aB-crystallin regulates oxidative stress-induced apoptosisin cardiac H9c2 cells via the PI3K/AKT pathway

Feng Xu • Haixia Yu • Jinyao Liu • Lu Cheng

Received: 15 July 2012 / Accepted: 20 November 2012 / Published online: 1 December 2012

� Springer Science+Business Media Dordrecht 2012

Abstract The present study was carried out to observe the

protective effects of aB-crystallin protein on hydrogen perox-

ide (H2O2)-induced injury in rat myocardial cells (H9c2) and to

investigate the mechanisms of these protective effects at the

cellular level, which could provide the experimental basis for

future applications of aB-crystallin in the treatment of cardio-

vascular disease. Western blotting was used to measure the

expression of aB-crystallin in cultured H9c2 cells in vitro. A

aB-crystallin recombinant expression vector, pcDNA3.1-Cry-

ab, was constructed to transfect H9c2 cells for the establishment

of cells that stably expressed aB-crystallin. A tetrazolium-

based colorimetric assay (MTT test) was used to measure

changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after

induced by 150 lM H2O2 to establish a model of H2O2 injury to

cells. H2O2 was applied to H9c2 cells that were stably trans-

fected with aB-crystallin, and the effect of aB-crystallin

overexpression on the viability of myocardial cells subjected to

H2O2-induced injury was measured by the MTT assay. The

effect of aB-crystallin overexpression on the H2O2-induced

injury of H9c2 cells was also analyzed by flow cytometry. The

mitochondrial components and cytoplasmic components of

H9c2 cells were separated, and western blotting was used to

measure the effect of aB-crystallin overexpression on the

release of cytochrome c from the mitochondria. Western blot-

ting was also used to measure the effect of aB-crystallin

overexpression on the expression of the anti-apoptosis protein

Bcl-2 and components of the phosphatidylinositol 3-OH kinase

(PI3K)/AKT pathway. The aB-crystallin recombinant

expression vector pcDNA3.1-Cryab successfully transfected

H9c2 cells, and H9c2 cells that were stably transfected with

aB-crystallin were established after G418 selection. The

measurements carried out by western blotting showed that

aB-crystallin proteins are expressed in normal H9c2 cells, but

the proteins’ expression was much higher in pcDNA3.1-

Cryab transfected cells (P \ 0.01). The MTT assays showed

that 4 h of H2O2 treatment induced significant injury in H9c2

cells (P \ 0.01), but aB-crystallin overexpression can effec-

tively antagonize the H2O2-induced injury to H9c2 cells

(P \ 0.05). The results of flow cytometry analysis showed

that aB-crystallin overexpression can significantly reduce

apoptosis in H2O2-injured H9c2 cells (P \ 0.05). The results

of western blotting showed that aB-crystallin overexpression

in myocardial cells can reduce the H2O2-induced release of

cytochrome c from the mitochondria (P \ 0.05), antagonize

the H2O2-induced downregulation of Bcl-2 (P \ 0.05) and

magnify the decrease in phosphorylated AKT levels induced

by H2O2 injury (P \ 0.05). The overexpression of aB-

crystallin has a protective effect on H2O2-injured H9c2 cells,

and aB-crystallin can play a protective role by reducing

apoptosis, reducing the release of cytochrome c from the

mitochondria and antagonizing the downregulation of Bcl-2

expression. The protective effects of aB-crystallin may be

related to the PI3K/AKT pathway.

Keywords aB-crystallin � Hydrogen peroxide �Myocardial cells � Mitochondria � PI3K/AKT

Introduction

Cardiovascular disease (CVD) is currently the leading

cause of death in the domestic population, and the

F. Xu (&) � J. Liu � L. Cheng

Department of Cardiovascular Medicine, The First Affiliated

Hospital of China Medical University, No. 155, Nanjing Street,

Shenyang 110001, China

e-mail: drfengxu@126.com

H. Yu

Department of Emergency, Chengde Central Hospital, Chengde

067000, China

123

Mol Biol Rep (2013) 40:2517–2526

DOI 10.1007/s11033-012-2332-2

morbidity and mortality rates increase every year [1, 2]. In

recent years, a large number of studies have shown that

myocardial cell apoptosis is involved in the development of

CVD, where oxidative stress/injury plays an important role

[3, 4]. Because myocardial cells are terminally differentiated

cells, the cumulative effects of apoptosis lead to decreases in

cell quantities and cardiac function and promote the devel-

opment of heart failure [5, 6]. Therefore, intervention in

myocardial cell apoptosis is an important topic in the

research of CVD prevention and control [7, 8].

