ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca2+ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F0F1ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca2+-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.
Citation: Sokolova N, Pan S, Provazza S, Beutner G, Vendelin M, Birkedal R, et al. (2013) ADP Protects Cardiac Mitochondria under Severe Oxidative Stress. PLoS ONE 8(12): e83214. https://doi.org/10.1371/journal.pone.0083214
Editor: Salvatore V Pizzo, Duke University Medical Center, United States of America
Received: May 15, 2013; Accepted: October 31, 2013; Published: December 13, 2013
Copyright: © 2013 Sokolova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NS was supported by Estonian Science Foundation grant no. ETF8041, Wellcome Trust grant no. 081755 and a travel grant (Estonian Science foundation Dora programme activity 6) from the Archimedes Foundation. S. Provazza was supported by HL-33333 and an ARRA supplement award. GB and SSS were supported by NIH grant RO1HL-033333, RO1HL-093671, and R21HL-110371. MV and RB were supported by Wellcome Trust grant no. 081755. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Ca2+ and ADP are the two major regulators of mitochondrial energy metabolism that function in coordination to keep the balance between the energy demand and supply. In cardiac muscle cells, during the excitation-contraction coupling, Ca2+ enters mitochondria to stimulate Krebs’ cycle. As such, the nicotinamide adenine dinucleotide redox potential and ATP synthesis required for cardiac workload are maintained . Concomitantly, ADP generated by ATPases and kinases enters the mitochondrial matrix via the adenine nucleotide translocase (ANT) and stimulates ATP-production by F1F0-ATPase [2,3]. Therefore, both Ca2+ and ADP have a positive impact on ATP generation under physiological conditions.
Ca2+ and ADP are also major modulators of mPTP [4–7]. But here, they function oppositely. Physiologically, the mPTP may open briefly, functioning as a mitochondrial Ca2+-release channel . Pathologically, mitochondrial Ca2+-overload triggers irreversible opening of mPTP, which is a major cause of cell death. ADP, on the contrary, is a potent inhibitor of mPTP [6,7].
The molecular identity of mPTP is still unsolved. Two hypotheses exist regarding the pore-forming component. Both involve cyclophilin D (CypD) and ADP as regulators. CypD is a peptidyl-prolyl cis-trans isomerase, which binds to several proteins including ANT, the mitochondrial phosphate carrier (mPiC) and F1F0 ATPase, and increases mPTP Ca2+-sensitivity . Irrespective of its exact site of action, it was shown that cyclosporine A (CsA) binding to CypD inhibits mPTP opening by unmasking an inhibitory Pi-binding site . Some suggest that mPiC is the pore-forming component and mainly regulated by CypD and ANT . ANT in the “c” (cytosol) or “m” (matrix) conformation increases or decreases mPTP Ca2+-sensitivity, respectively. ADP decreases Ca2+-sensitivity, because its binding shifts ANT to the “m” conformation . Others suggest that dimers of F1F0-ATPase are responsible for the formation of mPTP . CypD also binds and inhibits F1F0-ATPase activity , and ADP is a potent inhibitor of the channel activity of F0F1-ATPase dimers .
Until today, little is known about the effect of ADP on mPTP in diseased mitochondria, which experience increased oxidative stress, Ca2+-load, and energy deficiency. ADP-binding to ANT is reduced by oxidative stress , which might reduce the inhibiting effect of ADP on mPTP. In this paper, we wanted to address the potency of ADP as an mPTP inhibitor in diseased mitochondria with the hope to obtain clues about its mechanism of action. As noted above, ADP may exert its function by binding to either ANT or the F0F1-ATPase. But ADP may also enhance Ca2+-sequestration in the form of Ca2+-phosphate precipitates [16,17]. Furthermore, it may be speculated that part of the ADP-effect on Ca2+-uptake capacity is due to its reduction of ROS production . Indeed, as the substrate of F1F0-ATPase, which uses the electrochemical energy stored in the proton gradient to produce ATP, ADP should reduce ROS production.
In this study, we assessed at the level of isolated mitochondria from mouse hearts how chronic and acute oxidative stress affects the effect of ADP on CRC and ROS production. As models of long-term oxidative stress, we used old mice and diabetic mice. As models of acute oxidative stress, we used IR and exposure to a low dose of t-BH.
All procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by an Institutional Animal Care and Use Committee (University Committee on Animal Resources (UCAR) protocol 2010–030).
Animals and models of disease and oxidative stress
Control mice: 6-8 weeks old male C57BL6 mice (n=64). Aging: 12-15-month old male C57BL6 mice (n=8). Diabetes: To induce type I diabetes, 5 weeks old male C57BL6 mice (n=12) were injected intraperitoneally with 150 mg/kg streptozotocin dissolved in 0.1 mol/L sodium citrate buffer, pH 4, prepared within 5 min of administration. Mice were given drinking water supplemented with 7.5 % sucrose for 2.5 days to avoid severe hyperglycemia. After 5 weeks the mice were diabetic and used for experiments. Exposure to t-BH: Mitochondria from 6-8 weeks old C57BL6 male mice (n=44) were exposed to 5 µmol/L t-BH for 10 min before recording Ca2+-uptake or ROS production. Ischemia-reperfusion injury: Male C57BL6 mice, 6-8 weeks old (n=20) were anesthetized with freshly prepared Avertin (2,2,2-tribromoethanol, 0.5 mg/kg injected intraperitoneally). Isolated hearts were retrogradely perfused in Langendorff mode under constant flow (4 ml/min) with Krebs–Henseleit buffer as in . After 10 min of equilibration, hearts were subjected to 15 min of global ischemia followed by 60 min of reperfusion.
