Mitochondria play a critical role in mediating the cellular response to oxidants formed during acute and chronic cardiac dysfunction. It is widely assumed that, as cells are subjected to stress, mitochondria are capable of drawing upon a ‘reserve capacity’ which is available to serve the increased energy demands for maintenance of organ function, cellular repair or detoxification of reactive species. This hypothesis further implies that impairment or depletion of this putative reserve capacity ultimately leads to excessive protein damage and cell death. However, it has been difficult to fully evaluate this hypothesis since much of our information about the response of the mitochondrion to oxidative stress derives from studies on mitochondria isolated from their cellular context. Therefore the goal of the present study was to determine whether ‘bioenergetic reserve capacity’ does indeed exist in the intact myocyte and whether it is utilized in response to stress induced by the pathologically relevant reactive lipid species HNE (4-hydroxynonenal). We found that intact rat neonatal ventricular myocytes exhibit a substantial bioenergetic reserve capacity under basal conditions; however, on exposure to pathologically relevant concentrations of HNE, oxygen consumption was increased until this reserve capacity was depleted. Exhaustion of the reserve capacity by HNE treatment resulted in inhibition of respiration concomitant with protein modification and cell death. These data suggest that oxidized lipids could contribute to myocyte injury by decreasing the bioenergetic reserve capacity. Furthermore, these studies demonstrate the utility of measuring the bioenergetic reserve capacity for assessing or predicting the response of cells to stress.
- extracellular flux
- lipid peroxidation
- oxidative stress
Defining the role of mitochondria in various cardiovascular pathologies is currently an area of great interest, with many studies focusing on the properties of the organelle isolated from diseased or stressed cardiac cells. These studies now point to the importance of understanding how changes in isolated mitochondria translate to changes in bioenergetic events that take place in the myocardium during ischaemia/reperfusion and heart failure. Cardiac tissue is rich in mitochondria, which are capable of dynamically responding to energy demand for increased work. This oxidative phenotype allows for rapid and substantial ATP production for cardiac function. It is clear from experiments using 31P-NMR that, even under an increased work load in the physiological range, mitochondria appear to have a substantial ‘reserve capacity’ which is depleted under conditions of severe stress such as pressure overload or ischaemia [1,2]. However, this concept has proved technically difficult to examine using direct indices of mitochondrial function in intact myocytes.
Results from mitochondria isolated from hearts subject to stress typically show changes in the activity of the respiratory chain, but the functional impact remains unclear once the organelles are removed from their cellular context [3,4]. It is frequently the case that the activity of respiratory chain complexes of isolated mitochondria such as complex I are decreased in the diseased heart [5,6]. It is challenging to extend these changes to the cellular setting since the control over metabolism is lost in isolated mitochondria. For example, it is known that the maximal capacity for oxidative phosphorylation is higher than that used under normal conditions . This raises the question of the role of this reserve capacity and leads to the hypothesis that it is required by cells to respond to stress and that pathological events occur when this bioenergetic reserve is depleted.
The requirement of the reserve capacity for the response to stress, such as occurs in the ischaemic and failing heart, has not been examined in intact cardiovascular cells or tissues. Since the diseased heart is associated with increased oxidative stress, exposure of cells to reactive species generated during pathology can be used as a model of pathology and to test the role of oxidative stress in myocyte dysfunction. The significance of these reactive species is revealed in studies showing that overexpression of antioxidant enzymes such as Mn-SOD (superoxide dismutase) , catalase , extracellular-SOD  or glutathione peroxidase  protects the heart from ischaemia/reperfusion injury. Moreover, partial deficiency of Mn-SOD  or the complete absence of glutathione peroxidase  or CuZn-SOD  renders the heart more sensitive to injury. Interestingly, volume and pressure overload [14–16] as well as ischaemia [17,18] result in an increased ability of myocytes to consume oxygen, which might suggest a mechanism for further depletion of the bioenergetic reserve capacity in an environment where oxygen availability may be already limiting.
