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Qo site of mitochondrial complex III is the source of increased superoxide after transient exposure to hydrogen peroxide
Journal of Molecular and Cellular Cardiology 49 (2010) 875–885
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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
Original article
Q o site of mitochondrial complex III is the source of increased superoxide after transient exposure to hydrogen peroxide Helena M. Viola, Livia C. Hool ⁎ School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia
a r t i c l e
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Article history: Received 9 July 2010 Accepted 23 July 2010 Available online 2 August 2010 Keywords: Hydrogen peroxide Calcium L-type Ca2+ channel Mitochondria Reactive oxygen species
a b s t r a c t Transient exposure of cardiac myocytes to hydrogen peroxide (H2O2) results in further production of superoxide by the mitochondria as a result of increased influx of calcium through the L-type Ca2+ channel and increased calcium uptake by the mitochondria. The response persists as a result of positive feedback on the channel and induces alterations in protein synthesis and cell size consistent with the development of myocyte hypertrophy. The aim of this study was to investigate the site of increased superoxide production within the mitochondria. Exposure of myocytes to 30 μM H2O2 (5 min) then 10 U/mL catalase (5 min) increased dihydroethidium (DHE) signal by 58.7 ± 12.0% (n = 4) compared to myocytes exposed to 0 μM H2O2 for 5 min followed by 10 U/mL catalase (n = 9). Complex I inhibitors DPI (n = 5) and rotenone (n = 7) attenuated the increase in DHE signal due to H2O2. Complex III inhibitors myxothiazol (n = 16) and stigmatellin (n = 5) also attenuated the increase in DHE signal due to H2O2. However, antimycin A (inhibitor of Q i site of complex III) had no effect. We “isolated” complex III in the intact cell by applying succinate in the patch pipette and exposing the cell to rotenone and antimycin A. Myxothiazol and TCA cycle inhibitors α-keto-β-methyl-n-valeric acid (KMV) and 4-hydroxynonenal (4-HNE) completely attenuated the increase in DHE signal. Direct activation of the L-type Ca2+ channel by voltage-step mimicked the increase in DHE signal after transient exposure to H2O2 (47.6 ± 17.8%, n = 6) while intracellular application of catalase attenuated the increase in DHE signal due to H2O2 (n = 6). We propose that elevated superoxide production after transient exposure to H2O2 occurs at the Q o superoxide generation site of complex III in cardiac myocytes and that an increase in TCA cycle activity plays a significant role in mediating the response. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Elevated intracellular calcium and increases in reactive oxygen species (ROS) have been implicated in the development of pathology such as cardiac hypertrophy [1,2]. However the mechanisms by which calcium and ROS participate early in the development of cardiac hypertrophy are not fully understood. An increase in intracellular calcium can mediate hypertrophy as a result of activation of calciumdependent signaling pathways such as NFAT, serine–threonine and tyrosine kinases, CaMK and MAPK [3,4]. In addition, at sub-lethal concentrations ROS can activate a number of hypertrophic signaling kinases and transcription factors [2,5]. We previously investigated the effect of extracellular exposure of guinea-pig ventricular myocytes to hydrogen peroxide (H2O2) at a concentration insufficient to cause cell death. This experimental design was intended to mimic the effect of a transient oxidative stress in vivo in humans associated with ischemia– reperfusion injury. Exposure of myocytes to 30 μM H2O2 for 5 min ⁎ Corresponding author. Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: + 61 8 6488 3307; fax: + 61 8 6488 1025. E-mail address: [email protected] (L.C. Hool). 0022-2828/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2010.07.015
(followed by 10 U/mL catalase to degrade the H2O2) caused further production of superoxide from the mitochondria [6]. The increase in superoxide occurred as a result of increased uptake of calcium by the mitochondria and was not due to increased superoxide generated by NADP(H)-oxidase, xanthine oxidase or nitric oxide [6]. Intracellular calcium was increased as a result of direct activation of the L-type Ca2+ channel by H2O2. The response persisted for at least 8 h after the initial 5 min exposure to H2O2 in the absence of apoptosis or necrosis. We proposed that the response persisted due to positive feedback between increased L-type Ca2+ channel activity, increased intracellular calcium and mitochondrial calcium uptake, and increased superoxide production by the mitochondria [6]. We examined whether the response may be pathological and found that 5 min exposure of ventricular myocytes to 30 μM H2O2 is sufficient to increase cell size and protein synthesis consistent with the development of myocyte hypertrophy 48 h later [7]. In addition the majority of proteins with altered expression were mitochondrial in origin [7]. Increased mitochondrial calcium triggers activation of three key dehydrogenases of the tricarboxylic acid (TCA) cycle including isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase [8]. Activation of the TCA cycle enhances the production of NADH, that triggers movement of electrons down
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complexes I through to IV of the electron transport chain (ETC) by initially donating electrons to complex I [9,10]. Electrons are also fed into the ETC via complex II as a result of the conversion of succinate to fumarate within the TCA cycle. Some of the electrons passing down the ETC leak out and react with molecular oxygen to form superoxide. Under normal physiological conditions, this has been demonstrated to occur at mitochondrial complexes I and III [11,12]. In the current study we sought to identify the site of increased superoxide production within the mitochondria after transient exposure of cardiac myocytes to 30 μM H2O2. We used a series of pharmacological inhibitors designed to block electron flow at various sites of the electron transport chain and assessed changes in superoxide using the fluorescent indicator DHE. We also designed experiments in which we “isolated” the Q o site of mitochondrial complex III in the intact cell by applying succinate in the patch pipette while activating the channel using voltage-step. We find that the Q o site of complex III is the predominant source of increased superoxide generation in response to transient exposure of cardiac myocytes to H2O2. In addition, the TCA cycle appears to play an important role in mediating this response.
2. Materials and methods 2.1. Measurement of superoxide Adult guinea-pig ventricular myocytes were used for all studies. Myocytes were isolated as described previously [13,14]. Superoxide was measured using the fluorescent indicator DHE (5 μM, 515– 560 nm ex filter, 590 long pass em, Molecular Probes) as previously described [6,14,15]. Each n represents an independent experiment from a myocyte for each treatment group. Myocytes from at least 3 animals were used for each treatment group. In some experiments changes in DHE fluorescence were assessed simultaneously with changes in L-type Ca2± channel currents in the myocytes using the whole-cell configuration of the patch-clamp technique. Microelectrodes contained internal solution supplemented with 5 mM succinate and 5 μM DHE. Modified extracellular Tyrode's solution was supplemented with 1 μM rotenone, 7 nM antimycin A and 5 μM DHE. All experiments were performed at 37 °C. Macroscopic currents were recorded using an Axopatch 200B voltage-clamp amplifier and an IBM compatible computer with a Digidata 1322A
Fig. 1. Mitochondrial complex I inhibitors DPI and rotenone attenuate the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of DPI as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for all myocytes as indicated shown at right. Inset above left: DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of ruthenium red (RR) as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for all myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of rotenone. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right.
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interface and pClamp9 software. Once the whole-cell configuration was achieved, the holding potential was set at −80 mV. Na± channels and T-type Ca2± channels were inactivated by applying a 50 ms prepulse to −30 mV immediately before each test pulse. The time course of changes in Ca2± conductance was monitored by applying a 100 ms test pulse to ±10 mV once every 10 s. For further details see the Supplementary material and [6,14,15].