Unless otherwise stated, the mice were euthanized by CO2 inhalation and sacrificed by cervical dislocation.
Isolation of heart mitochondria
Mitochondria from 2-4 mouse hearts were isolated using the protocol of Rehncrona et al with modifications . The minced heart tissue was subjected to protease treatment: it was incubated with 5 mg nagarse dissolved in 10 ml medium A for 8 minutes at room temperature while gently stirring. The protease reaction was stopped by adding 1 ml of 0.2 mg/ml of bovine serum albumin dissolved in medium A. The tissue was then homogenized with a Potter-Elvehjem homogenizer, and mitochondria were isolated by differential centrifugation. The final mitochondrial pellet was suspended in isolation medium B.
For mitochondrial ROS generation measurements, mitochondria were Ca2+ depleted to minimize possible signalling of Ca2+ on ROS generation . This procedure consisted of 15 min incubation at room temperature in Ca2+-depletion buffer. The mitochondria were subsequently washed several times in a Ca2+-depletion buffer without NaCl, EGTA and succinate. The isolated mitochondria were kept on ice and used within 4 hours. Protein concentration was determined by the Lowry method using BSA as a standard.
Ca2+ uptake measurements with arsenazo III
Ca2+ uptake was measured with arsenazo III at room temperature as a difference in absorbance at 662 nm and the background at 692 nm using Beckman Coulter DU 800 UV-Vis spectrophotometer (Beckman Coulter Inc., Brae, CA). Isolated mitochondria (~1 mg/ml of mitochondrial protein) were added to 1 ml Ca2+-uptake buffer. The absorbance change upon Ca2+ addition was determined every 15 sec and followed for 30-70 min. Varying amounts of free Ca2+ were added every 2 minutes. Free Ca2+ concentrations were calculated using the MaxChelator program (http://www.stanford.edu/~cpatton/maxc.html).
Measurement of mitochondrial ROS production
Mitochondrial superoxide production was determined indirectly by coupling the dismutation of superoxide to H2O2. H2O2 was detected fluorimetrically using Amplex red (10-acetyl-3,7-dihydroxyphenoxazine), which reacts with H2O2 in a 1:1 stochiometry in the presence of horseradish peroxidase (HRP), producing highly fluorescent resorufin. For these experiments, mitochondria (~0.5 mg/ml of mitochondrial protein) were added to 2 ml ROS buffer. Fluorescence was recorded at room temperature using a Cary Eclipse fluorescence spectrophotometer (Varian Inc., Walnut Creek, CA). The excitation wavelength was 563 nm and the emitted fluorescence was detected at 587 nm. A calibration signal was generated with known amounts of H2O2 at the end of each experiment.
Measurement of mitochondrial oxygen consumption
Mitochondrial oxygen consumption was measured at room temperature using a Clark-type oxygen electrode from Hansatech (PP Systems, Boston MA).The measurements were carried out in 1 ml of respiration medium. The basal rate of respiration (State 2) was initiated by the addition of 5 mmol/L glutamate and 5 mmol/L malate as substrates. Maximal respiration rate (State 3) was measured in the presence of 1 mmol/L ADP. Respiration rates were expressed as nmol O2 min-1 mg mitochondrial protein-1. The respiratory control index (RCI) was calculated as RCI = State3/State2. At the end of each experiment, 8 μmol/L cytochrome c and 30 μmol/L atractyloside were added to test the intactness of the outer and inner mitochondrial membrane, respectively.
Krebs–Henseleit buffer for Langendorff perfusion and IR (in mmol/L): 118 NaCl, 4.7 KCl, 1.2 MgSO4 , 24 NaHCO3 , 1.2 KH2PO4, 2.5 CaCl2 , 11 D-glucose.
For isolation of mitochondria, medium A contained (in mmol/L): 225 mannitol, 70 sucrose, 1 EGTA and 10 HEPES, pH 7.2. Medium B contained (in mmol/L): 225 mannitol, 70 sucrose, and 10 HEPES, pH 7.2.
Ca2+-depletion buffer contained (in mmol/L): 195 mannitol, 25 sucrose, 40 HEPES, 10 NaCl, 1 EGTA, 5 succinate, pH 7.2
For recording mitochondrial CRC, the Ca2+-uptake buffer contained (in mmol/L): 120 KCl, 70 mannitol, 25 sucrose, 5 KH2PO4, 0.5 EGTA, 10 HEPES, pH 7.2 in the presence of 5 mmol/L malate and 5 mmol/L glutamate as substrates and 100 μmol/L arsenazo III.
For recording ROS production, the ROS buffer contained (in mmol/L): 120 KCl, 70 mannitol, 25 sucrose, 5 KH2PO4, 0.5 EGTA, 10 HEPES, pH 7.2, 10 μmol/L Amplex® red, 1 U/ml type II HRP, and 80 U/ml Cu/Zn superoxide dismutase.
For respiration experiments, respiration medium contained (in mmol/L): 120 KCl, 70 mannitol, 25 sucrose, 5 KH2PO4, 3 MgCl2, 0.5 EGTA, 20 HEPES, pH 7.2.
