Differential effects of a green tea-derived polyphenol ...

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Jan 30, 2008 - Abstract (−)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against ...
Pflugers Arch - Eur J Physiol (2008) 456:787–800 DOI 10.1007/s00424-008-0456-y

CARDIOVASCULAR PHYSIOLOGY

Differential effects of a green tea-derived polyphenol (−)-epigallocatechin-3-gallate on the acidosis-induced decrease in the Ca2+ sensitivity of cardiac and skeletal muscle Ying-Ming Liou & Shih-Chang Kuo & Shih-Rong Hsieh

Received: 4 December 2007 / Revised: 12 January 2008 / Accepted: 14 January 2008 / Published online: 30 January 2008 # Springer-Verlag 2008

Abstract (−)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined its effects on the acidosis-induced change in the Ca2+ sensitivity of myofilaments in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. EGCg (0.1 mM) significantly inhibited the decrease caused by lowering the pH from 7.0 to 6.0 in the Ca2+ sensitivity of myofibrillar ATPase activity in cardiac muscle, but not in skeletal muscle. Studies on recombinant mouse cardiac troponin C (cTnC) and chicken fast skeletal troponin C (sTnC) using circular dichroism and intrinsic and extrinsic fluorescence spectroscopy showed that EGCg bound to cTnC with a dissociation constant of ∼3–4 μM, but did not bind to sTnC. By presumably binding to the cTnC C-lobe, EGCg decreased Ca2+ binding to cTnC and overcame the depressant effect of protons on the Ca2+ sensitivity of the cardiac contractile response. To demonstrate isoform-specific effects of the action of EGCg, the pH sensitivity of the Ca2+ response was examined in cardiac myofibrils in which endogenous cTnC was replaced with exogenous sTnC or cTnC and in skeletal myofibrils in which the endogenous sTn complex was replaced with whole cardiac Tn complex (cTn). The results suggest that

Y.-M. Liou (*) : S.-C. Kuo Department of Life Sciences, National Chung-Hsing University, 250 Kuokang Road, Taichung 402, Taiwan e-mail: [email protected] S.-R. Hsieh Department of Cardiovascular Surgery, Taichung Veterans General Hospital, Taichung 407, Taiwan

the binding of EGCg to the cardiac isoform-specific TnC or Tn complex alters the effect of pH on myofilament Ca2+ sensitivity in striated muscle. Keywords EGCg . Green tea polyphenols . pH sensitivity . Ca2+ sensitivity . Troponin C

Introduction (−)-Epigallocatechin-3-gallate (EGCg) is a polyphenol that makes up 15–32% of the solids in green tea [1–3]. This compound has attracted much medicinal attention in the prevention of cardiovascular diseases [4–6]. Studies using an animal model of ischemia/reperfusion have shown that ischemia induces myocardial apoptosis via the STAT-1 (signal transducers and activators of transcription) signaling pathway [7], and that EGCg can inhibit intracellular STAT1 activation and protect the myocardium from apoptosis induced by ischemia [8]. However, it is not known whether green tea polyphenols can improve the cardiac function by the direct effects on the cardiac contractile apparatus. Troponin is a molecular switch which controls the Ca2+dependent activation of myofilaments in cardiac (cTn) and skeletal (sTn) muscle [9, 10]. The Tn complex contains three subunits: troponin C (TnC), a Ca2+-binding subunit; troponin I (TnI), an inhibitory subunit; and troponin T (TnT), a subunit which binds the complex to tropomyosin (TM). cTn complex, which can undergo covalent and noncovalent modification, plays an essential role in the modulation of myocardium performance in both normal and diseased hearts [11, 12]. Covalent modification largely through the phosphorylation of cTnI and cTnT by protein kinases A and C, modulates myofilament Ca2+ sensitivity and cross-bridge cycling in the myocardium [13]. Non-

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covalent modification of cardiac Tn occurs, e.g., the Cterminal region of cTnI is sensitive to intracellular acidification [14–16]. Such modification by acidosis has a marked depressant effect on myofilament Ca2+ sensitivity and the efficiency of the contractility associated with myocardial ischemia. Both sTnC and cTnC have two globular Ca2+-binding domains connected by a linker helix. The N-terminal domain of sTnC contains sites I/II that bind Ca 2+ exclusively but with a lower affinity (∼105 M−1) than the C-terminal sites III/IV, which also bind Mg2+. Upon Ca2+ binding to sites I and II the N-lobe of sTnC switches from a closed to an open conformation such that Ca2+ binding overcomes the inhibitory action of sTnI on the actin– myosin interaction [17, 18]. Since site I in cTnC is not functional due to some amino acid changes [19], binding of Ca2+ to site II in cTnC does not induce a similar opening. The fully open conformation of the N-lobe of cTnC requires the complexation with cTnI and the binding of Ca2+ to site II [20]. Sites III and IV in the C-terminal domain in both sTnC and cTnC bind Ca2+ with high affinity (∼107 M−1) and also bind Mg2+ (∼103 M−1). The occupation of these sites facilitates the binding of TnC to the thin filament. This C-terminal domain has been considered to be a structural domain [21]. In this study, we found that EGCg acts by modulating the pH-induced change in myofilament Ca2+ sensitivity in cardiac muscle but not in skeletal muscle. This muscle-type specific effect was shown to be related to the specific binding of EGCg to cTnC, presumably at the C-terminal sites. We suggest a novel mechanism whereby EGCg might act by modification of signaling around the C-lobe of cTnC in cardiac muscle.

