reperfusion injury through the modulation of oxidative stress and intracellular Ca2 + homeostasis

International Journal of Cardiology 185 (2015) 167–176

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Elatoside C protects the heart from ischaemia/reperfusion injury through the modulation of oxidative stress and intracellular Ca2 + homeostasis Min Wang a, Gui-bo Sun a,⁎, Jing-yi Zhang a, Yun Luo a, Ying-li Yu a, Xu-dong Xu a, Xiang-bao Meng a, Miao-di Zhang b, Wen-bin Lin b, Xiao-bo Sun a,⁎ a b

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China Harbin University of Commerce, Harbin 150076, Heilongjiang, PR China

a r t i c l e

i n f o

Article history: Received 16 October 2014 Received in revised form 27 January 2015 Accepted 11 March 2015 Available online 12 March 2015 Keywords: Elatoside C Ischaemia/reperfusion Oxidative stress Calcium overload

a b s t r a c t Background: We have previously shown that Elatoside C reduces cardiomyocyte apoptosis during ischaemia/reperfusion (I/R). Here, we investigated whether Elatoside C improves heart function in isolated rat hearts subjected to I/R and elucidated the potential mechanisms involved in Elatoside C-induced protection. Methods and results: Isolated rat hearts were subjected to global ischaemia followed by reperfusion in the absence or presence of Elatoside C. We found that Elatoside C significantly attenuated cardiac dysfunction and depressed oxidative stress induced by I/R. Consistently, Elatoside C prevented I/R-induced mitochondrial dysfunction, which was evident by the inhibition of mitochondrial ROS production, mitochondrial permeability transition pore (mPTP) opening, cytochrome c release from the mitochondria and Bax translocation. Moreover, Elatoside C improved abnormal calcium handling during I/R, including increasing sarcoplasmic reticulum Ca2+ ATPase (SERCA2) activity, alleviating [Ca2+]ER depletion, and reducing the expression levels of ER stress protein markers. All of these protective effects of Elatoside C were partially abolished by the PI3K/Akt inhibitor LY294002, ERK1/2 inhibitor PD98059, and JAK2/STAT3 inhibitor AG490. Further assessment in isolated cardiomyocytes showed that Elatoside C maintained the Ca2+ transients and cell shortening against I/R. Conclusions: Elatoside C protects against cardiac injury during I/R by attenuating oxidative stress and [Ca2+]i overload through the activation of both the reperfusion injury salvage kinase (RISK) pathway (including PI3K/ Akt and ERK1/2) and the survivor activating factor enhancement (SAFE) pathway (including JAK2/STAT3) and, subsequently, inhibiting the opening of mPTPs. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Ischaemia/reperfusion (I/R) injury is a major clinical problem that causes increased myocardial dysfunction and further cardiomyocyte death after cardiac surgery and myocardial infarctions [1]. The mechanisms of I/R injury are complex and mainly include excessive ROS production and intracellular calcium overload [2,3]. Recent studies show that these mechanisms are interrelated with each other, where ROS overproduction can induce abnormal Ca2+ handling by depressing endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) Ca2+ uptake and release activities [4,5]. In turn, dramatic increases in cytosolic calcium concentrations can enhance ROS production [6,7]. A substantial rise in ROS and Ca2+ release associated with I/R triggers contractile abnormalities and massive opening of the mitochondrial permeability transition pore (mPTP), whose opening eventually leads to cell death [3,8]. Thus,

⁎ Corresponding authors at: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, PR China. E-mail addresses: [email protected] (G. Sun), [email protected] (X. Sun).

http://dx.doi.org/10.1016/j.ijcard.2015.03.140 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.

