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Keywords:

  • cardiomyocytes;
  • cell damage;
  • hypoxia;
  • mitochondrial permeability transition pore;
  • tumour necrosis factor receptor-associated protein 1

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Tumour necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone that plays a role in maintaining mitochondrial function and regulating cell apoptosis. The opening of the mitochondrial permeability transition pore (MPTP) is a key step in cell death after hypoxia. However, it is still unclear whether TRAP1 protects cardiomyocytes from hypoxic damage by regulating the opening of the pore. In the present study, primary cultured cardiomyocytes from neonatal rats were used to investigate changes in TRAP1 expression after hypoxia treatment as well as the mechanism and effect of TRAP1 on hypoxic damage. The results obtained showed that TRAP1 expression increased after 1 h of hypoxia and continued to increase for up to 12 h of treatment. Hypoxia caused an increase in cell death and decreased cell viability and mitochondrial membrane potential; overexpressing TRAP1 prevented hypoxia-induced damage to cardiomyocytes. The silencing of TRAP1 induced an increase in cell death and decreased both cell viability and mitochondrial membrane potential in cardiomyocytes under normoxic and hypoxic conditions. Furthermore, cell damage induced by the silencing of TRAP1 was prevented by the mitochondrial permeability transition pore inhibitor, cyclosporin A. These data demonstrate that hypoxia induces an increase in TRAP1 expression in cardiomyocytes, and that TRAP1 plays a protective role by regulating the opening of the mitochondrial permeability transition pore.


Abbreviations
Ad-TRAP1

recombinant adenovirus vector for TRAP1 overexpression

CsA

cyclosporin A

CypD

cyclophilin D

GFP

green fluorescent protein

HSP

heat shock protein

MPTP

mitochondrial permeability transition pore

ROS

reactive oxygen species

siRNA

small interfering RNA

TRAP1

tumour necrosis factor receptor-associated protein 1

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Hypoxia is one of the main causes of myocardial damage after the receipt of a burn. In the early stages after a severe burn, myocardial damage not only causes cardiac insufficiency, but also induces or aggravates burn shock, which can cause or aggravate ischaemic/hypoxic injury to other organs [1,2]. Hence, it is important to protect cardiomyocytes from hypoxic damage. Mitochondria are the primary target of hypoxic damage in cardiomyocytes. Several inter-related factors, including calcium overload, an increase in reactive oxygen species (ROS) and a decrease in adenine nucleotides, contribute to mitochondrial impairment during hypoxia and ischaemia [3]. Mitochondrial dysfunction in cardiomyocytes can also directly lead to cell death after hypoxia. The mitochondrial permeability transition pore (MPTP) is a nonspecific pore that opens during the time of calcium overload, oxidative stress, adenine nucleotide depletion and elevated phosphate levels. Many studies have demonstrated the role of MPTP opening during an ischaemia/reperfusion injury to the heart and other organs [4–6]. We have also demonstrated that more MPTPs open in cardiomyocytes after hypoxia compared to normoxic conditions [7]. Once the pore opens, the membrane potential and pH gradient dissipate, preventing ATP generation by oxidative phosphorylation. Ultimately, these changes lead to cell death through the activation of phospholipases, nucleases and proteases [8]. Indeed, the irreversible mitochondrial injury caused by MPTP opening is the key step in cell death that occurs during hypoxia and other conditions [9].

Tumour necrosis factor receptor-associated protein 1 (TRAP1) localizes to the mitochondria and its targeting sequence, which is found in the N-terminus of the protein, is for mitochondria matrix. An analysis of the cDNA sequences reveals that TRAP1 is identical to heart shock protein (HSP) 75, which is a member of the HSP90 family [10]. HSP90 comprises an important molecular chaperone that is involved in many cellular processes. After hypoxia treatment, HSP90 expression increases, and this plays a protective role against damage [11]. However, the changes in TRAP1 in cardiomyocytes under hypoxic conditions remain unclear. TRAP1 comprises a mitochondrial chaperone that is critical for importing proteins into the mitochondrial matrix [12]. A previous study showed that up-regulation of TRAP1 expression suppressed arsenite-induced apoptosis in lung epithelium cells [13]. Apoptogenic inducers, such as the protein-tyrosine kinase inhibitor β-hydroxyisovalerylshikonin or the topoisomerase II inhibitor VP16, can decrease TRAP1 expression [14]. At the same time, TRAP1 antagonizes ROS production and protects tumour cells from granzyme M-mediated apoptosis [15]. A recent study also demonstrated that TRAP1 overexpression preserves the mitochondrial membrane potential (Δψ) and maintains ATP levels and cell viability during ischaemic-like injury in vivo [16]. These data suggest that TRAP1 may play an important role in maintaining mitochondrial function. As noted above, MPTP is recognized as a key player in cell death. However, whether TRAP1 can protect cells from hypoxic damage by regulating MPTP opening in cardiomyocytes has remained unclear until now.

