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PRE-CLINICAL RESEARCH

Effects of (–)-Epicatechin on Myocardial Infarct Size and Left Ventricular Remodeling After Permanent Coronary Occlusion

Katrina Go Yamazaki, PhD^_*, Pam R. Taub, MD^_*, Maraliz Barraza-Hidalgo, BS^_*, Maria M. Rivas, BS^_*, Alexander C. Zambon, PhD^_*, Guillermo Ceballos, MD, PhD and Francisco J. Villarreal, MD, PhD^_*,_*

^_* Departments of Medicine and Pharmacology, University of California, San Diego, La Jolla, California
Escuela Superior de Medicina del Instituto Politécnico Nacional, Seccion de Posgrado, Mexico City, Mexico

Manuscript received September 1, 2009; revised manuscript received January 7, 2010, accepted January 12, 2010.

^_* Reprints requests and correspondence: Dr. Francisco Villarreal, Department of Cardiology, University of California, San Diego, 9500 Gilman Drive, 0613J, BSB 4028, La Jolla, California 92093 (Email: fvillarr{at}ucsd.edu).

	Abstract

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Objectives: We examined the effects of the flavanol (–)-epicatechin on short- and long-term infarct size and left ventricular (LV) structure and function after permanent coronary occlusion (PCO) and the potential involvement of the protective protein kinase B (AKT)/extracellular signal-related kinase (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 (1 mg/kg daily) treatment was administered via oral gavage to 250 g male rats for 10 days before PCO and was continued afterward. The PCO controls received water. Sham animals underwent thoracotomy and treatment in the absence of PCO. Immunoblots assessed AKT/ERK involvement 2 h after PCO. The LV morphometric features and function were measured 48 h and 3 weeks after PCO.

Results: In the 48-h group, treatment reduced infarct size by 52%. There were no differences in hemodynamics among the different groups (heart rate and 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 and body weight, and LV chamber diameter versus sham. The PCO plus (–)-epicatechin group values were comparable with those of the sham plus (–)-epicatechin group. Treatment resulted in a 33% decrease in myocardial infarction size. The LV pressure-volume curves demonstrated a right shift in control PCO animals, whereas the (–)-epicatechin curves were comparable with those of the sham group. The 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 and function. The cardioprotective mechanism(s) of (–)-epicatechin seem to be unrelated to AKT or ERK activation. (–)-epicatechin warrants further investigation as a cardioprotectant.

Key Words: cardioprotection • catechins • flavanols • infarction • ischemia

Abbreviations and Acronyms   AKT = protein kinase B   CVD = cardiovascular disease   ERK = extracellular signal-related kinase   IR = ischemia–reperfusion   LV = left ventricle/ventricular   MI = myocardial infarction   PCO = permanent coronary occlusion   RISK = reperfusion injury salvage kinase

There are clues as to mechanisms that may explain cacao flavanol effects. Consumption of cacao flavanols leads to nitric oxide synthesis-dependent vasodilation (10,11). Flavanoids can also act as antioxidants and can inhibit platelet adhesion, low-density lipoprotein oxidation, inflammation, reactive oxygen species generation, and eicosanoid synthesis and can 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 previously reported on the cardioprotective effects of (–)-epicatechin before treatment using a rodent model of ischemia–reperfusion (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 and decreases in matrix metalloproteinase activity and tissue oxidative stress. The activation of the reperfusion injury salvage kinase (RISK) pathway (a term given to a group of prosurvival protein kinases that include protein kinase B [AKT] and extracellular signal-related kinase [ERK] 1/2), has been demonstrated to confer cardioprotection (15). Pre-clinical studies have demonstrated that agents such as insulin, erythropoietin, adenosine, volatile anesthetics, and statins can reduce MI size through RISK pathway activation (15). Because cardioprotection was observed with (–)-epicatechin in the setting of IR injury, we speculated that AKT activation, ERK activation, or both may be involved in mediating these effects.

Permanent coronary occlusion (PCO) models of MI also have 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 and 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 end points 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, may improve long-term LV structure and function. We also wished to examine the effects of (–)-epicatechin on AKT activation, ERK activation, or both in the setting of a PCO to identify possible cardioprotective mechanisms.

	Methods

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Surgical procedures.   All procedures were approved by the Institutional Animal Care and Use Committee and conform to published National Institutes of Health guidelines for animal research. Adult male Sprague Dawley rats (Harlan, Indianapolis, Indiana) weighing 250 to 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 was opened, and the left anterior descending coronary artery was occluded. The chest was closed and the animals were allowed to recover. Shams were subjected to the surgical procedure described above and the suture was placed without occluding the coronary artery.

