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(Circulation. 2001;103:2617.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Mitochondria as Targets for Nitric Oxide–Induced Protection During Simulated Ischemia and Reoxygenation in Isolated Neonatal Cardiomyocytes

Roby D. Rakhit, MD, MRCP; Mart H. Mojet, PhD; Michael S. Marber, PhD, FRCP; Michael R. Duchen, PhD, MRCP

From the Departments of Cardiology, Kings College London, St Thomas’ Hospital (R.D.R., M.S.M.), and Physiology, University College London (M.H.M., M.R.D.), UK. The first 2 authors contributed equally to this work.

Correspondence to Michael R. Duchen, Department of Physiology, University College London, Gower St, London, WC1E 6BT, UK. E-mail m.duchen{at}ucl.ac.uk

	Abstract

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Background—As shown previously, exposure to NO donors initiates protective mechanisms in cardiomyocytes that persist after removal of the donor, a form of pharmacological preconditioning. Because NO also affects mitochondrial respiration, we studied the effect of NO on mitochondrial Ca²⁺ uptake.

Methods and Results—Neonatal rat ventricular myocytes in primary culture were exposed to 1 hour of simulated ischemia and 1 hour of reoxygenation (sI/R). Pretreatment with the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (1 mmol/L for 90 minutes), followed by washing and incubation for 10 to 30 minutes, reduced sI/R-induced cell death to 25.4% compared with control (propidium iodide exclusion assay, P<0.001). Short (10-second) exposures to SNAP reversibly suppressed mitochondrial respiration without a detectable change in mitochondrial potential. In contrast, treatment with SNAP for 90 minutes caused a modest but sustained mitochondrial depolarization, as judged by JC-1 fluorescence. SNAP pretreatment limited cellular Ca²⁺ overload during ischemia (fura-2 ratio rose to 226±40% versus 516±170% of baseline, n=5, P<0.05) and prevented loss of cell membrane integrity during reoxygenation. SNAP pretreatment also significantly reduced the ability of mitochondria to accumulate Ca²⁺ in the face of a similar cytosolic Ca²⁺ load (peak rhod-2 fluorescence 133±4% versus 166±7% of baseline at similar fluo-3 levels, P=0.0004, n=52 and 25, respectively).

Conclusions—Pretreatment with an NO donor induces a modest, sustained mitochondrial depolarization and protects cardiomyocytes from sI/R injury. The demonstrated reduction in mitochondrial Ca²⁺ uptake possibly reduces cytosolic Ca²⁺ overload, providing a likely mechanism for NO-induced protection.

Key Words: nitric oxide • cells • calcium • ischemia • reperfusion

	Introduction

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Cellular Ca²⁺ overload is an important determinant of ischemic myocardial injury.¹ As early as 1972, Shen and Jennings² studied myocardial injury and described mitochondrial swelling and prominent granular mitochondrial dense bodies attributable to Ca²⁺ deposits. Since then, mitochondrial Ca²⁺ overload has been thought to be critical in the pathogenesis of irreversible ischemic cell death. During ischemia, mitochondrial Ca²⁺ uptake limits the increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). As a result, the mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) rises in far greater proportion than [Ca²⁺]_c.³ Furthermore, cell studies suggest that the extent of rise in [Ca²⁺]_m during ischemia determines the likelihood of reoxygenation-induced hypercontracture and cell death.⁴ Therefore, limiting mitochondrial Ca²⁺ accumulation during ischemia may represent a potential therapeutic target.

NO is a regulator of cell physiology and may play an important role in cytoprotection, including protection during ischemia/reoxygenation injury mediated through the phenomenon of ischemic preconditioning.^{5 6 7} For example, using MTT bioreduction assay and LDH release in a cellular model, we showed that the protection against simulated ischemia-reoxygenation (sI/R) provided by a 90-minute period of sublethal simulated ischemia is prevented by the nonspecific NO synthase inhibitor N^G-monomethyl-L-arginine and mimicked by 90 minutes of incubation with 1 mmol/L S-nitroso-N-acetyl-penicillamine (SNAP), a slow-release NO donor.⁶

NO can exert dual effects on mitochondrial energetics in the heart through both reversible and irreversible inhibition of respiratory chain complexes.^{8 9} This interaction may result in depolarization of the mitochondrial inner membrane potential (_m),¹⁰ which in turn may affect subcellular Ca²⁺ homeostasis.¹¹ Therefore, we hypothesized that NO-induced modulation of mitochondrial function underlies the basis of the observed cytoprotection. In this study, we modified our cellular model of sI/R to study changes in [Ca²⁺] and mitochondrial function using fluorescence microscopy. Our data suggest that pretreatment with the slow-release NO donor SNAP results in a modest mitochondrial depolarization that outlasts the SNAP application and may protect cardiomyocytes against subsequent sI/R injury by limiting mitochondrial Ca²⁺ accumulation, providing a mechanism to account for NO-induced cardioprotection.

