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Cardiovascular Research 2001 51(4):691-700; doi:10.1016/S0008-6363(01)00330-3
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Copyright © 2000, European Society of Cardiology

Mitochondrial KATP channel opening protects a human atrial-derived cell line by a mechanism involving free radical generation

Richard Carrolla, Vanya A Gantb and Derek M Yellona,*

aThe Hatter Institute and Centre for Cardiology, University College Hospital and Medical School, Grafton Way, London WC1E 6DB, UK
bThe Department of Medical Microbiology, University College Hospital and Medical School, Grafton Way, London WC1E 6DB, UK

hatter-institute{at}ucl.ac.uk

* Corresponding author. Tel.: +44-203-809-888; fax: +44-203-885-095

Received 12 January 2001; accepted 20 April 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 5 Summary
 References
 
Objectives: The mechanism by which the mitochondrial KATP channel openers confer protection against ischemia/reperfusion injury is debated. Evidence suggests that rather than solely being an end effector, opening of these channels may act by a trigger mechanism. We examined the effects of the mitochondrial KATP channel opener, diazoxide on parameters of mitochondrial function with specific reference to reactive oxygen species (ROS) generation in a human atrial derived cell line model of simulated ischemia/reperfusion (LSI/R). Methods and results: Propidium iodide (PI) exclusion was used to assess survival. Diazoxide treatment conferred protection against LSI/R (13.9±0.9% vs. 36.9±4.5% controls) that was abolished by pre-treatment with the mitoKATP channel blocker, 5-hydroxydecanoate (5-HD) (33.3±3.6%) and with the free radical scavenger, 2-mercaptopropionylglycine (MPG) (29±4.0%). Diazoxide caused increased oxidation of the ROS probe, reduced mitotracker orange (1.3 vs. 1.0 arbitrary units for control; P<0.01 vs. control) that was abrogated by either 5-HD or MPG (1.07 and 1.07 arbitrary units, respectively). At the same time there was no change in orange fluorescent signal from the membrane potential sensitive probe, JC-1 indicating no change in mitochondrial membrane potential. Changes in light scattering, reflecting changes in mitochondrial volume, occurred during treatment with diazoxide. Conclusion: These results demonstrate for the first time that the mitoKATP channel opener diazoxide can act as a trigger of preconditioning by a mechanism involving mitochondrial swelling and the generation of ROS.

KEYWORDS Mitochondria; K-ATP channel; Free radicals; Preconditioning

This article is referred to in the Editorial by H.H. Patel and G.J. Gross (pages 633–636) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 5 Summary
 References
 
Myocardial preconditioning describes the induction of resistance to a normally lethal ischemic insult in myocardium exposed to preceding, brief episodes of ischemia/reperfusion [1]. The initial protection conferred by preconditioning is short-lived, but after 24 h, this protection reappears and lasts for up to 72 h [2]. This delayed cardioprotection or second window of protection (SWOP) has been demonstrated in every species studied to date [3], and has therapeutic clinical potential [4].

Experimental evidence supports the mitochondrial ATP-sensitive K-channel (mitoKATP channel) as having a central role in the mechanisms of both classic and delayed cardioprotection [5] in several models and mediated by a variety of diverse triggers such as adenosine and opioid receptor activation. The opening of this mitoKATP channel may be a possible mediator of protection [6]. In addition, mitoKATP channel blockers, such as 5-hydroxydecanoate sodium and glibenclamide abolish the protection afforded by a variety of triggers such as simulated ischemia and exogenous adenosine [7], opioids [8] and acetylcholine [9]. Inhibition is seen at concentrations of 5-HD that had no effect on the sarcolemmal KATP channel and protection can be mimicked by diazoxide, a potent opener of mitoKATP channels, at a concentration that has no effect on sarcolemmal KATP channels [10]. As such, the mechanism of the protection afforded by mitoKATP channel opening is not fully understood.

Transient exposure to reactive oxygen species (ROS) such as the superoxide moiety can trigger preconditioning per se in both the classical [11,12] and delayed settings [13]. Similarly the generation of endogenous reactive oxygen intermediates from cardiomyocytes appear important in preconditioning, and it appears that these species are generated in the mitochondria of the myocyte [14]. Yao et al. have shown a link between both ROS generation and mitoKATP channel activation in the protection mediated by acetylcholine [9]. Furthermore, the interaction between another established trigger of protection, namely, opioids, and the KATP channel and ROS has also recently been established [8]. It has also been demonstrated that the cardioprotective effect of diazoxide can be abolished by prior incubation with the non-specific free radical scavenger N-acetyl cysteine (NAC) [15], this work being repeated by Downey et al. using 2-mercaptopropionyl glycine (MPG) in a similar model [16]. Opening of the mitoKATP channel causes influx of the K+ cation accompanied by anion, making it electroneutral; the overall effect being a change in volume of the matrix [17]. It is conceivable that this change in volume could cause "functional uncoupling" of electron transport and oxidative phosphorylation in the mitochondrion and this will lead to generation of ROS primarily from complex III enzymes [18,19]. As such, this may herald a common pathway between ischemic preconditioning and diazoxide-mediated cardioprotection.

