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Cardiovascular Research 2003 57(2):405-415; doi:10.1016/S0008-6363(02)00675-2
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Copyright © 2003, European Society of Cardiology

The cardioprotective and mitochondrial depolarising properties of exogenous nitric oxide in mouse heart

Robert M Bell, Helen L Maddock and Derek M Yellon*

The Hatter Institute for Cardiovascular Studies, University College Hospitals and Medical School, University College, London, London WC1E 6DB, UK

hatter-institute{at}ucl.ac.uk

* Corresponding author.: Tel.: +44-20-7380-9888; fax: +44-20-7388-5095.

Received 21 March 2002; accepted 16 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Nitric oxide (NO) is reported to be both protective and detrimental in models of myocardial ischaemia/reperfusion injury, which may be concentration dependent. Our objective was to characterise this dichotomy using the nitric oxide donor, S-nitroso N-acetyl penicillamine (SNAP) in isolated perfused mouse heart and isolated mouse cardiac mitochondria. Methods: To determine the effect of nitric oxide concentration on myocardial viability, isolated mouse hearts were subjected to 35 min global ischaemia and 30 min reperfusion in the presence of SNAP (0.02–20 µM). To determine whether NO mediated protection was via opening of the putative mitochondrial KATP channel and/or free radical synthesis, SNAP perfused hearts were also treated with the mitochondrial KATP channel blocker, 5-hydroxy decanoate (5-HD) and the free-radical scavenger, N-(2-mercaptopropionyl)-glycine (MPG). This data was correlated with mitochondrial membrane potential ({Delta}{Psi}m), measured with the potentiometric dye, tetra-methyl rhodium methyl ester (TMRM), in isolated mitochondria,by flow cytometry. Results: SNAP dose-dependently attenuated infarct size, with maximal protection observed at 2 µM (17±4% versus controls 32±3%, P<0.01). At greater concentrations however, protection was lost with infarct sizes tending towards control at 20 µM (29±3%). These results were paralleled by changes in {Delta}{Psi}m in the isolated mitochondria: {Delta}{Psi}m depolarisation peaking with 1 µM SNAP (26±4% shift in TMRM fluorescence, P<0.01); at greater concentrations, this relationship was lost. The mitochondrial KATP channel blocker, 5-HD, resulted in both abrogation of SNAP infarct size reduction and concomitant loss of {Delta}{Psi}m depolarisation in the mitochondria. MPG however did not influence the cardioprotective properties of SNAP. Conclusion: We demonstrate that nitric oxide can mediate cardioprotection in a dose-dependent fashion by an effect that may be related to {Delta}{Psi}m. Both cardioprotection and {Delta}{Psi}m changes are sensitive to 5-HD and the cardioprotection appears independent of free-radical synthesis.

KEYWORDS Free radicals; Infarction; K-ATP channel; Mitochondria; Nitric oxide


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Nitric oxide (NO) is associated with many aspects of cellular biology, involved in neuronal signalling, maintenance of vasodilatory tone in numerous vascular beds, cell growth, regulation of platelet aggregation and leukocyte binding to endothelium [1,2]. Modest production of nitric oxide, as associated with preconditioning, has been linked to myocyte survival [3–6]. However, nitric oxide synthesis has also been associated with myocyte death [7,8] following ischaemia/reperfusion injury. Moreover, the significant induction of nitric oxide synthesis in conditions such as myocarditis [9] and allograft failure [10] has also been found to be deleterious to myocyte survival. The mechanisms of cardioprotection versus nitric oxide mediated injury are unclear. It has certainly been shown that nitric oxide, through the generation of the free-radical, peroxynitrite, can inhibit mitochondrial respiration through irreversibly binding to various components of the electron transport chain, that may in turn lead to free-radical mediated cellular injury [11]. Moreover, mitochondrial damage may be potentiated by nitric oxide, leading to activation of the apoptotic-signalling cascade [12]. Whilst the mitochondria appear to be a target for nitric oxide mediated injury, the mitochondria may also be a potential target for protection.