In recent years, many studies have shown that a multitude

of factors, including myocardial ischemia/reperfusion injury,

hyperlipidemia, hypertension, diabetes, aging, inflammation

and certain drugs, can induce myocardial cell apoptosis

through oxidative stress. Such factors can injury myocardial,

vascular smooth muscle and endothelial cells; interfere with

electron transport in the mitochondrial respiratory chain;

promote lipid peroxidation; consume intracellular antioxidant

proteins; and increase the generation of reactive oxygen spe-

cies [9–13]. These changes in intracellular redox states can

lead to cardiovascular cell apoptosis through the activation of

apoptosis signal transduction pathways.

The mechanism of apoptosis involves two main signal

transduction pathways: the mitochondrial pathway and the

membrane death receptor pathway [14–16]. In recent years,

the mitochondrial apoptosis pathway has become a topic of

great interest and the frontier of apoptosis research [17]. It

has been shown that such factors as oxidative stress can

increase the permeability of the outer mitochondrial mem-

brane and promote the release of pro-apoptotic molecules,

such as cytochrome c, Smac/DIABLO, apoptosis-inducing

factor (AIF), endonuclease G, and a variety of caspases, into

the membrane gap. These pro-apoptotic molecules subse-

quently induce apoptosis through various downstream

mechanisms [18, 19]. However, the mitochondrial signaling

pathway is also affected and regulated by the Bcl-2 family of

proteins, transcription factors such as p53 and NF-jB and

heat shock proteins [20, 21].

A large number of recent studies have shown that various

heat shock proteins, such as HSP70, HSP90 and HSP27, can

have anti-apoptotic functions at different levels on distinct

steps of apoptotic signaling pathways in the cell [22, 23]. As

a key member of the small heat shock protein family, the

relatively strong expression of aB-crystallin protein in adult

cardiac tissue suggests that it has biological significance [23,

24]. Recent studies in tumor cells, crystal cells and cardiac

tissue have found that aB-crystallin also has an anti-

apoptotic function. The mechanism of aB-crystallin’s anti-

apoptotic function is related to its binding of the apoptotic

protease caspase-3 to inhibit caspase-3 activation [25, 26].

However, it is not clear whether aB-crystallin can carry out

its anti-apoptotic function at an early stage in the process of

oxidative stress-induced apoptosis of myocardial cells

in vitro, whether its mechanism is related to an effect on the

mitochondrial signal transduction pathway or whether aB-

crystallin can interact with apoptosis-related proteins and if

it can, what the significance of such interactions might be.

The present study uses the reactive oxygen species hydrogen

peroxide (H2O2) to induce rat myocardial cell apoptosis as a

model in which to systematically study these questions in

depth. It provides new scientific evidence for endogenous

protective mechanisms of the cardiovascular system and

possibilities for CVD prevention and treatment.

Materials and methods

Reagents

The rat myocardial cell line H9c2 was purchased from

American Type Culture Collection (ATCC CRL-1446).

DMEM/F12, trypsin and fetal bovine serum were pur-

chased from Invitrogen. Cell culture plates were purchased

from Corning.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-

um bromide) and H2O2 were purchased from Sigma-Aldrich.

Annexin V and propidium iodide (PI) were purchased from

Roche. G418, pcDNA3.1, a Lipofectamine 2000 liposome

transfection kit, Trizol reagent and a reverse transcription kit

were purchased from Invitrogen. A Qproteome Mitochondria

Isolation Kit was purchased from QIAGEN.

The aB-crystallin (GenBank accession number:

NM_012935.3) upstream primer P1 was 50-CG GAA TTC

GAC ATA GCC ATC CAC CAC CCC-30; it contained an

EcoRI restriction site, which is underlined. The downstream

primer P2 was 50-GG GGT ACC CTT CTT AGG GGC TGC

AGT GAC-30; it contained a KpnI restriction site, which is

underlined. Primers for the internal reference gene glycer-

aldehyde-3-phosphate dehydrogenase (GAPDH) (GenBank

accession number: NM_002046.3) were upstream primer P1

of sequence 50-CCA CAG TCC ATG CCA TCA CT-30 and

downstream primer P2 of sequence 50-TCC ACC ACC CTG

TTG CTG TAG-30. All of the primers were synthesized by

the Shanghai Invitrogen Biotechnology Company.

