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Home / H2o2 Continuous Vs Acute Exposure on Cells

H2o2 Continuous Vs Acute Exposure on Cells

Reactive oxygen species (ROS) can act as signaling molecules able to stimulate and modulate a variety of biochemical and genetic systems, including the regulation of signal transduction pathways, gene expression, proliferation, and cell death by apoptosis.1 The regulation of signaling pathways by hydrogen peroxide (H2O2) and superoxide has been linked to the development of various cardiovascular diseases including ischemic heart disease, hypertension, cardiomyopathies, cardiac hypertrophy, and congestive heart failure.2–4

Mitochondria play an integral role in cellular metabolism and oxidative phosphorylation but are also a source of superoxide and an important determinant of the fate of a cell. Increased ROS production by mitochondria has been reported after exposing mitochondria to ROS in cardiac myocytes.5,6 The synchronized release of ROS by the mitochondria has been shown to induce oscillations in action-potential duration and life-threatening postischemic arrhythmias.5,7,8 A persistent increase in intracellular ROS is associated with pathological remodeling and myocardial dysfunction.2,4,9

It has been suggested that increases in mitochondria-derived ROS are attributable to a direct effect of ROS on mitochondrial function.5,6 Another possible explanation for increased production of ROS by the mitochondria is enhanced Ca2+ uptake attributable to altered L-type Ca2+ channel (I Ca-L) function. It is reasonable to postulate an involvement of the channel in oxidative stress because the α1C subunit of the channel protein contains a number of cysteines that could be modified under oxidizing conditions. In support of this, channel function can be acutely modified by thiol-oxidizing compounds including H2O2.10–13 Acute exposure to H2O2 or thiol-oxidizing agents has been shown to increase macroscopic basal I Ca-L.10,11,13,14 In addition, adrenergic regulation of the channel is modified in response to alterations in the ROS production of the cell. A decrease in cellular production of superoxide or H2O2 has been shown to increase the sensitivity of the channel to β-adrenergic receptor stimulation.10,11,15 Therefore, there is good evidence that the activity of I Ca-L is responsive to alterations in the redox state of the cell.

In this study, we sought to understand the effects of H2O2 on myocyte function at a concentration insufficient to cause apoptosis or necrosis. We found that transient exposure to H2O2 induces an increase in mitochondria-derived superoxide in ventricular myocytes. The increase in cellular superoxide is reversible and is associated with increased intracellular Ca2+ and influx of Ca2+ into the mitochondria as a result of an increase in basal I Ca-L density. The increase in basal I Ca-L persisted for several hours after exposure to H2O2. We propose a model to explain the data and the persistent response. We suggest that this may be a possible mechanism for pathophysiological remodeling associated with transient oxidative stress that involves elevated intracellular Ca2+ and ROS.

Materials and Methods

Detection of Superoxide

Guinea pig ventricular myocytes and rat neonatal ventricular myocytes were isolated using a collagenase digestion method, as previously described.10,16 Generation of superoxide was assessed in 24-hour guinea pig myocytes or 36-hour contracting neonatal myocytes using the fluorescent indicator dihydroethidium (DHE) (5 μmol/L, 515- to 560-nm excitation filter, 590 long pass emission; Molecular Probes), as previously described.17 Application of the superoxide scavengers N-tert-butyl-α-phenyl-nitrone and superoxide dismutase significantly decreased DHE signal, confirming specificity of the indicator for superoxide (n=4).17 Comparisons using inhibitors were made with cells exposed to 30 μmol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes and were performed on the same day. For further details regarding cell isolation and detection of superoxide, see the online data supplement, available at http://circres.ahajournals.org.

Quantitation of Apoptosis/Caspase 3 Activity Assay

Caspase 3 activity was measured using Ac-DEVD-AMC (Promega) as a substrate.18,19 For further details, see the online data supplement.

Determination of Intracellular Ca2+

Intracellular Ca2+ was monitored using the fluorescent indicator Fura-2 acetoxymethyl ester (1 μmol/L, Molecular Probes). Fluorescent ratios at 340/380 nm excitation, 510 nm emission were measured over 50 ms at 1-minute intervals on a Hamamatsu Orca ER digital camera attached to an inverted Nikon TE2000-U microscope. Metamorph 6.3 was used to quantify the signal by manually tracing myocytes. An equivalent region not containing cells was used for background and was subtracted. The fluorescent ratios recorded over 3 minutes before and immediately after addition of H2O2 and catalase were averaged. We performed calibrations to determine intracellular Ca2+ concentrations in guinea pig ventricular myocytes as previously described (see the online data supplement).

