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Cardiovascular Research 2005 67(4):624-635; doi:10.1016/j.cardiores.2005.04.025
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Copyright © 2005, European Society of Cardiology

Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca2+ channel function during acute hypoxia

Livia C. Hoola,c,*, Carla A. Di Mariab,c, Helena M. Violaa,c and Peter G. Arthurb,c

aDiscipline of Physiology, The University of Western Australia, Crawley, WA, Australia
bDiscipline of Biochemistry, The University of Western Australia, Crawley, WA, Australia
cThe Western Australian Institute for Medical Research, Australia

* Corresponding author. Physiology M311, The University of Western Australia, 35 Stirling Highway, Crawley, WA, 6009, Australia. Tel.: +61 8 6488 3307; fax: +61 8 6488 1025. Email address: lhool{at}cyllene.uwa.edu.au

Received 9 February 2005; revised 21 April 2005; accepted 22 April 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The role of NAD(P)H oxidase in regulating cellular production of reactive oxygen species (ROS) and the L-type Ca2+ channel during acute hypoxia was examined in adult ventricular myocytes from guinea pig.

Methods: The fluorescent indicator dihydroethidium (DHE) was used to detect superoxide and the response of the L-type Ca2+ channel to β-adrenergic receptor stimulation was used as a functional reporter since hypoxia increases the sensitivity of the L-type Ca2+ channel (ICa-L) to isoproterenol (Iso).

Results: Hypoxia caused a 41.2 ± 5.2% decrease in the rate of the DHE signal (n = 21; p<0.01). Of the classical NAD(P)H oxidase inhibitors, DPI but not apocynin mimicked the effect of hypoxia on the sensitivity of ICa-L to Iso. However, the potent NAD(P)H oxidase agonist angiotensin II had no effect on cellular superoxide or the sensitivity of ICa-L to Iso. Although DPI inhibits NAD(P)H oxidase, it also decreased superoxide in isolated mitochondria in a concentration-dependent manner. Partial inhibition of mitochondrial function with nanomolar concentrations of FCCP or myxothiazol mimicked the effect of hypoxia on cellular superoxide and the sensitivity of ICa-L to Iso. In addition, hypoxia caused a 69.3 ± 0.8% decrease in superoxide in isolated mitochondria (n = 4; p<0.01), providing direct evidence for a role for the mitochondria.

Conclusions: Our data suggest that mitochondria appear to be involved in oxygen sensing, regulation of cellular ROS, and the function of ICa-L during acute hypoxia in cardiac myocytes and NAD(P)H oxidase does not appear to contribute substantially.

KEYWORDS Hypoxia; NADPH-oxidase; Ca-channel; Mitochondria

This article is referred to in the Editorial by R.T. Mallet (pages 578–580) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Reactive oxygen species (ROS) are well recognised as important mediators of cardiovascular pathology including hypertrophy, hypertension, atherosclerosis and heart failure [1]. The one electron-reduction of oxygen leads to the formation of superoxide that readily dismutates to hydrogen peroxide (H2O2), a stable and diffusible signalling molecule. Both mitochondria and NAD(P)H oxidase are capable of participating directly in the reduction of oxygen and producing superoxide. In vascular tissue the NAD(P)H oxidase is recognised as the predominant source of superoxide [2].

Recent studies are providing evidence for a role for NAD(P)H oxidase in cardiac pathology. Mice lacking gp91 phox subunit do not develop angiotensin II (Ang II)-induced cardiac hypertrophy [3,4]. In addition, NAD(P)H oxidase expressed in guinea pig cardiac myocytes is a major source of superoxide in pressure overload hypertrophy [5]. NAD(P)H oxidase could also be responsive to changes in oxygen concentration as oxygen is a substrate for NAD(P)H oxidase. Hypoxia would be expected to cause a decrease in superoxide if NAD(P)H oxidase is the primary site of production of superoxide in a cell [6]. Of importance physiologically therefore is whether the oxidase plays a role in the rapid regulation of cellular levels of superoxide in cardiac myocytes in response to changing oxygen concentrations. Identifying the mechanisms responsible for this is important in cardiac myocytes because rapid changes in cellular oxygen can contribute to electrophysiological instability in the myocardium and the development of arrhythmias [7–9].