Secondary products of oxidative stress such as HNE (4-hydroxynonenal) are normally detoxified by energy-requiring processes; however, under pathological conditions, these detoxification pathways fail, leading to accumulation of oxidized lipids  that can damage key proteins in the mitochondrial respiratory chain. In humans and animal models, downstream products of oxidative stress such as oxidized lipids are abundant in the ischaemic [19,20] and failing [21,22] heart. Therefore, the generation of lipid peroxidation products capable of reacting with cellular nucleophiles could be primary instigators of tissue injury . The α,β-unsaturated aldehydes (such as HNE) are likely to be the most significant because they modify proteins which affect energy production [24–30] and cell death pathways [31,32]. This is corroborated by studies showing that activation of a key enzyme required for the mitochondrial detoxification of HNE  protects the heart from injury [33,34].
These findings raise two important questions. (i) Does bioenergetic reserve capacity exist in cardiac myocytes? (ii) Does it modulate the response to stress-associated pathology? The recent availability of technology which allows for the non-invasive measurement of mitochondrial respiration and glycolysis offers the opportunity to address these questions. Therefore we hypothesized that maintenance of mitochondrial function and the availability of a bioenergetic reserve capacity is critical to combat oxidative stress and that, when exceeded, protein damage and cell death occurs. To test this hypothesis, we used an emerging technology, high-throughput XF (extracellular flux) analysis, to quantify the bioenergetic changes that occur in intact cardiac myocytes exposed to HNE. By measuring XF, we were able to measure oxygen consumption and proton production, indicative of oxidative phosphorylation and glycolysis respectively, in intact NRVMs (neonatal rat ventricular myocytes) exposed to HNE.
Our results from the present study support the hypothesis that pathologically relevant concentrations of oxidized lipids exhaust the reserve capacity of mitochondria. When this capacity is depleted, cellular injury occurs accompanied by decreased mitochondrial oxygen consumption, decreased efficiency due to proton leak and increased protein–HNE adduct formation. These results directly demonstrate the presence of a bioenergetic reserve capacity in intact myocytes. Furthermore, these findings suggest that oxidized lipids such as HNE, which accumulate in the heart during ischaemia and heart failure, could cause, accelerate or worsen myocardial injury by diminishing this reserve capacity.
MATERIALS AND METHODS
Reagent HNE was obtained from Calbiochem. All materials and reagents for the XF assays were from Seahorse Biosciences. Nonanal, oligomycin, thiazoyl blue tetrazolium, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and antimycin A were from Sigma. Secondary HRP (horseradish peroxidase)-linked antibodies and antibodies against actin were from Cell Signaling Technology. Anti-protein–HNE antibodies were a gift from Dr Sanjay Srivastava (Division of Cardiology, University of Louisville, Louisville, KY, U.S.A). ECL® (enhanced chemiluminescence) Plus reagents were from GE Healthcare.
NRVM primary cultures
All animal experiments were approved by the University of Alabama Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85–23, Revised 1996). Primary cultures of NRVMs were obtained from 2–3-day-old neonatal Sprague–Dawley rats and were cultured as described previously . NRVMs were seeded at 75000 cells/well on to collagen-coated Seahorse Bioscience V7 culture plates in growth medium containing 15% FBS (fetal bovine serum) on the first day. The next day, medium was replaced, and cells were grown in the culture growth medium without FBS. Within 1–2 days of isolation, a confluent monolayer of spontaneously beating NRVMs formed, and cells were used as described below.
An XF24 Analyzer (Seahorse Biosciences) was used to measure bioenergetic function in intact NRVMs. The XF24 creates a transient 7 μl chamber in specialized microplates that allows for OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) or PPR (proton production rate) to be monitored in real time . For all bioenergetic measurements, the culture medium was changed 1 h prior to the assay run to unbuffered DMEM (Dulbecco's modified Eagle's medium, pH 7.4) supplemented with 4 mM L-glutamine (Gibco). First, the optimum number of cells needed for these experiments was determined. NRVMs were seeded to a density of 25000, 50000 or 75000 cells/well. Oxygen consumption in these cells was linear with respect to cell number within this range (Figure 1A), and a seeding density of 75000 cells/well was chosen for the remainder of the experiments.