the disruption of this vicious cycle of damage by an excess of ROS production and Ca2+ overload during I/R may have important implications in reducing cardiac injury. Aralia elata (Miq) Seem, a well-known adaptogenic plant, has been traditionally used as a tonic medicine to increase energy and improve the body's hypoxia ability [9,10]. The total saponins of A. elata (AS), which are considered as the main pharmacologically active ingredient extracted from A. elata, have been shown to exhibit anti-myocardial ischaemic and anti-hypoxic activities [11,12]. Moreover, A. elata Xinmaitong capsules (Clinical Trial Approval Number 2003L01111 by China Food and Drug Administration), which we developed for the treatment of coronary heart disease, are mainly composed of AS and have successfully completed Phase III clinical trials in China [13]. Thus, it is interesting to further explore the cardio-protective potentials of the active compound from AS. Elatoside C (Supplementary material, Supplementary Fig. 1) is one of the major triterpenoid saponins in AS [14]. A previous report demonstrated that it has strong antisuperoxide activity [14]. We recently found that Elatoside C can alleviate hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis [15]. However, the effects of Elatoside C on I/R injury in isolated perfused hearts remain unknown.

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Fig. 1. Effects of different concentrations of Elatoside C on cardiac function and lactate dehydrogenase (LDH) release in isolated rat hearts subjected to 45 min of no-flow global ischaemia followed by 60 min of reperfusion. Hearts were exposed to Elatoside C (E-C) at concentrations of 2–50 nmol/L for 15 min prior to ischaemia. (A–D) Time-course of cardiac functional indexes in isolated hearts during I/R. (A) Left ventricular systolic pressure (LVSP); (B) heart rate; (C) maximum rate of LV pressure development (+dP/dtmax); (D) maximum rate of LV pressure decline (−dP/dtmax); and (E) LDH leakage during I/R. n N 10 in each group, ##P b 0.01 versus control; *P b 0.05 versus I/R; **P b 0.01 versus I/R.

Great attention has focused on the innate protective pathways, such as the reperfusion injury salvage kinases (RISKs) and survivor activating factor enhancement (SAFE) pathways, which confer myocardial protection against I/R injury [1,16]. The RISK pathway involves the kinases Akt and ERK1/2 [17]. Janus kinase/signal transducer and activator of transcription (JAK2/STAT3) signalling is part of the SAFE pathway [18]. All of these pathways have been suggested to converge on the mitochondria to inhibit the open probability of the mPTP and to mediate protection [16]. Elatoside C was found to protect cultured cardiomyocytes against hypoxia/ reoxygenation injury via the activation of the STAT3 pathway [15]. Whether these signalling pathways contribute to the cardioprotection of Elatoside C against I/R in hearts, however, has not been investigated. Therefore, the aim of the present study was to (1) evaluate whether Elatoside C would improve cardiac dysfunction in I/R hearts; (2) investigate the potential effects of Elatoside C on oxidative stress and abnormal Ca2 + regulation by I/R injury; and (3) determine the roles of the SAFE and RISK signalling components, such as JAK2/STAT3, Akt and ERK1/2, in Elatoside C-induced cardioprotection.

2. Methods 2.1. Animals Male Sprague–Dawley rats (180 g to 250 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The animals were housed under standard laboratory conditions (25 ± 1 °C, 60% humidity, with a 12 h photoperiod) and provided free access to sterile food and water. All of the procedures were approved by the Laboratory Animal Ethics Committee of the Institute of Medicinal Plant Development, Peking Union Medical College, and conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1996). 2.2. Isolated rat heart Langendorff perfusion Male Sprague–Dawley rats were anesthetised with sodium pentobarbital and heparinised. Then, the heart was rapidly excised and cannulated on a Langendorff perfusion system through the aorta and perfused with oxygenated Krebs–Henseleit (KH) buffer (95% O2 + 5% CO2) containing (mmol/L) 120 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4,