Materials and methods All reagents used were ACS or MB grade. EGCg was purchased from Sigma. 7-Diethyl-amino-3-[4′-maleimidylphenyl]4-methylcoumarin (CPM), was purchased from Molecular Probes. Chromatographic reagents (DEAE-Sephadex A-25 and A-50, CM-Sephadex C-50, HisTrap HP, Phenyl-Sepharose 6 Fast Flow, and HiPre 26/60 Sephacryl S-200 high resolution) were purchased from Amersham Pharmacia Biotech Asia Pacific. Bicinchoninic acid protein assay reagent was obtained from Pierce Chemicals. A 10 mM stock solution of EGCg was prepared by dissolving the compound in de-ionized water. Protein preparation and fluorescent labeling Mouse cTnC and chicken fast sTnC cDNAs, generated by RT-PCR, were cloned, respectively, into the pET32a or pT7-5 expression vector and overexpressed in E. coli BL

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21 (DE3) as described previously [22]. Cardiac troponin complex (cTn) was prepared from porcine heart as described by Potter [23]. CPM fluorescent labeling of recombinant cTnC was described previously [24]. The amount of fluorescence bound was 1.4–2.0 mol of fluorophore per mole of protein. CD measurements Circular dichroism (CD) spectra were obtained on a JASCO J-815 spectrometer (JASCO International Co Ltd., Tokyo, Japan) at a constant temperature of 25°C. cTnC or sTnC (0.25 mg/ml) was dissolved in 10 mM phosphate buffer, pH 7.0, and EGCg was added as indicated. For each data point, three scans were averaged and corrected for base line by subtraction of the spectrum for buffer alone. The CD data obtained at each wavelength, λ, were converted to mean residue ellipticity (½ql ) using the equation described by Gusev and Barskaya [25]:  ½θλ ¼ ðθλ  M Þ ð10  l  cÞ deg cm2 dmol1

ð1Þ

where θλ is the observed ellipticity at wavelength λ in degrees, l the optical path length in centimeters, c the concentration of the protein in grams per milliliter, and M the mean residue weight, 115. The fraction helix, fH, was estimated using the following empirical equation, according to Van Eyk et al. [26]:   fH ¼ ½θ222 ½θ1 ð2Þ H ð1  k=nÞ where [θ]222 is the mean molar residue ellipticity at 222 nm, ½q 1 H the mean molar residue ellipticity for an infinite helix (−37,400 deg), k the chain length dependence factor (2.5), and n the number of residues (9) in a typical helix. The number of helical residues in cTnC and sTnC was then calculated by multiplying fH by the number of amino acids in the protein (161 and 163, respectively). Fluorescence measurements The protein intrinsic fluorescence spectra of tyrosine in cTnC and phenylalanine in sTnC were measured using a PerkinElmer LS 50B spectrofluorimeter (Beaconsfield, Buckinghamshire, England) at a constant temperature of 25°C. cTnC or sTnC was dissolved at a concentration of 16.6 μM in pH 7.0 buffer (100 mM MOPS, 90 mM KCl, and 2 mM EGTA) or pH 6.5 buffer (100 mM MES, 90 mM KCl, and 2 mM EGTA), with EGCg, Ca2+, or Mg2+ being added where indicated. EGCg has a major absorption peak at 231 nm, but no detectible fluorescence emission from 270 to 420 nm. In all cases, the corrected tyrosine fluorescence for cTnC in the presence and absence of EGCg was obtained by excitation at 276 nm and emission scanning from 270 to 430 nm.

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Phenylalanine fluorescence for sTnC was elicited by excitation at 257 nm and emission was scanned from 270 to 320 nm. The slit widths for excitation and emission filters were 5 and 10 nm, respectively. For measurements of the CPM fluorescence spectra, 1– 2 μM labeled cTnC was dissolved in pH 7.0 buffer (100 mM MOPS), pH 6.5 buffer (100 mM MES), or pH 6.0 buffer (100 mM MES), each containing 90 mM KCl, 2 mM EGTA, with EGCg, Ca2+, or Mg2+ added where indicated. The solution was excited at 387 nm and the fluorescence emission was scanned from 420 to 520 nm, with slit widths of 5 and 10 nm for excitation and emission, respectively. The unlabeled or CPM-labeled protein solution was placed in a 1 ml semi-micro cell and EGCg or Ca2+ titrations carried out by adding aliquots of a stock solution of 10 mM EGCg or a commercial standard solution of 0.1 M CaCl2, respectively. The total volume change caused by the EGCg and Ca2+ additions did not exceed 2%. For data analysis, the tyrosine fluorescence of cTnC, phenylalanine fluorescence of sTnC, or CPM fluorescence of labeled cTnC with emission at 306, 287, or 470 nm, respectively, was measured as a function of the EGCg/protein molar ratio and the pCa (−log [Ca2+]) at different pH. In the case of EGCg, the measured fluorescence without EGCg was used to normalize the subsequent fluorescence change (ΔF) caused by EGCg titration (Δ[EGCg]). The relationship between 1/ΔF and 1/Δ[EGCg] was plotted as a regression line and the slope and the y intercept obtained were used to estimate the dissociation constant (Kd =slope/y intercept) for the interaction of cTnC with EGCg. To evaluate Ca2+ binding, the measured fluorescence at a given pCa was subtracted from that at pCa 8 and the fluorescence value (Fx) normalized to the value at saturating Ca2+ (pCa 4.0; F0). If the normalized fluorescence (U=Fx /F0) is used, a straight line is obtained when log [U/(1−U)] is plotted versus the logarithm of the Ca2+ concentration. The data were fitted to the Hill equation: log½U =ð1  U Þ ¼ nðlog ½Cax Þ þ log k

