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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 6  |  Issue : 3  |  Page : 284-294

Ginsenoside Rb1 pretreatment attenuates myocardial ischemia by reducing calcium/calmodulin-dependent protein kinase II-medicated calcium release


1 Research Center for Traditional Chinese Medicine Complexity System, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China
2 AntiCancer, Inc., San Diego, USA

Date of Submission29-Oct-2019
Date of Acceptance10-Feb-2020
Date of Web Publication05-Aug-2020

Correspondence Address:
Prof. Shi-Bing Su
Research Center for Traditional Chinese Medicine Complexity System, 1200 Road Cailun, Shanghai
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_24_20

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  Abstract 


Objective: The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinase II (CaMKII).Methods: A myocardial ischemia (IS) rat. model and a myocardial H9C2 cell hypoxia model were established. MI was induced by occluding the left anterior descending artery for 120 min. Ginsenoside Rb1 (10 mg/kg) was administered 30 min before ischemia induction, and the treatment continued for 7 days. Results: In the rat IS injury model, ginsenoside Rb1 reduced myocardial infarct size, mean left ventricular diastolic pressure, incidence of arrhythmia, and levels of serum creatine kinase, lactate dehydrogenase, and malondialdehyde. However, the mean left ventricular systolic pressure, and maximal rising and falling rates of ventricular pressure (±dp/dtmax) increased. In the myocardial H9C2 cell hypoxia model, ginsenoside Rb1 reduced intracellular calcium concentrations ([Ca2+ ]i) during hypoxia, and markedly reversed the hypoxia-induced decrease in cell survival. Ginsenoside Rb1 was involved in the downregulation of CaMKII and the ryanodine receptor, as well as hypoxia-induced H9C2 cell survival. Conclusion: The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats, partially through the downregulation of CaMKII expression.

Keywords: Calcium/calmodulin-dependent protein kinase II, Ginsenoside Rb1, myocardial ischemia, ryanodine receptor


How to cite this article:
Zhou WJ, Li JL, Zhou QM, Cai FF, Chen XL, Lu YY, Zhao M, Su SB. Ginsenoside Rb1 pretreatment attenuates myocardial ischemia by reducing calcium/calmodulin-dependent protein kinase II-medicated calcium release. World J Tradit Chin Med 2020;6:284-94

How to cite this URL:
Zhou WJ, Li JL, Zhou QM, Cai FF, Chen XL, Lu YY, Zhao M, Su SB. Ginsenoside Rb1 pretreatment attenuates myocardial ischemia by reducing calcium/calmodulin-dependent protein kinase II-medicated calcium release. World J Tradit Chin Med [serial online] 2020 [cited 2020 Sep 26];6:284-94. Available from: http://www.wjtcm.net/text.asp?2020/6/3/284/290079




  Introduction Top


Ischemic heart disease is one of the several clinical problems resulting in myocardial damage, arrhythmia, and atrial stunning.[1] Globally, it is a widespread disease, especially in Western countries, with seven million fatalities annually reported due to coronary artery disease caused by myocardial ischemia (MI).[2] Severe ischemic heart disease can lead to localized myocardial infarction or even sudden death. The impact of this disease has become increasingly serious with the growing incidence of coronary heart disease. Therefore, it is crucial to prevent the occurrence of ischemic heart disease and improve the survival rate. However, currently, there are no effective clinical drugs for the prevention of ischemic heart disease.

During the process of ischemia-reperfusion (I/R), excess intracellular Ca2+ results in the death of cardiomyocytes.[3] The disruption of Ca2+ homeostasis, a principal consideration of I/R injury, is reportedly regulated by the calcium/calmodulin-dependent protein kinase II (CaMKII).[4],[5] CaMKII is a multifunctional protein belonging to the serine/threonine-protein kinase family.[6],[7] Its overexpression is a common phenomenon in heart failure caused by both ischemic and dilated cardiomyopathy.[8],[9],[10],[11] CaMKII overexpression can be observed at the beginning of reperfusion, resulting in the apoptosis of cardiomyocytes,[12],[13],[14] and often occurs in patients with heart diseases and arrhythmias.[15],[16] Studies have shown that CaMKII is involved in the signal transduction process in arrhythmias,[17],[18] cardiac hypertrophy,[19],[20] heart failure,[21] and other diseases.[22],[23] Ryanodine receptor 2 (RyR2) is the key protein to determine the intracellular calcium level. Ca2+ is released from the sarcoplasmic reticulum (SR) through the RyR2 receptors.[24] The abnormal expression or excessive phosphorylation of RyR2 can cause diastolic leakage and in turn, result in intracellular calcium overload.[25],[26] CaMKII, which can increase the RyR2 opening frequency and calcium release[27],[28] is directly associated with RyR2.