Next, an assay was developed to measure indices of mitochondrial function. Oligomycin, FCCP and antimycin A were injected sequentially through ports in the Seahorse Flux Pak cartridges to final concentrations of 1 μg/ml, 1 μM and 10 μM respectively. This allowed determination of the basal level of oxygen consumption, the amount of oxygen consumption linked to ATP production, the level of non-ATP-linked oxygen consumption (proton leak), the maximal respiration capacity and the non-mitochondrial oxygen consumption. As shown in Figure 1(B), three basal OCR measurements were recorded prior to injection of oligomycin. After mixing and recording the oligomycin-insensitive OCR, FCCP was injected and another OCR measurement was recorded. The OCR measured after FCCP injection represents the maximal capacity that cells have to reduce oxygen under the experimental conditions. Finally, antimycin A was injected to inhibit the flux of electrons through complex III, and thus no oxygen was further consumed at cytochrome c oxidase. The remaining OCR determined after this treatment is primarily non-mitochondrial and could have been due to cytosolic oxidase enzymes.
Protein–HNE and GSH measurements
Protein–HNE adducts were detected using a polyclonal anti-protein HNE antibody, as described in . Briefly, cells were lysed in the 24-well XF plates using 20 μl/well of lysis buffer containing 20 mM Hepes, pH 7.0, 1 mM DTPA (diethylenetriaminepenta-acetic acid), 1% Nonidet P40, 0.1% SDS and protease inhibitor cocktail (Roche). The lysates were then added to SDS Laemmli Buffer, and the proteins were separated on 10% polyacrylamide/SDS gels. After transfer on to PVDF membranes, HNE-modified proteins were detected using anti-protein–HNE primary and HRP-linked anti-rabbit secondary antibodies by chemifluorescence on a Typhoon Variable Mode Imager (GE Healthcare). Relative levels of protein–HNE adducts were quantified by densitometry using ImageQuantTL software (GE Healthcare). Total glutathione (GSH+GSSG) was measured in cell lysates with and without treatment with HNE using the Tietze recycling assay .
Cell death assays
Cell viability was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay as described previously  with the following modifications. NRVMs were seeded as described above into V7 culture plates at 75000 cells/well. At 8 and 16 h after HNE treatment, the assay medium was replaced with medium containing 0.4 mg/ml thiazoyl blue tetrazolium. The cells were allowed to incubate in a non-CO2 incubator at 37°C for an additional 2 h. The medium was removed, and the resulting formazan crystals were solubilized in 250 μl of DMSO. The absorbance was read at 550 nm and the results are expressed as a percentage of control.
Data are reported as means±S.E.M. Comparisons between two groups were performed with unpaired Student's t tests. Comparisons among multiple groups or between two groups at multiple time-points were performed by either one-way or two-way ANOVA, as appropriate. P<0.05 was considered statistically significant.
Protein HNE adducts and cell death increase with HNE treatment
NRVMs were seeded at 75000 cells/well and then treated with either HNE or the non-electrophilic C9 lipid analogue of HNE, nonanal, for 2 h. For these experiments, we used concentrations of HNE that have been shown to occur in the ischaemic and failing heart [19,21,40,41]. The cells were harvested and proteins were separated by SDS/PAGE followed by Western blotting with anti-protein–HNE antibodies. As shown in Figures 2(A) and 2(B), HNE adducts accumulated in a concentration-dependent manner. The non-protein reactive analogue of HNE, nonanal, was used as a structural control and did not promote the formation of protein–HNE adducts. Additionally, cell death as a result of HNE treatment was examined at 8 and 16 h by the MTT assay. HNE exhibited both a concentration- and time-dependent effect on NRVM viability (Figure 2C). HNE can be metabolized by a number of enzymes in the cell, including the GSH-dependent transferases [19,42]. Accordingly, we measured total glutathione (GSH+GSSG) levels in the lysates of cells treated with HNE for 90 min (Figure 2D). As anticipated, HNE treatment depleted glutathione, and this was maximal at the lowest concentration of HNE tested.