Animal groups and (–)-epicatechin treatment.   (–)-epicatechin (1 mg/kg daily; Sigma-Aldrich, St. Louis, Missouri) or vehicle (water) was administered by oral gavage once daily beginning 10 days before thoracotomy and continuing until the time of the terminal study (2 h, 48 h, or 3 weeks). Two-hour study groups included PCO (n = 6) and PCO plus (–)-epicatechin (n = 5). The 2-h time point 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 plus (–)-epicatechin (n = 5), PCO (n = 8), and PCO plus (–)-epicatechin (n = 11). The 48-h time point was selected to distinguish clearly regions of necrotic tissue from viable myocardium. For 3-week studies, groups included sham (n = 11), sham plus (–)-epicatechin (n = 8), PCO (n = 12), and PCO plus (–)-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 before the terminal studies. For hemodynamic measurements (heart rate and aortic and LV pressures), animals were anesthetized with 5% isoflurane and were maintained with 1% to 2% isoflurane. The LV and aortic pressures were measured with a micromanometer (2-F, 140 cm; Millar Instruments, Inc., Houston, Texas) introduced via the right carotid artery. Pressures were recorded digitally for subsequent analysis using WINDAQ software version 2.15 (DATAQ Instruments, Inc., Akron, Ohio).

LV sampling and morphologic features.   Hearts were exposed via medial sternotomy and were 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, 2,000 U/l) into the LV. The 3-week animal hearts were used for LV pressure-volume and pressure-strain studies (described in the following text). After arrest, hearts were excised and weighed and the right ventricle was removed. A 2-mm ring section was taken from the middle of the LV and stained for approximately 10 min using triphenyltetrazolium chloride. Computer-assisted image analysis was performed by a blinded operator. Results were expressed as percent infarct area of the LV. The images of stained rings also were 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 described previously (17). Briefly, a deflated balloon attached to a cannula and pressure transducer was inserted into the LV. A triangle of white titanium dots (approximately 3 to 4 mm of separation between dots) was painted on the surface of the LV within the scar to measure 2-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 0 pressure. Internal balloon pressure and infused volume were measured simultaneously as the balloon was inflated slowly at a rate of approximately 1 ml/min to a peak pressure of 25 mm Hg. Data were acquired after 2 to 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, Maryland). Two-dimensional epicardial scar strains were calculated with respect to the cardiac coordinate system yielding circumferential and longitudinal strains as described previously (17) by a blinded operator.

Western blotting.   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 diflouride membrane. Membranes were blocked in 5% bovine serum albumin in tris-buffered saline that contained 0.1% Tween 20 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 were developed using an ECL Plus detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). All antibodies used were obtained from Cell Signaling Technology (Danvers, Massachusetts).

Data analysis.   All data are reported as mean ± SEM. Statistical analyses were performed using a Student unpaired t test, 1- or 2-way analysis of variance, and Bonferroni post hoc test, as appropriate. Results were considered statistically significant at p 0.05.

