Effects of (−)-epicatechin on myocardial infarct size and left ventricular remodeling following permanent coronary occlusion

Abstract

Objectives

We examined the effects of the flavanol (−)-epicatechin on short and long-term infarct size and left ventricular (LV) structure/function after permanent coronary occlusion (PCO) and the potential involvement of the protective AKT/ERK signaling pathways.

Background

(−)-Epicatechin reduces blood pressure in hypertensive patients and limits infarct size in animal models of myocardial ischemia-reperfusion injury. However, nothing is known about its effects on infarction after PCO.

Methods

(−)-Epicatechin (1mg/kg/day) treatment (Tx) was administered via daily oral gavage to 250 g male rats for 10 days prior to PCO and continued afterwards. PCO controls received water. Sham animals underwent thoracotomy and treatment in the absence of PCO. Immunoblots assessed AKT/ERK involvement 2 h after PCO. LV morphometry and function were measured 48 h and 3 weeks post-PCO.

Results

In the 48 h group, Tx reduced infarct size by 52%. There were no differences in hemodynamics amongst the different groups (heart rate, aortic and LV pressures). Western blots revealed no differences in AKT or ERK phosphorylation levels. At 3 weeks, PCO control animals demonstrated significant increases in LV end-diastolic pressure, heart weight/body weight, and LV chamber diameter vs. sham. PCO + (−)-epicatechin group values were comparable to sham + (−)-epicatechin. Tx resulted in a 33% decrease in MI size. LV pressure-volume curves demonstrated a right shift in control PCO animals, whereas (−)-epicatechin were comparable to sham. LV scar area strains were significantly improved with (−)-epicatechin.

Conclusions

These results demonstrate the unique capacity of (−)-epicatechin to confer cardioprotection in the setting of a severe form of myocardial ischemic injury. Protection is sustained over time and preserves LV structure/function. The cardioprotective mechanism(s) of (−)-epicatechin appear unrelated to AKT or ERK activation. (−)-Epicatechin warrants further investigation as a cardioprotectant.

Introduction

Diet is one of the most important lifestyle factors that influence the incidence of CVD. Many plants and natural products contain significant amounts of flavonoids (1). Evidence indicates a negative correlation between consumption of flavonoids and incidence of CVD (2–5). Cacao beans contain very high amounts of flavonoids in particular, the flavanol subtype (6). Main cacao flavanols are catechin and (−)-epicatechin present in mono- and multimeric forms (3,6). Kuna Indians living on islands near Panama consume large amounts of a local cacao beverage and have a low incidence of CVD in particular, hypertension. Studies relate their low incidence of CVD to the substantial consumption of cacao and not other factors such as eating habits, physical activity or genetic background (7,8). Interestingly, data from a population based cohort study of 1169 patients links chocolate consumption with decreased mortality after MI (9).

There are clues as to mechanisms that may explain cacao flavanol effects. Consumption of cacao flavanols leads to NO synthesis-dependent vasodilation (10,11). Flavanoids can also act as anti-oxidants and inhibit platelet adhesion, low-density lipoprotein oxidation, inflammation, reactive oxygen species generation, eicosanoid synthesis and improve insulin resistance (for reviews see, (12)). In humans, the ingestion of (−)-epicatechin causes vasodilatation and reproduces the antioxidant and insulin sensitizing effects of cocoa (13). We have previously reported on the cardioprotective effects of (−)-epicatechin pre-treatment using a rodent model of IR injury (14). Our results showed a significant reduction in infarct size that was sustained up to 3 weeks after injury. Reductions in infarct size were accompanied by preserved myocardial inflammation, decreases in matrix metalloproteinase activity and tissue oxidative stress. The activation of the RISK pathway (a term given to a group of pro-survival protein kinases that include AKT and ERK 1/2), has been demonstrated to confer cardioprotection (15). Preclinical studies have demonstrated that agents such as insulin, erythropoietin, adenosine, volatile anesthetics and statins can reduce myocardial infarct size through RISK pathway activation (15). Since cardioprotection was observed with (−)-epicatechin in the setting of IR injury, we speculated that AKT and/or ERK activation may be involved in mediating these effects.