Data Acquisition for Patch-Clamp Studies

The whole-cell configuration of the patch-clamp technique was used to record L-type Ca2+ currents up to 9 hours after isolation of myocytes as described previously.10 For further details, see the online data supplement.

Results

Transient Exposure to H2O2 Is Associated With an Increase in DHE Signal

We examined the effect of a transient exposure to H2O2 on DHE signal in adult ventricular myocytes. We chose DHE because it is a good indicator of changes in intracellular superoxide and does not interact with H2O2.20 However, intracellular superoxide is rapidly dismutated to H2O2, and we have shown that increases in cellular superoxide parallel increases in cellular H2O2.15,17 We began the experiments by titrating increasing concentrations of H2O2 until we detected an increase in cellular superoxide. We found that 20 to 30 μmol/L H2O2 was the minimum concentration required to reproducibly induce an increase in cellular superoxide. Myocytes were exposed to 30 μmol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the peroxide. The concentration of catalase was sufficient to completely degrade extracellular H2O2 (n=5; see the online data supplement). In 45 cells, 30 μmol/L H2O2 caused a 66.4±8.6% increase in DHE signal (Figure 1A and 1B). The increase in superoxide did not cause necrosis in any of the 45 cells tested (assessed with propidium iodide uptake) or apoptosis determined by caspase 3 assay (Figure 1C).

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Figure 1. A transient exposure to H2O2 is associated with an increase in DHE signal without apoptosis. A, DHE recorded from a guinea pig ventricular myocyte before and after 5 minutes of exposure to 30 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase (30 μmol/L H2O2 followed by catalase) and from another guinea pig ventricular myocyte before and after 5 minutes of exposure to 0 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase (0 μmol/L H2O2 then catalase). Addition of drugs occurred during the pause in data acquisition. B, Means±SE of the ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 minutes over the slope of the signal measured at 0 to 20 minutes for guinea pig ventricular myocytes exposed to 0 μmol/L H2O2 then catalase and for guinea pig ventricular myocytes exposed to 30 μmol/L H2O2 followed by catalase, as indicated. C, Caspase 3 assay performed in guinea pig ventricular myocytes in the presence of 0 μmol/L H2O2 followed by catalase (No H2O2) after exposure to 30 μmol/L H2O2 followed by catalase (Post H2O2) and doxorubicin as a positive control (see Materials and Methods for details). *P<0.05 compared with cells exposed to 0 μmol/L H2O2 then catalase (No H2O2).

Source of Increased Production of Superoxide Is the Mitochondria

We examined different sources for the increase in superoxide. In vascular smooth muscle cells, NAD(P)H oxidase is a prominent source of superoxide.21 Cells were exposed to 50 μmol/L gp91ds-tat peptide, a concentration of the peptide that is sufficient to inhibit angiotensin II–induced superoxide production in aortic rings by preventing association of gp47 phox with gp91 phox in NAD(P)H oxidase.22 The cells were then exposed to 30 μmol/L H2O2 for 5 minutes followed by 10 U/mL catalase, and superoxide production was measured (Figure 2A). In 5 cells, 30 μmol/L H2O2 caused a 91.0± 26.9% increase in DHE signal. This was not significantly different from cells exposed to H2O2 in the absence of the peptide (66.4±8.6% increase, n=45). Similar results were obtained when cells were exposed to NAD(P)H oxidase inhibitor apocynin (300 μmol/L) followed by H2O2 (59.9±17.6% increase, n=5; P=NS versus H2O2 in the absence of apocynin).

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Figure 2. Inhibitors of NAD(P)H oxidase, xanthine oxidase, and nitric oxide do not alter the increase in DHE signal after exposure to H2O2. A, DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and from another guinea pig ventricular myocyte before and after exposure to 50 μmol/L gp91ds-tat peptide followed by H2O2, as indicated. Means±SE of the ratio of fluorescence for guinea pig ventricular myocytes exposed to H2O2 or gp91ds-tat peptide, as indicated, are shown at right. B, DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and from another guinea pig ventricular myocyte before and after exposure to 50 μmol/L allopurinol followed by H2O2, as indicated. Means±SE of the ratio of fluorescence for guinea pig ventricular myocytes exposed to H2O2 or allopurinol, as indicated, are shown at right. C, DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and from another guinea pig ventricular myocyte before and after exposure to 100 μmol/L L-NAME followed by H2O2, as indicated. Means±SE of the ratio of fluorescence for guinea pig ventricular myocytes exposed to H2O2 or L-NAME, as indicated, are shown at right.

