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European Journal of Heart Failure 2008 10(5):439-445; doi:10.1016/j.ejheart.2008.03.004
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© 2008 European Society of Cardiology

Positive inotropic effects of epigallocatechin-3-gallate (EGCG) involve activation of Na+/H+ and Na+/Ca2+ exchangers

Mario Lorenza, Niels Helligeb, Philipp Riedera, Hans-Tilmann Kinkela, Christiane Trimpertc, Alexander Staudtc, Stephan B. Felixc, Gert Baumanna, Karl Stangla and Verena Stangla,*

a Medizinische Klinik mit Schwerpunkt Kardiologie und Angiologie, Charité — Universitätsmedizin Berlin Campus Mitte, Germany
b Institut für Diagnostische Radiologie, Universitäts-Spital Zürich Zürich, Switzerland
c Klinik für Innere Medizin B, Ernst-Moritz-Arndt-Universität Greifswald Germany

* Corresponding author. Medizinische Klinik mit Schwerpunkt Kardiologie und Angiologie, Charité — Universitätsmedizin Berlin, CCM, Charitéplatz 1, D-10117 Berlin, Germany. Tel.: +49 30 450 513 153; fax: +49 30 450 513 932. E-mail address: verena.stangl{at}charite.de (V. Stangl).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: There is evidence that the tea catechin epigallocatechin-3-gallate (EGCG) modulates myocardial contractility. However, the underlying mechanisms remain to be determined.

Aims: To study potential signalling pathways involved in EGCG-induced contractile parameters.

Methods and results: EGCG increased fractional shortening in rat cardiac myocytes and enhanced intracellular systolic Ca2+ concentrations. In isolated rat hearts, perfusion with EGCG resulted in significant, dose-dependent increase in peak systolic left ventricular pressure, as well as in contraction and relaxation velocities. Heart rate did not change. Inhibition of the β1-receptor with metoprolol had no influence on the contractile effects of EGCG. Furthermore, levels of cAMP and phosphorylation of phospholamban did not change with EGCG, indicating that the beta-receptor pathway is not involved. The L-type Ca2+ channel inhibitors, nifedipine and gallopamil, failed to modulate EGCG-induced increase in contractility. However, the myocardial effects and intracellular calcium transients stimulated by EGCG were significantly reduced by the antagonist of the Na+/H+ exchanger (NHE) methyl-N-isobutyl amiloride (MIA), and by blocking of the reverse mode of the Na+/Ca2+ exchanger (NCX) by KB-R7943.

Conclusion: These results indicate that Ca2+-dependent positive inotropic and lusitropic effects of EGCG are mediated in part via activation of the Na+/H+ exchanger and the reverse mode of the Na+/Ca2+ exchanger in the rat myocardium.

Key Words: EGCG • Myocardium • Contractility • Inotropic • Rat

Received September 4, 2007; Revised January 23, 2008; Accepted March 6, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Consumption of tea lowers the risk of heart disease and reduces cardiovascular mortality [1-3]. Tea is rich in a group of flavonoids, the catechins, of which epigallocatechin-3-gallate (EGCG) is the major and most potent representative [4]. A fast growing number of beneficial cardiovascular effects for EGCG and tea extracts have been reported. EGCG has been shown to induce endothelial-dependent vasorelaxation by activating endothelial nitric oxide synthase [5,6]. Local delivery of green tea extract containing primarily EGCG, as well as pure EGCG itself, inhibited neointimal formation in a rat carotid artery injury model [7]. In addition, green tea extract prevented the development of atherosclerosis in apoE-deficient mice [8].

Moreover, favourable myocardial effects of tea components have also been demonstrated. EGCG prevented experimentally induced hypertrophy in rats [9,10]. Orally administered tea catechins in rats attenuated chronic ventricular remodelling after myocardial ischaemia [11], and improved left ventricular dysfunction in autoimmune myocarditis [12]. Furthermore, ischaemia/reperfusion-mediated cardiac cell death was prevented by reduction of STAT-1 activity [13], and reperfusion-induced myocardial damage in rat hearts was attenuated by EGCG [14].