PCO models of MI have also been used to examine the cardioprotective potential of candidate agents and to determine the long-term effects that the compounds may exert on LV structure/function. To our knowledge, no compounds have demonstrated a sustained, long term reduction in MI size in the setting of a PCO. The effects that cocoa flavanols have on these endpoints are unknown. Given the cardioprotective effects of (−)-epicatechin noted in the setting of myocardial IR injury and the reported pleiotropic actions of flavanols, we hypothesized that pre-treatment with the compound may yield sustained reductions in infarct size and as a consequence improve, long term LV structure/function. We also wished to examine the effects of (−)-epicatechin on AKT and/or ERK activation in the setting of a PCO in order to identify possible cardioprotective mechanisms.

Material and Methods

Surgical procedures

All procedures were approved by the Institutional Animal Care and Use Committee and conform to published National Institutes of Health guidelines for animals research. Adult male Sprague Dawley rats (Harlan, Indianapolis, IN) weighing 250–300 g were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), intubated, and ventilated with room air. The heart was exposed via a left thoracotomy, the pericardium opened and left anterior descending coronary artery occluded. The chest was closed and animals were allowed to recover. Shams were subjected to the surgical procedure described above and the suture placed without occluding the coronary artery.

Animal groups and (−)-epicatechin treatment

(−)-Epicatechin (1 mg/kg/day; Sigma-Aldrich, St. Louis, MO) or vehicle (water) was administered by oral gavage one time per day beginning 10 days before thoracotomy and continuing until the time of the terminal study (2 h, 48 h or 3 wk). Two hour study groups included PCO (n=6) and PCO + (−)-epicatechin (n=5). The 2 h timepoint was selected as optimal for the evaluation of the RISK pathway activation as per published reports (16). For 48 h studies, groups included sham (n=5), sham + (−)-epicatechin (n=5), PCO (n=8), and PCO + (−)-epicatechin (n=11). The 48 h time point was selected in order to clearly distinguish regions of necrotic tissue from viable myocardium. For 3 wk studies, groups included sham (n=11), sham + (−)-epicatechin (n=8), PCO (n=12), and PCO + (−)-epicatechin (n=14). Six animals from each group were used to measure passive mechanics of the LV.

Hemodynamics

In vivo hemodynamic measurements were obtained from all animals used in the study just prior to the terminal studies. For hemodynamic measurements (heart rate, aortic and LV pressures) animals were anesthetized with 5% isoflurane and maintained with 1–2% isoflurane. LV and aortic pressures were measured with a micromanometer (2 French, 140 cm; Millar Instruments Inc. Houston, Tx) introduced via the right carotid artery. Pressures were digitally recorded for subsequent analysis using WINDAQ software (version 2.15, DATAQ Instruments Inc.).

LV sampling and morphology

Hearts were exposed via medial sternotomy and arrested with an apical injection of ice-cold arrest solution (NaCl, 4 g/L; KCL, 4.48 g/L, NaHCO₃, 1 g/L; glucose, 2 g/L; 2,3-butanedione monoximine, 3 g/L; heparin, 2000 U/L) into the LV. The 3 week animal hearts were used for LV pressure-volume/pressure-strain studies (described below). Following arrest, hearts were excised, weighed, and the right ventricle removed. A 2 mm ring section was taken from the middle of the LV and stained for ~10 min using TTC. Computer-assisted image analysis was performed by a blinded operator. Results were expressed as % infarct area of the LV. The images of stained rings were also used to measure internal and external LV chamber diameters and anterior and septal wall thicknesses.

LV pressure-volume and pressure-strains

Ex vivo passive mechanics of the LV and mature scar were analyzed in 3 week subjects as previously described (17). Briefly, a deflated balloon attached to a cannula and pressure transducer was inserted into the LV. A triangle of white titanium dots (about 3–4 mm separation between dots) was painted on the surface of the LV within the scar in order to measure two-dimensional epicardial scar strains with changing LV pressure. A video camera was set up to record the position of the dots during balloon inflation. Water was infused into the balloon to reach zero pressure. Internal balloon pressure and infused volume were simultaneously measured as the balloon was slowly inflated at a rate of ~1 ml/min to a peak pressure of 25 mmHg. Data was acquired after 2–3 cycles of inflation and deflation of the balloon. For pressure-strain analyses, video frames at defined pressures were digitized using Scion Image (Scion Corp., Fredrick, MD). Two-dimensional epicardial scar strains were calculated with respect to the cardiac coordinate system yielding circumferential (E₁₁), and longitudinal (E₂₂) strains as previously described (17) by a blinded operator.