ð3Þ

where [Cax] is the actual Ca concentration, n (Hill coefficient) is the slope, and k is the x-axis intercept of the fitted line. By using the constants derived from the Hill equation, the curves of the normalized fluorescence changes (Fx /F0) versus pCa were determined using a computer with the equation: 2+

ðFx =F0 Þ ¼ ½Cax n =ððEC50 Þn þ ½Cax n Þ

ð4Þ

where EC50 is the Ca2+ concentration giving 50% activation of fluorescence changes. Free Ca2+ concentrations in EGTA buffers were calculated on the basis of constants tabulated by Fabiato and Fabiato [27]. The pCa values were calculated by the computer program EQCAL (Biosoft, Cambridge, UK).

ATPase activity assay Myofibrils were prepared from porcine cardiac muscle and chicken pectoral muscles as described previously [28–29]. Actomyosin ATPase activity was measured by analyzing inorganic phosphate release. The relationship between myofibrillar ATPase activity (nanomole Pi per minute per milligram protein) and the pCa was determined using the Hill plot as described previously [30]. From the plot, the Hill coefficient (n), as a measure of cooperativity, and the Ca2+ concentration giving half-maximal activation (pCa1/2) were obtained for the Ca2+-activated myofibrillar ATPase activity. Extraction of cTnC from, and reinsertion of sTnC or cTnC into, cardiac myofibrils Extraction of endogenous cTnC from and exogenous cTnC or sTnC re-incorporation into porcine cardiac myofibrils was described by Liou and Chang [31]. The degree of extraction and re-incorporation was determined by SDSPAGE and by the loss and recovery of Ca2+-activated ATPase activity. Exchange of the troponin complex (Tn) in skeletal myofibrils Exchange of the whole troponin complex (Tn) in chicken pectoral skeletal myofibrils with purified porcine cTn was carried out at 25°C according to She et al. [32]. Based on the difference in the molecular weight in the different tissues, the efficiency of cTn complex replacement was determined by SDS-PAGE, followed by Western blotting for TnI and TnT using monoclonal antibodies against TnI (Mab 1691, Chemicon, Minipore) or TnT (JLT-12, Sigma), respectively. Statistics Quantitative values are expressed as the mean ± SEM. Comparisons were performed using Student’s t test with P values less than 0.05 being considered significant.

Results Effects of EGCg on the acidic pH-induced decrease in Ca2+dependent myofibrillar ATPase activity in cardiac and skeletal muscle Intracellular acidosis is an important factor in the depressed contractility associated with myocardial ischemia. Figure 1 shows that EGCg (0.1 mM) reduced the acidic pH-induced decrease in Ca2+-dependent myofibrillar ATPase activity in

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Fig. 1 Effects of EGCg on the acidic pH-induced decrease in Ca2+-dependent myofibrillar ATPase activity in a cardiac or b skeletal muscle. c Quantitative analysis of the effects of EGCg on the pH-induced decrease in the Ca2+ sensitivity (pCa1/2) of myofibrillar ATPase activity in porcine cardiac or chicken pectoral muscle. Actomyosin ATPase activity was measured by suspending myofibrils (0.2 mg) in pH 7.0 buffer (100 mM MOPS), pH 6.5 buffer (100 mM MES), or pH 6.0 buffer (100 mM MES), all containing 90 mM KCl, 5 mM MgCl2, and 2 mM EGTA, in the presence of 0.1 mM EGCg (bottom) or absence of EGCg (top). The measured ATPase activity at the actual pCa was subtracted from the activity at pCa 8. The subtracted activity was normalized to the activity value at pCa 4. The relationship between the normalized ATPase activity and the pCa was determined as described in the “Materials and methods” section. Each value is the mean ± SEM of five measurements

Pflugers Arch - Eur J Physiol (2008) 456:787–800

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cardiac muscle. On lowering the pH from 7.0 to 6.0 (Fig. 1a), the Ca2+ sensitivity (pCa1/2) of cardiac myofibrillar ATPase decreased in the absence of EGCg (Table 1), and this effect was reduced in the presence of 0.1 mM EGCg (Table 1). The degree of the decrease in the pCa1/2 (ΔpCa1/2) induced by lowering the pH (0.64 going from pH 7 to 6.5 and 1.27 for pH 6.5 to 6.0) was significantly reduced by EGCg (0.37 for pH 7 to 6.5 and 0.69 for pH 6.5 to 6.0; Fig. 1c), showing that EGCg reduced the acidic pHinduced decrease in the pCa1/2 for cardiac myofibrillar ATPase activity. In contrast, the acidic pH-induced decrease in the pCa1/2 for skeletal myofibrillar ATPase activity was not affected by EGCg (Fig. 1b, Table 1). These results

show that EGCg has a differential effect on the acidic pHinduced decrease in Ca2+ sensitivity of different muscles. Interaction of EGCg with cTnC or sTnC To demonstrate the interaction of EGCg with TnC, the effects of different concentrations of EGCg on the CD spectrum of cTnC were examined and are shown in Fig. 2a. As shown in Fig. 2a (left panel), cTnC alone showed a CD spectrum characteristic of α helices, with a negative peak with separate maxima of similar magnitude at 208 and 222 nm, and this spectrum was not affected by the addition of EGCg up to a molar ratio of 0.5 (shown as “0–0.5”). An