RCI of mitochondrial preparations
The quality of the mitochondrial preparations was controlled by recording their respiration rate in the presence of substrates alone, State 2, and after addition of 1 mmol/L ADP, State 3. RCI was calculated as State 3/State 2. These values are shown in Table 1. RCI in control mice was 7.6 ± 0.2, n=10. The respiratory parameters were not different in diabetic mice. Old mice had a lower State 2 (8.6 ± 0.8, P = 0.057, n=7) and State 3 (61.3 ± 6.7, P = 0.028, n=7), but RCI was the same as in control (7.0 ± 0.4, P = 0.222, n=7). This may be attributed to a fraction of the mitochondria having already undergone mPTP. However, their CRC was the same as in control (see below). The low concentration of t-BH (5 µmol/L for 10 min) does not affect glutamate and malate-dependent respiration , and this was confirmed in our recordings, where State 2 and 3 and RCI was not significantly different from control. RCI was significantly lower after IR (5.3 ± 0.6, P = 0.009, n=4), consistent with an inhibition of electron transport chain activities after severe stress.
|n||State 2||State 3||RCI|
|Control||10||13.1 ± 1.4||100.8 ± 12.0||7.6 ± 0.2|
|Aging||7||8.6 ± 0.8||61.3 ± 6.7*||7.0 ± 0.4|
|Diabetes||3||14.1 ± 2.8||83.8 ± 11.3||6.2 ± 0.9|
|IR||4||9.3 ± 1.1||48.8 ± 6.4||5.3 ± 0.6**|
|t-BH||3||9.3 ± 1.4||64.1 ± 8.6||6.9 ± 0.4|
The protective effect of ADP is specific to [ADP]
The inhibition of mPTP by ADP has been widely reported including the seminal studies by Haworth and Hunter [23–25], in which the mPTP phenomenon was discovered. When adding ADP to the solution with isolated mitochondria in the presence of substrates, the majority will be converted into ATP. After a short period of time, an equilibrium between [ADP] and [ATP] will be reached. Oligomycin inhibits the conversion of ADP into ATP by F1F0-ATPase. Thus, [ADP] will be higher in the presence of oligomycin. We addressed the question whether the total amount of adenine nucleotides ([ADP] + [ATP]) or [ADP] specifically is important for the inhibition of mPTP. Figures 1A and B show representative traces of Ca2+-uptake recordings. Figure 1C summarizes the mitochondrial Ca2+-uptake capacity under various conditions as indicated below each column. Ca2+-uptake capacity for control and 500 μmol/L ADP was 344 ± 32 nmol/mg protein and 774 ± 65 nmol/mg protein, respectively, confirming that ADP inhibited mPTP potently. 1 mmol/L ATP and 10 mmol/L creatine, which stimulate mitochondrial creatine kinase to generate ADP, had a similar effect as 500 μmol/L ADP on Ca2+-uptake capacity (P = 0.432). A smaller dose of ADP, 50 μmol/L, had a significantly smaller effect increasing CRC to only 458 ± 30 nmol/mg protein (Figure 1C) (P = 0.003 compared to 500 μmol/L ADP; P = 0.013 compared to 1 mmol/L ATP and creatine). Inhibition of the F1F0-ATPase with 5 μmol/L oligomycin had a negative effect on Ca2+-uptake capacity, which decreased to approximately 62% of control (P = 0.016, Figure 1C). However, in the presence of oligomycin, 50 µmol/L ADP had a significantly larger effect on CRC, which increased from 213 ± 17 nmol/mg protein to 536 ± 49 nmol/mg protein. Thus, [ADP] is the most important inhibitor of mPTP.
A-B. Representative raw traces of the Ca2+-uptake experiments. Mitochondria were incubated with Arsenazo III to follow extramitochondrial Ca2+ spectrophotometrically. Consecutive pulses leading to an increase of 40 μmol/L free Ca2+ were added as indicated by arrow heads. The CRC was defined as the concentration at which the mitochondria failed to accumulate more Ca2+ and mPTP opened to release all Ca2+ so far accumulated by the mitochondria. A. Traces are shown with mitochondria from control mice, 6-8 weeks old, under control conditions (no additions; black), in the presence of 500 µmol/L ADP (blue), 1 mmol/L ATP and 10 mmol/L creatine (Cr) (red), and 0.2 µmol/L cyclosporine A (CsA) (turquoise). B. Representative raw traces of the Ca2+-uptake experiments in the presence of 5 µmol/L oligomycin (green), 50 µmol/L ADP (orange), 5 µmol/L oligomycin and 50 µmol/L ADP (violet). C. Column diagrams of the averaged results under the conditions indicated below. The amount of Ca2+ was normalized to the mitochondrial content (mg protein). All values are mean ± SEM. * and ** denote significant difference P < 0.05, and P<0.01, respectively, between treatment and control. The number of experiments is indicated below.
ADP recovers mitochondrial Ca2+-uptake capacity after acute oxidative stress
Figure 2 shows the CRC in the different models of oxidative stress. Ca2+ was added consecutively to isolated mitochondria until they reached their maximum uptake capacity, where mPTP opened to release all Ca2+, causing an increase in extramitochondrial Ca2+ as monitored by Arsenazo III absorbance. Control mitochondria were able to sequester up to 10 pulses of 40 µmol/L free Ca2+ with a mean value of 344 ± 32 nmol/mg protein (Figure 1A and Figure 1C). We were surprised to find that chronic oxidative stress did not affect the initial CRC or the effect of ADP (Figure 2C-D). In contrast, acute oxidative stress had an adverse effect on mitochondrial CRC by facilitating mPTP opening. After IR or t-BH exposure, the first addition of 40 µmol/L Ca2+ was able to trigger mPTP opening (Figure 2B, red trace). Therefore, 0.2 µmol/L and 5 µmol/L Ca2+ additions were used for assessing mitochondrial CRC after IR and t-BH treatments, respectively (Figure 2B). The CRC was dramatically decreased to 1 ± 0.2 nmol/mg protein (IR) and 32 ± 4 nmol/mg protein (t-BH) (Figure 2E-F).