Chinese herbal medicines have been utilized in the treatment of heart diseases for several years. Among Chinese herbal medicines, ginseng and Panax notoginseng saponin have been used for over 2000 years in Asian countries, and have demonstrated several pharmacological activities affecting the cardiovascular, endocrine, immune, and central nervous systems.[29] Among more than 30 different ginsenosides, ginsenoside Rb1 [Figure 1] possesses anti-inflammatory, antioxidant, and anti-apoptotic activities.[30] Recent studies have demonstrated that ginsenoside Rb1 can reduce oxidative damage in cardiomyocytes.[31] Moreover, it has demonstrated protective effects on MI and reperfusion injury in a diabetic rat model.[21],[23] However, the protective effect of ginsenoside Rb1 on IS injury in rats and on hypoxia-induced cardiomyocyte survival remains poorly understood. In this study, we investigated whether ginsenoside Rb1 pretreatment could attenuate MI injury in rats and whether it can demonstrate a beneficial effect on hypoxia-induced H9C2 cell survival through the downregulation of CaMKII expression.
Figure 1: Chemical structure, molecular formula, and molecular weight of ginsenoside Rb1

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  Methods Top


Myocardial ischemia model

The animals were anesthetized using an intraperitoneal injection of 3% pentobarbital sodium (50 mg/kg) and fixed on the operating table to perform the surgical procedures. Electrocardiogram (ECG) electrodes were subcutaneously placed on both front limbs, and the left-back limb, with lead II continuously monitored. A 14-gauge angiocatheter was inserted into the trachea through a neckline incision and then connected to a volume-controlled ventilator (C201 animal ventilator, Chengdu Instrument Factory, Chengdu, China). Heparin (500 U/kg) was intravenously administered, and the right carotid artery was catheterized into the left ventricle (LV). The JP01 pressure transducer was connected and the signal input was received into the RM6240 biological signal acquisition and processing system (Chengdu Instrument Factory, Chengdu, China) to continuously monitor hemodynamic parameters, including mean left ventricular diastolic pressure (mLVDP), T waves, arrhythmia ratio, and increase in mean left ventricular systolic pressure (mLVSP), and maximal rising and falling rates of ventricular pressure (±dp/dtmax). The ECG signal was displayed on the monitor of the RM6240 biological signal acquisition and processing system. A thoracotomy was performed at the fourth left intercostal space, and the pericardium was opened. Using a tapered needle, a 6-0 silk suture was passed under the left anterior descending coronary artery, 2 mm from the tip of the left auricle. The coronary artery was occluded for 120 min.

The rats were randomly assigned to three groups (n = 10 in each group), including (1) sham-operated group (sham group) without occlusion; (2) MI group (IS group) subjected to 120 min of MI; (3) Ginsenoside Rb1 pretreatment group treated with ginsenoside Rb1 (10 mg/kg) daily for 7 days. The last feeding time point was 10 min before coronary ischemia.

Cell hypoxia model

The myocardial H9C2 cell line, derived from the embryonic rat heart, was acquired from Shanghai cell bank, the Chinese Academy of Sciences. The H9C2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 5% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a water vapor-saturated atmosphere with 5% CO2 at 37°C. Cells were plated in 60-mm dishes (for western blot analysis) or 96-well culture plates (for cell viability and intracellular calcium concentration assays) at a density of 0.5 × 106 cells per well and grown to 90% confluency. For hypoxia treatment, cells were cultured in serum-free medium for 24 h and then exposed to the oxygen-glucose deprivation solution (Hanks' balanced salt solution: 125 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4 7H2O, 1.2 mM NaH2 PO4 2H2O, 1.8 mM CaCl2 2H2O, 8.0 mM NaHCO3, and 20 mM HEPES, pH 6.4). The solution was free of serum, glucose, and oxygen (gassed with N2 for 15 min). Then, the cells were transferred to the hypoxia box with N2. After 1 or 2 h, the cells were returned to normal medium and cultured under 5% CO2 at 37°C.

