- Email: [email protected]
Mechanisms underlying the cardioprotective effect of Salvianic acid A against isoproterenol-induced myocardial ischemia injury in rats: Possible involvement of L-type calcium channels and myocardial contractility
Author’s Accepted Manuscript Mechanisms underlying the cardioprotective effect of Salvianic acid A against isoproterenol-induced myocardial ischemia injury in rats: Possible involvement of l-type calcium channels and myocardial contractility Qiongtao Song, Xi Chu, Xuan Zhang, Yifan Bao, Yuanyuan Zhang, Hui Guo, Yang Liu, Hongying Liu, Jianping Zhang, Ying Zhang, Li Chu
PII: DOI: Reference:
www.elsevier.com/locate/jep
S0378-8741(16)30309-9 http://dx.doi.org/10.1016/j.jep.2016.05.038 JEP10176
To appear in: Journal of Ethnopharmacology Received date: 17 August 2015 Revised date: 28 March 2016 Accepted date: 16 May 2016 Cite this article as: Qiongtao Song, Xi Chu, Xuan Zhang, Yifan Bao, Yuanyuan Zhang, Hui Guo, Yang Liu, Hongying Liu, Jianping Zhang, Ying Zhang and Li Chu, Mechanisms underlying the cardioprotective effect of Salvianic acid A against isoproterenol-induced myocardial ischemia injury in rats: Possible involvement of l-type calcium channels and myocardial contractility, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.05.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mechanisms underlying the cardioprotective effect of Salvianic acid A against isoproterenol-induced myocardial ischemia injury in rats: possible involvement of L-type calcium channels and myocardial contractility
Qiongtao Song a, Xi Chu b, Xuan Zhang c, Yifan Bao d, Yuanyuan Zhang c, Hui Guo c, Yang Liu a, Hongying Liu e, Jianping Zhang c, Ying Zhang c, Li Chu a, c* a
Hebei Medical University, No.361, East Zhongshan Road, Shijiazhuang 050017,
Hebei, China. b
The Fourth Hospital of Hebei Medical University, No.12, Jiankang Road,
Shijiazhuang 050011, Hebei, China. c
Hebei University of Chinese Medicine, No.3, Xingyuan Road, Shijiazhuang 050200,
Hebei, China. d
Key Laboratory of Drug Metabolism & Pharmacokinetics, China Pharmaceutical
University, No. 1, Shennong Road of the Central Door, Nanjing 210038, Jiangsu, China.. e
Department of Infectious Diseases, Hebei General Hospital, Shijiazhuang, Hebei
050051, Shijiazhuang, China.
*
Corresponding author: Tel.: +86 311 89926718; fax: +86 311 89926718.
E-mail addresses: [email protected].
1
ABSTRACT Ethnopharmacological relevance: Salvianic acid A (SAA), which is the main water-soluble fraction in Radix Salviae Milthiorrhizae, has been widely applied for treating cardiovascular diseases in China. Aim of the study: To explore the effects of SAA against myocardial ischemia injury induced by isoproterenol (ISO) in rats and to clarify its underlying myocardial protective mechanisms based on L-type calcium channels and myocardial contractility. Materials and methods: The myocardial ischemia injured rat model was induced by administering ISO (85 mg/kg) subcutaneously at evenly spaced intervals throughout the day and night for 2 consecutive days. Serum cardiac biomarkers were analyzed, and heart tissues were isolated and prepared for histopathology assay. The regulatory effects of SAA on the L-type calcium current (ICa-L) in rat ventricular myocytes were observed by the patch clamp technique. The IonOptix Myocam detection system was used to observe the contractility of isolated rat ventricular myocytes. Results: SAA significantly ameliorated changes in heart morphology and electrocardiographic patterns and reduced serum levels of creatine kinase and lactate dehydrogenase in the ISO-induced myocardial ischemia injured rat model. Meanwhile, SAA reduced ICa-L in a concentration-time dependent way with an IC50 of 1.47 × 10−5 M, upshifted the current-voltage, activation, and inactivation curves of ICa-L, and 2
significantly inhibited the amplitude of the cell shortening. Conclusions: These results indicate that SAA exhibits significant cardioprotective effects against the ISO-induced myocardial ischemia injury, potentially through inhibiting ICa-L and decreasing myocardial contractility. Keywords: Radix Salviae Milthiorrhizae, Salvianic acid A, Isoproterenol, Myocardial ischemia injury, L-type calcium current, Myocardial contractility Abbreviations: ATP, adenosine triphosphate; CK, creatine kinase; Con, Control; H&E, hematoxylin and eosin; H-SAA, high-dose Salvianic acid A; ICa-L, L-type calcium current; IHD, ischaemic heart disease; ISO, isoproterenol; LDH, lactate dehydrogenase; L-SAA, low-dose Salvianic acid A; LTCCs, L-type calcium channels; SAA, Salvianic acid A; VER, Verapamil
1. Introduction Cardiovascular diseases still remain prominent causes of morbidity and mortality in industrialized countries (Guilbert, 2003), and the ischaemic heart disease (IHD) occupies a primary place in the cause of cardiovascular diseases (Aronow, 2006; Whellan, 2005). The main culprit in IHD is that the coronary blood supply does not meet the demand of cardiomyocytes, which leads to the rapid development of myocardial necrosis. Isoproterenol (ISO), which is a synthetic catecholamine and β-adrenergic agonist, gives rise to acute irreversible myocardial ischemia injury in rats once in an overdose situation, which is similar to myocardial infarction in humans (Anandan et al., 2007; Karthikeyan et al., 2007). Calcium overload, hypoxia, and 3
coronary hypotension are supposedly the main reasons leading to the development of ISO-induced myocardial ischemia injury (Mohanty et al., 2004). In traditional Chinese medicine, the dried roots or rhizomes of Salvia miltiorrhiza Bge are used as medicine to commonly treat cardiovascular diseases (Ji et al., 2000). The content of Radix Salviae Milthiorrhizae consists of lipid-soluble and water-soluble compounds. In recent studies, we found that injection of an aqueous extract of Radix Salviae Milthiorrhizae could aid in cardioprotection through inhibition of L-type calcium channels (LTCCs) and myocardial contractility similarly to LTCC blockers (Gao et al., 2014). As the main components of Radix Salviae Milthiorrhizae injection, Salvianic acid A (SAA) (Fig. 1) was reported to have powerful effects on suppressing myocardial ischemia reperfusion injury, decreasing lipid peroxidation, and scavenging free radicals (Tang et al., 2011; Wu et al., 2007; Yin et al., 2013; Zhao et al., 1996). However, the possible protective mechanisms of SAA on myocardial ischemia are not fully understood. In the ischemic myocardium, the anaerobic metabolism acts as the major source of energy production characterized by a reduction of myocardial adenosine triphosphate (ATP) stores which affects the trans-sarcolemmal Na+-K+ exchange and in turn elevates intracellular Na+ and intracellular Ca2+ through an enhanced Na+-Ca2+ exchange (Chouairi et al., 1995). Besides, Ca2+ uptake by the sarcoplasmic reticulum and extrusion of Ca2+ from cells decrease due to lowered ATP stores. The resultant augmented intracellular Ca2+ concentration causes mitochondrial Ca2+ overload, which interferes with the mitochondrial capacity to generate ATP, at the same time, 4
activation of intracellular Ca2+ ATPases augments ATP usage and activation of sarcolemmal phospholipases and proteases will destroy the integrity of the cell membrane (Opie, 1989). In addition, improving the levels of intracellular calcium ions can increase myocardial contractility and promote the associated pathology changes, such as myocardial hypertrophy and cell apoptosis (Chen et al., 2005; Frey and Olson, 2003), and increased contractility of ventricular myocytes is a well-known central feature that responds to myocardial ischemia injury (Bristow et al., 1985). LTCCs are the primary pathways of calcium entering cardiomyocytes and the LTCC blockers interfere with Ca2+ influx through LTCCs (Fan et al., 2000). Thus, it is generally accepted that the LTCC blockade may mitigate myocardial ischemic injury through inhibiting LTCCs and cardiac contractility. Therefore, drugs that have been found to specifically weaken L-type calcium current (ICa-L) are promising candidates to decrease myocardial contractility and play a role in myocardial protection. In a previous study, the myocardial protection mechanism of SAA was shown to be scattered, partial, and limited (Cui et al., 2013; Li et al., 2012; Yin et al., 2013). We speculate that SAA exerts its cardioprotective effects via inhibition of LTCCs and cardiac contractility. In the present study, we investigated the effects of SAA against myocardial ischemia injury induced by ISO in rats and used the whole-cell patch-clamp techniques and the IonOptix Myocam detection system to study the influences of SAA on ICa-L and contractility in rat ventricular myocytes under physiological conditions in order to clarify the cellular mechanisms of its cardioprotective effects. This research will not only contribute to a better 5
comprehension to the efficacies of Radix Salviae Milthiorrhizae in clinical treatments, but it will also provide experimental evidence for rational applications of Radix Salviae Milthiorrhizae.