The present study aimed to observe changes in TRAP1 expression after hypoxia treatment and to investigate the effect of TRAP1 on cell death and MPTP opening in primary cardiomyocytes.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Hypoxia increases TRAP1 expression in cardiomyocytes

Western blot analysis was used to investigate TRAP1 expression after hypoxia treatment in cardiomyocytes. TRAP1 content increased after 1 h of hypoxia and continued to increase until for up to 12 h compared to the normoxic group. At the same time, longer hypoxic treatments yielded higher TRPA1 expression (Fig. 1A,B). We then examined TRAP1 immunoreactivity with an immunofluorescence assay. After 1 h of hypoxia, TRAP1 fluorescence intensity was brighter in hypoxic cells than in normoxic cells, which meant that TRAP1 expression increased after 1 h of hypoxia (Fig. 1C,D). Furthermore, increases in TRAP1 fluorescence intensity became greater with an extension of hypoxic treatment time (Figs 1E–G and 2I). The results obtained were similar to those observed with the western blot.

image

Figure 1.  Effects of hypoxia on the TRAP1 levels in primary cultured cardiomyocytes. (A) Western blots show TRAP1 immunoreactivity in normoxic or hypoxic cells at the indicated times. β-actin was used as an internal control. (B) TRAP1 levels were normalized with β-actin under normoxic or hypoxic conditions. (C–G) TRAP1 expression detected by immunofluorescence under normoxic conditions (C) and hypoxic conditions for 1 h (D), 3 h (E), 6 h (F) and 12 h (G). TRAP1 primary antibody was omitted as a negative control (H). (I) Differences in fluorescence intensity of TRAP1 in normoxic or hypoxic cells. Data are the mean ± SEM. Scale bar = 25 μm. *P < 0.05 compared to the normoxic group. The experiment was repeated three times.

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image

Figure 2.  TRAP1 overexpression prevented the hypoxia-induced reductions in cell viability and Δψ in primary cultured cardiomyocytes. (A) Cardiomyocytes were infected with negative vector or Ad-TRAP1 for 48 h and then observed under a fluorescence microscope to determine the infection efficiency by visualizing expression of the gene for GFP. Scale bar = 200 μm. (B) Expression of TRAP1 levels in the uninfected control, negative vector-infected and Ad-TRAP1-infected cardiomyocytes as determined by western blotting. (C, D) Cardiomyocytes were infected with vector or Ad-TRAP1 for 48 h, starved, and then treated for 6 h under hypoxic conditions; cell viability was determined with a cell counting kit (C) and Δψ was determined with tetramethylrhodamine ethylester; and then one hundred cells from each group were randomly chosen to measure fluorescence intensity (D). Data are the mean ± SEM. *P < 0.05 compared to the normoxic group. #P < 0.05 compared with the hypoxic and hypoxia + vector groups. The experiment was repeated three times.

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TRAP1 overexpression decreases hypoxic damage to cardiomyocytes

Because TRAP1 expression of cardiomyocytes was increased after hypoxia treatment, we performed experiments to determine whether the increase in TRAP1 expression plays a protective role in hypoxic cardiomyocytes. We constructed a recombinant adenovirus vector for TRAP1 overexpression (Ad-TRAP1) and transfected the cardiomyocytes. After 48 h of infection, infection efficiency was visualized by the expression of green fluorescent protein (GFP), and more than 90% of the cardiomyocytes were infected (Fig. 2A). Protein was then harvested and the results obtained by western blotting revealed that TRAP1 expression increased significantly in cardiomyocytes infected with Ad-TRAP1 compared to the expression in negative vector-transduced cardiomyocytes and to endogenous TRAP1 levels in normoxic cells (Fig. 2B).

