Cardioprotective Role of Syzygium cumini Against Glucose-Induced Oxidative Stress in H9C2 Cardiac Myocytes

Neha Atale • Mainak Chakraborty • Sujata Mohanty •

Susinjan Bhattacharya • Darshika Nigam •

Manish Sharma • Vibha Rani

� Springer Science+Business Media New York 2013

Abstract Diabetic patients are known to have an inde-

pendent risk of cardiomyopathy. Hyperglycemia leads to

upregulation of reactive oxygen species (ROS) that may

contribute to diabetic cardiomyopathy. Thus, agents that

suppress glucose-induced intracellular ROS levels can have

therapeutic potential against diabetic cardiomyopathy.

Syzygium cumini is well known for its anti-diabetic

potential, but its cardioprotective properties have not been

evaluated yet. The aim of the present study is to analyze

cardioprotective properties of methanolic seed extract

(MSE) of S. cumini in diabetic in vitro conditions. ROS

scavenging activity of MSE was studied in glucose-stressed

H9C2 cardiac myoblasts after optimizing the safe dose of

glucose and MSE by 3-(4,5-dimethyl-thiazol-2-yl)-2,

5-diphenyl tetrazolium bromide. 20,70-dichlorfluorescein

diacetate staining and Fluorescence-activated cell sorting

analysis confirmed the suppression of ROS production by

MSE in glucose-induced cells. The intracellular NO and

H2O2 radical–scavenging activity of MSE was found to be

significantly high in glucose-induced cells. Exposure of

glucose-stressed H9C2 cells to MSE showed decline in the

activity of catalase and superoxide dismutase enzymes and

collagen content. 40,6-diamidino-2-phenylindole, propidi-

um iodide and 10-N-nonyl-3,6-bis (dimethylamino) acri-

dine staining revealed that MSE protects myocardial cells

from glucose-induced stress. Taken together, our findings

revealed that the well-known anti-diabetic S. cumini can

also protect the cardiac cells from glucose-induced stress.

Keywords Syzygium cumini � Glucose � Diabetic

cardiomyopathy � Cardiac hypertrophy � Reactive oxygen

species � Oxidative stress � Extracellular matrix remodeling

Introduction

Reactive oxygen species (ROS) such as superoxide anion

radical singlet oxygen, hydrogen peroxide and highly-

reactive hydroxyl radical are derived from the metabolism

of molecular oxygen and trapped by specific enzymes such

as catalase, superoxide dismutase, glutathione peroxidase

etc. to maintain balance with biochemical antioxidants in

all aerobic cells [1, 2]. Environmental stress increases the

levels of free radicals drastically, thereby disturbing the

equilibrium between free radical production and the anti-

oxidant capability causing oxidative stress because of

excess ROS, antioxidants depletion, or both [3]. Diabetic

cardiomyopathy, one of the major implications of increased

ROS, is the most vital cause of mortality and morbidity

among diabetic patients [4, 5]. The drugs often prescribed

for patients with diabetes have a limited ability to treat and

can create more problems in the long run in different

organs like eyes, kidneys, nerves and cardiovascular sys-

tem [6].

Natural and synthetic antioxidants have recently attracted

considerable attention due to its free radical–scavenging

activities that protects cells against oxidative stress–induced

N. Atale � M. Chakraborty � S. Mohanty � S. Bhattacharya �V. Rani (&)

Department of Biotechnology, Jaypee Institute of Information

Technology, A-10, Sector-62, Noida 210307, Uttar Pradesh,

India

e-mail: vibha.rani@jiit.ac.in

D. Nigam

Department of Biochemistry, School of Life Sciences,

Dr. B. R. Ambedkar University, Agra 282004, India

M. Sharma

Peptide and Proteomics Division, DIPAS, DRDO,

New Delhi 110054, India

123

Cardiovasc Toxicol

DOI 10.1007/s12012-013-9207-1

death in vivo and in vitro [7]. Several studies have reported

beneficial effects of a therapy with the antioxidant agents

including trace elements and dietary antioxidants against

the cardiovascular abnormalities inherent in diabetes [8].

Curcumin, vitamin E, beta carotene, catechins, gallic acid,

zinc, selenium, trolox, quercetin, etc. have potential health

benefits [9, 10]. However, clinical trials examining the

therapeutic efficacy of antioxidants have yielded conflict-

ing results. This could be owing to their poor bioavail-

ability, toxicity and gastric breakdown. Also, the

mechanism of action of these synthetic antioxidants has not

been fully understood [11]. Use of crude food extracts

rather than isolated compounds could be an approach to

increase the efficacy of the therapy. The presence of other

phytochemicals such as flavnoids and lycopenes provide

other beneficial effects as well. Therefore, study with plant

extracts as natural antioxidants offer a great hope for the

prevention of chronic human diseases and can enhance the

long-term health of a diabetic person [12]. Hence, the

present study was designed to investigate the cardiopro-

tective potential of a well-known Indian medicinal plant,

Syzygium cumini, against high glucose-induced stress in

cardiac H9C2 cells. The plant S. cumini was selected for its

antioxidative potential to scavenge free radicals and reduce

blood glucose levels in diabetes [13–15].

