Introduction
Myocardial ischemia and reperfusion generate a large amount of reactive oxygen species (ROS) in cardiomyocytes subject to injury. ROS assaults intracellular organelles, cell membranes, and biological macromolecules including nucleic acid, protein, and lipid, resulting in oxidative stress and cell apoptosis [
1,
2]. Catalase (CAT) is one of essential enzymes metabolizing oxygen free radical via breakdown of H
2O
2 into H
2O and O
2, and thus protects cells from oxidative damage. However, exogenous CAT does not enter living cells automatically because of its poor permeability and cell membrane selectivity. Its translational value in protecting cells from oxidative stress damage, therefore, is very limited.
A great deal of efforts have been made to deliver full-length proteins into mammalian cells. Morris Group has designed a new type of PEP-1 peptide carrier (KETWWETWWTEWSQPKKKRKV) that enables the entering of large proteins into living cells [
3]. In fact, several laboratories have successfully delivered full-length PEP-1 fusion proteins into cultured cells and nervous system by using this PEP-1 peptide carrier, including EGFP, β-Gal, antibodies, cyclophilin A, and human copper chaperone for Cu, Zn-SOD1 and CAT [
4‐
7]. Our previous studies have demonstrated that PEP-1-CAT fusion proteins can be transduced into myocardium and protect against myocardial injury induced by ischemia-reperfusion in rats [
8].
Cardiomyocyte apoptosis is an inevitable process during myocardial ischemia-reperfusion-induced injury [
9]. We have previously reported an anti-apoptotic effect of PEP-1-CAT on H9c2 cardiomyocytes [
10]. However, detailed mechanisms underlying the effect of PEP-1-CAT on H/R-induced H9c2 remain unknown. In the present study, we used the hypoxia-reoxygenation (H/R)-induced apoptosis model to investigate the mechanisms underlying the anti-apoptotic effect of PEP-1-CAT in H9c2 cells. H/R is a classic
in vitro model mimicking myocardial ischemia-reperfusion injury
in vivo. We found that PEP-1-CAT protected H9c2 from H/R-induced injury through blocking p38 MAPK activity and activating PI3K/Akt and Erk1/2 activity, which resulted in a blockade of Bax/Bcl-2/mitochondria apoptotic pathway and thus a reduction of cardiomyocyte apoptosis.
Materials and methods
Generation of biologically active PEP-1-CAT fusion protein
PEP-1-CAT fusion protein was isolated and purified as described by our laboratory previously [
11]. Briefly, two prokaryotic expression plasmids for CAT and PEP-1-CAT were constructed using TA-cloning method. Both recombinant proteins were tagged with six histidine residues (His-tag) at the amino terminus. The two proteins were expressed and purified separately as described [
11].
Cell culture
H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM,Invitrogen) with 5 g/L glucose supplemented with 15% (v/v) fetal bovine serum (FBS, Hangzhou sijiqing Biological Engineering Materials Co., Ltd., China). Cells were routinely grown to subconfluency (>90% by visual estimate) in 75 cm2 flasks at 37°C in a humidified atmosphere with 5% CO2 prior to passage and seeding for experiments. To observe the morphological alteration, H9c2 cells were grown on cover slips and observed using a microscope (Nikon, Japan). To examine the aberrant nuclei in apoptotic cells, H9c2 cells were stained with 4,6-Diamidino-2-phenylinole (DAPI), and the nuclei were observed using a fluorescent microscope.
Immunocytochemistry staining
H9c2 cells were grown to confluence in a 24-well plate and treated with purified PEP-1-CAT (2 μM) or CAT (2 μM). 6 h later, cells were washed twice with 1 × PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Immunocytochemistry staining was performed by using rabbit anti-Hisprobe (diluted 1:200) (Santa Cruz Biotechnology, USA) and mouse anti-Troponin T antibodies (diluted 1:200) (Santa Cruz Biotechnology, USA). Cells were then incubated with tetraethyl rhodamine isothiocyanate (TRITC)-conjugated rat anti-rabbit Ig G (diluted 1:250) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ig G (diluted 1:250) at 25°C for 1 h. After washing for 3 times with PBS, cells were incubated with DAPI (Sigma, USA) for 10 min. The immunostained cells were observed with a fluorescent microscope (Nikon, Japan).