Westerns

Western blots were used to determine increases in phosphorylated AKT, phosphorylated ERK relative to total AKT and total ERK protein levels. Briefly, equal amounts of total protein were separated by 10% SDS-PAGE gels under reducing conditions and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked in 5% bovine serum albumin in tris-buffered saline that contained 0.1% Tween 20 (TTBS) and were exposed to the proper primary antibody. Membranes were incubated for 1 h at room temperature with the respective secondary horseradish peroxidase-labeled antibody and then developed using an ECL Plus detection system (Amersham Pharmacia Biotech; Buckinghamshire, UK). All antibodies used were obtained from Cell Signaling Technology, Danvers, MA.

Data analysis

All data are reported as mean ± standard error of the mean. Statistical analyses were performed using a Student’s unpaired t-test, one-way or two-way ANOVA and Bonferroni’s post-hoc test, as appropriate. Results were considered statistically significant at p≤0.05.

Results

Hemodynamics

Hemodynamic parameters were measured to determine LV contractile function. No significant changes were observed in heart rate, LV end-diastolic or systolic pressure, or mean aortic pressure between groups 48 after PCO (Table 1). In 3 wk studies (Table 2), there was a significant increase in LV end-diastolic pressures in the PCO group compared to both sham groups (p<0.05). A significant, albeit small decrease in peak systolic pressure was noted in the PCO + (−)-epicatechin group vs. sham. No significant changes were observed in the other parameters measured.

Table 1.

Hemodynamic data obtained from either sham or PCO groups 48 h post-PCO

	Sham	Sham + epicatechin	PCO	PCO + epicatchin
HR (bpm)	319±12	334±10	333±17	342±8
LVPSP (mmHg)	100±2	102±2	98.4±4	107±4
LVEDP (mmHg)	6.8±0.9	4.3±1.2	8.2 ±4.2	5.8±1.2
MAP (mmHg)	83±3	83±2	76±6	91±5

Values are means ± SEM. PCO, permanent coronary occlusion; HR, heart rate; LVPSP, left ventricular peak systolic pressure; LVEDP, left ventricular end diastolic pressure; MAP, mean arterial pressure.

Table 2.

Hemodynamic and morphometry data obtained from either sham or 3 wk PCO groups

In vivo	Sham	Sham + epicatechin	PCO	PCO + epicatechin
HR (bpm)	294±11	303±10	273±9	268±10
LVPSP (mmHg)	112±6	118±4	104±2	102±3*
LVEDP (mmHg)	7.4±0.4	6.4±0.9	14±2*+	11±1
MAP (mmHg)	85±6	92±4	83±4	82±2
Ex vivo morphometry
HW/BW	3.7±0.2	3.8±0.3	5.0±1.2*+	4.4±0.7
Outer LV diam. (mm)	1.47±0.06	1.47±0.08	1.5±0.03	1.495±0.03
Inner LV diam. (mm)	0.51±0.02	0.49±0.05	0.65±0.03+	0.55±0.05
AW thickness (mm)	0.61±0.03	0.58±0.05	0.32±0.03^	0.45±0.04
SW thickness (mm)	0.38±0.04	0. 38±0.04	0.49±0.01	0.48±0.02

Values are means ± SE. PCO, permanent coronary occlusion; HR, heart rate; LVPSP, left ventricular peak systolic pressure; LVEDP, left ventricular end diastolic pressure; MAP, mean arterial pressure; HW/BW, heart weight-to-body weight ratio; LV, left ventricle; AW, anterior wall; SW, septal wall.

^*p<0.01 vs sham,

⁺p<0.01 vs sham + (−)-epicatechin,

^{^}p<0.01 vs PCO SW thickness (One way ANOVA, Bonferroni test, 4 comparisons).

Infarct size and morphometry

No differences in area at risk were identified between PCO groups at any of the time points studied. At 48 h after infarction (figure 1), the PCO and PCO + (−)-epicatechin groups had infarct areas 41 ± 5 and 20 ± 2% respectively (p<0.001). Figure 2 summarizes the results from the 3 wk studies. The infarct area of PCO animals was 43 ± 3 vs. 29 ± 5% (p<0.02). As shown in table 2, HW/BW ratio and inner LV diameter were significantly higher in PCO (p<0.01) vs. both sham groups, whereas PCO + (−)-epicatechin demonstrated no differences. Anterior wall vs. septal wall thicknesses yielded a statistical difference only in the vehicle-treated PCO group (p<0.01).