We examined whether xanthine oxidase was a possible source of superoxide by exposing the cells to 50 μmol/L allopurinol before H2O2. In 7 cells, 30 μmol/L H2O2 caused a 49.6±5.5% increase in DHE signal that was not significantly different from the increase in DHE signal recorded in the absence of allopurinol (66.4±8.6%, n=45; Figure 2B). Similarly when cells were exposed to the nitric oxide synthase inhibitor N G-nitro-l-arginine methyl ester (L-NAME) (100 μmol/L), there was no change in the DHE signal compared with cells exposed to H2O2 in the absence of L-NAME (74.4±22.6%, n=7 versus 66.4±8.6%, n=45; P=NS; Figure 2C).

We examined whether mitochondria were the source of superoxide by partially reducing mitochondrial membrane potential with 2 nmol/L carbonyl cyanide p-(trifluoromethoxy) phenyl-hydrazone (FCCP), an uncoupler of oxidative phosphorylation. In 10 cells, FCCP significantly attenuated the increase in DHE signal (P<0.05; Figure 3A). In addition, exposure of myocytes to 30 μmol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes caused a 11.6±5.6% increase in mitochondrial membrane potential assessed with the fluorescent indicator JC-1 (n=13; Figure 3B; see also the online data supplement). We performed additional experiments in which we partially inhibited the electron transport chain with 7 nmol/L myxothiazol. In 14 cells, myxothiazol significantly decreased DHE signal (P<0.05; Figure 3C). Consistent with previously published data, FCCP and myxothiazol decreased DHE signal in the absence of H2O2 (Figure 3A and 3C, inset, right).17 These data strongly suggest that the source of increase in superoxide following exposure to H2O2 is the mitochondria.

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Figure 3. The source of superoxide is mitochondrial. A, DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and from another guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and then 2 nmol/L FCCP. Means±SE of the ratio of fluorescence for all guinea pig ventricular myocytes exposed to H2O2 or FCCP, as indicated, are shown at right. *P<0.05 for 30 μmol/L H2O2 followed by catalase vs other treatments. B, JC-1 recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and from another guinea pig ventricular myocyte before and after exposure to 0 μmol/L H2O2 followed by catalase. To establish that the JC-1 signal was indicative of mitochondrial membrane potential, 20 μmol/L oligomycin and 4 μmol/L FCCP were added at the end of each experiment to collapse mitochondrial membrane potential where indicated. C, DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and from another guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and then 7 nmol/L myxothiazol. Means±SE of the ratio of fluorescence for all guinea pig ventricular myocytes exposed to H2O2 or myxothiazol, as indicated, are shown at right. *P<0.05 for 30 μmol/L H2O2 followed by catalase vs other treatments.

Increase in Cellular Superoxide Requires an Increase in Uptake of Ca2+ by the Mitochondria

We investigated whether an uptake of Ca2+ into the mitochondria is a requirement for the increase in superoxide. Ru360 (2 μmol/L), an inhibitor of the mitochondrial Ca2+ uniporter, applied before or after 30 μmol/L H2O2 significantly attenuated the increase in DHE signal (P<0.05; Figure 4A). To investigate the source of calcium, we exposed cells to the I Ca-L inhibitor nisoldipine (2 μmol/L) before or after 30 μmol/L H2O2. Nisoldipine significantly attenuated the increase in DHE signal (P<0.05; Figure 4B), suggesting that calcium influx through I Ca-L is a requirement for the increase in cellular superoxide. Oxidative stress can cause Ca2+ release from ryanodine receptors.23 However preventing ryanodine receptor activation with 20 μmol/L dantrolene did not change the DHE signal (n=6; P=NS versus exposure to H2O2 in the absence of dantrolene; Figure 4C).