To evaluate the role of TRAP1 overexpression in cardiomyocytes under hypoxic conditions, we investigated cell viability, Δψ and cell death. After 6 h of hypoxia, cell viability and Δψ were significantly lower in the uninfected and vector-infected cardiomyocytes compared to normoxic cells. By contrast, TRAP1 overexpression increased hypoxic cell viability (Fig. 2C) and preserved Δψ (Fig. 2D). Additionally, propidium iodide staining was used to investigate the effect of TRAP1 overexpression on cell death. As shown in Fig. 3, hypoxia treatment resulted in increased cell death, which was reduced by TRAP1 overexpression. At the same time, infection with the negative vector had no effect on hypoxia-induced cell death.

image

Figure 3.  TRAP1 overexpression decreased hypoxia-induced cell death in primary cultured cardiomyocytes. Cell death was determined by incubating normoxic cells, hypoxic cells, vector-infected hypoxic cells and Ad-TRAP1-infected cells after 6 h of hypoxia with Hoechst 33342 (10 μg·mL−1, blue) and propidium iodide (PI) (10 μg·mL−1, red). Scale bar = 50 μm. Graphs show the quantification of cell death (mean ± SEM) and 200–300 cells were counted for each group. *P < 0.05 compared to the normoxic group. #P < 0.05 compared to the hypoxic and hypoxic + vector groups. The experiment was repeated three times.

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Silencing of TRAP1 expression induces cardiomyocyte damage

After demonstrating that TRAP1 overexpression can prevent hypoxic damage in cardiomyocytes, we next examined whether silencing TRAP1 expression induced damage in cardiomyocytes. After infection with TRAP1-small interfering RNA (siRNA) or control vector adenovirus for 4 days, more than 90% of the cardiomyocytes were determined to be infected by observing GFP expression using a fluorescent microscope (Fig. 4A). The effective silencing of endogenous TRAP1 by TRAP1-siRNA adenovirus infection was also confirmed by western blotting (Fig. 4B).

image

Figure 4.  Silencing TRAP1 expression induced cell viability and Δψ in primary cultured cardiomyocytes. (A) Cardiomyocytes were infected with negative vector or TRAP1-siRNA for 4 days, and then a fluorescence microscope was used to observe the infection efficiency by visualizing expression of the gene for GFP. Scale bar = 200 μm. (B) Expression of TRAP1 levels in uninfected control, vector-infected and TRAP1-siRNA-infected cardiomyocytes as determined by western blotting. (C) Cardiomyocytes were infected with vector or TRAP1-siRNA for 4 days, starved, and then cell viability was determined under normoxic conditions. (D) Cardiomyocytes were infected with vector or TRAP1-siRNA for 4 days, starved, and then Δψ was determined under normoxic conditions or after 6 h of hypoxia. The results are shown as the mean ± SEM. *P < 0.05 compared to the normoxic and normoxic + vector groups. #P < 0.05 compared to the hypoxic and hypoxic + vector groups. The experiment was repeated three times.

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After TRAP1-siRNA infection, the viability of the cardiomyocytes was significantly decreased compared to that of normoxic cells and vector-infected cells (Fig. 4C). Furthermore, silencing TRAP1 expression induced a decrease in Δψ of cardiomyocytes under normoxic conditions and aggravated Δψ loss induced by hypoxia (Fig. 4D). As shown in Fig. 5, TRAP1 depletion also induced a significant increase in cardiomyocytes death, whereas there was very little cell death in the normoxic cardiomyocytes and vector-infected cardiomyocytes.

image

Figure 5.  Silencing TRAP1 expression induced cell death in primary cultured cardiomyocytes under normoxic conditions. Cell death was determined by incubating uninfected, vector-infected and TRAP1-siRNA-infected cardiomyocytes under normoxic conditions with Hoechst 33342 (10 μg·mL−1, blue) and propidium iodide (PI) (10 μg·mL−1, red). Scale bar = 50 μm. Graphs show the quantification of cell death (mean ± SEM) and 200–300 cells were counted for each group. *P < 0.05 compared to the normoxic and normoxic + vector groups. The experiment was repeated three times.