Ginsenoside Rb1, ODG, and KN-93 (Sigma, St. Louis, MO, USA) were added to the H9C2 cells for indicated periods. Cells were cultured in DMEM supplemented with antibiotics and 10% FBS. The cells were randomly assigned to four groups: (i) control group with no treatment; (ii) hypoxia group with hypoxia treatment for 1 or 2 h; (iii) Ginsenoside Rb1 pretreatment group (Rb1). Different concentrations of Rb1 (0.5 and 1.5 mmol/L) were added to the H9C2 cells for 24 h before exposing the cells to the ODG medium for 1 or 2 h; (iv) KN-93 pretreatment group (KN-93). KN-93(1 μmol/L) was added to the media for 30 min before the induction of hypoxia; (v) Rb1 + KN-93 group: KN-93(1 μmol/L) was added to the media for 30 min after the cells were treated with Rb1 for 24 h. Hypoxia was induced after these treatments.

Determination of plasma creatine kinase and lactate dehydrogenase levels

Blood samples were drawn from the abdominal aorta at the end of the ischemic period. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) levels were measured using an automatic microplate reader (Synergy2, BioTek, Winooski, VT, USA) according to the manufacturer's instructions.

Determination of superoxide dismutase and malondialdehyde levels

At the end of the ischemic period, the myocardial tissue was homogenized. After centrifugation (4000 rpm, 10 min at 4°C) and the supernatant was collected. The malondialdehyde (MDA) level in the supernatant was determined using the thiobarbituric acid reaction with a commercial kit.[28] In addition, superoxide dismutase (SOD) activity in the supernatant was evaluated by the inhibition of nitroblue tetrazolium reduction using the xanthine/xanthine oxidase system with a commercial kit.[32]

Determination of the infarct size

After 2 h of ischemia, the animals were sacrificed. The heart was dissected and placed in a small dish filled with ice. The coronary artery was reoccluded, and 1 mL of 0.9% sodium chloride was injected to flush the heart. After resecting the right ventricle, the LV was cut into five transverse slices. To determine the infarct area (IA) and the area at risk, the slices were incubated in 1% triphenyltetrazolium chloride (TTC) solution at 37°C for 25 min. Photographs were obtained under a dissecting microscope. The left ventricular area, area at risk, and IA were determined by planimetry using the MetaVue image analysis software (Molecular Devices, Sunnyvale, CA). The area of the myocardial tissue presenting a white color was defined as the IA, and the region in red was defined as the area at risk. The tissues were then photographed using a digital camera (Canon PowerShot AS1000, Japan) and weighed. The percentage of the myocardial infarct size was determined by dividing the weight of the myocardial ventricle with infarction to the weight of the whole LV. For each slice, the total LV area and the area lacking TTC staining (IA) were determined using a light microscope (Olympus, Japan). Furthermore, the ratio of IA/LV was calculated for each slice.

Immunohistochemical staining of ryanodine receptor 2

The expression of RyR2 was immunohistochemically assessed. Paraffin-embedded left ventricular tissues were sectioned to 3 μm slices. The sections were deparaffinized, rehydrated, treated with the target retrieval buffer, blocked with 3% hydrogen peroxide, washed with phosphate-buffered saline (PBS), and blocked with 5% normal goat serum in PBS for 30 min. The sections were then incubated overnight with the rabbit polyclonal anti-RyR2 antibody (1:100 dilution in 0.1% bovine serum albumin) (Boster Biotech, Shanghai, China), followed by incubation with biotin-conjugated secondary antibody (1:1000 dilution). Finally, the sections were incubated with the avidin-biotin complex kit (Boster Biotech, Shanghai, China) and detected using the diaminobenzidine reagent (Boster Biotech Shanghai, China). The slides were examined using light microscopy at ×200 magnification (Olympus BX 50 Microphotographic System, Japan). For each animal, three random tissue sections (five fields per se ction) were examined. Quantitative immunohistochemical assessments for myocardial endothelial nitric oxide synthase expression were performed as previously described. The staining signal intensity was measured by image cytometry performed with the HIPAS-2000 image analysis software (Qianli Technical Imaging, Shanghai, China).