In this study we examine whether NAD(P)H oxidase is involved in acute hypoxia responses in ventricular myocytes. Specifically we examine whether NAD(P)H oxidase alters cellular production of superoxide and whether it regulates the function of the L-type Ca2+ channel (ICa-L) during hypoxia. In addition, we used the response of the channel as a functional reporter of alterations in cellular levels of ROS. The justification for this is based on evidence that perfusing cardiac myocytes intracellularly with catalase (that specifically converts H2O2 to O2 and H2O) mimics the increase in the sensitivity of ICa-L to β-adrenergic receptor stimulation recorded during acute hypoxia [10]. Here we report that the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) mimics the effect of hypoxia on ICa-L. However our data suggest that alterations in production of superoxide by the mitochondria appear to mediate the changes in channel function. We conclude that NAD(P)H oxidase does not appear to be involved in acute oxygen sensing in adult ventricular myocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Data acquisition for patch-clamp studies
Cardiac myocytes were isolated as previously described [11,12] and approved by The Animal Ethics Committee of The University of Western Australia in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NH and MRC, 7th Edition, 2004 consistent with NIH guidelines). The whole-cell configuration of the patch-clamp technique was used to record currents up to 9 h after isolation of myocytes as described previously [11]. Microelectrodes with tip diameters of 3–5 µm and resistances of 0.5–1.5 M{Omega} contained (in mM): CsCl 115, HEPES 10, EGTA 10, tetraethylammonium chloride 20, MgATP 5, Tris–GTP 0.1, phosphocreatine 10, and CaCl2 1 (pH adjusted to 7.05 at 37 °C with CsOH). The composition of the internal solution was calculated to result in a free Ca2+ concentration of {approx}10 nM [13]. Currents were measured in extracellular modified Tyrode's solution that contained (in mM): NaCl 140, CsCl 5.4, CaCl2 2.5, MgCl2 0.5, HEPES 5.5 and glucose 11 (pH adjusted to 7.4 with NaOH).

2.2. Detection of superoxide and hydrogen peroxide
Using standard culture technique, cardiac myocytes were plated onto poly-D-lysine-coated culture dishes and allowed to become adherent with incubation overnight. Just prior to experimentation the culture dish media was changed to a solution containing (in mM): KCl 5.33, MgSO4 0.41, NaCl 139, Na2HPO4 5.63, glucose 5, HEPES 20, glutamine 2, Ca(NO3)2 0.42 and 1 ml/100ml penicillin/streptomycin (pH adjusted to 7.4 with NaOH). Generation of superoxide was assessed using the fluorescent indicator dihydroethidium (DHE, 5 µM, 515–560 nm ex filter, 590 long pass em; Molecular Probes) and hydrogen peroxide was assessed with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (DCF, 2 µM, 450–90 ex filter, 515 long pass em; Molecular Probes) [10]. Metamorph 5.2 was used to quantify the signal by manually tracing myocytes. An equivalent region not containing cells was used for background and was subtracted. The ratio of fluorescence was expressed as the slope of the signal measured at 35–45 min over the slope of the signal measured at 20–30 min within the same cell. We found no difference in the density of ICa-L or the sensitivity of ICa-L to Iso during hypoxia between freshly isolated myocytes and 24-h-old cultured myocytes in 6 cells tested.

2.3. Studies in isolated mitochondria
Mitochondria were isolated as described by Sayen et al. [14] after Langendorff perfusion and used at a final concentration of 50 µg protein/ml in respiration media at 30 °C. The respiration media contained (in mM): sucrose 200, MgCl2 3, HEPES 20, taurine 20, KH2PO4 10, and EGTA 0.5 (pH adjusted to 7.4 with KOH). Oxygen consumption was measured using a Clark type oxygen electrode after addition of complex I substrates 5 mM glutamate and 5 mM malate. ADP at 1 mM was added to initiate state III respiration. Respiratory control ratios above 8.0 indicated intact mitochondria. Superoxide was monitored using DHE in a Shimadzu RF-5000 Spectrofluorophotometer, with excitation and emission wavelengths of 480 (10 nm slit width) and 590 nm (30 nm slit width), respectively. Initial background measurements were obtained with media and DHE (10 µM) alone, before the addition of 5 mM glutamate, 5 mM malate and 10 mM malonate. A stable background was followed by the addition of freshly isolated intact mitochondria. The average rate of DHE oxidation was monitored over 5 min, before the addition of superoxide dismutase (100 U/ml) to obtain a final background rate (later subtracted).

