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Interactive Cardiovascular and Thoracic Surgery 3:612-615(2004)
© 2004 European Association of Cardio-Thoracic Surgery

Work in progress report - Cardiac general

A novel method of ‘preparative’ myocardial protection using green tea polyphenol in oral uptake

Senri Miwa, Kazuhiro Yamazaki, Suong-Hyu Hyon and Masashi Komeda*

Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan

* Corresponding author. Tel.: +81-75-751-3780; fax: +81-75-751-3098. (E-mail: masakom{at}kuhp.kyoto-u.ac.jp).

Received April 14, 2004; received in revised form June 16, 2004; accepted June 17, 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
Reperfusion injury has been one of the serious problems in cardiac surgery and medicine. We report a novel method of myocardial protection. Eight Sprague–Dawley rats, group A, were administrated green tea polyphenol orally. Eight other rats, group B, received no medication. Isolated hearts were perfused with a Langendorff's apparatus, and reperfused after 90min arrest. The left ventricular (LV) function, size, weight and the oxidative stress spaciotemporal analysis were performed. The LV size in group B became enlarged by 1.8±0.12 (mean±SD), but in group A, the LV enlarged only 1.1±0.08 times. The heart weight ratio was lighter (1.35±0.05) in group A than in group B (1.49±0.03, P<0.05). The LV end-systolic pressure volume relationship remained higher in group A. Oxidative stress as shown by 8-hydroxy-2'-deoxyguanosine was lower in group A (81.5±11.6) than in group B (226.9±35.6, P<0.0001). Polyphenol labeled FITC was located in the cell membrane of cardiomyocite. Polyphenol can protect a heart from oxidative stress, and maintain good LV function after ischemic arrest and reperfusion. This pre-treatment by polyphenol may add further benefit to current treatments of myocardial protection.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 
During cardiac surgery, protection of myocardium is paramount. Myocardial protection in surgery usually consists of using cardioplegic solution or other methods.

Historically, the topical cooling method was first used. Later the method of core-cooling using cold cardioplegic solution, e.g. St. Thomas Hospital's solution, was developed.

Recently, many scientific reports discussed other ways to protect the heart during surgery, for example, tepid cardioplegia, blood cardioplegia, and so on. But we have not yet been able to find the completely effective way to protect hearts from the damage of ischemia and reperfusion injury [1]. Actually, we are sometimes faced with serious damages of the heart and heart function after operation because of ischemic damages or reperfusion injuries.

Due to this primary concern, our team has previously focused and reported on oxidative stress sustained by the heart, specifically in the ischemic condition, and after reperfusion. We reported that oxidative stress, an important factor of reperfusion injury, continued from the peri-operative stage to the post-operative stage. Basically, oxidative stress is closely related to the balance between ischemic factors and defensive factors. We inferred that some pre-treatment to strengthen the defensive factors of the heart would be effective for myocardial protection during and after surgery. As there were only a few reports on medication or methods of myocardial protection that are used pre-operatively, we focused on the possible preventive effects of a natural compound, green tea polyphenol.

Polyphenol is a natural substance found in common food. A recent study reported that polyphenol was one of the free radical scavengers, protecting cells from ischemic damages [2].

We hypothesized that polyphenol pre-treatment, administered orally prior to surgery, could protect that heart from oxidative stress after reperfusion and avoid cell edema. Therefore polyphenol could present a new method of myocardial protection. Green tea, being rich in polyphenol, was a natural choice for our study.

Our hypothesis was that green tea polyphenol, which was given in pre-operative oral uptake method, could decrease heart damage and maintain heart function after ischemia and reperfusion, because we observed the green tea polyphenol protects the heart by remaining in the cell membrane of cardiomyocite. The purpose of this study was to explicate the effect of green tea polyphenol on reperfusion injury of heart and to propose the new method of heart protection.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgements
 References
 
2.1. Green tea polyphenol

Polyphenol extracted from green tea was purchased from PFI Inc., Kyoto, Japan. It was composed mainly of (–)-epigallo-catechin-3-O-gallate (28%), (–)-gallocatechin-3-O-gallate (11.6%), (–)-epicatechin-3-O-gallate (4.6%), (–)-epigallocatechin (15.0%), (+)-gallocatechin (14.8%), (–)-epicatechin (7.0%) and (+)-catechin (9.5%), and its purity exceeded 90%.

2.2. Models

Sixteen Sprague–Dawley (S.D.) rats were randomized. Eight S.D. rats had 10–3M green tea polyphenol in oral uptake (35ml/day in average, group A) for 14 days. Eight other rats had no medication (group B). All animals in this study received human care in compliance with ‘Principles of Laboratory Animals Care’ formulated by National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by Institute of Laboratory Animals Resources and published by the National Institute of Health.