Hypoxia-reoxygenation of H9c2 Cells
H9c2 cells were pretreated with or without PEP-1-CAT (2 μM) in low serum media (2% FBS) for 6 h followed by culturing in a low-oxygen condition (95% N
2 + 5% CO
2) for 21 h in a humidified hypoxia chamber (Stem Cell Technology, USA). After hypoxia incubation, the medium were replaced, and the cells were exposed to normal-oxygen condition (95% air + 5% CO
2) for reoxygenation for 6 h [
12]. Control cells were cultured in normoxic conditions. The supernatant and cells were collected separately for further analysis.
Measurement of lactate dehydrogenase (LDH) and malondialdehy (MDA) levels
H9c2 cells were treated with PEP-1-CAT, harvested and lysed as previously described LDH release and MDA content were measured using commercial kits (JianCheng Bioengineering Institute, China).
Superoxide anion production in H9c2
H9c2 cells were grown to confluence in a 24-well plate followed by H/R with CAT or PEP-1-CAT treatment. Cells were then split and cultured on cover slips and incubated with DHE (5 mM) (Beyotime Insitute of Brotechnology) at 37°C for 30 min. The DHE staining detecting superoxide anion production was observed using a fluorescent microscope (Nikon, Japan) or quantified by Flow Cytomety.
Annexin V and PI binding assay
Annexin V and PI fluorescein staining kit (Bender MedSystems, Austria) were utilized to measure H9c2 cell apoptosis by following the manufacturer’s instruction. Briefly, 1 × 106 cells were suspended in 200 μl 1 × binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Cells were then incubated with Annexin V (1:20) for 3 min followed by incubation with propidium iodide (PI, 1 mg/ml) for 15 min. Apoptosis rate was evaluated by Flow Cytometry.
Measurement of mitochondrial membrane potential
Mitochondrial transmembrane potential was assessed using a sensitive fluorescent dye, a lipophilic cationic probe JC-1 (Invitrogen, USA). H9c2 cells were grown on cover slips and incubated with 5 mM JC-1 dye (Molecular Probes) at 37°C for 15 min. Cells were then washed with PBS for three times and analyzed immediately with a fluorescent microscope. Red emission indicates membrane potential-dependent JC-1 aggregates in mitochondria. Green fluorescence reflects the monomeric form of JC-1 appearing in cytoplasm after mitochondrial membrane depolarization.
Quantitative reverse transcription polymerase chain reaction (qPCR)
Total RNA from H9c2 cells was extracted using TRIZOL Reagent (Invitrogen). RNA concentration was determined by UV spectrophotometry. qRT-PCR was performed using Thunderbird SYBR Master Mix (TOYOBO, Japan). Primer sequences were: Bcl-2: 5′-CGA CTT TGC AGA GAT GTC CA-3′ (forward), 5′-ATG CCG GTT CAG GTA CTC AG-3′ (reverse); Bax: 5′- CTG CAG AGG ATG ATT GCT GA-3′ (forward), 5′- GAT CAG CTC GGG CAC TTT AG-3′ (reverse); β-actin: 5′-GTC CAC CGC AAA TGC TTC TA-3′ (forward), 5′-TGC TGT CAC CTT CAC CGT TC-3′ (reverse). qPCR was performed on a Real-time PCR Detection System (Slan, Hongshi) with the following cycles: 95°C for 1 min, followed by 95°C for 15 s, 58°C for 15 s, and 72°C for 45 s for 40 cycles. β-actin expression was used as an internal control.
Western blot analysis
Western blot was carried out to detect protein expression using following primary antibodies: rabbit anti-Bax (Santa Cruz Biotechnology), mouse anti-Bcl-2 (Santa Cruz Biotechnology), rabbit anti-Caspase-3 (Santa Cruz Biotechnology), rabbit anti-PARP-1 (Santa Cruz Biotechnology), rabbit anti-phospho-p38 MAPK (cell signaling technology), and rabbit anti-p38 MAPK (cell signaling technology). The protein expression levels were visualized using enhanced chemiluminescence method.
Statistical analysis
All data are expressed as means ± SEM unless indicated otherwise. Differences among groups were determined by ANOVA. Differences between groups were determined by Student’s t-test with P < 0.05 considered statistically significant.