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Figure 4. A, Ru360 attenuates the increase in DHE signal when applied before or after exposure to H2O2. DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase, from a guinea pig ventricular myocyte before and after exposure to 2 μmol/L Ru360 followed by H2O2 and then catalase, and from another guinea pig ventricular myocyte before and after exposure to H2O2 followed by catalase and then 2 μmol/L Ru360. Means±SE of the ratio of fluorescence for all guinea pig ventricular myocytes exposed to H2O2 or Ru360, as indicated, are shown at right. *P<0.05 for 30 μmol/L H2O2 followed by catalase vs all other treatments. B, Nisoldipine attenuates the increase in cellular superoxide when applied before or after exposure to H2O2. DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase, from a guinea pig ventricular myocyte before and after exposure to H2O2 followed by catalase and then 2 μmol/L nisoldipine (Nisol), and from another guinea pig ventricular myocyte before and after exposure to 2 μmol/L nisoldipine followed by H2O2 and then catalase, as indicated. Means±SE of the ratio of fluorescence for all guinea pig ventricular myocytes exposed to H2O2 or nisoldipine, as indicated, are shown at right. *P<0.05 for 30 μmol/L H2O2 followed by catalase vs all other treatments. C, Dantrolene does not alter the increase in cellular superoxide applied before exposure to H2O2. DHE recorded from a guinea pig ventricular myocyte before and after exposure to 30 μmol/L H2O2 followed by catalase and from a guinea pig ventricular myocyte before and after exposure to 20 μmol/L dantrolene followed by H2O2 and then catalase. Means±SE of the ratio of fluorescence for all guinea pig ventricular myocytes exposed to H2O2 or dantrolene, as indicated, are shown at right.

Transient Exposure to H2O2 Is Associated With Sustained Alteration in L-Type Ca2+ Channel Function

Alterations in cellular production of H2O2 influence I Ca-L. Exposing cardiac myocytes to hypoxia is associated with a decrease in cellular production of superoxide and H2O2 by the mitochondria that results in a decrease in basal channel activity, while increasing the sensitivity of the channel to β-adrenergic receptor stimulation.10,11,15,17 In addition, perfusing myocytes intracellularly with catalase (that specifically converts H2O2 to H2O and O2) mimics the increase in sensitivity of the channel to β-adrenergic receptor stimulation during hypoxia.15 We therefore used the response of the channel to β-adrenergic receptor stimulation as a functional reporter of a persistent oxidative stress. We exposed the cells to 30 μmol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade extracellular H2O2 and recorded the sensitivity of the channel to increasing concentrations of the β-adrenergic receptor agonist isoproterenol (Iso). In the absence of H2O2, 0.003 μmol/L and 0.01 μmol/L Iso elicited currents that were 28.8±7.1 and 74.1±8.6% of the current elicited by 1 μmol/L Iso, a maximally stimulating concentration of the β-adrenergic receptor agonist within the same cell (n=5; Figure 5A). However, after exposure to H2O2, 0.003 μmol/L and 0.01 μmol/L Iso elicited currents that were only 0.67±0.4 and 22.4±9.3% of the current recorded in response to 1 μmol/L Iso (n=9; Figure 5B). The K 0.5 for activation of the channel by Iso was significantly increased from 5.8±0.3 to 27.8±0.1 nmol/L (P<0.05; Figure 6A). There was no difference in the response of the channel to a maximally stimulating concentration of Iso (1 μmol/L) before or after exposure to H2O2 (Figure 6B). However, the activity of I Ca-L under non–β-adrenergic-stimulated conditions (basal channel activity) was significantly increased from 5.4±0.5 (n=7) to 8.9±0.7 pA/pF (n=25) at +10 mV following exposure to H2O2 (P<0.05). These data were recorded, on average, 4 hours after exposure to H2O2. The decrease in sensitivity of the channel to Iso and the increase in basal current activity persisted for at least 9 hours after exposure to H2O2 (and degradation of extracellular H2O2 with10 U/mL catalase). These data suggest that a transient exposure to H2O2 is associated with a persistent alteration in I Ca-L. It would appear that existing channels are persistently activated with increased current after exposure to H2O2.