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Table 1 Effects of EGCg on the half-maximal Ca2+ concentrations (pCa1/2) and Hill coefficient (n) for Ca2+-regulated myofibrillar ATPase activity of cardiac (control, CDTA extraction, sTnC substitution, and cTnC substitution) and skeletal myofibrils (control, cTn reconstitution) at pH 7.0, 6.5, and 6.0 pH 7.0

Cardiac myofibrils Control CDTA extraction sTnC substitution cTnC substitution Control + EGCg sTnC substitution + EGCg cTnC substitution + EGCg Skeletal myofibrils Control Control + EGCg cTn reconstitution cTn reconstitution + EGCg

pH 6.5

pH 6.0

pCa1/2

n

pCa1/2

n

pCa1/2

n

6.38±0.05 4.59±0.57 6.07±0.05 6.34±0.12 5.79±0.03 6.07±0.05 5.45±0.08

1.49±0.08 0.11±0.00 1.63±0.19 1.33±0.13 1.78±0.19 1.63±0.19 1.29±0.11

5.74±0.02 4.06±0.55 5.54±0.17 5.50±0.06 5.31±0.04 5.58±0.06 5.80±0.07

2.04±0.43 0.06±0.02 2.13±0.27 1.60±0.16 1.69±0.10 2.33±0.40 2.13±0.38

4.47±0.09 2.03±0.19 5.16±0.13 4.70±0.09 4.78±0.06 5.12±0.14 5.26±0.07

1.50±0.07 0.06±0.01 1.87±0.13 1.62±0.31 1.65±0.18 1.30±0.12 1.44±0.1

6.70±0.02 6.54±0.07 6.59±0.10 6.03±0.12

3.37±0.22 2.53±0.39 1.46±0.05 1.35±0.15

6.06±0.03 5.93±0.01 5.63±0.05 5.82±0.04

2.62±0.04 2.17±0.14 1.43±0.24 1.50±0.08

5.44±0.02 5.16±0.05 5.14±0.12 5.34±0.02

1.64±0.34 2.08±0.15 1.82±0.26 1.82±0.05

Each value is the mean ± SEM with five measurements.

increase in the EGCg/cTnC molar ratio from 0.5 to 1.5 caused changes in the CD spectrum, with a small right shift of the peak maximum at 208 nm and an increase in the negative ellipticity at 222 nm ([θ222]; further evidence for an increase in α helix). No further changes were seen on increasing the ratio to 2.0. The effects on the [θ222] are summarized in Fig. 3a. In contrast, EGCg had no effect on the CD spectrum for sTnC (Figs. 2a and 3a). The number of helical residues (HR) calculated according to Van Eyk et al. [26] was 102 or 116, respectively, for cTnC alone or in the presence of saturating levels of EGCg, while only a small difference was seen with sTnC (HR=122 for sTnC alone or 124–126 with EGCg). This shows that EGCg binds to cTnC, but not to sTnC, and only elicits structural changes in cTnC.

interaction at a pCa of 4 or 8 and a pH of 7 or 6.5 were linear, suggesting that the binding of EGCg to cTnC was non-cooperative (Fig. 3b, right panel). The Kd values (dissociation constants=slope/y intercept) for the interaction of cTnC with EGCg at pH 7 and 6.5, estimated from Fig. 3d, were 3–4 and 6–7 μM, respectively. The binding of EGCg to cTnC was twice as strong at pH 7.0 than at pH 6.5. At both pH, there was only a small increase in the Kd value for the EGCg–cTnC interaction in the presence of Ca2+ (3.88 and 4.18 μM at pH 7 and pCa of 8 or 4, respectively; 6.17 and 7.72 μM at pH 6.5 and pCa 8 or 4, respectively). In contrast, addition of EGCg to sTnC with or without Ca2+ caused only a small change (less than 10%) in the intrinsic phenylalanine fluorescence of sTnC (Fig. 2b, right panel and Fig. 3b, left panel).

Effects of EGCg on the intrinsic fluorescence spectrum of TnC

Effects of EGCg on the fluorescence spectrum of cTnC labeled with CPM at Cys-35 and Cys-84

cTnC contains three tyrosine residues (Tyr-5, Tyr 111, and Tyr-150). It was shown that Ca2+ binding to the C-terminal sites caused a significant increase in tyrosine fluorescence while binding at the N-terminal sites caused no further change [31, 33]. To verify the EGCg-induced structural changes in cTnC, analyses of the intrinsic fluorescence spectra of tyrosine in cTnC were carried out to examine the binding of EGCg to cTnC at different molar ratios at a pCa (−log [Ca2+]) of 8.0 and 4.0 (Fig. 2b). Increasing the molar ratio of EGCg to cTnC from 0.5 to 2.0 caused a dosedependent decrease in the tyrosine fluorescence of cTnC, with almost a maximal effect at a 1:1 molar ratio of EGCg to cTnC (Fig. 2b, left panel). Double reciprocal plots (1/ΔF versus 1/[EGCg]) of the data for the EGCg and cTnC

The presence of the two cysteines (Cys-35 and Cys-84) in the N-terminal domain of cTnC allows the attachment of conformational probes to this region [24, 28, 31]. To distinguish between the effects of EGCg on the N-terminal and Cterminal domains of cTnC, we labeled the two N-terminal thiol groups of cTnC with a fluorescence probe (CPM) to monitor conformational changes in this region upon EGCg binding. As shown in Fig. 2c, in conditions when no metal ions were bound, Mg2+ was bound to the C-terminal high affinity sites (5 mM Mg2+ and pCa 8), or Mg2+/Ca2+ was bound to the C-terminal high affinity sites and Ca2+ to the Nterminal sites (5 mM Mg2+ and pCa 4), varying the EGCg/ cTnC molar ratio from 0 to 2 caused no significant change in the CPM fluorescence spectrum of labeled cTnC. The