Cell viability assay

First, H9C2 cells were treated with Rb1for 24 h. Three different concentrations (10, 100, and 150 μmol/L) of Rb1 were added to the cells 30 min before the ODG change. Then, the cells were placed in the hypoxia box with N2 for 1 or 2 h. Cell survival was evaluated using the crystal violet nuclear staining assay, as previously described.[33] Briefly, cells were washed with DMEM and fixed with MTT (5 mg/mL) for 4 h. Next, dimethyl sulfoxide (20 μl per well) was added. After 10 min of shaking, the percentage of cell survival was determined by measuring the absorbance at 490 nm using the microplate reader Synergy2.

Intracellular Ca2+ assay

Cells were loaded with fluorescent Ca2+ dye Fluo-3 AM[34] and incubated at 37°C for 40 min. The cells were then washed twice with PBS. The fluorescence signal was detected using excitation and emission wavelengths at 506 nm and 505 nm, respectively. Image analyses were performed using the IDL software (Research System Inc., Exelis Vis Inc., the US). The fluorescence intensity was measured using the Synergy2 microplate reader with the excitation at 485 ± 20 nm and emission at 528 ± 20 nm.

Western blot for calcium/calmodulin-dependent protein kinase II and ryanodine receptor 2

Endochylema and cellular proteins were extracted from H9c2 cells using a protein extraction kit according to the manufacturer's instructions. Equal amounts of proteins were loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and run at 100 V for 3 h. After electrophoresis, proteins were transferred onto PVDF membranes. The membranes were incubated overnight at 4°C with rabbit anti-CaMKII or anti-RyR2 polyclonal antibodies (1:1000 dilution) in Tris phosphate-buffered sodium (TBS-T) containing 5% skim milk. After washing three times with TBS-T, membranes were incubated with anti-rabbit IgG conjugated to HRP (1:10,000 dilution) in TBS-T containing 5% skim milk for 2 h at room temperature. The protein bands were visualized with enhanced chemiluminescence and captured on X-ray films. The bands were quantified with the Band Scan imaging analysis system.

Statistical analysis

The mean ± standard deviation values were calculated for all measurements acquired. One-way ANOVA and Duncan's multiple range tests were used to compare the differences among groups. A P < 0.05 was considered statistically significant.


  Results Top


Effects of ginsenoside Rb1 on creatine kinase and lactate dehydrogenase levels in the myocardial ischemia rat

To investigate whether ginsenoside Rb1 could reduce cardiomyocyte necrosis, the activities of serum CK and LDH (U/L) were measured at the end of the ischemic period. As shown in [Figure 2]a, in the IS group, serum levels of CK and LDH were markedly increased compared to those of the control group (P < 0.01). After pretreatment with ginsenoside Rb1, CK and LDH levels were significantly reduced compared to those in the IS group (P < 0.05). These results indicated that IS-induced myocardial injury and ginsenoside Rb1 protected against myocardial injury by preventing the production of CK and LDH.
Figure 2: Effects of ginsenoside Rb1 pretreatment on levels of creatine kinase, lactose dehydrogenase, superoxide dismutase, and malondialdehyde after 120 min of myocardial ischemia (IS). creatine kinase, lactate dehydrogenase, and malondialdehyde levels in the IS group were significantly increased (P < 0.05), whereas superoxide dismutase activity decreased, compared to the corresponding values in the control group. Treatment with 10 mg/kg ginsenoside Rb1 (Rb1) for 7 days before IS significantly decreased serum levels of lactate dehydrogenase and creatine kinase (a) as well as serum levels of malondialdehyde (b) (P < 0.05). Results are shown as means ± standard deviation (n = 10). *P < 0.05 versus sham group,#P < 0.05 versus IS group

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Effects of ginsenoside Rb1 on superoxide dismutase and malondialdehyde levels in myocardial ischemia rat

To examine whether ginsenoside Rb1 could alleviate myocardial oxidative stress, SOD and MDA levels were measured in the ischemia rat model. As shown in [Figure 2]b, MDA levels in the IS group markedly increased compared to the control group levels, whereas SOD activity decreased. After pretreatment with ginsenoside Rb1, MDA levels was significantly compared to those in the IS group (P < 0.05), whereas SOD activity was restored to normal (P > 0. 05).

