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Mitochondrial Na⁺ overload is caused by oxidative stress and leads to activation of the caspase 3- dependent apoptotic machinery

KUN-TA YANG, SHWU-FEN PAN, CHUNG-LIANG CHIEN^*, SU-MING HSU, YUNG-ZU TSENG, SEU-MEI WANG^*,1 and MEI-LIN WU¹

Departments of Physiology and
^* Anatomy and Cell Biology, College of Medicine, Departments of
Pathology and
Internal Medicine, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan; and the
Department of Biotechnology, Ming Chuan University, Gwei-Shan, Taoyuan County, Taiwan

¹ Correspondence: Department of Physiology (M.-L.W.) and Department of Anatomy and Cell Biology (S.-M.W), College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd., Taipei, Taiwan. E-mail: mlw{at}ha.mc.ntu.edu.tw

SPECIFIC AIMS

In many cell types, oxidative stress and mitochondrial Ca²⁺([Ca]_m) overload lead to the opening of the permeability transition pore (PTP), resulting in persistent loss of mitochondrial potential (_m), cytochrome c (cytC) release, and apoptosis. We used treatment with H₂O₂, a widely accepted model system for studying oxidative stress-induced apoptosis in many cell types and in cardiovascular disease. Using time-lapse confocal recording of live cardiomyocytes, we found that H₂O₂ caused a marked increase in Na⁺ and Ca²⁺ levels in cytosol ([Na]_cyt, [Ca]_cyt) and mitochondria ([Na]_m, [Ca]_m). The H₂O₂-induced intracellular Na⁺ ([Na]_i) overload was involved in the H₂O₂-induced [Ca]_cyt/[Ca]_m overload via activation of the reverse mode of the Na/Ca exchanger (rNCX). Furthermore, the H₂O₂-induced [Na]_m overload is an important upstream signal for the apoptotic machinery.

PRINCIPAL FINDINGS

1. Exposure of myocytes to H₂O₂ induces marked intracellular Ca²⁺ ([Ca]_i) and [Na]_i overload
Upon exposure to 100 µM H₂O₂ for 40–50 min in normal medium, a sustained increase in the [Ca]_i (1050±50 nM, n=27) and [Na]_i (65.9±3.5 mM, n=26) was seen. The H₂O₂-induced [Ca]_i and [Na]_i overloads were completely inhibited, respectively, in Ca-free medium ([Ca]_i=30±0.1 nM, n=6) or Na-free medium ([Na]_i=3.0±0.1 mM, n=7), showing that the overload was due to influx of external Ca²⁺ or Na⁺ ions. When the H₂O₂ concentration was reduced to 30 or 50 µM, [Ca]_i overload was similar to that seen using 100 µM H₂O₂, whereas [Na]_i overload was reduced from 65.9 mM to 50 mM. H₂O₂-induced [Ca]_i and [Na]_i overloads were inhibited using iron chelators or cell-permeable OH• scavengers, suggesting OH• was involved in the [Ca]_i/[Na]_i overload.

2. H₂O₂-induced [Na]_i overload is involved in producing the [Ca]_i overload: role of the rNCX
After 20 min in Na-free medium, the [Na]_i was low ([Na]_i=3 mM) and the H₂O₂-induced [Na]_i overload was completely inhibited. Under conditions of complete inhibition of the rNCX, H₂O₂-induced [Ca]_i overload was significantly reduced (460±40 nM vs. 1050±50 nM in Na_o-containing medium). Moreover, KB-R7943, a bi-directional NCX inhibitor, markedly inhibited the H₂O₂-induced [Ca]_i overload.

Where does the H₂O₂-induced Na_i overload originate? The patch-clamp technique showed that activation of the nonselective cation channel (NSCC) may be involved in the H₂O₂-induced Na_o influx (unpublished results).

3. H₂O₂ treatment causes cardiomyocyte apoptosis; inhibition of the [Na]_i overload prevents cardiac apoptosis
After the end of H₂O₂ treatment and transfer to normal medium, the percentage of TUNEL-positive myocytes increased from 3.6% at 0 h, to 55% at 4.5 h, and 85% at 16 h, confirming that H₂O₂ induced apoptosis. When the cells were treated with H₂O₂ in Na-free medium, generation of apoptotic myocytes at 4.5 and 16 h was completely blocked, showing that inhibition of the [Na]_i overload had both short- and long-term protective effects; in Ca-free medium, H₂O₂-induced apoptosis at 4.5 and 16 h was reduced, respectively, by only 15% and 30%.