A-B: Representative raw traces of the Ca2+-uptake from different models under control conditions. A. Control mice (black); Aging (orange); Diabetes (violet). Pulses of 40 μmol/L free Ca2+ were added as indicated by arrow heads. B. Ischemia-reperfusion, IR (green); exposure to 5 μmol/L tert-butyl hydroperoxide, t-BH, (blue). Pulses of 40 μmol/L (red), 0.2 μmol/L (green), and 5 μmol/L (blue) free Ca2+ were added respectively as indicated by arrow heads. C-F: Column diagrams of the averaged results under the conditions indicated below. The final concentrations of ADP and CsA are 500 μmol/L and 0.2 μmol/L, respectively. The amount of Ca2+ was normalized to the mitochondrial content (mg protein). All values are mean ± SEM. * and ** denote significant difference P < 0.05 and P < 0.01 respectively, between treatment and control within an animal group. # and ## denote significant difference P < 0.05 and P<0.01, respectively, between animal groups with similar treatment.
CsA is a well-known inhibitor of CypD and protects against ischemia-reperfusion damage at a concentration of 0.2 µmol/L . Indeed, this concentration of CsA increased CRC in all models (Figure 2). 500 μmol/L ADP had a similar effect and restored CRC to 447 ± 105 nmol/mg protein after IR and to 514 ± 103 nmol/mg protein after t-BH exposure, respectively (Figure 2E-F). The recovered CRC was not significantly different from that of control mice in the absence of ADP (P = 0.109 for t-BH and P = 0.689 for IR, respectively, Figure 2E-F). Thus, ADP is a potent inhibitor of mPTP – also in mitochondria, which have been exposed to severe oxidative stress.
Similar effect of ADP irrespective of initial Ca2+-uptake capacity
Figure 3 is a column diagram showing the effect of ADP – i.e. how much does ADP increase CRC. It illustrates, as noted above, that 50 µmol/L ADP increased CRC much more, when F1F0-ATPase was inhibited by oligomycin (P = 0.008). But what is in our opinion more remarkable, is the fact that although CRC was drastically lowered by acute oxidative stress (IR and t-BH) to the extent that mitochondrial Ca2+-buffering was almost absent (Figure 2E-F), ADP exerted a similar effect as in mitochondria from control mice. In control as well as all model of oxidative stress, 500 µmol/L ADP increased CRC by 360-460 nmol/mg protein (Figure 3).
In mitochondria from control mice, the increase in Ca2+-uptake capacity induced by 50 µmol/L ADP in the absence and presence of oligomycin, and 500 µmol/L ADP alone and in the presence of cyclosporine A (CsA) are shown as indicated. In models of chronic and acute oxidative stress, the increase induced by 500 µmol/L ADP is shown as indicated below the columns. The increase induced by 500 µmol/L ADP is remarkably consistent in control and the different models of oxidative stress.
ADP attenuates ROS generation in control, but not after exposure to t-BH
In addition to Ca2+, ROS are also potent activators of mPTP. To determine whether ADP-mediated ROS decrease contributes to the inhibition of mPTP, we measured ROS production in the preparations. Amplex Red was used to record ROS production with three consecutive steps: 1) mitochondria alone, 2) after addition of substrates (glutamate and malate), and 3) after the addition of one pulse of a submaximal Ca2+-concentration to trigger partial opening of mPTP. The data on ROS production in old and diabetic mice and after IR did not give any new information. Therefore, we show only the results from control and after t-BH exposure. Figure 4A shows original traces of recordings from control mice and the results are summarized in Figure 4C. As expected, ROS production increased upon addition of substrates, as indicated by the increase in the slope of Amplex Red fluorescence trace. Addition of 250 μmol/L Ca2+ increased ROS production further (Figure 4A and C, control). ADP or ATP and creatine, which stimulate F1F0-ATPase to utilize the proton gradient, both significantly abolished the substrate- and Ca2+-induced increase in ROS generation (Figure 4C). CsA abolished mostly the Ca2+-induced increase in ROS generation, but the net effects were significantly smaller than those of ADP (Figure 4C, P = 0.003 and P = 0.002 for substrate- and Ca2+-induced increase in ROS generation, respectively).
A-B: Representative raw traces of the ROS-recordings. The rate of hydrogen peroxide (H2O2) production was followed spectrofluorometrically by its reaction with Amplex Red in the presence of horseradish peroxidase. The rate of ROS production was recorded in three consecutive steps: After addition of mitochondria alone, after addition of substrates (5 mM glutamate and 5 mM malate) and after addition of one submaximal dose of Ca2+. Traces are shown under control conditions (no additions; black), in the presence of 500 µmol/L ADP (blue), 0.2 µmol/L CsA (green), and 1mmol/L ATP + 10 mmol/L creatine (Cr) (red). A: control mice; B: after exposure to 5 μmol/L t-BH. C-D: Column diagrams of the averaged results under the conditions indicated below each column. Steady-state H2O2 production was recorded in the mitochondria alone (rate between 1-3 min; white columns), after addition of substrates (rate between 6.5-8.5 min; light grey columns), and after addition Ca2+ (rate between 11-15 min; dark grey columns). Treatments within each group are indicated below the columns. All values are mean ± SEM. † and †† denote significant effect of substrates and/or Ca2+, P < 0.05, P < 0.01 respectively. *, ** denote significant difference P < 0.05, and P < 0.01, respectively, between control and different treatments within the group. #and ## denote significant difference (P < 0.05 and P<0.01, respectively) between control and t-BH with the same treatment. The number of experiments is indicated below.
In mitochondria exposed to t-BH, ROS-production was much higher than in control (Figure 4B and D). Neither Ca2+ nor ADP and CsA had a profound effect on ROS-production. This implies that the ADP-induced increase in mitochondrial Ca2+-uptake capacity is not related to a decrease in ROS-production in mitochondria treated with t-BH.