Recently, positive inotropic effects of EGCG on isolated rat and guinea pig atria had been demonstrated [15,16]. The increase in left ventricular developed pressure was associated with elevated cytosolic Ca2+ as well as with transient nitric oxide (NO) levels in guinea pig hearts [15]. However, no further mechanistic insights were provided. Therefore, in the present study we investigated the signalling pathways of the cardiac effects of EGCG in isolated rat hearts and adult rat cardiomyocytes.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Fractional shortening and intracellular Ca2+ in isolated cardiac myocytes
Adult rat cardiac myocytes were isolated as described previously [17,18]. For measurement of fractional shortening and intracellular calcium concentrations, cells were plated on 4-well chamber glass slides in Krebs-Henseleit buffer (pH 7.2) containing in mM: 117 NaCl, 2.8 KCl, 0.6 MgCl2, 1.2 KH2PO4, 1.2 CaCl2, 20 glucose, and 10 HEPES. After an attachment period of 60 min at 4 °C, the buffer was exchanged for a staining solution containing 0.1% dimethyl sulfoxide (DMSO) and 5 {varepsilon}M cell-permeable Fura-2 AM (Sigma-Aldrich, Deisenhofen, Germany) for 10 min at RT. After incubation, the probe solution was replaced with fresh buffer, and incubation with inhibitors continued for an additional 30 min. The cells were constantly perfused in a chamber slide with a volume of 500 {varepsilon}l, and were field-stimulated (1 Hz) at RT. Fluorescence measurements were recorded with a dual excitation, single-emission fluorescence photomultiplier system (IonOptix, Milton, Massachusetts) at 340 and 380 nm, respectively. Changes in intracellular calcium transients were inferred from the ratio of the fluorescence intensity at these 2 wavelengths. A video imaging edge detector system (IonOptix) was used for measurements of cell length.

2.2. Isolated rat heart perfusion
Male Wistar rats with body weights of 200-300 g were used in all experiments. All animal experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985) and were approved by the Charité University Hospital Ethics Committee. The perfused heart model has been described previously [19]. After intraperitoneal anaesthesia (pentobarbital sodium 50 mg/kg body weight), the hearts were quickly excised and perfused according to the Langendorff technique, with a modified Krebs-Henseleit solution (in mM: 127.1 NaCl, 4.7 KCl, 1.1 MgSO4, 1.19 KH2PO4, 24.9 NaHCO3, 1.26 CaCl2, 8.93 glucose, 10 HEPES), equilibrated with 95% O2 and 5% CO2 at 37 °C, pH 7.4. Left ventricular pressure was recorded by means of a fluid-filled latex balloon inserted through the mitral valve and attached to a pressure transducer and chart recorder. Coronary flow rate was monitored by an ultrasonic flow meter (Transsonic Systems, Ithaca, NY, USA) connected to a flow probe installed in the aortic arch. Coronary perfusion pressure was recorded by a pressure transducer attached to the aortic perfusion cannula. All parameters were continuously displayed. Recordings included systolic left ventricular (sLVP) and diastolic left ventricular pressure (dLVP), coronary flow rate (CF), and coronary perfusion pressure (CPP). Heart rate (HR) as well as LV contraction (+dP/dt) and LV relaxation velocity (–dP/dt) were derived from the left ventricular pressure signal.

After basal steady-state conditions had been achieved, the effects of EGCG were assessed at concentrations of 0.5, 1, and 4 {varepsilon}M at constant flow perfusion (10-12.5 ml/min). A washout period (5 min) followed each EGCG dose until steady-state conditions were reached. The subsequent steady state was used as the basal value for the following EGCG dose. The inhibitors of nitric oxide synthase (N-nitro-L-arginine methyl ester, L-NAME), the β1-receptor (metoprolol), the L-type Ca2+ channel (nifedipine and gallopamil), the Na+/H+ exchanger (methyl-N-isobutyl amiloride), and the reverse mode of the Na+/Ca2+ exchanger (KB-R7943) were added to the perfusate 15 min before administration of EGCG. Isoprenaline served as positive control in selected experiments.

