Total Glycosides of Ranunculus Japonius Prevent Hypertrophy in Cardiomyocytes via Alleviating Chronic Ca2+Overload

Hong-liang Dai1?*, Gui-zhi Jia2?, and Song Zhao

1Department of Community Health Nursing,2Department of Biochemistry and Molecular Biology,3Centre of Scientific Experiment,

Liaoning Medical University, Jinzhou, Liaoning 121001, China

Total Glycosides of Ranunculus Japonius Prevent Hypertrophy in Cardiomyocytes via Alleviating Chronic Ca2+Overload

Hong-liang Dai1?*, Gui-zhi Jia2?, and Song Zhao3

1Department of Community Health Nursing,2Department of Biochemistry and Molecular Biology,3Centre of Scientific Experiment,

Liaoning Medical University, Jinzhou, Liaoning 121001, China

total Glycosides of Ranunculus Japonius; cardiac hypertrophy; isoproterenol; angiotensin Ⅱ; Ca2+; sacroplasmic/endoplasmic reticulum Ca2+ATPase 2a

ObjectiveTo evaluate the in vitro anti-hypertrophic effect of total Glycosides of Ranunculus Japonius (TGRJ).

MethodsNeonatal rat cardiomyocytes were cultured and hypertrophy was induced by administrating isoproterenol (ISO, 10 μmol/L) or angiotensin Ⅱ (Ang Ⅱ, 1 μmol/L) for 48 hours. In the treatment groups, cells were pretreated with TGRJ (0.3 g/L) for 30 minutes prior to hypertrophic stimuli. The anti-hypertrophic effects of TGRJ were examined by measuring cell size, total protein content, and protein synthesis. Intracellular free Ca2+concentration ([Ca2+]i) was evaluated using fluorescence dye Fura-2/AM. Sacroplasmic/endoplasmic reticulum Ca2+ATPase 2a (SERCA2a), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC) protein expression levels were measured by Western blotting. SERCA2a activity was assayed by p-nitrophenal phosphate disodium salt hexahydrate method.

ResultsIncreased cell size, total protein content, and protein synthesis following ISO or Ang Ⅱstimulation were significantly inhibited by pretreatment with TGRJ (all p＜0.05). This anti-hypertrophic effect of TGRJ was confirmed by its suppressing effect on elevated expression of the three hypertrophic related genetic markers, ANP, BNP, and β-MHC. In addition, TGRJ inhibited ISO or Ang Ⅱ induced up-regulation of [Ca2+]iunder chronic but not acute conditions. And ISO or Ang Ⅱ induced down-regulation of SERCA2a expression and activity was also effectively rectified by TGRJ pretreatment.

ConclusionsThe results of present study suggested that TGRJ could prevent ISO or Ang Ⅱ induced cardiac hypertrophy through improving chronic [Ca2+]idisorder, might via normalizing SERCA2a expression and activity.

Chin Med Sci J 2015; 30(1)：37-43

VENTRICULAR myocardium in response to mechanical1and neurohormonal stimuli like isoproterenol (ISO),2angiotensin Ⅱ (Ang Ⅱ),3endothelin-1,4etc. will undergo a hypertrophic growth characterized by an increment of cell size, increased protein synthesis, and re-expression of a series of fetal cardiac genes.5Initially, it may be adaptive to normalize wall stress and improve cardiac pump function, acting as a compensatory mechanism under a variety of pathological stress signals.6However, this abnormal growth may gradually become decompensated and strongly associated with increased risk of heart failure and other severe cardiovascular disorders.7Thus, anti-hypertrophic therapy has become one of the major strategies in the treatment of heart diseases.

Ranunculus Japonius Thunb is a herb widely growing in China and has been extensively used in Chinese folk medicine as an agent for the treatment of such disorders as migraine, jaundice, malaria, stomachache, arthralgia, and crane-like arthropathy, etc.8As one of the main ingredients of Ranunculus Japonius Thunb, total Glycosides of Ranunculus Japonius (TGRJ) have been reported to possess analgesic and anti-inflammatory bioactivity.9More recently, TGRJ was found to be capable of improving hemodynamics,10indicating that TGRJ might have potential cardioprotective effects. Therefore, we designed this study to investigate whether TGRJ could prevent ISO or Ang Ⅱ from inducing hypertrophy of cultured cardiac myocytes of neonatal rats, and if so, further explore its mechanism of action.