The most notable results from this study are that 1) ADP increases CRC after IR and t-BH exposure to an extent that is remarkably similar to that in control, and 2) the effect of ADP on CRC in mitochondria exposed to t-BH is not the result of a decreased ROS-production.
Inhibition of mPTP relates to the concentration of ADP
The main mechanism through which ADP decreases mitochondrial Ca2+-sensitivity has been attributed to the binding of ADP to ANT, which stabilizes the transporter in the m-state and prevents pore opening . It was demonstrated that ANT is not a pore-forming component of the mPTP, as mPTP opening is still triggered in mice lacking ANT1 and ANT2 . However, the regulating role of ANT cannot be disputed, as inhibition of ANT in different conformations by bongkrekic acid or atractyloside has opposite effects on mitochondrial Ca2+-sensitivity . Haworth and Hunter proposed that ADP exerts its effect by binding to an internal site as well as to ANT . Recent studies suggest that either the mitochondrial phosphate carrier or F1F0-ATPase could be one of the key components for mPTP [13,14,29,30]. Both cases include a role for ADP [13,29]. In the latter case, ADP affects mPTP via direct binding to F0F1-ATPase. This, however, does not exclude an additional effect via ADP-binding to ANT.
The present study showed that the effect of ADP on CRC relates to its concentration. 500 μmol/L ADP had a signifcantly larger effect than 50 μmol/L ADP (Figure 1). Also, 50 μmol/L ADP had a significantly larger effect in the presence of oligomycin to inhibit ADP-turnover by F1F0-ATPase (Figure 1B-C). Thus, the effect of ADP is specific to [ADP] and not the total concentration of adenine nucleotides.
It is noticed that oligomycin on its own has a negative effect on mitochondrial Ca2+-uptake capacity (Figure 1B-C). The mitochondrial membrane potential decreases as the mitochondria take up a significant amout of Ca2+. It is conceivable that in the absence of oligomycin, the mitochondria will hydrolyze ATP to ADP in order to maintain their membrane potential. However, oligomycin renders the mitochondria unable to rescue their membrane potential, so that the critical potential at which mPTP opens is reached at a lower Ca2+-concentration.
Ca2+-sequestration has no significant effect on CRC
ADP can buffer Ca2+ by itself , and could have an additional effect on mitochondrial Ca2+ buffering such as the maintenance of high matrix Pi concentrations. It has been shown that ATP-Mg2-/Pi2- and HADP2-/Pi2- can increase matrix Ca2+ buffering via Ca2+-Pi precipitates and thus desensitize the mPTP [32,33]. Mitochondrial Ca2+-sequestration is proportional to the ADP-uptake . If Ca2+-sequestration played a significant role in the effect of ADP, we would expect that the Ca2+-uptake capacity is increased more by 500 µmol/L ADP than by 50 µmol/L ADP in the presence of oligomycin. Our results suggest that this is not the case (Figures 1 and 3). However, the role of Ca2+-sequestration should be studied further by quantifying intra- and extramitochondrial total and free [Ca2+] as mitochondria take up Ca2+ after successive Ca2+-pulses.
Similar effect of ADP on CRC in all models of oxidative stress
Oxidative stress has an unfavourable effect on mitochondrial CRC . We studied the effect of ADP on CRC in different models of increased oxidative stress. Chronic oxidative stress by aging and diabetes had no significant impact on CRC or the effect of ADP. However, the acute oxidative damage caused by IR and t-BH drastically lowered CRC to the extent that mitochondrial Ca2+-uptake was almost absent. It is interesting that ADP was able to restore this function (Figures 2E-F) and with an effect that was similar to that in control (Figure 3). It appears that irrespective of the healthiness of mitochondria, a certain amount of ADP increases CRC by a certain amount. This is interesting, because ADP-binding to ANT is reduced by oxidative stress . Therefore, our results suggest that although ADP to some extent inhibits mPTP opening via binding to ANT, significant inhibition also occurs by some other mechanism.
ADP does not increase CRC via an effect on ROS-production in mitochondria exposed to t-BH
In addition to Ca2+, ROS also effectively trigger mPTP openings. One of the aims of this study was to see whether ADP might affect mitochondrial CRC via ROS decrease in the models of oxidative stress. In our study, we used Amplex red and horseradish peroxidase as a H2O2 detection system. This allowed us to use the efflux of H2O2 from mitochondria as a measure of net superoxide production in the matrix. Under physiological conditions, the production of ROS and their consumption in the form of H2O2 is finely tuned. ROS are mainly produced as superoxide by complexes I and III in the mitochondrial electron transport chain . Superoxide is dismutated to H2O2 by manganese superoxide dismutase (MnSOD) in the mitochondrial matrix and copper/zinc superoxide dismutase (Cu/ZnSOD) in the mitochondrial intermembrane space and cytosol. The resulting H2O2 can easily diffuse across membranes to be detected by the assay. However, H2O2 can also be consumed by matrix enzymes, e.g. glutathione peroxidases, and cause an underestimate of superoxide production by competing with the efflux of H2O2 . By a series of redox reactions, H2O2 is converted to H2O, while glutathione (GSH) is oxidized to glutathione disulfide (GSSG). GSSG is reduced back to GSH by the oxidation of pyridine nucleotides (NADH/NADPH). The GSH/GSSG ratio provides an estimate of cellular redox buffering capacity, thus playing a key role in maintaining redox homeostasis .
