPO activity using a colorimetric assay (14). As shown in figure 3, in both the 48

h IR and IR + 10 d epicatechin groups, MPO in the infarct region was higher when compared to

the RV (p<0.05), indicating infiltration of inflammatory cells into the border zone and ischemic

regions. No differences in MPO levels were noted between IR and IR + 10 d epicatechin at any

of the analyzed regions.

Myocardial Oxidative Stress

We determined if epicatechin ameliorates the generation of oxidative stress within the

myocardium of rats subjected to IR. Results are shown as a ratio of oxidized glutathione to

reduced glutathione (GSSG/GSH), which is a measure of total tissue oxidative stress. Oxidative

stress was significantly increased (p<0.05) in the infarct region compared to the RV in the IR

group, and this increase was significantly attenuated (p<0.05) in the border zone and ischemic

regions of the IR + 10 d epicatechin group (figure 4).

Zymography Results

We determined if epicatechin decreases levels of MMP-2 and/or MMP-9 activity. Gelatin

zymography of tissue homogenates from the RV, border, and infarct region only revealed bands

corresponding to 92 kDa MMP-9 and 75 kDa MMP-2 (Figure 5a). The 86 kDa MMP-9 and 72

kDa MMP-2 were not visible. Densitometric analysis revealed that 92 kDa MMP-9 levels were

significantly increased (p<0.01) in the infarct region compared to the RV in the IR group, but not

in the IR + 10 d epicatechin group (Fig. 5B). As observed in the gel image and by densitometric

analysis, no notable differences were identified in 75 kDa MMP-2 levels.

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Discussion

Our results indicate that 10 d pre- and continuous epicatechin Tx significantly reduced

infarct size in rats 48 h after IR injury. The smaller infarct size observed in treated animals is

accompanied by significant reductions in tissue oxidative stress at the infarct region. These

results were accompanied by a preservation of local inflammation and lesser degrees of MMP-9

activity in treated, injured myocardium. More importantly, a sustained reduction in infarct size

was observed three weeks after IR injury.

Since the length of exposure to cocoa flavanols appears to determine bioactivity, we first

examined if short term pre-treatment with epicatechin could confer cardioprotective effects. Two

days of epicatechin pre-treatment did not significantly reduce MI size. These results contrast to

those of others where cardioprotection with flavanols can be generated immediately upon

treatment. Wang et al demonstrated the capacity of a synthetic flavanol, administered at 5

mg/kg just prior to reperfusion to reduce MI size in sheep while also decreasing ROS

generation, improving coronary blood flow and NO metabolite levels (42). Aneja et al reported

similar effects with epigallocatechin-3-gallate (EGCG) at 10 mg/kg given just prior to reperfusion

to rats (1). An important difference arises when comparing our 2 day results with these studies.

Flavanol doses used by these investigators were significantly higher than those of our study

(~5-10 fold). It is well known that higher doses of flavanols attain greater antioxidant effects

thus, effectively mitigating (with increased efficacy) the damaging effects of ROS during IR. This

observation is supported by data that indicates that serum concentrations of the order of ~0.01

mM allow flavonoids to act as effective in vivo ROS scavengers (17). The serum concentration

reported in animals treated with 1 mg/kg/day would only approximate 1/10th of this. The fact that

short term treatment with 1 mg/kg/day epicatechin does not confer cardioprotection suggests

that changes in myocardial gene expression and/or protein levels may need to develop with

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longer Tx times (11, 40). Alternatively, changes in epicatechin metabolism may be required to

generate IR protection (11).

In studies performed in patients suffering from hypertension, sustained reductions in

blood pressure were observed when Tx was given with high flavanol cocoa for at least 7 days

(15). Our results demonstrate that in animals with 10 days of pre- and continuous epicatechin

Tx, a significant, ~50%, reduction in infarct size was observed. A potential mechanism by which

epicatechin may decrease MI size is a reduction in afterload. Ten days of epicatechin treatment

in normal and IR animals did not reduce blood pressure and thus, changes in afterload fail to

explain the observed effects.

In the setting of myocardial ischemia, the lack of oxygen and associated ATP production

can lead to cell death (necrosis). Upon reperfusion, the reintroduction of blood flow to the

previously ischemic area leads to the activation of an injury pathway associated with

reoxygenation (4, 45). Reperfusion injury activates an inflammatory response, which can lead to

loss of contractile function, arrythmias, and may promote cell death in ischemic myocytes (4).