4. [Na]_i or [Ca]_i overload per se can induce apoptosis
Addition of 30–100 µM H₂O₂ evoked an [Na]_i overload of 50–65.9 mM. To investigate whether different levels of [Na]_i resulted in different levels of apoptosis, [Na]_i was clamped at 65.9, 52, or 30 mM without altering [Ca]_i by incubating the cells for 40 min in Ca-free medium containing 65.9, 52, or 30 mM Na⁺ ions plus Na⁺ ionophore cocktail (5 µM gramicidin D, 40 µM monensin, and 100 µM strophanthidine); cells were incubated for 4.5 h in Ca-free medium and [Na]_i-dependent apoptosis was seen. When H₂O₂-induced [Ca]_i overload was simulated by clamping the cells for 40 min at a [Ca]_i = 1050 nM/Na-free + ionomycin medium, then for 4.5 h in Na-free medium alone, 32% of the myocytes were TUNEL-positive, confirming that [Ca]_i overload per se induced apoptosis. Since the H₂O₂-induced [Ca]_i overload was reduced to 460 nM in Na-free conditions, when apoptosis was completely inhibited, we clamped the [Ca]_i at 460 nM using Na-free medium and found that apoptosis was indeed inhibited. These effects were not due to a nonspecific toxic effect of the ionophores, since the addition of ionophores per se (Na⁺ ionophore cocktail in Na-free medium or ionomycin in Ca-free medium) had little effect.

5. Identification of the signaling pathway involved in [Na]_i-dependent apoptosis
1) Caspase activation: immediate induction of caspase activation was seen after exposure to H₂O₂, [Ca]_i = 1050 nM/Na-free or [Na]_i = 65.9 mM/Ca-free medium. 2) H₂O₂, [Ca]_i = 1050 nM or [Na]_i = 65.9 mM medium induces an increase in the [Na]_m and/or [Ca]_m in live myocytes

Using time-lapse confocal recording, treatment with 100 µM H₂O₂ in normal medium induced an 8-fold increase in both cytosolic ([Ca]_cyt) and mitochondrial ([Ca]_m) Ca²⁺ levels (supplemental data: doi: 10.1096/fj.03-1038fje, Video Fig. 1 ). Treatment with [Ca]_i = 1050 nM/Na-free + ionomycin medium gave a similar result. [Ca]_cyt and [Ca]_m were both reduced by 55–60% in H₂O₂/Na-free medium, suggesting that [Na]_i overload is an important upstream signal of the H₂O₂-induced [Ca]_m overload.

View larger version (62K): [in this window] [in a new window]   Figure 1. Measurement of cytosolic and mitochondrial Na+ levels by time-lapse confocal imaging. Increases in the [Na]cyt and/or [Na]m in a single CoroNa red-loaded myocyte induced by 30–100 µM H2O2 in normal medium (a, b, f; see also supplemental data Video Fig. 2 ), by [Na]i = 65.9 mM in Ca-free medium (c), or by [Na]i = 65.9 mM in La3+- (200 µM) or RU360- (10 µM) pretreated medium (d–f). La3+ and RU360 (VDAC inhibitors) both selectively inhibited the H2O2-induced [Na]m increase (f). Mean value for five responding mitochondria in each myocyte was calculated; values shown are mean ± SE for n = 4–7 experiments. *P< 0.05 compared, respectively, to the 100 µM H2O2 group or [Na]i = 65.9 mM group using Wilcoxon’s rank sum test.

After exposure to 100 µM H₂O₂ in normal medium (Fig. 1 a; see also supplemental data Video Fig. 2 ) or [Na]_i = 65.9 mM/Ca-free + Na⁺ ionophore cocktail medium (Fig. 1c ), [Na]_cyt and [Na]_m were increased to 60–65 mM (Fig. 1f ).