	Results

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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 h after PCO (Table 1). In 3-week studies (Table 2), there was a significant increase in LV end-diastolic pressures in the PCO group compared with both sham groups (p < 0.05). A significant, albeit small, decrease in peak systolic pressure was noted in the PCO plus (–)-epicatechin group versus sham. No significant changes were observed in the other parameters measured.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 1 IA as a Function of LV Area 48 h After PCO in Rats Subjected to 10 Days of Vehicle or (–)-Epicatechin Pre-Treatment (A) Representative left ventricular (LV) equatorial ring sections of control and (–)-epicatechin–treated animals stained with triphenyltetrazolium chloride. (B) Dispersion plot of the infarct area (IA) in permanent coronary occlusion (PCO) (n = 8) and PCO plus (–)-epicatechin (n = 11) groups. Values are mean ± SE. *p < 0.001 versus PCO.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 2 IA as a Function of LV Area 3 Weeks After PCO in Rats Subjected to 10 Days of Vehicle or (–)-Epicatechin Pre-Treatment (A) Representative LV equatorial ring sections of control and (–)-epicatechin–treated animals stained with triphenyltetrazolium chloride. (B) Dispersion plot of the IA in PCO (n = 12) and PCO plus (–)-epicatechin (n = 12) groups. Values are mean ± SE. *p < 0.02 versus PCO. Abbreviations as in Figure 1.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Average Passive LV Pressure Volume Relations for Sham, Sham plus (–)-Epicatechin, PCO, and PCO Plus (–)-Epicatechin After 3 Weeks Solid circles = sham (n = 6). Open circles = sham plus (–)-epicatechin (n = 6). Solid triangles = PCO (n = 6). Open triangles = PCO plus (–)-epicatechin (n = 6). Two-way analysis of variance 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. *p < 0.02 versus sham control. #p < 0.02 versus sham (–)-epicatechin. Abbreviations as in Figure 1.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Average 2-Dimensional Circumferential and Longitudinal Strains in the Scar Area as Functions of Inflation Pressure After 3 Weeks (A) Circumferential (E11) and (B) longitudinal (E22) strains. Solid circles = sham (n = 6). Open circles = sham plus (–)-epicatechin (n = 6). Solid triangles = PCO (n = 6). Open triangles = PCO plus (–)-epicatechin (n = 6). Two-way analysis of variance followed by Bonferroni's test (30 comparisons) indicates that PCO plus (–)-epicatechin yields a stiffer scar in the E11 direction relative to PCO. No interactive effect between treatment and pressure was detected. *p < 0.05 versus sham. **p < 0.05 versus PCO control. Abbreviations as in Figure 1.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 5 ERK and AKT Levels in Animals Subjected to 10 Days of Vehicle or (–)-Epicatechin Pre-Treatment and 2 h of PCO Densitometric analysis of Western blots for (A) phosphorylated extracellular signal-related kinase (ERK), (B) phosphorylated protein kinase B (AKT), (C) total ERK, and (D) total AKT in PCO (n = 6) and PCO plus (–)-epicatechin (n = 5) animals. Abbreviations as in Figure 1.

	Discussion

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Previous studies have attempted to identify compounds capable of reducing MI size in the setting of a PCO. Kingma and Yellon (18) reported on the capacity of verapamil given 5 min after embolization to reduce 48-h infarct size in a closed-chest canine model. Smith et al. (19) reported the capacity of propanolol given 1 min before PCO to reduce tissue creatinine kinase levels 48 h after infarction. Kaga et al. (20) demonstrated that the flavanoid resveratrol is capable of reducing MI size in a rat 24 h after PCO. In this study, the authors suggested that resveratrol effects may be secondary to an early angiogenesis process, but relevant end points were examined only 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 end point (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 of PCO. Our results indicate that although the compounds yield short-term (24 to 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, nor resveratrol are reported as capable of reducing MI size in the magnitude achieved by 1 mg/kg daily (–)-epicatechin at 48 h (approximately 50%). Because (–)-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 was able to reduce diastolic and systolic blood pressure noticeably (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 morphometric features. Results yield a reduction in scar (infarct) size of approximately 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 and function is critical, because little is known about the pleiotropic effects of flavanols on post-MI wound healing and remodeling. As 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 and 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 and function, including heart and body weight, wall thickness, chamber size, and LV pressure-volume and pressure-strain curves. In contrast, treatment with steroids yielded a worsening of these end points versus untreated or doxycycline-treated MI animals. Results from (–)-epicatechin treatment of animals subjected to PCO indicate improvements in all these end points 3 weeks after MI. Hearts were smaller, LV pressure-volume and pressure-strain curves were comparable with those of shams, and there was a preservation of infarct wall thickness. Thus, the use of (–)-epicatechin does not seem to compromise the ability of the myocardium to heal and function properly. Regarding the mechanisms responsible for the improved long-term outcome, it follows that a smaller infarct would improve 3-week scar size and LV structure and function. However, we cannot exclude other effects, because the use of other flavanoids (epicatechin gallate) has been reported to improve tissue healing and 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 by 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 after PCO. Thus, (–)-epicatechin likely exerts its cardioprotective actions through other mechanisms. Interestingly, there is controversy as to the cardioprotective roles exerted by AKT and ERK, because 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 examined further, such as p70 ribosomal S6 protein kinase and glycogen synthase kinase 3b (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 up-regulation of proteins involved in apoptosis proteins, growth (vascular endothelial growth factor, H11 kinase), and cytoprotection (heat shock protein 70, hypoxia induced factor 1a, glucose transporter 1) (34). The up-regulation of these proteins by (–)-epicatechin may allow the cells to sustain ischemia better when present. Subsequently, ischemic-hibernating myocardium may recover on the reestablishment of blood flow via vessel recruitment, angiogenesis, or both. 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 treatment 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 used 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 daily (–)-epicatechin did not confer cardioprotection, whereas 10 days yielded reduced infarct size. These data suggest that changes in myocardial gene expression, protein levels, or both 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 pleiotropic, because they possess antioxidant (38,39), anti-inflammatory (40–42), and antithrombotic properties (43). As noted, flavonoids also can induce nitric oxide-mediated vasodilation via endothelial nitric oxide synthase activation (11,44). In a recent report by Gundewar et al. (21), metformin was demonstrated to reduce infarct size in a mouse model of myocardial IR injury. The effects were associated with increases in 5' adenosine monophosphate protein kinase and endothelial nitric oxide synthase phosphorylation. Indeed, it is widely accepted that endothelial nitric oxide synthase activation is recognized as being cardioprotective (45). Further investigation is warranted 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. Just as the regular consumption of moderate amounts of wine has been suggested to yield a beneficial impact on CVD, a similar argument eventually may be validated for the consumption of products enriched for flavanols. There is a need to develop flavanol-enhanced products that are low in calories, because regular commercial products are high in fat and sugar. Many commercial companies (e.g., Natraceuticals, Inc., Valencia, Spain) have begun to generate such products and to 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 to identify key underlying mechanisms of action of (–)-epicatechin's effects, which are likely complex. The reproducibility of these observations by independent groups is warranted. In addition, further MI pre-clinical 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 sex. Nonetheless, the results presented provide support toward the consideration of (–)-epicatechin as a possible therapeutic agent intended to prevent or limit the development of ischemic heart disease. Recent reports on the association of chocolate consumption to decreases in post-infarct mortality (9), on the reversal of vascular dysfunction in diabetic patients (46), and on the beneficial effects on vascular function in humans (44) suggest that cocoa indeed may have protective effects on the ischemic heart. However, caution must be exercised, because 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.