Effect of HNE on aerobic metabolism in intact neonatal cardiomyocytes
To determine if bioenergetic derangements preceded cell death due to HNE, we assessed oxygen consumption in NRVMs treated under the same conditions as shown in Figure 2. After basal oxygen consumption measurements, HNE was injected to give final concentrations of 0–30 μM. Control wells received vehicle (ethanol) or the structural control for HNE, nonanal (10 μM), in DMEM. As shown in Figures 3(A) and 3(B), HNE initially increased OCR in a concentration-dependent manner. Myocytes treated with the lowest concentration of HNE tested (5 μM) resulted in a steady increase in the rate of mitochondrial oxygen consumption which plateaued after approx. 150 min of HNE exposure. Exposure to higher concentrations of HNE (10–30 μM) resulted in a maximal OCR that declined thereafter. Apparent maximal increases in OCR were reached with 10, 20 and 30 μM HNE approx. 130, 70 and 35 min after injection respectively. Interestingly, the rate of increase in OCR (i.e. the slope of the initial stimulation of OCR by HNE) increased linearly with HNE concentration (Figure 3B). Nonanal was used as a structural control for non-electrophilic effects of HNE and did not affect oxygen consumption by the myocytes (results not shown).
To quantify the overall increase in the amount of oxygen consumed by the myocytes due to each treatment, the OCR area under the curve for each group was calculated by multiplying the group average by the time interval for that rate and subtracting the baseline rate. The resulting value can be expressed in pmoles of oxygen consumed during the assay after injection of vehicle, nonanal or HNE. As shown in Figure 3(C), exposure of the myocytes to HNE at 0–20 μM concentrations resulted in up to approx. 20 nmoles of oxygen consumed during the course of the experiment. The highest concentration of HNE used, i.e. 30 μM, showed no overall increase in oxygen consumption. At this concentration, OCR increased transiently, followed by a rapid decrease in oxygen consumption that was well below baseline (Figure 3A).
Effects of HNE on glycolysis
In addition to OCR, the XF assay also allowed the measurement of protons that were produced by the cells, which reflects lactate production and is therefore an index of glycolysis [36,43]. Figure 4(A) shows the ECAR profile of control myocytes and myocytes treated with 0–30 μM HNE. Similar to OCR, HNE increased the ECAR in a concentration- and time-dependent manner. Analogous to the OCR results (Figure 3A), ECAR showed a biphasic response in the 10 and 20 μM HNE groups, indicating severe damage to bioenergetic components at later time points. As shown in Figure 4(B), the initial rate of increase in ECAR was linear and dependent on the HNE concentration. Similar to that shown for OCR in Figure 3(C), the area under the curve of the PPR was calculated to determine the overall increase in proton production over the time course of the experiment. HNE treatment resulted in an increase in proton production, indicative of an increase in glycolysis, over the course of the experiment (Figure 4C). The maximal rate of proton production occurred in cells treated with 7.5–10 μM HNE.
Identification of mitochondrial defects incurred by HNE
Approx. 70 min after HNE addition (indicated by the dotted line in Figure 5B), the ECAR and OCR were used to obtain a ‘metabolic image’ of the cells in each treatment group. The image is divided into four quadrants, which relate the relative activities of the glycolytic and aerobic metabolism in response to HNE. As shown in Figure 5(A), HNE concentration-dependently stimulates both glycolysis and the aerobic consumption of oxygen, consistent with an increased energy demand of the cells as they respond to this stress.
