Mitochondria-Derived Superoxide Links to Tourniquet-Induced Apoptosis in Mouse Skeletal Muscle

Our previous study has reported that superoxide mediates ischemia-reperfusion (IR)-induced necrosis in mouse skeletal muscle. However, it remains poorly understood whether IR induces apoptosis and what factors are involved in IR-induced apoptosis in skeletal muscle. Using a murine model of tourniquet-induced hindlimb IR, we investigated the relationship between mitochondrial dysfunction and apoptosis in skeletal muscle. Hindlimbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Compared to sham treatment, tourniquet-induced IR significantly elevated mitochondria-derived superoxide production, activated opening of mitochondrial permeability transition pore (mPTP), and caused apoptosis in the gastrocnemius muscles. Pretreatment with a superoxide dismutase mimetic (tempol, 50 mg/kg) or a mitochondrial antioxidant (co-enzyme Q10, 50 mg/kg) not only decreased mitochondria-derived superoxide production, but also inhibited mPTP opening and apoptosis in the IR gastrocnemius muscles. Additionally, an inhibitor of mPTP (cyclosporine A, 50 mg/kg) also inhibited both mPTP opening and apoptosis in the IR gastrocnemius muscles. These results suggest that mitochondria-derived superoxide overproduction triggers the mPTP opening and subsequently causes apoptosis in tourniquet-induced hindlimb IR.


Introduction
Exsanguinating injury of the extremity is a major cause of battlefield deaths and an important cause of preventable trauma fatalities in civilian medicine [1][2][3][4][5][6]. As an effective means of arresting life-threatening limb hemorrhage, tourniquet is commonly used in both civilian and battlefield settings [7][8][9]. However, stopping the blood flow in the traumatized limb with a tourniquet, and following reperfusion can cause the ischemiareperfusion (IR) injury [10]. Therefore, an understanding of the pathomechanisms responsible for the tourniquet-induced IR injury can lead to novel therapeutic interventions to minimize the skeletal muscle IR injury induced by tourniquet.
The cell death secondary to IR is a mixture of cell necrosis and apoptosis [11,12]. The major characteristics of necrosis are cell swelling and irreversible rupture of the plasma membrane [11,12]. The major characteristics of apoptosis are cell shrinkage, DNA damage, chromatin condensation and fragmentation [11,12]. Our previous study has shown that tourniquet-induced IR significantly causes cell necrosis (infarct size) in mouse gastrocnemius muscle; and superoxide overproduction and reduced antioxidant activity contribute to this IR injury [13]. Although apoptosis has been extensively investigated in many other tissues as a major trigger for IRinduced cell death [14][15][16][17][18][19], a few studies reported IR-induced apoptosis in skeletal muscle [20,21]. More importantly, it is unclear whether tourniquet-induced IR can cause apoptosis and what mechanisms are involved in this type of cell death in the skeletal muscles. Using a model of tourniquet-induced acute murine hindlimb IR, therefore, our present study investigated IR-induced apoptosis and potential mechanisms responsible for the apoptosis of the skeletal muscles.

Animals
Male C57BL6 mice (10-12 weeks of age, 27-34 g, n = 102, Charles River Laboratory) were housed under controlled temperature and humidity and a 12:12-h dark-light cycle, and were provided water and mouse chow ad libitum. Experiments were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Acute Hindlimb IR Model
Mice were anesthetized with an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine, given as an intraperitoneal injection (0.01 ml/g body weight). The level of anesthesia was continuously monitored by observing the respiratory patterns and toe pinch reflex. Anesthesia was maintained throughout the duration of experiments with additional anesthetic cocktail (0.1 ml) as needed. The animals were restrained on a heating pad to maintain body temperature at 37uC.
Unilateral hind limb ischemia was induced by placing an orthodontic rubber band at the hip joint using a McGivney hemorrhoidal ligator [13,23]. After 3 h ischemia, the orthodontic rubber band tourniquet was released and the hindlimb underwent 4 h reperfusion. Sham-operated animals were subjected to the same procedure except for the application of the orthodontic rubber band (i.e., no ischemia). During the entire procedure, mice were kept hydrated with intraperitoneal injection of 0.2 ml normal saline every 2 h. Tourniquet-induced IR was identified by measuring blood flow to the gastrocnemius muscle, as described previously [13]. Blood flow dropped to about 2% of baseline after placement of tourniquet and remained steady during 3 h ischemia. Upon tourniquet release, a rapid and transient increase in the blood flow to approximately 50% of baseline was observed, which was followed by a decline to a steady state of about 30% of baseline.