Figure 1. Infarct area (IA) as a function of LV area 48 h post-permanent coronary occlusion (PCO) in rats subjected to 10 days vehicle or (−)-epicatechin pretreatment.

A: Representative LV equatorial ring sections of control and (−)-epicatechin treated animals stained with TTC. B: dispersion plot of the IA in PCO (n=8) and PCO + (−)-epicatechin (n=11) groups. Values are means ± SE. *p<0.001 vs PCO.

Figure 2. Infarct area (IA) as a function of LV area 3 wks post-permanent coronary occlusion (PCO) in rats subjected to 10 days vehicle or (−)-epicatechin pretreatment.

A: Representative LV equatorial ring sections of control and (−)-epicatechin treated animals stained with TTC. B: dispersion plot of the IA in PCO (n=12) and PCO + (−)-epicatechin (n=12) groups. Values are means ± SE. *p<0.02 vs PCO.

LV pressure-volume studies

Figure 3 show mean LV pressure-volume (P-V) curves obtained from treated and untreated PCO groups and compared to those obtained from shams. Untreated PCO yielded a significant right-shifted P-V curve when compared to shams (p<0.02). (−)-Epicatechin treated animals produced a PV curve comparable to both sham groups. No significant interaction between treatment and pressure was detected.

Figure 3. Average passive left ventricular pressure volume relations for sham (n=6; closed circles), sham + (−)-epicatechin (n=6, open circles), permanent coronary occlusion (PCO, n=6, closed triangles), and PCO + (−)-epicatechin (n=6, open triangles) after 3 weeks.

Two-way ANOVA followed by Bonferroni’s test (30 comparisons) revealed significant differences between PCO and shams (p<0.02). No interactive effect between treatment and pressure was detected.

LV pressure strains

Figure 4 shows mean LV pressure scar strains obtained in untreated and treated PCO groups. Irrespective of treatment, scar tissue is more compliant in the circumferential (E₁₁) direction. Untreated PCO had a significantly more compliant scar when compared to both sham groups and the PCO + (−)-epicatechin groups (p<0.01) in the E₁₁ direction. Notable differences in E₂₂ were also detected between untreated PCO and both sham groups (p<0.05). Two-way ANOVA revealed no interaction between treatment and pressure.

Figure 4. Average two-dimensional circumferential (E₁₁) (A) and longitudinal (E₂₂) (B) strains in the scar area as functions of inflation pressure for sham (n=6; closed circles), sham + (−)-epicatechin (n=6, open circles), permanent coronary occlusion (PCO, n=6, closed triangles), and PCO + (−)-epicatechin (n=6, open triangles) after 3 weeks.

Two-way ANOVA followed by Bonferroni’s test (30 comparisons) indicates that PCO + (−)-epicatechin yields a stiffer scar in the E₁₁ direction relative to PCO. No interactive effect between treatment and pressure was detected.

Westerns

The phosphorylation of AKT and ERK was measured 2 h after PCO with or without (−)-epicatechin. Densitometry analysis indicated that 10 d (−)-epicatechin pre-treatment led to no significant differences in AKT or ERK phosphorylation (Figure 5). Total AKT and ERK levels were unaltered.

Figure 5. ERK and AKT levels (AU=arbitrary units) in animals subjected to 10 days vehicle or (−)-epicatechin pretreatment and 2 h of permanent coronary occlusion (PCO).

A: Densitometric analysis of westerns for phosphorylated ERK (A), phosphorylated AKT (B), total ERK (C), and total AKT (D) in PCO (n=6) and PCO + (−)-epicatechin (n=5) animals.

Discussion

Unique findings derived from this study indicate that pre-treatment of animals with the flavanol (−)-epicatechin can substantially reduce infarct size 48 h after a PCO. The effect is sustained at 3 weeks and is accompanied with left shifted LV pressure-volume curves, reduction in epicardial scar strains and improvements in LV post-infarction morphometry. To our knowledge, this is the first time a compound has demonstrated significant and sustained long-term beneficial outcomes in a model of MI secondary to a PCO. A potential mechanism by which (−)-epicatechin may decrease MI size is a reduction in workload. As with our previous findings, (−)-epicatechin treatment in sham or PCO animals did not reduce blood pressure or heart rate and thus, changes in hemodynamics fail to explain the observed cardioprotective effects. The protective kinases, AKT and/or ERK at the assessed time point, appeared unaltered.