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In addition, we also observed the effect of silencing TRAP1 expression on cardiomyocyte damage under hypoxic conditions. It was found that hypoxia induced more injuries in cardiomyocytes in terms of both viability and cell death after TRAP1-siRNA infection (Fig. 6A,B).

image

Figure 6.  CsA prevented hypoxic damage after TRAP1-siRNA infection in primary cardiomyocytes. CsA (2 μm) was added into vector-infected and TRAP1-siRNA-infected cardiomyocytes after 2 days of infection. The cells were then starved, and subjected to hypoxia for 6 h after 4 days of infection. (A) Effects of CsA on cell death in uninfected, vector-infected and TRAP1-siRNA-infected cells under hypoxic conditions. In each group, 200–300 cells were counted. (B) Effects of CsA on cell viability in uninfected, vector-infected and TRAP1-siRNA-infected cells under hypoxic conditions. *P < 0.05 compared to the normoxic group. #P < 0.05 compared to the hypoxic and hypoxic + vector groups. **P < 0.05 compared to the hypoxic and hypoxic + vector groups. ##P < 0.05 compared to the hypoxic + TRAP1-siRNA group (data are the mean ± SEM). The experiment was repeated three times.

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MPTP mediates the TRAP1 effect

TRAP1 is a mitochondria chaperon and plays a role in maintaining mitochondrial homeostasis, whereas MPTP opening is a key step in the process of cell death. Therefore, we aimed to determine whether MPTP opening mediates TRAP1 behaviour. After cardiomyocytes were infected with TRAP1-siRNA or negative vector for 2 days, cyclosporin A (CsA; 2 μm), a selective inhibitor of MPTP opening, was added to the cardiomyocytes. Cells were then infected for an additional 2 days (4 days in total). Treatment with CsA prevented the decrease in cardiomyocyte viability and the increase in cell death induced by TRAP1-siRNA infection under normoxic conditions (Fig. 7). However, there were no differences between vector-infected cells and vector-infected cells after CsA treatment (Fig. 7).

image

Figure 7.  CsA prevented the cell damage induced by silencing TRAP1 in primary cardiomyocytes under normoxic conditions. CsA (2 μm) was added to vector-infected and TRAP1-siRNA-infected cardiomyocytes after 2 days of infection. The cells were then subjected to cell viability and cell death assay after 4 days of infection. (A) Effects of CsA on cell death in uninfected, vector-infected and TRAP1-siRNA-infected cells. In each group, 200–300 cells were counted. (B) Effects of CsA on cell viability in uninfected, vector-infected and TRAP1-siRNA-infected cells. *P < 0.05 compared to the normoxic and normoxic + vector groups. #P < 0.05 compared to the normoxic + TRAP1-siRNA group (data are the mean ± SEM). The experiment was repeated three times.

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Because silencing TRAP1 expression aggravated hypoxic damage of cardiomyocytes, we next investigated the effect of CsA on cell viability and cell death after TRAP1-siRNA infection under hypoxic conditions. After 6 h of hypoxia, treatment with CsA abolished cardiomyocyte damage induced both by hypoxia and silencing TRAP1 under hypoxic conditions (Fig. 6). On the basis of the results described above, we conclude that silencing TRAP1 expression induces MPTP opening in cardiomyocytes, resulting in cell injury. Furthermore, the up-regulation of TRAP1 expression may play a protective role in hypoxic cardiomyocytes by reducing MPTP opening.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

In the present study, we found that TRAP1 expression of cardiomyocytes increases after hypoxia and that TRAP1 overexpression protects cardiomyocytes from hypoxic damage. At the same time, silencing TRAP1 expression causes cell damage under normoxic and hypoxic conditions. Our data also indicate that TRAP1 plays a role in cardiomyocytes by regulating MPTP opening.