Syzygium cumini (commonly known as Jamun, Jambul,

Jambula) is a well-known Indian medicinal plant tested for

many therapeutic properties and is used as a viable treat-

ment for diabetes [16]. The plant belongs to the Myrtaceae

family and is native to the south Asian continent [17]. The

plant possesses acetyl oleanolic acid, triterpenoids, ellagic

acid, isoquercitin, quercetin, kaempferol and myricetin

[18]. S. cumini plant (leaves, stem, bark and fruit pulp) has

been evaluated extensively for antimicrobial, anti-diabetic,

anti-inflammatory, hepatoprotective, antihyperlipidemic

and diuretic properties [19]. The therapeutic properties of

S. cumini have been medically valued since ancient system

of medications like ayurveda and unani systems. According

to Ayurveda, its acrid and sweet juice is digestive and

astringent to the bowels, antihelmintic and prevents thirst,

sore throat, bronchitis, dysentery and ulcers. The seeds are

also suggested for use as an alternative natural healing

system in the Ayurvedic, Unani and Chinese medicines.

The seeds and bark are used in the Far East for the treat-

ment of dysentery and in control of hyperglycemia and

glycosuria in diabetic patients. Additionally, the ash of

burnt leaves is curative for gum and teeth [20, 21].

In our previous study, the phytochemical screening of

aqueous, ethanolic, methanolic, chloroform, n-hexane,

benzene and di-ethyl ether seed extracts of S. cumini was

performed among which aqueous, ethanolic and methanolic

extracts were found to be rich in phytocontents. However, it

was observed that methanolic seed extract contained the

highest amount of phytochemicals and reducing power

potential as compared to the aqueous and ethanolic seed

extracts [22]. Methanolic extract of S. cumini seeds have

been reported for its anti-arthritic and immunomodulatory

activities, but its cardioprotective effect in hyperglycemic

conditions is not investigated till date [23]. Therefore, in the

present study, we limited our research experiments to MSE,

and evaluated the cardio-protective potential of MSE in

hyperglycemic conditions in H9C2 cardiac muscle cells.

Materials and Methods

Chemicals

Methanol, hydrogen peroxide, phosphate buffered saline

(PBS), glacial acetic acid, phenazine methosulfate, nico-

tinamide adenine dinucleotide, 20,70-dichlorfluorescein

diacetate (DCFH-DA), 4-(2-hydroxyethyl)-1-piper-

azineethanesulfonic acid (HEPES), sodium chloride, glyc-

erol, sodium nitroprusside, griess reagent, Dulbecco’s

modified eagle’s medium (DMEM), dimethyl sulfoxide,

fetal bovine serum (FBS), trypsin–EDTA, insulin-trans-

ferrin-selenium supplement (ITS), ethylene diamine tetra

acetic Acid (EDTA), TRITON-X 100, protease inhibitor

cocktail, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetra-

zolium bromide (MTT), 40,6-diamidino-2-phenylindole

(DAPI), collagen, Picrosirius red, propidium iodide (PI),

10-N-nonyl-3,6-bis (dimethylamino) acridine (NAO),

giemsa, nitroblue tetrazolium bromide (NBT), riboflavin,

methionine, catalase (CAT) and superoxide dismutase

(SOD) were purchased from Sigma–Aldrich, USA.

Seed Collection

Seeds were collected from Noida, Uttar Pradesh, India, in

the month of June and were identified and authenticated by

Dr. Anshu Rani, Department of Botany, Govt. P.G. College,

Abu Road, Rajasthan, India. Seeds were washed with water

thoroughly and air dried. The dried seeds were ground and

the powder was collected for the preparation of methanolic

seed extract (MSE).

Preparation of Methanolic Extract of S. cumini Seeds

Methanolic extract of S. cumini seeds was prepared by

using Soxhlet extraction method. A total of 20 g of seed

powder was mixed with the 200 ml of methanol. The

temperature was set at its boiling point and 12–14 cycles

were run for complete extraction. The rotary vacuum

concentrator was used for further drying the extract and the

dried mass of methanolic extract was weighed and pre-

pared at a concentration of 1 mg/ml.

Cardiovasc Toxicol

123

Cell Culture

H9C2 was used as a model system in this study as it has

been earlier described to show similar responses as shown

by primary rat cardiac myocytes. Heart-derived H9C2

cardiomyoblast cells were obtained from the National

Centre for Cell Science (NCCS), Pune, India. H9C2 cells

were cultured with Dulbecco’s modified Eagle’s Medium

(DMEM) supplemented with penicillin, streptomycin,

gentamycin, amphotericin B, glucose, L-glutamine, sodium

bi-carbonate and 10 % fetal bovine serum (FBS) in

humidified CO2 incubator (New brunswick) with 5 % CO2

at 37 �C [24]. Cells were routinely subcultured at a split

ratio of 1:3. Glucose was used for induction of stress

[25, 26].

