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iTRAQ-based proteomic analysis reveals recovery of impaired mitochondrial function in ischemic myocardium by Shenmai formula Yi Wang, Yu Zhao, Wei Jiang, Xiaoping Zhao, Guanwei Fan, Han Zhang, Peiqiang Shen, Jiangmin He, and Xiaohui Fan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00450 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 5, 2018

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Journal of Proteome Research

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5

iTRAQ-based proteomic analysis reveals recovery of impaired mitochondrial function in ischemic myocardium by Shenmai formula

6

Yi Wang1, Yu Zhao1, Wei Jiang1, Xiaoping Zhao2*, Guanwei Fan3, Han

7

Zhang3, Peiqiang Shen4, Jiangmin He4, Xiaohui Fan1*

2 3 4

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1

9

Zhejiang University, Hangzhou, P.R. China;

Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences,

10

2

11

Hangzhou 310053, P.R. China;

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3

13

of Traditional Chinese Medicine, Tianjin 300193, P.R. China;

14

4 Chiatai

15

China;

16

CORRESPONDING AUTHOR: Xiaoping Zhao [email protected],

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Xiaohui Fan [email protected]

College of Preclinical Medicine, Zhejiang Chinese Medical University,

Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University

Qingchunbao Pharmaceutical Co., Ltd. Hangzhou, 310023, P.R.

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ACS Paragon Plus Environment

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Abbreviations:

2 3

Myocardial infarction, MI; oxygen consumption rate, OCR; Shenmai formula,

4

SM; Red ginseng, RG; Radix Ophiopogonis, OP; Hypoxia, Hyp;

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2


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1

ABSTRACT

2

Shenmai formula (SM) is a traditional medicinal remedy for treating cardiovascular

3

diseases in China since 800 years ago, however its mechanism of action remains

4

unclear. To explore the mechanism underlying cardioprotective effects of SM, iTRAQ-

5

based proteomic approach was applied to analyze protein of myocardium in rats with

6

myocardial ischemic injury. Upon treatment with SM and its two major components

7

Red ginseng (RG) and Radix Ophiopogonis (OP), 101 differentially expressed proteins

8

were filtered from a total of 711 detected and annotated proteins. They can be

9

classified according to their locations and functions, whilst most of them are located in

10

intracellular organelle, participating in cellular metabolic process. The function of them

11

are mostly associated with mitochondrial oxidative phosphorylation/respiration. The

12

differentially expressed proteins were validated by LC−MS/MS and Western blotting

13

(ATP5D, NDUFB10, TNNC1). Further in vitro experiments found that SM could

14

attenuate hypoxia induced impairment of mitochondrial membrane potential and

15

cellular ATP concentration in neonatal rat ventricular myocytes. Interestingly, the result

16

of quantitative mitochondrial biogenesis assays revealed that SM had dominant

17

positive effects on the maximum respiration, ATP-coupled respiration, and spare

18

capacity of mitochondria in response to hypoxia. Hence, our findings suggested that

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SM promoted mitochondrial function to protect cardiomyocytes against hypoxia which

20

provides a possible illustration for conventional botanical therapy on molecular level.

21 22

KEYWORDS

23

Comparative

24

Traditional Chinese Medicine; Network Pharmacology;

proteomic;

Shenmai

Formula;

Mitochondrial

Biogenesis;

25

3



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1. INTRODUCTION

2

Shenmai formula (SM), derived from a venerable prescription recorded in

3

ancient Chinese medical literature more than 800 years ago, still has been

4

widely used for treating coronary artery disease, viral myocarditis, and other

5

cardiovascular diseases in China. According to a recent system review of

6

clinical trials, SM plus conventional medicine treatment shows significant

7

improvement in New York Heart Association classification of clinical status for

8

chronic pulmonary heart disease 1. There are also several reports suggested

9

that SM can improve the cardiac function of patients with chronic heart failure

10

2.