Fig. 1. Chemical structure of SAA
2. Materials and methods 2.1. Reagents SAA was obtained from Beijing SLF Chemical Research Institute (Beijing, China); Isoproterenol (ISO) was obtained from Hefeng Pharmaceutical Co., Ltd. (Shanghai, China); and Verapamil (VER) was obtained from Harvest Pharmaceutical Co., Ltd. (Shanghai, China). Stock solutions were made in DMSO and were diluted into the proper concentration before implement of experiment. Except as otherwise noted, other laboratory reagents were acquired from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Induction of myocardial ischemia injury and protocol 40 adult male Sprague-Dawley rats (180-220 g) were caged under the environment of 22 ± 2 °C and a 12-hour day and night cycle with adequate food and water supplies. After acclimatization of 1 week, all rats were randomly assigned into 5
6
groups (n = 8 per group) named Control (Con), ISO, low-dose SAA (L-SAA), high-dose SAA (H-SAA), and VER groups. L-SAA, H-SAA, and VER groups were given SAA (3 mg/kg/day), SAA (10 mg/kg/day), and VER (2 mg/kg/day), respectively. Con and ISO groups were given distilled water. All treatments were administrated intraperitoneally. After 7 consecutive days of pretreatment, rats were intoxicated with ISO (85 mg/kg except for the control group) by subcutaneous injection on 2 consecutive days. The animal experiments were carried out according to the Guidelines of Animal Experiments from the Committee of Medical Ethics in China and the Ethics Committee for Animal Experiments of Hebei Medical University (approval number: HEBMU-2014-9; approval date: September 25, 2014). 2.3. Electrocardiogram An electrocardiogram test was conducted in anesthetized rats after finishing the final injection of ISO. The needle electrodes were linked to the right arm, left arm, and left leg of the rats, and the electrocardiographic-patterns were recorded by the BL-420S biological Function Experiment System. The J-point elevation and heart rate of rats were recorded. 2.4. Activities of creatine kinase (CK) and lactate dehydrogenase (LDH) After the experimental protocol, the serum was separated from the rat blood by centrifugation, and the diagnostic marker enzymes CK and LDH in serum were determined by standard commercial kits (Jian Cheng Biological Engineering Institute, Nanjing, China).
7
2.5. Analysis of histopathology Heart samples of Hematoxylin-Eosin (H&E) treatment were obtained according to the following steps: small pieces of the heart samples were prepared and fixed in 4% paraformaldehyde, then the samples were hydrated, cleared, and finally embedded in paraffin. After H&E staining, sections (5 μm thick) were evaluated through the light microscope (Leica DM4000B, Solms, Germany). 2.6. Adult rat ventricular myocytes separation Single ventricular myocytes were harvested through enzymolysis (Li et al., 2001; Tao et al., 2004). Rats were injected with 1,500 IU heparin and anesthetized with ethylcarbamate (4 mg/kg) intraperitoneally; we quickly took out the heart and put it into 0 °C Ca2+-free Tyrode's solution (135 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl2, 10 mM glucose and 10 mM HEPES, pH 7.4). The heart was retrogradely perfused with Ca2+-free Tyrode's solution to remove the blood with a Langendorff apparatus. After clearance of the blood, the enzyme solution containing 4 g/L collagenase type II (GIBCO, Invitrogen, Carlsbad, CA, USA), 4 g/L taurine, 10 g/L bovine serum albumin (Roche, Basel, Switzerland) perfused for 20-30 minutes until the heart was flaccid. Then, enzymes were washed out with Kreb`s buffer solution (KOH 80 mM, KCl 40 mM, KH2PO4 25 mM, L-glutamic acid 50 mM, taurine 20 mM, HEPES 10 mM, EGTA 1 mM, glucose 10 mM and MgSO4 3 mM, pH 7.2) after enzymatic digestion. All solutions used during the perfusion were oxygenated with 100% O2. At the end of perfusion, the ventricular tissue was minced and filtered in order to harvest 8
the rat ventricular myocytes. Cells were used within 9 hours after gradually adding 1.8 mM Ca2+ to the Kreb`s buffer solution. 2.7. Electrical Recordings The patch clamp technique was used to test ICa-L in the rat ventricular myocytes. Briefly, under the action of the pipette puller (Model P-97, Sutter Instruments), borosilicate glass electrodes received 2-5 MΩ resistance after filling with pipette solution (Mg-ATP 5 mM, CsCl 120 mM, HEPES 10 mM and EGTA 10 mM, pH 7.2). After establishing the whole-cell configuration by gigaseal and membrane rupture, ICa-L was recorded under the condition of compensating the electrode resistance in voltage-clamp mode. An axon patch 200B amplifier (Axon Instruments, Union City, CA, USA) and pClamp 10.2 software (Molecular Devices, Sunnyvale, CA) were used to record the data. 2.8. Measurements of cell shortening Cells were placed on the bottom of a glass chamber with normal Tyrode’s solution flowing at the velocity of 1 ml/min. Cell activity could be observed by an inverted microscope and a detection system placed on the glass chamber. Field stimulation at 0.5 Hz (2 msec duration) induced cell shortening, and we could ascertain the effects of SAA on myocardial contractility by measuring the cell shortening under the treatment of different concentration of SAA.
9
2.9. Statistics All data were expressed as means ± S.E.M. Data were analyzed and fitted using the Clampfit 10.2 (Molecular Devices, Sunnyvale, CA) and Origin 7.5 (OriginLab Corp., Northampton, MA) software. Differences between groups were determined by the Student’s t test or one-way ANOVA. P < 0.05 was considered to be significant.
3. Results 3.1. Attenuation of ISO-induced myocardial ischemia injury by SAA 3.1.1 .Effects of SAA on electrocardiography Compared with Con (Table. 1), heart rate and J-point elevation were elevated significantly in ISO-induced rats (P < 0.05 or P < 0.01), according to the electrocardiography study. A significant reduction of the heart rate and J-point elevation (P < 0.05 or P < 0.01) was achieved by SAA groups compared to ISO. Table. 1. Effects of SAA on electrocardiography. Values are given as mean ± SEM (n = 8). * P < 0.05 vs. Con; ** P < 0.01 vs. Con; # P < 0.05 vs. ISO; ## P < 0.01 vs. ISO.
Groups
J-point elevation (mV)
Heart rate (beats/min)
Con
0.045 ± 0.007
340.158 ± 8.751
ISO
0.078 ± 0.006**
417.968 ± 30.604*
L-SAA
0.044 ± 0.005##
336.760 ± 18.265#
H-SAA
0.035 ± 0.006##
304.754 ± 20.668##
VER
0.045 ± 0.005##
343.836 ± 10.862#
10
3.1.2. Effects of SAA on CK and LDH Myocardial damage was evaluated by measuring plasma CK and LDH (Fig. 2). A significant increase was detected in the content of CK and LDH in ISO compared with Con (P < 0.01); however, there was a significant decrease in the SAA and VER groups compared with the ISO group (P < 0.05 or P < 0.01).
Fig. 2. Effects of SAA on the activities of CK (A) and LDH (B). Rats were treated with L-SAA, H-SAA, or VER for 7 consecutive days before ISO exposure. Blood samples were collected 12 hours after ISO exposure, and the activities of CK and LDH were measured. Values are given as mean ± S.E.M.
**
P < 0.01 vs. Con; # P <
0.05 vs. ISO; ## P < 0.01 vs. ISO.
3.1.3. Effects of SAA on histopathological change Light microscopy was used to evaluate the histopathology of rat hearts (Fig. 3). A normal myofibrillar structure was revealed in tissue sections in Con (Fig. 3A), while the ISO group showed obvious myocardial cell swelling, degeneration, and loss of transverse striations (Fig.3B). The L-SAA, H-SAA, and VER groups revealed approximately normal structure with clear transverse striations, and they showed presence of slight edema and few vacuoles as compared to ISO (Fig.3C, 3D, and 3E).
11
Fig. 3. Effects of SAA on histopathological changes of rat heart stained with H&E. Representative sections (magnification: 40×) are from the heart of Con (A), ISO (B), L-SAA (C), H-SAA (D), and VER (E). Histopathological changes are indicated by green (edema), blue (vacuole) and red (degeneration) arrows. The scale bar is 50 μm. 3.2. Reduction of ICa-L and cell shortening by SAA 3.2.1 Confirmation of ICa-L As a specific LTCCs blocker, 0.1 mM VER almost entirely inhibited the currents induced by the steady-state activation protocol (P < 0.01, compared with Con), demonstrating the induced currents were ICa-L (Fig. 4).
12
Fig. 4. VER (0.1 mM) completely inhibited ICa-L in rat ventricular myocytes. (A) Representative traces under the treatment of VER. (B) Summary data of A. Values are given as means ± S.E.M. (n = 5 cells). ** P < 0.01 vs. Con. 3.2.2. Reversible effects of SAA on ICa-L ICa-L was decreased under the treatment of 3×10-4 M SAA with inhibition rate of 35.5 ± 0.9% (P < 0.01 vs. Con). After SAA treatment, ICa-L almost returned back to the level of Con (19.8 ± 2.7%) (n = 5) (Fig. 5).
Fig. 5. Reversible effects of SAA on ICa-L in rat ventricular myocytes. Exemplary traces (A) and pooled data (B) of ICa-L were recorded under the treatment of 3×10-4 M SAA and during wash out. Values are given as means ± S.E.M. (n = 5 cells). ** P < 0.01 vs. Con.