2.3. Immunoblotting
After treatment of perfused rat hearts with EGCG or isoprenaline, hearts were immediately freeze-clamped and proteins were extracted in lysis buffer containing in mM: 20 HEPES (pH 7.9), 100 NaCl, 1 Na3VO4, 4 sodium pyrophosphate, 10 EDTA, 1 phenylmethylsulfonyl fluoride, 10 NaF, 0.1 okadaic acid, and 1% Triton X-100. Total protein (30 {varepsilon}g per lane) was subjected to gradient SDS-PAGE, and membranes were probed with anti-phospho-phospholamban (Ser16) 1:5000, anti-phospho-phospholamban (Thr17) 1:5000, and with anti-SERCA2a 1:5000 antibodies from Badrilla (Leeds, UK). Bands were visualized using the ECL detection system (Amersham, Freiburg, Germany).

2.4. Intracellular cAMP and cGMP levels
Levels of intracellular cAMP and cGMP in isolated hearts were determined using an enzyme immunoassay kit (Sigma-Aldrich, Deisenhofen, Germany). After treatment of perfused rat hearts with EGCG, isoprenaline, or water as control, hearts were immediately freeze-clamped and extracts were isolated with 0.1 M HCl. Protein concentrations were measured using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). 300 {varepsilon}g (for cAMP) and 500 {varepsilon}g (for cGMP) of proteins were used in the immunoassays. Samples for cGMP analysis were acetylated to increase sensitivity of measurements.

2.5. Reagents
EGCG, isoprenaline, metoprolol, L-NAME, nifedipine, gallopamil, methyl-N-isobutyl amiloride (MIA), and all other reagents and media not otherwise specified were purchased from Sigma-Aldrich (Deisenhofen, Germany). KB-R7943 was obtained from Calbiochem (San Diego, CA, USA). Theaflavin-3,3'-digallate (TF3) was kindly provided by Mitsui Norin Food Research Laboratories (Japan). Collagenase II was obtained from Worthington Biochemical (Freehold, NJ, USA).

2.6. Data analysis
Data are given as mean±SEM. Differences were compared by Student's t-test and statistical significance was accepted at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. EGCG effects in isolated cardiac myocytes
Treatment of cardiac myocytes with 2.5 {varepsilon}M EGCG resulted in significantly increased fractional shortening (127.5±3.9%, P<0.05) compared to control. A further increase in fractional shortening was observed at 5 {varepsilon}M EGCG (140.3±5.5%; P<0.05) (Fig. 1A). Diastolic cell length remained unchanged at 2.5 {varepsilon}M EGCG (99.2±0.2%) and decreased only slightly at 5 {varepsilon}M EGCG (97.7±0.9%).


Figure 01
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  		Fig. 1 EGCG increases fractional shortening and calcium concentrations in isolated rat cardiomyoctyes. (A) Dose-dependent increase in fractional shortening after treatment of cells with the indicated doses of EGCG. (B) Changes in intracellular calcium concentration. Shown are the relative changes in systolic (Ca systole) and diastolic (Ca diastole) calcium transients as well as the difference between systolic and diastolic (Ca amplitude) intracellular calcium at the indicated doses of EGCG. Data are means±SEM from n=7 experiments; *P<0.05 versus control for (A) and (B).