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1.2 MgSO4, 1.25 CaCl2, and 11 glucose (37 °C, pH 7.4) under a constant pressure of 80 mm Hg. The left ventricular pressure and heart rate (HR) were measured via a pressure transducer (AD Instruments, Sydney, NSW, Australia) that was connected to a water-filled wrap balloon inserted into the left ventricle achieving a left ventricular end-diastolic pressure (LVEDP) between 2 and 8 mm Hg. All of the data were recorded using PowerLab and analysed using Chart V 7.3.3 (ADInstruments). The index of the myocardial function was determined by the left ventricular systolic pressure (LVSP), maximum derivatives of the ventricular pressure (+ dP/dtmax) and the heart rate. All of the hearts were stabilised with KH buffer for a period of 30 min before the application of the experimental protocols. 2.3. Experimental protocols The following experimental treatments were performed (n = 25 hearts/group): (1) Control, the isolated hearts were perfused for 120 min with oxygenated KHB. (2) I/R, the isolated hearts were perfused with KHB for 15 min and then subjected to 45 min of no-flow global ischaemia and 60 min of reperfusion. (3) Elatoside C + I/R, the isolated hearts were perfused with KHB containing 50 nM Elatoside C for 15 min and then subjected to I/R. (4) Elatoside C + I/R + AG490 (AG, inhibitor of JAK), the isolated hearts were perfused with KHB containing 50 nM Elatoside C and 5 μM AG for 15 min and then subjected to I/R. (5) Elatoside C + I/R + LY294002 (LY, inhibitor of PI3K), the isolated hearts were perfused with KHB containing 50 nM Elatoside C and 10 μM LY for 15 min and then subjected to I/R. In the group Elatoside C + I/R + PD98059 (PD, inhibitor of ERK), the isolated hearts were perfused with KHB containing 50 nM Elatoside C and 20 μM PD for 15 min and then subjected to I/R. The concentrations of inhibitors used, which had no effect on the cardiac function of the I/R-injured hearts, were selected based on preliminary experiments and the data found in the literature. 2.4. Determination of oxidative stress-related indicators in isolated hearts The left ventricle was harvested, and myocardial homogenates were prepared for the detection of MDA, CAT, GSH-Px, and SOD by the corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing,

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China) per the manufacturer's instructions. The activities of NADPH oxidase were investigated by NADPH Oxidase Activity Detection kit (Genmed Scientifics Inc., Shanghai, China). ROS levels were detected by the corresponding ELISA kit (Rapid Bio Lab, Calabasas, CA, USA). 2.5. Isolation of cardiac mitochondria Mitochondria were extracted from heart tissue using a Tissue Mitochondria Isolation Kit (Beyotime Institute of Biotechnology, China) according to the manufacturer's instructions. 2.6. Determination of mitochondrial permeability transition pore (mPTP) opening A mPTP fluorescence detection kit (Genmed Scientifics Inc., Shanghai, China) was used to assess mPTP opening. Calcein was used to stain the mitochondria. This dye selectively aggregates inside the mitochondria, resulting in green fluorescence. The dye is released from the mitochondria when the mPTP open. The change in mitochondrial fluorescence thus reflects the degree of mPTP opening. The fluorescence intensity of the mitochondria was determined using a microplate reader (TECAN Infinite M1000, Austria) at the excitation wavelength of 488 nm and emission wavelength of 505 nm. The data were normalised to control fluorescence. 2.7. Determination of mitochondrial reactive oxygen species (ROS) production The production of mitochondrial ROS was measured using the Genmed mitochondrial ROS highly sensitive fluorescence detection kit (Genmed Scientifics INC., Shanghai, China). Isolated mitochondria were stained with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA). CM-H2DCFDA (excitation/emission: 490/530 nm) is a cell-permeable indicator for ROS and remains non-fluorescent until acetate groups are removed by intracellular esterases and oxidation occurs. Thus, the levels of ROS were detected by changes in the DCF fluorescence intensity using a microplate reader (TECAN Infinite M1000, Austria).

Fig. 2. Effect of Elatoside C (E-C, 50 nmol/L) on cardiac function from isolated hearts during I/R with inhibitors: AG490 (AG), LY-294002 (LY), and PD98059 (PD). (A) LVSP; (B) heart rate; (C) +dP/dtmax; and (D) −dP/dtmax. n N 10 in each group, *P b 0.05 versus I/R; **P b 0.01 versus I/R.

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Fig. 3. Effects of Elatoside C on oxidative damage in I/R-injured hearts. E-C, Elatoside C. n N 10 in each group, #P b 0.05 versus control; *P b 0.05 versus I/R.