Effects of ginsenoside Rb1 on hemodynamics in myocardial ischemia rat

To examine whether ginsenoside Rb1 could improve the myocardial systolic and diastolic function, mLVSP, mLVDP, and ± dp/dtmax were determined in the ischemia heart tissue. As shown in [Figure 3], 120 min following ischemia induction, mLVSP and ± dp/dtmax levels were significantly decreased in the IS group compared to those in the sham group (P < 0.01), whereas the mLVDP level in the IS group was markedly increased (P < 0.01). After pretreatment with ginsenoside Rb1, mLVSP (mmHg) increased significantly compared to that in the IS group after 30 and 60 min of ischemia, whereas mLVDP (mmHg) partially recovered (P < 0.05). The value of +dp/dtmax(% mmHg/s: the level of the treated group divided by the baseline level) increased significantly compared to that in the IS group after 0 and 120 min of ischemia, whereas −dp/dtmax(% mmHg/s) was partially recovered after 0 and 60 min of ischemia (P < 0.05). These results indicated that IS-induced heart injury, while ginsenoside Rb1 alleviated ischemic injury.
Figure 3: Effects of ginsenoside Rb1 pretreatment on hemodynamics during 120 min of myocardial ischemia. After 120 min of ischemia, mean left ventricular systolic pressure and ±dp/dtmax levels were significantly decreased in the IS group compared to those in the sham group (P < 0.01), whereas mean left ventricular systolic pressure was markedly increased (P < 0.01). Ginsenoside Rb1 pretreatment (10 mg/kg) 7 days prior to IS markedly increased mean left ventricular systolic pressure (a), +dp/dtmax (c), and -dp/dtmax (d), and decreased mean left ventricular diastolic pressure (b) at different time points of myocardial ischemia compared to the corresponding values in the IS group (P < 0.05). Results are shown as means ± standard deviation (n = 10). *P < 0.05 versus sham group,#P < 0.05 versus IS group

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Effects of ginsenoside Rb1 on arrhythmia incidence in myocardial ischemia rat

To investigate whether ginsenoside Rb1 could reduce the incidence of arrhythmias, the duration and frequency of ventricular tachycardia (VT), and the frequency of ventricular ectopic beats (VEB) were measured 10 min after ischemia was induced. As shown in [Figure 4]a, the frequency (per 10 min) of VT was markedly increased after ischemia was induced. After pretreatment with ginsenoside Rb1, the frequencies of VT and VEB were significantly reduced when compared to those in the IS group (P < 0.05), whereas the mean duration time of VT partially recovered (P > 0.05). These results indicated that VT was induced by MI injury, and ginsenoside Rb1 could alleviate these symptoms by decreasing the incidence of VT.
Figure 4: Effects of ginsenoside Rb1 pretreatment on ventricular tachycardia, ventricular ectopic beats, and infarct size expressed as the percent of the left ventricular area in rats subjected to 120 min of ischemia. The ventricular tachycardia frequency (per 10 min) was markedly increased after ischemia induction. Treatment with 10 mg/kg ginsenoside Rb1 7 days prior to IS markedly decreased the frequencies of ventricular tachycardia and ventricular ectopic beats (a), and decreased the infarct size (b), when compared to the corresponding values in the IS group (P < 0.05). Results are shown as means ± standard deviation (n = 10). *P < 0.05 versus sham group,#P < 0.05 versus IS group

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Effects of ginsenoside Rb1 on the infarct size in the myocardial ischemia rat

Infarct sizes were measured in IS rats with or without ginsenoside Rb1 pretreatment. As shown in [Figure 4]b, the infarct size was 15.0 ± 3.0% in the IS group. Pretreatment with ginsenoside Rb1 reduced the infarct size to 6.0 ± 1.0% (P < 0.05 vs. IS group). These results indicated that the myocardial infarct was induced by MI, and ginsenoside Rb1 could protect the ischemic myocardium by decreasing the infarct size.