13
3.2.3. Concentration-dependent effects of SAA on ICa-L ICa-L was progressively suppressed by increasing concentrations of SAA (3×10-6, 10-5, 3×10-5, 10-4, and 3×10-4 M SAA) (Fig. 6A). Fig. 6B represented current traces elicited from the test potential of –80 mV to 0 mV at different SAA concentrations. The inhibition rates of 3×10-6, 10-5, 3×10-5, 10-4, and 3×10-4 M SAA were 9.5 ± 0.9%, 17.6 ± 1.1%, 29.6 ± 1.8%, 34.6 ± 1.9%, and 39.5 ± 1.9%, respectively (n = 5) (Fig. 6C).
Fig. 6. Effects of SAA at different concentrations on ICa-L. (A) Time-histories of ICa-L in exposure to 3×10-6, 10-5, 3×10-5, 10-4, 3×10-4 M SAA, and 0.1 mM VER. (B) Current traces of SAA and VER mentioned above. (C) Concentration-response curve of SAA. Values are given as means ± S.E.M. (n = 5-7 cells). 3.2.4. Effects of SAA on current-voltage (I-V) relationship of ICa-L The average I-V relationship was shown under the treatment of 3×10-6, 3×10-5, 14
3×10-4 M SAA, and 0.1 mM VER (Fig. 7). I-V curves shifted upward after the application of SAA, indicating SAA inhibited ICa-L in a concentration-time dependent way. However, activated potential and peak potential of ICa-L were significantly unchanged.
Fig. 7. Effects of SAA on the I-V relationship of ICa-L. Exemplary traces (A) and pooled data (B) are shown under the treatment of Con (□), SAA at 3×10-6 M (○), SAA at 3×10-5 M (△) and SAA at 3×10-4 M (▽), and VER at 0.1 mM (◇). Values are given as means ± S.E.M and n = 5 cells. 3.2.5. Effects of SAA on steady-state activation and inactivation of ICa-L SAA at 3×10-6, 3×10-5, 3×10-4 M SAA did not change the activation and inactivation of ICa-L (Fig. 8). Values at V1/2/slope factor (k) of the normalized activation conductance curves after application of 0, 3×10-6, 3×10-5, 3×10-4 M SAA were -10.40 ± 0.64 mV/7.03 ± 0.58, -9.53 ± 0.69 mV/6.99 ± 0.62, -8.16 ± 0.69 mV/6.99 ± 0.61, -8.61 ± 0.69 mV/6.88 ± 0.61, respectively. Values at V1/2/k of the normalized inactivation conductance curves after application of 0, 3×10-6, 3×10-5, 15
3×10-4 M SAA were -32.03 ± 0.15 mV/4.62 ± 0.13, -31.34 ± 0.08 mV/ 4.84 ± 0.08, -30.75 ± 0.01 mV/4.98 ± 0.01, -30.94 ± 0.02 mV/5.08 ± 0.02, respectively.
Fig. 8. Effects of SAA on steady-state activation and inactivation of ICa-L. Activation kinetics (A) and inactivation kinetics of ICa-L (B) are shown under the treatment of Con (□), SAA at 3×10-6 M (○), SAA at 3×10-5 M (△), and SAA at 3×10-4 M (▽). Values are given as means ± S.E.M and n = 5 cells. 3.2.6. Effects of SAA on cell shortening Fig. 9 shows changes of SAA on myocyte shortening. The results indicate that SAA at the concentration of 3×10-7 M significantly inhibits cell shortening by 33.48 ± 0.75% (** P < 0.01 vs. Con).
16
Fig. 9. Effects of SAA on cell shortening. (A) Time parameters of cell shortening under the treatment of 3×10-7 M SAA. (B) Exemplary traces of A. (C) Summary data of B. Values are given as means ± S.E.M and n = 5 cells. ** P < 0.01 vs. Con. 4. Discussion IHD, in particular myocardial infarction, is a major cause of cardiovascular diseases. The mechanism underlying subcutaneous injection of supramaximal doses of ISO-inducing ischemia or hypoxia in a rat model includes aspects such as myocardial hyperactivity and cytosolic Ca2+ overload; also, the pathophysiological changes in a rat model are similar to the observed results in humans using overdose of ISO (Li et al., 2012; Wang et al., 2009). In China, Radix Salviae Milthiorrhizae has been clinically applied in the treatment of cardiovascular disease for many years (Zhou et al., 2005). The lipid-soluble content of Radix Salviae Milthiorrhizae such as Tanshinone IIA was found to affect the human cardiac KCNQ1/KCNE1 potassium 17
channels' kinetics to enhance the slowly activating delayed rectifier potassium current in human embryonic kidney 293 cells (Sun et al., 2008), which could be considered as the partial mechanism of cardioprotection of Radix Salviae Milthiorrhizae. In our previous study, the main constituents of Danshen injection, an aqueous extract of Radix Salviae Milthiorrhizae, were analyzed by HPLC-UV. The actual concentrations of the main component SAA, protocatechuic aldehyde, and salvianolic acid B are 2.15, 0.44 and 1.01 mg/ml, respectively (Zhang et al., 2013). While SAA and the putative active components of Radix Salviae Milthiorrhizae’s aqueous extract may be beneficial, the underlying mechanisms of its cardioprotective effects are not well known (Chen et al., 2011; Wang et al., 2011). The subchronic study by Gao et al. has shown that rats were tested by daily intraperitoneal injection of SAA even at the dose of 450 mg/kg for 90 days did not demonstrate any adverse effects (Gao et al., 2009). In the current study, we have explored the cardioprotective effects and its underlying mechanisms of SAA against ISO-induced myocardial ischemia injury. Our study indicates that subcutaneous administration of ISO causes increases in J-point elevation and heart rate, while pretreatment with SAA lowers J-point elevation and heart rate, suggesting its protective effects (Table 1). Moreover, we used biochemical markers such as CK and LDH to detect the cardiac cellular damage under the myocardial ischemia condition induced by ISO. As illustrated in Fig. 2, SAA can partially decrease the plasma CK and LDH which are related to cellular damage and loss of functional integrity (Constant, 1997; Sabeena Farvin et al., 2004), indicating that SAA effectively maintains cell functional integrity and restricts the leakage of 18
these cytosolic enzymes into circulation. The cardioprotective effect of SAA was furthermore shown by the significant improvement in the histopathological examination. The sections from heart tissues of rats pretreated with SAA showed fewer areas of degeneration compared with those in ISO (Figs. 3B, C and D). The results provide strong evidence to prove that SAA provides cardioprotection against ISO-induced myocardial injury. At the same time, SAA dose-dependently reduced ICa-L and contractility in rat ventricular myocytes (Fig. 6 and 9). However, neither the I-V relationship nor activation and inactivation of ICa-L were affected (Fig. 7 and 8). The excitation-contraction coupling process started with Ca2+ entering through LTCCs into cardiac myocytes (Wier, 1991). Because of activation of LTCCs by sarcolemmal depolarization, extracellular Ca2+ flows into the myocyte in a dose-dependent manner, which results in the release of Ca2+ from sarcoplasmic reticular stores (Fabiato, 1983; Williams, 1997) and then proceeds to the development of a myofilament cross-bridge and myocardial contractility (Williams, 1997). As Ca2+ influx plays a vital role in excitation-contraction coupling in cardiomyocytes, drugs inhibiting ICa-L can directly decrease the release of sarcoplasmic reticular Ca2+ and reduce myocardial contractility; thus, they can consequently decrease the myocardial oxygen consumption, which has benefits for ischemic myocardial protection (Crystal et al., 2013; Sonnenblick et al., 1965). In addition, mitochondria are important subcellular organelles for cellular oxidative process and the major source of energy for contraction comes from the oxidative metabolism of mitochondria in the myocardial cell. The decrease in oxygen supply during myocardial ischemia impairs 19
energy production by mitochondria (Kumaran and Prince, 2010). From the above, a rapid rise in intracellular Ca2+ can trigger/worsen myocardial ischemia due to an increased oxygen demand, and foster fuel deprivation from an energy starved heart. In our present study, we found that SAA significantly inhibited ICa-L and could be regarded as the cardioprotective agent by inhibiting ICa-L and decreasing myocardial contractility. In addition, we observe that SAA seems to inhibit cell shortening much more than ICa-L. This phenomenon can be explained due to the complex process of myocardial contraction, which is not only related to intracellular Ca2+ concentration but is also related to intracellular proteins involved in contraction (actin and myosin) or regulation (troponin, tropomyosin, and tropomodulin) (Adamcova et al., 2006). The more detailed mechanisms of the effects of SAA on myocardial contraction need to be explored. In this study, we focus on the underlying mechanism of SAA on cardiovascular protection and offer a theoretical basis for the further study and clinical application of SAA. This study systematically demonstrated the cardioprotective effect of SAA from myocardial ischemia injury in vivo and clarified its underlying myocardial protective mechanisms in adult rat cardiomyocytes in vitro. Sufficient proof has linked SAA to cardioprotection by decreasing J-point elevation and heart rate, reducing CK and LDH, inhibiting ICa-L and myocardial contractility, and thereby reducing myocardial oxygen consumption. 5. Conclusion 20