Isolation of Mitochondria and Cytosol in Gastrocnemius Muscle
At the end of sham or tourniquet-induced IR protocol (3 h ischemia and 4 h reperfusion), gastrocnemius muscle was immediately harvested and washed with a cold isolation buffer A (in mM): 70 sucrose, 210 mannitol, 1 EDTA, 50 Tris-HCl (pH 7.4). After the adherent fascias, fats, and tendons were removed, the muscle was finely minced with scissors and then homogenized in the isolation buffer A using a Kontes tissue grinder. The homogenate was centrifuged at 1,300 g for 3 min. The supernatant was poured through cheesecloth and centrifuged at 10,000 g for 10 min. The supernatant was transferred into a fresh tube and kept on the ice as cytosolic fraction. The pellet was suspended in an isolation buffer B (in mM): 70 sucrose, 210 mannitol, 0.1 EDTA, 50 Tris-HCl (pH 7.4) and centrifuged at 6800 g for 10 min. The final pellets was diluted into an appropriate volume and kept on the ice as mitochondrial fraction. Mitochondrial or cytosolic protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce; Rockford, IL). The fresh mitochondrial and cytosolic fractions were used for the following measurements.

Western Blot Analysis for Lactate Dehydrogenase (LDH) and Cytochrome C Oxidase IV (COX IV)
The sample of the isolated mitochondria or cytosol was mixed with loading buffer containing b-mercaptoethanol and heated at 100uC for 5 min. Equal amounts of the protein were loaded. Protein was fractionated in a 13% polyacrylamide gel along with molecular weight standards and transferred to PVDF membrane. The membrane was probed with rabbit anti-LDH (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-COX IV (Abcam, Cambridge, MA) antibody and a peroxidase-conjugated goat anti-rabbit IgG (Pierce Chemical, Rockford, IL). The signal was detected using enhanced chemiluminescence substrate (Pierce Chemical, Rockford, IL) and the bands analyzed using UVP BioImaging Systems.

In situ Detection of Mitochondrial Superoxide
Mitochondria-derived superoxide was measured using the mitochondria-targeted, superoxide-sensitive fluorogenic probe MitoSOX Red (Invitrogen), as described previously [24,25]. Briefly, the fresh isolated mitochondria were loaded with MitoSOX Red (5 mM) and MitoTracker green (200 nM, a mitochondria marker; Invitrogen) at 36uC for 10 min. The images were captured using a Leica fluorescent microscope and quantified using Adobe Photoshop CS3 Extended (Adobe Systems).

Lucigenin Chemiluminescent Assay for Superoxide Measurement
Superoxide anion production was measured in the isolated mitochondria of gastrocnemius muscles using the lucigenin chemiluminescence method described previously [13,26]. The isolated mitochondria (0.2 ml) was placed in 0.5 ml microfuge containing dark-adapted lucigenin (5 mM) and NADH (100 mM), and then read in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). Light emission was recorded for 5 min and expressed as mean light units (MLU)/5 min/100 mg protein.
Total protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce; Rockford, IL).  Ca ++ -induced Mitochondrial Permeability Transition Pore (mPTP) Opening [27] The fresh isolated mitochondria (0.25 mg) are suspended in 0.5 ml reaction buffer (150 mM sucrose, 50 mM KCl, 2 mM KH 2 PO 4 , 5 mM succinic acid, 100 mM NADH, 5 mM HEPES with pH 7.4) at 36uC. After 3 min of equilibrium, different concentration of CaCl 2 (0-50 mM) was added into the reaction buffer. The absorbance was continuously measured using a spectrophotometer (Genesys 6, Thermo Scientific, Waltham, MA) at 540 nm during 3 min of equilibrium and 2 min of calcium treatment. Ca ++ -induced decrease in absorbance was used to express the Ca ++ -induced mPTP opening.