	Acknowledgments

 
The authors thank Michelle Cruz for the analysis of the pressure-volume and pressure-strain curves.

	Footnotes

 
This study was supported by predoctoral award T32-HL007444 to Dr. Yamazaki and by National Institutes of Health grants HL-43617 and AT-004277 to Dr. Villarreal. Dr. Taub is supported by an American College of Cardiology/Merck Fellowship. Dr. Zambon is supported by an NIGMS IRACDA program. Dr. Ceballos is supported by a visiting professor CONACYT fellowship from Mexico. W. H. Wilson Tang, MD, served as Guest Editor for this paper.

	References

Top Abstract Methods Results Discussion References

 
1. Middleton Jr E. Effect of plant flavonoids on immune and inflammatory cell function Adv Exp Med Biol 1998;439:175-182.[Web of Science][Medline]

2. Keen CL, Holt RR, Oteiza PI, Fraga CG, Schmitz HH. Cocoa antioxidants and cardiovascular health Am J Clin Nutr 2005;81:298S-303S.[Abstract/Free Full Text]

3. Kris-Etherton PM, Keen CL. Evidence that the antioxidant flavonoids in tea and cocoa are beneficial for cardiovascular health Curr Opin Lipidol 2002;13:41-49.[CrossRef][Web of Science][Medline]

4. Knekt P, Jarvinen R, Reunanen A, Maatela J. Flavonoid intake and coronary mortality in Finland: a cohort study Bmj 1996;312:478-481.[Abstract/Free Full Text]

5. Hertog MG, Kromhout D, Aravanis C, et al. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study Arch Intern Med 1995;155:381-386.[Abstract/Free Full Text]

6. Fisher ND, Hollenberg NK. Flavanols for cardiovascular health: the science behind the sweetness J Hypertens 2005;23:1453-1459.[Web of Science][Medline]

7. McCullough ML, Chevaux K, Jackson L, et al. Hypertension, the Kuna, and the epidemiology of flavanols J Cardiovasc Pharmacol 2006;47(Suppl 2):S103-S109discussion 119–21.[CrossRef][Web of Science][Medline]

8. Hollenberg NK, Martinez G, McCullough M, et al. Aging, acculturation, salt intake, and hypertension in the Kuna of Panama Hypertension 1997;29:171-176.[Abstract/Free Full Text]

9. Janszky I, Mukamal KJ, Ljung R, Ahnve S, Ahlbom A, Hallqvist J. Chocolate consumption and mortality following a firstacute myocardial infarction: the Stockholm Heart Epidemiology Program J Int Med 2009;266:248-257.[CrossRef][Web of Science][Medline]