This study indicated that SAA treatment effectively protected ISO-induced myocardial ischemia injury. The underlying mechanism of SAA against myocardial ischemia is potentially connected with its diverse abilities in inhibiting ICa-L and suppressing myocardial contractility. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (81573669), Natural Science Funds for Youths in Higher Education of Hebei province (No.QN2016035) and the Nature Fund of Hebei Province, China (No. C 2011206025 and No. H 2014206244). References Adamcova, M., Sterba, M., Simunek, T., Potacova, A., Popelova, O., Gersl, V., 2006. Myocardial regulatory proteins and heart failure. European journal of heart failure 8, 333-342. Anandan, R., Mathew, S., Sankar, T.V., Viswanathan Nair, P.G., 2007. Protective effect of n-3 polyunsaturated fatty acids concentrate on isoproterenol-induced myocardial infarction in rats. Prostaglandins, leukotrienes, and essential fatty acids 76, 153-158. Aronow, W.S., 2006. Epidemiology, pathophysiology, prognosis, and treatment of systolic and diastolic heart failure. Cardiology in review 14, 108-124. Bristow, M.R., Kantrowitz, N.E., Ginsburg, R., Fowler, M.B., 1985. Beta-adrenergic function in heart muscle disease and heart failure. Journal of molecular and cellular cardiology 17 Suppl 2, 41-52. Chen, X., Zhang, X., Kubo, H., Harris, D.M., Mills, G.D., Moyer, J., Berretta, R., Potts, S.T., Marsh, J.D., Houser, S.R., 2005. Ca2+ influx-induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis in ventricular myocytes. Circulation research 97, 1009-1017. Chen, Y.C., Cao, W.W., Cao, Y., Zhang, L., Chang, B.B., Yang, W.L., Liu, X.Q., 2011. Using neural networks to determine the contribution of danshensu to its multiple cardiovascular activities in acute myocardial infarction rats. Journal of ethnopharmacology 138, 126-134. Chouairi, S., Carrie, D., Puel, J., 1995. Myocardial protection with calcium-channel blockers during ischaemia and reperfusion by PTCA. European heart journal 16 Suppl H, 3-8. Constant, J., 1997. Alcohol, ischemic heart disease, and the French paradox. Clin Cardiol 20, 420-424. Crystal, G.J., Silver, J.M., Salem, M.R., 2013. Mechanisms of increased right and left ventricular oxygen uptake during inotropic stimulation. Life sciences 93, 59-63. Cui, G., Shan, L., Hung, M., Lei, S., Choi, I., Zhang, Z., Yu, P., Hoi, P., Wang, Y., Lee, S.M., 2013. A 21
novel Danshensu derivative confers cardioprotection via PI3K/Akt and Nrf2 pathways. International journal of cardiology 168, 1349-1359. Fabiato, A., 1983. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. The American journal of physiology 245, C1-14. Fan, I.Q., Chen, B., Marsh, J.D., 2000. Transcriptional regulation of L-type calcium channel expression in cardiac myocytes. Journal of molecular and cellular cardiology 32, 1841-1849. Frey, N., Olson, E.N., 2003. Cardiac hypertrophy: the good, the bad, and the ugly. Annual review of physiology 65, 45-79. Gao, Y., Liu, Z., Li, G., Li, C., Li, M., Li, B., 2009. Acute and subchronic toxicity of danshensu in mice and rats. Toxicology mechanisms and methods 19, 363-368. Gao, Y., Zhang, K., Zhu, F., Wu, Z., Chu, X., Zhang, X., Zhang, Y., Zhang, J., Chu, L., 2014. Salvia miltiorrhiza (Danshen) inhibits L-type calcium current and attenuates calcium transient and contractility in rat ventricular myocytes. Journal of ethnopharmacology 158 Pt A, 397-403. Guilbert, J.J., 2003. The world health report 2002 - reducing risks, promoting healthy life. Education for health (Abingdon, England) 16, 230. Ji, X.Y., Tan, B.K., Zhu, Y.Z., 2000. Salvia miltiorrhiza and ischemic diseases. Acta pharmacologica Sinica 21, 1089-1094. Karthikeyan, K., Bai, B.R., Devaraj, S.N., 2007. Cardioprotective effect of grape seed proanthocyanidins on isoproterenol-induced myocardial injury in rats. International journal of cardiology 115, 326-333. Kumaran, K.S., Prince, P.S., 2010. Caffeic acid protects rat heart mitochondria against isoproterenol-induced oxidative damage. Cell stress & chaperones 15, 791-806. Li, H., Xie, Y.H., Yang, Q., Wang, S.W., Zhang, B.L., Wang, J.B., Cao, W., Bi, L.L., Sun, J.Y., Miao, S., Hu, J., Zhou, X.X., Qiu, P.C., 2012. Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats. PloS one 7, e48872. Li, S., Blaschke, M., Heubach, J.F., Wettwer, E., Ravens, U., 2001. Effects of azelastine on contractility, action potentials and L-type Ca(2+) current in guinea pig cardiac preparations. European journal of pharmacology 418, 7-14. Mohanty, I., Arya, D.S., Dinda, A., Talwar, K.K., Joshi, S., Gupta, S.K., 2004. Mechanisms of cardioprotective effect of Withania somnifera in experimentally induced myocardial infarction. Basic & clinical pharmacology & toxicology 94, 184-190. Opie, L.H., 1989. Reperfusion injury and its pharmacologic modification. Circulation 80, 1049-1062. Sabeena Farvin, K.H., Anandan, R., Kumar, S.H., Shiny, K.S., Sankar, T.V., Thankappan, T.K., 2004. Effect of squalene on tissue defense system in isoproterenol-induced myocardial infarction in rats. Pharmacol Res 50, 231-236. Sonnenblick, E.H., Ross, J., Jr., Covell, J.W., Kaiser, G.A., Braunwald, E., 1965. Velocity of contraction as a determinant of myocardial oxygen consumption. The American journal of physiology 209, 919-927. Sun, D.D., Wang, H.C., Wang, X.B., Luo, Y., Jin, Z.X., Li, Z.C., Li, G.R., Dong, M.Q., 2008. Tanshinone IIA: a new activator of human cardiac KCNQ1/KCNE1 (I(Ks)) potassium channels. European journal of pharmacology 590, 317-321. Tang, Y., Wang, M., Le, X., Meng, J., Huang, L., Yu, P., Chen, J., Wu, P., 2011. Antioxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydroxyphenyl)-2-hydroxy-propanoic acid from Salvia miltiorrhiza) on isoproterenol-induced myocardial hypertrophy in rats. Phytomedicine : 22
international journal of phytotherapy and phytopharmacology 18, 1024-1030. Tao, J., Xu, H., Yang, C., Liu, C.N., Li, S., 2004. Effect of urocortin on L-type calcium currents in adult rat ventricular myocytes. Pharmacological research : the official journal of the Italian Pharmacological Society 50, 471-476. Wang, S.B., Tian, S., Yang, F., Yang, H.G., Yang, X.Y., Du, G.H., 2009. Cardioprotective effect of salvianolic acid A on isoproterenol-induced myocardial infarction in rats. European journal of pharmacology 615, 125-132. Wang, X., Wang, Y., Jiang, M., Zhu, Y., Hu, L., Fan, G., Wang, Y., Li, X., Gao, X., 2011. Differential cardioprotective effects of salvianolic acid and tanshinone on acute myocardial infarction are mediated by unique signaling pathways. Journal of ethnopharmacology 135, 662-671. Whellan, D.J., 2005. Heart failure disease management: implementation and outcomes. Cardiology in review 13, 231-239. Wier, W.G., 1991. Intracellular calcium during excitation-contraction coupling in mammalian ventricle. Medicine and science in sports and exercise 23, 1149-1156. Williams, A.J., 1997. The functions of two species of calcium channel in cardiac muscle excitation-contraction coupling. European heart journal 18 Suppl A, A27-35. Wu, L., Qiao, H., Li, Y., Li, L., 2007. Protective roles of puerarin and Danshensu on acute ischemic myocardial
injury
in
rats.
Phytomedicine
:
international
journal
of
phytotherapy
and
phytopharmacology 14, 652-658. Yin, Y., Guan, Y., Duan, J., Wei, G., Zhu, Y., Quan, W., Guo, C., Zhou, D., Wang, Y., Xi, M., Wen, A., 2013. Cardioprotective effect of Danshensu against myocardial ischemia/reperfusion injury and inhibits apoptosis of H9c2 cardiomyocytes via Akt and ERK1/2 phosphorylation. European journal of pharmacology 699, 219-226. Zhang, J.P., Zhang, Y.Y., Zhang, Y., Gao, Y.G., Ma, J.J., Wang, N., Wang, J.Y., Xie, Y., Zhang, F.H., Chu, L., 2013. Salvia miltiorrhiza (Danshen) injection ameliorates iron overload-induced cardiac damage in mice. Planta medica 79, 744-752. Zhao, B.L., Jiang, W., Zhao, Y., Hou, J.W., Xin, W.J., 1996. Scavenging effects of salvia miltiorrhiza on free radicals and its protection for myocardial mitochondrial membranes from ischemia-reperfusion injury. Biochemistry and molecular biology international 38, 1171-1182. Zhou, L., Zuo, Z., Chow, M.S., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. Journal of clinical pharmacology 45, 1345-1359.