To determine the specific mitochondrial derangements that occur in response to HNE, we used the mitochondrial function assay described in Figure 1(B). For these experiments, myocytes treated with HNE to final concentrations of 0–20 μM were exposed to oligomycin, FCCP and antimycin A at the time points indicated (Figure 5B). The measurements taken after each injection were used to calculate the ATP-linked OCR, proton leak, reserve capacity and non-mitochondrial OCR at 2 h after the addition of HNE. As shown in Figure 6(A), OCR was stimulated in response to HNE by 263.1±22.5 pmol/min above the baseline levels. Upon the addition of oligomycin, Figure 6(B) shows that the oligomycin-insensitive OCR in the presence of HNE increased significantly by up to 2.5-fold. To determine the individual parameters of mitochondrial function, the data shown in Figure 5(B) were analysed as described in Figure 1. Proton leak was increased by HNE in a concentration-dependent manner. Interestingly, the rate of ATP-linked oxygen consumption showed a biphasic response (Figure 6D), where it increased by approx. 3-fold with 10 μM HNE and decreased at 20 μM. These results suggest that HNE increases OCR both by increasing proton leak (thereby decreasing mitochondrial efficiency) and by increasing energy demand.
Next, the maximal respiratory rate was determined from the FCCP-stimulated rate. As shown in Figure 5(B), HNE at 0–10 μM concentrations had no effect on this maximal respiratory rate. However, concentrations of HNE in excess of 10 μM resulted in an inhibition of oxygen consumption even after FCCP addition, suggesting overt damage to mitochondrial respiratory complexes (Figure 5B). The reserve capacity was calculated based on the OCR immediately prior to oligomycin addition and the maximal respiratory rate. As shown in Figure 6(E), HNE decreased the mitochondrial reserve capacity in a concentration-dependent manner consistent with damage to the respiratory chain associated with increased protein adducts (Figure 2A). The rate of oxygen consumption due to non-mitochondrial sources was determined in the presence of HNE using antimycin A and was unchanged (Figures 5B and 6F).
Using XF analyses, we evaluated the effects of the electrophilic lipid HNE on bioenergetic function in intact myocytes. We show for the first time that oxidized lipids, at concentrations that accumulate under pathophysiological conditions in the heart, increase cardiomyocyte oxygen consumption and deplete the bioenergetic reserve capacity. We found that HNE stimulated the rate of cellular oxygen consumption in a concentration-dependent manner. This increase in oxygen consumption was due to both increased bioenergetic demand placed on the cardiac myocytes and decreased mitochondrial efficiency (i.e. proton leak). Glycolytic flux was also increased in response to HNE treatment, underscoring the integrated nature of the metabolic response to energy demand and expenditure in the presence of reactive lipid species. The HNE-induced increase in oxygen consumption was not compensated by an increase in the maximal respiratory capacity of the myocytes; consequently, HNE treatment resulted in depletion of the bioenergetic reserve capacity, overt respiratory failure and cell death.
In the heart, mitochondria make up at least 20% of the myocyte volume [44,45] and provide the unremitting energy required for contraction. Aerobic respiration is therefore indispensable to sustain cardiac function and viability. At rest, the heart consumes up to 0.15 ml of oxygen/min per g of tissue, which increases several fold with vigorous exercise by calling upon the reserve capacity of the mitochondria and glycolysis . Interestingly, the diseased heart requires more oxygen to meet energy needs, which has led to the hypothesis that a state of energy starvation may underlie myocardial pathology . In support of this concept, myocardial oxygen uptake is increased 2-fold in patients with left ventricular hypertrophy over normal subjects ; there is also an increase in the myocardial uptake of oxygen in experimental hypertrophy, congestive heart failure and diabetes [15,16,48]. Furthermore, the post-ischaemic heart uses more oxygen than the pre-ischaemic heart , and myocytes subjected to hypoxia/reoxygenation have an increased demand for oxygen . It is reasonable to conclude, therefore, that bioenergetic dysfunction and the imbalance of oxygen supply against demand is a basic defect in myocardial pathologies. However, the mechanisms underlying this increased demand for oxygen in the diseased heart are unknown. Increased tissue levels of reactive species such as HNE are associated with both acute and chronic cardiac disease [19–21,41]. If, as shown in the present study, such reactive species increase the rate of oxygen consumption and decrease bioenergetic reserve capacity in the diseased heart, this could account for the contractile dysfunction associated with pathology.