Myocardial mitochondria are sensitive to ischaemia and reperfusion mediated injury. Ischaemia is associated with mitochondrial swelling, progressive amorphism, disruption and loss of cristae, and reduction of matrix volume [13–16]. In turn, reperfusion is associated either with resolution of the ultrastructural changes associated with reversible myocyte injury [14], or with considerable mitochondrial accumulation of calcium [17–20], which is pathognomonic of irreversible injury. Calcium entry into mitochondria appears to be closely linked with the opening of the non-specific mitochondrial transition pore (or permeability transition pore, PTP) [21]. Opening of the PTP is associated with the release of cytochrome-c from the mitochondrial inter-membrane space, into the sarcoplasm [22], with subsequent activation of cell death cascades through binding to Apaf-1 and activation of caspase-9 [23]. Exogenous nitric oxide has been shown to attenuate reperfusion triggered calcium loading of the mitochondria [24,25], and reduce the PTP open probability [26]. Additionally, recent work has suggested that mitochondrial calcium overload may be attenuated by opening of the mitochondrial KATP channel [27,28], a putative end effector of the preconditioning phenomenon, whose opening is associated with cardioprotection [29]. Interestingly, nitric oxide has been linked to an increase of mitochondrial KATP channel open probability [30]. Thus, it is attractive to hypothesise that the cardioprotective effects of nitric oxide are potentially mediated through the alteration of mitochondrial function and the attenuation of calcium overload during reperfusion, possibly via a direct effect upon the mitochondrial KATP channel.

Curiously, whilst there is evidence for nitric oxide acting upon and interacting with the mitochondria, surprisingly little data exists to support the nitric oxide/mitochondrial hypothesis of myocardial infarct size limitation, although some data does exist linking the clinical therapeutic exogenous nitric oxide donor, glyceryl trinitrate and the mitochondrial KATP channel with improved post-ischaemic functional recovery and attenuated lactate dehydrogenase (LDH) release [31].

The objectives of the current study were two-fold. First was to determine the optimal cardioprotective concentration of nitric oxide in hearts subjected to ischaemia and reperfusion, and to determine the concentration at which the exogenous nitric oxide donor would prove to be deleterious. Secondly, we wished to investigate whether exogenous nitric oxide has a direct, dose-dependent, effect upon mitochondrial membrane potential. If this was indeed the case, we wanted to determine whether this effect was linked to the opening of the putative mitochondria KATP channel, as has been previously postulated, and/or associated with the generation of free-radical species.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
All work was conducted in accordance with the Guidelines on the Operation of the Animals (Scientific Procedures) Act 1986, published by The Stationery Office (London, U.K.). S-nitroso N-acetyl penicillamine, 5 hydroxy decanoate and N-(2-mercaptopropionyl)-glycine were purchased from Sigma (Poole, UK). C57 BL6 mice were obtained from the breeding colony kept at University College, London.

2.1 Langendorff perfusion
Male and female C57 BL6 mice (3–4 months of age, 20–30 g weight) were used, and randomly attributed to either control or treatment groups. Heart isolation and Langendorff perfusion (with modified Krebs–Henseleit buffer consisting of NaCl 118 mmol/l, NaCO3 24 mmol/l, –Glucose 10 mmol/l, KCl 4 mmol/l, NaH2PO4 1.0 mmol/l, Na2EDTA 0.5 mmol/l, MgCl2 1.2 mmol/l, CaCl2 2.5 mmol/l) were performed as previously described [5,6]. In brief, mice were anaesthetised with an intra-peritoneal injection of 60 mg/kg pentobarbitone. Hearts were then harvested via a para-medial sternotomy and rapidly transferred to a dissection dish filled with ice cold Krebs–Henseleit buffer. The aorta was cannulated with a 20 gauge murine cannula and transferred to the Langendorff rig. Hearts were paced throughout the stabilisation period and during the final 20 min of reperfusion (600 beats per minute). All hearts were subjected to 35 min of global ischaemia and 30 min of reperfusion. In preliminary experiments, we found that 30 min of reperfusion resulted in identical infarct size by triphenyl tetrazolium staining as 120 min (Fig. 1A), and thus all subsequent experiments were performed using this reperfusion period.