23
24
PII: DOI: Reference:
www.elsevier.com/locate/jep
S0378-8741(16)30309-9 http://dx.doi.org/10.1016/j.jep.2016.05.038 JEP10176
To appear in: Journal of Ethnopharmacology Received date: 17 August 2015 Revised date: 28 March 2016 Accepted date: 16 May 2016 Cite this article as: Qiongtao Song, Xi Chu, Xuan Zhang, Yifan Bao, Yuanyuan Zhang, Hui Guo, Yang Liu, Hongying Liu, Jianping Zhang, Ying Zhang and Li Chu, Mechanisms underlying the cardioprotective effect of Salvianic acid A against isoproterenol-induced myocardial ischemia injury in rats: Possible involvement of l-type calcium channels and myocardial contractility, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.05.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mechanisms underlying the cardioprotective effect of Salvianic acid A against isoproterenol-induced myocardial ischemia injury in rats: possible involvement of L-type calcium channels and myocardial contractility
Qiongtao Song a, Xi Chu b, Xuan Zhang c, Yifan Bao d, Yuanyuan Zhang c, Hui Guo c, Yang Liu a, Hongying Liu e, Jianping Zhang c, Ying Zhang c, Li Chu a, c* a
Hebei Medical University, No.361, East Zhongshan Road, Shijiazhuang 050017,
Hebei, China. b
The Fourth Hospital of Hebei Medical University, No.12, Jiankang Road,
Shijiazhuang 050011, Hebei, China. c
Hebei University of Chinese Medicine, No.3, Xingyuan Road, Shijiazhuang 050200,
Hebei, China. d
Key Laboratory of Drug Metabolism & Pharmacokinetics, China Pharmaceutical
University, No. 1, Shennong Road of the Central Door, Nanjing 210038, Jiangsu, China.. e
Department of Infectious Diseases, Hebei General Hospital, Shijiazhuang, Hebei
050051, Shijiazhuang, China.
*
Corresponding author: Tel.: +86 311 89926718; fax: +86 311 89926718.
E-mail addresses: [email protected].
1
ABSTRACT Ethnopharmacological relevance: Salvianic acid A (SAA), which is the main water-soluble fraction in Radix Salviae Milthiorrhizae, has been widely applied for treating cardiovascular diseases in China. Aim of the study: To explore the effects of SAA against myocardial ischemia injury induced by isoproterenol (ISO) in rats and to clarify its underlying myocardial protective mechanisms based on L-type calcium channels and myocardial contractility. Materials and methods: The myocardial ischemia injured rat model was induced by administering ISO (85 mg/kg) subcutaneously at evenly spaced intervals throughout the day and night for 2 consecutive days. Serum cardiac biomarkers were analyzed, and heart tissues were isolated and prepared for histopathology assay. The regulatory effects of SAA on the L-type calcium current (ICa-L) in rat ventricular myocytes were observed by the patch clamp technique. The IonOptix Myocam detection system was used to observe the contractility of isolated rat ventricular myocytes. Results: SAA significantly ameliorated changes in heart morphology and electrocardiographic patterns and reduced serum levels of creatine kinase and lactate dehydrogenase in the ISO-induced myocardial ischemia injured rat model. Meanwhile, SAA reduced ICa-L in a concentration-time dependent way with an IC50 of 1.47 × 10−5 M, upshifted the current-voltage, activation, and inactivation curves of ICa-L, and 2
significantly inhibited the amplitude of the cell shortening. Conclusions: These results indicate that SAA exhibits significant cardioprotective effects against the ISO-induced myocardial ischemia injury, potentially through inhibiting ICa-L and decreasing myocardial contractility. Keywords: Radix Salviae Milthiorrhizae, Salvianic acid A, Isoproterenol, Myocardial ischemia injury, L-type calcium current, Myocardial contractility Abbreviations: ATP, adenosine triphosphate; CK, creatine kinase; Con, Control; H&E, hematoxylin and eosin; H-SAA, high-dose Salvianic acid A; ICa-L, L-type calcium current; IHD, ischaemic heart disease; ISO, isoproterenol; LDH, lactate dehydrogenase; L-SAA, low-dose Salvianic acid A; LTCCs, L-type calcium channels; SAA, Salvianic acid A; VER, Verapamil
1. Introduction Cardiovascular diseases still remain prominent causes of morbidity and mortality in industrialized countries (Guilbert, 2003), and the ischaemic heart disease (IHD) occupies a primary place in the cause of cardiovascular diseases (Aronow, 2006; Whellan, 2005). The main culprit in IHD is that the coronary blood supply does not meet the demand of cardiomyocytes, which leads to the rapid development of myocardial necrosis. Isoproterenol (ISO), which is a synthetic catecholamine and β-adrenergic agonist, gives rise to acute irreversible myocardial ischemia injury in rats once in an overdose situation, which is similar to myocardial infarction in humans (Anandan et al., 2007; Karthikeyan et al., 2007). Calcium overload, hypoxia, and 3
coronary hypotension are supposedly the main reasons leading to the development of ISO-induced myocardial ischemia injury (Mohanty et al., 2004). In traditional Chinese medicine, the dried roots or rhizomes of Salvia miltiorrhiza Bge are used as medicine to commonly treat cardiovascular diseases (Ji et al., 2000). The content of Radix Salviae Milthiorrhizae consists of lipid-soluble and water-soluble compounds. In recent studies, we found that injection of an aqueous extract of Radix Salviae Milthiorrhizae could aid in cardioprotection through inhibition of L-type calcium channels (LTCCs) and myocardial contractility similarly to LTCC blockers (Gao et al., 2014). As the main components of Radix Salviae Milthiorrhizae injection, Salvianic acid A (SAA) (Fig. 1) was reported to have powerful effects on suppressing myocardial ischemia reperfusion injury, decreasing lipid peroxidation, and scavenging free radicals (Tang et al., 2011; Wu et al., 2007; Yin et al., 2013; Zhao et al., 1996). However, the possible protective mechanisms of SAA on myocardial ischemia are not fully understood. In the ischemic myocardium, the anaerobic metabolism acts as the major source of energy production characterized by a reduction of myocardial adenosine triphosphate (ATP) stores which affects the trans-sarcolemmal Na+-K+ exchange and in turn elevates intracellular Na+ and intracellular Ca2+ through an enhanced Na+-Ca2+ exchange (Chouairi et al., 1995). Besides, Ca2+ uptake by the sarcoplasmic reticulum and extrusion of Ca2+ from cells decrease due to lowered ATP stores. The resultant augmented intracellular Ca2+ concentration causes mitochondrial Ca2+ overload, which interferes with the mitochondrial capacity to generate ATP, at the same time, 4
activation of intracellular Ca2+ ATPases augments ATP usage and activation of sarcolemmal phospholipases and proteases will destroy the integrity of the cell membrane (Opie, 1989). In addition, improving the levels of intracellular calcium ions can increase myocardial contractility and promote the associated pathology changes, such as myocardial hypertrophy and cell apoptosis (Chen et al., 2005; Frey and Olson, 2003), and increased contractility of ventricular myocytes is a well-known central feature that responds to myocardial ischemia injury (Bristow et al., 1985). LTCCs are the primary pathways of calcium entering cardiomyocytes and the LTCC blockers interfere with Ca2+ influx through LTCCs (Fan et al., 2000). Thus, it is generally accepted that the LTCC blockade may mitigate myocardial ischemic injury through inhibiting LTCCs and cardiac contractility. Therefore, drugs that have been found to specifically weaken L-type calcium current (ICa-L) are promising candidates to decrease myocardial contractility and play a role in myocardial protection. In a previous study, the myocardial protection mechanism of SAA was shown to be scattered, partial, and limited (Cui et al., 2013; Li et al., 2012; Yin et al., 2013). We speculate that SAA exerts its cardioprotective effects via inhibition of LTCCs and cardiac contractility. In the present study, we investigated the effects of SAA against myocardial ischemia injury induced by ISO in rats and used the whole-cell patch-clamp techniques and the IonOptix Myocam detection system to study the influences of SAA on ICa-L and contractility in rat ventricular myocytes under physiological conditions in order to clarify the cellular mechanisms of its cardioprotective effects. This research will not only contribute to a better 5
comprehension to the efficacies of Radix Salviae Milthiorrhizae in clinical treatments, but it will also provide experimental evidence for rational applications of Radix Salviae Milthiorrhizae.