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2.4. Statistical analysis Results are reported as mean± SEM. Statistical comparisons of responses between unpaired data were made using the student's t-test or between groups of cells using one-way ANOVA and the Tukey's posthoc test (GraphPad Prism version 3.02). 3. Results
2.2. Measurement of mitochondrial NADH and mitochondrial membrane potential
3.1. The increase in superoxide after transient exposure to H2O2 occurs downstream from complex I
Mitochondrial NADH was measured using autofluorescence (ex 365 nm and em 460/535 nm). Mitochondrial membrane potential (Ψm) was measured using the fluorescent indicator JC-1 (200 nM, ex 480 nm, em 590/520 nm, Molecular Probes). Each n represents an independent experiment from a myocyte for each treatment. Myocytes from at least 3 animals were used for each treatment group. For further details see the Supplementary material and [6,14,15].
Consistent with our previous results, exposing myocytes to 30 μM H2O2 for 5 min followed by 10 U/mL catalase (to degrade extracellular H2O2) caused a 58.7 ± 12.0% increase in superoxide production compared to myocytes exposed to 0 μM H2O2 for 5 min followed by 10 U/mL catalase, assessed as changes in DHE fluorescence (Fig. 1(A)) [6]. Exposing myocytes to mitochondrial calcium uniporter blocker ruthenium red significantly attenuated the increase in DHE signal (Fig. 1(A), inset). These data are consistent with our previous findings demonstrating that the increase in DHE signal after transient exposure to H2O2 is dependent on increased mitochondrial calcium uptake [6]. We assessed whether complex I contributed to superoxide formation by applying pharmacological agents that block electron flow from complex I. Myocytes were exposed to 1 μM dibenziodolium chloride (DPI) that blocks electron flow prior to the superoxide generation site of complex I [16–18] then 30 μM H2O2 for 5 min followed by 10 U/mL catalase and changes in DHE signal were assessed. In 5 cells, DPI significantly attenuated the increase in DHE signal due to H2O2 (Fig. 1(A)). In another set of experiments we
2.3. Measurement of metabolic activity Cleavage of the tetrazolium salt 3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) to formazan crystals by the electron transport chain of the mitochondria was assessed in myocytes using a modified MTT assay as previously described [15]. Increased formazan production is representative of an increase in metabolic absorbance and therefore an increase in metabolic activity. For further details see the Supplementary material and [15].
Fig. 2. Myxothiazol and stigmatellin attenuate the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of myxothiazol as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of stigmatellin as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right.
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exposed myocytes to 1 μM rotenone that blocks electron flow after the superoxide generation site of complex I [19,20]. In 7 cells, rotenone significantly attenuated the increase in DHE signal due to H2O2 (Fig. 1(B)). To examine whether mitochondrial complex III is a source of elevated superoxide production, myocytes were exposed to 7 nM myxothiazol that blocks electron flow at the Q o superoxide generation site of complex III [21–25] or stigmatellin, that block electron flow at the Q o superoxide generation site of complex III [19,21–25] then 30 μM H2O2 for 5 min followed by 10 U/mL catalase. Myxothiazol significantly attenuated the increase in DHE signal due to H2O2 (Fig. 2(A)). Similar results were obtained when myocytes were exposed to 7 nM stigmatellin, that also blocks electron flow at the Q o superoxide generation site of complex III [19,21,23] (Fig. 2(B)). The increase in DHE signal was also attenuated when DPI, rotenone or myxothiazol were applied after addition of 30 μM H2O2 for 5 min followed by 10 U/mL catalase (see online Supplementary material) [6]. These data suggest that elevated superoxide production after transient exposure to H2O2 occurs at or downstream of the Q o superoxide generation site of complex III.
have used were due to alterations in Ψm. Exposure of myocytes to 1 μM DPI, 1 μM rotenone, 7 nM myxothiazol, or 7 nM stigmatellin did not alter Ψm assessed as changes in JC-1 fluorescence (Fig. 3(A)). In each instance, a collapse in JC-1 signal after exposure of cells to oligomycin and FCCP demonstrated that the signal was indicative of Ψm. Exposure of cells to oligomycin alone resulted in a 20.1 ± 3.4% increase in Ψm consistent with the inhibition of the ATP synthase (Fig. 3(A)). Therefore we examined the effects of DPI, rotenone, myxothiazol and stigmatellin on DHE signal in the presence of oligomycin to further confirm whether the inhibitors were attenuating superoxide production via alterations in the Ψm. Myocytes were exposed to 10 μM oligomycin followed by 1 μM DPI, 1 μM rotenone, 7 nM myxothiazol or 7 nM stigmatellin prior to exposure to 30 μM H2O2 for 5 min followed by 10 U/mL catalase. DPI, rotenone, myxothiazol and stigmatellin attenuated the increase in DHE signal after exposure of myocytes to 30 μM H2O2 in the presence of oligomycin (Fig. 3(B)). These results confirm that the inhibitors did not attenuate the DHE signal via a reduction in Ψm.
3.3. Q o superoxide generation site of complex III appears to be the source of superoxide production 3.2. Exposure of myocytes to DPI, rotenone, myxothiazol or stigmatellin attenuates the increase in DHE signal without altering Ψm Alterations in Ψm can influence ROS production [26,27]. We performed additional experiments to determine whether the concentrations of DPI, rotenone, myxothiazol and stigmatellin that we
We investigated the site of superoxide generation within complex III. We first exposed cardiac myocytes to antimycin A, that block electron flow at the Q i superoxide generation site of complex III [19,21,24,25]. The Q i site of complex III resides downstream from the Q o site. In 7 cells, in the presence of 7 nM antimycin A, exposure of
Fig. 3. DPI, rotenone, myxothiazol and stigmatellin do not alter Ψm. (A) JC-1 fluorescence recorded from untreated (control) myocyte and myocytes before and after exposure to inhibitors as indicated. Arrow indicates when inhibitors were added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) for myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to H2O2 then DPI, Rote, Myx or Stig as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right.
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myocytes to H2O2 increased DHE signal a further 66.4 ± 14.4% (Fig. 4 (A)). This was not significantly different from the increase recorded in response to 30 μM H2O2 in the absence of antimycin A (Fig. 4(A)). The concentration of antimycin A was sufficient to alter metabolic activity in the myocytes assessed as change in absorbance at 570 nm after formation of formazan from tetrazolium salt (Fig. 4(B)). In addition, exposure of myocytes to 7 nM antimycin A did not significantly alter Ψm (Fig. 4(C)). We conclude that Q o site appears to be contributing to the increased generation of superoxide. 3.4. Mitochondrial complex IV does not contribute to the increase in 2DHE We examined a possible role for complex IV in the production of superoxide. We exposed myocytes to 100 nM complex IV inhibitor sodium cyanide (NaCN) [28,29] followed by H2O2. In 6 cells, transient exposure to H2O2 increased DHE signal 64.8 ± 16.4% (Fig. 5(A)). This was not significantly different from the increase recorded after
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exposure of myocytes to 30 μM H2O2 in the absence of NaCN (58.7 ± 12.0%, Fig. 5(A)). However 100 nM NaCN significantly decreased absorbance at 570 nm by 80.6 ± 7.7% compared to untreated controls (Fig. 5(B)) without altering Ψm (Fig. 5(C)). These data suggest that 100 nM NaCN is sufficient to inhibit metabolic activity but it was unable to attenuate the increase in DHE signal. We conclude that mitochondrial complex IV does not contribute to elevated superoxide generation after transient exposure to H2O2. 3.5. Increased mitochondrial superoxide after transient exposure to H2O2 is associated with elevated TCA cycle activity Mitochondrial NADH production is dependent on TCA cycle activity [10,30]. We assessed changes in NADH autofluorescence in the myocytes after transient exposure to H2O2. Exposing myocytes to 30 μM H2O2 for 5 min followed by 10 U/mL catalase increased NADH signal 10.5 ± 2.3% compared to myocytes exposed to 0 μM H2O2 for 5 min followed by 10 U/mL catalase (Fig. 6(A)). The response was
Fig. 4. Mitochondrial complex III inhibitor antimycin A does not alter the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase and antimycin A as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. (B) MTT assay performed in untreated (control) myocytes and myocytes exposed to Anti. Slopes represent change in absorbance at 570 nm. Mean ± SEM of the ratio of slopes measured over 0–1 min for myocytes exposed to Anti as indicated shown at right. n: number of replicates of cultured cells for each treatment group from 2 animals. (C) JC-1 fluorescence recorded from untreated (control) myocyte and myocyte before and after exposure to Anti or Oligo. Arrow indicates when treatments were added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right.