The following primary antibodies were purchased

from Abcam: mouse anti-rat b-actin monoclonal antibody,

mouse anti-rat HSP60 monoclonal antibody, rabbit anti-rat

aB-crystallin polyclonal antibody, rabbit anti-rat cyto-

chrome c polyclonal antibody and rabbit anti-rat Bcl-2

polyclonal antibody. Rabbit anti-phospho-AKT (Ser473)

polyclonal antibody and rabbit anti-pan-AKT polyclonal

antibody were purchased from Cell Signaling Technology.

LY294002 was purchased from Calbiochem. The IRDye

800-conjugated affinity purified goat anti-mouse IgG and

IRDye 800-conjugated affinity purified goat anti-rabbit IgG

secondary antibodies were purchased from Odyssey.

2518 Mol Biol Rep (2013) 40:2517–2526

123

Construction of the rat aB-crystallin recombinant

expression vector

Rats were sacrificed, and the hearts were removed immedi-

ately. One hundred milligrams of heart tissue were crushed in

the grinding mortar under liquid nitrogen. Subsequently,

1 ml of Trizol reagent was added, and the mixture was

homogenized. Total RNA was extracted from the samples

according to the manufacturer’s instructions. Reverse tran-

scription reactions were performed with the reverse tran-

scription kit. First strand cDNA synthesis was carried out

according to the manufacturer’s instructions. The first cDNA

strands were stored at -20 �C.

The aB-crystallin and GAPDH primers were dissolved

in ddH2O and stored at -20 �C. cDNA in the amount of

2 ll was added to each 100 ll polymerase chain reaction

(PCR). The following amplification conditions were used:

denaturation for 5 min at 94 �C and 30 cycles of denatur-

ation for 30 s at 94 �C, annealing for 30 s at 56 �C and

extension for 30 s at 72 �C. A final extension of 10 min at

72 �C was carried out. The PCR products were examined

with 1.5 % agarose gel electrophoresis.

The PCR products and the empty vector pcDNA3.1

were double digested with EcoRI and KpnI, and the

digested products were collected. The digested products

were ligated with T4 ligase, and the ligation products were

screened to obtain recombinant plasmid pcDNA3.1-Cryab.

The successful formation of the recombinant expression

plasmid was verified by sequencing.

Measurement of of aB-crystallin expression in H9c2

cells by western blotting

H9c2 cells were cultured in 6-well plates to 95 % confluence

according to the instructions of the Lipofectamine 2000 lipo-

some transfection kit. The recombinant plasmid pcDNA3.1-

Cryab and the no-load plasmid pcDNA3.1 (4.0 lg) were

mixed with liposomes in a ratio of 1:3 to transfect the cells.

Transfected cells were transferred to normal growth medium

after 6 h and were cultured in selective medium after 48 h;

the G418 concentration was gradually increased to 1,000 lg/

ml. After 1 week, a single clone was selected for amplifi-

cation of the culture. Screening was continued until

4–6 weeks, and the total cellular proteins were subsequently

extracted for measurement.

RIPA lysis buffer [150 mM NaCl, 1 % NP40, 0.5 %

sodium deoxycholate, 0.1 % SDS, 50 mM Tris (pH 7.9),

10 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF),

and 19 protease inhibitors (Complete Protease Inhibitor

Cocktail Tablets, Roche)] in a quantity of 1 ml was added

into each well of the 6-well cell culture plate. The cell

lysates were then transferred to 1.5 ml centrifuge tubes and

centrifuged for 30 min at 160,0009g. The supernatants

were obtained to measure the protein concentrations with

the bicinchoninic acid (BCA) method. After 5 % stacking

gels and 12 % separating gels were prepared, 50 lg of the

protein extracts were loaded in each lane, electrophoreti-

cally separated, and wet-transferred to polyvinylidene

difluoride (PVDF) membrane (Bio-Rad Co., USA). The

membrane was later blocked for 1 h in 5 % nonfat dry milk

in TBST (10 mM Tris–HCl (pH 7.5), 150 mM NaCl, and

0.1 % Tween-20) blocking buffer at room temperature.

Rabbit anti-rat aB-crystallin polyclonal antibody (1:1,000

dilution) and mouse anti-rat b-actin monoclonal antibody

(1:1,000 dilution) were incubated with the membrane at

4 �C overnight. The corresponding IRDye 800-labeled

secondary antibody (1:2,000 dilution in PBS) was then

incubated with the membrane at 4 �C overnight. After

washing with TBST, the membrane was scanned with the

Odyssey Infrared Imaging System (Rockland Co.). The

relative content of aB-crystallin was expressed as an aB-

crystallin/b-actin grayscale ratio. The grayscale values were

analyzed with Quantity One software (Bio-Rad, USA).

