Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation Min Wang a,1, Xudong Xu a,1, Huibo Xu b, Fuchun Wen b, Xiaopo Zhang a, Hong Sun a, Fan Yao c, Guibo Sun a,n, Xiaobo Sun a,n a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, PR China b Academy of Chinese Medical Sciences of Jilin Province, Gongnongda road 1745, Changchun 130021, Jilin, PR China c School of Biological Sciences, University of Edinburgh, King's Buildings, Edinburgh EH9 3JU, UK

art ic l e i nf o

a b s t r a c t

Article history: Received 15 December 2013 Received in revised form 14 May 2014 Accepted 18 May 2014

Ethnopharmacological relevance: Total saponins of Aralia elata (Miq) Seem (AS) from the Chinese traditional herb Longya Aralia chinensis L. can improve cardiac function, although the active mechanism remains poorly understood. The present study aimed to determine the direct effect of AS on cardiac function in dogs and the effects on Ca2 þ transient and contractions in isolated rat cardiomyocytes. Material and Methods: In anesthetized dogs, hemodynamic indexes and myocardial oxygen consumption were determined before and after AS was administered. In isolated adult rat cardiomyocytes, contractile and intracellular Ca2 þ properties were determined simultaneously in real time by using an IonOptix MyoCam system. Results: Our results showed that AS directly induced a positive inotropic effect and improved coronary blood flow and energy metabolism, indicating that AS induced a beneficial effect to treat myocardial ischemia/reperfusion injury. Moreover, AS increased sarcomere shortening, maximal velocity of shortening/relengthening ( 7dL/dt), amplitude of [Ca2 þ ]i transients and SERCA activity in a concentrationdependent manner. PKCε was also activated after the cells were treated with AS. Conclusion: These findings revealed the positive inotropic effect of AS on canine myocardium and isolated rat cardiomyocytes. This effect was possibly associated with an increase in amplitude of the [Ca2 þ ]i transient and PKCε-dependent signaling pathway. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Total saponins Intracellular calcium concentration Positive inotropic effect (PIE) Cardiomyocyte

1. Introduction Aralia elata (Miq) Seem, which belongs to Araliaceae family, is a shrub widely distributed in Northeastern China, Far East Russia, Japan, and Korea (Li and Lu, 2009). The bark and roots have been traditionally used as a tonic, anti-arrhythmic, anti-arthritic, antihypertensive and anti-diabetic agent in traditional Chinese medicine (Xu et al., 1997; Li and Lu, 2009). Aralia elata is also a well-known adaptogenic plant used in Russia and the extract of Aralia was officially approved as a tonic for therapeutic use in 1957 (Wojcicki et al., 1977; Yance and Tabachnik, 2007). Aralia species contain some ginseng-like triterpenoid saponins (Aralosides) and the effect produced by aralosides is similar to that of panaxosides

n Correspondence to: 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. Tel./fax: þ 86 010 57833013. E-mail addresses: [email protected] (G. Sun), [email protected] (X. Sun). 1 These authors contributed equally to this work.

from ginseng (Baranov, 1982), which probably contributes to Aralia's ability to increase energy, strengthen body, and improve the body's hypoxia ability in regard to the cardiovascular system and to other parameters (Sololov et al., 1971; Baranov, 1982; Yance and Tabachnik, 2007). Total saponins of Aralia elata (AS), the main pharmacologically active ingredient extracted from Aralia elata (Xu et al., 1997), have been shown to stimulate heart activity (Sokolov, 1965; Xu et al., 1997), possess anti-myocardial ischemic and anti-hypoxic activities (Deng et al., 1988; Wen et al., 2005; Sun et al., 2006), exhibit a strong anti-arrhythmic effect (Maslov and Guzarova, 2007; Arbuzov et al., 2009; Maslov et al., 2009) and have protective effect against diabetic cardiomyopathy (Xi et al., 2009). However, the direct effects of AS on cardiac function in canines and its underlying mechanism of cardiac contractility remain largely unknown. Cardiac contraction is regulated by the excitation-contraction (EC) coupling (Fearnley et al., 2011). Ca2 þ cycling has an important function during this process. Contraction is initiated by Ca2 þ entry into cardiac myocytes via L-type Ca channels (LTCC). This process

http://dx.doi.org/10.1016/j.jep.2014.05.024 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Wang, M., et al., Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.024i