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Fig. 2 Effect of different doses of EGCg on its interaction with cTnC and sTnC. a Dose-dependent changes in the CD spectrum of cTnC (left panel) or sTnC (right panel) caused by the addition of EGCg. cTnC or sTnC (0.25 mg/ml) and EGCg at different molar ratios from 0 to 2 were dissolved in 10 mM phosphate buffer, pH 7.0. b Dose-dependent changes in the intrinsic tyrosine fluorescence spectrum of cTnC (left panel) or the phenylalanine fluorescence spectra of sTnC (right panel) in pH 7 buffer with a pCa of 8 caused by EGCg addition. cTnC or sTnC was dissolved at a concentration of 16 μM in 100 mM MOPS (pH 7.0) or 100 mM MES (pH 6.5) containing 90 mM KCl, 2 mM EGTA, and EGCg was added as indicated. c EGCg-independent changes in the fluorescence spectrum of CPM-labeled cTnC in conditions in which no metal ions are bound (upper left panel), Mg2+ is bound to the C-terminal high affinity sites (upper right panel), or Mg2+/ Ca2+ is bound to the C-terminal high affinity sites and Ca2+ to the N-terminal sites (bottom panel). The labeling ratio of cTnC with CPM was 1.4–2.0. Labeled cTnC (1–2 μM) was dissolved in pH 7.0 buffer (100 mM MOPS, 90 mM KCl, 2 mM EGTA), and EGCg, Ca2+, and Mg2+ were added as indicated

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Fig. 3 Quantitative analysis of the interaction of EGCg with cTnC or sTnC. a Dose-dependent effects of EGCg on the negative ellipticity at 222 nm ([θ222]) in the CD spectra of cTnC and sTnC. b EGCginduced changes in the relative fluorescence of cTnC tyrosine fluorescence emitted at 306 nm at pH 7.0 or 6.5 and pCa 4 or 8 and sTnC phenylalanine fluorescence at 287 nm at pH 7.0 and pCa 4 or 8 (left panel), and double reciprocal plots for analyzing the interaction of cTnC with EGCg (right panel). Fluorescence spectroscopy was performed as described in the “Materials and methods” section. The measured fluorescence without EGCg was used to normalize the subsequent fluorescence changes (ΔF) by EGCg titrations (Δ[EGCg]). The relationship between 1/ΔF and 1/Δ[EGCg] was

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plotted as a regression line, and the slope and y intercept were used to estimate the dissociation constant (Kd =slope/y intercept) for the interaction of cTnC with EGCg. Each point is the average of five measurements. c CPM fluorescence as different molar ratios of EGCg to labeled cTnC in conditions of no metal binding (1), Mg2+ bound to the C-terminal sites (2), or Mg2+/Ca2+ bound to the C-terminal sites and Ca2+ bound to the N-terminal sites (3). CPM fluorescence measurements were made as described in the “Materials and methods” section. The fluorescence emitted at 470 nm measured in the absence of EGCg was used to normalize the fluorescence changes caused by EGCg titrations. Each value is the mean ± SEM of five measurements

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Pflugers Arch - Eur J Physiol (2008) 456:787–800 R Fig. 4 Effects of EGCg on Ca2+-induced changes in cTnC tyrosine

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determination of the high-resolution structure of EGCg bound to cTnC.

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fluorescence at pH 7 and 6.5. a, b Ca2+ titration of the tyrosine fluorescence spectrum of cTnC in the presence of a 1:1 molar ratio of EGCg at pH 7 (a) or pH 6.5 (b). cTnC (16 μM) was dissolved in 100 mM MOPS (pH 7.0) containing 90 mM KCl, 2 mM EGTA in the presence or absence of 16 μM EGCg at pCa of 7.23 to 3.44 (a) or in 100 mM MES (pH 6.5) containing 90 mM KCl, 2 mM EGTA in the presence or absence of 16 μM EGCg at pCa of 7.07 to 3.84 (b). The corrected spectrum was obtained by subtraction of the spectrum of EGCg alone. c Tyrosine fluorescence–pCa curves of cTnC in the presence and absence of EGCg at pH 7 and 6.5. The relative tyrosine fluorescence of cTnC (at 306 nm) at each pCa was obtained by subtracting the fluorescence measured at each pCa from that at pCa 8, then normalizing the value to the difference at pCa 4. The curve was fitted to the data using the Hill equation as described in the “Materials and methods” section. Each point is the mean ± SEM of five measurements. d Ca2+ sensitivity (pCa1/2) of cTnC tyrosine fluorescence at pH 7 or 6.5 in the absence or presence of EGCg. The data are derived from c

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quantitative analysis of the CPM fluorescence as a function of the EGCg/labeled cTnC molar ratio is shown in Fig. 3c. This shows that EGCg did not cause structural changes in the N-terminal domain of cTnC, suggesting that it did not bind to this domain. However, this needs to be confirmed by