Previous studies have attempted to identify compounds capable of reducing MI size in the setting of a PCO. Kingma and Yellon reported on the capacity of verapamil given 5 min after embolization to reduce 48 h infarct size in a closed-chest canine model (18). Smith et al reported the capacity of propanolol given 1 min prior to PCO to reduce tissue CK levels 48 h after infarction (19). Kaga et al demonstrated that the flavanoid resveratrol is capable of reducing MI size in a rat 24 h after PCO (20). In this study, the authors suggested resveratrol effects may be secondary to an early angiogenesis process but relevant endpoints were only examined 4 days after MI. Other compounds such as metformin, erythropoietin, rosuvastatin and amlodipine have been examined for their potential for reducing MI size in the setting of PCO but did not improve this endpoint (21–24). We have examined the capacity of doxycycline and minocycline to yield sustained reductions in MI size both in the setting of IR and PCO. Our results indicate that while the compounds yield short-term (24–48 h) reductions in MI size they fail to sustain the effect in the long term (25–27). It is worth noting that neither verapamil, propanolol or resveratrol are as reported capable of reducing MI size in the magnitude achieved by 1 mg/kg/day (−)-epicatechin at 48 h (~50%). Since (−)-epicatechin can have vasodilatory effects, it was important to measure blood pressure which was not reduced by treatment in our animals. Interestingly, in a recent report, special formulation high flavanol cocoa given to spontaneously hypertensive rats (SHR) was able to notably reduce diastolic and systolic blood pressure (28). However, in control Wistar rats blood pressure remained stable suggesting that (−)-epicatechin has the property of exerting antihypertensive effects in a pathologic state with no hypotensive effects in normal subjects.

The purpose behind 3 week studies was to determine the extent to which there was a sustained reduction in MI with treatment and an improvement in passive mechanics and LV morphometry. Results yield a reduction in scar (infarct) size of ~33%. This result is indeed, unique in that no published studies have demonstrated the capacity of candidate cardioprotective agents to yield such sustained (long-term) effects in the setting of a PCO (29,30). The assessment of the effects of (−)-epicatechin on post-MI chamber structure/function was critical since little is known about the pleiotropic effects of flavanols on post-MI wound healing/remodeling. As it is well noted in the literature, the use of anti-inflammatory agents such as steroids in the setting of PCO leads to serious adverse effects on heart structure/function. We recently compared the effects of prednisone administration to those of doxycycline in an animal model of PCO induced MI (17). Doxycycline treatment yielded improvements in post-MI LV structure-function, including heart/body weight, wall thickness, chamber size, LV pressure-volume and pressure-strain curves. In contrast, treatment with steroids yielded a worsening of these endpoints vs. untreated or doxycycline MI animals. Results from (−)-epicatechin treatment of animals subjected to PCO, indicate improvements in all these endpoints 3 weeks after MI. Hearts were smaller, LV pressure-volume and pressure-strain curves were comparable to shams and there was a preservation of infarct wall thickness. Thus, the use of (−)-epicatechin does not appear to compromise the ability of the myocardium to heal and function properly. As to the mechanisms responsible for the improved long-term outcome, it follows that a smaller infarct would improve 3 week scar size and LV structure/function. However, we cannot exclude other effects since the use of other flavanoids (epicatechin gallate) has been reported to improve tissue healing/scarring in animals (31).

The activation of proteins belonging to the RISK pathway in the setting of myocardial IR injury has been associated with protective outcomes. Members of this group of kinases include AKT and ERK. In our previous IR study, we demonstrated that (−)-epicatechin treatment decreases infarct by 50% at 48 h and 32% by 3 weeks (14). In this study, the magnitudes of these decreases are the same at 48 h and 3 weeks when injury was induced via PCO. These results suggest a potential common mechanism(s) that may be responsible for cardioprotection. Recent reports have documented the capacity of (−)-epicatechin to activate signaling pathways known to be associated with cardioprotection (ERK and AKT) in neuronal cultures (32). Thus, we wished to explore the potential involvement of these kinases in mediating cardioprotective responses in the setting of PCO. Results fail to demonstrate changes in ERK and AKT phosphorylation levels at 2 h post-PCO. Thus, (−)-epicatechin likely exerts its cardioprotective actions through other mechanisms. Interestingly, there is controversy as to the cardioprotective roles exerted by AKT/ERK as they have been claimed to be involved in ischemic post-conditioning with ambiguous results (33). There are other members of the RISK pathway that also need to be further examined such as p70 ribosomal S6 protein kinase (P70S6K), and glycogen synthase kinase (GSK)3β(33).