 
To investigate whether inotropic effects of EGCG are caused by an increase in intracellular calcium concentrations, we performed experiments in isolated ventricular myocytes loaded with Fura-2AM. Compared to control, treatment with 2.5 {varepsilon}M and 5 {varepsilon}M EGCG significantly increased systolic calcium to 105±0.9% (P<0.05) and to 107±0.8%, (P<0.05), respectively. A slight increase in diastolic calcium was observed at 2.5 {varepsilon}M EGCG (102±0.8%, P<0.05) and at 5 {varepsilon}M EGCG (101.6±0.9%, n.s.). The difference in systolic and diastolic calcium (calcium amplitude) increased significantly at both EGCG concentrations (2.5 {varepsilon}M: 115.3±2.1%, P<0.05; 5 {varepsilon}M: 122.7±3.1%, P<0.05) (Fig. 1B).

3.2. Impact of EGCG on myocardial contractility in isolated rat hearts
EGCG infusion at concentrations of 1 and 4 {varepsilon}M resulted in increase of sLVP in a dose-dependent manner, concomitant with a rise in maximal velocity of contraction and relaxation. Lower doses of EGCG (0.5 {varepsilon}M) did not significantly affect cardiac performance (Fig. 2A). The time to reach steady state after administration of EGCG was about 1 to 3 min. After washout, the effects of EGCG were completely reversible. As summarized in Table 1, sLVP increased from 73.9±6.3 mmHg in untreated hearts to 130.6±10.4 mmHg at 4 {varepsilon}M EGCG (181±10%). In parallel, the LV +dP/dt increased from 2370±223 mmHg/s to 4099±343 mmHg/s (177±10%), and LV –dP/dt from 1300±152 to 2352±235 mmHg/s (181±14%) at 4 {varepsilon}M EGCG. Heart rate was 255.2±7.2 beats per minute at baseline and remained unchanged at doses up to 4 {varepsilon}M EGCG (258.6±8.3 beats per minute, 101±1%). Diastolic left ventricular pressure and coronary perfusion pressure did not change significantly.


Figure 02
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  		Fig. 2 Dose-dependent effects of EGCG and TF3 on cardiac function. (A) Relative changes in heart rate (HR), systolic left ventricular pressure (sLVP), left ventricular contraction (+dP/dt) and relaxation velocity (–dP/dt) after treatment of isolated rat hearts with the indicated doses of EGCG. Data are means±SEM from n=13 experiments. (B) Treatment of isolated rat hearts with various doses of TF3 had no influence on contractile parameters. Data are means±SEM from n=5 experiments; *P<0.05 versus control.

 


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  		Table 1 Dose-dependent effects of EGCG on cardiac function

 
In a previous study, the positive inotropic effects of EGCG in isolated guinea pig hearts were accompanied by elevated levels of nitric oxide [15]. To study whether production of NO is required for EGCG-induced changes, we pretreated perfused rat hearts with the NOS-inhibitor L-NAME (1 mM). However, pretreatment with L-NAME had no influence on myocardial effects of EGCG (data not shown). For the biological activities of tea polyphenols, the presence of galloyl groups in their 3' positions is thought to be important. We therefore tested the potential cardiac effects of the structurally similar black tea compound theaflavin-3,3'-digallate (TF3) [20]. However, doses of 0.5 to 4 {varepsilon}M TF3 did not affect myocardial contractile parameters (Fig. 2B). Similar results were obtained in isolated cardiac myocytes (data not shown).

3.3. β-adrenergic pathway
To investigate the potential β-receptor-dependent mechanisms of EGCG, we pretreated isolated hearts with the β1-receptor antagonist metoprolol. Although the effects of isoprenaline (0.5 {varepsilon}M) used as positive control were completely abolished after pretreatment of hearts with the β1-receptor blocker, the positive inotropic and lusitropic effects of EGCG (4 {varepsilon}M) were not modulated by pretreatment with 3 {varepsilon}M metoprolol (Fig. 3A). In addition, EGCG did not elevate intracellular cAMP levels in perfused hearts, whereas isoprenaline stimulated a 3-fold increase in cAMP (Fig. 3B). Moreover, levels of cGMP were not influenced by EGCG (data not shown). β-adrenergic stimulation leads to phosphorylation of phospholamban (PLB) that prevents the inhibitory effect of PLB on the activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA) during adrenergic activation [21]. Stimulation of hearts with isoprenaline (0.5 {varepsilon}M) induced marked phosphorylation of both Ser16 and Thr17 PLB phosphorylation sites. However, treatment of perfused hearts with EGCG (4 {varepsilon}M) did not change the phosphorylation status of PLB at either site (Fig. 3C).


