Cardiomyoblasts Exposed to Reactive Oxygen and Nitrogen Species
1. Water (see Note 1)
3.4. Determination of Mitochondrial
Transmembrane Potential
Alterations in mitochondrial transmembrane potential (m) in response to stressful stimuli (i.e., oxidative bursts) in cultured cells, can be determined using a JC-1, a cationic dye. The assay is based on accumulation of JC-1 in mitochondria which is depen-dent on m. The dye remains in the monomeric form in the cytoplasm and exhibits green fluorescence when the value ofm
is small. On the other hand, at high m, there is greater accu-mulation of JC-1 in the mitochondria forming J-aggregates, and the fluorescence shifts to red (Emax 590 nm). Thus, the changes in the emission patterns of JC-1 of this cyanine dye can be used as a sensitive measure of mitochondrial trans membrane potential.
1. Expose H9c2 cells to PN (10–20 M), SIN-1 (1–2 mM), or DEA NONOate (1–2 mM)) as described in Section 3.1, Steps 3 and 4.
2. Remove the culture medium after 8 h and wash the H9c2 cells twice with PBSG.
3. Add PBSG containing JC-1 at a final concentration of 10M (see Notes 12–13) and incubate for 30 min at 37◦C with 95% humidity and 5% CO2.
4. Remove the unbound JC-1 dye by washing the cells twice with PBSG and add fresh buffer (PBSG: 500L).
58 Sathishkumar et al.
0 2 5 5 0 7 5 1 0 0
10 µM 50 µM Peroxynitrite
1 mM 2 mM SIN-1
1 mM 2 mM DEA NONOate
Control
% Change in ΔΨm (relative to control)
Fig. 4.3. Changes in mitochondrial transmembrane potential in H9c2 cardiomyoblasts exposed to PN (10–50M), SIN-1 (1–2 mM), or DEA NONOate (1–2 mM), Dose- and time-dependent changes in the mitochondrial transmembrane potential were deter-mined 8 h following the exposure to PN, SIN-1, and DEA NONOate using JC-1 (see Section 3.4).
5. Using a Spectramax EM Gemeni spectrofluorometer, mea-sure the fluorescence at excitation and emission wavelengths of 488 and 590 nm, respectively.
6. The results are expressed as percentage decrease in m
compared to the untreated controls.
We measured changes inm after 8 h of exposure to PN, SIN-1, and DEA NONOate in the H9c2 cardiomyoblasts. The cationic dye JC-1 accumulates in the mitochondria of normal cells giving a maximal fluorescence at 590 nm (100%). In cells undergoing oxidative stress/cytotoxicity, the mitochondrial accu-mulation of the dye would be lower, provided there is a decrease in transmembrane potential. As shown in Fig. 4.3, we find evi-dence for lowering ofm in H9c2 cells exposed to PN, SIN-1, and DEA NONOate. Data are presented as mean± SEM of three separate experiments.
4. Notes
1. Unless specified otherwise, throughout this proce-dure “water” refers to ultrapure water with resistance
≥18.2 M.
2. These are immature embryonic cardiomyocytes with elec-trical and hormonal signaling pathways preserved as in the adult cardiac cells. About 95% of the cells are mononu-cleated and express L-type calcium channels typical of cardiomyocytes. Also, they express N-cadherin and other specific cardiac markers and maintain the phenotypic char-acteristics for several passages. Sipido and Marban (21)
Measurement Of Oxidative Stress in Cardiomyoblasts 59
argue that the H9c2 cells had been neglected as potential surrogates for primary cardiac cells.
3. Commercial preparations of H2O2 (30%) can be used as such provided the concentration based on UV absorbance at 240 nm ( = 41 M–1 cm–1) (16, 17) is around 9 M.
Where necessary, the preparation needs to be diluted with water.
4. Isoamyl nitrite is a hypotensive agent. No direct expo-sure or contact must be allowed. Gloves (double) and lab coat must be worn at all times. All experiments involving isoamyl nitrite must be performed behind a safety shield in a fume hood.
5. The concentration of isoamyl nitrite in the neat solution (96% w/v; density: 0.872 g/mL at 25◦C) is approximately 7.14 M.
6. Certain preparations of isoamyl nitrite contain Na2CO3up to 2% (w/v) as a stabilizing agent. As a precaution, wash the preparation with 3× 2 vol of water before using in the synthesis of PN (17).
7. Wash the MnO2 column with 20 mL each of water and 0.1 N NaOH before passing the solution of PN. The first few milliliters of the PN solution eluting from the MnO2 column should be discarded to minimize the dilution.
8. This helps to remove dissolved gases (O2 and N2O) and traces of organic solvent(s) used in the extraction process.
9. Peroxynitrite readily decomposes in aqueous media (t1/2≈ 1.5 s at pH 7.4). Therefore, the alkaline solutions of PN (pH≥ 12.0) must be introduced as bolus additions of 2–
10L and mixed in the shortest time possible. Also, cell cultures treated with decomposed PN (see Note 10) should be used as control and included in every experiment.
10. The decomposed solutions of PN (1–2 mM) can be pre-pared by incubating small volumes (10–20L) of PN stock solutions (∼100 mM) with 2 mL of PBS, pH 7.4, for 5 min at room temperature.
11. This helps avoiding interaction of O2•–, •NO, and the products of•NO–O2•–reaction (formed from the deposition of DEA NONOate and SN-1) with serum com-ponents.
12. During incubations, care should be taken to protect the plate from external light. This helps to minimize the loss of fluorescence, if any.
13. As it is difficult to completely solublize the JC-1 dye in the buffer, try to dissolve to the maximum possible by vigorous vortexing. Centrifuge briefly and remove the undissolved dye before addition to the culture.
60 Sathishkumar et al.
Acknowledgments
This publication was made possible by National Institutes of Health (NIH) Grants P20 RR16456 (from the BRIN Program of the National Center for Research Resources) and ES10018 (from the ARCH Program of the National Institute of Environmental Health Sciences), and US Department of Education (Title III, Part B – Strengthening Historically Black Graduate Institutions, HBGI; grant number: PO31B040030). Its contents are solely the responsibility of authors and do not necessarily represent the offi-cial views of the NIH, NSF, or US Department of Education.
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