In Vitro Cytotoxicity for Glucose and Methanolic Seed

Extract

Cell viability and proliferation was measured by 3-(4,

5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT) which is reduced to purple formazan by the action

of cellular enzymes present in the cytosol of living cells

[27]. For MTT Assay, 8000 cells were seeded in 96-well

plates. After treatment with glucose and MSE at different

concentrations, 10 ll MTT solution (5 mg/ml) was added

and incubated at 25 �C for 3 h. Supernatant was then

aspirated and formazan salt crystals were dissolved in

200 ll dimethylsulfoxide (DMSO). Samples were analyzed

in an ELISA plate reader (Biorad) at 570 nm. Cell viability

is defined relative to untreated control cells as follows: cell

viability = absorbance of treated sample/absorbance of

control. The cells were then treated with the optimized

doses and cultured in serum-free DMEM supplemented

with ITS.

Treatment of Cells with Glucose and MSE

Optimal concentrations of Glucose and MSE were calcu-

lated by MTT assay as above. H9C2 cells were cultured in

serum-free DMEM supplemented with ITS (insulin 50 mg/

ml, transferrin 27.5 mg/ml and selenium 0.025 mg/ml) and

treated with optimal concentration of glucose. In order to

see the effect of MSE, it was added in an optimal con-

centration under similar culture conditions simultaneously

along with Glucose in the culture media. These treatments

were carried out for 48 h along with an untreated control.

Morphological Analysis

Cells were visualized under inverted microscope for mor-

phological changes at 40X magnification [28]. Cells were

treated in four experimental sets- untreated control, glucose

induced (GI), MSE treated glucose induced (MSE ? GI)

and treated with MSE alone (MSE).

Giemsa Staining

Giemsa is a polychromatic stain used to observe cell

morphology. It stains nucleus pink or magenta, the nucleoli

dark blue and cytoplasm grayish blue [29]. For the deter-

mination of modulation in cell morphology, H9C2 cells

were seeded onto six-well plates and cultured overnight in

complete media. Cells were treated the next day as

explained earlier. The four experimental sets were

observed after 48 h of incubation after induction. There-

after, cells were washed with 1X PBS and fixed with

100 % cold methanol, and 5 % giemsa solution was added

to each well and incubated for 15 min at 25 �C to observe

the morphological changes as described earlier. Images

were captured under the inverted microscope at 40X

magnification. Cell size was also quantified by image J

software.

Evaluation of Oxidative Stress by DCFH-DA Staining

and FACS for ROS

For the evaluation of oxidative stress and reactive oxygen

species formation, 20, 70-dichlorofluorescin diacetate

(DCFH-DA) was added to cultures, since DCFH-DA enters

cells and the acetate group on DCFH-DA is cleaved by

cellular esterases, trapping the nonfluorescent DCFH inside

the cells. Subsequent oxidation by ROS, particularly H2O2

and hydroxyl radical, yields an increase in the fluorescent

product DCF, which is suggestive of H2O2 or hydroxyl

generation [30]. To study the oxidative stress, cells were

seeded onto glass cover slips and grown overnight in a six-

well plate. They were treated the next day in three exper-

imental sets- control, glucose-induced and MSE-treated

glucose-induced cells. After 48 h of treatment, the cells

were PBS washed and fixed in methanol. They were then

incubated with DCFH-DA dye at a concentration of 3 lg/

ml in PBS buffer and observed after 15 min under fluo-

rescent microscope (Olympus) at 40X magnification. For

the determination of ROS by flow cytometry, the mea-

surements were carried out using FACS Calibur flow

cytometer (BD biosciences, USA). Increase in oxidation of

DCFH to DCF was used a marker to demonstrate ROS

overproduction. After treatment under different experi-

mental conditions and methanol fixation, DCFH-DA dye

was added in a ratio of 1: 1000 in PBS buffer in each well.

It was then incubated for 10 min, and the fluorescence was

measured using the excitation at 480 nm and an emission

wavelength of 530 nm.

Cardiovasc Toxicol

123

Picrosirius Red staining for Estimation of Collagen

Content

The Sirius red stain is a dye that binds to the (Gly-x–y)

triple-helix structure found in all collagen fibers. This

property of Sirius red stain was utilized to assess collagen

in cardiac cells under bright field. Cells were treated in

three experimental sets as described. Followed by PBS

wash and methanol fixation, cells were treated with 0.1 %

Sirius Red F3BA in saturated picric acid (w/v) and stained

for 1 h at room temperature. The collagen-bound stain was

further eluted with 0.1 N NaOH for 5 min. The absorbance

of eluted stain was recorded at 540 nm in a microplate

reader (Bio-Rad Labs). A standard curve was plotted as

quantity of collagen versus absorbance, and the collagen

content of samples was estimated [31].