To determine whether the binding of EGCg to cTnC affected the Ca2+-binding properties of cTnC, we measured the relationship between the pCa and tyrosine fluorescence of cTnC in the presence and absence of EGCg at pH 7.0 and 6.5. We confirmed the previous observation [31, 33] that Ca2+ binding to the high affinity sites (III and IV) in the Cterminal domain of cTnC caused a significant increase in tyrosine fluorescence. In the presence of a 1:1 molar ratio of EGCg to cTnC and at pH 7.0, Ca2+ titration from pCa 7.23 to 3.44 caused changes in the tyrosine fluorescence spectrum (Fig. 4a). The pCa–tyrosine fluorescence relation was shifted to a lower pCa in the presence of EGCg (pCa1/2 =6.26±0.05; n=1.82±0.12) compared to in its absence (pCa1/2 =6.99± 0.04; n=1.41±0.24; Fig. 5c). At acidic pH, protons are known to interfere with Ca2+ binding to the C-terminal sites of cTnC and thus affect the Ca2+ sensitivity of the tyrosine fluorescence [31]. Consistent with this result, we found that, at pH 6.5 and in the absence of EGCg the Ca2+-dependent curve was shifted to a lower pCa (pCa1/2 =6.24±0.08; n= 1.53±0.08) compared to that at pH 7.0 (Fig. 4c). In contrast, in the presence of EGCg, the change in the fluorescence spectrum was more Ca2+ sensitive at pH 6.5 (Fig. 4b) and the Ca2+-dependent curve for the fluorescence change was shifted back to a higher pCa (pCa1/2 =6.68±0.07; n=1.15± 0.12; Fig. 4c). Figure 4d summarizes the effect of pH on Ca2+ binding to cTnC in the presence and absence of EGCg. This suggests that EGCg might affect Ca2+ binding to the high affinity sites III and IV in the C-terminal domain of cTnC and overcome the interference by protons on the Ca2+ binding to these sites in cTnC.

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Fig. 5 Effects of EGCg on the Ca2+-dependent fluorescence change in CPM-labeled cTnC at different pH. a, b Ca2+-dependent changes in the fluorescence spectra of CPM-labeled cTnC at pH 7.0 (a) or pH 6.5 (b) in the absence (left panel) or presence (right panel) of EGCg. c Ca2+dependent fluorescence change curves (left panel) and the pHdecreased Ca2+ sensitivity (pCa1/2; right panel) for CPMlabeled cTnC in the presence or absence of EGCg at pH 7 and 6.5. CPM-labeled cTnC was dissolved at a concentration of 1–2 μM in 100 mM MOPS (pH 7.0) or 100 mM MES (pH 6.5) buffer containing 90 mM KCl and 2 mM EGTA in the presence or absence of 1–2 μM EGCg and Ca2+ as indicated. The fluorescence emitted at 470 nm for the CPM-labeled cTnC at the actual pCa was subtracted from the fluorescence at pCa 8 and the value normalized to the fluorescence intensity at pCa 4. The relationship between the normalized fluorescence and pCa was plotted as described above. Each point is the mean ± SEM of five measurements

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Effects of EGCg on the Ca2+-dependent fluorescence changes of CPM-labeled cTnC at pH 7.0 and 6.5 Figure 5 shows the effects of EGCg on the Ca2+-induced fluorescence changes in cTnC labeled with CPM at the two N-terminal thiol groups. In the absence (Fig. 5a, left panel) or presence (Fig. 5a, right panel) of EGCg (1:1 molar ratio), binding of Ca2+ to sites III and IV (pCa 7.35–6.57) at pH 7 had no effect on the CPM fluorescence, while binding to site II (pCa 6.22–3.39) resulted in a significant increase in fluorescence. The Ca2+-dependent fluorescence change curves showed that the half-maximal Ca2+ concentration (pCa1/2) was 6.02±0.03 and 6.15±0.04 and the cooperativity (n) was 1.41±0.15 and 1.53±0.1 for labeled cTnC alone or in the presence of EGCg, respectively (Fig. 5c). Thus, at pH 7.0, EGCg only caused a small increase (∼0.1 pCa) in Ca2+ binding to site II in the N-terminal domain of cTnC. At pH 6.5, CPM-labeled cTnC in the absence (Fig. 5b, left panel) or presence (Fig. 5b, right panel) of EGCg showed decreased sensitivity of the Ca2+induced changes in the fluorescence spectrum. In addition, the Ca2+-dependent curves for the fluorescence changes of CPM-labeled cTnC in the absence or presence of EGCg shifted to higher Ca2+ concentrations when the pH was decreased from 7 to 6 (Fig. 5c). These results show that EGCg did not alter the effect of protons (acidic pH) on Ca2+ binding to N-terminal site II in cTnC. Effects of EGCg on the acidic pH-induced decrease in Ca2+dependent ATPase activity in cTnC- and/or sTnCreconstituted cardiac myofibrils To determine whether the differential effect of EGCg was due to the binding of EGCg to tissue-specific TnC isoforms in cardiac and skeletal muscle, we exchanged the TnC isoform in cardiac myofibrils. Ninety percent of the endogenous cTnC was removed from porcine cardiac myofibrils using low ionic strength CDTA solution and substantial amounts of either cTnC or sTnC inserted into the CDTA-treated cardiac myofibrils (Fig. 6a). At pH 7.0 (Fig. 6b), 6.5 (Fig. 6c), or 6.0 (Fig. 6d), CDTA extraction significantly reduced the regulation of myofibrillar ATPase activity by Ca2+, and insertion of either cTnC or sTnC restored the Ca2+-activated ATPase activity. The effect of acidic pH on the ΔpCa1/2 for myofibrillar ATPase was greater in cTnC-reconstituted (−0.84 for pH 7 → 6.5, −0.80 for pH 6.5 → 6.0) than in sTnC-reconstituted (−0.53 for pH 7 → 6.5, −0.38 for pH 6.5 → 6.0) cardiac myofibrils (Fig. 6e). Consistent with the results of a previous study of the ectopic expression of the sTnC in the heart of transgenic mice [34], sTnC reduces contractile sensitivity to acidosis in cardiac myocytes. In the presence of 0.1 mM EGCg, cTnC-reconstituted cardiac myofibrils showed a reduced