Other possible scenarios by which (−)-epicatechin may exert cardioprotective actions include the induction of a hibernating myocardium-like gene program. Hibernating myocardium is characterized by the upregulation of proteins involved in anti-apoptosis (IAP), growth (VEGF, H11 kinase), and cytoprotection (HSP70, HIF-1β, GLUT1) (34). The upregulation of these proteins by (−)-epicatechin may allow the cells to better sustain ischemia when present. Subsequently, ischemic-hibernating myocardium may recover upon the reestablishment of blood flow via vessel recruitment and/or angiogenesis. As noted above, flavanoids such as resveratrol have demonstrated the capacity to induce angiogenesis in ischemic myocardium (35), however, less is known about cocoa flavanols. There is indirect evidence to suggest that (−)-epicatechin actions indeed, may occur through changes in gene expression. In studies performed in patients with hypertension, sustained reductions in blood pressure were observed when Tx was given with high flavanol cocoa for at least 7 days indicating that the response requires a time dependent effect (36). In our previous study, we utilized 2 pre-treatment schemes to examine the effects of (−)-epicatechin on hearts subjected to IR injury. Pre-treatment was provided for either 2 or 10 days. Short term treatment with 1 mg/kg/day (−)-epicatechin did not confer cardioprotection whereas 10 days yielded reduced infarct size. These data suggest that changes in myocardial gene expression and/or protein levels may need to develop with longer treatment times (6,37). Alternatively, the accumulation of (−)-epicatechin or sustained changes in blood levels of (−)-epicatechin metabolites may be required to generate cardioprotection (6). It is also well established that flavonoids are pleiotrophic as they possess antioxidant (38,39), anti-inflammatory (40–42), and antithrombotic properties (43). As noted, flavonoids can also induce NO-mediated vasodilation via endothelial nitric oxide synthase (eNOS) activation (11,44). In a recent report by Gundewar et al, metformin was demonstrated to reduce infarct size in a mouse model of myocardial IR injury (21). The effects were associated with increases in AMPK and eNOS phosphorylation. Indeed, it is widely accepted that eNOS activation is recognized as being cardioprotective (45). Further investigation is warranted in order to determine if any of the mechanisms of action discussed above has relevance to (−)-epicatechin’s actions on limiting infarct size in the setting of a PCO.

As in our first study, experiments focused on the use of a pre-treatment scheme to induce cardioprotection. These results imply that the regular and sustained consumption of products high in flavanols may induce a prophylactic organ protective phenotype. As the regular consumption of moderate amounts of wine has been suggested to yield a beneficial impact on CVD a similar argument may eventually be validated for the consumption of products enriched for flavanols. Thus, the need to develop flavanol enhanced products that are low in calories, since regular commercial products are high in fat and sugar. Many commercial companies (e.g. Natraceuticals Inc.) have begun to generate such products and promote the apparent beneficial effects of cocoa consumption.

On the basis of the results presented, further studies need to be undertaken to validate these results and identify key underlying mechanisms of action of (−)-epicatechin effects which are likely complex. The reproducibility of these observations by independent groups is warranted. In addition, further MI preclinical studies need to be performed using small and large animal models of human disease such as those with diabetes and importantly, to verify the variability of the effects on the basis of age and gender. Nonetheless, the results presented provide support towards the consideration of (−)-epicatechin as a possible therapeutic agent intended to prevent and/or limit the development of ischemic heart disease. Recent reports on the association of chocolate consumption to decreases in post-infarct mortality (9), on reversal of vascular dysfunction in diabetic patients (46) and on beneficial effects on vascular function in humans (44) suggests cocoa may indeed have protective effects on the ischemic heart. However, caution must be exercised as the regular consumption of most commercially available chocolate or cocoa related products provide a high caloric content and likely vary as to their overall composition (as cocoa has many bioactive molecules) and in the amount of (−)-epicatechin present.

ABBREVIATIONS

CVD

Cardiovascular disease

NO

Nitric oxide

IR

Ischemia-reperfusion

PCO

Permanent coronary occlusion

LV

Left ventricle

MI

Myocardial infarction

RISK

Reperfusion injury salvage kinase

ERK

Extracellular signal related kinase

AKT

Protein kinase B

TTC

Triphenyltetrazolium chloride