Extraction of Total Cell Protein

The cell pellet was washed with ice-cold 1X PBS and lysed

in protein extraction buffer (20 mM HEPES, 20 % glyc-

erol, 500 mM NaCl, 0.2 mM EDTA, 0.1 % TRITON-X

and protease inhibitor cocktail) at 4 �C for 1 h. Total

protein was obtained after centrifugation at 13,000 rpm for

15 min in a refrigerated centrifuge. The supernatant was

transferred to a pre-chilled microfuge tube and stored at

-80 �C. The total protein obtained was estimated using

Bradford assay [32].

NO Scavenging Assay

At physiological pH, nitric oxide generated from aqueous

sodium nitroprusside (SNP) solution interacts with oxygen

to produce nitrite ions, which may be quantified by the

Griess Illosvoy reaction. The 30 lg/ml of total cell protein

extracted from control, glucose-induced and glucose-

induced ? MSE-treated cells was taken for the experi-

ment. Greiss reagent with sodium nitroprusside solution

was taken as control [33]. Greiss reagent and total cell

protein were taken in 1:1 ratio and the absorbance was

measured at 540 nm. The NO content was calculated in all

three sets using the following equation-

% Inhibition ¼ A0 � A1

A1

� 100

Where A0 is the absorbance of control reaction (without

sample) and A1 is the absorbance of sample/standard after

reaction has taken place.

H2O2 Scavenging Assay

The assay is based on quantification of the degradation

product of 2-deoxyribose by condensation with TBA.

Hydroxyl radical was generated by the Fe3? -ascorbate-

EDTA-H2O2 system (the Fenton reaction). H2O2 scav-

enging assay was performed in all the three experimental

sets. A total of 4 mM H2O2 solution (prepared in phosphate

buffer, pH—7.4) was taken as blank [34]. Equal amount of

H2O2 solution and 30 lg/ml of total cell protein were taken

and absorbance was measured at 230 nm. The H2O2 level

was calculated by the above-described equation.

Catalase Assay (CAT)

Catalase assay activity was analyzed by the method of Aebi

[35]. The assay is based on the reaction of the catalase

enzyme with H2O2. The absorbance of hydrogen peroxide

at 240 nm is measured directly to calculate the reaction

rate. Assay reaction mixture consisted of 100 mM potas-

sium phosphate buffer pH 7.0, 20 mM H2O2 and 30 lg/ml

of total cell protein from each of the experimental sets in a

total volume of 1 ml. Phosphate buffer and enzyme

extracts were added and the reaction was initiated by

adding H2O2. Continuous decrease of absorbance was

recorded till 2 min and activity was measured as U/mg of

protein.

Superoxide Dismutase Assay (SOD)

Superoxide Dismutase Assay activity was determined in

H9C2 cells using the method of Beauchamp and Fridovich

[36]. The superoxide anions reduce a tetrazolium salt,

NBT to a colored formazan product that absorbs light. SOD

scavenges superoxide anions, thereby reducing the rate of

formazan dye formation. The reaction mixture consisted of

100 mM potassium phosphate buffer pH 7.8, 0.01 lM

EDTA pH 8.0, 65 mM L-methionine, 750 lM NBT, 2 mM

riboflavin and 30 lg/ml of total cell protein from each of

the experimental sets. The samples were incubated for

30 min in light. The reaction was terminated by turning off

the light and the absorbance was measured at 560 nm. The

activity was expressed as U/mg of protein.

Nuclear Morphological Analysis

DAPI Staining

40, 6-diamidino-2-phenylindole (DAPI) is a blue fluores-

cent dye that preferentially stains dsDNA by strongly

binding to A–T rich regions. Binding of this stain produces

an enhanced florescence due to the displacement of water

molecules from both DAPI and the minor groove of the

DNA.

Cells were induced followed by methanol fixation.

Membrane permeable fluorescent dye, DAPI (50 ng/ml),

was added and incubated for 15 min at 25 �C. Cells were

Cardiovasc Toxicol

123

then observed under fluorescent microscope using the

DAPI filter at 40X magnification [37].

PI Staining

Propidium iodide is a red-fluorescence dye with excitation/

emission maxima *535/617 nm with bound DNA and is

permeant only to dead cells. PI is impermeable to cells with

an intact plasma membrane; hence, when the cell integrity

becomes compromised, it gains access to the nucleus where

it complexes with DNA rendering the nucleus highly

fluorescent. Cells were induced and fixed as described

above. The cells were stained with PI (2.5 ng/ll) solution

for 15 min in dark at 25 �C [38]. Slides were observed

under fluorescent microscope using the TRITC filter.

Images were captured at 40X magnification.