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of CsA, La3+, or RU360 on PTP opening and cytC release immediately after treatment with H2O2 or clamping of the [Ca]i or [Na]i at different levels. A) The m was measured using TMRM. The complete depolarization of the m (100% m, the control), produced using a mixture of FCCP (1 µM), oligomycin (5 µg/mL), and rotenone (10 µM), was CsA (5 µM) insensitive. B) After various treatments, cells were labeled with anti-cytC antibody and cytC release into the cytoplasm was examined. *P< 0.05 compared with the H2O2 group or the [Na]i = 65.9 mM or [Ca]i = 1050 nM group, as appropriate (n=4–37 experiments).

3)An increase in [Na]_m evokes PTP-sensitive cytC release. It has been suggested that the [Ca]_m overload induces cardiac apoptosis. Since H₂O₂ produced [Na]_m and [Ca]_m overload, to avoid the apoptotic effect induced by [Ca]_m overload, [Na]_i = 65.9 mM/Ca-free + Na⁺ ionophore cocktail medium was used. La³⁺ (200 µM) and RU360 (10 µM), two potent voltage-dependent anion channel (VDAC) inhibitors, blocked the [Na]_i = 65.9 mM medium-induced increase in the [Na]_m, but not that in the [Na]_cyt (Fig. 1d, f ).

TMRM was used to measure cyclosporin A (CsA) -sensitive _m depolarization, a good indicator of persistent PTP opening. Treatment with H₂O₂, [Na]_I = 65.9 mM, or [Ca]_I = 1050 nM, but not [Ca] = 460 nM, medium caused CsA-sensitive _m depolarization (Fig. 2 A). H₂O₂-induced _m depolarization was largely inhibited in Na-free but not in Ca-free medium. The CsA-sensitive _m depolarization induced by [Na]_i = 65.9 mM medium was inhibited by La³⁺ and RU360 (Fig. 2A ). These results show that prevention of [Na]_m overload blocks PTP opening.

Figure 2B shows cytC release. At a very low [Na]_i (Na-free medium) but not a very low [Ca]_i (Ca-free medium), H₂O₂-induced cytC release was largely inhibited. Exposure to different levels of [Na]_i (30, 52, or 65.9 mM) or [Ca]_i (460 or 1050 nM) evoked dose-dependent, CsA-sensitive cytC release. La³⁺ and RU360, which inhibited the [Na]_i = 65.9 mM medium-induced PTP opening (Fig. 2A ), completely inhibited cytC release (Fig. 2B ), further indicating that [Na]_m, but not [Na]_cyt, overload acts as the upstream signal for oxidative stress-induced apoptosis.

CONCLUSIONS AND SIGNIFICANCE

The present study shows that H₂O₂ treatment induces a marked increase in [Na]_cyt, [Na]_m, [Ca]_cyt, and [Ca]_m. Activation of the NSCC may be involved in H₂O₂-induced Na_o influx. H₂O₂-induced [Na]_i overload contributed significantly to the H₂O₂-induced [Ca]_cyt/[Ca]_m overload via activation of the rNCX. Moreover, [Na]_i-dependent apoptosis was seen and a reduction in the [Na]_m, but not [Na]_cyt, overload had a marked protective effect against H₂O₂-induced apoptosis (Fig. 3 ). An increase in the [Na]_m therefore is an important upstream signal for oxidative stress-induced, caspase 3-dependent apoptosis and may be involved in the cell death seen in many cell types and in ischemic heart disease.

View larger version (41K): [in this window] [in a new window]   Figure 3. Schematic diagram showing how oxidative stress (H2O2) induces [Na]cyt, [Na]m, [Ca]cyt, and [Ca]m overloads and apoptosis. The nonselective cation channel (NSCC) is probably involved in the H2O2-induced Nao/Cao influx (unpublished results). Part of the [Ca]cyt/[Ca]m overload is caused by the H2O2-induced [Na]cyt overload via activation of the rNCX. High [Na]m or [Ca]m per se resulted in CsA-sensitive PTP opening, cytC release, and caspase 3-dependent apoptosis. After inhibition of the [Na]m overload by La3+ or RU360 (VDAC inhibitors), the CsA-sensitive PTP opening and cytC release were both inhibited, showing that [Na]m overload, but not [Na]cyt overload, was the upstream signal for the apoptotic machinery. VDAC in the outer membrane, adenine nucleotide translocase (ANT) in the inner membrane, and cyclophilin D (Cy-D) in the matrix are three major components of the permeability transition pore (PTP).

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1038fje; doi: 10.1096/fj.03-1038fje

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