Fig. 1. Chemical structure of SAA
2. Materials and methods 2.1. Reagents SAA was obtained from Beijing SLF Chemical Research Institute (Beijing, China); Isoproterenol (ISO) was obtained from Hefeng Pharmaceutical Co., Ltd. (Shanghai, China); and Verapamil (VER) was obtained from Harvest Pharmaceutical Co., Ltd. (Shanghai, China). Stock solutions were made in DMSO and were diluted into the proper concentration before implement of experiment. Except as otherwise noted, other laboratory reagents were acquired from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Induction of myocardial ischemia injury and protocol 40 adult male Sprague-Dawley rats (180-220 g) were caged under the environment of 22 ± 2 °C and a 12-hour day and night cycle with adequate food and water supplies. After acclimatization of 1 week, all rats were randomly assigned into 5
6
groups (n = 8 per group) named Control (Con), ISO, low-dose SAA (L-SAA), high-dose SAA (H-SAA), and VER groups. L-SAA, H-SAA, and VER groups were given SAA (3 mg/kg/day), SAA (10 mg/kg/day), and VER (2 mg/kg/day), respectively. Con and ISO groups were given distilled water. All treatments were administrated intraperitoneally. After 7 consecutive days of pretreatment, rats were intoxicated with ISO (85 mg/kg except for the control group) by subcutaneous injection on 2 consecutive days. The animal experiments were carried out according to the Guidelines of Animal Experiments from the Committee of Medical Ethics in China and the Ethics Committee for Animal Experiments of Hebei Medical University (approval number: HEBMU-2014-9; approval date: September 25, 2014). 2.3. Electrocardiogram An electrocardiogram test was conducted in anesthetized rats after finishing the final injection of ISO. The needle electrodes were linked to the right arm, left arm, and left leg of the rats, and the electrocardiographic-patterns were recorded by the BL-420S biological Function Experiment System. The J-point elevation and heart rate of rats were recorded. 2.4. Activities of creatine kinase (CK) and lactate dehydrogenase (LDH) After the experimental protocol, the serum was separated from the rat blood by centrifugation, and the diagnostic marker enzymes CK and LDH in serum were determined by standard commercial kits (Jian Cheng Biological Engineering Institute, Nanjing, China).
7
2.5. Analysis of histopathology Heart samples of Hematoxylin-Eosin (H&E) treatment were obtained according to the following steps: small pieces of the heart samples were prepared and fixed in 4% paraformaldehyde, then the samples were hydrated, cleared, and finally embedded in paraffin. After H&E staining, sections (5 μm thick) were evaluated through the light microscope (Leica DM4000B, Solms, Germany). 2.6. Adult rat ventricular myocytes separation Single ventricular myocytes were harvested through enzymolysis (Li et al., 2001; Tao et al., 2004). Rats were injected with 1,500 IU heparin and anesthetized with ethylcarbamate (4 mg/kg) intraperitoneally; we quickly took out the heart and put it into 0 °C Ca2+-free Tyrode's solution (135 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl2, 10 mM glucose and 10 mM HEPES, pH 7.4). The heart was retrogradely perfused with Ca2+-free Tyrode's solution to remove the blood with a Langendorff apparatus. After clearance of the blood, the enzyme solution containing 4 g/L collagenase type II (GIBCO, Invitrogen, Carlsbad, CA, USA), 4 g/L taurine, 10 g/L bovine serum albumin (Roche, Basel, Switzerland) perfused for 20-30 minutes until the heart was flaccid. Then, enzymes were washed out with Kreb`s buffer solution (KOH 80 mM, KCl 40 mM, KH2PO4 25 mM, L-glutamic acid 50 mM, taurine 20 mM, HEPES 10 mM, EGTA 1 mM, glucose 10 mM and MgSO4 3 mM, pH 7.2) after enzymatic digestion. All solutions used during the perfusion were oxygenated with 100% O2. At the end of perfusion, the ventricular tissue was minced and filtered in order to harvest 8
the rat ventricular myocytes. Cells were used within 9 hours after gradually adding 1.8 mM Ca2+ to the Kreb`s buffer solution. 2.7. Electrical Recordings The patch clamp technique was used to test ICa-L in the rat ventricular myocytes. Briefly, under the action of the pipette puller (Model P-97, Sutter Instruments), borosilicate glass electrodes received 2-5 MΩ resistance after filling with pipette solution (Mg-ATP 5 mM, CsCl 120 mM, HEPES 10 mM and EGTA 10 mM, pH 7.2). After establishing the whole-cell configuration by gigaseal and membrane rupture, ICa-L was recorded under the condition of compensating the electrode resistance in voltage-clamp mode. An axon patch 200B amplifier (Axon Instruments, Union City, CA, USA) and pClamp 10.2 software (Molecular Devices, Sunnyvale, CA) were used to record the data. 2.8. Measurements of cell shortening Cells were placed on the bottom of a glass chamber with normal Tyrode’s solution flowing at the velocity of 1 ml/min. Cell activity could be observed by an inverted microscope and a detection system placed on the glass chamber. Field stimulation at 0.5 Hz (2 msec duration) induced cell shortening, and we could ascertain the effects of SAA on myocardial contractility by measuring the cell shortening under the treatment of different concentration of SAA.
9
2.9. Statistics All data were expressed as means ± S.E.M. Data were analyzed and fitted using the Clampfit 10.2 (Molecular Devices, Sunnyvale, CA) and Origin 7.5 (OriginLab Corp., Northampton, MA) software. Differences between groups were determined by the Student’s t test or one-way ANOVA. P < 0.05 was considered to be significant.
3. Results 3.1. Attenuation of ISO-induced myocardial ischemia injury by SAA 3.1.1 .Effects of SAA on electrocardiography Compared with Con (Table. 1), heart rate and J-point elevation were elevated significantly in ISO-induced rats (P < 0.05 or P < 0.01), according to the electrocardiography study. A significant reduction of the heart rate and J-point elevation (P < 0.05 or P < 0.01) was achieved by SAA groups compared to ISO. Table. 1. Effects of SAA on electrocardiography. Values are given as mean ± SEM (n = 8). * P < 0.05 vs. Con; ** P < 0.01 vs. Con; # P < 0.05 vs. ISO; ## P < 0.01 vs. ISO.
Groups
J-point elevation (mV)
Heart rate (beats/min)
Con
0.045 ± 0.007
340.158 ± 8.751
ISO
0.078 ± 0.006**
417.968 ± 30.604*
L-SAA
0.044 ± 0.005##
336.760 ± 18.265#
H-SAA
0.035 ± 0.006##
304.754 ± 20.668##
VER
0.045 ± 0.005##
343.836 ± 10.862#
10
3.1.2. Effects of SAA on CK and LDH Myocardial damage was evaluated by measuring plasma CK and LDH (Fig. 2). A significant increase was detected in the content of CK and LDH in ISO compared with Con (P < 0.01); however, there was a significant decrease in the SAA and VER groups compared with the ISO group (P < 0.05 or P < 0.01).
Fig. 2. Effects of SAA on the activities of CK (A) and LDH (B). Rats were treated with L-SAA, H-SAA, or VER for 7 consecutive days before ISO exposure. Blood samples were collected 12 hours after ISO exposure, and the activities of CK and LDH were measured. Values are given as mean ± S.E.M.
**
P < 0.01 vs. Con; # P <
0.05 vs. ISO; ## P < 0.01 vs. ISO.
3.1.3. Effects of SAA on histopathological change Light microscopy was used to evaluate the histopathology of rat hearts (Fig. 3). A normal myofibrillar structure was revealed in tissue sections in Con (Fig. 3A), while the ISO group showed obvious myocardial cell swelling, degeneration, and loss of transverse striations (Fig.3B). The L-SAA, H-SAA, and VER groups revealed approximately normal structure with clear transverse striations, and they showed presence of slight edema and few vacuoles as compared to ISO (Fig.3C, 3D, and 3E).
11
Fig. 3. Effects of SAA on histopathological changes of rat heart stained with H&E. Representative sections (magnification: 40×) are from the heart of Con (A), ISO (B), L-SAA (C), H-SAA (D), and VER (E). Histopathological changes are indicated by green (edema), blue (vacuole) and red (degeneration) arrows. The scale bar is 50 μm. 3.2. Reduction of ICa-L and cell shortening by SAA 3.2.1 Confirmation of ICa-L As a specific LTCCs blocker, 0.1 mM VER almost entirely inhibited the currents induced by the steady-state activation protocol (P < 0.01, compared with Con), demonstrating the induced currents were ICa-L (Fig. 4).
12
Fig. 4. VER (0.1 mM) completely inhibited ICa-L in rat ventricular myocytes. (A) Representative traces under the treatment of VER. (B) Summary data of A. Values are given as means ± S.E.M. (n = 5 cells). ** P < 0.01 vs. Con. 3.2.2. Reversible effects of SAA on ICa-L ICa-L was decreased under the treatment of 3×10-4 M SAA with inhibition rate of 35.5 ± 0.9% (P < 0.01 vs. Con). After SAA treatment, ICa-L almost returned back to the level of Con (19.8 ± 2.7%) (n = 5) (Fig. 5).
Fig. 5. Reversible effects of SAA on ICa-L in rat ventricular myocytes. Exemplary traces (A) and pooled data (B) of ICa-L were recorded under the treatment of 3×10-4 M SAA and during wash out. Values are given as means ± S.E.M. (n = 5 cells). ** P < 0.01 vs. Con.