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Fig. 5. Mitochondrial complex IV inhibitor NaCN does not alter the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of NaCN. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. (B) MTT assay performed in untreated (control) myocytes and myocytes exposed to NaCN. Slopes represent change in absorbance at 570 nm. Mean ± SEM of the ratio of slopes measured over 0–1 min for myocytes exposed to NaCN as indicated shown at right. n: number of replicates of cultured cells for each treatment group from 2 animals. (C) JC-1 fluorescence recorded from untreated (control) myocyte and myocyte before and after exposure to NaCN or Oligo. Arrow indicates when treatments were added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) for myocytes as indicated shown at right.
significantly attenuated when myocytes were exposed to 20 μM 4HNE, an inhibitor of TCA cycle enzyme isocitrate dehydrogenase [31] (Fig. 6(A)). We further examined the role of the TCA cycle by assessing alterations in DHE fluorescence in response to H2O2 in the presence and absence of TCA cycle enzyme inhibitors. In 7 cells, 4-HNE significantly attenuated the increase in DHE signal after exposure to H2O2 (Fig. 6(B)). Similar results were observed when myocytes were exposed to KMV, an inhibitor of α-ketoglutarate dehydrogenase [32] (Fig. 6(C)). Exposure of myocytes to 4-HNE or KMV did not alter Ψm (Figs. 6(B) and (C) insets). These data confirm that increased TCA cycle activity is associated with increased superoxide after transient exposure to H2O2. To examine whether increased electron flow into the ETC via complex II is involved in elevated superoxide generation, myocytes were exposed to 5 μM 2-thenoyltrifluoroacetone (TTFA) then 30 μM
H2O2 for 5 min followed by 10 U/mL catalase. In 5 cells, TTFA significantly attenuated the increase in DHE signal after exposure to H2O2 (Fig. 6(D)). Exposure of myocytes to TTFA did not alter Ψm (Fig. 6(D) inset). These data demonstrate that increased electron flow into the ETC via complex II is associated with increased superoxide after transient exposure to H2O2. 3.6. Confirmation of the role of the Q o site of complex III To further investigate the Q o site of complex III as the predominant source of superoxide production we designed experiments in which we “isolated” the Q o site of mitochondrial complex III in the intact cell. Using the patch-clamp technique we perfused cardiac myocytes intracellularly with complex II substrate succinate (in the patch pipette) while exposing the myocyte to extracellular solution supplemented with complex I inhibitor rotenone, complex III
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Fig. 6. TCA cycle inhibitors attenuate the increase in DHE signal. (A) NADH autofluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of 4-HNE. Addition of drugs occurred during the pause in data acquisition. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Increased NADH after addition of Oligo indicated an active TCA cycle. Decreased NADH following addition of FCCP indicated a collapse in Ψm. Mean ± SEM of changes in NADH (% increase) for myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of 4-HNE. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown below. Inset above: JC-1 fluorescence recorded from an untreated (control) myocyte and myocyte before or after exposure to 4-HNE or Oligo. Arrow indicates when 4-HNE was added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right. (C) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of KMV. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown below. Inset above: JC-1 fluorescence recorded from an untreated (control) myocyte and myocyte before and after exposure to KMV or Oligo. Arrow indicates when KMV was added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right. (D) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of TTFA. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. Inset above left: JC-1 fluorescence recorded from an untreated (control) myocyte and myocyte before and after exposure to TTFA or Oligo. Arrow indicates when TTFA was added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right.
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Q i site inhibitor antimycin A and DHE. We monitored alterations in DHE fluorescence after exposure to H2O2. This experimental design enabled us to assess the involvement of the Q o site of complex III because electron flow from complex I and beyond the Q i site of complex III were blocked. Exposing myocytes to H2O2 resulted in a 76.6 ± 20.2% increase in DHE in the presence of 1 μM rotenone, 7 nM antimycin A and 5 mM succinate (Fig. 7(A)). The response was similar to the increase in DHE signal recorded in cells not patch-clamped (Fig. 1(A)). In addition, antimycin A and rotenone did not directly alter the L-type Ca2+ current (Fig. 7(A) inset). The response was significantly attenuated when myocytes were exposed to 7 nM myxothiazol prior to 30 μM H2O2 (Fig. 7(B)) and 20 μM 4-HNE prior to 30 μM H2O2 (Fig. 7(C)). These data confirm that the Q o site of complex III is the predominant source of superoxide production after transient exposure to 30 μM H2O2 and that the TCA cycle plays a role in mediating this response. 3.7. Direct activation of the L-type Ca2+ channel increases superoxide production in cardiac myocytes The increase in superoxide production by the mitochondria after transient exposure to H2O2 occurs as a result of an increase in L-type Ca2+ channel activity and elevated intracellular calcium [6]. To confirm that activation of the channel is necessary for the response, we tested whether direct activation of the L-type Ca2+ channel by voltage-step could increase superoxide. Myocytes were patchclamped with the plasma membrane held at −80 mV while complex II substrate succinate was administered via the patch pipette supplemented with DHE, and 1 μM rotenone, 7 nM antimycin A and DHE administered extracellularly. Intracellular calcium was not buffered with EGTA and the internal solution contained 100 nM CaCl2. We voltage-stepped the plasma membrane to −30 mV for 50 ms followed by + 10 mV for 100 ms. DHE signal was significantly increased 47.6 ± 17.8% upon voltage-step to +10 mV compared to myocytes held at −30 mV only (Fig. 8(A)). These data confirm that activation of the L-type Ca2+ channel is sufficient to increase mitochondrial superoxide. We have previously proposed a positive feedback between increased L-type Ca2+ channel activity, increased intracellular calcium and increased superoxide production by the mitochondria [6]. Since superoxide is rapidly dismutated to H2O2, we investigated whether the signal activating the channel is H2O2. We perfused cells intracellularly with active or heat-inactivated catalase and assessed changes in DHE signal after transient exposure to H2O2. Cells were also perfused with complex II substrate succinate in the patch pipette, and 1 μM rotenone and 7 nM antimycin A administered extracellularly. In 5 cells the presence of 500 U/mL active catalase significantly attenuated the increase in DHE signal after exposure to 30 μM H2O2 compared to myocytes perfused intracellularly with 500 U/mL heat-inactivated catalase (Fig. 8(B)). These data suggest that an increase in intracellular H2O2 is necessary for activation of the L-type Ca2+ channel. 4. Discussion The mechanisms by which calcium and ROS participate early in the development of cardiac hypertrophy are not fully understood. Under normal physiological conditions, low levels of ROS (0.05–0.07 μM intracellular) are required to maintain cellular function [5]. Intracellular increases in ROS to 1–10 μM have been associated with mild oxidative stress in the absence of apoptosis or cell death [2–5]. Taking into account the antioxidant buffering capacity of the cell, extracellular exposure of myocytes to 30 μM H2O2 for 5 min will equate to approximately 3 μM H2O2 intracellular. We applied 30 μM H2O2 to mimic the effect of a transient oxidative stress in vivo in humans associated with ischemia–reperfusion injury. Since the response