All experiments were performed at 37 °C. Results are reported as means ± SEM. Student's t-test and ANOVA (with Tukey's posthoc test for multiple comparisons) were performed on GraphPad Prism. All hypoxia experiments were performed at ~17 mm Hg oxygen tension as determined by an oxygen-sensitive probe [11]. Normoxia (room oxygen) was determined as 150–160 mm Hg oxygen tension.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Hypoxia causes a decrease in cellular superoxide
We confirmed firstly that the myocytes express functional NAD(P)H oxidase protein by performing an NAD(P)H oxidase assay using NADPH as the substrate (Fig. 1A) [15]. We have shown previously that hypoxia causes a decrease in H2O2 in guinea pig ventricular myocytes [10]. To directly examine superoxide we used the fluorescent indicator DHE. Myocytes were exposed to normoxia (room oxygen; pO2 of 150–160 mm Hg) and fluorescence was recorded for 30 min. At 30 min the cells were exposed to solutions made hypoxic (pO2 of 15 mm Hg). Hypoxia caused a 41.2 ± 5.2% decrease in the rate of DHE signal in 21 cells (p<0.01) (Fig. 1B and C).


Figure 1
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  		Fig. 1 A. Adult guinea pig cardiac myocytes express functional NAD(P)H oxidase. NADPH consumption in the presence of 100 µM DPI and 500 µM apocynin (Apo) assessed by spectrophotometry from 5 hearts. B. Hypoxia is associated with a decrease in cellular superoxide. DHE fluorescence recorded from a cell exposed to normoxia only and in another cell exposed to normoxia followed by hypoxia (Normoxia/hypoxia); a.u.=arbitrary units. The superoxide scavenger N-tert-butyl-alpha-phenyl-nitrone (PBN) caused 90 ± 3% decrease in the rate of DHE signal (inset right). C. Ratio of fluorescence for cells exposed to normoxia only and for cells exposed to normoxia followed by hypoxia (Normoxia/hypoxia).

 
3.2. Of the classic NAD(P)H oxidase inhibitors, DPI but not apocynin mimics the effect of hypoxia on ICa-L
Oxygen is a substrate for NAD(P)H oxidase, so changes in oxygen concentrations could affect the rate at which NAD(P)H oxidase reduces oxygen to superoxide. Consequently, inhibiting NAD(P)H oxidase would be expected to mimic a hypoxic response. We tested whether the NAD(P)H oxidase inhibitors DPI and apocynin mimicked the effect of hypoxia on the response of ICa-L to Iso. Hypoxia causes a decrease in the K0.5 for activation of ICa-L by Iso from 5.3 ± 0.7 to 1.6 ± 0.1 nM [11,12]. We exposed cardiac myocytes to 3 and 10 nM Iso and compared the responses to the current elicited by a maximally stimulating concentration of the agonist (1 µM) in the same cell. In 11 cells 300 µM apocynin alone did not significantly alter basal ICa-L (1.9 ± 1.7% increase in ICa-L; Fig. 2A and C). In the continued presence of apocynin, the currents produced by 3 and 10 nM Iso were 43.2 ± 7.4% and 81.7 ± 9.3% of the response elicited by 1 µM Iso. These currents were not significantly different from currents elicited by 3 and 10 nM Iso in the absence of apocynin (28.7 ± 7.1% and 72.1 ± 8.6%, n = 5; Fig. 2C). We then exposed cells to 10–20 µM DPI, a concentration range typically used to inhibit NAD(P)H oxidase-mediated responses, and recorded ICa-L. In the absence of Iso, DPI had very little effect on basal ICa-L (1.5 ± 3.3% decrease in basal ICa-L). However in the continued presence of DPI, 3 and 10 nM Iso elicited currents that were 70.1 ± 12.5% and 98.1 ± 1.1% of the current activated by 1 µM Iso (n = 8; Fig. 2B). These responses were significantly different from currents elicited by the same concentrations of Iso in the absence of DPI (p<0.01) and similar to currents elicited by the same concentrations of Iso during hypoxia (Fig. 2C and [11,12]). In addition, there was no difference between the maximal responses recorded during 1 µM Iso and normoxia (10.2 ± 1.5 pA/pF), hypoxia (10.0 ± 0.8 pA/pF), DPI (9.4 ± 1.4 pA/pF) or apocynin (12.1 ± 1.7 pA/pF; ANOVA p = NS). In contrast to the results recorded with apocynin, these data indicate that inhibition of NAD(P)H oxidase activity with DPI mimics the effect of hypoxia on the response of ICa-L to Iso. The results with DPI but not apocynin suggest that NAD(P)H oxidase may mediate hypoxic responses in cardiac myocytes.