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Figure 5. Transient exposure to H2O2 alters the response of I Ca-L to Iso. Time course of changes in membrane current recorded in a guinea pig ventricular myocyte during exposure to Iso, as indicated, after 5 minutes of exposure to 0 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase (A) and after 5 minutes of exposure to 30 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase (B), including membrane currents recorded at the time points indicated (inset, left). Current/voltage (I-V) relationship measured in the guinea pig ventricular myocyte during voltage steps from −60 mV to +80 mV is shown (inset, right).
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Figure 6. A, Transient exposure to H2O2 decreases the sensitivity of I Ca-L to Iso. Concentration dependence of Iso activation of I Ca-L in guinea pig ventricular myocytes exposed to 0 μmol/L H2O2 for 5 minutes followed by 5 minutes of exposure to 10 U/mL catalase (no hydrogen peroxide; n=5 to 10 at each data point) and after 5 minutes of exposure to 30 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase (after hydrogen peroxide exposure; n=4 to 9 at each data point). The Ca2+ conductance (G Ca) measured at each concentration of Iso was normalized to G Ca measured in the presence of 1 μmol/L Iso (a maximally stimulating concentration) in the same cell. Data were fit to a logistic equation using a nonlinear least squares curve-fitting routine (GraphPad Prism). B, Transient exposure to H2O2 results in a persistent increase in basal current density but no change in the response of I Ca-L to 1 μmol/L Iso, measured up to 9 hours after H2O2. No H2O2 indicates 5 minutes of exposure to 0 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase; Post H2O2, 5 minutes of exposure to 30 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase.

Persistently Altered I Ca-L Is Mediated by Superoxide Produced by the Mitochondria

The persistently altered I Ca-L could have been attributable to irreversible oxidation of the channel protein following a transient exposure to H2O2. Alternatively, it may have been maintained in an oxidized state as a result of increased cellular H2O2. We perfused cells intracellularly with 2000 U/mL catalase (which converts H2O2 to H2O and O2) and measured the response of the channel to Iso. Catalase significantly attenuated the decrease in sensitivity of the channel to Iso and the increase in basal current activity (see the online data supplement). These data suggest that after a transient exposure to H2O2, channel function is altered as a result of elevated cellular H2O2.

We confirmed that the mitochondria were the source of production of superoxide. Following exposure to 30 μmol/L H2O2, when cells were perfused intracellularly with FCCP or myxothiazol, the decrease in sensitivity of the channel to Iso and increase in basal current were significantly attenuated (see the online data supplement). In addition, the source of superoxide did not involve NAD(P)H oxidase, xanthine oxidase, or nitric oxide (see the online data supplement). These results confirm that channel function is persistently altered after a transient exposure to H2O2 but can be reversed when mitochondrial production of superoxide is inhibited.

Transient Exposure to H2O2 Is Associated With Elevated Intracellular Ca2+

We measured intracellular Ca2+ before and after exposure of guinea pig ventricular myocytes to 30 μmol/L H2O2 using the fluorescent indicator Fura-2. Figure 7A illustrates the persistent increase in intracellular Ca2+ after exposure to H2O2 and catalase in a guinea pig myocyte. Exposure of cells to H2O2 caused a significant increase in 340/380 fluorescence that was attenuated by 2 μmol/L nisoldipine before exposure to H2O2 but not with prior exposure to 2 μmol/L Ru360 (P<0.05; Figure 7A, inset at right), indicating the source of calcium was I Ca-L.

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Figure 7. A, Transient exposure to H2O2 is associated with a persistent nisoldipine-sensitive increase in intracellular Ca2+ in quiescent guinea pig ventricular myocytes. Intracellular Ca2+ in a cell recorded before and during 30 μmol/L H2O2 followed by 10 U/mL catalase (extracellular calcium, 0.42 mmol/L). Means±SE of changes in intracellular Ca2+ after exposure to H2O2, nisoldipine or Ru360, as indicated, are shown at right. B, Activation of I Ca-L is required for the increase in DHE signal. DHE recorded from a guinea pig ventricular myocyte before and after exposure to 2 μmol/L Bay K, from a guinea pig ventricular myocyte before and after exposure to 2 μmol/L Ru360 and then 2 μmol/L Bay K, and from a guinea pig ventricular myocyte before and after exposure to 2 μmol/L nisoldipine (Nisol) and then 2 μmol/L Bay K. Means±SE of the ratio of fluorescence for guinea pig ventricular myocytes exposed to Bay K alone, Ru360 followed by Bay K, or nisoldipine followed by Bay K, as indicated, are shown at right. Control indicates continuous recording in the absence of drugs. *P<0.05 for Bay K vs all other groups.