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subsequently triggers a greater release of Ca2 þ from the sarcoplasmic reticulum (SR) via ryanodine receptors, thereby providing Ca2 þ to stimulate the myofilaments to induce contraction. In cardiac relaxation, Ca2 þ that enters the cell via LTCC is transported out of the cell mainly by the sarcolemmal Na þ /Ca2 þ exchanger (NCX). Ca2 þ released from the SR is reuptaken by SR Ca2 þ ATPase (SERCA), thereby providing available Ca2 þ for the next contraction cycle. This whole event determines the contraction and relaxation of the myocytes (Cheng and Lederer, 2008). However, the relationship between the cardiac effect of AS and calcium cycling has not been clarified. Studies have yet to determine whether or not the mechanism of the positive inotropic activity of AS is caused by an increase in the amplitude of [Ca2 þ ]i transient during EC coupling. Moreover, studies have shown that protein kinase C (PKC), a large family of serine/threonine protein kinases, is crucial in regulating contractility and Ca2 þ cycling in the heart (Rogers et al., 1990; Pi and Walker, 2000). Whether or not PKC are involved in AS-regulated cardiac contraction remain unknown. The present study aimed to examine the effect of AS on cardiac function in canines and evaluate the effect of AS on EC coupling by simultaneously determining the effects of AS on cell sarcomere shortening and intracellular Ca2 þ transients in isolated adult rat myocytes. We also studied the involvement of SERCA and PKC in these effects.

2. Materials and methods 2.1. Plant material The roots of AS were collected from Jilin Province of China in September 2010. The samples were identified by Professor ZhongKai Yan (Academy of Chinese Medical Sciences of Jilin Province). A voucher specimen (No. 20100920) was deposited in the same department.

2.2. Extraction and isolation of AS The roots of AS were refluxed thrice with 70% alcohol solution for 1.5 h at each time. The solution was filtered, subjected to a macroporous resin column, and eluted successively with deionized water, 20% and 80% ethanol. The solutions eluted by water and 20% ethanol were discarded. The remaining 80% ethanol solution was collected and evaporated to dryness under reduced pressure to obtain the total saponins (Zhang et al., 2013).

2.3. UPLC analysis The total saponin (0.01 g) was dissolved in methanol (5 mL  2) under ultrasonic irradiation. The sample was passed through a 0.22 mm filter prior to injection. Known amounts of the reference standards were weighed and dissolved in methanol to prepare the solutions at approximately 1 mg/mL. The standards were diluted 50-fold to prepare the working solutions before injection. The samples were separated on an Acquity UPLCTM system (Waters Corp., USA). Chromatographic analysis was conducted using a Waters phenyl column (100 mm  2.1 mm i.d., 1.7 mm) at 25 1C and a flow rate of 0.4 mL/min. The mobile phase consisted of acetonitrile (A) and water with 0.05% formic acid (B); the eluting gradient was used as follows: 0–3 min, 2.0–11.0%; 3–10 min, 11.0%; 10–16 min, 11.0–20.0%; 16–18 min, 20.0–24.0%; 18–22 min, 24.0%; 22–22.5 min, 24.0–30%; 22.5–26 min, 30.0%; 26–33 min, 30.0– 80.0%; and 33–35 min, 80.0–100.0%.