Although the idea that ROS (reactive oxygen species) play a fundamental role in cardiovascular disease is well accepted, the molecular mechanisms by which oxidative stress cause cardiac injury are not well defined. ROS can damage cellular components by direct oxidation; however, given the susceptibility of polyunsaturated fatty acids to oxidative attack, there is a strong rationale for invoking the involvement of secondary products of lipid oxidation such as HNE and acrolein in ROS-related injury. Indeed, adducts of such oxidized lipids with proteins have been detected in the diseased heart [19,20,28,30,41,49–51] and myocytes exposed to similar compounds capable of modifying proteins promote a phenotype akin to myocardial stunning . It remains unclear, however, whether oxidized lipids or their protein adducts are footprints of unquenched free radicals or if they indeed cause derangements in signalling or bioenergetics.
Recent studies showing that activation of enzymes critical for HNE detoxification protect the heart from ischaemic injury suggest that electrophiles are significant contributors to myocardial damage [33,34]. Many studies have shown that HNE damages electron transport chain complexes [3,25–30], affecting both respiration and critical events such as calcium-induced permeability transition [32,53]. These studies, however, were limited by their usage of isolated mitochondrial preparations. Although useful for understanding putative defects caused by HNE, experiments with isolated mitochondria are generally performed under saturating substrate conditions and are outside their normal intracellular environment. This removes all influence of cell signaling, ion fluxes and changes in intracellular substrate metabolism (e.g. glycolysis) on bioenergetic regulation. In contrast with the HNE-induced increase in oxygen consumption found in the present study, most studies to date report that HNE primarily inhibits mitochondrial respiration. This is probably due to the fact that the majority of studies have assessed HNE-induced changes in respiration using isolated mitochondrial preparations [19,25–27].
In the present study we demonstrate that HNE increases oxygen consumption and bioenergetic demand and decreases mitochondrial efficiency (Figures 3–6). These findings suggest that oxidized lipids contribute to myocardial injury by promoting an energy crisis. In support of this concept, we show that HNE at lower concentrations initially increased the OCR (Figure 3), which was due to both increased proton leak and bioenergetic demand (Figure 6). Using the process described in Figure 1, we can assess the impact of HNE on key bioenergetic parameters in the cell. We have assumed for the purposes of this calculation that the oligomycin-insensitive OCR is attributable to proton leak in the basal condition. However, this is not strictly correct, since oligomycin has been shown to increase mitochondrial membrane potential and the resulting OCR is likely to be an upper estimate of the contribution from proton leak. Although oligomycin-dependent hyperpolarization has no impact on the conclusion that HNE increases proton leak (Figure 6C), it will affect the value of the OCR which we attribute to ATP synthesis, primarily through an underestimation of this parameter. Nevertheless, it is clear that a significant portion of the stimulation of the basal OCR was due to increased bioenergetic demand. The increased proton leak across the inner mitochondrial membrane may be mediated by uncoupling proteins or leakage through damaged respiratory complexes. In support of this, studies using isolated mitochondria have shown that HNE promotes uncoupling and mitochondrial proton leak [24,54].