Figure 03
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  		Fig. 3 EGCG-stimulated changes in cardiac function in isolated rat hearts are not mediated via the β1-receptor pathway. (A) β1-receptor inhibition by metoprolol (Me, 3 {varepsilon}M) prevented cardiac effects of isoprenaline (Iso, 0.5 {varepsilon}M), but had no influence on the effects of EGCG (4 {varepsilon}M). Shown are relative changes of heart rate (HR), systolic left ventricular pressure (sLVP), left ventricular contraction (+dP/dt), and relaxation velocity (–dP/dt). Data are means±SEM from n=6 experiments; *P<0.05 versus control; #P<0.05 versus Iso alone. (B) Isoprenaline (Iso, 0.5 {varepsilon}M) increased cAMP levels, whereas EGCG (4 {varepsilon}M) had no effect. Data are means±SEM from n=5 experiments; *P<0.05 versus control. (C) Western blots of Thr17 and Ser16 phosphorylation sites of phospholamban. Treatment of isolated hearts with Iso (0.5 {varepsilon}M) induced the phosphorylation of both sites, whereas stimulation of hearts with EGCG (4 {varepsilon}M) had no effect. Total SERCA2a antibody served as control for equal loading of proteins.

 
3.4. Role of membrane transporters
We subsequently studied the involvement of the L-type Ca2+ channel on EGCG-mediated myocardial effects. Pretreatment of perfused hearts with the L-type Ca2+ channel antagonist nifedipine (100 nM) did not modulate the inotropic effects induced by EGCG (Fig. 4). Likewise, pretreatment of hearts with gallopamil (5 nM) had no influence on EGCG-mediated positive inotropic and lusitropic effects (data not shown).


Figure 04
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  		Fig. 4 Inhibition of the L-type Ca2+ channel does not impair the effects of EGCG on cardiac function. Pretreatment of isolated rat hearts with nifedipine (Nif, 100 nM) had no influence on EGCG-mediated changes in cardiac parameters. Relative changes of heart rate (HR), systolic left ventricular pressure (sLVP), as well as left ventricular contraction (+dP/dt) and relaxation velocity (–dP/dt) after treatment of hearts with EGCG (4 {varepsilon}M) are shown. Data are means±SEM from n=5 experiments; *P<0.05 versus control.

 
Inhibition of the cardiac Na+/H+ exchanger (NHE) in isolated hearts with methyl-N-isobutyl amiloride (MIA, 5 {varepsilon}M) reduced the EGCG-mediated increase of sLVP by 44%, +dP/dt by 42%, and –dP/dt by 35% (Fig. 5A). Furthermore, blocking of the reverse mode of the Na+/Ca2+ exchanger (NCX) with KB-R7943 (1 {varepsilon}M) significantly suppressed the EGCG-induced increase of sLVP by 62%, +dP/dt by 65%, and –dP/dt by 61%. However, simultaneous blockade of the Na+/H+ exchanger and the reverse mode of the Na+/Ca2+ exchanger with the combination of MIA and KB-R7943 (5 {varepsilon}M and 1 {varepsilon}M) did not lead to further inhibition of EGCG-induced cardiac effects as compared to KB-R7943 alone (Fig. 5A). In the absence of EGCG, the inhibitors MIA (5 {varepsilon}M), KB-R7943 (1 {varepsilon}M), and the combination of MIA and KB-R7943 had little effect on cardiac parameters (data not shown). Pretreatment of isolated cardiac myocytes with MIA (5 {varepsilon}M) and KB-R7943 (1 {varepsilon}M) diminished EGCG-stimulated contractility in cardiac myocytes. Similar to the results in perfused Langendorff hearts, the combination of MIA and KB-R7943 had no additional effect on cardiac contractility induced by EGCG (Fig. 5B).