MATERIALS AND METHODS

Preparation of TGRJ

Ranunculus Japonicas Thunb was obtained from Jinzhou market, Liaoning Province, Northeast China and was authenticated at Pharmaceutical College, Liaoning Medical University. TGRJ was obtained as previously described.11Briefly, shade-dried and powered Ranunculus Japonicas was extracted thrice with 85% ethanol reflux. The solutions were then subject to filtration through an 80-mesh sieve with the filtrate pooled. Pooled extracts were concentrated and dried in vacuo to obtain an EtOH extract. This extract was resuspended in distilled water, and repeatedly partitioned in a 1：1 ratio with ethyl acetate until upper layer (ethyl acetate) exhibited light green. The water fraction was then pooled and absorbed onto a D-101 macroporous resin column and eluted initially with water and subsequently with ethanol. The ethanol fraction, after evaporated to dryness, constituted TGRJ used in our investigations. This dried extract was dissolved in normal saline for use in the pharmacological studies. Our preliminary study showed that in the concentration range of 0.1-0.3 g/L, TGRJ significantly inhibited ISO or Ang Ⅱinduced upregulation of cell protein content, but higher concentration (0.4 and 0.5 g/L) of this agent did not produce any better effects. Based on this, we chose 0.3 g/L TGRJ for the following study.

Cell culture and treatment

Monolayer cultures of neonatal rat cardiomyocytes were prepared according to our previously used method.12In brief, hearts from 1-to-3-day-old neonatal Sprague-Dawley rats were quickly excised and immediately placed into ice-cold PBS. After minced, the tissue was dispersed by 0.8 g/L trypsin at 37°C. Then isolated cardiomyocytes were cultured in DMEM supplemented with 15% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO2. 5-Bromodeoxyuridine (0.1 mmol/L) was added into the culture medium to prevent multiplication of nonmyocardiocytes. After cell confluence, the content of FBS was reduced to 0.4% in order to minimize the influence of serum on research results. The cell cultures were incubated in this low serum containing medium for another 24 hours before use.

Cultured cardiomyocytes were randomly divided into five group： control group treated with normal saline; ISO group incubated with ISO (10 μmol/L) for 48 hours; Ang Ⅱ group incubated with Ang Ⅱ (1 μmol/L) for 48 hours; TGRJ+ISO group that was treated by ISO for 48 hours in the presence of 0.3 g/L TGRJ; TGRJ+Ang Ⅱ group that was treated by Ang Ⅱfor 48 hours in the presence of 0.3 g/L TGRJ. In the experiments for evaluating acute effects of TGRJ on intracellular free Ca2+concentration ([Ca2+]i), TGRJ was added 1 minute before ISO or Ang Ⅱ stimulation for additional 4 minutes.

Measurement of cell size

At the end of the experiment, ventricular cardiomyocytes were treated with 0.1% trypsin at 37°C for 10 minutes and the process were stopped with 10% FBS. Afterwards, digested cells were visualized using an inverted microscope and cell volume was quantified based on measurement of cell diameters.

Quantification of total protein content

Cell protein content determination has been described previously.12Dishes were washed rapidly for three times with Hank’s solution at the endpoint and the cells were dissolved in 1% sodium dodecylsulphate (SDS), and the protein content was quantified according to the instructionof commercial kit (Solarbio Science & Technology Co., Ltd, Beijing, China).

Incorporation of [3H] leucine

The medium (DMEM, low glucose) was aspirated from myocardial cells grown on 24-well plates and replaced with 3.7×104Bq [3H]-leucine. Drugs were added and incubation was continued for 48 hours. The medium was then aspirated, and cells were washed rapidly for three times with cold Hank’s solution. Next, the cells were lysed by the addition of 1 ml 1% SDS per well. Lysates were collected, precipitated by the addition of 1 ml 5% trichloroacetic acid, and applied to fiberglass GF/C filters. After washing three times with 5 ml Hank’s solution, the filters were dried and transferred to vials containing 4 ml scintillation fluid, and their radioactivity was determined by liquid scintillation counting. The radioactivity, which represented the [3H]-leucine incorporated into newly synthesized protein, was expressed as cpm per well.

Western blotting

Cell protein was extracted from cultured neonatal rat cardiac myocytes, and protein content was determined by BCA protein assay method. Sample containing 50 μg protein was subjected to SDS-PAGE and eletrophoretically transferred to PVDF membrane. The sample was incubated overnight at 4°C with the primary antibodies specific to atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta-myosin heavy chain (β-MHC) (all in 1：1000 dilution, Santa Cruz Biotechnology Inc., CA, USA) and sacroplasmic/endoplasmic reticulum Ca2+ATPase 2a (SERCA2a) (1：500 dilution, Sigma-Aldrich). Subsequently, the membrane was incubated with secondary antibodies (1：3000 dilution, Santa Cruz Biotechnology Inc.) diluted in TBS-T (20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4 and 0.1% Tween-20) for 2 hours at room temperature. Finally, the membrane was rinsed with PBS and visualized using enhanced chemiluminescence reagents (Thermo Scientific, IL, USA). Western blot bands were quantified using QuantityOne software by measuring the band intensity for each group and normalizing to β-actin as an internal control.