	Methods

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Isolation and Culture of Rat Ventricular Cardiomyocytes
Neonatal rat ventricular cardiomyocytes were prepared from 1- to 2-day-old Sprague-Dawley rats as described previously.¹² Briefly, neonatal rat ventricles were dispersed in a series of incubations at 37°C in HEPES-buffered salt solution containing 0.6 mg/mL pancreatin (Life Technologies, Inc) and 0.5 mg/mL collagenase (Worthington). To reduce fibroblast contamination to <5%, dispersed cells were preplated for 30 minutes, and the unattached cells were replated on gelatin-coated coverslips. Cells were cultured at 37°C in room air with 5% CO₂ in Dulbecco’s modified Eagle’s medium and Medium 199 (M199, at 4:1) supplemented with 10% horse serum, 5% FCS, and 100 U/mL penicillin/streptomycin for the first 24 hours (Life Technologies). Thereafter, cells were maintained in M199 supplemented with 1% FCS. Under these conditions, >80% of cells beat spontaneously for the duration of the experiment. Experiments were performed between days 2 and 3 in culture.

Materials, Solutions, and Dye Loading Protocols
Ischemia buffer was composed of (in mmol/L) NaCl 118, NaHCO₃ 24, KCl 16, KH₂PO₄ 1, CaCl₂ 2.5, MgCl₂ 1.2, Na⁺ EDTA 0.5, and Na⁺ lactate 20, pH 6.2. Control buffer (CB) had the same composition, except KCl was 4 mmol/L, lactate was replaced by 2 mmol/L Na⁺ pyruvate and 10 mmol/L D-glucose, and pH was 7.4. HEPES-buffered saline (HBS) contained (in mmol/L) NaCl 156, KCl 3, MgSO₄ 2, KH₂PO₄ 1.25, CaCl₂ 2, HEPES 10, and D-glucose 10, with pH adjusted to 7.35 with NaOH. PBS contained (in mmol/L) NaCl 155, Na₂HPO₄ 5, and KH₂PO₄ 1.5. Drugs included SNAP (Calbiochem), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; Sigma), sodium cyanide (CN^-; BDH), caffeine, and thapsigargin (Sigma).

Cells were loaded at room temperature with fluorescent indicator dye (Molecular Probes) after pretreatment with SNAP (unless otherwise indicated) as follows: tetramethylrhodamine ethyl ester (TMRE), 3 µmol/L for 15 minutes. Under these conditions, loss of _m causes a dequench of fluorescence and an increase in signal. JC-1, 10 µmol/L for 10 minutes at 37°C. JC-1 is a dual-emission indicator; monomers fluoresce at 530 nm, and accumulation in mitochondria results in formation of "J" complexes, which emit at 590 nm.^{13 14} Because monomer fluorescence is enhanced in a lipid environment¹³ and is variable between cells, we used the 590-nm signal only to indicate a loss of _m. Fura-2, 5 µmol/L of the AM ester for 30 minutes. Rhod-2, 10 µmol/L of the AM ester for 30 minutes at 37°C. Loading was carried out before SNAP pretreatment to allow rhod-2/AM to be partitioned and hydrolyzed in mitochondria. Fluo-3, 5 µmol/L of the AM ester for 30 minutes. After dye loading, cells were washed with HBS.

Experimental Setup for Fluorescence Imaging of Cardiomyocytes
Cardiomyocytes cultured on coverslips were placed into a purpose-built chamber on the stage of an inverted epifluorescence microscope (described in detail by Mojet et al¹⁵ ). Cells were perfused at 10 mL/min and 34°C to 36°C. Drugs were applied by pressure ejection with glass pipettes with an inner diameter of 2 µm, positioned within 100 µm of the cell. Fluorescence was elicited by illumination with a 100-W xenon arc lamp via a spinning filter wheel (Cairn Research) fitted with appropriate 10-nm bandpass filters. During sI/R experiments, cells were illuminated by opening a shutter for 2 seconds every minute to reduce photodynamic damage. Fluorescence signals were measured by use of 2 photomultiplier tubes after passing through bandpass filters appropriate for the fluorescence indicators used.