ADP can regulate mitochondrial ROS production through complexes I and III, as well as general redox state of the mitochondrial matrix (NADH/NADPH, GSH) via oxidative phosphorylation. Indeed, the addition of ADP caused a decrease in H2O2 export from control mitochondria (Figure 4A and C). However, in t-BH treated mitochondria the fine tuning of ROS production and consumption by ADP in mitochondria is disturbed (Figure 4B-D). t-BH treatment induces the oxidation of pyridine nucleotides  and GSH . GSSG is not exported from the mitochondria  and increased GSSG formation may decrease the activity of mitochondrial redox-sensitive proteins [40–46]. As a result, H2O2 export is increased in t-BH mitochondria (Figure 4B-D). Although ADP does reduce ROS, it is not able to bring it down to control level (Figure 4D). Thus, the t-BH results demonstrate that ADP regulates mitochondrial CRC not only via ROS reduction, but via some other mechanisms as well.
ADP is well known to desensitize the mPTP to intramitochondrial Ca2+, so that the mitochondria can take up more Ca2+, before opening of mPTP is triggered. In the present study, we found that ADP had the same effect in severely stressed mitochondria as in control. This suggests that ADP, in addition to its regulation via ANT, exerts significant regulation of mPTP via some other mechanism. Although ADP decreases ROS production in control mitochondria, its effect is minor in t-BH-treated mitochondria. Therefore, we conclude that at least in some cases e.g. t-BH treatment, the ADP-induced increase in mitochondrial Ca2+-uptake capacity is not related to a reduction in ROS. It is likely that significant regulation by ADP occurs via its internal binding site (reported in ), which could be on F1F0-ATPase .
From a more integrated point of view, it is very interesting that ADP is as potent an inhibitor of mPTP in severely stresssed mitochondria, and that its effect relates to the concentration. Namely, ADP concentrations increase in the diseased state due to less efficienct ATP production, depolarized mitochondrial membrane potential, Ca2+ overload, and a smaller pH gradient [47–49]. It is plausible that this increase in ADP concentration could protect against opening of mPTP and thus delay the injury and death of cells. This function of ADP as a regulator of mitochondrial ATP production, Ca2+ homeostasis, and ROS generation is shown schematically in Figure 5. Under physiological conditions, it serves as a substrate for ATP synthesis while ensuring the closure of mPTP and modest ROS generation. Under pathological conditions like IR injury, when the mitochondria are most vulnerable for mPTP opening, ADP can effectively delay mPTP opening so that mitochondrial membrane integrity can be preserved to maintain energy production. The ability of ADP to serve multiple roles as a potent mPTP blocker, a substrate for catalysing ATP generation, and an inhibitor for ROS generation, makes it a unique molecule to preserve mitochondrial function under pathophysiological conditions.
Under physiological conditions, optimal ADP concentrations in mitochondrial matrix serve effectively to maximize ATP generation while minimizing ROS. ROS production via ETC and annihilation via matrix scavenger systems (gluthatione/thioredoxin) is finely balanced. Under pathological conditions, mitochondrial Ca2+ and ROS increase, stimulate excessive mPTP opening and thus the signaling pathway for injury. These detrimental effects are countered by a concomitant increase in ADP, which allows the mitochondria to endure a larger load of Ca2+ and ROS, maintaining mitochondrial integrity and function when it is most critical.
ANTm – ANT in the “m”-conformation; ANTc – ANT in the “c”-conformation; CL - cardiolipin; CypD – cyclophilin D; ETC – electron transport chain; GR – glutathione reductase; GRx – glutathione peroxidase; GSH – reduced glutathione; GSSG – oxidized glutathione; MCCs – mitochondrial calcium channels; MnSOD – manganese superoxide dismutase; mPTP – mitochondrial permeability transition pore; Prx3 – peroxiredoxin 3; ROS – reactive oxygen species, TCA – tricarboxylic acid cycle; Trx2red - reduced thioredoxin 2; Trx2ox – oxidized thioredoxin 2; TrxR2 – thioredoxin reductase 2.
We thank Dr. Sergiy M Nadtochiy at the University of Rochester Medical Center (URMC) for his support on Langendorff perfusion of the mouse hearts, and Dr. Paul Brookes (URMC) for the use of spectrophotometer. We also thank the members of the Mitochondrial Research and Innovation Group (MRIG) at URMC for their comments.
Conceived and designed the experiments: SSS GB NS. Performed the experiments: NS S. Pan S. Provazza. Analyzed the data: NS MV RB. Contributed reagents/materials/analysis tools: SSS GB. Wrote the manuscript: RB NS S. Pan GB SSS. Prepared all the figures: NS.
- 1. Balaban RS (2002) Cardiac energy metabolism homeostasis: role of cytosolic calcium. J Mol Cell Cardiol 34: 1259–1271. PubMed: 12392982.
- 2. Vendelin M, Kongas O, Saks V (2000) Regulation of mitochondrial respiration in heart cells analyzed by reaction-diffusion model of energy transfer. Am J Physiol Cell Physiol 278: C747 –C764. PubMed: 10751324.
- 3. Brdiczka DG, Zorov DB, Sheu S-S (2006) Mitochondrial contact sites: their role in energy metabolism and apoptosis. Biochim Biophys Acta 1762: 148–163. doi:https://doi.org/10.1016/j.bbadis.2005.09.007. PubMed: 16324828.
- 4. Haworth RA, Hunter DR (2000) Control of the mitochondrial permeability transition pore by high-affinity ADP binding at the ADP/ATP translocase in permeabilized mitochondria. J Bioenerg Biomembr 32: 91–96. PubMed: 11768766.
- 5. Lemasters JJ, Theruvath TP, Zhong Z, Nieminen AL (2009) Mitochondrial calcium and the permeability transition in cell death. Biochim_Biophys_Acta 1787: 1395–1401.