2.4. Mass spectrometry Mass spectrometry detection was performed on a Synapt G2 MS system (Waters Corp., USA) equipped with an ESI source. Two data acquisition modes, or MSE, were selected to investigate precursor ions and product ions. Nitrogen gas was used for nebulization. The detection mode of the flying tube was selected as “V” pattern. The positive ion spectra of the column eluates were recorded at a range of m/z 100–1500. The optimized conditions of the ESI source were listed as follows: capillary voltage, 2.5 kV; sampling cone voltage, 40 V; extraction cone voltage, 3.0 V; ESI source temperature, 120 1C; desolvation temperature, 450 1C; cone gas flow, 30 L/h; desolvation gas flow, 800 L/h; collision gas flow, 0.5 mL/min; collision energy for MSE acquisition mode, 4.0 eV for low energy scan and 15–40 eV for high energy scan; and dynamic adjustment of the fragmentor voltage ranged from 25 V to 40 V for the MS/MS acquisition mode. The lock mass compound was leucine enkephalin (m/z 556.2771), and the interval scan time was 0.02 s. Masslynx 4.1 (Waters Corp.) software was used to control this instrument. 2.5. Animals Male and female adult mongrel dogs (15–17 kg) were obtained from Norman Bethune University of Medical Science. Male Sprague-Dawley rats (180–200 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The animals were housed under standard laboratory conditions (257 1 1C, 60% humidity, and 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 with the registration number: #IMPLAD2012112207. 2.6. Measurement of blood pressure, heart rate, ventricular function, and coronary artery blood flow in anesthetized dogs Twenty-four animals were randomly divided into four groups: a control group, two AS groups (30 and 60 mg/kg) and a Di-ao-xinxue-kang capsule (Di-ao capsule) group (86 mg/kg) as a positive drug, with n¼6 in each group. The dogs were anesthetized with sodium pentobarbital (30 mg/kg, iv) and then a ventilator was set up. A catheter connected to a pressure transducer was introduced into the femoral artery to record blood pressure and heart rate. After an incision was produced on the neck, a catheter connected to a pressure transducer was introduced into the left ventricle (LV) via the right carotid artery. LV systolic pressure (LVSP), end-diastolic pressure (LVEDP), and the maximal rates of pressure increase (þdp/ dt) and decrease ( dp/dt) were measured or calculated for each group. A left lateral thoracotomy was performed in the fourth intercostal space, the pericardium was incised, and the heart was suspended in a pericardial cradle. A probe was connected to the left circumflex coronary artery, and coronary blood flow (CBF) was recorded using an electromagnetic blood flow meter system (Nihon Konden, Japan). Myocardial flow (MF) was expressed as mL/min per 100 g of myocardium supplied by a coronary artery. All of these parameters were recorded at different times (15, 30, 45, 60, 90, 120, 180, 210, and 240 min) before and after different concentrations (30 and 60 mg/kg) of AS were duodenally administered. 2.7. Measurement of myocardial oxygen consumption in dogs Blood was collected from the femoral artery and the coronary sinus at different times (15, 45, 60, 90, 120, and 240 min) before and after AS was administered for blood gas analysis (DH-1300 blood gas analyzer, Nanjing Analytical Instrument

Please cite this article as: Wang, M., et al., Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.024i

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Factory Co., Ltd., Nanjing, China). The myocardial oxygen consumption was expressed as the amount of blood flow in the coronary artery  (arterial oxygen saturation–coronary sinus oxygen saturation). 2.8. Isolation of rat ventricular myocytes

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

Individual left ventricular myocytes were isolated using a modified enzymatic method (Ren and Wold, 2001). In brief, the hearts were rapidly removed from adult male Sprague-Dawley rats (180–200 g) anesthetized with pentobarbital sodium (140 mg/kg ip) and perfused via the aorta cannula with a series of different perfusion solutions at a rate of 6 mL/min. The hearts were perfused with Ca2 þ -containing Tyrode's solution with the following components: NaCl, 137 mM; KCl, 5.4 mM; MgCl2, 1.2 mM; HEPES, 10 mM; glucose, 10 mM; and CaCl2, 1.2 mM (pH 7.4, equilibrated with O2) for 2 min. The hearts were then perfused with Ca2 þ -free Tyrode's solution containing the following: NaCl, 137 mM; KCl, 5.4 mM; MgCl2, 1.2 mM; HEPES, 10 mM; and glucose, 10 mM (pH 7.4, equilibrated with O2) for 5 min. Afterward, the hearts were perfused with Ca2 þ -free Tyrode's solution containing 223 U/mL collagenase (Worthington Biochemical Corp., Freehold, NJ) for 20 min. After perfusion, the LVs were removed, minced, and filtered using nylon mesh (300 mm). The filtered myocytes were then washed with Ca2 þ -containing Tyrode's solution to restore the extracellular Ca2 þ back to 1.2 mM. Only rod-shaped ventricular myocytes with clear edges were used in this study.

2.11. Western blot analysis

2.9. Simultaneous measurement of Ca2 þ Transients and sarcomere shortening

UPLC/Q-TOF–MS analysis revealed that the active fraction contained high amounts of saponins. Approximately 30 visible peaks could be determined from the total ion current profile of the active fraction; among these peaks, 16 were identified. The compounds were characterized in terms of retention times and mass spectra; these compounds were then identified by comparing with published data or commercial standards. UPLC result of the AS sample is shown in Fig. 1. A complete list summarizing all of the compounds identified in the active fraction is shown in Table 1.