Confocal images were obtained on an LSM 510 (Carl Zeiss, Inc). Fluo-3 and rhod-2 were excited with the 488-nm argon laser line, and fluorescence was collected at 505 to 550 nm (fluo-3), at >585 nm (rhod-2), or with both channels (JC-1).

Experimental Protocols
Simulated Ischemia/Reoxygenation
An sI/R protocol was developed for microscopic study of isolated, superfused cardiomyocytes. Cells were subjected to 1 hour of ischemia by superfusion with ischemia buffer equilibrated with 95% argon/5% CO₂ and supplemented shortly before the experiment with sodium dithionite (final concentration 750 µmol/L) to reach PO₂ values <130 Pa (1 mm Hg), as assessed with a carbon fiber electrode.¹⁵ Subsequently, cells were reoxygenated for 1 hour with CB, equilibrated with 75% N₂, 20% O₂, and 5% CO₂.

Cell Viability Assay
After reoxygenation, cells were loaded with propidium iodide (15 µmol/L; Molecular Probes), which only stains the nuclei of membrane-compromised cells. To facilitate cell counting, Hoechst 33342 (10 µmol/L) was included to stain the nuclei of all cells. Viability (% cell death) is presented as the number of dead (propidium iodide–stained) cells/total number of cells (Hoechst 33342–stained). SNAP-induced reduction in cell death was then calculated from (SNAP-pretreated-perfusion control)/(control-treated-perfusion control).

Inhibition of Mitochondrial Energetics Using SNAP
Acute (10 seconds) administration of NO (Figure 3) was achieved by pressure-ejecting CB containing 1 mmol/L SNAP. Responses to SNAP were compared with those to the uncoupler FCCP (1 µmol/L) and the respiratory inhibitor CN^- (2 mmol/L). Alternatively (Figures 1 and 2 and 4 through 7), cells were pretreated with 1 mmol/L SNAP in CB for 90 minutes at 37°C in the incubator, washed with PBS, and dye-loaded for 10 to 30 minutes in HBS before the sI/R protocol was applied. SNAP at 1 mmol/L gives an effective concentration of 3 to 5 µmol/L (NO electrode measurements in incubation medium samples; for method, see Sharpe and Cooper¹⁶ ). As expected, this is only slightly higher than physiological concentrations of NO found in cardiovascular tissue (1.3 µmol/L¹⁷ and 1 µmol/L¹⁸ ). Control cells in the cell viability experiment (Figure 1, vehicle) were pretreated for 90 minutes in CB at 37°C without SNAP.

View larger version (12K): [in this window] [in a new window]   Figure 3. Short application of SNAP induced a small reversible inhibition of mitochondrial respiration. Records showing changes in NADH autofluorescence as percentage of prestimulus baseline in response to inhibition of respiration by 2 mmol/L CN- or 1 mmol/L SNAP, or to uncoupling by 1 µmol/L FCCP, which increases oxygen consumption and hence oxidation of NADH. Each agent was applied by pressure ejection for 10 to 20 seconds, and differences in time course of responses primarily reflect differences in time for drug to leave cell. Traces are displaced for clarity, with dashed lines indicating prestimulus levels.

View larger version (36K): [in this window] [in a new window]   Figure 1. SNAP pretreatment reduced sI/R-induced cell death. Percentage cell death was assessed by propidium iodide exclusion assay, as outlined in protocol 2. Data are mean±SEM percentage cell death, with n=24 or 32 (8 fields of view in 3 or 4 experiments, each from different cell preparations).

View larger version (20K): [in this window] [in a new window]   Figure 2. SNAP pretreatment prevented sI/R-induced changes in [Ca2+]c. Simultaneous recording of changes in [Ca2+]c and PO2 during ischemia/reoxygenation. Dotted line indicates control cells; solid line, after SNAP pretreatment. Inset shows return of [Ca2+]c oscillations after reoxygenation in control cells.