13
3.2.3. Concentration-dependent effects of SAA on ICa-L ICa-L was progressively suppressed by increasing concentrations of SAA (3×10-6, 10-5, 3×10-5, 10-4, and 3×10-4 M SAA) (Fig. 6A). Fig. 6B represented current traces elicited from the test potential of –80 mV to 0 mV at different SAA concentrations. The inhibition rates of 3×10-6, 10-5, 3×10-5, 10-4, and 3×10-4 M SAA were 9.5 ± 0.9%, 17.6 ± 1.1%, 29.6 ± 1.8%, 34.6 ± 1.9%, and 39.5 ± 1.9%, respectively (n = 5) (Fig. 6C).
Fig. 6. Effects of SAA at different concentrations on ICa-L. (A) Time-histories of ICa-L in exposure to 3×10-6, 10-5, 3×10-5, 10-4, 3×10-4 M SAA, and 0.1 mM VER. (B) Current traces of SAA and VER mentioned above. (C) Concentration-response curve of SAA. Values are given as means ± S.E.M. (n = 5-7 cells). 3.2.4. Effects of SAA on current-voltage (I-V) relationship of ICa-L The average I-V relationship was shown under the treatment of 3×10-6, 3×10-5, 14
3×10-4 M SAA, and 0.1 mM VER (Fig. 7). I-V curves shifted upward after the application of SAA, indicating SAA inhibited ICa-L in a concentration-time dependent way. However, activated potential and peak potential of ICa-L were significantly unchanged.
Fig. 7. Effects of SAA on the I-V relationship of ICa-L. Exemplary traces (A) and pooled data (B) are shown under the treatment of Con (□), SAA at 3×10-6 M (○), SAA at 3×10-5 M (△) and SAA at 3×10-4 M (▽), and VER at 0.1 mM (◇). Values are given as means ± S.E.M and n = 5 cells. 3.2.5. Effects of SAA on steady-state activation and inactivation of ICa-L SAA at 3×10-6, 3×10-5, 3×10-4 M SAA did not change the activation and inactivation of ICa-L (Fig. 8). Values at V1/2/slope factor (k) of the normalized activation conductance curves after application of 0, 3×10-6, 3×10-5, 3×10-4 M SAA were -10.40 ± 0.64 mV/7.03 ± 0.58, -9.53 ± 0.69 mV/6.99 ± 0.62, -8.16 ± 0.69 mV/6.99 ± 0.61, -8.61 ± 0.69 mV/6.88 ± 0.61, respectively. Values at V1/2/k of the normalized inactivation conductance curves after application of 0, 3×10-6, 3×10-5, 15
3×10-4 M SAA were -32.03 ± 0.15 mV/4.62 ± 0.13, -31.34 ± 0.08 mV/ 4.84 ± 0.08, -30.75 ± 0.01 mV/4.98 ± 0.01, -30.94 ± 0.02 mV/5.08 ± 0.02, respectively.
Fig. 8. Effects of SAA on steady-state activation and inactivation of ICa-L. Activation kinetics (A) and inactivation kinetics of ICa-L (B) are shown under the treatment of Con (□), SAA at 3×10-6 M (○), SAA at 3×10-5 M (△), and SAA at 3×10-4 M (▽). Values are given as means ± S.E.M and n = 5 cells. 3.2.6. Effects of SAA on cell shortening Fig. 9 shows changes of SAA on myocyte shortening. The results indicate that SAA at the concentration of 3×10-7 M significantly inhibits cell shortening by 33.48 ± 0.75% (** P < 0.01 vs. Con).
16
Fig. 9. Effects of SAA on cell shortening. (A) Time parameters of cell shortening under the treatment of 3×10-7 M SAA. (B) Exemplary traces of A. (C) Summary data of B. Values are given as means ± S.E.M and n = 5 cells. ** P < 0.01 vs. Con. 4. Discussion IHD, in particular myocardial infarction, is a major cause of cardiovascular diseases. The mechanism underlying subcutaneous injection of supramaximal doses of ISO-inducing ischemia or hypoxia in a rat model includes aspects such as myocardial hyperactivity and cytosolic Ca2+ overload; also, the pathophysiological changes in a rat model are similar to the observed results in humans using overdose of ISO (Li et al., 2012; Wang et al., 2009). In China, Radix Salviae Milthiorrhizae has been clinically applied in the treatment of cardiovascular disease for many years (Zhou et al., 2005). The lipid-soluble content of Radix Salviae Milthiorrhizae such as Tanshinone IIA was found to affect the human cardiac KCNQ1/KCNE1 potassium 17
channels' kinetics to enhance the slowly activating delayed rectifier potassium current in human embryonic kidney 293 cells (Sun et al., 2008), which could be considered as the partial mechanism of cardioprotection of Radix Salviae Milthiorrhizae. In our previous study, the main constituents of Danshen injection, an aqueous extract of Radix Salviae Milthiorrhizae, were analyzed by HPLC-UV. The actual concentrations of the main component SAA, protocatechuic aldehyde, and salvianolic acid B are 2.15, 0.44 and 1.01 mg/ml, respectively (Zhang et al., 2013). While SAA and the putative active components of Radix Salviae Milthiorrhizae’s aqueous extract may be beneficial, the underlying mechanisms of its cardioprotective effects are not well known (Chen et al., 2011; Wang et al., 2011). The subchronic study by Gao et al. has shown that rats were tested by daily intraperitoneal injection of SAA even at the dose of 450 mg/kg for 90 days did not demonstrate any adverse effects (Gao et al., 2009). In the current study, we have explored the cardioprotective effects and its underlying mechanisms of SAA against ISO-induced myocardial ischemia injury. Our study indicates that subcutaneous administration of ISO causes increases in J-point elevation and heart rate, while pretreatment with SAA lowers J-point elevation and heart rate, suggesting its protective effects (Table 1). Moreover, we used biochemical markers such as CK and LDH to detect the cardiac cellular damage under the myocardial ischemia condition induced by ISO. As illustrated in Fig. 2, SAA can partially decrease the plasma CK and LDH which are related to cellular damage and loss of functional integrity (Constant, 1997; Sabeena Farvin et al., 2004), indicating that SAA effectively maintains cell functional integrity and restricts the leakage of 18
these cytosolic enzymes into circulation. The cardioprotective effect of SAA was furthermore shown by the significant improvement in the histopathological examination. The sections from heart tissues of rats pretreated with SAA showed fewer areas of degeneration compared with those in ISO (Figs. 3B, C and D). The results provide strong evidence to prove that SAA provides cardioprotection against ISO-induced myocardial injury. At the same time, SAA dose-dependently reduced ICa-L and contractility in rat ventricular myocytes (Fig. 6 and 9). However, neither the I-V relationship nor activation and inactivation of ICa-L were affected (Fig. 7 and 8). The excitation-contraction coupling process started with Ca2+ entering through LTCCs into cardiac myocytes (Wier, 1991). Because of activation of LTCCs by sarcolemmal depolarization, extracellular Ca2+ flows into the myocyte in a dose-dependent manner, which results in the release of Ca2+ from sarcoplasmic reticular stores (Fabiato, 1983; Williams, 1997) and then proceeds to the development of a myofilament cross-bridge and myocardial contractility (Williams, 1997). As Ca2+ influx plays a vital role in excitation-contraction coupling in cardiomyocytes, drugs inhibiting ICa-L can directly decrease the release of sarcoplasmic reticular Ca2+ and reduce myocardial contractility; thus, they can consequently decrease the myocardial oxygen consumption, which has benefits for ischemic myocardial protection (Crystal et al., 2013; Sonnenblick et al., 1965). In addition, mitochondria are important subcellular organelles for cellular oxidative process and the major source of energy for contraction comes from the oxidative metabolism of mitochondria in the myocardial cell. The decrease in oxygen supply during myocardial ischemia impairs 19
energy production by mitochondria (Kumaran and Prince, 2010). From the above, a rapid rise in intracellular Ca2+ can trigger/worsen myocardial ischemia due to an increased oxygen demand, and foster fuel deprivation from an energy starved heart. In our present study, we found that SAA significantly inhibited ICa-L and could be regarded as the cardioprotective agent by inhibiting ICa-L and decreasing myocardial contractility. In addition, we observe that SAA seems to inhibit cell shortening much more than ICa-L. This phenomenon can be explained due to the complex process of myocardial contraction, which is not only related to intracellular Ca2+ concentration but is also related to intracellular proteins involved in contraction (actin and myosin) or regulation (troponin, tropomyosin, and tropomodulin) (Adamcova et al., 2006). The more detailed mechanisms of the effects of SAA on myocardial contraction need to be explored. In this study, we focus on the underlying mechanism of SAA on cardiovascular protection and offer a theoretical basis for the further study and clinical application of SAA. This study systematically demonstrated the cardioprotective effect of SAA from myocardial ischemia injury in vivo and clarified its underlying myocardial protective mechanisms in adult rat cardiomyocytes in vitro. Sufficient proof has linked SAA to cardioprotection by decreasing J-point elevation and heart rate, reducing CK and LDH, inhibiting ICa-L and myocardial contractility, and thereby reducing myocardial oxygen consumption. 5. Conclusion 20