Figure 2
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  		Fig. 2 DPI but not apocynin mimics the effect of hypoxia on ICa-L. Time course of changes in membrane current recorded in a cell during exposure to 300 µM apocynin and Iso (A) and 20 µM DPI and Iso (B) including membrane currents recorded at the time points indicated (inset left). Current–voltage (I–V) relationship measured in the cell during voltage steps from –60 to +80 mV is shown (inset right). C. Summarised mean ± SEM of basal currents recorded during hypoxia (H), apocynin (Apo) or DPI and currents elicited by 0.003 and 0.01 µM Iso normalised to current recorded in the presence of 1.0 µM Iso and hypoxia, normoxia, apocynin and DPI. *p<0.05 compared with currents recorded during normoxia or apocynin for the same concentration of Iso. **p<0.05 between the effect of hypoxia and apocynin or DPI on basal currents only.

 
3.3. The NAD(P)H oxidase agonist angiotensin II does not attenuate the effect of hypoxia on ICa-L
If NAD(P)H oxidase is involved in the effect of hypoxia on the sensitivity of ICa-L to Iso, then activation of NAD(P)H oxidase by Ang II should attenuate the effect of hypoxia on ICa-L. We firstly examined the effect of Ang II alone on basal ICa-L and the response of the channel to Iso. Ang II at 100 nM decreased basal ICa-L by 5.0 ± 3.2%. In the presence of Ang II, 3 and 10 nM Iso elicited currents that were 17.8 ± 9.6% and 69.2 ± 15.1% of currents elicited by 1 µM Iso (n = 6). These responses were not significantly different from currents recorded during the same concentrations of Iso in the absence of Ang II [10–12]. When myocytes were exposed to hypoxia and 100 nM Ang II, basal ICa-L was decreased 20.0 ± 3.6% (Fig. 3A). This was not significantly different from the effect of hypoxia alone on basal ICa-L (22.2 ± 1.7% decrease [10–12]). In the continued presence of hypoxia and Ang II, 3 and 10 nM Iso elicited currents that were 61.7 ± 11.9% and 84.6 ± 7.8% of the current recorded during exposure to 1 µM Iso (n = 10, Fig. 3A). These responses were not significantly different from currents recorded during exposure to the same concentrations of Iso during hypoxia alone (70.9 ± 10.7% and 94.4 ± 5.6%; n = 11)[10–12]. To examine this further myocytes were exposed to increasing concentrations of Iso and the concentration-dependence of ICa-L on Iso during hypoxia in the presence and absence of Ang II was determined (Fig. 3B). Ang II did not alter the sensitivity of ICa-L to Iso during hypoxia. The K0.5 for activation of ICa-L by Iso during exposure to hypoxia and Ang II was 1.7 ± 0.4 nM (p = NS vs hypoxia alone; [11]). There was no difference between the maximal responses recorded during 1 µM Iso and normoxia (10.2 ± 1.5 pA/pF), hypoxia (10.0 ± 0.8 pA/pF), or hypoxia plus Ang II (9.2 ± 1.3 pA/pF; ANOVA p = NS). Additional experiments were performed using the perforated patch method with pipettes containing amphotericin B at a concentration of 0.2 mg/ml pipette solution and free Ca2+ buffered to {approx}10 nM (see Methods). Under these conditions, 500 nM Ang II did not significantly alter basal ICa-L (1.3 ± 5.8% decrease) or the response of ICa-L to 3 and 10 nM Iso (34.0 ± 7.3% and 77.5 ± 10.2% of the current recorded during 1 µM Iso; n = 5, p = NS vs normoxia alone).


Figure 3
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  		Fig. 3 Ang II does not attenuate the effect of hypoxia on the sensitivity of ICa-L to Iso. A. Time course of changes in membrane current recorded in a cell during exposure to 100 nM Ang II alone followed by Ang II plus hypoxia and Ang II plus hypoxia and Iso. I–V shown from the same cell (inset left). Mean ± SE of currents exposed to Ang II alone (Ang II), Ang II plus hypoxia (+hyp) and Ang II plus hypoxia and 0.003 µM Iso, (+0.003), 0.01 µM Iso (+0.01) and 1.0 µM Iso (+1.0) are shown inset right. B. Concentration-dependence of Iso activation of ICa-L in cells exposed to normoxia (n = 5–10 at each data point), hypoxia alone (n = 5–11 at each data point) and 100 nM Ang II and hypoxia (n = 4–10 at each data point). Currents at each concentration were normalised to current elicited by 1 µM Iso in the same cell. Data were fit to a logistic equation using a non-linear least squares curve-fitting routine (GraphPad Prism).