Activation of I Ca-L Is Required for an Increase in Mitochondria-Derived Superoxide

We examined whether activation of I Ca-L was sufficient to increase intracellular superoxide. Application of 2 μmol/L Bay K, an L-type Ca2+ channel agonist, caused a 79.2±14.4% increase in DHE signal (n=7). This was comparable to the increase in DHE recorded after exposure to H2O2 (Figure 1A and 1B). The increase in DHE could be attenuated by application of 2 μmol/L Ru360 or 2 μmol/L nisoldipine before exposure to Bay K (Figure 7B). These data indicate that activation of I Ca-L is sufficient for an increase in mitochondrial uptake of calcium and increased superoxide production by the mitochondria.

Transient Exposure to H2O2 Is Associated With an Increase in DHE Signal and Elevated Intracellular Ca2+ in Active Cycling Myocytes

We examined the effect of a transient exposure to H2O2 on DHE signal in spontaneously contracting neonatal rat ventricular myocytes. Exposure to 30 μmol/L H2O2 for 5 minutes followed by 10 U/mL catalase for 5 minutes caused a 8.1-fold increase in DHE signal that could be attenuated by exposure of cells to 2 μmol/L Ru360 before H2O2 (Figure 8A). In addition, exposure of spontaneously contracting myocytes to 30 μmol/L H2O2 for 5 minutes resulted in a small increase in diastolic 340/380 fluorescence (10.5±5.1% increase in 4 of 19 cells), but after 10 minutes, the diastolic 340/380 fluorescence was increased 71.1±9.5% (Figure 8B), without causing apoptosis or necrosis, as determined by caspase 3 assay and lactate dehydrogenase release 24 hours later (see Figure III in the online data supplement). Application of 2 μmol/L nisoldipine before 30 μmol/L H2O2 prevented the increase in diastolic 340/380 fluorescence (Figure 8B). Consistent with the results recorded in quiescent guinea pig ventricular myocytes, a transient exposure to H2O2 is associated with a significant increase in cellular superoxide and diastolic Ca2+ in active calcium cycling myocytes.

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Figure 8. A, Transient exposure to H2O2 is associated with an increase in DHE signal in contracting neonatal rat ventricular myocytes. DHE recorded from a rat ventricular myocyte before and after 5 minutes of exposure to 30 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase (30 μmol/L H2O2 followed by catalase), from a rat ventricular myocyte before and after 5 minutes of exposure to 0 μmol/L H2O2 followed by 5 minutes of exposure to 10 U/mL catalase (0 μmol/L H2O2 followed by catalase), and from a rat ventricular myocyte before and after exposure to 2 μmol/L Ru360 followed by 5 minutes of exposure to 30 μmol/L H2O2 and then 5 minutes of exposure to 10 U/mL catalase (Ru360 and then 30 μmol/L H2O2 followed by catalase). Means±SE of the ratio of fluorescence for rat ventricular myocytes exposed to 30 μmol/L H2O2 followed by catalase, 0 μmol/L H2O2 followed by catalase, and Ru360 followed by H2O2 and then catalase, as indicated, are shown at right. *P<0.05 for 30 μmol/L H2O2 followed by catalase vs other groups. B, Transient exposure to H2O2 is associated with a nisoldipine-sensitive increase in intracellular Ca2+ in contracting neonatal rat ventricular myocytes. Ca2+ transients from a rat ventricular myocyte recorded before and after (indicated by pause in acquisition of transients) 10 minutes of exposure to 30 μmol/L H2O2, from a rat ventricular myocyte recorded before and after 10 minutes of exposure to 0 μmol/L H2O2, and from a rat ventricular myocyte recorded before and after 2 μmol/L nisoldipine (Nisol) followed by 10 minutes of exposure to 30 μmol/L H2O2. Means±SE of the percentage change in 340/380 fluorescence after exposure to H2O2 or nisoldipine, as indicated, are shown at right. *P<0.05 for 30 μmol/L H2O2 vs other groups. C, Proposed model explaining the persistent increase in intracellular ROS and intracellular Ca2+ after a transient H2O2 exposure (see Discussion).