11

asthma-induced airway smooth muscle cell by inhibiting the expression of

12

calcium channel protein 3. Our previous study also found that combined use of

13

active components from SM can significantly decrease the infarction size of

14

heart and serum malondialdehyde (MDA) level in rats with myocardial ischemia

15

4.

16

still unclear.

Pharmacological studies revealed SM may prevent the over-proliferation of

However, the mechanism of action of SM for its cardio protective effects is

17

The prescription of SM is composed of two herbs, red ginseng (RG, Panax

18

ginseng) and Radix Ophiopogonis (OP, Ophiopogon japonicus (L. f.) Ker-Gawl,

19

root). The chemical composition of SM have been identified by liquid

20

chromatography coupled with mass spectrometry5 to indicate that ginsenosides

21

(including ginsenoside Rg1, Rg2, Re, Rb1 and Rd, etc.) from RG and

22

ophiopogonin from OP were major constituents of SM. It has been reported that

23

ginseng can inhibit cardiomyocyte hypertrophy and heart failure by suppressing

24

NHE-1-dependent calcineurin activation 6, while ophiopogonin can ameliorate

25

H2O2-induced endothelial injury 7. Despite cardioprotective effects of several

26

active compounds from SM have been investigated, there is lack of scientific

27

evidences to characterize specific mechanism of SM in treating acute myocarial

28

injury.

4


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Due to its great capability for charactering global, holistic profiling of protein

2

expression, the comparative proteomic approach has been widely applied in

3

investigating therapeutic mechanism of drugs and active compounds 8. There

4

are numerous investigations used pharmacoproteomic approach to reveal

5

toxicological and pharmacological effects on molecular level

6

studies have been performed to reveal proteomic alteration after treatment of

7

multi-components drug or drug combinations. In contrast to studying individual

8

proteins by conventional biochemical techniques, the comparative proteomic

9

approach allows a more comprehensive and holistic survey of a great variety of

10

different proteins, which might suitable for characterizing the therapeutic effects

11

of two or more agents and their combination on the molecular level.

9, 10.

But few

12

To improve our understanding on cardioprotective effects afford by SM on

13

ischemic heart, we constructed a comparative proteomic study for dissecting

14

the contribution of mono-therapy of RG and OP as well as SM. Fig 1 shows the

15

representative flowchart of the present iTRAQ-based proteomic profiling to

16

investigate differentially expressed proteins of myocardium with ischemia after

17

the treatment of SM, RG and OP. The myocardial proteomes of ischemic rat

18

hearts treated with SM and two active components (RG&OP) were compared

19

to rats with myocardial infarction (MI). The differentially expressed proteins

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identified such as NADH dehydrogenase, Succinate dehydrogenase, rieske

21

proteins, kreb cycle related proteins and ATP synthesis enzymes were related

22

to energy metabolism and mitochondrial function, which were further validated

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by western blot. Further in vitro experiments indicated hypoxia induced

24

impairment of energy metabolism and mitochondrial function can be attenuated

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by SM in primary cultured cardiac myocytes. The findings suggested that SM

26

and its components can recover the energy metabolism and mitochondrial

27

function through multi-targeted activation or suppression on related protein

28

level. The results of present study revealed that comparative proteomic

29

approach might provide useful molecular insights into the action of mechanism 5



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of SM as well as other multi-components drug or drug combinations.

2 3

Fig 1. The representative diagram for dissecting cardioprotective effects of

4

Shenmai formula by iTRAQ-based proteomic approach.

5 6

2. MATERIALS AND METHODS

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2.1 Animal models and drug treatment

8

The animal model and myocardial tissue samples used in this study is parallel

9

to our transcriptomics study 11. Briefly, myocardial infarction model in rats were

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induced in male Sprague-Dawley rats by permanent occlusion of the left

11

anterior descending coronary artery. In sham-operated rats (Control group), the

12

suture was placed beneath the left coronary artery without ligation. The

13

experiment protocol was conducted in accordance with the Guidelines for the

14

Care and Use of Laboratory Animals of Zhejiang University.