Figure 05
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  		Fig. 5 The cardiac Na+/H+ and Na+/Ca2+ exchangers are involved in EGCG-stimulated cardiac function. (A) Inhibition of the Na+/H+ exchanger (NHE) by methyl-N-isobutyl amiloride (MIA, 5 {varepsilon}M) and of the reverse mode of the Na+/Ca2+ exchanger (NCX) by KB-R7943 (KBR, 1 {varepsilon}M) as well as the combination of KBR and MIA suppressed the effects of EGCG (4 {varepsilon}M) on systolic left ventricular pressure (sLVP), and left ventricular relaxation velocity (–dP/dt) in perfused rat hearts. Data are means±SEM from n=5 experiments; *P<0.05 versus control; #P<0.05 versus EGCG alone. (B) Pre-incubation of isolated cardiomyocytes for 30 min with methyl-N-isobutyl amiloride (MIA, 5 {varepsilon}M), KB-R7943 (KBR, 1 {varepsilon}M), and the combination of KBR and MIA inhibited the increase in fractional shortening by stimulation of cells with EGCG (5 {varepsilon}M). (C) Pre-incubation with MIA (5 {varepsilon}M), KB-R7943 (1 {varepsilon}M), and the combination of KBR and MIA for 30 min diminished EGCG-induced (5 {varepsilon}M) calcium transients in isolated cardiomyocytes. Data are means±SEM from n=7 experiments; *P<0.05 versus control; #P<0.05 versus EGCG alone for (B) and (C).

 
To further analyse a specific interaction of EGCG with the Na+/H+ exchanger and the reverse mode of the Na+/Ca2+ exchanger, we measured calcium transients in isolated cardiomyocytes after pretreatment of cells with the two inhibitors and EGCG. The increase in systolic calcium was slightly and EGCG-induced calcium amplitude was significantly diminished by both MIA and KB-R7943. Again, the combination of MIA and KB-R7943 had no additional effects as compared to either inhibitor alone (Fig. 5C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the present study, we characterised the effects of the tea catechin EGCG on myocardial contractility and studied the underlying mechanisms involved. In isolated ventricular rat cardiomyocytes, EGCG induced an increase in cell shortening and Ca2+ transients. In isolated perfused Langendorff rat hearts, EGCG dose-dependently enhanced peak systolic pressure, as well as contraction and relaxation velocities, without affecting heart rate. EGCG-mediated inotropic effects in perfused hearts involve activation of the cardiac Na+/H+ exchanger (NHE) as well as of the reverse mode of the Na+/Ca2+ exchanger (NCX).

The increase in fractional shortening by EGCG in cardiomyocytes was accompanied by a dose-dependent rise in intracellular Ca2+ levels, thus confirming previous results in isolated guinea pig hearts [15]. Of note, we found only a marginal elevation in diastolic intracellular Ca2+ levels. However, it should be noted that intracellular calcium handling varies significantly between species [22]. In an earlier study, EGCG was shown to increase NO levels in guinea pig hearts. Although the authors observed an increase in transient NO signals in isolated guinea pig hearts, in vivo pretreatment with the NOS-inhibitor L-NAME did not affect the EGCG-mediated increase in systolic pressure due to positive inotropy in rabbits [15]. In our study, treatment of perfused hearts with L-NAME did not impair EGCG-mediated myocardial effects. These data suggest that, although EGCG is able to stimulate NO production in hearts, the increase in NO levels is not a prerequisite for its positive inotropic effects.

To elucidate the underlying signal transduction pathways for the myocardial effects of EGCG, we initially focused on the β-adrenergic pathway. EGCG-mediated inotropic effects were not affected by beta-blockade. Furthermore, treatment of hearts with EGCG did not increase cAMP levels and failed to modulate phosphorylation sites of the β1 and β2 pathway of phospholamban [23]. Altogether, these data indicate that the β-adrenergic receptor pathway is likely not involved in EGCG-induced positive inotropic and lusitropic effects.