2.8. Isolation of sarcoplasmic reticulum vesicles

2.10. Determination of sarcoplasmic reticulum calcium content

Sarcoplasmic reticulum vesicles were prepared according to the methods described by Jones (25). In brief, ventricular tissue was homogenised in an ice-cold medium containing (in mM) 10 NaHCO3, 5 NaN3, 15 Tris–HCl (pH 6.8) and a cocktail of protease inhibitors (1 μmol/L leupeptin, 1 μmol/L pepstatin and 100 μmol/L phenylmethyl-sulfonyl fluoride). The homogenate was then sedimented twice at 14,000 ×g for 20 min, and the supernatant was re-centrifuged at 45,000 ×g for 30 min at 4 °C. The pellet obtained was suspended in a buffer containing 0.6 M KCl and 20 mM Tris–HCl (pH 6.8) and again centrifuged at 45,000 ×g for 45 min at 4 °C. The final pellet was washed and suspended in storage buffer containing 0.25 M sucrose and 10 mM histidine (pH 7.0), and this suspension was used as the SR vesicles.

The SR Ca2+ concentration ([Ca2+]SR) was determined according to the manufacturer's instructions (Genmed Scientifics Inc., Shanghai, China). In brief, sarcoplasmic reticulum vesicles (100 μg) were stained using Mag-Fluo-AM for [Ca2+]SR for 30 min at room temperature and then incubated at 37 °C for 30 min. The fluorescence was immediately performed using a microplate reader (TECAN Infinite M1000, Austria) at the excitation wavelength of 490 nm and the emission wavelength of 525 nm. The sarcoplasmic reticulum [Ca2+]i was calculated according to the equation: [Ca2+]SR = Kd[(F − Fmin) / (Fmax − F)], where Kd is the dissociation constant (22 μM), F is the fluorescence at intermediate Ca2+ levels, Fmin is the fluorescence intensity of the indicator in the absence of Ca2+ and is obtained by adding a negative solution, and Fmax is the fluorescence of the Ca2 +-saturated indicator and is obtained by adding a saturated solution.

2.9. Determination of sarcoplasmic reticulum Ca2 +-ATPase (SERCA) activity Using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), SERCA activity was measured according to the manufacturer's instructions.

2.11. Isolation of adult rat ventricular myocytes and treatment with simulated I/R Individual rat left ventricular myocytes were isolated using an enzymatic method as previously reported [13]. Only isolated rod-shaped

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Fig. 4. Effects of Elatoside C on apoptosis via the mitochondria pathway in I/R-injured hearts. (A) mPTP opening in cardiac mitochondria; (B) mitochondrial ROS production in cardiac mitochondria ER; (C) changes in the expression of cytochrome c and Bax in the mitochondrial (mito) and cytosolic fractions (cyto); and (D) changes in the expression of the apoptotic proteins Bax, Bcl-2, Caspase-9, and Caspase-3 in isolated rat hearts. E-C, Elatoside C. β-Actin expression was examined as the protein loading control. The data are expressed as the means ± SD from three independent experiments. #P b 0.05 versus control; ##P b 0.01 versus control; *P b 0.05 versus I/R; **P b 0.01 versus I/R.

myocytes accounting for N85% at the beginning of each experiment were considered satisfactory. Control recordings were made for 15 min in normal Tyrode's solution at 37 °C, pH 7.4. Subsequently,

the solution was switched to an ischaemic solution (in mM: 123 NaCl, 6 NaHCO3, 0.9 NaH2PO4, 8 KCl, 0.5 MgSO4, 20 Na-lactate, and 1.8 CaCl2 ; pH 6.8, and equilibrated with 90% N 2–10% CO 2 ) for

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Fig. 5. Effects of Elatoside C on Ca2+ release from the sarcoplasmic reticulum (SR) and ER stress in I/R-injured hearts. (A) SERCA2α activity; (B) calcium level in the ER; (C) changes in the expression of GRP78, CHOP and Caspase-12. E-C, Elatoside C. β-Actin expression was examined as the protein loading control. The data are expressed as the means ± SD from three independent experiments. #P b 0.05 versus control; ##P b 0.01 versus control; *P b 0.05 versus I/R; **P b 0.01 versus I/R.