2.3. Heart isolation and protocol of perfusion

Rats were anticoagulated with heparin (1000IU/kg i.v.). The isolated hearts were rapidly excited and perfused in a Langendorff's method at a constant pressure of 75mmHg with 37.5°C modified Krebs–Henseleit solution (in millimoles per liter: NaCl, 118; KCl, 4.7; MgSO4, 1.2, KH2PO4, 1.2; NaHCO3, 25; CaCl2, 2.5; glucose, 11) and gassed with a mixture of 95% oxygen and 5% carbon dioxide.

After 20min of equilibration, the hearts were arrested by 20ml/kg of St. Thomas solution. After 90min arrest with intermittent infusion of the cardioplegic solution (10ml/kg) every 30min, the hearts were reperfused. The left ventricular (LV) function was measured just before the arrest, 5 and 20min after reperfusion. At the same time, the coronary perfusion buffer was sampled from PA. After reperfusion, the hearts were evaluated with immunohistochemical studies on 8-hydroxy-2'-deoxyguanosine (8-OHdG) as an oxidative stress marker [3].

2.4. Immunohistochemistry

Cardiac tissues were fixed overnight in Bouin's solution immediately after dissection, followed by dehydration with 50 and 70% ethanol for 24h, respectively. The avidin–biotin complex method was used as previously described [3,4]. Briefly, after deparaffinization of the specimens, normal rabbit serum (diluted to 1:75; Dako, Kyoto) for the inhibition of nonspecific binding of the secondary antibody, a purified mouse monoclonal antibody against 8-OHdG (N45.1, 10µg/ml; Japan Institute for the Control of Aging, Fukuroi, Shizuoka, Japan), biotin-labeled rabbit anti-mouse IgG serum (diluted 1:100; Dako), and avidin–biotin complex (diluted 1:100; Vector Laboratories, Burlingame, CA) were sequentially applied. A substrate for alkaline phosphatase (black) was obtained from Vector.

Quantification of immunohistological data (8-OHdG index) was calculated as where X is the staining density indicated in gray scale, using NIH image and Photoshop [4].

2.5. Polyphenol-FITC

Six Sprague-Dawley rats were given green tea polyphenol labeled by FITC for 14days, and detected in same methods. And the sample of heart harvested was fixed frozen and determined by UV.

2.6. Statistical analysis

All values are expressed as mean±SD. The statistical analysis was performed using analysis of variance (ANOVA) and Student's t-test (StatView®, SAS Institute Inc., CA). The differences were considered as statistically significant at a P-value of less than 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgements
 References
 
3.1. LV function after reperfusion

{Delta}ESPVR (LV end systolic pressure volume relationship) was decreased 5min after reperfusion and returned to the level of pre-ischemic level in control group. However, it was increased in polyphenol group especially 20min after reperfusion (P=0.0002) (Fig. 1).



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  		Fig. 1 {Delta}ESPVR was increased in polyphenol group especially 20min after reperfusion.

 
3.2. Edema after reperfusion

{Delta}Heart Weight (HW 20min after reperfusion/HW just after harvest) in polyphenol group was 1.26±0.05. It was weighed less than the control group (1.49±0.09, P=0.0183). The LV size in control group became larger by 1.8±0.12 (mean±SD) times, but in polyphenol group, it enlarged only 1.1±0.08 times. In microscopic view, cystic change near nuclei of cardiomyocites was seen after the reperfusion. In polyphenol group, however, such a change was prevented (Fig. 2).



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  		Fig. 2 Photo A was in control group, and photo B was in polyphenol group. Cystic change near nuclei of cardiomyocites after the reperfusion was prevented in polyphenol group.

 
3.3. Oxidative stress

8-OHdG index was 81.5±11.6 in polyphenol group and 226.9±35.6 in control group (P<0.0001) (Fig. 3). The polyphenol group showed a low level of oxidative stress.



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  		Fig. 3 Photo A was in control group, and photo B was in polyphenol group. Oxidative stress in polyphenol group was less than in control group.

 
3.4. Location of polyphenol on myocardium

Polyphenol labeled FITC was located in the cell membrane of the cardiomyocite, shown by the green line (Fig. 4).



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  		Fig. 4 Polyphenol labeled FITC was located in cell membrane of the cardiomyocite.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgements
 References
 
Polyphenol is a natural substance, existing in a lot of common food such as apple juice, red wine, or green tea, and is a well known anti-oxidants [5]. Polyphenol has been reported to have several abilities of cell protection in many medical scientific fields. For example, polyphenol was reported as being able to preserve pancreatic islet long term [6]. Polyphenol is a well-known free radical scavenger [7,8]. In recent reports, polyphenol was shown to inhibit atherosclerosis [9], enhance nitric oxide release from endothelial cells [10,11], and protect against nitric oxide toxicity [12]. In the research of cardiology, Vinson reported that polyphenol has an anti-oxidative effect on heart disease [13], and Ray showed that polyphenol protects the heart from ischemia reperfusion injury [14].