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effect of acidic pH on the ΔpCa1/2 for myofibrillar ATPase (+0.35 for pH 7 Δ pCa 6.5, −0.54 for pH 6.5 → 6.0) compared to in its absence (−0.84 for pH 7 → 6.5, −0.80 for pH 6.5 → 6.0; Fig. 6e). In contrast, EGCg had only a small effect on the pH sensitivity of the Ca2+ response in sTnC-reconstituted cardiac myofibrils (Fig. 6e). This result shows that the TnC isoform determines the effect of EGCg on the acidic pH-dependent change in Ca2+ sensitivity in cardiac muscle. Effect of EGCg on the acidic pH-induced reduction of Ca2+ sensitivity of myofibrillar ATPase activity in skeletal myofibrils in which the endogenous Tn is replaced with cardiac Tn To verify the differential effect of the Tn isoform on the action of the EGCg, the endogenous sTn complex in chicken pectoral skeletal myofibrils was replaced with the porcine cardiac Tn complex, as shown in Fig. 7a. A significant decrease in cooperativity and a small right shift in the pCa1/2 for myofibrillar ATPase activity was found after cTn complex replacement (Fig. 7b, Table 1). A 0.1 mM EGCg caused a small decrease in the pCa1/2 and cooperativity in control pectoral muscle, but a greater decrease in the pCa1/2 and no change in cooperativity in cTn-replaced pectoral muscle (Fig. 7b, Table 1). These results show that the replacement of the endogenous sTn complex with the cTn complex altered the characteristics of Ca2+ regulation in chicken pectoral muscle. In addition, the pH-sensitive deactivation of Ca2+ sensitivity was greater in the cTn-replaced skeletal muscle than in control muscle (Fig. 7c–e). This is consistent with the notion that the muscle type-specific Tn isoform plays a key role in determining the pH dependence of Ca2+ sensitivity in cardiac and skeletal muscle [35]. These results show that EGCg acts on cTn, but not on sTn, and thus could have a differential effect on the pH-dependent changes in Ca2+ sensitivity in cardiac muscle.

Discussion There is increasing evidence that green tea-derived polyphenols (e.g., catechins and EGCg) may be promising compounds for protection against myocardial damage [1– 8]. Studies on myocardial ischemia/reperfusion injury have suggested that the myocardial protective effect of green tea polyphenols is associated with their antioxidant properties of scavenging active oxygen radicals, modulating redoxsensitive transcription factors (e.g., NFκB, AP-1), reducing STAT-1 activation and Fas receptor expression, and increasing NO production [4–8]. In this study, we showed that EGCg might bind to the cardiac isoform-specific TnC

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pH Fig. 6 Effects of EGCg on the acidic pH-induced decrease in Ca2+dependent ATPase activity in cardiac myofibrils reconstituted with cTnC or sTnC at pH 7, 6.5, or 6. a Typical SDS gel showing the removal of cTnC from porcine cardiac myofibrils by CDTA treatment and reconstitution of the CDTA-extracted cardiac myofibrils with either cTnC or sTnC. cTnC removal from cardiac myofibrils (lane 2) and reconstitution with cTnC (lane 3) or sTnC (lane 4) were performed as described in the “Materials and methods” section. Fifty to sixty micrograms of myofibrillar proteins or 10 μg of pure cTnC (lane C) or pure sTnC (lane S) was loaded on the gel. Lane 1 is control

cardiac myofibrils. The main constituents of myofibrils (e.g., actin, cTnT, Tm, cTnI, LC1, LC2, and cTnC) are indicated. b–d Comparison of the Ca2+-dependent ATPase activity in CDTA-extracted, cTnCreconstituted, and sTnC-reconstituted cardiac myofibrils in the presence and absence of EGCg at pH 7 (b), pH 6.5 (c), or pH 6.0 (d). e EGCg modulation of the pH-induced decrease in the pCa1/2 for myofibrillar ATPase activity of cTnC-reconstituted and sTnC-reconstituted cardiac muscle. In b–e, each value is the mean ± SEM of five measurements

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Fig. 7 Effects of EGCg on the acidic pH-induced decrease in the Ca2+ sensitivity of skeletal myofibrillar ATPase activity in chicken pectoral skeletal myofibrils in which the endogenous Tn complex was replaced with whole porcine cardiac Tn complex. a Exchange of whole Tn complex of chicken fast skeletal myofibrils with cTn complex was performed as described in the “Materials and methods” section, according to She et al. [32]. Fifty to sixty micrograms of myofibrillar proteins or 10 μg cTn complex (lane cTn) isolated from porcine cardiac muscle was loaded on the gel. The TnI isoform was detected using a mouse monoclonal antibody against TnI as described in the “Materials and methods” section and the type determined by the molecular weight of 21 kD for sTnI or 24 kD for cTnI. After the membranes were washed, they were developed with an ECL plus kit (Perkin-Elmer LAS,