TRAP1 was initially identified by the yeast two-hybrid system as a novel protein that interacted with the intercellular domain of the type 1 tumour necrosis factor receptor [17]. On the basis of the sequence of the homologue, TRAP1 was identified as a member of the HSP90 family. The ATPase activity of TRAP1 is inhibited by geldanamycin, which is a specific inhibitor of HSP90. Despite its ATP-binding activity, TRAP1 does not form a stable complex with the co-chaperones of HSP90, such as Hop and p23 [18]. Studies have shown that TRAP1 does not have a C-terminal EEVD sequence, which exists in HSP90 and is important for HSP90-Hop binding [19]. Thus, it appears that TRAP1 has specific functions that are different from those of other well-characterized HSP90 homologues. TRAP1 is up-regulated by glucose deprivation, oxidative stress and ultraviolet A irradiation, but cannot be induced by heat [16,20,21]. Furthermore, deferoxamine, an iron chelator, decreases TRAP1 levels in a dose- and time-dependent manner and induces mitochondrial dysfunction in human hepatocytes [22]. However, the changes induced in TRAP1 expression in cardiomyocytes after hypoxia treatment are still unclear. In the present study, we demonstrated that hypoxia treatment (for 1, 3, 6 and 12 h, respectively) induces a time-dependent increase in the levels of TRAP1 protein.

Hypoxia is a common pathophysiological process in diseases such as shock, stroke and heart failure. Hypoxic damage of the myocardium is relevant not only to coronary artery diseases, but also to hypertensive and cardiomyopathic heart disease [23,24]. Mitochondria are the most susceptible organelles to hypoxic damage in cardiomyocytes. Although hypoxia induced TRAP1 expression increases in cardiomyocytes, the role of that TRAP1 increase remains unclear. The question remains as to whether the hypoxia-induced TRAP1 increase is a protective reaction in cardiomyocytes. Because TRAP1 is a mitochondrial chaperone, it has an important role in regulating cell apoptosis and maintaining mitochondrial homeostasis and function. Silencing TRAP1 enhances cytochrome c release from the mitochondria and apoptosis induced by β-hydroxyisovalerylshikonin and VP16 [14]. TRAP1 depletion also sensitizes PC12 cells to oxidative stress-induced cytochrome c release and cell death, which means that TRAP1 play a role in the modulation of the mitochondrial apoptotic cascade [25]. Moreover, TRAP1 overexpression improves mitochondrial function after ischaemic injury in primary astrocytes in vitro [16]. In the present study, we found that TRAP1 overexpression abolishes the hypoxic damage in cardiomyocytes. Silencing TRAP1 expression not only induces cell damage under normoxic conditions, but it also aggravates hypoxic damage of cardiomyocytes.

MPTP is a channel consisting of several proteins that is usually in a low permeability or closed state. Some models have proposed the presence of other molecular components of the pore, although there is still no consensus regarding the exact components. However, cyclophilin D (CypD) is generally accepted as a critical regulatory component of MPTP and plays an important role in regulating MPTP opening [8,26]. CsA, a selective MPTP inhibitor, prevents MPTP opening by inhibiting the activity of the peptidyl-prolyl cis-trans isomerase of CypD [27,28]. The consequences of MPTP opening are cell necrosis and apoptosis and, even if MPTP opening is insufficient to cause necrosis, apoptosis can occur. After the MPTP opens, apoptogenic substrates (i.e. cytochrome c) are released into the cytoplasm and activate caspase-dependent apoptotic pathways. Because MPTP plays a critical role in cell necrosis and apoptosis, it is also involved in protecting cell against hypoxic and ischaemic damages [29,30]. MPTP not only contributes to the early and delayed protective effects of ischaemic preconditioning in rat or rabbit heart, but it is also relevant to ischaemic post-conditioning [31]. We had also previously demonstrated that adenosine A1 receptor activation reduces hypoxic damage by preventing MPTP opening in rat cardiomyocytes [7]. Many studies have demonstrated that Δψ loss iš accompanied by an increase in MPTP opening [32–34]. It is considered that Δψ reflects the state of MPTP opening indirectly. In the present study, we found that silencing TRAP1 induces Δψ loss in cardiomyocytes, and that overexpression of TRAP1 suppresses Δψ loss caused by hypoxia. Furthermore, our present data also show that CsA prevents the cell damage induced by TRAP1 depletion under normoxic and hypoxic conditions, which means that silencing TRAP1 expression can cause MPTP opening and lead to damage. Because the opening of MPTP increases after hypoxia treatment, and TRAP1 overexpression abolishes hypoxic damage, we therefore assume that TRAP1 overexpression may prevent MPTP opening and having a protective effect under hypoxic conditions in cardiomyocytes. In tumour cells, TRAP1 interacts with CypD, and the association of TRAP1 with CypD is prevented by CsA and not geldanamycin, suggesting that this association may be necessary for CypD activity [35].