To determine [Ca2 þ ]i, we incubated the myocytes with Fura-2 AM (2 uM), a Ca2 þ indicator, for 10 min and then washed twice with Ca2 þ -containing Tyrode's solution to remove residual Fura-2 AM before the study was initiated. These myocytes were subsequently placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus, IX-70) and superfused with Ca2 þ containing Tyrode's solution in the absence or presence of AS (1.25, 2.5, and 5 mg/mL) at a rate of 1.5 mL/min. After the field was stimulated (0.5 Hz for 2 ms at 16 V), sarcomere shortening and Ca2 þ transients were simultaneously assessed using a video-based sarcomere length and Ca2 þ acquisition module system (IonOptix Corporation, Milton, MA, USA) with intact myocytes at room temperature before and during the treatment with the agents when the response reached a steady level. Data were recorded and analyzed with IonWizard software (version 6.2.0.59) (Ren and Wold, 2001).

The cardiomyocytes were exposed to AS (5 mg/mL) at different durations (0–5 min), collected and then cytosolic and membrane proteins were prepared by using membrane protein extraction kits (BioVision). Western blot analysis was performed as previously described (Sun et al., 2011). 2.12. Statistical analysis Data from at least three independent experiments were expressed as mean 7standard deviation (SD). Comparisons among the groups or between different time points within a group were made using one-way analysis of variance (ANOVA). A value of Po 0.05 was accepted as statistically significant.

3. Results 3.1. Characterization of the saponins in the active fraction

3.2. Effects of AS on hemodynamic variables and myocardial oxygen consumption in dogs Blood pressure, heart rate and ventricular function determined in the four groups of dogs are shown in Fig. 2. All of the dogs were hemodynamically stable at the baseline measurement. At 30 mg/kg AS, blood pressure did not significantly change, but the heart rate was gradually decreased and the most significant effect was observed at 120 min after AS was administered (Po0.05). By

Fig. 1. Total ion current profile corresponding to the UPLC–ESI-MS analysis of the total saponins of Aralia elata (AS).

Please cite this article as: Wang, M., et al., Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.024i

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Table 1 List of saponins identified in the active fraction of Aralia elata. #Peak Chemical name 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Elatoside L Congmunsoides X Congmunsoides V Congmuosides XI Congmunsoides G Elatoside D Tarasaponin IV Elatoside C Elatoside K Aralosdie C Elatoside I Spinasaponin A 28-OGlc Araloside A Ginsenoside Rd Chikusetsusapnoin IV Chikusetsusaponin IV

[MþNa] þ

Fragments and their intensity (%)

1435.6212 1303.5857 (6.2), 1141.5377 (10.2), 965.5003 (18.2), 949.4755 (12.6), 729.3074 (8.6), 637.2259 (5.4), 509.1479 (7.4), 439.3572 (14.8) 1303.5846 1141.5305 (12.2),965.5045 (9.3), 817.4280 (8.6), 649.2185 (3.2), 509.1446 (10.6), 439.3559 (46.8) 1127.5171 965.4675 (5.2), 863.3168 (7.6), 537.2616 (16.3), 515.2830 (62.4), 493.1171 (37.8), 443.2600 (100) 1273.5757 1141.5369 (32.5),979.4843 (8.8), 949.4748 (8.6), 803.4537 (33.2), 493.1159 (4.6), 439.3557 (42.8) 1317.6060 1109.5093 (54.2), 807.4851 (100), 721.4186 (26.8), 670.2960 (35.4), 493.1150 (43.2), 369.1950 (52.0) 1141.5367 979.4832 (22.6), 795.4523 (5.6), 557.1013 (4.2), 523.1266 (45.4), 439.3564 (20.3) 1111.5275 949.4787 (6.6), 949.4739 (10.4), 641.4011 (5.2), 493.1174 (16.2), 439.3564 (26.2) 1111.5276 949.4750 (16.8), 641.4030 (8.2), 493.1162 (46.2), 439.3575 (6.8) 1111.5264 949.4754 (26.2), 641.4035 (5.2), 493.1164 (48.5), 439.3566 (26.8) 1111.5283 979.4855 (11.9), 949.4731 (26.8), 641.4010 (18.5), 493.1164 (36.4), 439.3566 (14.2) 979.4857 817.4307 (16.3), 641.4307 (8.4), 439.3553 (22.6), 361.0741 (21.4) 979.4856 817.4324 (16.3), 817.4307 (17.6), 641.4022 (18.8), 439.3566 (32.6), 361.0745 (31.4) 949.4743 969.5341 965.5048 949.4813