Discussion

In this study, we examined the effects of a brief exposure of cardiac myocytes to H2O2 at a concentration that did not cause apoptosis or necrosis. We found that 5 minutes of exposure of cardiac myocytes to 30 μmol/L H2O2 was sufficient to cause persistent alterations in cellular superoxide production, I Ca-L, and cellular Ca2+. The increase in cellular superoxide was dependent on activation of I Ca-L, and it was reversible. One current view regarding the increased production of ROS by mitochondria following oxidative stress is that the mitochondria are the target of ROS.6,8 Our results differ from these studies in that we specifically applied an oxidative stress externally that is likely to mimic a burst of ROS associated with ischemia/reperfusion in vivo. We attenuated the oxidative stress with catalase and examined the effect on cellular function. At low concentrations (30 μmol/L) of H2O2, an increase in the activity of I Ca-L was required for the increase in mitochondrial ROS production. Because Bay K alone increased cellular superoxide (Figure 7B), it would appear that the increase in superoxide occurs solely because of an increase in basal I Ca-L activity and that low concentrations of H2O2 do not have to directly affect mitochondria. Persistently elevated intracellular Ca2+ and ROS are associated with induction of calmodulin and NFAT pathways that lead to pathological states such as cardiac hypertrophy and failure.24,25 Chronic in vitro exposure to low concentrations of H2O2 (10 to 30 μmol/L) have been shown to induce protein synthesis in adult cardiac myocytes, without affecting survival.26 Our results may represent the mechanisms that contribute to the development of cardiac pathology.

The L-type Ca2+ channel is responsive to alterations in cellular redox state. When intracellular H2O2 is decreased with exposure of myocytes to hypoxia or intracellular application of catalase, the sensitivity of the channel to β-adrenergic receptor stimulation is increased.10,11,15 This response is mimicked when cells are exposed to the thiol-specific reducing agent dithiothreitol, suggesting that direct redox modification of the channel or redox modification of a signaling intermediate such as protein kinase A is responsible for the response. We found that the K 0.5 for activation of the channel by Iso significantly increased after exposure to H2O2, consistent with a response of the channel to an oxidized cellular environment (Figures 5B and 6A). The response persisted for many hours after the insult as a result of a persistent increase in production of superoxide by the mitochondria because the mitochondria inhibitors myxothiazol and FCCP attenuated the decrease in sensitivity of the channel to Iso. If direct oxidation of the channel is necessary for the increase in basal current density (as acute exposure to hydrogen peroxide or thiol-oxidizing agents would suggest10–13), then the persistent increase in cellular superoxide appears to be necessary to maintain the channel in an oxidized state. In support of this, we found that basal current density was increased many hours after the transient exposure to H2O2 (Figure 6B), and this was attenuated by intracellular catalase, FCCP, and myxothiazol.

Consistent with persistent L-type Ca2+ channel activation, intracellular Ca2+ was increased in the myocytes (Figures 7A and 8B). Our data indicate that Ca2+ influx through the L-type Ca2+ channel is required for the increase in superoxide by the mitochondria because nisoldipine attenuated the increase in DHE signal (Figure 4B) and the increase in intracellular Ca2+ (Figures 7A and 8B). In addition Bay K alone was sufficient to increase Ca2+ uptake into the mitochondria and increase superoxide (Figure 7B). Dantrolene, an inhibitor of ryanodine release of Ca2+ from sarcoplasmic reticulum stores, did not alter the increase in DHE signal after exposure to H2O2 (Figure 4C). We propose therefore that the increase in intracellular Ca2+ and superoxide persists because a positive feedback exists between increased basal channel activity and superoxide production by the mitochondria (Figure 8C). The model does not preclude a direct effect of H2O2 on the mitochondria. Our data show that increased L-type Ca2+ channel activity is required for the response at low H2O2 concentrations. The L-type Ca2+ channel appears to be an important regulator of cellular Ca2+ and cellular superoxide production under conditions of oxidative stress that do not involve apoptosis or necrosis and have the potential to mediate cardiac pathology.

Presented in part at the 3rd Annual Symposium of the American Heart Association Council on Basic Cardiovascular Sciences, July 31–August 3, 2006, Keystone, Colo (Circ Res 2006;99:E24).

Original received May 31, 2006; resubmission received November 8, 2006; revised resubmission received February 7, 2007; accepted February 27, 2007.

Sources of Funding

This study was supported by a grant from the National Health and Medical Research Council of Australia. L.C.H. is the recipient of a National Health and Medical Research Council Career Development Award.

Disclosures

None.

Footnotes

Correspondence to Dr Livia Hool, Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, University of Western Australia, 35 Stirling Hwy, Crawley, WA, 6009, Australia. E-mail [email protected]

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Source: https://www.ahajournals.org/doi/10.1161/01.res.0000263010.19273.48