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 (CoQ10) 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.) 
. Although apoptosis has been extensively investigated in many other tissues as a major trigger for IR-induced cell death 
, 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 
. Using isolated rat spinotrapezius muscle preparation, Suzuki, et al. found that an increased incidence of DNA fragmentation occurred during IR 
. However, in a rat lower limb tourniquet-induced IR model, two research groups 
did not find that TUNEL-positive nuclei appear in skeletal muscle during IR. Wang, et al. 
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 
. 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 (, ), which clearly confirmed that tourniquet-induced IR causes apoptosis in skeletal muscle, besides necrosis 
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 
. In particular, mPTP may be involved in IR-induced cell death 
. 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 
.In the present study, tourniquet-induced IR promoted mPTP opening (); and mPTP inhibitor (CsA) significantly inhibited IR-induced mPTP opening and subsequent apoptosis (, , ). 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 
. 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 
. 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 
. In the present study, mitochondria-derived superoxide was overproduced in the gastrocnemius muscles with tourniquet-induced IR (, ). A permeable superoxide dismutase mimetic (tempol) and a mitochondrial antioxidant (CoQ10
) 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, CoQ10
, and CsA attenuate tourniquet-induced apoptosis in the skeletal muscles. However, we do realize that the protective roles of tempol, CoQ10
, and CsA in muscle contractility and long-term limb functions need to be further confirmed in future in vivo
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.
Recently, connexin-43, a major gap junction protein is thought to be involved in preconditioning-induced cardioprotection 
. MPAK- and PKC-dependent mitochondrial translocation and phosphorylation of connexin-43 stimulate cardioprotection 
. Connexin-43 inhibition with specific RNA interference significantly decreased cardiomyocyte survival 
, whereas mitochondria-specific overexpression of connexin-43 improved stem cell survival during heart cell therapy 
. Additionally, phosphorylated connexin 43 inhibits the production of reactive oxygen species and mPTP opening 
. Therefore, it is possible that alteration of connexin-43 also play a role in tourniquet-induced skeletal muscle apoptosis, which needs to be explored in our future studies.
In conclusion, mitochondria-derived superoxide is overproduced in skeletal muscle during tourniquet-induced IR. The elevation of mitochondria-derived superoxide contributes to IR-induced 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.