Measurement of Caspase 9 Activity
Caspase-9 colorimetric activity assay kit (EMD Millipore, Billerica, MA) was used to measure caspase-9 activity [28]. At the end of sham or tourniquet-induced IR protocol, gastrocnemius muscle was homogenized and centrifuged at 1,300 g for 3 min. The supernatant was added to assay mixture and incubated at 37uC for 2 h, and the absorbance at 405 nm wavelength was measured using Infinite M200 microplate reader (Tecan US, Durham, NC). According to the manufacturer's instructions, caspase 9 activity (unit/mg protein) was calculated with a standard curve generated for each experiment.
In situ Measurement of Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-end Labeling (TUNEL) TUNEL was used to measure apoptosis of gastrocnemius muscles [29,30]. At the end of sham or tourniquet-induced IR protocol, gastrocnemius muscles were frozen and cut into 10 mmthick sections in a freezing cryostat at 220uC. To detect apoptotic DNA strand breaks, a fluorometric TUNEL detection kit was used according to the manufacturer's instructions (Trevigen, Gaithersburg, MD). Briefly, muscle sections were fixed with 4% formaldehyde in PBS (pH 7.4) at room temperature for 20 min, permeabilized with protein K at room temperature for 30 min, and incubated with the labeling reaction mixture in a humidified chamber at 37uC for 1h. Pre-digestion of tissue section with nuclease was served as positive control. The sections were then processed with standard immunocytochemical staining procedure to incubate with antibody against laminin (a marker for sarcolemma; Sigma-Aldrich, St.Louis, MO) and DAPI (a cell nucleus marker; Invitrogen, Carlsbad, CA). Images were captured using a Leica fluorescent microscope, and the number of TUNEL and DAPI positive nuclei was counted. Percentage of TUNEL positive nuclei in total nuclei (DAPI positive nuclei) was used to express apoptosis.

Data Analysis
All data are presented as mean 6 SE. Sigmaplot 12 (Systat Software, Chicago, IL) was used for data analysis. A two-way ANOVA, with a Bonferroni procedure for post hoc was used in the comparison of all parameters. Normal distribution of data was confirmed with Kolmogorov-Smirnov test and equal variance with Levene's test. Statistical significance was accepted when P,0.05.

Purity of the Fresh Isolated Mitochondria
In order to measure the alterations of the mitochondrial functions (including superoxide production and mPTP opening), mitochondria of the gastrocnemius muscles were isolated and the purity of the fresh isolated mitochondria was evaluated by western blot analysis. As shown in Fig. 1, LDH (a cytosolic protein marker) significantly expressed in cytosolic fractions and was not detectable in mitochondrial fractions in sham and tourniquet-induced IR gastrocnemius muscles. This result demonstrates that mitochondria were purely isolated from the gastrocnemius muscles. COX IV (a mitochondrial protein marker) not only expressed in mitochondrial fractions but also mildly presented in cytosolic fractions. Additionally, expression of COX IV in cytosolic fractions from IR gastrocnemius muscles was higher than that from sham gastrocnemius muscles (Fig. 1). Furthermore, we also found that the content of mitochondria purely isolated from IR gastrocnemius muscles (0.09860.007 mg mitochondrial protein/ mg muscle protein, n = 13 mice) was lower than that from sham gastrocnemius muscles (0.18060.013 mg mitochondrial protein/ mg muscle protein, n = 13 mice, *p,0.05).

Mitochondria-derived Superoxide Production in Gastrocnemius Muscles
Superoxide-sensitive probe MitoSOX Red is thought to be a useful tool for measuring mitochondria-derived superoxide [24,25]. In Fig.2, tourniquet-induced IR significantly enhanced MitoSOX Red fluorescent images in the isolated mitochondria, which indicates an increase in mitochondria-derived superoxide in the gastrocnemius muscles. In vivo pretreatment of tempol (a superoxide dismutase mimetic) or CoQ 10 (a mitochondrial antioxidant) markedly decreased mitochondria-derived superoxide production in the isolated mitochondria from tourniquet-induced IR gastrocnemius muscles but not sham gastrocnemius muscles.
We also investigated the effects of tempol and CoQ 10 on mitochondria-derived superoxide production in the isolated mitochondria from sham and tourniquet-induced IR gastrocnemius muscles using lucigenin chemiluminescence method. Similarly, both tempol and CoQ 10 normalized IR-enhanced mitochondria-derived superoxide production in the gastrocnemius muscles (Fig. 3). However, these chemicals did not affect the mitochondria-derived superoxide production in the isolated mitochondria from sham gastrocnemius muscles (Fig. 3).