Effects of ginsenoside Rb1 on calcium/calmodulin- dependent protein kinase II protein expression in the myocardial ischemia rat

To examine whether ginsenoside Rb1 could influence the protein expression of CaMKII in the MI rat, western blot analysis was performed. As shown in [Figure 5], with the development of ischemia, CaMKII expression was increased. After pretreatment with ginsenoside Rb1 (50 μmol/L), CaMKII expression decreased significantly compared to that in the IS group 2 h after ischemia induction. These results indicated that the increased CaMKII protein expression was induced by ischemia in rats, and ginsenoside Rb1 downregulated CaMKII expression (P < 0.05), suggesting that CaMKII may mediate the protective effects of ginsenoside Rb1.
Figure 5: (a) Effects of ginsenoside Rb1 pretreatment on calcium/ calmodulin-dependent protein kinase II expression at 2 h in the myocardial ischemia rat. calcium/calmodulin-dependent protein kinase II expression was increased in the ischemic rat. After pretreatment with ginsenoside Rb1 (50 μmol/L), calcium/calmodulin-dependent protein kinase II expression decreased significantly compared to that in the IS group. (b) Results are shown as means ± standard deviation (n = 3). *P < 0.05 versus sham group,#P < 0.05 versus IS group

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Effects of ginsenoside Rb1 on ryanodine receptor 2 protein expression in the myocardial ischemia rat

As shown in [Figure 6], RyR2 expression was minimally detected in the myocardial tissue of the sham group. Significantly higher RyR2 expression was observed in the IS group (P < 0.05, vs. Sham). The Rb1 group showed significantly lower RyR2 expression than the IS group (P < 0.05).
Figure 6: (a)Effects of ginsenoside Rb1 on the protein expression of ryanodine receptor 2. After 2 h of ischemia, ryanodine receptor 2 expression was increased. Following pretreatment with ginsenoside Rb1 (50 μmol/L), ryanodine receptor 2 expression decreased significantly compared to that of the IS group. (b)Results are shown as means ± standard deviation (n = 3). *P < 0.05 versus sham group,#P < 0.05 versus IS group

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Effects of ginsenoside Rb1 on the survival of H9C2 cells

Cell survival was determined in the H9C2 cells with or without ginsenoside Rb1 pretreatment. As shown in [Figure 7]a, hypoxia caused a decrease in cell survival. After pretreatment with different concentrations of ginsenoside Rb1 (12.5–200 μmol/L), the cell survival (percentage, the number of living cells in the treated group divided by that in the sham group) increased significantly compared to that in the IS group (P < 0.05). The effect of 100 and 200 μmol/L Rb1 was less prominent, and 12.5 μmol/L ginsenoside Rb1 caused adverse effects. These results indicated that cell death was caused by hypoxia, and ginsenoside Rb1 (50 μmol/L) could protect H9C2 cells from hypoxia. As shown in [Figure 7]b, both ginsenoside Rb1 (50 μmol/L) and the CaMKII inhibitor (KN-93, 1 μmol/L) increased cell survival compared to that in the IS group. Cotreatment (R11 + KN-93) with ginsenoside Rb1 and KN-93 exerted protective effects synergistically. These results indicated that ginsenoside Rb1 may increase cell survival by inhibiting CAMK II activity.
Figure 7: Effects of ginsenoside Rb1 on cell survival. (a) Effects of different ginsenoside Rb1 concentrations on the survival of H9C2 cells. Treatment with 50, 100, and 150 μmol/L ginsenoside Rb1 increased cell survival compared to that in the IS group, whereas 12.5 and 200 μmol/L ginsenoside Rb1 decreased cell survival. (b) After pretreatment with ginsenoside Rb1 (50 μmol/L) and the calcium/calmodulin-dependent protein kinase II inhibitor (KN-93, 1 μmol/L), cell survival increased significantly compared to that in the IS group. Treatment with both Rb1 and KN-93 (Rb1 + KN-93) markedly improved cell survival (P < 0.05). Results are shown as means ± standard deviation#P < 0.05 versus IS group

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Effects of ginsenoside Rb1 on intracellular calcium in H9C2 cells