10. Heiss C, Dejam A, Kleinbongard P, Schewe T, Sies H, Kelm M. Vascular effects of cocoa rich in flavan-3-ols JAMA 2003;290:1030-1031.[Free Full Text]

11. Fisher ND, Hughes M, Gerhard-Herman M, Hollenberg NK. Flavanol-rich cocoa induces nitric-oxide-dependent vasodilation in healthy humans J Hypertens 2003;21:2281-2286.[CrossRef][Medline]

12. Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, van Norren K, van Leeuwen PA. Flavonoids: a review of probable mechanisms of action and potential applications Am J Clin Nutr 2001;74:418-425.[Abstract/Free Full Text]

13. Corti R, Flammer AJ, Hollenberg NK, Luscher TF. Cocoa and cardiovascular health Circulation 2009;119:1433-1441.[Abstract/Free Full Text]

14. Yamazaki KG, Romero-Perez D, Barraza-Hidalgo M, et al. Short- and long-term effects of (–)-epicatechin on myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol 2008;295:H761-H767.[Abstract/Free Full Text]

15. Hausenloy DJ, Yellon DM. Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection Heart Fail Rev 2007;12:217-234.[CrossRef][Medline]

16. Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning protects by activating prosurvival kinases at reperfusion Am J Physiol Heart Circ Physiol 2005;288:H971-H976.[Abstract/Free Full Text]

17. Garcia RA, Go KV, Villarreal FJ. Effects of timed administration of doxycycline or methylprednisolone on post-myocardial infarction inflammation and left ventricular remodeling in the rat heart Mol Cell Biochem 2007;300:159-169.[CrossRef][Web of Science][Medline]

18. Kingma Jr JG, Yellon DM. Limitation of myocardial necrosis with verapamil during sustained coronary occlusion in the closed-chest dog Cardiovasc Drugs Ther 1988;2:313-323.[CrossRef][Web of Science][Medline]

19. Smith 3rd EF, Egan JW, Griswold DE. Effect of propranolol on ischemic myocardial damage and left ventricular hypertrophy following permanent coronary artery occlusion or occlusion followed by reperfusion Pharmacology 1989;38:298-309.[Web of Science][Medline]

20. Kaga S, Zhan L, Matsumoto M, Maulik N. Resveratrol enhances neovascularization in the infarcted rat myocardium through the induction of thioredoxin-1, heme oxygenase-1 and vascular endothelial growth factor J Mol Cell Cardiol 2005;39:813-822.[CrossRef][Web of Science][Medline]

21. Gundewar S, Calvert JW, Jha S, et al. Activation of AMP-activated protein kinase by metformin improves left ventricular function and survival in heart failure Circ Res 2009;104:403-411.[Abstract/Free Full Text]

22. Hagar JM, Newman LG, Kloner RA. Effects of amlodipine on myocardial infarction, infarct expansion, and ventricular geometry in the rat Am Heart J 1992;124:571-580.[CrossRef][Web of Science][Medline]

23. Hale SL, Sesti C, Kloner RA. Administration of erythropoietin fails to improve long-term healing or cardiac function after myocardial infarction in the rat J Cardiovasc Pharmacol 2005;46:211-215.[CrossRef][Web of Science][Medline]

24. Weinberg EO, Scherrer-Crosbie M, Picard MH, et al. Rosuvastatin reduces experimental left ventricular infarct size after ischemia-reperfusion injury but not total coronary occlusion Am J Physiol Heart Circ Physiol 2005;288:H1802-H1809.[Abstract/Free Full Text]

25. Romero-Perez D, Fricovsky E, Yamazaki K, et al. Cardiac uptake of minocycline and mechanisms for in vivo cardioprotection J Am Coll Cardiol 2008;52:1086-1094.[Abstract/Free Full Text]

26. Villarreal FJ, Griffin M, Omens J, Dillmann W, Nguyen J, Covell J. Early short-term treatment with doxycycline modulates postinfarction left ventricular remodeling Circulation 2003;108:1487-1492.[Abstract/Free Full Text]

27. Griffin MO, Jinno M, Miles LA, Villarreal FJ. Reduction of myocardial infarct size by doxycycline: a role for plasmin inhibition Mol Cell Biochem 2005;270:1-11.[CrossRef][Web of Science][Medline]