persisted for many hours after the transient exposure to H2O2, we propose that the brief exposure may be sufficient to induce pathology. In support of this argument, we have demonstrated that 5 min exposure of ventricular myocytes to 30 μM H2O2 is sufficient to cause an increase in cell size and protein synthesis consistent with the development of myocyte hypertrophy 48 h later [7]. The persistent oxidative stress triggered by increased mitochondrial ROS is consistent with “ROS-induced ROS-release” reported previously [33,34]. However unlike the previous studies, we specifically applied H2O2 in the extracellular solution and find that persistent oxidative stress occurs as a result of increased calcium influx through the L-type Ca2+ channel. Under normal physiological conditions, mitochondrial superoxide generation has been demonstrated to occur at mitochondrial complexes I and III [11,12]. In this study we sought to identify the source of increased superoxide production within the mitochondria in response to transient H2O2 exposure. Inhibition of electron flow both before and after the superoxide generation site of complex I attenuated the increase in superoxide (Figs. 1(A) and (B)). Inhibition of electron flow at the Q o superoxide generation site of complex III also attenuated the increase in superoxide (Figs. 2(A) and (B)). However inhibition of electron flow at the Q i superoxide generation site of complex III that resides downstream from the Q o site did not alter the increase in superoxide (Fig. 4(A)). These data suggest the Q o site is the predominant source of superoxide generation. In support of these findings, the complex IV inhibitor NaCN did not alter superoxide production (Fig. 5(A)). Inhibition of the TCA cycle enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase attenuated the increase in mitochondrial NADH and superoxide generation after transient exposure of cardiac myocytes to H2O2 (Figs. 6(A)–(C)). Inhibition of electron flow at complex II also attenuated the increase in superoxide generation after transient exposure of cardiac myocytes to H2O2 (Fig. 6(D)). Under normal physiological conditions, electrons flow down the ETC from complex I. Electrons are also fed into the ETC via complex II as a result of conversion of succinate to fumarate within the TCA cycle. Therefore an increase in TCA cycle activity will result in an increase in electron flow to complex II. We find that increased superoxide generation occurs at complex III as a result of an increase in electron flow from the TCA cycle to complex II after exposure to H2O2. To further confirm the findings, cells were patch-clamped with the complex II substrate succinate and superoxide production was assessed in the presence of complex I and III inhibitors (Fig. 7). The experiments were designed to “isolate” complex III as the site of generation of superoxide and mimic isolated mitochondrial studies in the intact cell. Under these conditions myxothiazol and 4-HNE attenuated the increase in DHE signal (Figs. 7(B) and (C)). We also confirmed that direct activation of the channel by voltage-stepping the plasma membrane could mimic the effect of H2O2 (Fig. 8(A)). However voltage-step of the membrane per se does not appear to be required for the response since the dihydropyridine receptor agonist Bay K8684 can mimic the effect of H2O2 on mitochondrial superoxide production [15]. Increased intracellular H2O2 is necessary for the persistent activation of the channel since catalase attenuated the increase in superoxide (Fig. 8(B)). Therefore our data demonstrate that H2O2 is the signal responsible for altered calcium homeostasis and further oxidative stress in cardiac myocytes. H2O2 is a well recognised signal molecule capable of altering protein function as a result of modification of critical cysteines on proteins [5]. We propose that a transient oxidative stress can activate the L-type Ca2+ channel and cause a further increase in mitochondrial superoxide from Q o site of complex III as a result of increased calcium uptake by the mitochondria and elevated TCA cycle activity (Fig. 8(C)). The L-type Ca2+ channel, TCA cycle and Q o superoxide generation site of complex III may represent possible sites to target in the prevention of the development of cardiac hypertrophy associated with mild oxidative stress.
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Fig. 7. Myxothiazol and 4-HNE attenuate the increase in DHE signal when complex III is “isolated” in the patch-clamped cell. (A) DHE fluorescence recorded from a patch-clamped myocyte before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase. External solution (ES) was supplemented with rotenone (rote) and antimycin A (Anti). Internal solution (IS) was supplemented with succinate. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 15 to 19 min over the slope at 0 to 4 min for myocytes exposed to H2O2 and catalase as indicated shown at right. Inset above left: current traces recorded from an untreated (control) myocyte and in the same myocyte exposed to Anti and rote. (B) DHE fluorescence recorded from individual patch-clamped myocytes before and after exposure to 0 or 30 μM H2O2 in the presence and absence of myxothiazol (myx) as indicated. ES was supplemented with rote and Anti. IS was supplemented with succinate. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 15 to 19 min over the slope at 0 to 4 min for myocytes as indicated shown at right. (C) DHE fluorescence recorded from individual patch-clamped myocytes before and after exposure to 0 or 30 μM H2O2 in the presence or absence of 20 μM 4-HNE. ES was supplemented with Rote and Anti. IS was supplemented with succinate. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 15 to19 min over the slope at 0 to 4 min for myocytes as indicated shown at right.
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Fig. 8. (A) Activation of the L-type Ca2+ channel mimics the increase in DHE. DHE fluorescence recorded from a patch-clamped myocyte held at −30 mV, and a myocyte held initially at −30 mV then voltage-stepped to + 10 mV as indicated with dashed arrow. External solution (ES) was supplemented with rotenone (rote) and antimycin A (Anti). Internal solution (IS) was supplemented with succinate. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 5 to 9 min over the slope at 0 to 4 min for myocytes held at −30 mV (not voltage-stepped) or held initially at −30 mV then voltage-stepped to + 10 mV (voltage-stepped) shown at right. (B) H2O2 is the signal activating the channel. DHE fluorescence recorded from a myocyte before and after exposure to 30 μM H2O2 while patch-clamped with 500 U/mL active or heat-inactivated catalase containing IS. IS was also supplemented with succinate. ES was supplemented with Rote and Anti. Addition of H2O2 occurred as indicated with arrow. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 5 to 9 min over the slope at 0 to 4 min for myocytes exposed to H2O2 in the presence of inactive or active catalase as indicated shown at right. (C) Proposed model explaining the mechanism for increased production of superoxide from the mitochondria after transient exposure to H2O2. Exposure of cardiac myocytes to 30 μM H2O2 for 5 min causes increased activation of the L-type Ca2+ channel and increased Ca2+ entry into the cytosol. Calcium entry through the mitochondrial calcium uniporter (MCU) into the mitochondrial matrix triggers increased activation of the TCA cycle. Increased electron flow to the Qo superoxide generation site of complex III (via complex II) results in elevated superoxide production from this site (see also Discussion).
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Acknowledgments This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC). Helena Viola is the recipient of a Biomedical Postgraduate Research Scholarship from NHMRC and National Heart Foundation of Australia. Livia Hool is the recipient of a NHMRC Career Development Award.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yjmcc.2010.07.015.