20 min, followed by a 30-min reperfusion with normal Tyrode's solution.

emitted at 340 and 380 nm was recorded as an indicator of [Ca2 +]i. The data were recorded and analysed with IonWizard software (version 6.2.0.59).

2.12. Simultaneous measurement of Ca2 + transients and sarcomere shortening

2.13. Western blot analysis

Ca2+ transients and sarcomere shortening were detected simultaneously using a video-based sarcomere length and Ca2 + acquisition module system (IonOptix Corporation, Milton, MA, USA) as previously described [13]. Cardiomyocytes were incubated with Fura-2 AM (2 μM for 20 min at 37 °C; Invitrogen). The loaded cells were electrically stimulated at 0.5 Hz. The ratio of fluorescence

Heart tissues lysate preparation and western blot analyses were performed as previously described [19]. The primary antibodies were against p-ERK1/2, ERK1/2, p-Akt, Akt, cytochrome c, GRP78, CHOP, Caspase-3, Caspase-12, Caspase-9, Bax, Bcl-2, β-actin (Santa Cru, CA, USA), phospho-tyrosine705-STAT3 and STAT3 (Abcam, Cambridge, UK). Specific bands were visualised after incubation

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Fig. 6. Effect of Elatoside C on sarcomere contraction of adult rat cardiomyocytes during I/R. (A) Resting sarcomere length; (B) peak shortening (% of resting cell length); (C) maximal velocity of shortening (+dL/dtmax); (D) maximal velocity of re-lengthening (−dL/dtmax); (E) time-to-90% re-lengthening (TR90); and (F) time-to-peak shortening (TPS). E-C, Elatoside C. All data are expressed as the means ± SD, n = 28 to 35 cells from three rats per group, #P b 0.05 versus control; ##P b 0.01 versus control; *P b 0.05 versus I/R; **P b 0.01 versus I/R.

with horseradish peroxidase-conjugated secondary antibodies by chemiluminescence using an ECL kit. 2.14. Statistical analyses The results are expressed as the means ± standard deviation. Comparisons between N 2 groups or multiple groups over time were performed using simple or repeated-measures ANOVA (Prism 5.00 software) as appropriate. A Newman–Keuls post hoc test was used except in the analysis of Langendorff data, where Bonferroni's analysis was used. Statistical significance was set at P b 0.05. All data are the result of at least three independent experiments. 3. Results 3.1. Elatoside C improved cardiac function of the I/R hearts In the present study, compared with the I/R group, Elatoside C from 2 to 50 nmol/L concentration dependently improved the

functional recovery of the I/R hearts, which was demonstrated by significant increases in LVSP, ± dP/dt max , and heart rate throughout the reperfusion period, although the baseline mechanical parameters with Elatoside C were not significantly different compared with those of the control condition (Fig. 1A–D). Next, we investigated whether Elatoside C affects myocardial injury by detecting LDH release. A significant concentrationdependent reduction in LDH release in the 2 to 50 nmol/L groups (Fig. 1E) was observed compared with that in the I/R group. Thus, we used a concentration of 50 nmol/L Elatoside C for the following assessments. However, as shown in Fig. 2, during I/R, the inhibition of JAK2, PI3K, or ERK with AG, LY, or PD, respectively, significantly attenuated the beneficial effect of Elatoside C on cardiac function. In the additional groups, we found that Elatoside C or the inhibitor (AG, LY, or PD) treatment alone did not have a significant effect on the mechanical parameters after 120 min of perfusion compared with those of the control group (data not shown).