Figure 1
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  		Fig. 1 (A) showing preliminary data, whereby 30 min of reperfusion results in equivalent infarct size to 120 min reperfusion in Langendorff perfused mouse heart stained with TTC. (B and C) showing representative heart slices from control and 2 µmol/l SNAP treated hearts, respectively.

 
2.2 Infarct size analysis
At the end of each infarct-size experimental protocol, the hearts were infused with 5 ml of phosphate buffered (pH 7.4) triphenyl tetrazolium chloride solution (TTC), prior to removal and incubation in TTC for 10 min at 37 °C. The hearts were then frozen at –20 °C before slicing (perpendicular to the long atrio-ventricular axis of the heart at <1 mm intervals). The heart slices were then de-stained in 10% formaldehyde solution over night prior to digital photography using a custom built photographic apparatus, connected to a Macintosh PowerPC. Each slice was mounted between two plates of transparent perspex of a known distance apart, so that volumes could be readily calculated once the areas of the slice were determined. The images were imported into a graphics package (NIH Image 1.62) and the infarct size determined using a custom-written infarct detecting macro programme. The infarct to risk zone ratio was calculated by determining the volumes of necrotic, pale staining tissue, as a proportion of viable, brick red stained myocardium. The risk zone, in this globally ischaemic model, was the total myocardial volume. Representative heart slices from control and SNAP treated hearts are shown in Fig. 1B and C.

2.3 Contractile function measurement
Contractile function was assessed by monitoring septal shortening, by inserting a tie through the apex of the heart, and attaching the heart to a linear force transducer (Scame, France). The transducer was mounted on a micromanipulator that enabled the resting tension over the aorto-apical axis to be adjusted to obtain the optimal contractile force without excessive end diastolic tension. The mean resting tension in each group is summarised in Table 1. The contractile function, a secondary outcome measure in the present study, was monitored throughout the experimental protocol. End stabilisation function for each study group is summarised in Table 1.


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  		Table 1 Morphometrics and baseline functional data

 
2.4 Mitochondrial preparation
Mitochondria were isolated from hearts of female C57, BL6 mice (body weight 20–25 g) using a method previously described [32]. In brief, hearts were removed from terminally anaesthetised mice and briefly infused with cold MSTEB buffer, containing mannitol 210 mM, sucrose 70 mM, TRIS 10 mM (pH 7.2 with hydrochloric acid), EGTA 1 mM and BSA 0.5 mg/ml. The heart was then homogenised in MSTEB buffer. After centrifugation (450 g, 5 min), two thirds of the supernatant was decanted into fresh, pre-chilled tubes. Mitochondria were isolated by further centrifugation (5800 g, 10 min). The mitochondrial pellet was resuspended in cold MST (containing mannitol 210 mM, sucrose 70 mM and TRIS 10 mM) and the previous centrifugation step repeated. Mitochondrial concentration was determined by protein assay using the BCATM protein assay kit, measuring absorbance on a photospectrometer at 562 nm.

2.5 Flow cytometry
Mitochondria were assessed using the flurometric probe, tetramethyl rhodamine methyl ester (TMRM, Molecular Probes Inc., Leiden, The Netherlands), a non-toxic, mono-valent cation, that reversibly accumulates according to membrane potential with a Nernstian distribution [33].

For each experiment a mitochondrial aliquot of 100 µl (diluted to 0.5 mg mitochondrial protein/ml) was re-suspended in a KCl buffer (containing KCl 45 mM, potassium acetate 25.4 mM, TES 5 mM, EGTA 0.1 mM, pH 7.4 and MgCl2 1 mM). Each sample was incubated with substrates for respiration (glutamate 5 mM and malate 2 mM) and TMRM (200 nM [34]) at room temperature for 2 min. Where the experiment required the administration of a drug, this was added immediately prior to cytometry.