We have previously demonstrated the ability of simulated ischemic preconditioning and diazoxide to protect a human atrial cardiocyte-derived cell line against simulated ischemia reoxygenation injury [20]. We have also shown that both of these interventions resulted in significant generation of ROS in the absence of a change of membrane potential [21]. The present study was specifically designed to address whether diazoxide causes generation of ROS, and whether these ROS were responsible for the observed cytoprotection. In order to achieve this, two distinct experimental groups were compared. Firstly, diazoxide was used in the standard cardioprotective manner, by incubation immediately prior to simulated ischemia, or, secondly, by using diazoxide administered as a "pulse", 15 min prior to simulated ischemia to examine opening the mitoKATP channel as a possible "trigger" mechanism. In order to understand the mechanisms of this phenomenon, we measured mitochondrial membrane potential, mass and changes in matrix volume.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 5 Summary
 References
 
2.1 Chemicals
All chemicals and solvents were obtained from Sigma (Poole, UK), except for dihydroethidium, nonyl acridine orange and reduced mitotracker orange, which were purchased from Molecular Probes, Eugene, Oregon. All compounds were dissolved in ultra-pure water (Analar, BDH, Poole, UK) except diazoxide, which was dissolved as a stock solution in dimethyl sulfoxide and then diluted in media immediately prior to use. All molecular probes were dissolved in ultra pure anhydrous DMSO immediately prior to use.

2.2 Tissue culture
Cultured human atrial derived cardiocytes were purchased from the European Collection of Cell Cultures (Salisbury, UK) and maintained in Minimal Essential Medium supplemented with foetal calf serum (10%), non-essential amino acids (1%), glutamine (1%) and penicillin/streptomycin (1%), (Gibco BRL, Paisley, Scotland), referred to as full growth medium (FGM). Cells were obtained at passage 529 and used up to passage 540 with no visible phenotypic change. Cells were maintained in culture and passaged every 48–72 h and plated at an initial density of 3x105 cm–2 in 24 or 6 well plates (Starstedt, Milton Keynes, UK). When grown to confluence, cells were treated as described.

2.3 Lethal simulated ischemia (LSI)
Lethal simulated ischemia consisted of 6 h incubation in hypoxic buffer, modified from Esumi et al. [22] with high lactate (20 mM), physiological potassium concentration and a lower pH of 6.3, in a custom built hypoxic chamber demonstrated to reduce pO2 to below 5 Torr. Interventions were performed either 15 min prior to LSI ("late" groups) or 30 min prior to LSI ("early" groups). Cells were treated with diazoxide, (30 µM) alone or following prior incubation with 5-hydroxydecanoate sodium, (50 µM), 2-mercaptopropionyl glycine, (400 µM) or N-acetyl cysteine, (20 mM). Following lethal simulated ischemia, cells were washed and allowed to reoxygenate for 1 h in normal growth medium at atmospheric pO2.

2.4 Flow cytometry assessment of propidium iodide exclusion
At the end of the lethal simulated ischemic period, medium from each well was aspirated and saved. Cells were washed twice with warm Dulbecco's PBS and then incubated for 2 min with 0.25% trypsin in 1 mM EDTA (Gibco BRL, Paisley, Scotland). Detached cells were resuspended in the saved medium and then centrifuged at 3000 rpm for 4 min in a pre-cooled benchtop centrifuge at 4°C. The resulting cell pellet was then resuspended in ice-cold PBS containing 1 µg/ml propidium iodide. The cell suspension containing approximately 2x106 cells ml–1 was then analysed by flow cytometry (Bryte-HS, Bio-Rad Microscience, UK). The number of cells "propidium iodide positive" were expressed as a percentage of total cell count and considered oncotic. Data were collected from a minimum of 1x104 cells per sample. Untreated dishes of cells treated in a similar way were analysed and the experiment was excluded if >2% control cells were positive at the end of the assay protocol.

2.5 Flow cytometric quantification of ROS generation
Cells were loaded with either dihydroethidium (5 µM) or reduced mitotracker orange (200 nM) for 15 min before washing twice in fresh medium. Cells were excited with a bandpass wavelength of 470–490 nm using an arc source Bryte HS FCM and fluorescence signals were collected at a wavelength >520 nm. Amplifiers for forward scatter (FS) (<1.5°) and orthogonal scatter (SS) (<1.5°) were set in linear mode and threshold settings excluding sub-cellular particles were set on the FS channel. Any signal in the forward scatter amplifier over the trigger threshold was considered an event. A region of interest was defined in the forward scatter versus orthogonal scatter cytogram in order to gate out signals from sub-cellular debris. A minimum of 10 000 events in the region of interest were measured for each sample. Fluorescence detectors with bandwiths of 520–565 nm (FL1), 565–605 nm (FL2), and >605 nm (FL3) were set in logarithmic gain mode. PMT voltage was adjusted in order that all unlabelled control cells were ascribed to the first logarithmic decade.