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3.2. Elatoside C reduced I/R-induced myocardial oxidative stress Oxidant stress has long been linked to heart injury in response to I/R [2]. As shown in Fig. 3, we found that the activities of the major cellular antioxidants GSH-Px, SOD and CAT in the rat myocardium were decreased in the I/R group and significantly elevated in the Elatoside C + I/R group. Consistently, significantly reduced levels of MDA (lipid peroxidation), ROS and NADPH oxidase were also observed in the Elatoside C + I/R group compared with the I/R group. However, the effects of Elatoside C against I/R-induced oxidative stress in isolated hearts were almost abolished by pretreatment with AG, LY, or PD. 3.3. Elatoside C inhibited I/R-induced mitochondrial injury and the mitochondrial apoptosis pathway It has been demonstrated that mitochondrial permeability transition pore (mPTP) opening is a major determinant of cell death in the progression of I/R injury that is correlated with the release of cytochrome c (Cyt c) after Bax and enhanced ROS levels [20]. The opening of the mPTP was examined using calcein-AM staining combined with CoCl2. The results showed that the calcein fluorescence intensity in the I/R group was significantly lower than that of the control group, suggesting that I/R led to increased mPTP opening. Elatoside C effectively inhibited I/R-induced mPTP opening, which was evidenced by the increased calcein fluorescence (Fig. 4A). In addition, there was an increased generation of mitochondrial ROS in I/R hearts, and Elatoside C treatment significantly reduced this increase (Fig. 4B). To further investigate the molecular mechanism of Elatoside C in response to I/R, we performed western blot analyses using different antibodies for apoptotic protein makers. Fig. 4C showed that Elatoside C down-regulated the expression of Bax in the mitochondrial fraction and inhibited mitochondrial cytochrome c release into the cytosol, which were induced by I/R. Elatoside C also significantly reduced the expression of the pro-apoptotic proteins Caspase-3 and Caspase-9, as well as increased the anti-apoptotic protein Bcl-2 caused by I/R (Fig. 4D).

However, the effects of Elatoside C on mitochondrial functions were abolished by the pathway inhibitors, as shown in Fig. 4. 3.4. Elatoside C maintained SERCA activity, attenuated [Ca2+]ER depletion and inhibited the ER stress induced-apoptosis pathway during I/R Recent findings indicated that I/R is associated with abnormal ER Ca2+ levels [21]. Normally, ER calcium is maintained by sarco/endoplasmic reticulum calcium ATPase (SERCA), which functions by pumping calcium into the ER lumen from the cytosol [22]. Therefore, the activity of SERCA2 and the [Ca2+]ER were examined. We found that I/R significantly decreased the activity of SERCA2, while this alteration was attenuated by pretreatment with Elatoside C. The content of [Ca2+]ER in the I/R group was significantly lower than that in the Elatoside C group, indicating that Elatoside C improved the depletion of the Ca2+ pool in the ER (Fig. 5A and B). The Ca2+ overload and reduced ER calcium stores during I/R can result in ER stress [23,24]. Excessive ER stress can trigger cellular apoptosis through activation of ER stress-associated apoptosis markers, such as GRP78, CHOP and Caspase-12 [24]. As shown in Fig. 5C, the expression of GRP78 increased after I/R treatment, and this promotion was significantly decreased by Elatoside C treatment. Moreover, Elatoside C also significantly inhibited the expressions of chop and Caspase-12 compared with the I/R group. The inhibition of the pathway abolished the effects of Elatoside C, suggesting that JAK2, PI3K, and ERK were involved in the mechanism. 3.5. Elatoside C improved the impairment of cardiomyocyte contractile and intracellular Ca2+ homoeostasis induced by I/R Our further assessment of the cardiomyocyte mechanics revealed that I/R markedly depressed the peak shortening (PS) amplitude and the maximal velocity of shortening/re-lengthening (±dL/dt), accompanied by a prolonged duration of re-lengthening (TR90) and duration of

Fig. 7. Effect of Elatoside C on Ca2+ transient of adult rat cardiomyocytes during I/R. (A) Resting intracellular Ca2+ levels; (B) amplitude of Ca2+ transients; (C) maximum decay velocity of Ca2+ transients; and (D) Ca2+ transient decay rate. E-C, Elatoside C. F340/F380, fluorescence ratio of 340 nm to 380 nm. All data expressed as the means ± SD, n = 28 to 35 cells from three rats per group, ##P b 0.01 versus control; *P b 0.05 versus I/R; **P b 0.01 versus I/R.