To examine whether ginsenoside Rb1 decreases [Ca2+]I, calcium fluorescence intensities were measured in H9C2 cells. As shown in [Figure 8], hypoxia increased the calcium fluorescence intensity. After pretreatment with ginsenoside Rb1 (50 μmol/L), calcium fluorescence intensity decreased significantly compared to that of the IS group. The CAMK II inhibitor (KN-93) exerted a similar effect as Rb1. RB1 + KN-93 cotreatment demonstrated a considerably significant effect (P < 0.05). These results indicated that the calcium overload was induced by hypoxia in H9C2 cells and that ginsenoside Rb1 could decrease [Ca2+]I by inhibiting CAMK II activity.
Figure 8: (a) Effects of ginsenoside Rb1 on intracellular calcium in hypoxic H9C2 cells. Hypoxia increased the calcium fluorescence intensity. After pretreatment with ginsenoside Rb1 (50 μmol/L) or KN-93 (1 μmol/L), the calcium fluorescence intensities decreased significantly compared to those observed in the IS group. The effects of RB1 + KN-93 treatment were also significant (P < 0.05). (b) Results are shown as means ± standard deviation *P < 0.05 versus sham group,#P < 0.05 versus IS group.

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Effects of ginsenoside Rb1 on calcium/calmodulin- dependent protein kinase II and ryanodine receptor 2 expression in H9C2 cells

To examine whether ginsenoside Rb1 could influence the protein expression of CaMKII, p-CaMKII, and RyR2 in H9C2 cells, western blot analysis was performed. As shown in [Figure 9], hypoxia increased CaMKII and RyR2 expression. After pretreatment with ginsenoside Rb1 (50 μmol/L), CaMKII expression decreased significantly 2 h after hypoxia induction compared to that in the IS group. The CaMKII inhibitor (KN-93) demonstrated a similar effect. Treatment with both Rb1 and KN-93 (RB1 + KN-93) demonstrated a substantial marked effect (P < 0.05). After pretreatment with ginsenoside Rb1, RyR2 expression decreased significantly compared to that in the IS group 2 h after hypoxia induction. The CaMKII inhibitor (KN-93) exerted a similar effect. The RB1 + KN-93 treatment showed marked effects. These results indicated that the elevated protein expression of CaMKII and RyR2 was induced by hypoxia. Ginsenoside Rb1 could downregulate the expression of CaMKII and RyR2, at least partly, by inhibiting CAMKII expression, suggesting that CaMKII and RYR2 may mediate the protective effects of ginsenoside Rb1.
Figure 9: Effects of ginsenoside Rb1 pretreatment on (a) calcium/calmodulin-dependent protein kinase II and (b) ryanodine receptor 2 expression. After 2 h of hypoxia induction, calcium/calmodulin-dependent protein kinase II and ryanodine receptor 2 expression levels increased in H9C2 cells. After pretreatment with ginsenoside Rb1 (50 μmol/L) and the calcium/calmodulin-dependent protein kinase II inhibitor (KN-93), calcium/calmodulindependent protein kinase II expression decreased significantly compared to that in the IS group. RB1 + KN-93 treatment was also significant (P < 0.05). (c) Results are shown as means ± standard deviation (n = 3). *P < 0.05 versus sham group,#P < 0.05 versus IS group.

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  Discussion Top


I/R injury increases the level of Ca2+, reactive oxygen species, and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine openings in the mitochondria, resulting in the death of myocardial cells. Previously, studies have shown that acute MI can compromise myocardial systolic and diastolic functions,[35],[36] and overexpression of the cytoplasmic CaMKIIδC isoform in mouse hearts results in profound contractile dysfunction and heart failure.[28],[37] The excessive activation of CaMKII can cause the abnormal functioning of RyR2, which results in the leakage of Ca2+ from RyR2 on the SR to the cytoplasm, increasing [Ca2+]i in the cytoplasm.[38] Concurrently, the storage of Ca2+ in the SR declines during the rest period. The leakage of Ca2+ can cause a decline in Ca2+ release during the systolic period, affecting myocardial contractility and elevating cytoplasmic Ca2+ during the diastolic period, and resulting in diastolic dysfunction. In this study, we observed that the cardiac function was depressed and CaMKII expression increased after acute MI. mLVSP and ± dp/dtmax levels were significantly decreased, whereas mLVDP and CaMKII expression were markedly increased in the IS group, compared to the values in the sham group. After pretreatment with ginsenoside Rb1, mLVSP and + dp/dtmax increased significantly, whereas mLVDP and − dp/dtmax levels partially recovered compared to those in the IS group. These results indicated that cardiac injury was induced by IS, but ginsenoside Rb1 could alleviate this effect. The protective effect of ginsenoside Rb1 could be closely related to the reduced protein expression of CaMKII in the ischemic myocardium. By inhibiting the overexpression of CaMKII in the ischemic myocardium, ginsenoside Rb1 could decrease the calcium leakage from RyR2. The resulting increase in SR calcium storage leads to an increase in myocardial contractility in the ischemia rats, and a decrease in [Ca2+]i during the diastolic phase, thus improving the diastolic function of the ischemic myocardium.