 
We examined the effect of thrombin, another agonist of NAD(P)H oxidase. Thrombin alone (5 units/ml) decreased basal ICa-L 10.0 ± 8.8%. In the presence of thrombin, hypoxia caused a further 8.7 ± 3.0% decrease in basal ICa-L. In the continued presence of thrombin and hypoxia, 3 and 10 nM Iso elicited currents that were 72.8 ± 16.1% and 91.2 ± 8.2% of the current recorded during 1 µM Iso (n = 6; p = NS vs hypoxia alone [11,12]). These data suggest that agonists of NAD(P)H oxidase do not acutely alter the effect of hypoxia on basal ICa-L or the sensitivity of ICa-L to Iso.

3.4. Angiotensin II does not alter cellular superoxide
We tested whether activation of NAD(P)H oxidase with Ang II could alter superoxide in the absence or presence of hypoxia. First cells were exposed to hypoxia for 20 min and then fluorescence was recorded. After 30 min of recording, Ang II was added to the hypoxic solution. In 12 cells, 5 µM Ang II did not acutely alter the rate of DHE signal during hypoxia (96.9 ± 4.1% of the superoxide recorded during hypoxia alone; p = NS; Fig. 4A). Consistent with this, we found that Ang II also did not significantly alter H2O2 during hypoxia using the fluorescent indicator DCF (95.2 ± 5.7% of H2O2 recorded during hypoxia alone, n = 13; p = NS). Similar results were obtained with exposure of 6 myocytes to 5 µM Ang II during room oxygen (Fig. 4B).


Figure 4
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  		Fig. 4 A. Ang II does not alter superoxide during exposure to hypoxia in cardiac myocytes. DHE fluorescence recorded from a cell exposed to hypoxia only and in another cell exposed to hypoxia followed by 5 µM Ang II (Hypoxia/Ang II). Ratio of fluorescence for cells exposed to hypoxia only and for cells exposed to hypoxia followed by Ang II (Hypoxia/Ang II) is shown inset right. B. Ang II does not alter superoxide during exposure to room oxygen in cardiac myocytes. DHE fluorescence recorded from a cell exposed to normoxia only and in another cell exposed to normoxia followed by addition of 5 µM Ang II at 30 min (Normoxia/Ang II). Ratio of fluorescence for cells exposed to normoxia only and for cells exposed to normoxia followed by Ang II (Normoxia/Ang II) is shown inset right. C, Ang II increases cellular superoxide in embryonic cortical neurons. DHE fluorescence recorded from a neuron exposed to normoxia only and in another neuron exposed to normoxia followed by 5 µM Ang II (Normoxia/Ang II). Ratio of fluorescence for cells exposed to normoxia only and for cells exposed to normoxia followed by Ang II (Normoxia/Ang II) is shown inset at right.

 
Since Ang II can mediate pressor effects via a NAD(P)H oxidase-dependent increase in superoxide in central neurons [16], we isolated embryonic cortical neurons [17] and examined whether Ang II could activate NAD(P)H oxidase in the neurons under our experimental conditions. In the presence of 5 µM Ang II, we recorded a 96.6 ± 31.4% increase in superoxide in the neurons (n = 16, p<0.01; Fig. 4C). This experiment indicates that our system is capable of detecting alterations in cellular superoxide caused by Ang II. We conclude that in adult ventricular myocytes Ang II does not alter cellular superoxide.

3.5. DPI causes a decrease in cellular and mitochondrial superoxide
We examined whether the effect of DPI on ICa-L was due to an alteration in cellular superoxide in cardiac myocytes. Cells were exposed to room oxygen for 30 min and fluorescence was recorded. At 30 min the cells were exposed to 1 µM DPI and the ratio of fluorescence was determined. DPI caused a 21.3 ± 4.7% decrease in the rate of DHE signal in 16 cells (p<0.05; Fig. 5A). In contrast, the ratio of the rate of increase in DHE signal was not different between normoxia alone (95.0 ± 5.5%, n = 14) and normoxia plus 300 µM apocynin (94.4 ± 5.2%, n = 5).