Evaluation of acute and chronic effects of TGRJ on [Ca2+]i

Cultured neonatal rat cardiac myocytes were stained by Fura-2/AM (5 μmol/L, Sigma-Aldrich, MO, USA) for 30 minutes at 37°C. Any unincorporated dye was removed by washing twice with saline solution (in mmol/L： NaCl 137, KCl 5, MgCl20.5, CaCl21.3, KH2PO40.44, NaHCO34, MgSO40.8 with glucose 10). Fluorescent intensity of Fura-2/AM-loaded sample was recorded by an Olympus IX71 Live cell imaging fluorescence microscope (Tokyo, Japan) with an excitation wave at 340 and 380 nm and emission wave at 510 nm.

To evaluate the acute effect of TGRJ, cells were incubated with Fura-2/AM and baseline measurements were read for 1 minute. TGRJ was then added to cells and fluorescent intensity was measured for another 1 minute. Subsequently, ISO or Ang Ⅱ was then added to cells in the presence of TGRJ. Measurements were taken for 4 minutes. To evaluate chronic effects, cultured cardiomyocytes were stimulated by ISO or Ang Ⅱ for 48 hours in the presence of TGRJ, directly followed by measurements of fluorescent intensity for 5 minutes.

Measurement of SERCA2a activity

SERCA2a activity assay was performed entirely according to previously reported method.13Briefly, cardiomyocytes were homogenized and centrifuged at 4°C for 20 minutes. For each phosphatase reaction, 20 μl supernant was preincubated for 10 minutes at 37°C with 100 μl buffer solution (in mmol/L： MgCl21.5, EGTA 1.5, KCl 150, HEPES 20, 0.01% TritonX-100 either with or without CaCl26.5; pH 7.4), and the reaction was triggered by the addition of 30 μl 100 mmol/L p-nitrophenal phosphate disodium salt hexahydrate (p-NPP, Sigma, MO, USA). This reaction was quenched after 30 minutes by the addition of 2 ml terminative agent (500 mmol/L Tris and 55 mmol/L EDTA). The liberated p-nitrophenol (p-NP) was measured by spectrophotometer at 405 nm and calibrated by standard p-NP (Sigma). The supernatant protein concentration was determined by Coomassie Plus Protein Assay Reagent kit (Pierce, IL, USA). The SERCA2a activity in the control group was designated as a value of one.

Statistical analysis

Statistical analysis was performed using SPSS for windows, version 13.0 (SPSS Inc., IL, USA). The data were represented as mean±SEM. One-way analysis of variance (ANOVA) followed by Post Hoc Least Significance Difference (LSD) test was used to evaluate differences between groups. P＜0.05 was considered statistically significant.

RESULTS

Effect of TGRJ on cardiac hypertrophy

ISO or Ang Ⅱ treated cardiomyocytes showed appreciable hypertrophic changes as demonstrated by enhanced cell size, protein content as well as de novo protein synthesis compared with normal control. Intriguingly, pretreatment with TGRJ significantly brought down the elevated these levels (P＜0.05) as shown in Fig. 1.

In order to confirm the anti-hypertrophic action, further experiments were carried out to examine the expression profiles of several hypertrophic markers including ANP, BNP, and β-MHC. As expected, these three markers’ expressions were all up-regulated to varying degree in response to ISO or Ang Ⅱ stimulation (all P＜0.05). TGRJ pretreatment, however, effectively inhibited their abnormal expression (all P＜0.05) as shown in Fig. 2.

Figure 1.Effects of TGRJ on ISO or Ang Ⅱ induced cardiac hypertrophy.

Figure 2.Effects of TGRJ on the expression of hypertrophic markers ANP, BNP, and β-MHC in ISO or Ang Ⅱ induced in vitro cardiac hypertrophic model.

Acute effect of TGRJ on [Ca2+]i

As seen in Fig. 3, ISO and Ang II triggered a rapid increase of Fura-2/AM fluorescence value under acute conditions (P＜0.05). TGRJ pretreatment took no effects on ISO or Ang Ⅱ induced these changes. TGRJ itself also had no influence on acute [Ca2+]ichanges.

Chronic effect of TGRJ on [Ca2+]i

ISO and Ang Ⅱ triggered a significant increase of Fura-2/AM fluorescence value under chronic conditions (P＜0.05). As shown in Fig. 4, TGRJ pretreatment greatly alleviated ISO or Ang Ⅱ induced fluorescence changes (P＜0.05). TGRJ itself had no influence on chronic [Ca2+]ichanges.