Major mediators of IR injury are neutrophils, oxygen radicals, proteases, and calcium overload

(4, 45). Compounds that target “single” mediators of IR related injury, such as antioxidants,

have shown to improve indicators of tissue injury in animal models. However, the translation of

these improvements into the clinical setting has not been successful (3, 24, 45). Similar failures

have been observed with strategies that target tissue inflammation (i.e. neutrophil infiltration) or

protease activity where again, promising results in animal models did not translate into the

clinical setting (3, 24, 45). This leads to the question of: Are small molecules that possess

pleiotropic actions potentially a better strategy to limit IR injury? Studies have demonstrated that

flavonoids including flavanols are pleiotrophic. Flavonoids possess antioxidant (17, 43, 44), anti-

inflammatory (1, 9, 36, 39), and antithrombotic properties (19, 31). Flavonoids can also induce

NO-mediated vasodilation (12, 22, 35). Interestingly, several flavonoid properties mirror those of

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adenosine, the only agent that has shown some promise as a cardioprotectant (3, 24). It is

interesting to note that for adenosine to act as a prophylactic cardioprotector, the doses required

would likely yield hypotension and thus, be impractical for daily use. However, studies using

flavonoids have yet to identify any toxic or significant side effects, including notable hypotension

in normotensive individuals (6, 12).

It is well established that during the acute phase of IR injury, inflammatory cells are

recruited to the site of injury either infiltrating the tissue and/or causing microvascular plugging

(i.e. no-reflow phenomenom). Inflammatory cells release proteases, such as MMPs, and

generate an oxidative burst (4, 45). Previous studies have investigated the effects of catechins

on tissue inflammation. Dona et al., examined the effects of epigallocatechin gallate (EGCG) to

modulate in vitro and in vivo lung inflammatory responses (9). EGCG reduced ROS activity,

inhibited apoptosis, and reduced chemokine-induced neutrophil chemotaxis in vitro. EGCG

significantly reduced pulmonary inflammatory responses and fibrosis. Aneja et al, also

investigated the effects of EGCG on neutrophil activation in rats subjected to IR (1).

Cardioprotection was associated with decreased IL-6 production and neutrophil infiltration as

measured by MPO activity. In our study, epicatechin treatment did not reduce in a significant

manner MPO activity in the infarct region, indicating that at 48 h post-IR there is a preservation

of acute inflammation. There is continuing controversy as to the wisdom of the potential benefit

to be derived from therapies intended to suppress acute inflammation in the setting of

myocardial infarction. While the reduction of neutrophil infiltration can reduce IR injury in

animals, the use of anti-inflammatory agents in patients has led to adverse outcomes, including

ventricular rupture (38). In a study performed in our laboratory, the continuous use of steroids

after coronary occlusion led post-MI adverse remodeling (14).

In the setting of IR injury, oxidative stress has a number of deleterious biochemical

effects on the heart. ROS produced in IR injury can contribute to mitochondrial damage, which

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can lead to cell death by necrosis and apoptosis (4, 45). ROS can also activate pro-enzymes,

such as pro-MMPs, and promote calcium overload (4, 45). There are several endogenous

antioxidants found in tissues, including reduced glutathione (GSH). It is known that GSH plays a

central role in intracellular endogenous antioxidant defenses as it is involved in all lines of

protection against ROS (28). In the course of IR, GSH is converted into its oxidized form,

GSSG, and the level of ROS exceeds the antioxidant capacity of the cell, leading to oxidative

stress (28). A study by Chen et al. investigated the effect of tea catechin on the Pb2+-induced

change of intracellular thiol levels (5). They demonstrated that Pb2+ significantly increased tissue

oxidative stress in a concentration-dependent manner. Supplementation of the cells with (-)-

epicatechin or (-)-epicatechin gallate resulted in reduced oxidative stress. These results match

ours in that we showed a significant increase in the GSSG/GSH ratio in the infarct regions

compared to the non-infarct regions in control hearts, with epicatechin significantly attenuating

this increase. In our study, we utilized doses of epicatechin that are low and would appear on

the basis of expected blood concentrations (~0.002 mM) to yield limited antioxidant activity (17).

Thus, the manner in which low doses of epicatechin significantly reduce tissue oxidative stress

needs further investigation as it raises a wide spectrum of possibilities. One such possibility is

that epicatechin over the course of several days is concentrated intracellularly several fold vs.

blood levels and allows it to act as an effective antioxidant.