- 6. Halestrap AP (2010) A pore way to die: the role of mitochondria in reperfusion injury and cardioprotection. Biochem Soc Trans 38: 841–860. doi:https://doi.org/10.1042/BST0380841. PubMed: 20658967.
- 7. Baines CP (2009) The mitochondrial permeability transition pore and ischemia-reperfusion injury. Basic Res Cardiol 104: 181–188. doi:https://doi.org/10.1007/s00395-009-0004-8. PubMed: 19242640.
- 8. Di Lisa F, Carpi A, Giorgio V, Bernardi P (2011) The mitochondrial permeability transition pore and cyclophilin D in cardioprotection. Biochim Biophys Acta 1813: 1316–1322. doi:https://doi.org/10.1016/j.bbamcr.2011.01.031. PubMed: 21295622.
- 9. Leung AW, Halestrap AP (2008) Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore. Biochim Biophys Acta 1777: 946–952. PubMed: 18407825.
- 10. Giorgio V, Soriano ME, Basso E, Bisetto E, Lippe G et al. (2010) Cyclophilin D in mitochondrial pathophysiology. Biochim Biophys Acta 1797: 1113–1118. PubMed: 20026006.
- 11. Juhaszova M, Wang S, Zorov DB, Nuss HB, Gleichmann M et al. (2008) The identity and regulation of the mitochondrial permeability transition pore: where the known meets the unknown. Ann N Y Acad Sci 1123: 197–212. doi:https://doi.org/10.1196/annals.1420.023. PubMed: 18375592.
- 12. Halestrap AP, Connern CP, Griffiths EJ, Kerr PM (1997) Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury. Mol Cell Biochem 174: 167–172. doi:https://doi.org/10.1023/A:1006879618176. PubMed: 9309682.
- 13. Giorgio V, Von Stockum S, Antoniel M, Fabbro A, Fogolari F et al. (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci U S A 110: 5887–5892. doi:https://doi.org/10.1073/pnas.1217823110. PubMed: 23530243.
- 14. Giorgio V, Bisetto E, Soriano ME, Dabbeni-Sala F, Basso E et al. (2009) Cyclophilin D modulates mitochondrial F0F1-ATP synthase by interacting with the lateral stalk of the complex. J Biol Chem 284: 33982–33988. doi:https://doi.org/10.1074/jbc.M109.020115. PubMed: 19801635.
- 15. McStay GP, Clarke SJ, Halestrap AP (2002) Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. Biochem J 367: 541–548. doi:https://doi.org/10.1042/BJ20011672. PubMed: 12149099.
- 16. Carafoli E (2010) The fateful encounter of mitochondria with calcium: how did it happen? Biochim Biophys Acta 1797: 595–606. doi:https://doi.org/10.1016/j.bbabio.2010.03.024. PubMed: 20385096.
- 17. Chinopoulos C, Adam-Vizi V (2010) Mitochondrial Ca2+ sequestration and precipitation revisited. FEBS J 277: 3637–3651. doi:https://doi.org/10.1111/j.1742-4658.2010.07755.x. PubMed: 20659160.
- 18. Saito A, Castilho RF (2010) Inhibitory effects of adenine nucleotides on brain mitochondrial permeability transition. Neurochem Res 35: 1667–1674. doi:https://doi.org/10.1007/s11064-010-0228-x. PubMed: 20652632.
- 19. Nadtochiy SM, Tompkins AJ, Brookes PS (2006) Different mechanisms of mitochondrial proton leak in ischaemia/reperfusion injury and preconditioning: implications for pathology and cardioprotection. Biochem J 395: 611–618. doi:https://doi.org/10.1042/BJ20051927. PubMed: 16436046.
- 20. Rehncrona S, Mela L, Siesjö BK (1979) Recovery of brain mitochondrial function in the rat after complete and incomplete cerebral ischemia. Stroke 10: 437–446. doi:https://doi.org/10.1161/01.STR.10.4.437. PubMed: 505482.
- 21. Hoffman DL, Salter JD, Brookes PS (2007) Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling. Am J Physiol Heart Circ Physiol 292: H101–H108. doi:https://doi.org/10.1152/ajpheart.00699.2006. PubMed: 16963616.
- 22. Endlicher R, Kriváková P, Rauchová H, Nůsková H, Cervinková Z et al. (2009) Peroxidative damage of mitochondrial respiration is substrate-dependent. Physiol Res 58: 685–692. PubMed: 19093725.
- 23. Hunter DR, Haworth RA (1979) The Ca2+-induced membrane transition in mitochondria. I. The protective mechanisms. Arch Biochem Biophys 195: 453–459. doi:https://doi.org/10.1016/0003-9861(79)90371-0. PubMed: 383019.
- 24. Haworth RA, Hunter DR (1979) The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch Biochem Biophys 195: 460–467. doi:https://doi.org/10.1016/0003-9861(79)90372-2. PubMed: 38751.
- 25. Hunter DR, Haworth RA (1979) The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release. Arch Biochem Biophys 195: 468–477. doi:https://doi.org/10.1016/0003-9861(79)90373-4. PubMed: 112926.
- 26. Griffiths EJ, Ocampo CJ, Savage JS, Stern MD, Silverman HS (2000) Protective effects of low and high doses of cyclosporin A against reoxygenation injury in isolated rat cardiomyocytes are associated with differential effects on mitochondrial calcium levels. Cell Calcium 27: 87–95. doi:https://doi.org/10.1054/ceca.1999.0094. PubMed: 10756975.
- 27. Klingenberg M (2008) The ADP and ATP transport in mitochondria and its carrier. Biochim Biophys Acta 1778: 1978–2021. doi:https://doi.org/10.1016/j.bbamem.2008.04.011. PubMed: 18510943.