According to the results of this study, polyphenol, which was given orally 14 days before the operation, was delivered and remained in the cell membrane of cardiomyocite. Our data show polyphenol bound to the cell membrane of the cardiomyocite and stayed during the ischemic phase and after reperfusion. Consequently, polyphenol protected the heart effectively from oxidative stress after reperfusion, avoided cell edema, and maintained good LV function after the ischemic arrest and reperfusion. These facts suggest that polyphenol is suitable for pre-treatment for ischemic heart condition during heart surgery.

According to our knowledge, this is the first report to suggest the benefit of polyphenol, administrated pre-operatively. Previously, several methods of cardiac protection were reported; however, most of them were intra-operative methods using some drugs or cardioplegic solutions. Our method is novel because it proposed a pre-treatment method. Oral uptake is a very simple and easy method, so it may be useful in clinical treatment. With respect to pre-treatment methods, Coghlan reported the effect of allopurinol [15]. Allopurinol is one of the anti-oxidative substances, and Coghlan showed a positive effect of allopurinol on heart function after surgery in a randomized clinical study. However, compared with allopurinol, natural polyphenol, including green tea polyphenol, has some advantages. Polyphenol is basically one of the natural substances in food, and no serious side effects of polyphenol have been reported, whereas allopurinol has some side effects, e.g. headache, drowsiness and so on. Therefore, we can propose not only a medical treatment but also a dietary cure method using polyphenol. From this point of view, polyphenol offers possibilities for broad health care for heart diseases.

This study was limited to Langendorff's model and therefore not equal to ischemic reperfusion in cardiac surgery. In future study, we will examine the effects of green tea polyphenol pre-treatment on larger animal model prior to a cardiopulmonary bypass.

In conclusion, this novel pre-treatment method, using polyphenol orally, may improve the current method of myocardial protection in present use and improve the recovery rate for heart diseases by diminishing the oxidative stress that caused ischemic arrest and reperfusion injury.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
References
 
We really appreciate Miss Mikiko Saito, the research assistant, for her help in collecting data for this study.

doi:10.1016/j.icvts.2004.06.013


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 References
 

  1. Schmitt JP, Schroder J, Schunkert H, Birnbaum DE, Aebert H. Role of apoptosis in myocardial stunning after open surgery Ann Thorac Surg 2002;73(4):1229-1235.[Abstract/Free Full Text]
  2. Roy M, Chakrabarty S, Sinha D, Bhattacharya RK, Siddiqi M. Anticlastogenic, antigenotoxic and apoptotic activity of epigallocatechin gallate: a green tea polyphenol Mutat Res 2003;523–524:33-41.
  3. Toyokuni S, Tanaka T, Hattori Y, Nishiyama Y, Ochi H, Hiai H, Uchida K, Osawa T. Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model Lab Invest 1997;76:365-374.[Medline]
  4. Miwa S, Toyokuni S, Nishina T, Nomoto T, Hiroyasu M, Nishimura K, Komeda M. Spaciotemporal alteration of 8-hydroxy-2'-deoxyguanosine levels in cardiomyocytes after myocardial infarction in rats Free Radic Res 2002;36(8):853-858.[Medline]
  5. Chandra S, De Mejia EG. Polyphenoilic compounds, antioxidant capacity, and quinone reductase activity of an aqueous extract of Ardisia compressa in comparison to mate and green teas J Agric Food Chem 2004;52:3583-3589.[CrossRef][Medline]
  6. Hyon SH, Kim DH. Long-term preservation of rat pancreatic islets under physiological conditions J Biotechnol 2001;85(3):241-246.[Medline]
  7. Saint-Cricq De Gaulejac N, Provost C, Vivas N. Comparative study of polyphenol scavenging activities assessed by different methods J Agric Food Chem 1999;47(2):425-431.[Medline]
  8. Imai K, Inagaki M, Saitoh Y, Yura A, Sakagami H, Suzuki M, Oguchi K. Related articles, links radical scavenging activity and cytotoxicity of phenethyl ether and cinnamic amide derivatives Anticancer Res 2002;22(3):1661-1666.[Medline]
  9. Vinson JA, Teufel K, Wu N. Red wine, dealcoholized red wine, and especially grape juice, inhibit atherosclerosis in a hamster model Atherosclerosis 2001;56(1):67-72.
  10. Leikert JF, Rathel TR, Wohlfart P, Cheynier V, Vollmar AM, Dirsch VM. Red wine polyphenols enhance endothelial nitric oxide synthase expression and subsequent nitric oxide release from endothelial cells Circulation 2002;106(13):1614-1617.[Abstract/Free Full Text]
  11. Khan NQ, Lees DM, Douthwaite JA, Carrier MJ, Corder R. Comparison of red wine extract and polyphenol constituents on endothelin-1 synthesis by cultured endothelial cells Clin Sci 2002;103:72S-75S.[Medline]
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  15. Coghlan JG, Flitter WD, Clutton SM, Panda R, Daly R, Wright G, Ilsley CD, Slater TF. Allopurinol pretreatment improves postoperative recovery and reduces lipid peroxidation in patients undergoing coronary artery bypass grafting J Thorac Cardiovasc Surg 1994;107:248-256.[Abstract/Free Full Text]




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