MTT measurement of H2O2-induced injury

in H9c2 cells

Normal rat myocardial H9c2 cells were cultured at 37 �C

in 5 % CO2 and DMEM/F12 medium with 10 % fetal

bovine serum (FBS). The H9c2 cells were seeded into a

96-well culture plate at a concentration of 2 9 105 cells/

ml. The volume of each well was 100 ll. After adhering to

the wall, the cells were moved to DMEM medium con-

taining H2O2 at a final concentration of 150 lM and placed

in a 37 �C, 5 % CO2 incubator. A 100 ll volume of a

0.5 mg/ml MTT solution was added to each well at 1, 2, 3

and 4 h, and the cells were incubated for 4 h in a 5 % CO2

incubator at 37 �C. A 100 ll volume of 20 % SDS (50 %

dimethyl formamide cosolvent) was added to each well,

and the cells were then incubated for 24 h at 37 �C. A

microplate reader (Bio-Tek) was used to measure the ODs

at 570 nm. There were 10 wells in each group, and the

experiment was repeated 3 times.

MTT measurement of aB-crystallin’s

effect on the viability of H9c2 cells subjected

to H2O2-induced injury

H9c2 cells in the logarithmic growth phase were trypsini-

zed with 0.25 % trypsin. The cell densities were adjusted to

2 9 105 cells/ml, and the cells were seeded into 96-well

culture plates. The volume of each well was 100 ll. The

following classification of experimental groups was used:

normal control group, H2O2-injured group, pcDNA3.1-

Cryab-transfected group (H2O2 ? pcDNA3.1-Cryab) and

pcDNA3.1-transfected group (H2O2 ? pcDNA3.1). Cells

Mol Biol Rep (2013) 40:2517–2526 2519

123

were incubated in a 5 % CO2 incubator at 37 �C for 4 h. After

a 4 h treatment with 150 lM H2O2, 100 ll of a 0.5 mg/ml

MTT solution was added to each well, and the cells were

incubated in a 5 % CO2 incubator at 37 �C for 4 h. A 100 ll

volume of 20 % SDS (50 % dimethyl formamide cosolvent)

was added to each well, and the cells were incubated for 24 h

at 37 �C. A microplate reader (Bio-Tek) was used to measure

the ODs at 570 nm. There were 10 wells in each group, and

the experiment was repeated 3 times.

Measurement of aB-crystallin’s effect on the apoptosis

of H2O2-injured H9c2 cells by flow cytometry

H9c2 cells were uniformly seeded into 6-well culture plates

at concentrations of 2 9 105 cells/ml. The volume of each

well was 100 ll. The following classification of experi-

mental groups was used: normal control group, H2O2-treated

group, pcDNA3.1-Cryab-transfected group (H2O2 ?

pcDNA3.1-Cryab) and pcDNA3.1-transfected group (H2O2 ?

pcDNA3.1). The cells were incubated in a 5 % CO2 incubator

at 37 �C. After a 4 h treatment with 150 lM H2O2, the cells

were washed in PBS 1–2 times, digested with trypsin, and then

suspended in 19 Binding Buffer. The cell densities were

adjusted to 1 9 106 cells/ml. One hundred microliters of cell

solution (1 9 105 cells) was drawn and put into a 5 ml cen-

trifuge tube; 5 ll of FITC Annexin V and 5 ll of PI were added.

The cells were mixed gently and then incubated for 15 min in

the dark at room temperature (25 �C). A 400 ll volume of

19 Binding Buffer was then added to each tube. Analysis with

a flow cytometer (BD, USA) was performed within 1 h.

Measurement of aB-crystallin’s effect on the release

of cytochrome c in H2O2-injured H9c2 cells by western

blotting

H9c2 cells in the normal control group, the H2O2-treated

group, the pcDNA3.1-Cryab-transfected group (H2O2 ?

pcDNA3.1-Cryab) and the pcDNA3.1-transfected group

(H2O2 ? pcDNA3.1) were uniformly seeded into 6-well

culture plates at concentrations of 2 9 105 cells/ml. After

4 h of 150 lM H2O2 treatment, the cells were washed in

PBS 1–2 times. The cells were then scraped, placed in 2-ml

centrifuge tubes and centrifuged at 4 �C and 8509g for

2 min. The supernatants were removed following the

centrifugations. The mitochondrial and cytosolic fractions

were isolated according to the instructions provided with

the Qproteome Mitochondria Isolation Kit (QIAGEN

China (Shanghai) Co., Ltd., Shanghai, China). The mito-

chondrial and cytosolic proteins were obtained after lysis

with cell lysis buffer.