28. Cienfuegos-Jovellanos E, Quinones, MDel M, Muguerza B, Moulay L, Miguel M, Aleixandre A. Antihypertensive effect of a polyphenol-rich cocoa powder industrially processed to preserve the original flavonoids of the cocoa beans J Agric Food Chem 2009;57:6156-6162.[CrossRef][Web of Science][Medline]

29. Bolli R, Becker L, Gross G, Mentzer Jr. R, Balshaw D, Lathrop DA. Myocardial protection at a crossroads: the need for translation into clinical therapy Circ Res 2004;95:125-134.[Abstract/Free Full Text]

30. Kloner RA, Rezkalla SH. Cardiac protection during acute myocardial infarction: where do we stand in 2004? J Am Coll Cardiol 2004;44:276-286.[Abstract/Free Full Text]

31. Kapoor M, Howard R, Hall I, Appleton I. Effects of epicatechin gallate on wound healing and scar formation in a full thickness incisional wound healing model in rats Am J Pathol 2004;165:299-307.[Web of Science][Medline]

32. Schroeter H, Bahia P, Spencer JP, et al. (–)-epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons J Neurochem 2007;101:1596-1606.[CrossRef][Web of Science][Medline]

33. Skyschally A, van Caster P, Boengler K, et al. Ischemic postconditioning in pigs: no causal role for RISK activation Circ Res 2009;104:15-18.[Abstract/Free Full Text]

34. Depre C, Kim SJ, John AS, et al. Program of cell survival underlying human and experimental hibernating myocardium Circ Res 2004;95:433-440.[Abstract/Free Full Text]

35. Penumathsa SV, Koneru S, Samuel SM, et al. Strategic targets to induce neovascularization by resveratrol in hypercholesterolemic rat myocardium: role of caveolin-1, endothelial nitric oxide synthase, hemeoxygenase-1, and vascular endothelial growth factor Free Radic Biol Med 2008;45:1027-1034.[CrossRef][Web of Science][Medline]

36. Grassi D, Necozione S, Lippi C, et al. Cocoa reduces blood pressure and insulin resistance and improves endothelium-dependent vasodilation in hypertensives Hypertension 2005;46:398-405.[Abstract/Free Full Text]

37. van Praag H, Lucero MJ, Yeo GW, et al. Plant-derived flavanol (–)-epicatechin enhances angiogenesis and retention of spatial memory in mice J Neurosci 2007;27:5869-5878.[Abstract/Free Full Text]

38. Waterhouse AL, Shirley JR, Donovan JL. Antioxidants in chocolate Lancet 1996;348:834.[Web of Science][Medline]

39. Hanasaki Y, Ogawa S, Fukui S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids Free Radic Biol Med 1994;16:845-850.[CrossRef][Web of Science][Medline]

40. Aneja R, Hake PW, Burroughs TJ, Denenberg AG, Wong HR, Zingarelli B. Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats Mol Med 2004;10:55-62.[CrossRef][Web of Science][Medline]

41. Selmi C, Mao TK, Keen CL, Schmitz HH, Eric Gershwin M. The anti-inflammatory properties of cocoa flavanols J Cardiovasc Pharmacol 2006;47(Suppl 2):S163-S171discussion S172–6.[CrossRef][Web of Science][Medline]

42. Steffen Y, Schewe T, Sies H. Myeloperoxidase-mediated LDL oxidation and endothelial cell toxicity of oxidized LDL: attenuation by (–)-epicatechin Free Radic Res 2006;40:1076-1085.[CrossRef][Web of Science][Medline]

43. Heptinstall S, May J, Fox S, Kwik-Uribe C, Zhao L. Cocoa flavanols and platelet and leukocyte function: recent in vitro and ex vivo studies in healthy adults J Cardiovasc Pharmacol 2006;47(Suppl 2):S197-S205discussion S206–9.[CrossRef][Web of Science][Medline]

44. Schroeter H, Heiss C, Balzer J, et al. (–)-epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans Proc Natl Acad Sci U S A 2006;103:1024-1029.[Abstract/Free Full Text]

45. Jones SP, Lefer DJ. Myocardial reperfusion injury: insights gained from gene-targeted mice News Physiol Sci 2000;15:303-308.[Abstract/Free Full Text]

46. Balzer J, Rassaf T, Heiss C, et al. Sustained benefits in vascular function through flavanol-containing cocoa in medicated diabetic patients J Am Coll Cardiol 2008;51:2141-2149.[Abstract/Free Full Text]

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