Mitochondrial Structure Analysis by NAO Staining

Nonyl acridine orange (NAO), a probe that stains mitochon-

dria independently of their energetic state, is an indicator of

mitochondrial mass. NAO interacts stoichiometrically with

intact, nonoxidized cardiolipin. For NAO staining, Cells were

induced and fixed by the above-described procedure. Nonyl

acridine orange (15 lM) solution was added to each well in

dark and incubated for 15 min at 25 �C. Images were

observed under fluorescence microscope using the FITC filter

after excitation at 490 nm and captured at 40X magnifi-

cation [39].

Measurement of Mitochondrial Membrane Potential

JC-1 dye exhibits potential dependent accumulation in

mitochondria and thus was employed to detect the change

in mitochondrial membrane potential. It aggregates at high

potential and can be excited at 488 nm. The emission shifts

from green (525 nm) to red (590 nm) when JC-1 aggre-

gates form [40]. H9C2 cells were cultured on coverslips in

three experimental sets, fixed with 3 % paraformaldehyde

in PBS. JC-1 dye was added to culture medium at 10 lg/ml

and incubated for 15 min at 37 �C. The medium was then

removed and washed three times with PBS. After being

mounted on slide, the cells were examined under fluores-

cent microscope at 409 magnification.

Statistical Analysis

For quantitative analysis, all data in triplicates were

expressed as mean ± SD. The significance of differences

in the data was evaluated by one-sided ANOVA. A value

of P \ 0.05 was considered statistically as significant and

the null hypothesis was rejected in that case.

Results

The viability of H9C2 cardiomyoblasts treated with dif-

ferent concentrations of MSE and glucose showed that the

concentration of 25 mM glucose and 9 lg/ml MSE repre-

sented significant cell viability after which viability of cells

reduced below 95 %. These optimized doses were further

used for induction and treatment of the cardiac cells

(Fig. 1a). Simultaneous treatment of glucose and MSE at

these concentrations did not reduce the cell viability below

95 %.

The study was initially conducted with four sets in

triplicates, (1) control cells, where H9C2 cells were cul-

tured without any treatment, (2) H9C2 cardiac myoblast

cells induced with 25 mM glucose alone (glucose-stressed

cells), (3) Glucose-stressed cells treated with 9 lg/ml

methanolic seed extract (MSE) and (4) cells treated with

9 lg/ml MSE alone. Microscopic analysis of the cells was

done with three different fields from the above sets,

respectively. After 48 h of treatment, the effect of MSE

significantly reversed the increase in cardiac cell size

induced by high glucose concentration comparable to the

control cells (Fig. 1b). This was further confirmed by

giemsa staining which showed the morphological changes

leading to hypertrophy in glucose-induced cells, while

MSE in combination with glucose-induced cells showed

protective effect of S. cumini. In addition, cells treated with

only MSE showed no adverse effect on the viability or

morphology of cells (Fig. 2 a, b). Therefore, we limited our

subsequent study to three experimental sets- control, glu-

cose-induced and glucose-induced ? MSE-treated H9C2

cardiac myoblast cells.

To further observe the effect of high glucose on intra-

cellular ROS overproduction, DCFH-DA assay was per-

formed. The formation of intracellular ROS was increased

in glucose-induced H9C2 cells as compared to the control

which decreased significantly in MSE-treated cells as

shown in Fig. 3a. This establishes the fact that MSE has the

ability to suppress the free radical upregulation under

hyperglycemic condition in cardiac cells. To further vali-

date the effect, we performed DCFH-DA Fluorescence-

activated cell sorting (FACS) to assess intracellular ROS

generation in all three experimental sets. This gave us

similar results where glucose-induced cells showed sig-

nificant increase in the DCFH-DA fluorescence as com-

pared to the control cells, and MSE treatment reduced the

fluorescence intensity (Fig. 3b).

We further tested the intracellular NO and H2O2 radical

scavenging ability of MSE in glucose-induced cardiac cells

with an equal amount of total cell protein (30 lg/ml). The

NO scavenging activity for control cells, glucose-induced

cells and MSE-treated glucose-induced cells were found to

be 94.67 % ± 1.49, 30.64 % ± 1.72 and 73.54 % ± 1.76,

Cardiovasc Toxicol

123

respectively. Mean scavenging activity from triplicates was

evaluated for each set of cells. The inhibition was lesser in

glucose-induced cells in comparison with control cells, and

scavenging increased after treatment with MSE under the

stressed condition. The H2O2 scavenging activity also shows

us similar results with 91 % ± 1.57, 33.22 % ± 1.98 and

Fig. 1 Cell viability assay and morphological analysis of H9C2 cells.