The activation of MMPs with IR injury has also been described. We and others have

documented the upregulation of MMP-2 and MMP-9 activity in infarcted myocardium (16, 27).

MMPs contribute to the loss of contractile function and damage the integrity of the extracellular

matrix. Our results showed significant increases in MMP-9 activity in the infarct region

compared to the non-infarct region in the vehicle group, and a lack of a significant increase in

the necrotic region with epicatechin Tx. This correlates with the findings of Dell’agli et al, who

studied the effects of EGCG on MMP-9. Results indicate that EGCG directly inhibited MMP-9

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activity (8). Interestingly, the reduction in tissue oxidative stress observed in our study may

have also led to decrease levels of MMP-9 activity via inhibition of the cysteine switch activation

mechanism, which is known to be ROS dependent (32).

The purpose behind the study of 3 weeks post-IR animals was to determine the extent to

which there was a sustained reduction in tissue injury with treatment. Results yield a reduction

in scar (infarct) size of ~32%. Our 3 week results are encouraging in that most studies have

failed to demonstrate the capacity of candidate cardioprotective agents to yield sustained (long-

term) effects (3, 24, 45). The observed preservation of post-MI chamber morphometry is also

important since little is known about what impact the pleiotropic effects of flavonoids may have

on post-MI wound healing/remodeling. To our knowledge as noted above, only the use of the

pleiotrophic agent adenosine has shown promise as a possible cardioprotector and promoter of

either preserved or reduced post-MI remodeling (7, 41).

Since the sustained beneficial effects of cocoa flavanols depend on their continued

intake it implies that their possible use as cardioprotective agents at the time of reperfusion or

soon afterwards would appear unlikely. However, cocoa induced vasodilation can be

reproduced by a select group of metabolites (35) thus, there is the possibility that future studies

using flavanol derivatives may allow for their examination as “immediate” cardioprotectors.

Alternatively, there is the possibility that higher does of flavanols may yield immediate

cardioprotection. This possibility will need to be explored. The concept of feasible prophylactic

cardioprotection has been proposed. Bolli et al., have noted that the most practical strategy for

limiting infarct size and improving clinical outcome may be to induce a chronically protected

cardiac phenotype where this property would be operative at the onset of ischemia (3). The

observation that Kuna Indians have low incidences of cardiovascular disease is supportive of

this idea. However, this population is not representative of the poorer health status observed in

developed countries general population. The concept of suggesting that humans consume

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traditional cocoa based products high in flavanol content is not a practical one given their high

caloric content. Alternatively, it is more likely to conceive of the production of cocoa based

“supplements” devoid of calories to be used for daily prophylactic use. This is a concept that has

been proposed as viable on the basis of observations derived from the use of the flavonoid

resveratrol on animals exposed to unhealthy diets (2).

On the basis of the results presented, more work needs to be performed to validate

these results and identify key underlying mechanisms of action of epicatechin. The

reproducibility of these observations by independent groups is warranted. In addition, further

preclinical studies need to be performed using small and large animal models of human disease

such as those with hypertension, diabetes, stroke prone, aging and importantly, to verify the

variability of the effects on the basis of gender. Nonetheless, the results presented provide

support towards the consideration of cocoa flavanols as possible therapeutic agents intended to

prevent and/or limit the development of ischemic heart disease.

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Acknowledgements

We wish to acknowledge the support of NIH HL-43617 and HL-67922 to Dr. F. Villarreal, a

CONACYT-UC MEXUS doctoral fellowship to D. Romero-Perez, an undergraduate diversity

supplement to B. Cortez-Gomez (HL-80049) and a pre-doctoral NIH fellowship (HL-07444) to K.

Yamazaki.

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Figure Legends.

Figure 1. Infarct area as a function of area at risk (IA/AAR) in IR rats subjected to 10d vehicle or

epicatechin treatment and 48h reperfusion. Infarct size was determined by staining hearts with

trypan blue and triphenyltetrazolium chloride. (A) Dispersion plot of the area at risk in IR (n=21)

and IR +10 d epicatechin (n=15) in rats subjected to 48 h reperfusion. (B) Bar graphs of the

area at risks. (C) Dispersion plot of the infarct area in IR (n=21), and IR + 2d epicatechin (n=15)

subjected to 48 h reperfusion. (D)Bar graphs of the infarct areas. Values are mean ± S.E.M.