Assessment of Mitochondrial Ca²⁺ Uptake
The capacity for mitochondria to take up Ca²⁺ was assessed with the cytosolic indicator fluo-3 and the predominantly mitochondrial dye rhod-2. Neonatal cardiomyocytes in culture show spontaneous Ca²⁺ signals (eg, Figure 2), impeding careful assessment of the mitochondrial response to a cytosolic Ca²⁺ load. Therefore, [Ca²⁺]_c oscillations were inhibited by bathing cells in Ca²⁺-free HBS (with 1 mmol/L EGTA).¹⁹ In addition, for these experiments, the sarcoplasmic reticulum (SR) Ca²⁺ pump was inhibited with thapsigargin (250 nmol/L)²⁰ to prevent SR Ca²⁺ uptake, to limit calcium-induced calcium release, and to avoid possible direct effects of NO on the SR Ca²⁺ pump.²¹ In this way (see Figure 5a), mitochondrial Ca²⁺ uptake was isolated to permit study of the specific effect of NO pretreatment on the ability of mitochondria to accumulate Ca²⁺ after a defined Ca²⁺ load induced by depolarization of the sarcolemma by extracellular application of 50 mmol/L K⁺ containing 2 mmol/L Ca²⁺.

View larger version (49K): [in this window] [in a new window]   Figure 5. Simultaneous measurement of [Ca2+]c and [Ca2+]m. A, Diagram showing protocol 4, to isolate mitochondrial Ca2+ uptake from other Ca2+ fluxes. B, Confocal images of neonatal rat ventricular cardiomyocytes coloaded with rhod-2 and fluo-3. C, Effect of a defined Ca2+ load induced by application of 50 mmol/L K+ on fluo-3 and rhod-2 fluorescence measured over a small cell volume containing a single identifiable mitochondrion. Squares indicate maximum change.

Statistical Analysis
Data are expressed as mean±SEM and were obtained from 4 to 7 separate experimental preparations. Where appropriate, data are expressed as percentage of baseline (ie, 100% x induced value/value before stimulation). Statistical significance (P<0.05) was determined with a 2-tailed Student’s t test or Mann-Whitney test (Graphpad Instat version 3.0).

	Results

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Pharmacological Preconditioning by SNAP in a Cellular Model of sI/R Injury Under Epifluorescent Microscopy
The sI/R protocol resulted in 48.5±2.0% cell death in control cells. Ninety minutes of SNAP pretreatment and 30 minutes of incubation before sI/R was significantly protective (Figure 1), producing a reduction in cell death to 34.5±1.3% (P<0.001, Student’s t test with Welch correction). Cell death in untreated cells exposed to 2 hours of perfusion with CB was 29.7±1.4%. This value agrees well with earlier findings,²² whereas Mackay and Mochly-Rosen²³ reported 16% cell death in their cultures before perfusion with basic salt solutions and possible fluorescent dye–related photodynamic damage. Therefore, SNAP pretreatment reduced sI/R-induced cell death to 25.4% of the ischemia control.

Measurement of [Ca²⁺]_c During sI/R
In control cells, simulated ischemia induced a progressive rise in [Ca²⁺]_c (Figure 2). On reoxygenation, [Ca²⁺]_c returned to baseline, and [Ca²⁺]_c oscillations that accompanied spontaneous beating returned. Usually a secondary, exponential rise in fura-2 ratio developed, followed eventually by loss of cell membrane integrity. SNAP pretreatment did not affect the resting [Ca²⁺]_c levels but dramatically reduced the magnitude of the [Ca²⁺]_c increase during ischemia, from 516±170% to 226±40% (n=5, P<0.05). In addition, SNAP pretreatment suppressed the secondary [Ca²⁺]_c increase and cellular integrity was maintained to the end of the experiment. The apparent difference in latency until the start of the anoxia-induced increase in [Ca²⁺]_c was not significant.

Effect of SNAP on Mitochondrial Energetics
Acute Administration
NO-induced inhibition of mitochondrial respiration was assessed in single cells by NADH autofluorescence. A brief (10-second) application of SNAP caused a reproducible and rapidly reversible rise in NADH autofluorescence (106.9±1.9% of baseline, P=0.012, n=7; see Figure 3). The response to SNAP was small compared with the responses to CN^- or FCCP, which indicate the full range of changes in NADH redox state (123.8±4.2%, n=6, and 80.1±3.4%, n=9, respectively). Consistent with its modest inhibitory effect on redox state, SNAP did not affect _m (assessed with TMRE; data not shown), possibly because the change in _m was too small to detect.