Discussion
Myocardial apoptosis is a significant pathophysiological event in myocardial ischemia-reperfusion injury [
9]. It is widely acknowledged that intervention of myocardial apoptosis is a very important approach to the prevention of myocardial ischemia-reperfusion injury [
15]. Reperfusion causes myocardium to produce a large amount of ROS including superoxide anion (O
2-·), hydroxyl radical (OH
-), and hydrogen peroxide (H
2O
2), etc [
16]. CAT, one of most important enzymes, can protect cells from oxidative damage. But its potential to be used to protect myocardium from H/R-induced injury is hindered by the poor permeability and the selectivity of cell membrane. By fusing CAT with a PEP-1 peptide, we were able to efficiently transduce PEP-1-CAT into H9c2 cells and protect myocardium from H/R-induced injury [
10]. The present study advanced our previous finding by identifying novel mechanisms underlying PEP-1-CAT function in protecting cardiomyoctyes. We have found that PEP-1-CAT protects H/R-induced injury of H9c2 cells by restoring H/R-induced alteration of H9c2 morphology, inhibiting H/R-induced production of O
2-·, and blocking LDH release and MDA production, the two indicators for hypoxia-reoxygenation injury [
17,
18].
ROS causes damages to intracellular macromolecules such as DNA breakage and lipid membrane peroxidation, leading to cell apoptosis [
19]. Our data demonstrate that PEP-1-CAT blocks H/R-induced H9c2 apoptosis by regulating mitochondria-related apoptotic pathways. Recent studies have shown that H/R injury induces mitochondria to produce a high level of ROS [
20,
21]. Excessive ROS damages mitochondria, opens its permeability transition pore (PTP) and thus induces mitochondrial permeability transition (MPT), leading to mitochondrial depolarization and outer membrane rupture, which causes cell apoptosis or death [
22,
23]. Our studies indicate that H/R induces a decreased mitochondrial membrane potential, suggesting an impairment of mitochondria function. PEP-1-CAT transduction, however, restores mitochondrial membrane potential. These data demonstrate that PEP-1-CAT protects H9c2 cells from H/R-induced apoptosis by maintaining mitochondria membrane integrity and function of cardiomyocytes. Moreover, previous studies indicate that Bcl-2 family is upregulated during the opening of PTP [
24]. Our results demonstrate that PEP-1-CAT regulates the expression of Bcl-2 family. PEP-1-CAT significantly increases Bcl-2 while decreasing Bax protein levels that are altered by H/R injury.
PEP-1-CAT prevents cardiomyocyte from H/R-induced injury by regulating multiple signaling pathways. Although a number of signaling pathways are involved in H/R-induced myocardial injury and apoptosis, PEP-1-CAT protects cardiomyocytes through down-regulation of p38 MAPK and activation of PI3K and Erk1/2 signaling pathways. PEP-1-CAT transduction inhibits p38 MAPK phosphorylation, suggesting that p38 MAPK mediates, at least in part, the function of PEP-1-CAT. Blockade of PI3K and Erk1/2 signaling significantly attenuates PEP-1-CAT-mediated reduction of H9c2 apoptosis, indicating that PI3K and Erk1/2 signaling pathways are essential for PEP-1-CAT activity in protecting cardiomocytes.
In summary, PEP-1-CAT transduction efficiently protects cardiomyocyte from H/R-induced apoptosis by blocking ROS production in mitochondria, which maintains mitochondria membrane integrity and inhibits the activation of Bcl2/Bax apoptotic pathway. Moreover, PEP-1-CAT blocks cardiomyocyte apoptosis by blocking p38 MAPK while activating PI3K and Erk1/2 MAPK signaling pathways. How these signaling pathways interact with each other in mediating PEP-1-CAT function will be a interesting subject for future study. Nevertheless, our study provides novel information and rationale for developing PEP-1-CAT as a therapeutic agent for treating or preventing myocardial ischemia-reperfusion injury.
Acknowledgements
This study was supported by grants from National Natural Science Foundation of China (81170095), Hubei Education Department Science Foundation (T200811, T201112), China and National Institutes of Health (HL093429 and HL107526 to S.Y.C.).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LZ and SW designed and performed the experiments, collected the data and analyzed the results. JNW and JMT participated in the experimental design and interpretation of the results. LYG made fusion protein and evaluated the apoptosis by Flow Cytometry. FZ carried out Western blot. XK performed part of the in vitro experiments. JYY and YZH carried out the immunoassays. SYC assisted with writing the manuscript. All the authors have read and approved the final manuscript.