- 28. Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J et al. (2004) The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore. Nature 427: 461–465. doi:https://doi.org/10.1038/nature02229. PubMed: 14749836.
- 29. Leung AWC, Halestrap AP (2008) Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1777: 946–952. Available online at: https://doi.org/10.1016/j.bbabio.2008.03.009
- 30. Bonora M, Bononi A, De Marchi E, Giorgi C, Lebiedzinska M et al. (2013) Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition. Cell Cycle 12: 674–683. doi:https://doi.org/10.4161/cc.23599. PubMed: 23343770.
- 31. Michailova A, McCulloch A (2001) Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte. Biophys J 81: 614–629. doi:https://doi.org/10.1016/S0006-3495(01)75727-X. PubMed: 11463611.
- 32. CARAFOLI E, Rossi CS, LEHNINGER AL (1965) UPTAKE OF ADENINE NUCLEOTIDES BY RESPIRING MITOCHONDRIA DURING ACTIVE ACCUMULATION OF CA++ AND PHOSPHATE. J Biol Chem 240: 2254–2261. PubMed: 14299656.
- 33. Traba J, Del Arco A, Duchen MR, Szabadkai G, Satrústegui J (2012) SCaMC-1 promotes cancer cell survival by desensitizing mitochondrial permeability transition via ATP/ADP-mediated matrix Ca(2+) buffering. Cell Death Differ 19: 650–660. doi:https://doi.org/10.1038/cdd.2011.139. PubMed: 22015608.
- 34. Turrens JF (2003) Mitochondrial formation of reactive oxygen species. J Physiol (Lond) 552: 335–344. doi:https://doi.org/10.1113/jphysiol.2003.049478.
- 35. Treberg JR, Quinlan CL, Brand MD (2010) Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production--a correction using glutathione depletion. FEBS J 277: 2766–2778. doi:https://doi.org/10.1111/j.1742-4658.2010.07693.x. PubMed: 20491900.
- 36. Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30: 1191–1212. doi:https://doi.org/10.1016/S0891-5849(01)00480-4. PubMed: 11368918.
- 37. Nieminen AL, Byrne AM, Herman B, Lemasters JJ (1997) Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species. Am J Physiol 272: C1286–C1294. PubMed: 9142854.
- 38. Sies H, Moss KM (1978) A role of mitochondrial glutathione peroxidase in modulating mitochondrial oxidations in liver. Eur J Biochem 84: 377–383. doi:https://doi.org/10.1111/j.1432-1033.1978.tb12178.x. PubMed: 25178.
- 39. Olafsdottir K, Reed DJ (1988) Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment. Biochim Biophys Acta 964: 377–382. doi:https://doi.org/10.1016/0304-4165(88)90038-4. PubMed: 3349102.
- 40. Hurd TR, Costa NJ, Dahm CC, Beer SM, Brown SE et al. (2005) Glutathionylation of mitochondrial proteins. Antioxid Redox Signal 7: 999–1010. doi:https://doi.org/10.1089/ars.2005.7.999. PubMed: 15998254.
- 41. Taylor ER, Hurrell F, Shannon RJ, Lin T-K, Hirst J et al. (2003) Reversible glutathionylation of complex I increases mitochondrial superoxide formation. J Biol Chem 278: 19603–19610. doi:https://doi.org/10.1074/jbc.M209359200. PubMed: 12649289.
- 42. Fratelli M, Demol H, Puype M, Casagrande S, Villa P et al. (2003) Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells. Proteomics 3: 1154–1161. doi:https://doi.org/10.1002/pmic.200300436. PubMed: 12872216.
- 43. Garcia J, Han D, Sancheti H, Yap L-P, Kaplowitz N et al. (2010) Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates. J Biol Chem 285: 39646–39654. doi:https://doi.org/10.1074/jbc.M110.164160. PubMed: 20937819.
- 44. Odin JA, Huebert RC, Casciola-Rosen L, LaRusso NF, Rosen A (2001) Bcl-2-dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis. J Clin Invest 108: 223–232. doi:https://doi.org/10.1172/JCI10716. PubMed: 11457875.
- 45. Nulton-Persson AC, Starke DW, Mieyal JJ, Szweda LI (2003) Reversible inactivation of alpha-ketoglutarate dehydrogenase in response to alterations in the mitochondrial glutathione status. Biochemistry 42: 4235–4242. doi:https://doi.org/10.1021/bi027370f. PubMed: 12680778.
- 46. Nulton-Persson AC, Szweda LI (2001) Modulation of mitochondrial function by hydrogen peroxide. J Biol Chem 276: 23357–23361. doi:https://doi.org/10.1074/jbc.M100320200. PubMed: 11283020.
- 47. Metelkin E, Demin O, Kovács Z, Chinopoulos C (2009) Modeling of ATP-ADP steady-state exchange rate mediated by the adenine nucleotide translocase in isolated mitochondria. FEBS J 276: 6942–6955. doi:https://doi.org/10.1111/j.1742-4658.2009.07394.x. PubMed: 19860824.
- 48. Chinopoulos C, Adam-Vizi V (2010) Mitochondria as ATP consumers in cellular pathology. Biochim Biophys Acta 1802: 221–227. doi:https://doi.org/10.1016/j.bbadis.2009.08.008. PubMed: 19715757.
- 49. Wu KLH, Hsu C, Chan JYH (2009) Nitric oxide and superoxide anion differentially activate poly(ADP-ribose) polymerase-1 and Bax to induce nuclear translocation of apoptosis-inducing factor and mitochondrial release of cytochrome c after spinal cord injury. J Neurotrauma 26: 965–977. doi:https://doi.org/10.1089/neu.2008.0692. PubMed: 19473058.