Ca ++ -induced mPTP Opening
Ca ++ -induced mPTP opening in isolated mitochondria was monitored by a decrease in 540 nm absorbance (Fig. 4). In mitochondria isolated from sham gastrocnemius muscles, 50 mM CaCl 2 slightly induced the mPTP opening with time-dependent manner. The same concentration of CaCl 2 significantly enhanced the mPTP opening in mitochondria isolated from IR gastrocnemius muscles, compared to sham gastrocnemius muscles (Fig. 4A). Additionally, the mPTP opening was markedly greater in mitochondria isolated from IR gastrocnemius muscles than from sham gastrocnemius muscles at all CaCl 2 concentrations (5-50 mM) (Fig. 4B). Tempol, CoQ 10 , and cyclosporine A (CsA, an inhibitor of mPTP) each inhibited IRenhanced mPTP opening (Fig. 4B). However, these chemicals did not influence Ca 2+ -induced mPTP opening in mitochondria isolated from sham gastrocnemius muscles (data not shown).
Effects of Tempol, CoQ 10 , and CsA on Caspase 9 Activity in Sham and IR Gastrocnemius Muscles A significant increase of caspase 9 activity was found in IR gastrocnemius muscles compared to sham gastrocnemius muscles (Fig. 5). In vivo pretreatment of tempol, CoQ 10 , or CsA markedly blunted IR-increased caspase 9 activity (Fig. 5); whereas they did not affect caspase 9 activity in sham gastrocnemius muscles (data not shown).

Effects of Tempol, CoQ 10 , and CsA on TUNEL Staining in Sham and IR Gastrocnemius Muscles
TUNEL staining was performed to identify apoptotic nuclei (Fig. 6). We did not find TUNEL-positive nuclei in sham gastrocnemius muscles. Tourniquet-induced IR significantly increased the number of TUNEL-positive nuclei in gastrocnemius muscles (Fig. 6). Although tempol, CoQ 10 , and CsA did not alter the TUNEL-positive nuclei in sham gastrocnemius muscles, they partially decreased the number of TUNEL-positive nuclei in IR gastrocnemius muscles (Fig. 6).