Prolonged SNAP Pretreatment
Mitochondria of control cells loaded with JC-1 showed red fluorescence, attributable to _m-dependent JC-1 accumulation and subsequent J-complex formation. In cells pretreated with SNAP for 90 minutes, the mean red fluorescence was reduced to 54.9% of control (P<0.0001, n=16 and 15, respectively; see Figure 4b). The SNAP-induced changes in JC-1 fluorescence were further characterized both morphologically and functionally. Confocal microscopy clearly showed that the red fluorescence originated from mitochondria (Figure 4c), whereas SNAP pretreatment reduced the overall red fluorescence by 51.9% (P<0.001, n=7 fields). To assess _m-dependent mitochondrial staining, the change in signal was examined with photomultipliers. We found that the red fluorescence gradually decreased with time (Figure 4d). Application of FCCP, however, caused a greater change in red fluorescence in control cells than in SNAP-pretreated cells. To quantify this effect, the FCCP-induced change was expressed relative to the extrapolation of preapplication decay. SNAP pretreatment reduced the FCCP response from 32.1±1.8% to 22.1±3.5% (n=24 and 27, respectively, P=0.016, Student’s t test with Welch correction). These data strongly suggest that the mitochondria were already partially depolarized in SNAP-pretreated cells and that this effect persisted for 1 hour after the donor was removed. The partial inhibition of mitochondrial respiration was not associated with any compromise in cell viability up to 24 hours later (MTT bioreduction assay).⁶

View larger version (36K): [in this window] [in a new window]   Figure 4. Incubation with SNAP for 90 minutes induced a modest, sustained mitochondrial depolarization. A, Linear protocol; B, chart showing total cellular red fluorescence in JC-1–stained control and SNAP-pretreated cells. To characterize JC-1 fluorescence in control and SNAP-pretreated cells under otherwise identical conditions, C shows example confocal images, and D shows time-dependent changes, integrated with photomultiplier tubes. FCCP 1 µmol/L was applied by pressure ejection to eliminate all m-dependent fluorescence. At 3 minutes after FCCP application, mean m-dependent fluorescence in SNAP-pretreated cells was compared with that in control treated cells, both expressed relative to fluorescence derived by extrapolation (dotted lines) of a single exponential fit to data before FCCP application. arb.u. indicates arbitrary units.

Effect of SNAP on Mitochondrial Ca²⁺ Uptake
A moderate but sustained reduction of _m might be expected to reduce mitochondrial Ca²⁺ uptake, which is driven by the electrochemical potential for Ca²⁺ across the mitochondrial inner membrane. To assess the capacity for mitochondria to take up Ca²⁺ independently of other cellular Ca²⁺ fluxes, we developed protocol 4 (see also Figure 5a). Cells were coloaded with rhod-2 and fluo-3 to measure changes in [Ca²⁺]_m and [Ca²⁺]_c, respectively, from the same volume of the cell by confocal microscopy.²⁴ Figure 5b shows that the rhod-2 fluorescence was clearly localized to mitochondria, whereas the fluo-3 fluorescence was diffusely distributed throughout the cell. Changes in fluo-3 and rhod-2 fluorescence also differed in time course. Brief depolarization of the sarcolemma induced a transient increase in [Ca²⁺]_c and a slower and sustained increase in [Ca²⁺]_m (Figure 5c).

Having established the methodology, we then studied the effect of SNAP pretreatment on the mitochondrial Ca²⁺ uptake (Figure 6). In SNAP-pretreated cells, the mitochondrial signal was harder to identify clearly, and the increase in [Ca²⁺]_m after depolarization was markedly reduced (compare Figure 6b with 6a), suggesting impaired mitochondrial Ca²⁺ uptake. Mitochondrial Ca²⁺ uptake, however, depends on local [Ca²⁺]_c, and it is therefore necessary to evaluate the peak increase in [Ca²⁺]_m as a function of the peak local [Ca²⁺]_c (Figure 7). Figure 7a shows that mitochondria in SNAP-pretreated cells accumulated less Ca²⁺ than mitochondria in control cells when exposed to an equivalent [Ca²⁺]_c load. As expected from evidence that NO inhibits sarcolemmal voltage-dependent Ca²⁺ channels,²⁵ the average [Ca²⁺]_c rose by 268±12% in SNAP-pretreated cells compared with 343±10% in control cells, a modest but significant decrease (P<0.0001, n=58; Mann-Whitney; Figure 7b). Excluding all cells with a low increase in [Ca²⁺]_c (threshold 250% of baseline, average [Ca²⁺]_c 349±17% versus 357±9%, P=0.39, n=25 and 52, respectively, Mann-Whitney), however, it was clear that the rise in [Ca²⁺]_m was significantly reduced from 166±7% in control cells to 133±4% in SNAP-pretreated cells (P=0.0004, n=52 and 25, respectively, Mann-Whitney).