To demonstrate normal mitochondrial depolarisation, we used the mitochondrial uncoupler, carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 10 µM) as a positive control; FCCP causes near complete depolarisation of the mitochondria. In the absence of these membrane potential changes, the mitochondria were discarded.

Cytofluorimetric analysis was performed using a Partec PAS flow cytometer (Partec, Germany) equipped with a 488 nm argon ion-laser. The TMRM signal was analysed in the FL2 channel, which was equipped with a bandpass filter at 560±20 nm. Data were acquired on a logarithmic scale. Arithmetic mean values of the median fluorescent intensities were determined for each sample for subsequent graphical representation. Experiments were performed on mitochondria isolated from six individual mice, each experiment representing 15 000 mitochondria. Mitochondrial depolarisation is represented as percentage shifts in the mean FL2 fluorescence peak from control values in untreated, control quiescent mitochondria.

2.6 Experimental protocols
The study was divided into two phases: phase (1): isolated mouse heart subjected to ischaemia and reperfusion, phase (2): isolated mouse heart mitochondria in the presence or absence of SNAP.

Phase 1 isolated mouse hearts were randomly allocated into one of four groups (Fig. 2). Group I control hearts perfused with normal Krebs–Henseleit buffer. Group II: hearts subjected to ischaemia and reperfusion in the presence of incremental concentrations of SNAP ranging from 0.02 to 20 µmol/l. Group III: hearts perfused in the presence of 5-HD, 100 µmol/l±SNAP 1 µmol/l. Group IV: hearts perfused in the presence of MPG 300 µmol/l±SNAP 2 µmol/l.


Figure 2
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  		Fig. 2 Experimental protocols. Groups (I–IV) Langendorff perfused mouse hearts, subjected to 35 min global ischaemia and 30 min reperfusion. Groups (V and VI) Isolated mitochondria; membrane potential assessed by incubation with TMRM, in the presence or absence of treatments prior to analysis by flow cytometry.

 
Isolated mitochondria in phase 2 of the study were incubated with TMRM in the presence or absence of drug. Thus, in group V, the mitochondria were exposed to incremental concentrations of SNAP (from 0.01 to 100 µmol/l) and in group VI, 100 µmol/l 5-HD±1 µmol SNAP.

2.7 Statistical analysis
All values are expressed as mean±S.E.M. Differences in continuously distributed variables between predetermined experimental groups were analysed using one-way ANOVA followed by Fisher's protected test of least significant difference. Differences between continuous measures were determined by factorial ANOVA. Fisher's protected test of least significant difference was used as the post hoc test. P-values of 0.05 were considered to be at the limit of statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Baseline body and heart mass are summarised in Table 1, along with baseline functional parameters. Although SNAP is a potent vasodilator, in this model there was no statistical significance in coronary flow rates at the end of the stabilisation period.

3.1 Is exogenous nitric oxide cardioprotective or cardiotoxic in ischaemia/reperfusion injury?
Using the spontaneous nitric oxide donor SNAP, we used incremental concentrations to build a dose–response curve to determine the optimal cardioprotective concentration of exogenous nitric oxide. Here we show that SNAP results in significant reduction of infarct size over controls at 1 and 2 µmol/l (21±1 and 17±4%, respectively, versus control, 32±3%, P<0.01, Fig. 3). At concentrations greater than 2 µmol/l, we found that infarct size tended back towards control values; cardioprotection was lost (24±2 and 29±3% with 6 and 20 µmol/l SNAP, respectively, P0.05). A similar dose–response relationship was found in relation to SNAP concentration at contractile recovery (Fig. 4A–C), whereby again, contractile function recovery was greater with concentrations of SNAP peaking at 2 µmol/l (high logarithythmic correlation, r2=0.99, P<0.001, Fig. 4C). At higher concentrations, however, the benefit of exogenous nitric oxide administration was lost. Interestingly, SNAP had no significant impact upon reperfusion coronary flow rates (data not shown).