Mitochondrial proteins and cytosolic proteins were

quantified by the BCA method, separated by electrophoresis

and wet-transferred to PVDF membrane. After blocking for

1 h in a 5 % nonfat dry milk in TBST blocking buffer at room

temperature, rabbit anti-rat cytochrome c polyclonal anti-

body (1:1,000 dilution), mouse anti-rat HSP60 monoclonal

antibody (1:1,000 dilution) and mouse anti-rat b-actin

monoclonal antibody (1:1,000 dilution) were then incubated

with the membrane at 4 �C overnight. The corresponding

IRDye 800-labeled secondary antibody (1:2,000 dilution in

PBS) was incubated with the membrane at 4 �C overnight.

After washing with TBST, the membrane was scanned with

the Odyssey Infrared Imaging System (Rockland Co.). The

relative content of cytochrome c in the mitochondria and

cytoplasm was expressed as cyt c/HSP60 and cyt c/b-actin

grayscale ratios, respectively. The grayscale values were

analyzed with Quantity One software (Bio-Rad, USA).

Measurement of aB-crystallin’s effect on Bcl-2

in H2O2-injured H9c2 cells by western blotting

Cells in each group were treated with 150 lM H2O2 for 4 h

and then washed in PBS 1–2 times. The cell proteins were

harvested, quantified by BCA, separated by electrophoresis

and wet-transferred to PVDF membrane. After blocking for

1 h in a 5 % nonfat dry milk TBST blocking buffer at room

temperature, rabbit anti-rat Bcl-2 polyclonal antibody

(1:1,000 dilution) was incubated with the membrane at

4 �C overnight. The corresponding IRDye 800-labeled

secondary antibody (1:2,000 PBS dilution) was then incu-

bated with the membrane at 4 �C overnight. After washing

with TBST, the membrane was scanned with the Odyssey

Infrared Imaging System (Rockland Co.). The relative

content of Bcl-2 was expressed as a Bcl-2/b-actin grayscale

ratio. The grayscale values were analyzed with Quantity

One software (Bio-Rad, USA).

The effect of aB-crystallin on the PI3K/AKT pathway

in H2O2-injured H9c2 cells

H9c2 cells in the normal control group, the pcDNA3.1-

Cryab-transfected group (H2O2 ? pcDNA3.1-Cryab), the

pcDNA3.1-transfected group (H2O2 ? pcDNA3.1) and the

PI3K/AKT-inhibited group (25 lM LY294002 ? H2O2 ?

pcDNA3.1-Cryab) were uniformly seeded into 6-well cul-

ture plates at concentrations of 2 9 105 cells/ml. Cells in

each group were treated with 150 lM H2O2 for 4 h and

then washed in PBS 1–2 times. The cell proteins were

harvested, quantified by BCA, separated by electrophoresis

and wet-transferred to a PVDF membrane. After blocking

for 1 h in a 5 % nonfat dry milk TBST blocking buffer at

room temperature, rabbit anti-phospho-AKT (Ser473)

polyclonal antibody (1:1,000 dilution), rabbit anti-pan-

AKT polyclonal antibody (1:1,000 dilution), and mouse

anti-rat b-actin monoclonal antibody (1:1,000 dilution)

were incubated with the membrane at 4 �C overnight. The

2520 Mol Biol Rep (2013) 40:2517–2526

123

corresponding IRDye 800-labeled secondary antibody

(1:2,000 PBS dilution) was later added and incubated with the

membrane at 4 �C overnight. After washing with TBST, the

membrane was scanned with the Odyssey Infrared Imaging

System (Rockland Co.). The relative contents of p-AKT and

t-AKT were expressed as AKT/b-actin and t-AKT/b-actin

grayscale ratios, respectively. The grayscale values were

analyzed with Quantity One software (Bio-Rad, USA).

Statistical analysis

The experimental results were analyzed with Stata 7.0

statistical software. v2 analyses and t tests were used.

P \ 0.05 was considered to be statistically significant.

Results

Measurement of aB-crystallin expression in H9c2 cells

by western blotting

The results of the measurements by western blotting showed

that a small amount of endogenous aB-crystallin is expres-

sed in normal H9c2 cells and in empty vector pcDNA3.1-

transfected H9c2 cells. However, in H9c2 cells that had been

transfected with the aB-crystallin recombinant expression

vector pcDNA3.1-Cryab, the expression of aB-crystallin

was significantly increased (P \ 0.01) (Fig. 1).