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Glossary 4-HNE: 4-hydroxynonenal Anti: antimycin A ATP: adenosine triphosphate Ca2+: calcium Cat: catalase DHE: dihydroethidium DPI: dibenziodolium chloride ETC: electron transport chain FCCP: carbonyl cyanide p-(trifluromethoxy)phenyl-hydrazone H2O2: hydrogen peroxide JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide KMV: α-keto-β-methyl-n-valeric acid MCU: mitochondrial calcium uniporter MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide Myx: myxothiazol NaCN: sodium cyanide NADH: reduced nicotinamide adenine dinucleotide Oligo: oligomycin ROS: reactive oxygen species Rote: rotenone Stig: stigmatellin TCA: tricarboxylic acid TTFA: 2-thenoyltrifluoroacetone Ψm: mitochondrial membrane potential
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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
Original article
Q o site of mitochondrial complex III is the source of increased superoxide after transient exposure to hydrogen peroxide Helena M. Viola, Livia C. Hool ⁎ School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia
a r t i c l e
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Article history: Received 9 July 2010 Accepted 23 July 2010 Available online 2 August 2010 Keywords: Hydrogen peroxide Calcium L-type Ca2+ channel Mitochondria Reactive oxygen species
a b s t r a c t Transient exposure of cardiac myocytes to hydrogen peroxide (H2O2) results in further production of superoxide by the mitochondria as a result of increased influx of calcium through the L-type Ca2+ channel and increased calcium uptake by the mitochondria. The response persists as a result of positive feedback on the channel and induces alterations in protein synthesis and cell size consistent with the development of myocyte hypertrophy. The aim of this study was to investigate the site of increased superoxide production within the mitochondria. Exposure of myocytes to 30 μM H2O2 (5 min) then 10 U/mL catalase (5 min) increased dihydroethidium (DHE) signal by 58.7 ± 12.0% (n = 4) compared to myocytes exposed to 0 μM H2O2 for 5 min followed by 10 U/mL catalase (n = 9). Complex I inhibitors DPI (n = 5) and rotenone (n = 7) attenuated the increase in DHE signal due to H2O2. Complex III inhibitors myxothiazol (n = 16) and stigmatellin (n = 5) also attenuated the increase in DHE signal due to H2O2. However, antimycin A (inhibitor of Q i site of complex III) had no effect. We “isolated” complex III in the intact cell by applying succinate in the patch pipette and exposing the cell to rotenone and antimycin A. Myxothiazol and TCA cycle inhibitors α-keto-β-methyl-n-valeric acid (KMV) and 4-hydroxynonenal (4-HNE) completely attenuated the increase in DHE signal. Direct activation of the L-type Ca2+ channel by voltage-step mimicked the increase in DHE signal after transient exposure to H2O2 (47.6 ± 17.8%, n = 6) while intracellular application of catalase attenuated the increase in DHE signal due to H2O2 (n = 6). We propose that elevated superoxide production after transient exposure to H2O2 occurs at the Q o superoxide generation site of complex III in cardiac myocytes and that an increase in TCA cycle activity plays a significant role in mediating the response. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Elevated intracellular calcium and increases in reactive oxygen species (ROS) have been implicated in the development of pathology such as cardiac hypertrophy [1,2]. However the mechanisms by which calcium and ROS participate early in the development of cardiac hypertrophy are not fully understood. An increase in intracellular calcium can mediate hypertrophy as a result of activation of calciumdependent signaling pathways such as NFAT, serine–threonine and tyrosine kinases, CaMK and MAPK [3,4]. In addition, at sub-lethal concentrations ROS can activate a number of hypertrophic signaling kinases and transcription factors [2,5]. We previously investigated the effect of extracellular exposure of guinea-pig ventricular myocytes to hydrogen peroxide (H2O2) at a concentration insufficient to cause cell death. This experimental design was intended to mimic the effect of a transient oxidative stress in vivo in humans associated with ischemia– reperfusion injury. Exposure of myocytes to 30 μM H2O2 for 5 min ⁎ Corresponding author. Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: + 61 8 6488 3307; fax: + 61 8 6488 1025. E-mail address: [email protected] (L.C. Hool). 0022-2828/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2010.07.015
(followed by 10 U/mL catalase to degrade the H2O2) caused further production of superoxide from the mitochondria [6]. The increase in superoxide occurred as a result of increased uptake of calcium by the mitochondria and was not due to increased superoxide generated by NADP(H)-oxidase, xanthine oxidase or nitric oxide [6]. Intracellular calcium was increased as a result of direct activation of the L-type Ca2+ channel by H2O2. The response persisted for at least 8 h after the initial 5 min exposure to H2O2 in the absence of apoptosis or necrosis. We proposed that the response persisted due to positive feedback between increased L-type Ca2+ channel activity, increased intracellular calcium and mitochondrial calcium uptake, and increased superoxide production by the mitochondria [6]. We examined whether the response may be pathological and found that 5 min exposure of ventricular myocytes to 30 μM H2O2 is sufficient to increase cell size and protein synthesis consistent with the development of myocyte hypertrophy 48 h later [7]. In addition the majority of proteins with altered expression were mitochondrial in origin [7]. Increased mitochondrial calcium triggers activation of three key dehydrogenases of the tricarboxylic acid (TCA) cycle including isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase [8]. Activation of the TCA cycle enhances the production of NADH, that triggers movement of electrons down
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complexes I through to IV of the electron transport chain (ETC) by initially donating electrons to complex I [9,10]. Electrons are also fed into the ETC via complex II as a result of the conversion of succinate to fumarate within the TCA cycle. Some of the electrons passing down the ETC leak out and react with molecular oxygen to form superoxide. Under normal physiological conditions, this has been demonstrated to occur at mitochondrial complexes I and III [11,12]. In the current study we sought to identify the site of increased superoxide production within the mitochondria after transient exposure of cardiac myocytes to 30 μM H2O2. We used a series of pharmacological inhibitors designed to block electron flow at various sites of the electron transport chain and assessed changes in superoxide using the fluorescent indicator DHE. We also designed experiments in which we “isolated” the Q o site of mitochondrial complex III in the intact cell by applying succinate in the patch pipette while activating the channel using voltage-step. We find that the Q o site of complex III is the predominant source of increased superoxide generation in response to transient exposure of cardiac myocytes to H2O2. In addition, the TCA cycle appears to play an important role in mediating this response.
2. Materials and methods 2.1. Measurement of superoxide Adult guinea-pig ventricular myocytes were used for all studies. Myocytes were isolated as described previously [13,14]. Superoxide was measured using the fluorescent indicator DHE (5 μM, 515– 560 nm ex filter, 590 long pass em, Molecular Probes) as previously described [6,14,15]. Each n represents an independent experiment from a myocyte for each treatment group. Myocytes from at least 3 animals were used for each treatment group. In some experiments changes in DHE fluorescence were assessed simultaneously with changes in L-type Ca2± channel currents in the myocytes using the whole-cell configuration of the patch-clamp technique. Microelectrodes contained internal solution supplemented with 5 mM succinate and 5 μM DHE. Modified extracellular Tyrode's solution was supplemented with 1 μM rotenone, 7 nM antimycin A and 5 μM DHE. All experiments were performed at 37 °C. Macroscopic currents were recorded using an Axopatch 200B voltage-clamp amplifier and an IBM compatible computer with a Digidata 1322A
Fig. 1. Mitochondrial complex I inhibitors DPI and rotenone attenuate the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of DPI as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for all myocytes as indicated shown at right. Inset above left: DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of ruthenium red (RR) as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for all myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of rotenone. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right.
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interface and pClamp9 software. Once the whole-cell configuration was achieved, the holding potential was set at −80 mV. Na± channels and T-type Ca2± channels were inactivated by applying a 50 ms prepulse to −30 mV immediately before each test pulse. The time course of changes in Ca2± conductance was monitored by applying a 100 ms test pulse to ±10 mV once every 10 s. For further details see the Supplementary material and [6,14,15].
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2.4. Statistical analysis Results are reported as mean± SEM. Statistical comparisons of responses between unpaired data were made using the student's t-test or between groups of cells using one-way ANOVA and the Tukey's posthoc test (GraphPad Prism version 3.02). 3. Results
2.2. Measurement of mitochondrial NADH and mitochondrial membrane potential
3.1. The increase in superoxide after transient exposure to H2O2 occurs downstream from complex I
Mitochondrial NADH was measured using autofluorescence (ex 365 nm and em 460/535 nm). Mitochondrial membrane potential (Ψm) was measured using the fluorescent indicator JC-1 (200 nM, ex 480 nm, em 590/520 nm, Molecular Probes). Each n represents an independent experiment from a myocyte for each treatment. Myocytes from at least 3 animals were used for each treatment group. For further details see the Supplementary material and [6,14,15].