Effect of TGRJ on the expression of SERCA2a

Since TGRJ affected [Ca2+]ionly under chronic conditions, we reckoned that certain genomic mechanism might be involved in TGRJ mediated [Ca2+]iregulation. As shown in Fig. 5, our data showed that under exposure to ISO or Ang Ⅱ for 48 hours, cultured cardiomyocytes exhibited markedly decreased expression of SERCA2a (P＜0.05). However, TGRJ pretreatment potently elevated SERCA2a expression (P＜0.05).

Figure 3.Acute effect of TGRJ on [Ca2+]iin cultured neonatal rat cardiomyocytes.

Effect of TGRJ on the activity of SERCA2a

To further confirm that SERCA2a is involved in TGRJ’s effect of chronic [Ca2+]iregulation, the relative activity of this protein was also determined. As shown in Fig. 6, the SERCA2a activity was reduced by 45% and 55% in cardiomyocytes exposed to ISO and Ang Ⅱ (all P＜0.05), respectively. Treatment with TGRJ significantly prevented this decrease in protein activity (all P＜0.05).

DISCUSSION

Ang Ⅱ and ISO are of well established pro-hypertrophic factors and commonly used as tools to induce cardiac hypertrophy models.14,15The characteristic hypertrophic changes of cardiomyocytes primarily include increased cell size, protein content, and expressions of the hypertrophicmarkers such as ANP, BNP, and β-MHC. We also found a consistent hypertrophic phenotype following Ang Ⅱ or ISO stimulation. More interestingly, we also found that TGRJ counteracted the hypertrophic effects induced by Ang Ⅱ and ISO, a novel hitherto unappreciated cardioprotective function of this natural herbal extract.

Figure 4.Chronic effect of TGRJ on [Ca2+]iin cultured neonatal rat cardiomyocytes.

Figure 5.Effects of TGRJ on the expression of SERCA2a.

TGRJ (0.3 g/L) significantly alleviated both ISO and Ang Ⅱ induced decrease of SERCA2a activity. Data were expressed as mean±SEM from four individual experiments.*P＜0.05 compared with the control group;#P＜0.05 compared with the ISO or Ang Ⅱ group.

Since TGRJ could indifferentially block ISO and Ang Ⅱinduced hypertrophy, the anti-hypertrophic mechanism of TGRJ might not be related to receptor inhibition. And we reckoned that TGRJ probably targeted a shared molecule(s) with ISO and Ang Ⅱ to modulate cardiac hypertrophy. Ca2+plays an essential role in the development of cardiac hypertrophy, including under ISO and Ang Ⅱ stimulation.16Morevore, TGRJ exhibited Ca2+-antagonizing effect in A7r5 cells in response to Ang Ⅱ stimulation, as demonstrated by a recent study.17All these facts prompted us to reckon that TGRJ’s anti-hypertrophic effect might originate from its Ca2+-antagonizing effect. As predicted, we herein reported that TGRJ was able to alleviate chronic [Ca2+]i overload in the context of ISO and Ang Ⅱ stimulation, although no effects on acute [Ca2+]iwere found.

We next attempted to investigate the molecular mechanisms underlying the inhibitory effects of TGRJ on chronic [Ca2+]ioverload. Intracellular sarcoplasmic/ endoplasmic reticulum (SR) Ca2+uptake using SERCA2a plays a critical role to remove Ca2+from the cytosol.18SERCA2a is a Ca2+-ATPase in SR and has been reported to play a key role in improving heart function in the failing heart.19,20We subsequently accordingly focused on the expression and activity of this protein. Our results showed cardiomyocytes under ISO and Ang Ⅱ stimulation were associated with decreased expression and activity of SERCA2a protein. However, pretreatment with TGRJ significantly prevented these decreases. Thus, our data support the concept that treatment of cardiomyocytes with TGRJ normalizes the level of SERCA2a protein, thus preserves its activity and thereby prevents [Ca2+]ioverload and subsequent cardiac hypertrophy. Undoubtedly, our current study further provided novel evidence of TGRJ in the aspects of cardiovascular protection.

In conclusion, it was apparent from the above results that TGRJ exerted anti-hypertrophic effects. These effects may be mediated by rectifying SERC2a expression and subsequent Ca2+overload. This finding will provide us the supportive information of using TGRJ as an alternative medicine in anti-hypertrophic therapy, although the effective ingredients representing the anti-hypertrophic activity of TGRJ still need to be clarified.

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for publication July 25, 2014.

?These authors contributed equally to this work.

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