MTT measurement of H2O2-induced injury

in H9c2 cells

MTT measurements showed that H9c2 cell viability is

lower than the normal control group 3 and 4 h after H2O2

injury (P \ 0.05). Especially after a 4-h treatment with

H2O2, the cell viability significantly decreased, and the OD

values were significantly different from the values of the

normal control group (P \ 0.01) (Fig. 2). Based on these

results, H9c2 cells treated with 4 h of H2O2 was selected as

the model for H2O2-injured cells.

MTT measurement of aB-crystallin’s effect

on the viability of H2O2-injured H9c2 cells

The MTT assay was used to observe the protective effects of

aB-crystallin on H2O2-injured H9c2 cells. The MTT mea-

surements showed that the cell viabilities of the H2O2-injured

group and the pcDNA3.1-transfected group (H2O2 ?

pcDNA3.1) were significantly decreased (P \ 0.01) com-

pared with the normal control group, and the cell viability of

the pcDNA3.1-Cryab-transfected group (H2O2 ? pcDNA3.1-

Cryab) was significantly higher than the cell viability of the

H2O2-injured group (P \0.05) (Fig. 3).

Measurement of aB-crystallin’s effect on the apoptosis

of H2O2-injured H9c2 cells by flow cytometry

The effect of aB-crystallin on the apoptosis of H2O2-injured

H9c2 cells was analyzed by FITC Annexin V and PI double-

labeling flow cytometry. The results showed that the propor-

tion of apoptotic H9c2 cells was relatively low in the normal

control group but higher in the pcDNA3.1-transfected group

(H2O2 ? pcDNA3.1) and the pcDNA3.1-Cryab-transfected

group (H2O2 ? pcDNA3.1-Cryab). The proportion of early

Fig. 1 a Western blot analysis ofaB-crystallin expression in H9c2 cells in

each group. b The relative change in aB-crystallin expression in each

group of H9c2 cells (compared with the normal control group, **P\0.01)

Fig. 2 Statistical analysis of the MTT measurements of H9c2 cell

viability at 1, 2, 3 and 4 h after H2O2 treatment (compared with the

normal control group, *P \ 0.05 and **P \ 0.01)

Mol Biol Rep (2013) 40:2517–2526 2521

123

apoptotic cells was significantly lower in the pcDNA3.1-

Cryab-transfected group (H2O2 ? pcDNA3.1-Cryab) than

the H2O2-injured group (P \ 0.05) (Fig. 4). The results sug-

gest that aB-crystallin can effectively prevent the H2O2

injury-induced apoptosis of H9c2 cells.

Measurement of aB-crystallin’s effect on the release

of cytochrome c in H2O2-injured H9c2 cells by western

blotting

Western blotting showed that the cytosolic cytochrome

c content increased significantly (P \ 0.01) after H2O2

treatment; in the pcDNA3.1-transfected group (H2O2 ?

pcDNA3.1) and the pcDNA3.1-Cryab-transfected group

(H2O2 ? pcDNA3.1-Cryab), the release of cytochrome c

from the mitochondria to the cytoplasm was significantly

higher. However, when compared with the H2O2-injured

group, the level of cytochrome c release from the mito-

chondria to the cytoplasm was lower in the pcDNA3.1-

Cryab-transfected group (H2O2 ? pcDNA3.1-Cryab) (P \0.05) to a certain extent (Fig. 5).

Measurement of aB-crystallin’s effect on Bcl-2

in H2O2-injured H9c2 cells by western blotting

The measurements by western blotting showed that after

H2O2 treatment, the level of Bcl-2 expression decreased in

the H2O2-injured group, the pcDNA3.1-transfected group

(H2O2 ? pcDNA3.1) and the pcDNA3.1-Cryab-transfec-

ted group (H2O2 ? pcDNA3.1-Cryab). However, com-

pared with the H2O2-injured group, the decrease in Bcl-2

expression in the pcDNA3.1-Cryab-transfected group

(H2O2 ? pcDNA3.1-Cryab) was slightly less (P \ 0.05)

(Fig. 6).