Notably, CaMKII overexpression associated with arrhythmias[39],[40],[41],[42],[43] can disturb the balance of myocardial intracellular calcium homeostasis, which is an important pathological mechanism of ventricular arrhythmias.[14] Earlier studies have revealed that reperfusion arrhythmias may result from early-after depolarizations (EADs),[35],[44] and have demonstrated the association between EAD initiation, L-type Ca2+ current facilitation, and CaMKII activation.[39] CaMKII contributes to EADs upon reperfusion through the facilitation of the L-type Ca2+ current, which could favor the SR Ca2+ overload and appearance of depolarizing inward current (DAD).[39],[40] Moreover, recent reports have described a close association between spontaneous Ca2+-oscillations, DADs, and reperfusion arrhythmias,[41],[42] involving spontaneous diastolic Ca2+ release from cardiac RyR2 on the SR. CaMKII overexpression can alter RyR function, leading to more SR Ca2+ leakage and reduced SR Ca2+ content.[43],[44] Here, we observed that the most common arrhythmias were VT and VEB. Additionally, the increased CaMKII expression during this process is consistent with a previous report. After pretreatment with ginsenoside Rb1, the number of VT and VEB and the CaMKII expression were significantly reduced. These results indicated that arrhythmias could be induced by IS, and ginsenoside Rb1 could improve arrhythmias. The cardioprotective effect of ginsenoside Rb1 could be related to the lowered expression of CaMKII. Upon ginsenoside Rb1 treatment, the incidence of arrhythmias and sudden death after ischemia could be reduced. Thein vitro experiments demonstrated that ginsenoside Rb1 downregulated RyR2 expression in H9C2 cells.

Previously, findings in several species demonstrated that ischemia disrupted diastolic and systolic Ca2+, which might be associated with a series of changes in Ca2+ transients in the myocardium.[45],[46],[47] CaMKII contributes to the SR Ca2+ overload through the phosphorylation of RyR at both Ser2809 and Ser2815 sites[48],[49] which can increase its opening frequency and calcium release[12],[28] and facilitate the L-type Ca2+ current upon reperfusion.[39],[40] In this study, we observed that the intracellular calcium concentration increased, and cell survival decreased after acute hypoxia in the H9C2 cell line, which is consistent with a previous report. Our data showed that calcium fluorescence intensity increased, and cell survival decreased compared to the values observed in the sham group. After pretreatment with ginsenoside Rb1, calcium fluorescence intensity decreased, and cell survival increased significantly compared to those in the IS group. Ginsenoside Rb1 could improve cell survival and decrease intracellular calcium concentration by inhibiting CaMKII activity and RyR2 phosphorylation. This could decrease the opening frequency of the calcium channel, resulting in reduced calcium release. Furthermore, ourin vitro experiments demonstrated that ginsenoside Rb1 could directly downregulate RyR2 expression in the H9C2 cells. Further study is needed to investigate whether CaMKII also decreases the intracellular calcium concentration by utilizing other Ca2+ transport proteins.


  Conclusion Top


In summary, ginsenoside Rb1 pretreatment limited the MIS and decreased mLVDP, the incidence of arrhythmia, and levels of CK, LDH, and MDA, as well as the intracellular calcium concentration. In contrast, mLVSP, ±dp/dtmax, and cell survival were increased following pretreatment. These results suggested that the ginsenoside Rb1 pretreatment could alleviate the severity of heart injury induced by MI in rats. Furthermore, the results indicated that ginsenoside Rb1 was able to protect the ischemic heart, partly, by downregulating the protein expression of CaMKII.

Financial support and sponsorship

This study was supported by the National Natural Science Funds (81073134), 085First-Class Discipline Construction Innovation Science and Technology Support Project of Shanghai University of TCM (085ZY1206) and E-institutes of Shanghai Municipal Education Commission (No E 03008).

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]



 

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