Discussion
Our present study reports that tourniquet-induced IR (3 h ischemia and 4 h reperfusion) increases mitochondria-derived superoxide production, causes mPTP opening, and induces apoptosis in mouse gastrocnemius muscles. A permeable superoxide dismutase mimetic (tempol) and a mitochondrial antioxidant (CoQ 10 ) significantly inhibit IR-induced mitochondria-derived superoxide overproduction, mPTP opening, and apoptosis.
Additionally, an inhibitor of mPTP (CsA) also blunts IR-induced mPTP opening and apoptosis. These results indicate that mitochondria-derived superoxide overproduction is involved in IR-induced apoptosis through promoting mPTP opening in mouse gastrocnemius muscles.
Apoptosis is a form of cell death, which is characterized by morphological and biochemical alterations including cell shrinkage, DNA damage, chromosomal condensation and fragmentation, and activation of caspases (caspase 3, 8, 9, etc.) [11,12,31]. Although apoptosis has been extensively investigated in many other tissues as a major trigger for IR-induced cell death [14][15][16][17][18][19], it is not clear whether apoptosis is involved in IR-induced cell death in skeletal muscle. The findings about IR-induced apoptosis in skeletal muscle are controversial. Using the primary cells isolated from rat skeletal muscles, Wang, et al. have demonstrated that IR causes both necrosis and apoptosis [20]. Using isolated rat spinotrapezius muscle preparation, Suzuki, et al. found that an increased incidence of DNA fragmentation occurred during IR [32]. However, in a rat lower limb tourniquet-induced IR model, two research groups [33,34] did not find that TUNEL-positive nuclei appear in skeletal muscle during IR. Wang, et al. [20] think there are two possibilities to explain the different findings from above two research groups' observations. First, the tourniquet might not completely stop arterial blood flow during ischemia, and might induce microcirculatory no-flow during reperfusion. Second, false positive or negative results of TUNEL staining sometimes make this technique unable to reflect cellular apoptosis in vivo. However, we do not think either of these is likely to occur in our present study. In the tourniquet-induced mouse IR model used in the present study, blood flow drops to about 2% of baseline during ischemia and recovers to approximately 30% of baseline during reperfusion in the gastrocnemius muscles [13]. Additionally, we combined a biochemical variable (caspase 9 activity) with TUNEL staining to evaluate apoptosis in all experiments. From our results, we found that tourniquet-induced IR increased the number of TUNEL-positive nuclei and caspase 9 activity in the gastrocnemius muscles (Figs. 5, 6), which clearly confirmed that tourniquet-induced IR causes apoptosis in skeletal muscle, besides necrosis [13].
Although the mechanisms responsible for IR-induced apoptosis are still unclear, much evidence has demonstrated that mitochondrial dysfunction could play a central role in cell death leading to both necrosis and apoptosis in many tissues [35][36][37]. In particular, mPTP may be involved in IR-induced cell death [38]. The mPTP is a non-selective channel to span the inner mitochondrial membrane and predominantly is in a closed state under the physiological condition. Although the role of the mPTP in healthy cells remains unclear, a major consequence of mPTP opening is found that the inner mitochondrial membrane no longer maintains a barrier to protons and all small molecular weight molecules, which dissipates the proton electrochemical gradient, inhibits ATP production, causes the mitochondrial swelling and rupture, finally initiates the apoptotic processes [39][40][41][42][43].In the present study, tourniquet-induced IR promoted mPTP opening (Fig. 4); and mPTP inhibitor (CsA) significantly inhibited IRinduced mPTP opening and subsequent apoptosis (Figs. 4,5,6). These data indicate that mPTP opening also mediates IR-induced cell apoptosis in the skeletal muscles.
Mitochondria as the major source of superoxide have been found in the skeletal muscle [44][45][46][47]. Although there has been considerable debate about the site(s) of superoxide generation within mitochondria, the most data indicate that complexes I and III of the electron transport chain are the main sites of mitochondrial superoxide production [47,48]. Even if there is no information about role of superoxide in skeletal muscle mitochondrial dysfunction including mPTP opening, superoxide as a major factor contributes to mPTP opening in cardiac IR [49]. In the present study, mitochondria-derived superoxide was overproduced in the gastrocnemius muscles with tourniquet-induced IR (Figs. 1,  2). A permeable superoxide dismutase mimetic (tempol) and a mitochondrial antioxidant (CoQ 10 ) markedly reduced mitochondria-derived superoxide overproduction, and inhibited mPTP opening and apoptosis in IR gastrocnemius muscles. Thus, mitochondria-derived superoxide is thought to be a major trigger for IR-induced apoptotic cell death in the skeletal muscles. Additionally, our present study clearly demonstrates that tempol, CoQ 10 , and CsA attenuate tourniquet-induced apoptosis in the skeletal muscles. However, we do realize that the protective roles of tempol, CoQ 10 , and CsA in muscle contractility and long-term limb functions need to be further confirmed in future in vivo studies.
There are several different types of cells in the skeletal muscles including skeletal myocytes, vascular endothelial cells, smooth muscle cells, infiltrated inflammatory cells, etc. In addition to skeletal myocytes, other types of cells might also produce superoxide and contribute to the apoptosis in skeletal muscle. However, compared to other types of cells, the skeletal myocytes are mitochondria-rich cells, which determines that the skeletal myocytes play a central role in mitochondria-derived superoxide production and apoptosis in the present study.
For the isolation of mitochondria, we found that content of pure mitochondria in IR gastrocnemius muscles was lower than that in sham gastrocnemius muscles. Additionally, we obtained mitochondria-contaminated cytosolic fraction even if pure mitochondrial fraction was isolated. One possibility is that mitochondria are broken by tissue homogenization and tourniquet-induced IR. The broken mitochondria could contaminate the cytosolic fraction in western blot analysis.
In conclusion, mitochondria-derived superoxide is overproduced in skeletal muscle during tourniquet-induced IR. The elevation of mitochondria-derived superoxide contributes to IRinduced apoptosis in skeletal muscle through activating mPTP opening. These data further our understanding of the factors responsible for the tourniquet-induced apoptosis. More importantly, an improved understanding of the role of mitochondrial dysfunction in apoptosis may allow us to design effective therapeutic interventions and to improve tourniquet application in the civilian and battlefield setting.