15

Extracts of SM and its two components RG and OP were obtained from Chiatai

16

Qingchunbao Pharmaceutical Co., Ltd. (Hangzhou, China). SM extract was

17

given via intraperitoneal injection for 7 days in rats with myocardial ischemia

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with dose of 10 mL/kg, which was calculated in accordance with clinical

19

consumption. The dosage of RG and OP was equal to their proportion in SM to

20

ensure that SM was used as a combination of RG and OP. The vehicle was 6


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given to the sham-operated rats (Sham group) and rats with myocardial

2

infarction (MI group). On the eighth day, rats were sacrificed and the border

3

between the infarct and non-infarct left ventricle area in myocardium were

4

harvested to extract proteins. Samples from three different rats in each group

5

were sent for proteomic analysis. To characterize chemical composition of SM,

6

high performance liquid chromatography coupled with mass spectrometry was

7

utilized to putatively identify more than fifty compounds, which ensure batch-to-

8

batch consistency of SM extracts (Supporting Information 1). Another heart was

9

rapidly fixed in 10% formalin and embedded in paraffin. Sections of hearts were

10

stained with hematoxylin and eosin for histologic evaluation.

11

2.2 Preparation of protein samples and iTRAQ labeling

12

Tissue samples were homogenized with lysis buffer by needle sonication.

13

The lysis buffer is composed of RIPA lysis buffer (containes 1% NP-40, 0.5%

14

deoxycholate and 0.1% SDS) (Beyotime) and 1mM phenylmethanesulfonyl

15

fluoride (PMSF) (Beyotime). The solubilized samples were centrifuged at

16

16,000 × g for 10 min at 4 °C and the total protein in the supernatant was

17

quantified using BCA assay. Protein extracts (100 mg each) were re-suspended

18

in 200 mL of 0.25M triethylammonium bicarbonate (TEAB), pH 8.5, 0.1% SDS,

19

reduced with 1mM Tris (2-carboxyethyl)-phosphine (60 ºC, 1 h) and alkylated

20

with 1mM methyl methanethiosulfonate (room temperature, 10 min). Proteins

21

were digested overnight at 37℃ with 5 mg trypsin (Promega). Samples were

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dried down in a vacuum centrifuge and reconstituted with 30 mL of 500mM

23

TEAB. Each sample was labeled using 8-plex iTRAQ kit as the manufacturer’s

24

instructions (Applied Biosystems, CA). In this study, five groups of control,

25

myocardial ischemic rats without treatment (MI), myocardial ischemic rats

26

treated with RG, OP, and SM were labeled with 116, 117, 118, 119, and 121

27

iTRAQ reagents respectively, and three independent biological replications

28

were performed. iTRAQ reagent labels (116, 117, 118, 119 and 121) were re-

29

suspended in a final concentration of 70% v/v ethanol, added to the respective 7



Page 8 of 43

1

samples and incubated at room temperature for 1 h. The reaction was

2

quenched by adding 100 mL of ultrapure water to each sample. The iTRAQ

3

labeled samples were mixed in equal ratios, dried in a vacuum centrifuge and

4

stored at -20℃.

5 6

2.3 2D LC/MS/MS Analysis

7

The combined peptide mixture was fractionated by strong cation exchange

8

(SCX) chromatography on a 20AD high-performance liquid chromatography

9

(HPLC) system (Shimadzu; Kyoto, Japan) using a polysulfoethyl column (2.1 ×

10

100 mm, 5 μm, 200 Å; The Nest Group, Southborough, MA). The mixed sample

11

was diluted with a loading buffer (10 mM KH2PO4 in 25% ACN, pH 2.6) and

12

loaded onto the column. Buffer A was identical in composition to the loading

13

buffer, and buffer B was buffer A containing 350 mM KCl. Separation was

14

performed using a linear binary gradient of 0–80% buffer B in buffer A at a flow

15

rate of 200 μL/min for 60 min. The absorbance at 214 nm and 280 nm was

16

monitored, and a total of 20 SCX fractions were collected along the gradient.