Figure 3
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  		Fig. 3 SNAP dose–response curve. Up to concentrations of 2 µmol/l, SNAP dose-dependently attenuates infarct size. At greater concentrations however, the protective properties of SNAP are lost. (** P<0.01 versus control hearts).

 

Figure 4
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  		Fig. 4 (A) Contractile recovery following ischaemia; SNAP dose dependently improves contractile recovery up to 2 µmol/l—as summarised here by mean function. Mean contractile recovery is significantly greater than controls at 1 and 2 µmol/l (P<0.05) (B) shows the logarithmic relationship between functional recovery and SNAP concentration at concentration ranges up to 2 µmol/l.

 
3.2 Does nitric oxide affect mitochondria?
To determine whether exogenous nitric oxide administration is associated with alterations in mitochondrial membrane potential ({Delta}{Psi}M), we incubated isolated mitochondria with SNAP (SNAP concentration ranging from 0.01 to 100 µmol/l). As with the cardioprotective concentrations of SNAP in the isolated heart, exposure of isolated mitochondria to SNAP demonstrated a dose-dependent reduction of TMRM fluorescence, indicative of loss of {Delta}{Psi}M (depolarisation). The depolarisation peaked with 1 µmol/l SNAP (9.7±5.3, 19.2±3.8, and 25.8±4.4% reduction of mean TMRM fluorescence with 0.01, 0.1 and 1 µmol/l SNAP, respectively, P<0.01 1 mmol/l SNAP versus control, Fig. 5). At concentrations greater than 1 µmol/l however, the dose-dependent reduction of membrane potential was lost (3.7±2.1 and 14.1±2.8% at 10 and 100 µmol/l SNAP, respectively).


Figure 5
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  		Fig. 5 Membrane potential SNAP dose–response curve. Incremental concentrations of SNAP resulted in progressive reductions of TMRM fluorescence, indicative of mitochondrial depolarisation. At concentrations greater than 1 µmol/l, this relationship was lost.

 
3.3 Does the putative mitochondrial KATP channel mediate the membrane potential changes?
Given the potential association between nitric oxide and the mitochondrial KATP channel [30], and opening of the mitochondrial KATP channel with infarct sparing [35] and loss of {Delta}{Psi}M [36], we wished to examine this relationship further. Interestingly, both the infarct sparing and the loss of {Delta}{Psi}M had strong, significant logarithmic associations up to the optimal concentration of SNAP to effect both protection and membrane depolarisation (Fig. 6A and B).


Figure 6
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  		Fig. 6 (A) Cardioprotective concentrations of SNAP resulted in a strong logarithmic relationship between dose and infarct limitation (P<0.01). (B) Cardioprotective concentrations of SNAP resulted in mitochondrial depolarisation with a strong correlation coefficient (P<0.01). (C) Membrane depolarisation associated with SNAP administration abrogated by the co-administration of the KATP channel blocker, 5-HD. (D) Cardioprotection associated with SNAP blocked by the KATP channel blocker, 5-HD.

 
Next, we went on to test the hypothesis that nitric oxide results in increased open probability of the mitochondrial KATP channel, leading to mitochondrial depolarisation, to protect the myocardium against ischaemia/reperfusion injury. Thus, we proposed that the mitochondrial selective mitochondrial KATP channel blocker, 5-HD [35,37], at a concentration of 100 µmol/l, would abrogate the cardioprotective properties and the mitochondrial depolarising effects of 1 µmol/l SNAP. Whilst 5-HD on its own had no independent effect upon either infarct size (36±3%) or mitochondrial membrane potential, 5-HD nonetheless ameliorated SNAP-induced reduction of TMRM fluorescence (0.0±0.2% versus SNAP 25.8±4.4%, P<0.01, Fig. 6C) and abrogated cardioprotection (38±3%, P0.05 versus control, 32±3%, and P<0.05 versus SNAP, 21±1%, Fig. 6D).