787.4208 (11.5), 641.4016 (22.3), 439.3569 (32.6) 789.4738 (33.5), 425.3737 (6.8), 407.3669 (12.3) 831.4465 (2.8), 509.1452 (14.5), 491.2415 (15.6), 439.3557 (26.2) 795.4448 (22.7), 493.1172 (34.9), 439.3530 (21.6)

Fig. 2. Effect of total saponins of Aralia elata (AS) on blood pressure, heart rate and ventricular function in anesthetized dogs. LVSP, the left ventricular systolic function; LVEDP, the end-diastolic pressure; þdp/dt, the maximal rates of pressure rise;  dp/dt, the maximal rates of pressure fall. (●), control, n ¼6; (■), AS 30 mg/kg, n¼6; (▲), AS 60 mg/kg, n¼ 6; (▼), Di-ao capsule 86 mg/kg, n¼ 6. All data expressed as mean 7 SD, aP o0.05, bPo 0.01 compared to before administration.

contrast, 60 mg/kg AS significantly slowed the heart rate from 60 min to 150 min and elicited a hypotensive effect from 45 min to 120 min after AS was administered. The effects of Di-ao capsule on blood pressure and heart rate were a little weaker than those of AS (60 mg/kg). For the indexes of ventricular function, þ dp/dtmax was not significantly changed at 30 mg/kg AS. By contrast, this parameter was markedly increased from 30 min to 120 min at 60 mg/kg AS; meanwhile,  dp/dtmax was also significantly increased at the 45 min point after AS administration. LVSP was decreased at 30 and 60 mg/kg AS, but LVEDP remained unaffected. Di-ao capsule did not significantly change both LVSP and LVEDP. Fig. 3 shows that AS (30 mg/kg and 60 mg/kg) as well as Di-ao capsule significantly increased the coronary blood flow (CBF), myocardial flow (MF), cardiac output, cardiac index, stroke index, and stroke volume, but the total peripheral resistance (TPR) and coronary vascular resistance (CVR) were decreased. The data on myocardial oxygen consumption are summarized in Fig. 4. Both AS (30 and 60 mg/kg) and Di-ao capsule significantly decreased the content and the index of oxygen consumption of the

myocardium (P o0.01) as well as the myocardial oxygen uptake rate (P o0.01) compared with the control group. However, the effects of AS (60 mg/kg) were more potential than those of Di-ao capsule. 3.3. Effect of AS on contractions in isolated myocytes In Fig. 5, the exposure to AS caused a significant increase in of the amplitude of sarcomere shortening (Fig. 5(B)) in a concentrationdependent manner without affecting the resting sarcomere length (Fig. 5(A)). This effect lasted for at least 5 min. The sarcomere return velocity þ dL/dt (Fig. 5(C)) and the departure velocity  dL/dt (Fig. 5(D)) were also increased in AS-exposed cells. These data indicated that AS could enhance myocardial contraction. 3.4. Effect of AS on intracellular Ca2 þ transients in isolated myocytes In Fig. 6(A)–(C), AS from 1.25 mg/mL to 5 mg/mL increased the amplitude of [Ca2 þ ]i transients in a concentration-dependent

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Fig. 3. Effect of total saponins of Aralia elata (AS) on blood flow and cardiac function in anesthetized dogs. CBF, coronary blood flow; MF, myocardial flow; CVR, coronary vascular resistance; TPR, total peripheral resistance; CI, cardiac index; CO, cardiac output; SV, stroke volume; SI, stroke index. (●), control, n¼ 6; (■), AS 30 mg/kg, n¼ 6; (▲), AS 60 mg/kg, n¼ 6; (▼), Di-ao capsule 86 mg/kg, n¼ 6. All data expressed as mean 7 SD, aP o 0.05, bPo 0.01 compared to before administration.

