Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide--characterization of morphological features of cell death

Abstract

Background: When exposed to oxidative conditions, cells suffer not only biochemical alterations, but also morphologic changes. Oxidative stress is a condition induced by some pro-oxidant compounds, such as by tert-butylhydroperoxide (tBHP) and can also be induced in vivo by ischemia/reperfusion conditions, which is very common in cardiac tissue. The cell line H9c2 has been used as an in vitro cellular model for both skeletal and cardiac muscle. Understanding how these cells respond to oxidative agents may furnish novel insights into how cardiac and skeletal tissues respond to oxidative stress conditions. The objective of this work was to characterize, through vital imaging, morphological alterations and the appearance of apoptotic hallmarks, with a special focus on mitochondrial changes, upon exposure of H9c2 cells to tBHP.

Results: When exposed to tBHP, an increase in intracellular oxidative stress was detected in H9c2 cells by epifluorescence microscopy, which was accompanied by an increase in cell death that was prevented by the antioxidants Trolox and N-acetylcysteine. Several morphological alterations characteristic of apoptosis were noted, including changes in nuclear morphology, translocation of phosphatidylserine to the outer leaflet of the cell membrane, and cell blebbing. An increase in the exposure period or in tBHP concentration resulted in a clear loss of membrane integrity, which is characteristic of necrosis. Changes in mitochondrial morphology, consisting of a transition from long filaments to small and round fragments, were also detected in H9c2 cells after treatment with tBHP. Bax aggregates near mitochondrial networks were formed after short periods of incubation.

Conclusion: Vital imaging of alterations in cell morphology is a useful method to characterize cellular responses to oxidative stress. In the present work, we report two distinct patterns of morphological alterations in H9c2 cells exposed to tBHP, a pro-oxidant agent frequently used as model to induce oxidative stress. In particular, dynamic changes in mitochondrial networks could be visualized, which appear to be centrally involved in how these cells respond to oxidative stress. The data also indicate that the cause of H9c2 cell death following tBHP exposure is increased intracellular oxidative stress.

Figure 1

tBHP induced and increase intracellular oxidative stress in H9c2 cells. (Top panel) H9c2 cells were exposed to 0 (panel A-B) or 50 μM tBHP (panel C-D) for 1 hour. Increase in intracellular oxidative stress was detected by oxidation of dichlorofluorescein. Fluorescence observed in panel B and D is proportional to the degree of oxidation. The corresponding DIC images can be observed in panels A and C. Images are representative from 3 different cell preparations. A-D are the same magnification; scale bar in A = 20 μm. (Bottom panel) Quantification of cell DCF fluorescence intensity. Four different fields were analyzed in each experiment. Data represents the mean ± SEM of 3 different cell preparations. *p < 0.05 vs control.

Figure 2

Cytotoxicity of tBHP and the effects of NAC, Trolox, Cyclosporin-A was analyzed by sulforhodamine B dye-binding assay. Effects of NAC and Trolox (top pannel) or cyclosporin-A (bottom panel) on tBHP-induced cell loss. Cell treatment and analysis were performed as described in the methods section. Data represent the mean ± SEM of 3 different cell preparations. *p < 0.05 vs respective control (tBHP without inhibitor); ** p < 0.05 vs control (no tBHP added).

Figure 3

Alterations in cellular and mitochondrial morphology induced by tBHP. (A) DIC images from untreated cells, showing normal membrane morphology. (B) Laser scanning confocal microscopy image from untreated cells showing triple labeling with Hoechst 33342 (blue), calcein (green) and TMRM (red). (C, D) Laser scanning confocal microscopy showing the fluorescence of the three dyes followed with the time, (C) 45 min, and (D) 90 minutes, after treatment with 80 μM tBHP. (E) Magnification of the cell indicated by an arrow in panel C and (F) magnification of the cell indicated by an arrow in panel D. (G) DIC images from H9c2 cells treated with 25 μM tBHP for three hours. (H) Laser scanning confocal microscopy (LSCM) image from cells treated with 25 μM tBHP during three hours. Images are representative from 3 different cell preparations. Panels B-D and G, H are the same magnification. Scale bar corresponds to 20 μm.

Figure 4

tBHP induces formation of BAX clusters close to the mitochondrial network. (Top panel) Control and tBHP treated cells were incubated with 125 nM Mitotracker red (red) for 30 min previous to fixation and subsequently labeled with an antibody to BAX (green) and counterstained with Hoechst (blue). (A-C) Control cells showing a homogeneous BAX distribution throughout the cell and filament-shaped mitochondria. (D-F) and (G-I) H9c2 cells treated with 50 μM and 100 μM tBHP, respectively, during 1 hour, showing BAX aggregates located close to the mitochondrial network and a change in mitochondria morphology from a filament-like form to small round mitochondria. (J) and (K) Magnification of the cell indicated by an arrow in Panel E (J-BAX immunolabeling; K-Mitotracker red labeling). Images are representative from 3 different cell preparations. Scale bar corresponds to 20 μM. (Bottom panel) Quantification of the cell fluorescence intensity of Hoechst, Bax immunolabeling and Mitotracker red. Data represent the mean ± SEM of 3 different cell preparations. *p < 0.05 vs 0 μM tBHP, ^# p < 0.05 vs 50 μM tBHP.

Figure 5

tBHP induces the translocation of phosphatidylserine to the outer leaflet of the cellular membrane. (Top panel) After treatment with 0, 50 and 100 μM tBHP, for 1 hour, H9c2 cell were labeled with Annexin V and propidium iodide. Left panels: DIC images of H9c2 cells, treated with 0, 50 and 100 μM tBHP. Right panels: corresponding epifluorescence microscopy images of propidium iodide (red) and Annexin V (green). Images are representative from 3 different experiments. All images are the same magnification; scale bar = 20 μm. (Bottom panel) Statistical analysis of the population of control cells or cells treated with increasing concentrations of tBHP, showing the relation between treatment and annexin V/propidium iodide labeling. Analysis was performed by counting cells without labeling (healthy cells), annexin V labeling (apoptotic cells) and PI/Annexin V double labeling (necrotic cells). Data represent the mean ± SEM of 3 different cell preparations.

Figure 6

Quantitation of apoptotic cells by Hoechst labeling methods. (Top panel) Epifluorescence microscopy images of nuclei showing H9c2 cells treated with 0, 50 and 100 μM tBHP during 3 and 6 hours. Changes in nuclear morphology characteristic of apoptosis (arrows) were detected in Hoechst 33342-stained cells. Scale bar = 20 μm. (Bottom panel) The numbers of apoptotic nuclei were counted and expressed as the percentage of total cells counted (approximately 200 cells per coverslip). Data represents the mean ± SEM of 5 different experiments (bars indicate standard error). *p < 0.05 vs control.

Figure 7

Real-time epifluorescence microscopy of calcein and ethidium homodimer fluorescence after treatment with 100 μM tBHP. DIC (left panels), calcein (right panels) and EH-1(middle panels) epifluorescence microscopy images, collected every two minutes after treatment with 100 μM of tBHP. Images are representative of still micrographs obtained from the time-lapse of cells treated with tBHP and labeled with calcein-AM and EH-1.