Consistent with our previous results, exposing myocytes to 30 μM H2O2 for 5 min followed by 10 U/mL catalase (to degrade extracellular H2O2) caused a 58.7 ± 12.0% increase in superoxide production compared to myocytes exposed to 0 μM H2O2 for 5 min followed by 10 U/mL catalase, assessed as changes in DHE fluorescence (Fig. 1(A)) [6]. Exposing myocytes to mitochondrial calcium uniporter blocker ruthenium red significantly attenuated the increase in DHE signal (Fig. 1(A), inset). These data are consistent with our previous findings demonstrating that the increase in DHE signal after transient exposure to H2O2 is dependent on increased mitochondrial calcium uptake [6]. We assessed whether complex I contributed to superoxide formation by applying pharmacological agents that block electron flow from complex I. Myocytes were exposed to 1 μM dibenziodolium chloride (DPI) that blocks electron flow prior to the superoxide generation site of complex I [16–18] then 30 μM H2O2 for 5 min followed by 10 U/mL catalase and changes in DHE signal were assessed. In 5 cells, DPI significantly attenuated the increase in DHE signal due to H2O2 (Fig. 1(A)). In another set of experiments we
2.3. Measurement of metabolic activity Cleavage of the tetrazolium salt 3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) to formazan crystals by the electron transport chain of the mitochondria was assessed in myocytes using a modified MTT assay as previously described [15]. Increased formazan production is representative of an increase in metabolic absorbance and therefore an increase in metabolic activity. For further details see the Supplementary material and [15].
Fig. 2. Myxothiazol and stigmatellin attenuate the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of myxothiazol as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of stigmatellin as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right.
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exposed myocytes to 1 μM rotenone that blocks electron flow after the superoxide generation site of complex I [19,20]. In 7 cells, rotenone significantly attenuated the increase in DHE signal due to H2O2 (Fig. 1(B)). To examine whether mitochondrial complex III is a source of elevated superoxide production, myocytes were exposed to 7 nM myxothiazol that blocks electron flow at the Q o superoxide generation site of complex III [21–25] or stigmatellin, that block electron flow at the Q o superoxide generation site of complex III [19,21–25] then 30 μM H2O2 for 5 min followed by 10 U/mL catalase. Myxothiazol significantly attenuated the increase in DHE signal due to H2O2 (Fig. 2(A)). Similar results were obtained when myocytes were exposed to 7 nM stigmatellin, that also blocks electron flow at the Q o superoxide generation site of complex III [19,21,23] (Fig. 2(B)). The increase in DHE signal was also attenuated when DPI, rotenone or myxothiazol were applied after addition of 30 μM H2O2 for 5 min followed by 10 U/mL catalase (see online Supplementary material) [6]. These data suggest that elevated superoxide production after transient exposure to H2O2 occurs at or downstream of the Q o superoxide generation site of complex III.
have used were due to alterations in Ψm. Exposure of myocytes to 1 μM DPI, 1 μM rotenone, 7 nM myxothiazol, or 7 nM stigmatellin did not alter Ψm assessed as changes in JC-1 fluorescence (Fig. 3(A)). In each instance, a collapse in JC-1 signal after exposure of cells to oligomycin and FCCP demonstrated that the signal was indicative of Ψm. Exposure of cells to oligomycin alone resulted in a 20.1 ± 3.4% increase in Ψm consistent with the inhibition of the ATP synthase (Fig. 3(A)). Therefore we examined the effects of DPI, rotenone, myxothiazol and stigmatellin on DHE signal in the presence of oligomycin to further confirm whether the inhibitors were attenuating superoxide production via alterations in the Ψm. Myocytes were exposed to 10 μM oligomycin followed by 1 μM DPI, 1 μM rotenone, 7 nM myxothiazol or 7 nM stigmatellin prior to exposure to 30 μM H2O2 for 5 min followed by 10 U/mL catalase. DPI, rotenone, myxothiazol and stigmatellin attenuated the increase in DHE signal after exposure of myocytes to 30 μM H2O2 in the presence of oligomycin (Fig. 3(B)). These results confirm that the inhibitors did not attenuate the DHE signal via a reduction in Ψm.
3.3. Q o superoxide generation site of complex III appears to be the source of superoxide production 3.2. Exposure of myocytes to DPI, rotenone, myxothiazol or stigmatellin attenuates the increase in DHE signal without altering Ψm Alterations in Ψm can influence ROS production [26,27]. We performed additional experiments to determine whether the concentrations of DPI, rotenone, myxothiazol and stigmatellin that we
We investigated the site of superoxide generation within complex III. We first exposed cardiac myocytes to antimycin A, that block electron flow at the Q i superoxide generation site of complex III [19,21,24,25]. The Q i site of complex III resides downstream from the Q o site. In 7 cells, in the presence of 7 nM antimycin A, exposure of
Fig. 3. DPI, rotenone, myxothiazol and stigmatellin do not alter Ψm. (A) JC-1 fluorescence recorded from untreated (control) myocyte and myocytes before and after exposure to inhibitors as indicated. Arrow indicates when inhibitors were added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) for myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to H2O2 then DPI, Rote, Myx or Stig as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right.
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myocytes to H2O2 increased DHE signal a further 66.4 ± 14.4% (Fig. 4 (A)). This was not significantly different from the increase recorded in response to 30 μM H2O2 in the absence of antimycin A (Fig. 4(A)). The concentration of antimycin A was sufficient to alter metabolic activity in the myocytes assessed as change in absorbance at 570 nm after formation of formazan from tetrazolium salt (Fig. 4(B)). In addition, exposure of myocytes to 7 nM antimycin A did not significantly alter Ψm (Fig. 4(C)). We conclude that Q o site appears to be contributing to the increased generation of superoxide. 3.4. Mitochondrial complex IV does not contribute to the increase in 2DHE We examined a possible role for complex IV in the production of superoxide. We exposed myocytes to 100 nM complex IV inhibitor sodium cyanide (NaCN) [28,29] followed by H2O2. In 6 cells, transient exposure to H2O2 increased DHE signal 64.8 ± 16.4% (Fig. 5(A)). This was not significantly different from the increase recorded after
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exposure of myocytes to 30 μM H2O2 in the absence of NaCN (58.7 ± 12.0%, Fig. 5(A)). However 100 nM NaCN significantly decreased absorbance at 570 nm by 80.6 ± 7.7% compared to untreated controls (Fig. 5(B)) without altering Ψm (Fig. 5(C)). These data suggest that 100 nM NaCN is sufficient to inhibit metabolic activity but it was unable to attenuate the increase in DHE signal. We conclude that mitochondrial complex IV does not contribute to elevated superoxide generation after transient exposure to H2O2. 3.5. Increased mitochondrial superoxide after transient exposure to H2O2 is associated with elevated TCA cycle activity Mitochondrial NADH production is dependent on TCA cycle activity [10,30]. We assessed changes in NADH autofluorescence in the myocytes after transient exposure to H2O2. Exposing myocytes to 30 μM H2O2 for 5 min followed by 10 U/mL catalase increased NADH signal 10.5 ± 2.3% compared to myocytes exposed to 0 μM H2O2 for 5 min followed by 10 U/mL catalase (Fig. 6(A)). The response was
Fig. 4. Mitochondrial complex III inhibitor antimycin A does not alter the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase and antimycin A as indicated. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. (B) MTT assay performed in untreated (control) myocytes and myocytes exposed to Anti. Slopes represent change in absorbance at 570 nm. Mean ± SEM of the ratio of slopes measured over 0–1 min for myocytes exposed to Anti as indicated shown at right. n: number of replicates of cultured cells for each treatment group from 2 animals. (C) JC-1 fluorescence recorded from untreated (control) myocyte and myocyte before and after exposure to Anti or Oligo. Arrow indicates when treatments were added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right.