17

SCX fractions were reconstituted in 2% ACN, 1% TFA in dI water. Seven less

18

abundant fractions, as determined by inspection of SCX chromatograms, were

19

pooled resulting in a total of 14 fractions for RP chromatography. These

20

samples were injected into a Tempo LC MALDI Spotting System, a single-stage

21

HPLC and spotter (Applied Biosystems), equipped with a ProteCol (C18, 3 mm,

22

120 Å, 300 mm × 610 mm) trap column (SGE) and an Onyx Monolithic (C18,

23

150 mm × 60.1 mm) analytical column (Phenomenex, Torrance, CA). Samples

24

eluting at 2 uL/min during a 98% Buffer A (2% ACN, 0.1% TFA) to 45% Buffer

25

B (98% ACN, 0.1% TFA) gradient over 30 min were mixed 1:1 with 7 mg/mL

26

CHCA spiked with 10 mM ammonium phosphate and spotted every 8 s onto

27

Opti-TOF LC/MALDI 123 mm × 81 mm plates (Applied Biosystems). Four

28

hundred ninety-nine spots were collected per fraction with a maximum of two

29

SCX fractions on each plate. 8


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1 2

2.4 Protein Identification by MALDI-TOF-TOF and Data Analysis

3

MALDI plates were analyzed on a 5800 MALDI TOF/TOF Analyzer (Applied

4

Biosystems). MS spectra were acquired in positive ion reflector mode over a

5

mass range of 700–3600 m/z with 1000 laser shots per spot. MS data were

6

processed with internal calibration and filtered through the following criteria to

7

determine a maximum of 50 precursors for MS/MS per spot: minimum

8

signal/noise of 25 and fraction-to-fraction mass tolerance of 200 ppm. Tandem

9

MS spectra were acquired with 2500 laser shots and a 100 resolution (full width

10

at half maximum, FWHM) mass window per precursor. Fragmentation was

11

induced with 2 kV collision energy. MS/MS data were processed with default

12

calibration. The resulting peak lists were submitted for database sequence

13

searches using ProteinPilot Software (version 4.5, Applied Biosystems). The

14

following parameters were set in the searching: trypsin as enzyme, fixed

15

modification of methyl methanethiosulfonate, iTRAQ as sample type, no special

16

factors,

17

parameters, such as tryptic cleavage specificity (default), precursor ion mass

18

accuracy (100ppm), and fragment ion mass accuracy (0.6Da) were built-in

19

functions of ProteinPilot Software, and the Paragon method was adopted. The

20

MS data of missing values and shared peptides have been discarded by

21

ProteinPilot inserted function after uploading the raw data into the software. The

22

results showed the average ratio of different peptides and were further analyzed.

23

The relative quantification was calculated based on the ratio of the reporter ions

24

intensities at m/z 117.1, 118.1, 119.1, and 121.1 corresponding to the

25

myocardial ischemia condition as well as treated with RG, OP, and SM, over

26

the reporter ion intensity at m/z 116.1 corresponding to sham-operated

27

condition. Criteria to identify significantly differentially expressed proteins were

28

set as: greater than 1.50-fold change in iTRAQ ratio, p < 0.05 (Student’s t test,

29

assuming unequal variance), and at least 2 peptides with confidence >95%

30

detected. All identified proteins with these expression ratios are reported in

biological

modification,

thorough

identification

search.

Other

9



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1

Supporting Information 2.The data has been uploaded into Proteomexchange

2

database

3

ftp://massive.ucsd.edu/MSV000081517).

with

registration

number

MSV000081517

with

(URL

4 5 6

2.5 Protein Function Analysis and Pathway Analysis The DAVID software tool (http://david.abcc.ncifcrf.gov)

12, 13

was used to

7

annotate the function of the confidently identified proteome. Cellular localization

8

and functional processing categories were generated based on the annotations

9

of Gene Ontology. Ingenuity Pathway Analysis (IPA) software (Ingenuity

10

Systems, http://analysis.ingenuity.com) was employed to perform enrichment

11

analyses of biological and canonical functions of the differentially abundant

12

proteins. STRING v10.5 was used to generate altered protein interaction data

13

and Cytoscape 3.3.0 was used to interpret the altered protein network.