Many factors are involved in inducing MPTP opening, especially calcium overload and oxidative stress [36,37]. ROS increases could lead to the MPTP opening persistently. However, TRAP1 also shows an important role in regulating ROS generation. ROS production is decreased by TRAP1 overexpression and promoted by silencing TRAP1 expression [15,16,38]. Because TRAP1 plays a role against cell damage by MPTP, further studies are needed to determine whether ROS are mediators between TRAP1 and MPTP in cardiomyocytes.

In summary, hypoxia increases the level of TRAP1 in cardiomyocytes, which may protect cells from hypoxic damage by regulating MPTP opening. These results provide us with a deeper understanding of the protective role of TRAP1 in cardiomyocytes and offer new considerations for myocardial protection after burn shock.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Cardiomyocyte culture and hypoxia treatment

Primary cardiomyocyte cultures were prepared from the ventricles of neonatal Sprague-Dawley rats (days 1–3) and trypsinized as described previously [39] in accordance with a protocol approved by the Animal Care and Use Committee of the Third Military Medical University. The cultures were grown in a DMEM/F12 medium (Hyclone, Logan, UT, USA) with 10% (v/v) fetal bovine serum (Hyclone), 0.1 mm bromodeoxyuridine (Sigma-Aldrich, St Louis, MO, USA), 100 U·mL−1 penicillin and 100 U·mL−1 streptomycin. Cells were maintained in a 5% CO2 incubator at 37 °C. Before hypoxia treatment, the cardiomyocytes were deprived of serum for 12 h.

Hypoxic conditions were prepared by using an anaerobic jar (Mitsubishi, Tokyo, Japan) and a vacuum glove box (Chunlong, Lianyungang, China). Serum-free medium was placed in the vacuum glove box filled with a mixed gas containing 94% nitrogen, 5% CO2 and 1% O2 overnight and allowed to equilibrate with the hypoxic atmosphere. Cardiomyocytes were then subjected to hypoxic conditions by replacing the normoxic medium with hypoxic medium and placing the cultures in an anaerobic jar. All procedures were performed in vacuum glove box.

Recombinant adenovirus vector for TRAP1 overexpression

Ad-TRAP1 and a negative adenovirus vector were produced by Shanghai GeneChem, Co. Ltd (Shanghai, China). The vectors encoded the GFP sequence, which served as a marker gene. A high titre adenovirus stock was made after several rounds of amplification in HEK293A (American Type Culture Collection, Manassas, VA, USA). All recombinant adenoviruses were tested for transgene expression in cardiomyocytes by western blotting. Cardiomyocytes were infected with Ad-TRAP1 or a negative vector at a multiplicity of infection of 10 for 48 h and then subjected to experiments after being deprived of serum for 12 h.

Recombinant adenovirus vector for silencing of TRAP1 expression

The recombinant adenovirus vector for silencing of TRAP1 expression (TRAP1-siRNA) was purchased from Shanghai GeneChem, Co. Ltd. The targeting sequence of the siRNA against rat TRAP1 was 5′-CAACAGAGATTGATCAAAT-3′. A negative control adenovirus vector containing nonspecific siRNA was constructed in the same way (nonspecific vector, 5′-TTCTCCGAACGTGTCACGT-3′). All vectors contained the gene for GFP, which served as a marker. Cardiomyocytes were infected with TRAP1-siRNA or control vector by the addition of adenovirus to the cell culture at a multiplicity of infection of 10. After 4 days of infection, the cells were serum starved for 12 h and then treated.