The biphasic nature of the HNE-induced changes in oxygen consumption (Figures 3A and 5B) suggest at least two factors contributing to the effects of HNE. Initially, HNE increased the oxygen consumption rate in a concentration-dependent manner (Figure 3B). We propose that this is an initial adaptive response to cellular stress induced by HNE and does not involve damage to the respiratory chain. The second phase, where the OCR decreased at the higher concentrations of HNE, is probably due to damage to respiratory complexes. Another possibility is that endogenous substrates became limiting as OCR increased. This is unlikely since 20 and 30 μM HNE, which quickly reached a peak respiratory capacity in the cells (Figure 3A), resulted in a lower overall consumption of oxygen compared with myocytes treated with 5 and 10 μM HNE (Figure 3C) and decreased the ability of the cells to respire maximally when treated with the uncoupler, FCCP (Figure 5B). Furthermore, the concentrations of HNE that caused a transient breach of the reserve capacity (i.e. 10–30 μM HNE) were associated with substantially increased protein–HNE adducts, and this resulted in complete cell death (Figures 2A and 2B). As anticipated, glutathione was rapidly depleted by HNE and was maximal at the lowest concentration of HNE tested. Since the major bioenergetic changes induced by HNE occurred without a significant change in these repressed glutathione levels, it is unlikely that glutathione depletion plays a major role in the HNE-dependent bioenergetic dysfunction reported in the present study. From these results, therefore, it is likely that extensive formation of protein–HNE adducts caused damage to respiratory complexes that resulted in inhibition of electron transport.
As discussed above, HNE levels are substantially increased in multiple myocardial pathologies [19–21,41,51], suggesting that HNE promotes tissue damage and dysfunction by decreasing the bioenergetic reserve capacity. Interestingly, myocardial oxygen consumption in the pressure-overloaded heart is increased to the maximal dinitrophenol-stimulated rate during high workloads produced by catecholamine infusion , suggesting that reserve capacity is depleted in the diseased heart under conditions of stress. This is particular interesting since even low dose catecholamine treatment promotes lipid peroxidation and the formation of protein–HNE adducts [22,51]. Although of particular relevance to cardiovascular disease, reduced bioenergetic reserve capacity has also been recognized in neurodegeneration. Specifically, reduction in the spare respiratory capacity has been shown to regulate glutamate excitotoxicity in neurons [55,56]. Those studies suggested that dysfunction occurs when the ATP demand exceeds the maximal ATP supply put forth by glycolysis and oxidative phosphorylation, thereby implicating that the spare respiratory capacity is critical for maintaining ATP generation under conditions of increased demand . Similarly, we show that the ability of cardiomyocyte mitochondria to respire collapses when the maximal reserve capacity is depleted due to the increase in oxygen consumption by HNE (Figure 6C).
Taken as a whole, these results support the view that products of lipid peroxidation, in the concentration range reported under pathological conditions, contribute to myocardial injury by promoting bioenergetic stress. By evaluating mitochondrial function in intact cells, our findings reveal for the first time a dynamic response of cardiomyocytes to HNE, where oxygen consumption is increased through increased energy demand as well as via non-ATP-linked oxygen sinks (i.e. proton leak). Furthermore, these studies provide insight into a fundamental mechanism critical to the evolution of myocyte injury, namely the oxidized lipid-induced increase in oxygen consumption and depletion of the bioenergetic reserve capacity. Consequently, these findings have implications not only for our understanding of the pathophysiological processes underlying cardiac disease, but also in other disease states associated with increased oxidative stress.
Bradford Hill, Brian Dranka and Luyun Zou performed the experiments. Bradford Hill, Brian Dranka, John Chatham and Victor Darley-Usmar designed experiments and analysed the data. John Chatham and Victor Darley-Usmar provided financial support for the study. All authors contributed to the preparation of the manuscript.
This work was supported by the National Institutes of Health (NIH) [grant number ES10167] and Seahorse Biosciences (to V. D. U.), [grant numbers HL079364 and HL67464 (to J. C. C.)], and by NIH training grants [grant numbers T32 HL07457 (to B. G. H.) and T32 HL007918 (to B. P. D.)].
Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ECAR, extracellular acidification rate; FBS, fetal bovine serum; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HNE, 4-hydroxynonenal; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NRVM, neonatal rat ventricular myocyte; OCR, oxygen consumption rate; PPR, proton production rate; ROS, reactive oxygen species; SOD, superoxide dismutase; XF, extracellular flux
- © The Authors Journal compilation © 2009 Biochemical Society