Figure 5
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  		Fig. 5 A. DPI is associated with a decrease in cellular superoxide. DHE fluorescence recorded from a cell exposed to normoxia only and in another cell exposed to normoxia followed by DPI (Normoxia/DPI). Ratio of fluorescence for cells exposed to normoxia only and for cells exposed to normoxia followed by DPI (Normoxia/DPI) is shown inset at right. B. DPI is associated with a decrease in superoxide in mitochondria isolated from guinea pig ventricular myocytes. DHE fluorescence recorded from isolated mitochondria exposed to normoxia alone and in isolated mitochondria exposed to normoxia +100 µM DPI; mitos added=time point at which mitochondria were added. SOD added=time point at which superoxide dismutase was added. Mean ± SEM of the slope of fluorescence recorded in isolated mitochondria exposed to 0, 1, 10 and 100 µM DPI normalised to fluorescence recorded in the absence of DPI is shown inset at right. C. DPI inhibits oxygen consumption in isolated mitochondria from guinea pig ventricular myocytes. Oxygen consumption in isolated mitochondria exposed to normoxia alone and in isolated mitochondria exposed to normoxia +100 µM DPI. Mean ± SEM of the slope of oxygen consumption recorded in isolated mitochondria exposed to 0, 1, 10 and 100 µM DPI normalised to oxygen consumption recorded in the absence of DPI is shown at right. Mean oxygen consumption in the absence of DPI was 165 ± 28.3 nmol O2/min/mg protein.

 
We examined whether the effect of DPI on ICa-L could be explained by a direct effect of DPI on the mitochondria [18,19]. Firstly we examined whether DPI altered superoxide in mitochondria isolated from guinea pig ventricular myocytes from 4 hearts. DPI caused a dose-dependent decrease in superoxide (Fig. 5B). We also determined the effect of DPI on ADP-stimulated oxygen consumption in isolated mitochondria. In preparations from 4–6 hearts, DPI caused a significant decrease in oxygen consumption at 1, 10 and 100 µM DPI (Fig. 5C). These data indicate that the concentration of DPI used experimentally (1–20 µM) was sufficient to partially inhibit mitochondrial function and cause a decline in superoxide from mitochondria. We conclude that DPI does not exclusively inhibit NAD(P)H oxidase activity in cardiac myocytes and probably inhibits mitochondrial production of superoxide.

3.6. Partial inhibition of mitochondrial function with myxothiazol or FCCP decreases cellular superoxide and mimics the hypoxic response
Since DPI appeared to alter channel function via effects on the mitochondria, we examined further whether the effects of hypoxia were being mediated via alterations in mitochondrial superoxide. Since superoxide production has been shown to be dependent on mitochondrial membrane potential [20] we partially inhibited the electron transport chain with 4 nM myxothiazol. Complete inhibition of electron transport with higher concentrations of myxothiazol caused rapid myocyte contraction and death. Fluorescence was recorded in room oxygen and then after addition of 4 nM myxothiazol to the myocytes. Myxothiazol caused a 23.3 ± 3.8% decrease in the rate of DHE signal in 38 cells (Fig. 6A). When cells were dialyzed with 4 nM myxothiazol, 3 and 10 nM Iso elicited currents that were 77.8 ± 13.7% and 90.0 ± 7.2% of currents recorded in the presence of 1 µM Iso (n = 6; Fig. 6B). These were similar to currents recorded during hypoxia alone [10–12]. There was no difference between maximal responses recorded during 1 µM Iso and normoxia (10.2 ± 1.5 pA/pF), hypoxia (10.0 ± 0.8 pA/pF) or myxothiazol (9.8 ± 2.1 pA/pF; ANOVA p = NS).


Figure 6
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  		Fig. 6 A. Myxothiazol mimics the effect of hypoxia on superoxide. DHE fluorescence recorded from a cell exposed to normoxia only and in another cell exposed to normoxia followed by 4 nM myxothiazol (Normoxia/myx). Ratio of fluorescence for cells exposed to normoxia only and for cells exposed to normoxia followed by myxothiazol (Normoxia/myx) is shown inset at right. B. Myxothiazol mimics the effect of hypoxia on the sensitivity of ICa-L to Iso. Time course of changes in membrane current recorded in a cell while being perfused intracellularly with 4 nM myxothiazol. I–V shown from the same cell (inset left). Mean ± SE of currents exposed to 0.003 (+0.003), 0.01 (+0.01) and 1.0 µM Iso (+1.0) while being perfused intracellularly with myxothiazol are shown inset at right. *Statistical significance (p<0.05) compared with cells not perfused with myxothiazol in room oxygen.