View larger version (43K): [in this window] [in a new window]   Figure 6. Example of SNAP-induced reduction in mitochondrial Ca2+ uptake. Confocal images of rhod-2 fluorescence in control (A) and SNAP-pretreated (B) cells before (i) and after (ii) a cytosolic Ca2+ load was induced, using protocol in Figure 5a. To demonstrate spatial and temporal change in rhod-2 fluorescence, surface plots (iii) were obtained by plotting time-dependent changes in intensity profile along a line across cell. Note that application of 50 mmol/L K+ (arrow) induced very little change in rhod-2 fluorescence over nucleus (*). Scale bars=10 µm. arb. u. indicates arbitrary units.

View larger version (30K): [in this window] [in a new window]   Figure 7. SNAP pretreatment reduces capacity of mitochondria to take up Ca2+. A, Maximum change in [Ca2+]m as a function of maximum increase in [Ca2+]c (see squares in Figure 5c) obtained using protocol 4 (Figure 5a). B, SNAP pretreatment reduced mean increase in [Ca2+]c. C, At similar [Ca2+]c, SNAP pretreatment reduced load-induced increase in [Ca2+]m.

These data strongly suggest that the capacity of mitochondria to accumulate Ca²⁺ is attenuated in SNAP-pretreated cells.

	Discussion

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Mitochondrial Ca²⁺ uptake plays an integral part in subcellular calcium homeostasis, most notably modulating (1) intracellular calcium signaling pathways²⁶ and (2) the function of mitochondrial dehydrogenases.²⁷ Large increases in [Ca²⁺]_m, however, can be detrimental to the cell.²⁸ Indeed, accumulating evidence suggests that [Ca²⁺]_m may play a critical role in sI/R injury. For example, [Ca²⁺]_m rises significantly during ischemia,³ and the magnitude of this rise determines the outcome of ischemia/reoxygenation.⁴ Because mitochondrial Ca²⁺ uptake via the uniporter²⁹ is driven primarily by _m and a low intramitochondrial [Ca²⁺], a decrease of _m may limit mitochondrial Ca²⁺ accumulation, providing a potential therapeutic target.

NO modulates mitochondrial function through both irreversible and reversible interactions with respiratory chain complexes,^{9 30} which may explain its dichotomous cytotoxic and cytostatic effects. The latter may play an important beneficial role during ischemia.^{31 32 33} The effect of prolonged NO-induced suppression of mitochondrial respiration is depolarization of _m, which also may or may not be detrimental to the cell.¹⁰ The results of this study further demonstrate a cardioprotective role for NO against sI/R injury.

We have shown that prolonged exposure to SNAP results in a modest depolarization of _m. Remarkably, this effect appears to leave a "memory," in that it persists despite removal of the donor. Using cellular techniques that simulate the physiological microenvironment of ischemia (anoxia, high lactate, high K⁺, low pH, absence of glucose and pyruvate), we showed that SNAP pretreatment had striking effects on limiting the cellular [Ca²⁺] increase during simulated ischemia and prevented the secondary increase in [Ca²⁺]_c and loss of membrane integrity during reoxygenation. Interestingly, modest mitochondrial depolarization with FCCP was also shown to limit sI/R injury in other cellular models.^{34 35} Finally, we showed that pretreatment with SNAP significantly reduced the capacity of mitochondria to take up Ca²⁺. In view of the persistent effect of SNAP on _m and the close relationship between _m and mitochondrial Ca²⁺ uptake, we propose that the suppression of mitochondrial Ca²⁺ entry during ischemia underlies the basis of the observed NO-induced cardioprotection. This appears to provide a novel mechanism for the basis of cardioprotection by NO, linking the modulation of mitochondrial energetics to subcellular ionic homeostasis. A greater understanding of the cytostatic nature of NO should herald the reassessment and development of new pharmacological strategies for drugs with NO-modulating properties in the clinical management of ischemic heart disease.

	Acknowledgments

 
This work was supported by the Wellcome Trust. We are grateful to Drs Nicola Smart and Martyn Sharpe for help with the NO concentration measurements and Lilian Patterson for technical assistance.

Received October 5, 2000; revision received January 24, 2001; accepted January 26, 2001.

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