3.4 If cardioprotection is mediated via a 5-HD sensitive mechanism, can the effects of exogenous nitric oxide be attenuated with a free radical scavenger?
Given recent evidence linking KATP channels with free radical generation and protection [38], and nitric oxide triggered protection to the synthesis of radical species [39], we wished to determine whether nitric oxide mediated protection could be abrogated by the co-administration of a free radical scavenger, MPG. MPG has been shown to be an excellent hydroxyl and peroxynitrite [40] radical scavenger. Administration of MPG alone had no impact upon infarct size, nor an effect upon nitric oxide mediated protection (32±3 and 19±4%, respectively, versus control and SNAP treated hearts, 32±3 and 17±4%, P0.05 against respective MPG treated groups, Fig. 7).


Figure 7
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  		Fig. 7 The effect of free radical scavenging on nitric oxide mediated protection. MPG had no effect upon infarct size on its own nor against the protection observed with 2 µmol/l SNAP.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The role of nitric oxide during ischaemia and reperfusion is controversial. Whilst nitric oxide has been shown to elicit and/or mediate preconditioning in both its early [5,39] and delayed phases [3,4,6], it has also been shown to be capable of triggering cell death in pathological states [9,10]. One explanation of this apparent dichotomy is concentration dependent, a hypothesis that can be illustrated by the effect of nitric oxide concentration upon mitochondrial electron-chain transport. Low concentrations of nitric oxide reversibly bind to the heme a3 component of cytochrome-c oxidase/complex IV [41–44], reversibly inhibiting cellular oxygen consumption, a process that has been proposed to be cardioprotective [45]. Conversely, higher concentrations of nitric oxide, through the generation of the free-radical peroxynitrite, inhibit mitochondrial respiration irreversibly by binding to components of the electron transport chain, triggering cellular injury [11]. Moreover, high concentrations of nitric oxide are known to increase the open probability of the mitochondrial permeability transition pore, releasing pro-apoptotic signalling initiators such as cytochrome C from the mitochondria [46], as well as directly leading to nuclear DNA damage and energy depletion through poly (ADP–ribose) polymerase activation [47].

The dose-dependent nitric oxide hypothesis of protection versus injury appears to be supported by the data we present here. We show that the administration of low-concentration nitric oxide, bracketing both ischaemia and reperfusion, results in a dose-dependent reduction of infarct size. Beyond a concentration threshold level (2 µmol/l of the nitric oxide donor, SNAP), the protection is lost.

We demonstrate too that protection associated with low-dose nitric oxide is abrogated by the concomitant administration of the mitochondrial KATP channel blocker, 5-HD, implicating a potential link between nitric oxide mediated protection and mitochondrial function. This mitochondrial link with cardioprotection is strengthened by two observations in isolated mouse heart mitochondria. First is that SNAP results in a dose-dependent depolarisation of isolated mitochondria (which under the conditions of this model is co-incidentally concurrent with the concentration range that elicits protection in the isolated heart). Second is that the SNAP induced change in {Delta}{Psi}M is inhibited by 5-HD, which appear to parallel the observations with the loss of cardioprotection in the whole heart. In both heart and mitochondria, supra-maximal concentrations of SNAP are not associated with either cardioprotection, or continuation of the initial dose-dependent reduction of mitochondrial membrane potential.

Thus, on the basis of our results with 5-HD, it appears that the mitochondrial KATP channel is a pivotal target for the protective effects of nitric oxide. Aside from the problems associated with using pharmacological inhibitors for a channel that has yet to be cloned, there is evidence to support a role for this mitochondrial channel in mediating cardioprotection. Mitochondrial calcium overload is pathognomic of irreversible ischaemia/reperfusion injury—the calcium paradox [17–20]. Interestingly, mitochondrial KATP channel openers attenuate the calcium paradox [28] and limit calcium accumulation in mitochondria by altering mitochondrial calcium homeostasis [27]. Given that opening of the mitochondrial permeability transition pore (PTP) is associated with triggering of apoptotic cell-death cascades, and that high calcium leads to opening of the PTP [22,48], the finding that low-dose exogenous nitric oxide attenuates both the calcium paradox and opening of the PTP is of great interest [26]. Since exogenous nitric oxide has been demonstrated to increase the open probability of mitochondrial KATP channels [30], it is attractive to postulate that cardioprotective low-dose nitric oxide is mediated via the mitochondrial KATP channel, a hypothesis supported by our findings in both isolated heart and mitochondria.