 
We performed additional experiments in which we partially reduced mitochondrial membrane potential with 2 nM carbonyl cyanide p-(trifluromethoxy)phenyl-hydrazone (FCCP), an uncoupler of oxidative phosphorylation. Completely uncoupling the electron transport chain with higher concentrations of FCCP caused rapid myocyte contraction and death. Myocytes were exposed firstly to normoxia and fluorescence was recorded. At 30 min FCCP was added to the cells. FCCP caused a 31.3 ± 4.5% decrease in the rate of DHE signal in 23 cells (Fig. 7A). We also perfused cells intracellularly with 5 nM FCCP in the presence of 5 mM ATP and recorded the response of ICa-L to Iso. In the presence of FCCP, 3 and 10 nM Iso elicited currents that were 59.0 ± 8.8% and 86.6 ± 5.9% of the current recorded in the presence of 1 µM Iso (n = 7; Fig. 7B). These were similar to currents recorded during hypoxia alone (Fig. 2C, [10–12]). There was no difference between maximal responses recorded during 1 µM Iso and normoxia (10.2 ± 1.5 pA/pF), hypoxia (10.0 ± 0.8 pA/pF) or FCCP (11.6 ± 0.9 pA/pF; ANOVA p = NS). These data indicate that partial disruption of mitochondrial function mimics the effect of hypoxia on cellular superoxide and channel function.


Figure 7
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  		Fig. 7 A. FCCP mimics the effect of hypoxia on superoxide. DHE fluorescence recorded from a myocyte exposed to normoxia only and in another cell exposed to normoxia followed by 2 nM FCCP (Normoxia/FCCP). Ratio of fluorescence for cells exposed to normoxia only and for cells exposed to normoxia followed by FCCP (Normoxia/FCCP) is shown inset at right. B. FCCP mimics the effect of hypoxia on the sensitivity of ICa-L to Iso. Time course of changes in membrane current recorded in a cell while being perfused intracellularly with 5 nM FCCP. I–V shown from the same cell (inset left). Mean ± SEM of currents exposed to 0.003 (+0.003), 0.01 (+0.01) and 1.0 µM Iso (+1.0) while being perfused intracellularly with FCCP are shown inset at right. *p<0.05 compared with cells not perfused with FCCP in room oxygen. C. Oligomycin and FCCP significantly attenuate production of superoxide. DHE fluorescence recorded from a myocyte exposed to normoxia followed by 20 µM oligomycin alone (Normoxia/Oligo) and another cell exposed to normoxia followed by 20 µM oligomycin plus 0.5 nM FCCP (Normox/Oligo+FCCP). Ratio of fluorescence for cells exposed to normoxia and oligomycin only (Normox/Oligo) and for cells exposed to normoxia followed by oligomycin plus FCCP (Normox/Oligo+FCCP) is shown inset at right.

 
The addition of FCCP causes a decline in mitochondrial membrane potential and this may have caused the reversal of the ATP synthase. Consequently, the results obtained with FCCP alone may be confounded by a decline in cytosolic ATP concentrations or maintenance of mitochondrial membrane potential through the reversal of the ATP synthase. To test this, we exposed myocytes to 20 µM oligomycin to prevent the reversal of ATP synthase followed by addition of FCCP. Oligomycin alone did not affect superoxide production as assessed with DHE (Fig. 7C), but addition of 0.5–10 nM FCCP caused a 68.3 ± 5.7% decrease in DHE signal (n = 18; p<0.01). These data indicate that mitochondria are the predominant source of cellular superoxide.

3.7. Hypoxia causes a decrease in superoxide in isolated mitochondria
We directly assessed whether hypoxia could affect superoxide from mitochondria in adult guinea pig ventricular myocytes. Isolated mitochondria were exposed to room oxygen and baseline fluorescence was recorded for 5 min followed by hypoxia for 10 min. In mitochondria from 4 hearts, hypoxia caused a 69.3 ± 0.8% decrease in superoxide (Fig. 8). These data indicate that hypoxia directly affects mitochondrial production of superoxide.