This study indicated that SAA treatment effectively protected ISO-induced myocardial ischemia injury. The underlying mechanism of SAA against myocardial ischemia is potentially connected with its diverse abilities in inhibiting ICa-L and suppressing myocardial contractility. Conflict of interest The authors declare no conflicts of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (81573669), Natural Science Funds for Youths in Higher Education of Hebei province (No.QN2016035) and the Nature Fund of Hebei Province, China (No. C 2011206025 and No. H 2014206244). References Adamcova, M., Sterba, M., Simunek, T., Potacova, A., Popelova, O., Gersl, V., 2006. Myocardial regulatory proteins and heart failure. European journal of heart failure 8, 333-342. Anandan, R., Mathew, S., Sankar, T.V., Viswanathan Nair, P.G., 2007. Protective effect of n-3 polyunsaturated fatty acids concentrate on isoproterenol-induced myocardial infarction in rats. Prostaglandins, leukotrienes, and essential fatty acids 76, 153-158. Aronow, W.S., 2006. Epidemiology, pathophysiology, prognosis, and treatment of systolic and diastolic heart failure. Cardiology in review 14, 108-124. Bristow, M.R., Kantrowitz, N.E., Ginsburg, R., Fowler, M.B., 1985. Beta-adrenergic function in heart muscle disease and heart failure. Journal of molecular and cellular cardiology 17 Suppl 2, 41-52. Chen, X., Zhang, X., Kubo, H., Harris, D.M., Mills, G.D., Moyer, J., Berretta, R., Potts, S.T., Marsh, J.D., Houser, S.R., 2005. Ca2+ influx-induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis in ventricular myocytes. Circulation research 97, 1009-1017. Chen, Y.C., Cao, W.W., Cao, Y., Zhang, L., Chang, B.B., Yang, W.L., Liu, X.Q., 2011. Using neural networks to determine the contribution of danshensu to its multiple cardiovascular activities in acute myocardial infarction rats. Journal of ethnopharmacology 138, 126-134. Chouairi, S., Carrie, D., Puel, J., 1995. Myocardial protection with calcium-channel blockers during ischaemia and reperfusion by PTCA. European heart journal 16 Suppl H, 3-8. Constant, J., 1997. Alcohol, ischemic heart disease, and the French paradox. Clin Cardiol 20, 420-424. Crystal, G.J., Silver, J.M., Salem, M.R., 2013. Mechanisms of increased right and left ventricular oxygen uptake during inotropic stimulation. Life sciences 93, 59-63. Cui, G., Shan, L., Hung, M., Lei, S., Choi, I., Zhang, Z., Yu, P., Hoi, P., Wang, Y., Lee, S.M., 2013. A 21
novel Danshensu derivative confers cardioprotection via PI3K/Akt and Nrf2 pathways. International journal of cardiology 168, 1349-1359. Fabiato, A., 1983. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. The American journal of physiology 245, C1-14. Fan, I.Q., Chen, B., Marsh, J.D., 2000. Transcriptional regulation of L-type calcium channel expression in cardiac myocytes. Journal of molecular and cellular cardiology 32, 1841-1849. Frey, N., Olson, E.N., 2003. Cardiac hypertrophy: the good, the bad, and the ugly. Annual review of physiology 65, 45-79. Gao, Y., Liu, Z., Li, G., Li, C., Li, M., Li, B., 2009. Acute and subchronic toxicity of danshensu in mice and rats. Toxicology mechanisms and methods 19, 363-368. Gao, Y., Zhang, K., Zhu, F., Wu, Z., Chu, X., Zhang, X., Zhang, Y., Zhang, J., Chu, L., 2014. Salvia miltiorrhiza (Danshen) inhibits L-type calcium current and attenuates calcium transient and contractility in rat ventricular myocytes. Journal of ethnopharmacology 158 Pt A, 397-403. Guilbert, J.J., 2003. The world health report 2002 - reducing risks, promoting healthy life. Education for health (Abingdon, England) 16, 230. Ji, X.Y., Tan, B.K., Zhu, Y.Z., 2000. Salvia miltiorrhiza and ischemic diseases. Acta pharmacologica Sinica 21, 1089-1094. Karthikeyan, K., Bai, B.R., Devaraj, S.N., 2007. Cardioprotective effect of grape seed proanthocyanidins on isoproterenol-induced myocardial injury in rats. International journal of cardiology 115, 326-333. Kumaran, K.S., Prince, P.S., 2010. Caffeic acid protects rat heart mitochondria against isoproterenol-induced oxidative damage. Cell stress & chaperones 15, 791-806. Li, H., Xie, Y.H., Yang, Q., Wang, S.W., Zhang, B.L., Wang, J.B., Cao, W., Bi, L.L., Sun, J.Y., Miao, S., Hu, J., Zhou, X.X., Qiu, P.C., 2012. Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats. PloS one 7, e48872. Li, S., Blaschke, M., Heubach, J.F., Wettwer, E., Ravens, U., 2001. Effects of azelastine on contractility, action potentials and L-type Ca(2+) current in guinea pig cardiac preparations. European journal of pharmacology 418, 7-14. Mohanty, I., Arya, D.S., Dinda, A., Talwar, K.K., Joshi, S., Gupta, S.K., 2004. Mechanisms of cardioprotective effect of Withania somnifera in experimentally induced myocardial infarction. Basic & clinical pharmacology & toxicology 94, 184-190. Opie, L.H., 1989. Reperfusion injury and its pharmacologic modification. Circulation 80, 1049-1062. Sabeena Farvin, K.H., Anandan, R., Kumar, S.H., Shiny, K.S., Sankar, T.V., Thankappan, T.K., 2004. Effect of squalene on tissue defense system in isoproterenol-induced myocardial infarction in rats. Pharmacol Res 50, 231-236. Sonnenblick, E.H., Ross, J., Jr., Covell, J.W., Kaiser, G.A., Braunwald, E., 1965. Velocity of contraction as a determinant of myocardial oxygen consumption. The American journal of physiology 209, 919-927. Sun, D.D., Wang, H.C., Wang, X.B., Luo, Y., Jin, Z.X., Li, Z.C., Li, G.R., Dong, M.Q., 2008. Tanshinone IIA: a new activator of human cardiac KCNQ1/KCNE1 (I(Ks)) potassium channels. European journal of pharmacology 590, 317-321. Tang, Y., Wang, M., Le, X., Meng, J., Huang, L., Yu, P., Chen, J., Wu, P., 2011. Antioxidant and cardioprotective effects of Danshensu (3-(3, 4-dihydroxyphenyl)-2-hydroxy-propanoic acid from Salvia miltiorrhiza) on isoproterenol-induced myocardial hypertrophy in rats. Phytomedicine : 22
international journal of phytotherapy and phytopharmacology 18, 1024-1030. Tao, J., Xu, H., Yang, C., Liu, C.N., Li, S., 2004. Effect of urocortin on L-type calcium currents in adult rat ventricular myocytes. Pharmacological research : the official journal of the Italian Pharmacological Society 50, 471-476. Wang, S.B., Tian, S., Yang, F., Yang, H.G., Yang, X.Y., Du, G.H., 2009. Cardioprotective effect of salvianolic acid A on isoproterenol-induced myocardial infarction in rats. European journal of pharmacology 615, 125-132. Wang, X., Wang, Y., Jiang, M., Zhu, Y., Hu, L., Fan, G., Wang, Y., Li, X., Gao, X., 2011. Differential cardioprotective effects of salvianolic acid and tanshinone on acute myocardial infarction are mediated by unique signaling pathways. Journal of ethnopharmacology 135, 662-671. Whellan, D.J., 2005. Heart failure disease management: implementation and outcomes. Cardiology in review 13, 231-239. Wier, W.G., 1991. Intracellular calcium during excitation-contraction coupling in mammalian ventricle. Medicine and science in sports and exercise 23, 1149-1156. Williams, A.J., 1997. The functions of two species of calcium channel in cardiac muscle excitation-contraction coupling. European heart journal 18 Suppl A, A27-35. Wu, L., Qiao, H., Li, Y., Li, L., 2007. Protective roles of puerarin and Danshensu on acute ischemic myocardial
injury
in
rats.
Phytomedicine
:
international
journal
of
phytotherapy
and
phytopharmacology 14, 652-658. Yin, Y., Guan, Y., Duan, J., Wei, G., Zhu, Y., Quan, W., Guo, C., Zhou, D., Wang, Y., Xi, M., Wen, A., 2013. Cardioprotective effect of Danshensu against myocardial ischemia/reperfusion injury and inhibits apoptosis of H9c2 cardiomyocytes via Akt and ERK1/2 phosphorylation. European journal of pharmacology 699, 219-226. Zhang, J.P., Zhang, Y.Y., Zhang, Y., Gao, Y.G., Ma, J.J., Wang, N., Wang, J.Y., Xie, Y., Zhang, F.H., Chu, L., 2013. Salvia miltiorrhiza (Danshen) injection ameliorates iron overload-induced cardiac damage in mice. Planta medica 79, 744-752. Zhao, B.L., Jiang, W., Zhao, Y., Hou, J.W., Xin, W.J., 1996. Scavenging effects of salvia miltiorrhiza on free radicals and its protection for myocardial mitochondrial membranes from ischemia-reperfusion injury. Biochemistry and molecular biology international 38, 1171-1182. Zhou, L., Zuo, Z., Chow, M.S., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. Journal of clinical pharmacology 45, 1345-1359.
23
24