14 15

2.6 Western Blotting

16

Total proteins of each sample (20μg) were separated by 15% (w/v) SDS-

17

PAGE, and transferred to a PVDF membrane (Millipore, Bedford, MA). After

18

blocking with 10% skim milk, the membrane were incubated (4 °C overnight)

19

with the corresponding primary antibodies, including rabbit polyclonal antibody

20

to MDH1, TNNC1 (Proteintech Group, Inc., Chicago, IL), mouse monoclonal

21

antibody to Tublin Actin(Beyotime Biotechnology, Haimen, China). After

22

extensive washing with TPBS for three times, the membranes were incubated

23

with HRP-conjugated anti-rabbit or anti-mouse secondary antibody. Proteins

24

were visualized using SuperSignal West Femto Maximum Sensitivity Substrate

25

(Thermo Scientific). The optical density (OD) was analyzed using Quantity One

26

software (v4.5.0, Bio-Rad).

27

2.7 Measurement of mitochondrial membrane potential and intercellular

28

ATP in hypoxic cardiac myocytes

29

Neonatal rat ventricular myocytes (NRVMs) was isolated from neonatal

30

Sprague-Dawley rats according to the previously described method with slight 10


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14.

1

modification

Briefly, 1- to 2-day-old neonatal Sprague-Dawley rats were

2

sacrificed for their cardiac ventricles. The ventricles were minced on ice and

3

digested in a step-wise manner in 0.05% collagenase Ⅱ containing 0.03%

4

tryspin dissolved in D-Hanks. The dissociated cells were transferred into a

5

75cm2 culture flask with 10% FBS, 90% DMEM-F12, 100U/ml penicillin, 100

6

μg/ml streptomycin and after one and a half hours incubation, the non-adherent

7

cells were moved to a new culture flask adding 0.1mM 5-Bromo-2’-deoxyuridine

8

in the medium under the environment of 37℃, 5% CO2.

9

To test the effects of RG, OP, and SM on energy metabolism and

10

mitochondrial function of ischemic myocardium, intercellular ATP contents and

11

mitochondrial membrane potential (ΔΨm) in hypoxic NRVMs were measured.

12

After incubated with SM, RG, and OP with 20μL/mL, hypoxia was performed by

13

placing plates containing cardiomyocytes with serum-free DMEM in an

14

anaeronic chamber (Billups-Rothenberg. Inc) filled with 95% N2 and 5% CO2

15

for 24 hours. In control normoxia experiments, cells were incubated with fresh

16

culture growth medium in an incubator atmosphere of 5% CO2 for 24 h.

17

The dissipation of ΔΨm was measured using confocal microscopy analysis

18

of NRVMs stained with 5,5’,6,6’-tetrachloro-1,1’,3,3-tetraethylbenzimidazole

19

carbocyan ideiodine (JC-1, Beyotime Biotechnology), which is a lipophilic cation

20

that can enter into mitochondria in proper conditions and a voltage-sensitive

21

fluorescent indicator, as previously described15. The image was captured by

22

Zeiss LSM510 META confocal laser scanning microscope (Zeiss, Thornwood,

23

NY), and laser excitation of 488nm and 543nm were used for green and red

24

fluoresence. The quantitative data of thirteen fields of each group from triplicate

25

experiments were processed using MetaMorph software.

26

The intercellular ATP was measured by luciferin-luciferase assay (ATP

27

Luminescence Kit, Beyotime Biotechnology) as previously described with

28

slightly modification

29

incubated with SM, RG, and OP (20μL/mL) were subjected to hypoxia for 24h,

16, 17.