The present data would appear to suggest that nitric oxide is capable of acting directly upon the mitochondria: isolated mitochondria are removed from the intermediary signalling pathways found in the sarcolemma and sarcoplasm, and so would suggest that cyclic GMP related signalling pathways may not be required for this effect. It should also be noted however, that whilst nitric oxide may be capable of acting directly upon isolated mitochondria, this phenomenon does not preclude the recruitment of cellular signalling by nitric oxide in intact cells. Indeed it is conceivable, that these nitric oxide-recruited signalling pathways may be associated with toxic effects of higher concentrations of exogenous nitric oxide in the whole heart—a hypothesis that requires further investigation.

As with high dose (greater than 2 µmol/l SNAP) nitric oxide in the heart, high-dose SNAP treatment of isolated mitochondria resulted in loss of the dose-dependent depolarisation. Indeed, the mitochondria appeared to partially repolarise at 10 µmol/l SNAP. The reason for this occurrence is not clear. High concentrations of nitric oxide donors (greater that 20 µmol/l) have been shown to directly nitrosylate the mitochondrial PTP, leading to increased open probability and subsequent activation of the apoptotic signalling cascade [12,46]. However, PTP opening would be expected to result in significant mitochondrial depolarisation, far greater than that seen in our preparations. This suggests that there may be a compensating membrane-polarisation mechanism, perhaps through reversal of the F1F0 ATP synthase as suggested by work from Moncada's lab [49]. Here, Beltran et al. demonstrated that exogenous nitric oxide maintains cellular viability in serum-free conditions for longer, concomitant with maintained in-situ mitochondrial membrane potential. Whether the loss of nitric oxide mediated cardioprotection against ischaemia/reperfusion injury correlates with a reversal of the mitochondrial F1F0 ATP synthase and whether the mitochondrial depolarisation associated with high-dose nitric oxide is due to opening of the PTP is unclear. Both observations require further investigation.

With respect to free-radicals, our results seem to indicate that the protective effects of low-dose nitric oxide in the whole heart are not mediated by their generation, which appears contrary to a previous report using exogenous nitric oxide used as a trigger of preconditioning [39]. The discrepancy would suggest that exogenous nitric oxide as a cardioprotective agent mediates protection via different mechanisms to those recruited by transient exposure of the heart to nitric oxide to trigger preconditioning; preconditioning requires up-stream signalling to result in a cardioprotective ‘memory.’ In the paradigm presented in this report, nitric oxide is free to act directly upon end-effector targets downstream of preconditioning-recruited pathways, without the need to recruit preconditioning memory, and therefore no need for free-radicals to achieve this state.

In summary, we have shown that low-dose exogenous nitric oxide can dose-dependently protect the myocardium against ischaemia/reperfusion injury. This result is paralleled in isolated mitochondria, where low-dose nitric oxide results in dose-dependent mitochondrial depolarisation. Moreover, both the cardioprotection associated with low-dose nitric oxide and the mitochondrial depolarisation are both abrogated by the co-administration of mitochondrial KATP channel blocker, 5-HD. The protection appears not to be mediated by the generation of free-radicals, as the protection is not lost by concomitant administration of MPG. The optimal concentration of the nitric oxide donor, SNAP against ischaemia/reperfusion injury is 1–2 µmol/l. At high-concentration nitric oxide administration, both cardioprotection and the effect upon membrane potential are lost, suggestive of cardiotoxicity through an effect upon mitochondria.

Time for primary review: 28 days.


   Acknowledgements
 
This work was supported by the British Heart Foundation.


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

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