Figure 8
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  		Fig. 8 Hypoxia directly alters superoxide in mitochondria isolated from guinea pig ventricular myocytes. A. DHE fluorescence recorded from isolated mitochondria exposed to normoxia alone and in isolated mitochondria exposed to hypoxia; mitos added=time point at which mitochondria were added. SOD added=time point at which superoxide dismutase was added. B. Mean ± SE of the slope of fluorescence recorded in isolated mitochondria exposed to hypoxia normalised to fluorescence recorded during normoxia alone.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the current study we examined the roles of NAD(P)H oxidase and the mitochondria in the generation of superoxide during acute hypoxia in ventricular myocytes. The fluorescent indicators DHE and DCF were used to detect reactive oxygen species in single adult ventricular myocytes and isolated mitochondria and electrophysiological recordings of ICa-L were used as a functional indicator. Consistent with previous reports [21–25], we show that hypoxia is associated with a decrease in cellular superoxide (Fig. 1B and C) but the predominant source of production of superoxide does not appear to be the NAD(P)H oxidase complex, instead it originates in the mitochondria. Despite the fact that DPI mimicked the effect of hypoxia on channel function (Fig. 2B and C) the effect of DPI could be mediated via an inhibition of mitochondrial function as determined by a decrease in superoxide and depressed oxygen consumption in isolated mitochondria (Fig. 5), rather than an effect of DPI on the oxidase. We examined the effect of DPI over concentrations that are typically used to inhibit the oxidase since the Ki for inhibition of NAD(P)H oxidase by DPI is 5.6 µM [26]. Within the limitations of the sensitivity of our techniques, we did not find any additional evidence for a role for NAD(P)H oxidase in modulating cellular superoxide or altering channel function during acute hypoxia despite the fact that the myocytes expressed functional NAD(P)H oxidase (Fig. 1A). The data suggest that the primary source of production of superoxide in adult cardiac myocytes would appear to be different from the primary source in vascular smooth muscle. A number of studies have confirmed the importance of the oxidase and ROS generated by the oxidase in vascular pathology [27]. However, consistent with our results, NAD(P)H oxidase is not central to hypoxic responses in all cells [28–33]. For example, in pulmonary vascular smooth muscle, hypoxic pulmonary vasoconstriction is preserved in mice lacking gp91phox [34]. In addition, hypoxia causes renal arterioles to dilate and pulmonary arteries to constrict because of differential expression of mitochondrial components in renal mitochondria compared with pulmonary vascular mitochondria [35]. With respect to the heart, it would appear that NAD(P)H oxidase contributes to pathological growth under conditions where the oxidase is activated over many hours, days or weeks and involves alterations in transcription and translation of proteins including the NAD(P)H oxidase subunits [36,5,37,3,38].

The data presented in this study have implications with respect to understanding acute oxygen sensing in ventricular myocytes. Although both NAD(P)H oxidase and the mitochondria are capable of producing superoxide our data indicate that in quiescent adult ventricular myocytes the mitochondria appear to be responsible for responding to changes in oxygen tension. We found that hypoxia (pO2 of 15–17 mm Hg) caused a significant decrease in superoxide in single myocytes (Fig. 1) and isolated mitochondria (Fig. 8) and modifying mitochondrial function (myxothiazol and FCCP) mimicked the effect of hypoxia on superoxide generation and the sensitivity of ICa-L to Iso (Figs. 6 and 7Go). The data reflect responses to acute changes in hypoxia (immediate to 30 min) consistent with acute hypoxic events. The duration and level of hypoxia appears to be critical to the response since longer periods of hypoxia (greater than 1 h) may result in increased generation of superoxide [39,40]. In addition, since ATP production does not become compromised until oxygen tension falls below 2 mm Hg [41,42] and we controlled intracellular ATP, glucose and Ca2+ concentrations in pipette solutions in patch-clamp experiments (see Methods), the effects observed did not reflect changes in protein function due to cellular metabolic inhibition.

In this study hypoxia decreased superoxide in isolated mitochondria and low concentrations of specific inhibitors of mitochondrial function mimicked the effect of hypoxia on the sensitivity of the channel to Iso. The results of this study confirm that the mitochondria and not NAD(P)H oxidase appear to be the predominant source of reactive oxygen species regulating L-type Ca2+ channel function during hypoxia in cardiac myocytes. We have shown previously that pre-exposing myocytes to hydrogen peroxide attenuates the effect of hypoxia on basal ICa-L and the sensitivity of the channel to Iso [10] implicating hydrogen peroxide as the mediator. Enhancement of the L-type Ca2+ current results in the generation of arrhythmogenic early afterdepolarisations [43,44]. The modulation of production of hydrogen peroxide by the mitochondria in cardiac myocytes may be an important determinant of triggering of arrhythmia during hypoxia.


   Acknowledgements
 
We gratefully acknowledge the assistance of Gina Ravenscroft for isolation of cardiac myocytes and Graeme Matich for providing embryonic cortical neurons. This study was supported by a grant from the National Health and Medical Research Council of Australia and a grant from the Western Australian Institute for Medical Research. In addition, Dr Hool is the recipient of a National Health and Medical Research Council Career Development Award.


   Notes
 
Time for primary review 14 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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