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shortening (TPS). However, all of these effects were significantly attenuated by pretreatment with Elatoside C (Fig. 6). I/R induced a significant increase in resting Ca2+ transients' levels that represents the diastolic cytosolic Ca2 + content, but this increase was markedly attenuated by Elatoside C (Fig. 7A). The amplitude of the Ca2 + transients was decreased after I/R, while Elatoside C maintained the amplitude of Ca2 + transients close to the control level (Fig. 7B). After I/R, the maximum upstroke velocity (Vmax) of Ca2 + transients was significantly reduced, whereas the decrease was restored by Elatoside C treatment (Fig. 7C). The decay of [Ca2+]i, representing the speed of Ca2+ removal from the cytoplasm mainly via SERCA, was significantly prolonged during I/R. Elatoside C reduced the decay of [Ca2+]i nearly to the control level, suggesting an increase in SERCA activity (Fig. 7D). These results demonstrate that the protective effects of Elatoside C against I/R injury are associated with improved Ca2+ transients and contractile functioning. 4. Discussion Elatoside C, a major triterpenoid saponin of A. elata, has been known to possess antioxidant and anti-H/R properties [14,15]. However, the cardioprotective effects of Elatoside C on I/R hearts are largely unknown. In the present study, we found that Elatoside C suppresses cardiac dysfunction during I/R. Furthermore, Elatoside C depresses oxidative stress and mitochondrial injury induced by I/R. On the other hand, Elatoside C attenuates I/R-induced abnormal Ca2+ handling and ER stress related to apoptosis. Using survival kinase inhibitors, we demonstrate that the cardioprotective effects of Elatoside C are dependent on the activation of the RISK (PI3K/Akt and ERK1/2) and the SAFE (JAK2/STAT3) pathways (Supplementary Fig. 2), which subsequently inhibit mPTP opening. Moreover, Elatoside C improves cell contraction and Ca2 + transients in isolated cardiomyocytes during I/R. These findings provide novel evidence for the cardioprotective effects of Elatoside C against I/R injury. Oxidative stress, defined as an imbalance between the generation of reactive oxygen species (ROS) and the antioxidant defence systems [25], is widely considered a dominant mechanism during I/R injury [2]. It has long been recognised that membrane lipid oxidation is one of the primary events in oxidative damage, and MDA has been widely used as a convenient biomarker for lipid peroxidation [25]. NADPH oxidase, which is the primary source of superoxide radicals, has been reported to be the major enzymatic source of ROS in I/R hearts [26]. Our study revealed that Elatoside C decreased the levels of oxidative stress markers including MDA, NADPH oxidase, and intracellular ROS as well as increased the activities of antioxidant enzymes consisting of GSHPx, SOD and CAT during I/R (Fig. 3), indicating that the cardioprotective effect of Elatoside C is partly caused by oxidative stress suppression. Cardiac mitochondria, which are the major producers of reactive oxygen species (ROS), suffer severe damage during I/R [27]. Emerging evidence has indicated that mitochondrial oxidative damage induced by I/R may impair the efficiency in mitochondrial function, which, conversely, further contributes to mitochondrial oxidative damage and ultimately promotes the opening of mPTPs and thereby induces mitochondria-dependent apoptosis [7,20,27]. In this study, the inhibition of mitochondrial ROS was observed in Elatoside C-pretreated hearts. Consistently, mitochondrial function was markedly improved by Elatoside C, as indicated by suppressed mPTP opening, cytochrome c release from mitochondria and Bax translocation to the mitochondria. The mitochondrial translocation of Bax has been shown to increase the mitochondrial membrane permeability and consequently enhances the release of cytochrome c [20]. This release of cytochrome c in turn activates Caspase-9 and subsequently Caspase-3, eventually leading to cell death [28]. Our finding of decreased levels of Caspase-9 and Caspase-3 provides further evidence of the Elatoside C-induced anti-apoptotic response during I/R. Thus, the protective effects of Elatoside C are most likely achieved through alleviating oxidative stress and maintaining

