Tert-butyl hydroperoxide (tBHP) is a membrane-permeant pro-oxidant agent used in several cell lines as a model to study the effects of oxidative stress on cellular function and cell death pathways [18, 19, 21, 22]. Once inside cells, tBHP generates tert-butoxy radicals inducing several physiological alterations with consequent loss of cell viability via apoptosis or necrosis . The type of effect is dependent on the cell line or primary culture type, tBHP concentration, and exposure period. Lipid peroxidation , depletion of intracellular reduced glutathione , modification of protein thiols  and cytosolic calcium deregulation  are some of the most common alterations.
In cardiomyocytes, tBHP induced loss of cell shape, depletion of ATP, and formation of adenosine . Other studies demonstrated that treatment with 1 mM tBHP lead to a rise in intracellular calcium concentration, hyper-contracture and loss of membrane integrity in cardiac myocytes isolated from rat ventricles . To the best of our knowledge, however, morphological alterations in cardiomyocytes or myoblasts during tBHP-induced cell death has not been reported.
In the present study, tBHP was used as a model compound to characterize morphologic changes in H9c2 cells resulting from oxidative stress. It was found that 1 hour treatment with 50 μM tBHP induces an increase in the oxidation of the probe DCF, which indicates the presence of intracellular oxidative stress. The data also indicates that after 6 hours treatment with 50 and 100 μM tBHP, cell death and detachment occurred. These effects were prevented by N-acetylcysteine (NAC) and Trolox. NAC is the acetylated form of the amino acid L-cysteine and a source of sulfhydryl (SH) groups. In the body, NAC is converted into metabolites capable of stimulating glutathione (GSH) synthesis and can also act directly as a free radical scavenger, due to its nucleophilic and antioxidant properties [32, 33]. Trolox is a water-soluble analogue of vitamin E lacking the phytyl chain, with strong antioxidant properties . As reported in Figure 2 (top panel) both antioxidant compounds prevented the cytotoxic effect induced by tBHP, supporting the hypothesis that ROS production in H9c2 cells is responsible for the cell death that occurs following tBHP treatment. Moreover, vital imaging studies demonstrate that some features of apoptosis, including cell rounding, membrane blebbing, and chromatin condensation, occur in cells undergoing tBHP-induced oxidative stress. However, cells also remain calcein-positive, and mitochondrial membrane potential can persist for quite some time in these apoptotic cells.
Apoptosis is a cell death program that is dependent on ATP, most of which is normally produced by mitochondria under aerobic conditions. Although tBHP-treated apoptotic cells displayed significant changes in mitochondrial morphology, many altered mitochondrial remained polarized. On the other hand, most necrotic cells lacked polarized mitochondria. The results suggest that functional mitochondria are necessary for tBHP-induced apoptosis in H9c2 cells. Nevertheless, although morphologically compromised, the mitochondrial network retains sufficient function to continue producing enough ATP to drive apoptosis in oxidatively damaged cells. Transformation of filamentous mitochondria to small spherical forms, as observed in this study, has been termed the "thread-grain transition" and has been proposed to represent a mechanism to isolate a damaged part of the mitochondrial system from the rest of the mitochondrial network . According to this hypothesis, thread-grain transitions represent an obligatory step in mitochondrial-mediated apoptosis. In addition to thread-grain transitions, we noted that polarized mitochondria became concentrated near the nuclear region upon treatment with tBHP. Skulachev et al.  proposed that small mitochondria around the nucleus may serve to more rapidly direct some apoptotic proteins to their nuclear targets. Alternatively, concentrating polarized mitochondria around the nucleus could be an important mechanism to supply the energy needed by the nucleus during the apoptotic program.
Immunocytochemistry of H9c2 cells revealed the formation of Bax aggregates near the mitochondrial network after tBHP treatment (Figure 4). This labeling appeared to be most pronounced in areas of the mitochondrial network that displayed the weakest Mitotracker Red labeling. Mitotracker Red is a widely used mitochondrial marker, which is membrane potential dependent (according to the manufacturer). In these experiments, control and tBHP-treated cultures were processed at the same time and under identical conditions for Mitotracker, Bax and Hoechst labeling. Both control and experimental samples were photographed in the same session using identical microscope and camera settings. Visual examination showed consistent differences in fluorescence intensities of these probes between control and tBHP-treated groups, and statistical analyses of fluorescence values from digitized images demonstrated a significant inverse relationship between Mitotracker Red and Bax immunolabeling (Figure 6, lower panel). We also observed a direct association between Bax immunolabeling and Hoechst staining, showing that increased Bax labeling accompanies the stronger Hoechst labeling, associated with chromatin condensation in apoptotic tBHP-treated H9c2 cells.
Bax has been suggested to be translocated from the cytosol of cardiomyocytes in two distinct phases . The first phase involves the Bax-induced release of cytochrome c, and the second phase involves packaging of Bax monomers close to mitochondria. Our results are consistent with this model in that we observed a translocation of Bax around mitochondria upon treatment with tBHP, which is further evidence that tBHP is inducing apoptosis. Considering that Bax translocation to mitochondria can be accompanied by morphological alterations in these organelles, we hypothesized that the mitochondrial permeability transition (MPT) could be involved as well. It has been reported by other authors that the mitochondrial permeability transition is an oxidative stress-dependent mechanism . As tBHP induces oxidative stress in H9c2 cells, we also suspected that the MPT could be another reason for tBHP-induced H9c2 cytotoxicity. In order to test this hypothesis, we examined whether tBHP cytotoxicity could be prevented with cyclosporin-A, a MPT inhibitor. The results (Figure 2, bottom panel) showed that cyclosporin-A did not prevent cell death induced by tBHP, indicating that the MPT does not appear to be cause for the cytotoxic effect induced by tBHP under our experimental conditions.
Changes in plasma membrane asymmetry are one of the earliest features of cells undergoing apoptosis. In apoptotic cells, phosphatidylserine (PS) is translocated from the inner to the outer leaflet of plasma membrane, which serves as a recognition signal for macrophages . Annexin V binds PS, and is commonly used to detect externalized PS in apoptotic cells. However, because Annexin V can also label non-externalized PS in necrotic cells with compromised plasma membranes, propidium iodide (PI) is commonly used together with annexin V to identify and distinguish necrotic from apoptotic cells . Our results demonstrate that tBHP-treatment is able to induce the externalization of PS, as evidenced by the presence of annexin V-positive but PI-negative cells. Additionally, treatment with lower concentrations of tBHP for short periods of time induced an increase in the number of nuclei showing condensed chromatin, characteristic of apoptosis (Figure 6).
Cell membrane behavior after treatment with tBHP was also evaluated by DIC imaging (Figures 3, 5 and 7). tBHP-treated H9c2 cells shrank in size while undergoing membrane blebbing and releasing apoptotic bodies. Nevertheless, during this period, cell membrane integrity is maintained as seen by the maintenance of calcein fluorescence inside the cells and PI exclusion (Figure 7). In many cells, this apoptotic period was followed by necrosis, and release of cellular contents.