In brief, NRVMs were cultured in 6-well plate. Cells

11



Page 12 of 43

1

while cells cultured in normal condition served as control. Subsequently, cells

2

were lysised for determining protein contents and ATP concentration. Standard

3

curve was generated to calculate ATP content, while protein content of each

4

sample was used for normalization.

5 6

2.8 Measurement of cellular energetics in normal and hypoxic cardiac

7

myocytes

8

Bioenergetic functions of cardiomyocytes following RG, OP, and SM

9

treatment were measured using a Seahorse Bioscience XF24 extracellular flux 18, 19.

10

analyzer (Billerica, MA) as previously described

11

seeded to a density of 75, 000 cells/well. After pretreatment of RG, OP, and SM

12

with 1μL/mL for 6 hours in normal or hypoxia condition, the medium were

13

washed out and changed to assay media, and the cells were then loaded into

14

the XF24. OCR (oxygen consumption rate, pmol/min) and PPR (proton

15

production rate, H+/min) of NRVMs were monitored. After baseline

16

measurements,

17

trifluoromethoxyphenylhydrazone (FCCP), antimycin A and rotenone with final

18

concentration of 1μM were sequentially injected, which allowed to determine

19

the basal level of oxygen consumption, the amount of oxygen consumption that

20

is ATP-linked, the maximal oxygen consumption, and the non-mitochondrial

21

oxygen consumption respectively.

22

2.9 Statistics

oligomycine,

Briefly, NRVMs were

carbonylcyanide-p-

23

All values are expressed as the means ± S.E.M. Unpaired Student t-test with

24

Welch’s correction was used to perform statistical analysis. P values of less

25

than 0.05 were considered statistically signiﬁcant.

26 27

3. Results and Discussion

28

3.1 Cardioprotective effects of SM on ischemic rat heart

29

Botanical drugs have been widely used for treating ischemic heart diseases,

30

the clinical benefits of SM have been found in patients with congestive or 12


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20, 21.

1

chronic heart failure

Our previous study also found that SM extracts can

2

attenuate the infarct size of heart in rats with acute ischemia 4. In present study,

3

the rats had intraperitoneal injection of SM for seven days before the permanent

4

occlusion of the left anterior descending coronary artery surgery. Histology

5

analysis was assessed in Sham, MI and SM treatment group, whilst valsartan

6

treated rats with myocardial infarction were serves as positive control. The

7

representative images of myocardial infarction section suggest that SM-treated

8

rats exhibited a modest reduce in infiltration of inflammatory cells as well as

9

fibrotic phenomenon compared to MI group.

10

11

Fig 2. Representative images of heart section. H&E (A) and Masson’s

12

trichrome(B) staining images showing the protective effects of SM in

13

myocardial infarction myocardium.

14 15

3.2 Identification of differentially expressed proteins and functional

16

classification

17

In order to have a global observation and better understanding of the proteins

18

regulated by SM in treating MI rats, a proteomic analysis based on iTRAQ 13



Page 14 of 43

1

technique was carried out as previously mentioned.22,

23

2

extracted from rats of Sham group, MI group and SM, RG or OP treatment

3

group. Then they were labeled by different iTRAQ reagment and subjected to

4

LC-MALDI for analysis. After quantification, differentially expressed proteins

5

were identified from ischemic myocardium of rats treated with SM, OP, and RG

6

compared to untreated rats with myocardial infarction, follwed by the

7

bioinformatics and functional classification and protein-protein network analysis.

Proteins were

8

Using iTRAQ-based proteomics analysis, totals of 1117, 1192, 1061

9

proteins were detected in three biological replicates (>95% confidence)

10

respectively, 711 proteins were commonly identified and quantified after

11

combined different peptides related to same protein accession. Differentially

12

expressed proteins which exhibited a 1.5-fold (or greater) or 1/1.5 fold (or lower)

13

expression were chosen for further analysis. Finally, we got a total of 101

14

differentially expressed proteins to be classified into different functional groups

15

according to the David Functional Annotation (p

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