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mitochondrial function, thereby inhibiting mitochondria-dependent apoptosis. Accompanying oxidative stress injury, another major mechanism of I/R injury is calcium overload [21,29]. Ca2+ homeostasis plays a central role in normal heart function through excitation–contraction coupling as well as Ca2+-mediated signalling [30,31]. Reperfusion can lead to a dramatic rise of [Ca2+]i due to abnormal Ca2+ handling [29,30]. Here, we show that cardiac dysfunction induced by I/R is significantly attenuated by Elatoside C. Moreover, Elatoside C significantly improves cell contraction and Ca2+ transients in isolated cardiomyocytes during I/R. SERCA2α is the pivotal regulator of Ca2+ homeostasis and contractile activity in cardiac SR [22]. The present data show that Elatoside C could preserve SERCA activity and increase the SR Ca2+ content during I/R, which explains the Elatoside C-maintained [Ca2 +]i homeostasis during I/R. Notably, it has been proposed that oxidative stress can interfere with SR function, such as the inhibition or oxidative modification of SERCA [5]. Thus, we speculate that the beneficial effects of Elatoside C in improving cardiac performance and modulation of intracellular calcium during I/R may also be related to its attenuation of ROS generation. The ER is essential for calcium storage. Ca2+ overload and disruption of ER calcium homeostasis by I/R result in ER stress, and prolonged ER stress can induce apoptosis [23]. To further investigate whether ER stress-induced apoptosis is related to the cardioprotective effects of Elatoside C, we detected three major ER stress proteins: GRP78, CHOP and Caspase-12. We found that the expression levels of ER stress markers were increased by I/R, and Elatoside C significantly attenuated this enhancement, which was consistent with the findings from our previous study on cardiomyocytes [15]. In addition, ER stress is reported to enhance mitochondria ROS production and trigger the mPTP opening [8,32,33]. Our results also showed that Elatoside C could prevent the mitochondrial injury during I/R, suggesting that the cardioprotection of Elatoside C in response to I/R may be associated with the inhibition of ER stress. Extensive studies have demonstrated that the activation of the RISK pathway, which incorporates the activation/phosphorylation of several pro-survival kinases such as PI3K/Akt and ERK1/2, is required for protection with ischaemic preconditioning and various pharmacological mediators [16,34]. In our present study, inhibition of the Akt or ERK1/ 2 signalling pathway by LY-294002 or PD98059 abrogated the cardioprotective effects of Elatoside C, indicating that RISK pathway activation is involved in the cardioprotective effects of Elatoside C. In addition, the SAFE pathway, which recognises STAT3 activation as a central point, is also found to be activated during I/R and appears to provide protection independent of the RISK pathway [18]. As already outlined, we have demonstrated that the STAT3 pathway plays an important role in Elatoside C-induced cardioprotection against H/R [15]. In the present study, we obtained further data consistent with this result, that the inhibition of STAT-3 using AG490 abolished the cardioprotective effects of Elatoside C. Although the exact mechanism is not yet fully clear, the most plausible interpretation of our observations is that Elatoside C-induced protection against myocardial I/R injury can at least partially be attributed to the activation of the RISK/SAFE pathways (Supplementary Fig. 2). Similar results were also observed in several other pharmacological mediator studies [35–37]. In conclusion, our findings suggest that Elatoside C reduces cardiac injury and improves myocardial performance during I/R. Such cardioprotection seems to be largely due to the attenuation of ROS overproduction and the maintenance of [Ca2+]i homeostasis in which both the SAFE (JAK2/STAT3) and the RISK (Akt and ERK1/2) pathways were involved, subsequently leading to the inhibition of mPTP opening. These results suggest that Elatoside C may be potentially beneficial in preventing myocardial I/R injury during cardiac surgery and ischaemic heart disease. Nevertheless, this study is still in its initial stages, and detailed investigations are required to establish the cross-talk that exists between these cell signalling pathways in terms of the mechanisms of Elatoside C-induced cardioprotection.

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M. Wang et al. / International Journal of Cardiology 185 (2015) 167–176

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2015.03.140.

[16]

Conflict of interest The authors report no relationships that could be construed as a conflict of interest.

[17]

[18]

Acknowledgements [19]

This study was supported by the National Natural Science Foundation of China (Grant Nos. 81173589 and 81473380), the Natural Science Foundation of Beijing (Grant No. 7142108), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20121106110033), and the Program for Innovative Research Team in IMPLAD (Grant No. IT1301).

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[21]

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