(a) Dose optimization for Glucose and MSE based on the formazan

product formation using MTT assay as described in methods. Cells

incubated with different doses of glucose and MSE for 48 h are

represented. (b) H9C2 cells were cultured for 48 h with/without

glucose in different experimental sets (1) untreated control, (2) glucose

induced (GI): induced with 25 mM glucose, (3) GI ? MSE: treated

with glucose (25 mM) and MSE (9 lg/ml) and (4) treated with MSE

(9 lg/ml) alone. Arrows indicate glucose-induced cells undergoing

stress (increase in cell size) which is observed to be reduced upon

treatment with MSE. Images were captured at 40X magnification

Fig. 2 Giemsa staining for H9C2 cells. (a) H9C2 cells were cultured

for 48 h in different experimental sets as described in Fig. 1. Staining

showed similar results to the conventional light-field microscopy

where glucose-induced cells exhibit stress while MSE reverses it as

represented by arrows. (b) The results were quantified by image J

software from images taken in different fields at 40X magnification.

Each value represents the mean ± SEM of triplicates, (*P B 0.05)

Cardiovasc Toxicol

123

65.48 % ± 1.74 inhibition in control cells, glucose-induced

cells and MSE-treated glucose-induced cells, respectively

(Fig. 4a).

We further measured the effect of MSE on the activity of

two biologically important antioxidative enzymes CAT and

SOD in previously mentioned experimental sets. The CAT

Fig. 3 Effect of MSE on the release of ROS in H9C2 cardiac

myoblasts. (a) DCFH-DA assay for assessment of intracellular ROS

in (1) control, (2) GI and (3) GI ? MSE-treated cells. Images were

captured at 40X magnification. Arrows increased DCFH-DA

fluorescence in glucose-induced cells and suppression of fluorescence

in GI ? MSE-treated cells. (b) Flow cytometery analysis for DCFH-

DA relative intensity. The red signal represents the ROS overpro-

duction in GI cells which is reduced on treatment with MSE

Fig. 4 Antioxidative assays in

H9C2 cells. (a) Free radical

scavenging assays: Comparison

of NO and H2O2 content from

control, GI and GI ? MSE-

treated cells. The amount of NO

and H2O2 was increased in

H9C2 cells induced with

glucose H9C2 and declined on

MSE treatment.

(b) Antioxidative enzyme

assays: Catalase (CAT) and

Superoxide dismutase (SOD)

activity in control, glucose-

induced (GI) and glucose-

induced MSE-treated cells

(GI ? MSE). Values indicated

as U/mg protein extract for CAT

and SOD activity. (*P B 0.05)

Cardiovasc Toxicol

123

activity was found to be 176 ± 1.29 U/mg of protein extract

for control cells, 272 ± 1.68 U/mg of protein extract for

glucose-induced cells and 139 ± 1.78 U/mg of protein

extract for MSE-treated glucose-induced cells, showing a

significant increase in glucose-induced cells. Treatment with

MSE controlled the increased CAT activity in stressed cells.

Super oxide Dismutase (SOD) activity was also determined

for control, glucose-induced and MSE-treated glucose-

induced cells which were 1,249 ± 1.89, 1,353 ± 1.29 and

1,239 ± 1.97 U/mg of protein extract respectively, further

establishing antioxidative potential of MSE in hyperglyce-

mia-induced cardiac stress (Fig. 4b).

Qualitative analysis of cells stained with Picrosirius col-

lagen stain indicated enhanced collagen content in cells trea-

ted with 25 mM glucose as compared to the control, whereas

MSE treatment reduced the collagen content as comparable to

control cells (Fig. 5a). The eluted yield of collagen was found

to be 11.45 ± 0.59 lg/ml in control, 24.25 ± 0.88 lg/ml in

glucose-induced cells and 14.89 ± 0.45 lg/ml in glucose-

induced ? MSE-treated cells further validating our qualita-

tive interpretation (Fig. 5b).

To further observe the effect of glucose-induced ROS

damage on nucleus, DAPI and PI staining were performed

after 48 h of treatment. The glucose-induced cells showed

changes in nuclear morphology than MSE-treated glucose-

induced cells. Glucose-stressed cells (25 mM concentra-

tion) showed a hypertrophic response such as increase in

nuclear size, whereas glucose-induced cells treated with

MSE showed reversal of this hypertrophy almost compa-

rable to control (Fig. 6a, b). However, higher doses of

glucose (100 mM) transits the cell from hypertrophy to

apoptosis leading to cell death (data not shown). It could be

hypothesized that the altered nuclear morphology is due to

a combined effect of increase in over all cell size and ROS

level in the cell.

Further, experiments were done to study the role of

glucose on mitochondrial content of cells by staining cells

with NAO. With mitochondrial damage or loss of mem-

brane potential, the dye cannot accumulate in the mito-

chondria, which is indicated by a lack of orange

fluorescence. The cells when induced with 25 mM glucose

showed increase in their organelle size and intense green

stain, whereas the 25 mM glucose-induced cells treated

with MSE showed on par organelle size to that of control

cells and greenish orange–stained areas under 40X mag-

nification, suggesting the role of effect of glucose-induced

ROS in mitochondrial dysfunction in cardiac cells, and

MSE has the potential to reverse the ROS-mediated mito-

chondrial stress (Fig. 7).