Fig. 4. Effect of total saponins of Aralia elata (AS) on myocardial oxygen consumption in anesthetized dogs. (●), control, n¼ 6; (■), AS 30 mg/kg, n¼6; (▲), AS 60 mg/kg, n¼ 6; (▼), Di-ao capsule 86 mg/kg, n ¼6. All data expressed as mean7 SD, aPo 0.05, bP o0.01 compared to before administration.

manner compared with the control group (Fig. 6(B)), but AS did not affect the resting [Ca2 þ ]i during steady-state twitches compared with the control group (Fig. 6(A)). Fig. 6(C) shows that the decay rate of [Ca2 þ ]i transients was reduced by AS. These data indicated that AS enhanced myocardial contraction by increasing the amplitude of Ca2 þ transients. 3.5. Effect of AS on SERCA activity The SERCA plays a pivotal role in regulating the Ca2 þ homeostasis and contractility in cardiac muscle (Xu et al., 2007). To study whether SERCA is involved in the AS-induced PIE, the present study examined SERCA activity in SR vesicles extracted

from myocytes. As shown in Fig. 7(A), AS increased the SERCA activity in myocytes versus control in a concentration-dependent manner. 3.6. Effects of AS on the activation of PKC Studies have reported that PKC activation increases myocardial contractility (Pi and Walker, 2000; Kang and Walker, 2005). We then examined whether or not PKCε activation is involved in ASinduced PIE. The translocation of PKCs from the cytosol to membranous sites is considered as a hallmark of PKC activation (Duquesnes et al., 2011). Our data further showed that AS exposure significantly induced PKCε activation (Fig. 7(B)).

Please cite this article as: Wang, M., et al., Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.024i

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Fig. 5. Effects of total saponins of Aralia elata (AS; 1.25–5 mg/mL for 5 min) on sarcomere contractile function in adult rat cardiomyocytes. (A) Resting sarcomere length; (B) amplitude of sarcomere shortening; (C) sarcomere return velocity þ dL/dt; (D) sarcomere departure velocity  dL/dt. All data expressed as mean7 SD, n¼ 28 to 35 cells from three rats per group, **p o 0.01 vs. control value.

Fig. 6. Effects of total saponins of Aralia elata (AS; 1.25–5 mg/mL for 5 min) on intracellular Ca2 þ concentration in adult rat cardiomyocytes. (A) Resting intracellular Ca2 þ levels; (B) amplitude of Ca2 þ transients; (C) [Ca2 þ ]i transient decay rate. [Ca2 þ ]i, intracellular Ca2 þ concentration; 360/380, fluorescence ratio of 360 nm to 380 nm. All data expressed as mean 7 SD, n¼28 to 35 cells from three rats per group, **po 0.01 vs. control value.

Fig. 7. Effects of total saponins of Aralia elata on the SERCA activty and PKCε activation (A) Effect of AS (1.25–5 mg/mL for 5 min) on SERCA activity. (B) Representative (upper panel) and summarized immunoblots (low panel) for the effect of AS (5 mg/mL for 0–5 min) on PKCε translocating to the membrane fraction. PKCε, protein kinase C epsilon. All data expressed as mean 7 SD of three independent experiments; *Po 0.05, **P o0.01 vs. control value.

Please cite this article as: Wang, M., et al., Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.024i