Inc, MA, USA), following the manufacturer’s recommendations. Typical stained SDS gel (upper panel) and Western blotting analysis (lower panel) showing the tissue-specific isoforms TnT, TnI, and TnC in chicken fast skeletal myofibrils (S), porcine cardiac myofibrils (C), and chicken fast skeletal myofibrils with an exchanged Tn complex (Re). Lane M is molecular weight markers. b–d Comparison of the Ca2+-dependent myofibrillar ATPase activity of control and cTnexchanged chicken pectoral skeletal myofibrils in the presence or absence of EGCg at pH 7.0 (b), pH 6.5 (c), or pH 6.0 (d). e EGCg modulation of the pH-induced decrease in the pCa1/2 for myofibrillar ATPase activity of control and cTn-reconstituted chicken pectoral muscle. In b–e, each value is the mean ± SEM of five measurements

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and hence alter the effect of pH on myofilament Ca2+ sensitivity in cardiac muscle. There are three routes for the regulatory process of cardiac excitation–contraction (E–C) coupling in intact myocardium: an upstream mechanism that increases intracellular Ca2+ mobilization; a central mechanism that triggers cross-bridge cycling by Ca2+ binding to cTnC; and a downstream mechanism that involves the thin filament regulation of cTnC or cross-bridge cycling and direct activation of the cross bridge [36]. Using isolated guinea pig Langendorff hearts subjected to ischemia and reperfusion, Hirai et al. [4] and Hotta et al. [5] showed that at low concentration (10∼30 μM) EGCg increased the left ventricular developed pressure (LVDP) with a simultaneous elevation of a Ca2+ transient, but at high concentration (0.1 mM) EGCg induced a maximum LVDP without an increase in intracellular Ca2+ in a manner similar to the Ca2+- sensitizer pimobendan. At a low concentration, EGCg may act by increasing intracellular Ca2+ mobilization and hence Ca2+ transients to elicit a positive inotropic effect on the heart muscle. In contrast, EGCg at a high dose may act through other regulatory mechanism to enhance myocardial contractility by generating more force without an increase in cytosolic-free Ca2+. In this study, we found that at both concentrations of 10 μM (data not shown) and 0.1 mM (Fig. 1) EGCg reduced the Ca2+ sensitivity of cardiac myofibrillar ATPase activity in cardiac myofibrils. This might be due to the removal of the membrane causing the disappearance of the positive inotropic effect and Ca2+ sensitizer of EGCg in skinned cardiac muscle preparations. A preliminary study by Tadano et al. [37] on the effect of EGCg on the force–pCa relationship in skinned porcine cardiac muscle fibers also found that EGCg acts by desensitizing Ca2+-dependent cardiac muscle contraction by binding to cTnC. Apparently, after membrane removal of cardiac muscle cells EGCg has the direct effects on the Ca2+ desensitization of the contractile apparatus. The results obtained with the labeling of CPM at the two N-terminal thiol groups of cTnC (Figs. 2 and 3) showed that EGCg did not cause any significant change in the fluorescence of CPM-labeled cTnC, suggesting that EGCg might not bind to the N-terminal domain of the molecule. This is consistent with the findings of the NMR study by Tadano et al. [37] showing that EGCg binds to the C-lobe of cTnC and that this might alter Ca2+ binding to the C-terminal sites, and our study further shows that this binding reduces the acidic pH-induced decrease in Ca2+ sensitivity (Figs. 4 and 5). In this study, we also determined the pH sensitivity of the myofilament response to Ca2+ in cardiac myofibrils in which the endogenous cTnC was replaced with exogenous sTnC or cTnC (Fig. 6) and in skeletal myofibrils in which the whole sTn complex was replaced with cardiac Tn complex (cTn; Fig. 7). The results showed that EGCg has a differential

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effect on changes in the pH sensitivity of the myofilament response to Ca2+ in cardiac and skeletal muscle which depends on the cardiac-specific isoform of TnC or whole Tn complex. A study of the effect of EMD 57033, a thiadiazinone-derived Ca2+ sensitizer, on cardiac and skeletal muscle showed that it had a marked effect on the rate of modulation of force development in guinea pig cardiac muscle, but only a small effect in frog skeletal muscle [38]. In addition, another study conducted by the same research group showed that following replacement of endogenous cTnC in the skinned cardiac muscle bundle with exogenous cTnC or sTnC, EMD 57033 had an effect on cTnC-, but not sTnC-, substituted cardiac muscle [39]. This differential effect of EMD 57033 on the modulation of force development in cardiac and skeletal muscle has been ascribed to the binding of EMD 57033 to cTnC and subsequent alteration of the interaction between cTnC and cTnI [39]. The structure of the Tn complex has been determined in human cardiac muscle [40] and chicken pectoral muscle [41]. Although the global organization of the subunits in the skeletal Tn complex (sTn) is similar to that in the cardiac Tn complex (cTn), the central helix of sTnC involved in binding to the inhibitory segment of sTnI has a distinct long extended structure, while, in the cardiac Tn complex model, the central helix also binds the inhibitory segment of cTnI, but has a disordered configuration. Thus, the structural difference in the central linker of cTnC (85–92) and sTnC (92–103) might account for the difference in EGCg binding. A high-resolution structure for EGCg bound cTnC would be useful. In summary, the data reported here suggest a novel action for green tea-derived polyphenols in protecting the myocardium from damage caused by intracellular acidosis by modulation of Ca2+ signaling through isoform-specific protein–protein interactions among troponin subunits in cardiac muscle. Acknowledgments This work was partly supported by the National Science Council of Taiwan government (NSC 95-2320-B-005-005) and cooperative projects between the Taichung Veterans General Hospital and the NCHU (TCVGH-NCHU 967602).

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