Alterations in mitochondrial membrane potential were

detected using JC-1 dye. Untreated control cells stained

with fluorescent dye JC-1 exhibited numerous brightly

stained mitochondria that emits reddish orange fluores-

cence, representing JC-1 aggregates that accumulates at

normally hyperpolarized membrane potential. Cells after

glucose treatment exhibited green JC-1 monomers, indi-

cating gradual dissipation of mitochondrial membrane

potential. While treatment of MSE showed orange aggre-

gates that elicits its protective role on mitochondria

(Fig. 8).

Fig. 5 Analysis of collagen

content in H9C2 cells by

Picrosirius staining.

(a) Comparison of collagen

content in control, glucose-

induced (GI) and glucose-

induced MSE-treated

(GI ? MSE) H9C2 cells by

light microscopy at 40X

magnification. (b) Absorbance

of the eluted stain from different

samples was used to calculate

the collagen concentration from

standard curve and was plottedas a histogram (*P \ 0.05)

Cardiovasc Toxicol

123

Fig. 6 Flourescence

micrographs (40X

magnification) of stained H9C2

nuclei under different

experimental conditions to

observe nuclear alterations by

(a) DAPI staining (b) PI

staining. Representative images

showed the altered nuclear

morphology in glucose-induced

cells while MSE treatment

exerts a protective effect as

indicated by arrows

Fig. 7 H9C2 Mitochondrial NAO staining. H9C2 cells were stained by NAO in above-mentioned experimental sets. Lack of orange florescenceindicates the mitochondrial deformities in glucose-induced cells.—All the images were captured at 40X optical magnification (*P \ 0.05)

Fig. 8 Localization of JC-1 in H9C2 cells by fluorescence micros-

copy. Reddish orange fluorescence observed in untreated control cells

and MSE-treated glucose-induced cells indicates formation of

J-aggregates while green fluorescence in glucose-induced cells

indicates formation of monomers leading to loss of mitochondrial

membrane potential. All the images were captured at 40X optical

magnification (*P \ 0.05)

Cardiovasc Toxicol

123

Discussion

The cellular and molecular mechanisms of glucose-induced

stress as well as the effect of MSE on glucose-induced

cardiomyocytes has not been well documented. The result

obtained from this study demonstrates that H9C2 cardiac

cell line undergoes stress upon exposure to high glucose,

and S. cumini can protect cardiac myocytes from glucose-

induced stress.

Diabetes ranks among the risk factor for the develop-

ment of heart failure and death [35, 36]. Diabetic cardio-

myopathy, a diabetic specific complication, refers to a

disorder that eventually leads to left ventricular hypertro-

phy and is related directly to hyperglycemia. In recent

studies, hyperglycemia-induced ROS in cardiomyocytes

has been linked to diabetic cardiomyopathy [37]. Reactive

oxygen species generation has been detected in cells

exposed to high glucose concentration, and ROS-induced

cell death play a critical role in development of cardiac

complications. Thus, hyperglycemia seems to be linked to

cell death in cardiac myocytes [38].

Cardiac myocytes are terminally differentiated cell types

which do not divide further once they have achieved adult

phenotype [39]. However, under various stress such as high

glucose, cardiac myocytes increase in size and undergo

hypertrophy but prolonged hypertrophy leads to death of

cardiac cells and ultimately heart failure [40]. Increase in

cell size upon glucose stress is evident from microscopic

analysis of the cells in our study. We observed the increase in

size of the cardiac myocytes in hyperglycemic conditions.

Giemsa staining was also applied to observe the morpho-

logical changes of H9C2 cells under hyperglycemic stress.

Recently, it has been found that H9C2 cells show almost

identical stress responses to those observed in primary

cardiomyocytes and thus can be used as a model for in vitro

studies for cardiac stress [41]. Cellular levels may fluctuate

according to balance between ROS production and antiox-

idant regeneration [42]. In situations of high glucose, cel-

lular levels of ROS overwhelms the antioxidant capability of

cellular components, such as DNA, lipids and proteins [43].

Antioxidant enzymes (CAT, SOD) impairment contributes

significantly the impairment of ROS that leads to cardiac

myocyte remodeling and failure [44]. Concentrations of

glucose approaching 10 mM are pre-diabetic levels and

glucose concentrations above 10 mM are analogous to a

diabetic condition within the cell culture system [45]. This is

important because the same processes that can affect cells

and molecules in vivo can occur in vitro. The consequence to

growing cells under conditions that are essentially diabetic is

that cells and cell products are modified by the processes of

glycation and glyoxidation. Therefore, the present study was

done to evaluate the effect of MSE on increased ROS pro-

duction in H9C2 cells induced with high glucose, where

25 mM glucose concentration was selected as it leads to

diabetic condition and 95 % confluency of cardiac cells can

be obtained [46]. ROS-mediated cardiac complications were

found to be the major factor of response in heart-related

death in diabetes [47]. In consistence with the earlier report,

in the present study, we confirmed that high glucose induces

ROS in cardiac myoblast cells. Accumulating evidences

suggest that ROS like H2O2 and NO may act as signaling

molecule for the initiation and execution of high glucose–

mediated complications. Furthermore, activated signals

cause a significant increase in intracellular ROS and oxidant

levels which aggravates the consequences. The present

study shows that there is a marked increase in intracellular

ROS generation in cells treated with diabetic concentration

of glucose (25 mM) in cardiac cells. Further, a drastic

increase in H2O2 and NO levels was also observed in glu-

cose-stressed cells. Understanding the contribution of free

radicals in the pathogenesis allowed us to study the devel-

opment of oxidative stress which was generated in cardiac

cells due to imbalance between cellular production of oxi-

dant molecules and the availability of antioxidative species.