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4. Discussion Myocardial infarction (MI) caused by ischemia/reperfusion (IR) injury is considered as one of the leading causes of mortality and morbidity worldwide (Gerczuk and Kloner, 2012). During the past decade, the use of hundreds of potential cardioprotective agents in clinical trials have met with little success (Miura and Miki, 2008; Gerczuk and Kloner, 2012). Therefore, there remains an urgent need to further develop cardioprotective strategies to improve MI. Our previous study demonstrated that AS can decrease the myocardial infarct size and decrease the activities of serum creatine kinase and lactate dehydrogenase as well as the contents of serum free fatty acid and lipid peroxidation; by contrast, AS can increase the activities of serum superoxide dismutase and glutathione peroxidase in an acute myocardial ischemic dog model after the occlusion of the left anterior descending coronary artery, indicating that AS can considerably enhance anti-oxidant enzyme activities against MI (Shi et al., 2006). According to the hemodynamic results presented in this study, the possible mechanisms of AS in the treatment of MI may also be attributed to increasing CBF and MF as well as decreasing TPR and CVR. Moreover, AS could improve energy metabolism by reducing the content and index of myocardial oxygen consumption and myocardial oxygen uptake rate. Our results demonstrated that AS directly slowed down the heart rate and increased LV pressure and LV 7dp/dtmax in the canine myocardium, which are in accordance with earlier hemodynamic studies in rats (Ge et al., 2001). This result further suggested that AS induced both positive inotropic effect (PIE) and negative chronotropic effect. Di-ao capsule is a complex of steroidal saponins which are mainly extracted from rhizomes of Dioscorea panthaica Prain et Burkill and Dioscorea nipponica Makino (Jia et al., 2012). It is a second-class new drug of China and is now widely used for treating coronary heart disease and myocardial infarction (Zhang, 1995). To evaluate the cardioprotective effects of AS, we introduced Di-ao capsule as the positive control drug in our study because of its proven protective effects on myocardial tissue affected by MI (Zhang, 1995). In this study, the results showed that some important indices, including blood pressure, total peripheral resistance, left ventricular systolic function, and myocardial oxygen consumption were also significantly changed by Di-ao capsule. However, the effects of AS (60 mg/kg) were superior to those of Diao capsule. All these results indicate that AS may be potentially useful in the treatment of MI through multiple ways. Studies have not yet investigated the direct effects of AS on myocyte shortening in an individual adult rat myocyte. In the present study, AS increased cardiomyocyte contractile function as indicated by a significant dose-dependent increase in single sarcemere shortening amplitude and 7dL/dtmax. This result is consistent with that in our previous study on the cardiac positive inotropic action of AS in a whole rat heart Langendorff apparatus setting (Zhao et al., 2002). Notably, we also found that AS exhibited a negative inotropic effect (NIE) at higher concentrations (data not shown). However, the mechanisms of NIE observed at higher AS concentrations should be further studied. The PIE of AS could be mainly attributed to a Ca2 þ -dependent mechanism and/or an increase in the myofilament responsiveness to Ca2 þ (Endoh, 1998). In our study, further shortening induced by AS was accompanied by the increased amplitude of Ca2 þ transients. The decay rate of [Ca2 þ ]i transient is commonly used to characterize the speed of Ca2 þ clearance in the cytoplasm (Wang et al., 2008). In cardiomyocytes, SERCA is responsible for 75–92% reuptake of intracellular Ca2 þ immediately after contraction (Gianni et al., 2005). Our results showed that decay rate of [Ca2 þ ]i transient was decreased by AS, indicating that SERCA activity was likely enhanced. In our study, we examined SERCA activity by measuring the inorganic phosphate liberated from ATP hydrolysis

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(Xu et al., 2007), and we found that AS does increase the SERCA activity. The PKC family comprises a group of highly related protein kinases that also regulate myocardial function (Steinberg, 2012). PKCε, a Ca2 þ -independent PKC isoform, is one of the commonly expressed PKCs in the cardiomyocytes of human and rodent hearts (Duquesnes et al., 2011). The direct activation of PKCε provides cardiac protection before an ischemic event or at reperfusion (Duquesnes et al., 2011; Mochly-Rosen et al., 2012). Studies have shown that the activation or overexpression of PKCε results in enhanced cardiac contractility (Kang and Walker, 2005; Li et al., 2008). Our results revealed that the treatment with AS caused an increase in PKCε expression in the membrane fraction of cardiomyocytes in a time-dependent manner, indicating that PKCε may be involved in AS-induced PIE. Given that PKCε activation mediates a Ca2 þ -independent PIE, our findings suggested that AS may function in myocyte contraction via Ca2 þ -independent mechanisms in addition to Ca2 þ -dependent pathways. However, this result should be further investigated. In conclusion, using the established dog model, we demonstrated that AS directly induced PIE and improved CBF and energy metabolism. Our results also demonstrated that AS increased cardiomyocyte contractile function in individual isolated rat cardiac myocytes possibly by increasing the amplitude of [Ca2 þ ]i transient during EC coupling and by enhancing SERCA activity. Another mechanism could be via a PKC-dependent pathway. These results helped elucidate the protective function of AS in myocardial IR injury. Nonetheless, further studies should be conducted to examine the precise mechanism of AS-induced increase in cardiomyocyte contractile function.

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Please cite this article as: Wang, M., et al., Effect of the total saponins of Aralia elata (Miq) Seem on cardiac contractile function and intracellular calcium cycling regulation. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.024i