The effect of aB-crystallin on the PI3K/AKT pathway

in H2O2-injured H9c2 cells

The results of western blotting showed that H2O2 injury did

not significantly affect AKT expression in H9c2 cells

(P [ 0.05) but significantly lowered the intracellular

expression of p-AKT (P \ 0.01). Compared with the normal

Fig. 3 The analysis of aB-crystallin’s effect on the viability of H2O2-

injured H9c2 cells (compared with the normal control group, *P \ 0.05

and **P \ 0.01; compared with the H2O2-injured group, #P \ 0.05)

Fig. 4 a Effect of aB-crystallin

on the apoptosis of H2O2-

injured H9c2 cells. b The results

of the statistical analysis of the

apoptotic cells in each group

(compared with the normal

control group, *P \ 0.05 and

**P \ 0.01; compared with the

H2O2-injured group, #P \ 0.05)

2522 Mol Biol Rep (2013) 40:2517–2526

123

control group, the overexpression of aB-crystallin signifi-

cantly increased the intracellular p-AKT expression in

H2O2-injured cells (P \ 0.05). After the AKT inhibitor

LY294002 was added to the pcDNA3.1-Cryab-transfected

group, the levels of phosphorylation were further decreased

(Fig. 7).

Discussion

The aB-crystallin gene is located at chromosome position

11q12-q23, is 522 bp long and encodes a 175-amino-acid

protein [27]. aB-crystallin belongs to the small heat shock

protein family [28]. This protein can maintain necessary

protein conformations in the cell during the stress response

and plays an important function in protein aggregation,

protein folding, transmembrane transport, translocation and

cytoskeleton stability [29, 30]. In recent years, the func-

tions and mechanisms of several major heat shock proteins

have become the focuses of cardiovascular, neurobiologi-

cal and tumor research [24, 31, 32].

aB-crystallin is widely expressed in a variety of tissues

and cells; the highest expression levels are found in the lens

and striated muscle (heart, skeletal muscle) [33]. aB-crys-

tallin plays an important role in stabilizing the quantity and

quality of proteins in the intracellular environment.

Recently, it has been found that aB-crystallin is involved in a

variety of processes in cell physiology and the stress

response and, in some respects, demonstrates protective

functions in anti-tissue cell injury [34]. In the present study, a

rat aB-crystallin recombinant expression vector was con-

structed. The results of western blotting showed that a small

Fig. 5 a Western blot analysis

of the expression of

mitochondrial cytochrome c and

cytosolic cytochrome c in H9c2

cells of each group. b Changes

in the expression of cytochrome

c relative to mitochondrial

proteins in H9c2 cells of each

group. c Changes in the

expression of cytochrome c

relative to cytosolic proteins in

H9c2 cells of each group (mitomitochondrial proteins; cytocytosolic proteins; compared

with the normal control group,

*P \ 0.05 and **P \ 0.01;

compared with the H2O2-injured

group, #P \ 0.05)

Fig. 6 a Western blot analysis results of Bcl-2 expression in H9c2

cells of each group. b The relative expression of Bcl-2 in H9c2 cells

of each group (compared with the normal control group, *P \ 0.05

and **P \ 0.01; compared with the H2O2-injured group, #P \ 0.05)

Mol Biol Rep (2013) 40:2517–2526 2523

123

quantity of endogenous aB-crystallin is expressed in normal

H9c2 cells and empty vector pcDNA3.1-transfected H9c2

cells. However, in the aB-crystallin recombinant expres-

sion vector pcDNA3.1-Cryab-transfected H9c2 cells,

aB-crystallin expression was significantly increased (P\0.01).

Many previous studies have found that aB-crystallin

protein in the muscle rapidly translocates from the cyto-

plasm to the cytoskeletal structure under such stresses as

tissue hypoxia, ischemia, reperfusion and an increased

mechanical load. Thus, the presence of aB-crystallin pro-

tein in the myocardial tissue has long been considered to be

related to the stability of the cytoskeleton during the stress

response [35]. In the present study, an H2O2-injured rat

myocardial H9c2 cell model was constructed. The MTT

measurements showed that the cell viability of the

pcDNA3.1-Cryab- transfected group (H2O2 ? pcDNA3.1-

Cryab) was significantly higher than the cell viability of the

H2O2-injured group (P \ 0.05), suggesting that aB-crys-

tallin overexpression can effectively antagonize the

decrease in H9c2 cell viability caused by H2O2 injury.

Velotta et al. [25] have studied the protective effects of

aB-crystallin on the mouse heart during ischemic reper-

fusion. Shin et al. [36] found that aB-crystallin can pro-

tect C6 glial cells during H2O2-induced injury, which is

consistent with previous studies.