Figure 2. Infarct area as a function of area at risk (IA/AAR) in IR rats subjected to 10d vehicle or

epicatechin treatment and 3 weeks reperfusion. Infarct size was determined by staining hearts

with trypan blue and triphenyltetrazolium chloride. (A) Area at risk in IR (n=7) and IR +10 d

epicatechin (n=8) in rats subjected to 3 weeks reperfusion. (B) Infarct area in IR (n=7), and IR +

10 d epicatechin (n=8) subjected to 3 week of reperfusion. Values are mean ± S.E.M.

Figure 3. Myocardial myeloperoxidase (MPO) activity in IR (n=8) and IR + 10 d epicatechin

(n=7) hearts was determined by a recently described colorimetric assay. Values are mean ±

S.E.M.

Figure 4. Myocardial levels of oxidative stress (GSSG/GSH) in IR (n=8) and IR + 10 d

epicatechin (n=7) infarcted hearts were detected in IR hearts using the fluorophore o-

pthalaldehyde. Values are mean ± S.E.M.

Figure 5. Myocardial MMP-2 and MMP-9 levels in IR (n=8) and IR + 10 d epicatechin hearts

(n=7) as determined by gelatin zymography. (A) Representative gelatin zymograms of

myocardial MMP-2 and MMP-9 levels in IR and IR + 10 d epicatechin. (B) Densitometric

analysis of 92 kDa MMP-9 zymographic activity. Values are mean ± S.E.M. ‘Std’ = human MMP-

2/-9 standard.

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Table 1. Hemodynamic data obtained from either normal or 48 h ischemia-reperfusion (IR) groups. Epicatechin groups were pretreated for 10 d. Values shown are mean ± S.E.M. *p<0.05 vs normal.

Normal

Normal + epicatechin

IR

IR + epicatechin

Group Size 6 6 8 8

HR (bpm) 298 ± 17 346 ± 8 368 ± 12* 373 ± 17*

LVPSP (mmHg)

125 ± 8 112 ± 14 102 ± 6 110 ± 5

LVEDP (mmHg)

7.6 ± 0.9 6.7 ± 1.3 5 ± 0.3 4.9 ± 0.5

MAP (mmHg) 101 ± 7 99 ± 10 89±3 90 ± 5

HR = heart rate, LVPSP = left ventricular peak systolic pressure, LVEDP = left ventricular end diastolic pressure, MAP = mean arterial pressure

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Table 2. Hemodynamic and morphometry data obtained from either sham or 3 week ischemia-reperfusion (IR) groups. Epicatechin groups were pretreated for 10 d. Values shown are mean ± S.E.M. *p<0.01 vs. sham; +p<0.01 vs. IR SW thickness.

HEMODYNAMICS

Sham

Sham + epicatechin

IR

IR + epicatechin

Group Size 5 4 7 8

HR (bpm) 318 ± 12 328 ± 11 290±6 302 ± 18

LVPSP (mmHg)

119 ± 2.2 116 ± 6.2 108 ± 2 108 ± 4

LVEDP (mmHg)

6.9 ± 1.1 2.6 ± 0.2 6.1 ± 0.8 5.5 ± 1.5

MAP (mmHg) 94 ± 4.7 92 ± 11 89 ± 2 85±4

MORPHOMETRY

Group Size 5 4 7 8

HW/BW 3.2 ± 0.2 3.2 ± 0.5 4.5 ± 0.2* 4.5 ± 0.2*

Outer LV diam. (mm)

1.6 ± 0.1 1.6 ± 0.1 1.6 ± 0.06 1.6 ± 0.04

Inner LV diam. (mm)

0.53 ± 0.02 0.52 ± 0.08 0.52 ± 0.06 0.55 ± 0.05

AW thickness

(mm)

0.52 ± 0.05 0.53 ± 0.05 0.4 ±0.03+ 0.41 ± 0.02

SW thickness

(mm)

0.49 ± 0.05 0.47 ± 0.04 0.62 ± 0.04 0.49 ± 0.03

HR = heart rate, LVPSP = left ventricular peak systolic pressure, LVEDP = left ventricular end diastolic pressure, MAP = mean arterial pressure, HW/BW = heart weight/body weight, AW = anterior wall, SW = septal wall.