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Fig. 5. Mitochondrial complex IV inhibitor NaCN does not alter the increase in DHE signal. (A) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of NaCN. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. (B) MTT assay performed in untreated (control) myocytes and myocytes exposed to NaCN. Slopes represent change in absorbance at 570 nm. Mean ± SEM of the ratio of slopes measured over 0–1 min for myocytes exposed to NaCN as indicated shown at right. n: number of replicates of cultured cells for each treatment group from 2 animals. (C) JC-1 fluorescence recorded from untreated (control) myocyte and myocyte before and after exposure to NaCN or Oligo. Arrow indicates when treatments were added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) for myocytes as indicated shown at right.
significantly attenuated when myocytes were exposed to 20 μM 4HNE, an inhibitor of TCA cycle enzyme isocitrate dehydrogenase [31] (Fig. 6(A)). We further examined the role of the TCA cycle by assessing alterations in DHE fluorescence in response to H2O2 in the presence and absence of TCA cycle enzyme inhibitors. In 7 cells, 4-HNE significantly attenuated the increase in DHE signal after exposure to H2O2 (Fig. 6(B)). Similar results were observed when myocytes were exposed to KMV, an inhibitor of α-ketoglutarate dehydrogenase [32] (Fig. 6(C)). Exposure of myocytes to 4-HNE or KMV did not alter Ψm (Figs. 6(B) and (C) insets). These data confirm that increased TCA cycle activity is associated with increased superoxide after transient exposure to H2O2. To examine whether increased electron flow into the ETC via complex II is involved in elevated superoxide generation, myocytes were exposed to 5 μM 2-thenoyltrifluoroacetone (TTFA) then 30 μM
H2O2 for 5 min followed by 10 U/mL catalase. In 5 cells, TTFA significantly attenuated the increase in DHE signal after exposure to H2O2 (Fig. 6(D)). Exposure of myocytes to TTFA did not alter Ψm (Fig. 6(D) inset). These data demonstrate that increased electron flow into the ETC via complex II is associated with increased superoxide after transient exposure to H2O2. 3.6. Confirmation of the role of the Q o site of complex III To further investigate the Q o site of complex III as the predominant source of superoxide production we designed experiments in which we “isolated” the Q o site of mitochondrial complex III in the intact cell. Using the patch-clamp technique we perfused cardiac myocytes intracellularly with complex II substrate succinate (in the patch pipette) while exposing the myocyte to extracellular solution supplemented with complex I inhibitor rotenone, complex III
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Fig. 6. TCA cycle inhibitors attenuate the increase in DHE signal. (A) NADH autofluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase in the presence or absence of 4-HNE. Addition of drugs occurred during the pause in data acquisition. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Increased NADH after addition of Oligo indicated an active TCA cycle. Decreased NADH following addition of FCCP indicated a collapse in Ψm. Mean ± SEM of changes in NADH (% increase) for myocytes as indicated shown at right. (B) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of 4-HNE. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown below. Inset above: JC-1 fluorescence recorded from an untreated (control) myocyte and myocyte before or after exposure to 4-HNE or Oligo. Arrow indicates when 4-HNE was added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right. (C) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of KMV. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown below. Inset above: JC-1 fluorescence recorded from an untreated (control) myocyte and myocyte before and after exposure to KMV or Oligo. Arrow indicates when KMV was added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right. (D) DHE fluorescence recorded from individual myocytes before and after exposure to 0 or 30 μM H2O2 then 10 U/mL catalase in the presence or absence of TTFA. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 30 to 50 min over the slope at 0 to 20 min for myocytes as indicated shown at right. Inset above left: JC-1 fluorescence recorded from an untreated (control) myocyte and myocyte before and after exposure to TTFA or Oligo. Arrow indicates when TTFA was added. To establish that the JC-1 signal was indicative of Ψm 20 μM Oligo and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Mean ± SEM of changes in JC-1 fluorescence (% of pre-treatment) as indicated shown at right.
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Q i site inhibitor antimycin A and DHE. We monitored alterations in DHE fluorescence after exposure to H2O2. This experimental design enabled us to assess the involvement of the Q o site of complex III because electron flow from complex I and beyond the Q i site of complex III were blocked. Exposing myocytes to H2O2 resulted in a 76.6 ± 20.2% increase in DHE in the presence of 1 μM rotenone, 7 nM antimycin A and 5 mM succinate (Fig. 7(A)). The response was similar to the increase in DHE signal recorded in cells not patch-clamped (Fig. 1(A)). In addition, antimycin A and rotenone did not directly alter the L-type Ca2+ current (Fig. 7(A) inset). The response was significantly attenuated when myocytes were exposed to 7 nM myxothiazol prior to 30 μM H2O2 (Fig. 7(B)) and 20 μM 4-HNE prior to 30 μM H2O2 (Fig. 7(C)). These data confirm that the Q o site of complex III is the predominant source of superoxide production after transient exposure to 30 μM H2O2 and that the TCA cycle plays a role in mediating this response. 3.7. Direct activation of the L-type Ca2+ channel increases superoxide production in cardiac myocytes The increase in superoxide production by the mitochondria after transient exposure to H2O2 occurs as a result of an increase in L-type Ca2+ channel activity and elevated intracellular calcium [6]. To confirm that activation of the channel is necessary for the response, we tested whether direct activation of the L-type Ca2+ channel by voltage-step could increase superoxide. Myocytes were patchclamped with the plasma membrane held at −80 mV while complex II substrate succinate was administered via the patch pipette supplemented with DHE, and 1 μM rotenone, 7 nM antimycin A and DHE administered extracellularly. Intracellular calcium was not buffered with EGTA and the internal solution contained 100 nM CaCl2. We voltage-stepped the plasma membrane to −30 mV for 50 ms followed by + 10 mV for 100 ms. DHE signal was significantly increased 47.6 ± 17.8% upon voltage-step to +10 mV compared to myocytes held at −30 mV only (Fig. 8(A)). These data confirm that activation of the L-type Ca2+ channel is sufficient to increase mitochondrial superoxide. We have previously proposed a positive feedback between increased L-type Ca2+ channel activity, increased intracellular calcium and increased superoxide production by the mitochondria [6]. Since superoxide is rapidly dismutated to H2O2, we investigated whether the signal activating the channel is H2O2. We perfused cells intracellularly with active or heat-inactivated catalase and assessed changes in DHE signal after transient exposure to H2O2. Cells were also perfused with complex II substrate succinate in the patch pipette, and 1 μM rotenone and 7 nM antimycin A administered extracellularly. In 5 cells the presence of 500 U/mL active catalase significantly attenuated the increase in DHE signal after exposure to 30 μM H2O2 compared to myocytes perfused intracellularly with 500 U/mL heat-inactivated catalase (Fig. 8(B)). These data suggest that an increase in intracellular H2O2 is necessary for activation of the L-type Ca2+ channel. 4. Discussion The mechanisms by which calcium and ROS participate early in the development of cardiac hypertrophy are not fully understood. Under normal physiological conditions, low levels of ROS (0.05–0.07 μM intracellular) are required to maintain cellular function [5]. Intracellular increases in ROS to 1–10 μM have been associated with mild oxidative stress in the absence of apoptosis or cell death [2–5]. Taking into account the antioxidant buffering capacity of the cell, extracellular exposure of myocytes to 30 μM H2O2 for 5 min will equate to approximately 3 μM H2O2 intracellular. We applied 30 μM H2O2 to mimic the effect of a transient oxidative stress in vivo in humans associated with ischemia–reperfusion injury. Since the response