Preparation of cell lysates

Cells were washed three times with ice-cold NaCl/Pi at the appropriate time after treatment, and lysed in radioimmunoprecipitation assay (Sigma-Aldrich) lysis buffer that contained 2 μg·mL−1 aprotinin, 2 μg·mL−1 pepstatin, 2 μg·mL−1 leupeptin and 100 μg·mL−1 phenylmethanesulfonyl fluoride. Cells were then scraped, and the resulting lysate was ultrasonicated and centrifuged at 12 000 g for 20 min at 4 °C. The supernatant was subjected to western blot analysis.

Western blot analysis

Protein concentrations were determined by the RC DC assay (Bio-Rad, Hercules, CA, USA). Thirty micrograms of proteins were fractionated by 10% SDS-PAGE and then transferred to a poly(vinylidene difluoride) membrane (Roche, Rotkreuz, Switzerland). The membrane was blocked with 5% (w/v) skim milk in TBST [20 mm Tris-HCl (pH 8.0), 150 mm NaCl and 0.1% (v/v) Tween-20] for 2 h at room temperature. Next, the membrane was probed with a 1 : 500 dilution of primary anti-TRAP1 serum (BD Biosciences, San Jose, CA, USA) in blocking buffer at 4 °C overnight. The membrane was washed four times with TBST and incubated with a horseradish peroxidase-conjugated antibody against mouse IgG for 1 h at room temperature. The membrane was then rinsed with TBST, and the protein bands were visualized with ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA). The images were analysed with quantity one 4.1 software (Bio-Rad). The experiment was repeated three times, and the same results were obtained.

Immunofluorescence assay

Cardiomyocytes were grown on coverslips. After hypoxia treatment, the cells were fixed with 4% (w/v) formaldehyde in NaCl/Pi for 10 min and permeabilized with 0.2% (v/v) Triton X-100 for 15 min at room temperature. Nonspecific binding sites were blocked by incubating the coverslips with 10% (v/v) goat serum in NaCl/Pi for 1 h. Cells were probed with primary anti-TRAP1 serum at a 1 : 100 dilution overnight at 4 °C, washed with NaCl/Pi, and incubated in the dark at 37 °C for 1 h with fluorescein isothiocyanate-conjugated IgG. The cells were then washed again with NaCl/Pi and stained with 0.4 mg·mL−1 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) for 10 min at room temperature. Microscopic images were acquired using a Leica Confocal Microscope (Leica Microsystems, Wetzlar, Germany). In the negative control, the primary antibody was omitted.

Detection of cardiomyocyte viability

Cardiomyocyte viability was determined with a cell counting kit (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan). Cells were cultured in 96-well plates (10 000 cells per well) and the original medium was removed after 6 h of hypoxia. Then, 10 μL of CCK-8 solution and 100 μL of DMEM/F12 medium were added to each well, and the cells were incubated at 37 °C in the dark for 1 h in accordance with the manufacturer’s instructions. The value of D450 was determined (n = 3) and the experiment was repeated three times.

Cell death assays

Cell death was quantified in Hoechst 33342 (10 μg·mL−1; Sigma-Aldrich) and propidium iodide (10 μg·mL−1; Sigma-Aldrich)-labelled cells. Propidium iodide readily penetrates cells with compromised plasma membranes (dead cells) but does not cross intact plasma membranes. Hoechst is a cell-permeable nucleic acid stain that labels both live and dead nuclei.

Mitochondrial membrane potential

Δψ was monitored by tetramethylrhodamine ethylester (Sigma-Aldrich). Cells cultured in a serum-free medium were incubated in the dark with 200 nmol·L−1 tetramethylrhodamine ethylester at 37 °C for 15 min. Cells were then washed with NaCl/Pi and observed using a laser scanning confocal microscope. The experiment was repeated three times.

Statistical analysis

All values were expressed as the mean ± SEM. spss, version 11.0 (SPSS Inc., Chicago, IL, USA) was used to conduct analyses of variance and Tukey’s tests. P < 0.05 was considered statistically significant.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

This work was supported by the Key Project of China National Programs for Basic Research and Development (2005CB522601), the Key Program of National Natural Science Foundation of China (30430680), the Program for Changjiang Scholars, and the Innovative Research Team in University (IRT0712). We thank Sun Wei and Wang Li-ting (Central Library of The Third Military Medical University) for their technical assistance with the laser scanning confocal microscope. The authors declare that there are no conflicts of interest.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References
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