Previously, it was thought that the main form of myo-

cardial cell injury was necrosis. However, in recent years,

many studies have shown that apoptosis is also one of the

main forms of myocardial cell injury. Many scholars have

found that there are large quantities of apoptotic myocar-

dial cells in the central area and periphery of lesions

during myocardial infarction in heart disease patients and

experimental animal models. Both apoptosis and necrosis

promote the expansion of the infarct area, and in certain

situations, myocardial cell apoptosis can also be trans-

formed into cell necrosis [4, 37]. In the present study, the

results of flow cytometry analysis showed that the ratio of

apoptotic cells to living cells in the early stage of cell

injury is significantly lower in the pcDNA3.1-Cryab-

transfected group (H2O2 ? pcDNA3.1-Cryab) than in the

H2O2-injured group (P \ 0.05). The results suggest that

aB-crystallin can effectively prevent the H2O2 injury-

induced apoptosis of H9c2 cells. The study of Velotta et al.

[25] found that aB-crystallin protein can reduce cell

apoptosis during the ischemic reperfusion of endothelial

cells. The results of the present study are consistent with

previous studies.

Studies on the mechanisms of myocardial cell apoptosis

have found evidence that oxidative injury can activate the

mitochondrial signaling pathway and induce myocardial

cell apoptosis [38]. H2O2 can promote the release of cyto-

chrome c and myocardial cell apoptosis by increasing the

expression of the pro-apoptotic protein p53 and the trans-

location of Bax and Bad from the cytosol to the mito-

chondria [39]. It has also been found that reactive oxygen

species can induce myocardial cell apoptosis by inhibiting

the expression of the anti-apoptotic protein Bcl-2 [40]. The

results of the present study showed that after H2O2 treat-

ment, the cytosolic cytochrome c content increased signif-

icantly (P \ 0.01). Compared with the H2O2-injured group,

the level of cytochrome c release from the mitochondria to

the cytoplasm was lower in the pcDNA3.1-Cryab-trans-

fected group (H2O2 ? pcDNA3.1-Cryab) (P \ 0.05). The

results indicate that aB-crystallin inhibits the H2O2-induced

release of cytochrome c from the mitochondria to the

cytoplasm. After H2O2 treatment, the level of Bcl-2

expression decreased in the H2O2-injured group, the

pcDNA3.1-transfected group (H2O2 ? pcDNA3.1) and the

pcDNA3.1-Cryab-transfected group (H2O2 ? pcDNA3.1-

Cryab) (P \ 0.05). However, compared with the H2O2-

injured group, the level of Bcl-2 expression in the

pcDNA3.1-Cryab transfected group (H2O2 ? pcDNA3.1-

Cryab) did not decrease as markedly (P \ 0.05). The results

indicate that aB-crystallin inhibits the decrease in H2O2-

induced Bcl-2 expression and thus reduces H2O2-induced

apoptosis.

Many cytokines, growth factors, and physical stimuli

can induce AKT phosphorylation through PI3K activation.

Fig. 7 a Western blot analysis of p-AKT expression and AKT

expression in H9c2 cells of each group. b Changes in the relative

expression of p-AKT/AKT in H9c2 cells of each group (compared

with the normal control group, *P \ 0.05 and **P \ 0.01; compared

with the pcDNA3.1-Cryab-transfected group, star P \ 0.05)

2524 Mol Biol Rep (2013) 40:2517–2526

123

The activated AKT can cause a variety of cellular activities

and biological effects, including downstream phosphory-

lation cascades; interactions between target proteins; cell

growth regulation, survival, proliferation and apoptosis;

sugar metabolism; gene transcription; angiogenesis; cell

migration; and cell cycle regulation [41, 42]. Many studies

have provided evidence that the PI3K/AKT pathway plays

an important role in the protection against myocardial

ischemic reperfusion injury. Under certain conditions,

PI3K/AKT pathway activation can have a protective effect

by effectively inhibiting myocardial cell apoptosis [43, 44].

The results of the present study showed that aB-crystallin

overexpression can significantly promote p-AKT expres-

sion in H2O2-injured cells (P \ 0.05) compared with the

normal control group.

In summary, aB-crystallin overexpression has a pro-

tective effect against the H2O2 injury of H9c2 cells. aB-

crystallin plays a protective role by reducing apoptosis,

reducing the release of cytochrome c from mitochondria

and antagonizing the downregulation of Bcl-2 expression.

The protective effect of aB-crystallin may be related to the

PI3K/AKT pathway.

Conflict of interest The authors declare no conflicts of interest.

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