persisted for many hours after the transient exposure to H2O2, we propose that the brief exposure may be sufficient to induce pathology. In support of this argument, we have demonstrated that 5 min exposure of ventricular myocytes to 30 μM H2O2 is sufficient to cause an increase in cell size and protein synthesis consistent with the development of myocyte hypertrophy 48 h later [7]. The persistent oxidative stress triggered by increased mitochondrial ROS is consistent with “ROS-induced ROS-release” reported previously [33,34]. However unlike the previous studies, we specifically applied H2O2 in the extracellular solution and find that persistent oxidative stress occurs as a result of increased calcium influx through the L-type Ca2+ channel. Under normal physiological conditions, mitochondrial superoxide generation has been demonstrated to occur at mitochondrial complexes I and III [11,12]. In this study we sought to identify the source of increased superoxide production within the mitochondria in response to transient H2O2 exposure. Inhibition of electron flow both before and after the superoxide generation site of complex I attenuated the increase in superoxide (Figs. 1(A) and (B)). Inhibition of electron flow at the Q o superoxide generation site of complex III also attenuated the increase in superoxide (Figs. 2(A) and (B)). However inhibition of electron flow at the Q i superoxide generation site of complex III that resides downstream from the Q o site did not alter the increase in superoxide (Fig. 4(A)). These data suggest the Q o site is the predominant source of superoxide generation. In support of these findings, the complex IV inhibitor NaCN did not alter superoxide production (Fig. 5(A)). Inhibition of the TCA cycle enzymes isocitrate dehydrogenase and α-ketoglutarate dehydrogenase attenuated the increase in mitochondrial NADH and superoxide generation after transient exposure of cardiac myocytes to H2O2 (Figs. 6(A)–(C)). Inhibition of electron flow at complex II also attenuated the increase in superoxide generation after transient exposure of cardiac myocytes to H2O2 (Fig. 6(D)). Under normal physiological conditions, electrons flow down the ETC from complex I. Electrons are also fed into the ETC via complex II as a result of conversion of succinate to fumarate within the TCA cycle. Therefore an increase in TCA cycle activity will result in an increase in electron flow to complex II. We find that increased superoxide generation occurs at complex III as a result of an increase in electron flow from the TCA cycle to complex II after exposure to H2O2. To further confirm the findings, cells were patch-clamped with the complex II substrate succinate and superoxide production was assessed in the presence of complex I and III inhibitors (Fig. 7). The experiments were designed to “isolate” complex III as the site of generation of superoxide and mimic isolated mitochondrial studies in the intact cell. Under these conditions myxothiazol and 4-HNE attenuated the increase in DHE signal (Figs. 7(B) and (C)). We also confirmed that direct activation of the channel by voltage-stepping the plasma membrane could mimic the effect of H2O2 (Fig. 8(A)). However voltage-step of the membrane per se does not appear to be required for the response since the dihydropyridine receptor agonist Bay K8684 can mimic the effect of H2O2 on mitochondrial superoxide production [15]. Increased intracellular H2O2 is necessary for the persistent activation of the channel since catalase attenuated the increase in superoxide (Fig. 8(B)). Therefore our data demonstrate that H2O2 is the signal responsible for altered calcium homeostasis and further oxidative stress in cardiac myocytes. H2O2 is a well recognised signal molecule capable of altering protein function as a result of modification of critical cysteines on proteins [5]. We propose that a transient oxidative stress can activate the L-type Ca2+ channel and cause a further increase in mitochondrial superoxide from Q o site of complex III as a result of increased calcium uptake by the mitochondria and elevated TCA cycle activity (Fig. 8(C)). The L-type Ca2+ channel, TCA cycle and Q o superoxide generation site of complex III may represent possible sites to target in the prevention of the development of cardiac hypertrophy associated with mild oxidative stress.
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Fig. 7. Myxothiazol and 4-HNE attenuate the increase in DHE signal when complex III is “isolated” in the patch-clamped cell. (A) DHE fluorescence recorded from a patch-clamped myocyte before and after exposure to 0 or 30 μM H2O2 (5 min) then 10 U/mL catalase. External solution (ES) was supplemented with rotenone (rote) and antimycin A (Anti). Internal solution (IS) was supplemented with succinate. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 15 to 19 min over the slope at 0 to 4 min for myocytes exposed to H2O2 and catalase as indicated shown at right. Inset above left: current traces recorded from an untreated (control) myocyte and in the same myocyte exposed to Anti and rote. (B) DHE fluorescence recorded from individual patch-clamped myocytes before and after exposure to 0 or 30 μM H2O2 in the presence and absence of myxothiazol (myx) as indicated. ES was supplemented with rote and Anti. IS was supplemented with succinate. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 15 to 19 min over the slope at 0 to 4 min for myocytes as indicated shown at right. (C) DHE fluorescence recorded from individual patch-clamped myocytes before and after exposure to 0 or 30 μM H2O2 in the presence or absence of 20 μM 4-HNE. ES was supplemented with Rote and Anti. IS was supplemented with succinate. Addition of drugs occurred during the pause in data acquisition. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 15 to19 min over the slope at 0 to 4 min for myocytes as indicated shown at right.
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Fig. 8. (A) Activation of the L-type Ca2+ channel mimics the increase in DHE. DHE fluorescence recorded from a patch-clamped myocyte held at −30 mV, and a myocyte held initially at −30 mV then voltage-stepped to + 10 mV as indicated with dashed arrow. External solution (ES) was supplemented with rotenone (rote) and antimycin A (Anti). Internal solution (IS) was supplemented with succinate. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 5 to 9 min over the slope at 0 to 4 min for myocytes held at −30 mV (not voltage-stepped) or held initially at −30 mV then voltage-stepped to + 10 mV (voltage-stepped) shown at right. (B) H2O2 is the signal activating the channel. DHE fluorescence recorded from a myocyte before and after exposure to 30 μM H2O2 while patch-clamped with 500 U/mL active or heat-inactivated catalase containing IS. IS was also supplemented with succinate. ES was supplemented with Rote and Anti. Addition of H2O2 occurred as indicated with arrow. Mean ± SEM of ratio of fluorescence expressed as the slope of the signal measured at 5 to 9 min over the slope at 0 to 4 min for myocytes exposed to H2O2 in the presence of inactive or active catalase as indicated shown at right. (C) Proposed model explaining the mechanism for increased production of superoxide from the mitochondria after transient exposure to H2O2. Exposure of cardiac myocytes to 30 μM H2O2 for 5 min causes increased activation of the L-type Ca2+ channel and increased Ca2+ entry into the cytosol. Calcium entry through the mitochondrial calcium uniporter (MCU) into the mitochondrial matrix triggers increased activation of the TCA cycle. Increased electron flow to the Qo superoxide generation site of complex III (via complex II) results in elevated superoxide production from this site (see also Discussion).
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Acknowledgments This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC). Helena Viola is the recipient of a Biomedical Postgraduate Research Scholarship from NHMRC and National Heart Foundation of Australia. Livia Hool is the recipient of a NHMRC Career Development Award.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yjmcc.2010.07.015.
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Glossary 4-HNE: 4-hydroxynonenal Anti: antimycin A ATP: adenosine triphosphate Ca2+: calcium Cat: catalase DHE: dihydroethidium DPI: dibenziodolium chloride ETC: electron transport chain FCCP: carbonyl cyanide p-(trifluromethoxy)phenyl-hydrazone H2O2: hydrogen peroxide JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide KMV: α-keto-β-methyl-n-valeric acid MCU: mitochondrial calcium uniporter MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide Myx: myxothiazol NaCN: sodium cyanide NADH: reduced nicotinamide adenine dinucleotide Oligo: oligomycin ROS: reactive oxygen species Rote: rotenone Stig: stigmatellin TCA: tricarboxylic acid TTFA: 2-thenoyltrifluoroacetone Ψm: mitochondrial membrane potential