Whereas the myocardial effects of EGCG remained unchanged after blocking of the L-type Ca2+ channel with two independent inhibitors, pretreatment of hearts with an antagonist of the Na+/H+ exchanger (NHE) significantly diminished the increase of sLVP and –dP/dt. Furthermore, inhibition of the reverse mode of the Na+/Ca2+ exchanger (NCX) significantly reduced the EGCG-mediated increase of sLVP, +dP/dt, and –dP/dt in perfused hearts. However, simultaneous blockade of the Na+/H+ exchanger (NHE) and of the reverse mode of the Na+/Ca2+ exchanger (NCX) did not further inhibit the myocardial effects of EGCG. Similar results were obtained after stimulation of isolated hearts with apelin [24]. The suppression of the EGCG-induced increase in fractional shortening and of EGCG-mediated calcium transients in isolated cardiac myocytes by MIA and KB-R7943 further supports our finding of an interaction of EGCG with the Na+/H+ exchanger (NHE) and with the reverse mode of the Na+/Ca2+ exchanger (NCX) in our study.

Activation of the reverse mode of NCX is responsible for Ca2+ influx into cells in exchange for Na+. It has been suggested that the increase in intracellular Na+ by NHE subsequently leads to elevated intracellular Ca2+ levels via activation of the reverse mode of NCX [25,26]. This may serve as an explanation for the rise in systolic Ca2+ levels in cardiomyocytes by EGCG, as observed in our study.

A number of positive inotropic substances such as apelin and endothelin-1 act by activation of NHE and NCX [24,25]. In addition to effects on myocardial contractility, increased activation of the predominant cardiac isoform NHE-1 has been associated with cardiac hypertrophy [27]. In contrast, although EGCG exerts potent positive inotropic effects partially mediated by activation of NHE, there is compelling evidence that EGCG actually prevents cardiac hypertrophy [9,10]. The reasons for these discrepant findings are not known at present. It could be speculated that the modes of NHE and NCX activation differ between the various agonists; for example whereas EGCG did not affect cAMP levels in our study, incubation of rat cardiomyocytes with ET-1 stimulated cAMP production in these cells [28]. In addition, other EGCG-mediated mechanisms involved in hypertrophy prevention, such as antioxidative effects and blocking of growth factor receptors by EGCG, may counteract hypertrophic signal transduction pathways [10]. Further studies are needed to clarify the detailed mechanisms of the cardiac actions of EGCG and to elucidate the additional factors involved.

In this study and in our previous study, the effective dose of EGCG required to exert physiological effects in isolated organs was around 1 {varepsilon}M; a concentration that can be achieved in human plasma [29]. In confirmation of the in vivo relevance of positive inotropic effects, orally applied purified catechins have been shown to improve left ventricular fractional shortening in a rat autoimmune myocarditis model [12].

A number of studies suggest that the biological activities of tea polyphenols involve the presence of galloyl groups in their 3' positions. The compound theaflavin-3,3'-digallate (TF3) from black tea possesses similar galloyl groups to EGCG. In our study, however, TF3 failed to affect cardiac performance in perfused rat hearts and isolated cardiomyocytes, indicating that structurally similar tea polyphenols may differ in their physiological properties in different cell types or tissues.

In conclusion, we have shown a direct effect of EGCG on cardiac performance via an intracellular increase of systolic Ca2+, and at least partial involvement of the Na+/H+ exchanger and Na+/Ca2+ exchanger. In contrast to other non-catecholamine-like positive inotropic agents, EGCG also leads to positive lusitropy. The combination of positive inotropism and lusitropism, in conjunction with the absence of an increase in heart rate and with beneficial effects on the vascular system, gives EGCG an advantageous haemodynamic profile.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We wish to thank Kerstin Daemen, Angelika Vietzke, and Wanda Michaelis for their excellent technical assistance.


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

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