The antioxidant enzymes regulate the ROS level by main-

taining superoxide and H2O2 at low levels [48]. Our study

shows an increase in activity of antioxidative enzymes, SOD

and catalase in high glucose–stressed cardiac cells. The

increased activity may be to suppress the production of

intracellular free radical generation, but the increased

activity is not sufficient to normalize the upregulated ROS

levels.

The increased accumulation of extracellular matrix pro-

teins is the key feature of stress in cardiac myocytes [49].

Among the ECM proteins, collagen family is considered the

major component and determinant of the modulated myo-

cardial structural integrity. In our study, hyperglycemia-

induced ROS production is sufficient to generate stress on

cardiac myocytes and lead to increase in the collagen con-

tent. The increase in ECM is initially to compensate the

glucose stress, but prolonged stress leads to cardiac cell

death that can be characterized by nuclear deformities [50].

Using myoblast H9C2 cells, direct exposure to high levels of

glucose induces significant stress and nuclear alterations as

key feature that can be detected by DAPI and PI nuclear

staining. During cardiac cell apoptosis, the increase in cell

membrane permeability leads to more uptake of nuclear

stains such as DAPI and PI [51]. In our study, the nuclear

morphology of normal H9C2 cells was found to be round,

clear edged and uniformly stained, but glucose-stressed cells

showed irregular edges around the nucleus, chromosome

condensation and intense staining.

The left ventricular cardiac hypertrophy is hallmarked

by elevated levels of oxidative stress leading to mito-

chondrial dysfunction due to increased production and/or

impaired scavenging of reactive oxygen species. The

Cardiovasc Toxicol

123

present work was aimed to determine whether the cell

death induced by high glucose is of mitochondrial-

dependent or mitochondrial-independent mechanism, since

cardiolipins present in inner membrane of mitochondria

participates in several mitochondria-dependent apoptotic

steps in various diseases [52]. The NAO staining was used

to study the mitochondrial involvement and dysfunction,

wherein the dye specifically binds to cardiolipin content of

inner mitochondrial membrane and emits an excitation

florescence that can be visualized microscopically [53]. We

demonstrated that more cardiolipin is present in glucose-

induced cells by NAO staining, suggesting the oxidative

stress generated in diabetic cardiac myopathies leading to

mitochondrial dysfunction and eventually to cell death and

heart failure. Our data suggest that glucose-mediated car-

diac stress is leading to change in mitochondrial membrane

potential, suggesting mitochondrial-mediated pathway

critical for progressive stress. It is also likely that the

mechanism underlying mitochondrial membrane potential

breakdown is a consequence of direct damaging effect on

mitochondrial membrane caused by ROS produced in

mitochondria present in H9C2 cells.

The drug-induced toxicity poses a serious concern for

diabetic patients, like some of the commonly used synthetic

antidiabetic drugs (metformin, pioglitazone etc.) imposes

additional stress on heart and is suggested to be a ROS-

mediated mechanism [54]. Therefore, the natural agent that

hampers ROS load, anti-diabetic as well as cardioprotective

can be used as a potential drug against diabetic cardiomy-

opathies. In summary, our study demonstrates that the seeds

of Syzygium cumini exert cardioprotective action by reduc-

ing ROS generation, preventing the increase in cell size and

extracellular matrix components in hyperglycemic condi-

tion. This establishes the dual effective role of S. cumini as

anti-diabetic and cardioprotective. However, the present

study needs to be further validated by use of primary rat

cardiac myocytes or in vivo systems. Our recent unpublished

data shows that S. cumini methanolic seed extract is rich in

gallic acid derivatives, suggesting use of gallic acid as a

positive control in our future studies to know the signaling

mechanism involved in the cardioprotection by MSE in

glucose-induced stress. A model can therefore be proposed

that an increase in oxidative stress due to ROS results in

myocardial remodeling and increase in overall collagen

content in diabetic cardiomyopathies and MSE is having

ability to significantly reduce this ROS-mediated events,

thereby minimizing the glucose-mediated cardiac stress.

Acknowledgments We acknowledge Jaypee Institute of Informa-

tion Technology, Deemed to be University for providing the infra-

structural support.

Conflict of interest Authors declare that there is no conflict of

interest.

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