Superoxide generation was measured by detecting the conversion of the blue fluorescent dihydroethidium to ethidium. This intercalates with dsDNA to form a far red fluorescent complex with a large Stokes shift and peak emission at 610 nm. This was measured in the FL3 detector. In the case of reduced mitotracker orange (CM-H2TMRos), excitation was provided with a bandpass of 510–560 nm and subsequent emission was collected using a dichroic mirror with a longpass >590 nm. CM-H2TMRos is oxidized by a variety of ROS and was used to measure "total" ROS generation by formation of the red/orange fluorescent CMTMRos, emission maximum 576–600 nm, measured in FL2 detector.

Data were saved on high density magnetic disks (Iomega) for later off-line analysis.

2.6 Assessment of changes in mitochondrial volume
The change in the ratio of FS V 0.6xSS was calculated on a cell by cell basis off-line using Winlist software (Verity, NY, USA) and plotted on a linear scale for control cells and compared with each of the experimental interventions. Light scatter parameters were measured with excitation between 520 and 540 nm.

2.7 Assessment of changes in mitochondrial membrane potential
Cells were loaded with the molecular probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; CBIC2(3)) at a concentration of 5 µM for 15 min, washed and submitted to experimental interventions before analysis by FCM.

2.8 Measurement of cell volume
To assure that a change of cell volume was not responsible for the changes in FS or SS, event flight time (WID) parameters were compared within and between experimental groups.

2.9 Measurement of mitochondrial mass
Cells were incubated with the probe nonyl acridine orange, (100 nM) in fresh medium for 15 min before analysis by FCM. Cells were exited using a wavelength of 470–490 nm and emission was collected using a 520–560 bandpass filter.

2.10 Protocol for determination of the effects of diazoxide
For each parameter measured, cells were either allowed to remain in growth medium for 15 min (control) incubated with diazoxide for 15 min and immediately harvested ("late group") or allowed to remain in growth medium for 30 min (control), incubated with diazoxide for 15 min, washed then held in growth medium for a further 15 min before harvesting ("early group").

2.11 Statistical analysis
For comparison of changes in fluorescence intensity, individual experiments were compared off line using transformation analysis and then expressed as mean±S.E.M. For measurements of cell viability, results are expressed as mean±S.E.M. Results were compared using one-way ANOVA with post hoc analysis using Tukey's test using Prism 2.0 statistical analysis package (Graph Pad Software Inc., NY, USA).


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 5 Summary
 References
 
3.1 Effect of free radical scavengers on cardioprotection
Fig. 1 shows the effect of preincubation of cells with diazoxide in the presence or absence of MPG or NAC. Incubation with diazoxide (30 µM) for 15 min immediately prior to simulated ischemia/reoxygenation resulted in significant protection against cell death (13.9±0.9% pI positive vs. 36.9±4.5% pI positive controls, P<0.001). This protection was abolished by prior incubation with either 2-MPG or NAC (29±4.0% cell death and 30.1±2.6%, respectively). Similarly incubation with the mitoKATP channel blocker 5-HD abolished protection (cell death 33.3±3.6% vs. diazoxide 13.9±0.9%, P<0.001). Neither 5-HD, 2-MPG or NAC had an independent effect of cell viability (see Fig. 1).


Figure 1
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  		Fig. 1 Effects on cell viability of administration of diazoxide for 15 min immediately prior to simulated ischaemia ("late"). I/R: simulated ischemia/reoxygenation; DZ: diazoxide 30 µM; 5-HD: 5-hydroxydecanoate sodium 50 µM, MPG: 2-mercaptopropionyl glycine 400 µM, NAC: N-acetylcysteine 2 mM. Data are mean±S.E.M. *=P<0.001 vs. I/R.

 
Fig. 2 demonstrates the effect of a "pulse" of preincubation with diazoxide in the presence or absence of MPG or NAC. Cells were incubated with diazoxide (30 µM) for 15 min followed by washing and a 15 min interval prior to simulated ischemia/reoxygenation. This resulted in significant protection against cell death (17.8±1.8% pI positive vs. 36.9±4.5% pI positive controls, P<0.001). This protection was abolished by prior incubation with either 2-MPG or NAC (31.7±3.5% cell death and 31.8±2.7%, respectively). Similarly incubation with the mitoKATP channel blocker 5-HD abolished protection (cell death 38.3±3.9% vs. diazoxide 17.8±1.8%, P<0.001). This indicates that as well as a cardioprotective role as mediator or end-effector, activation of the mitoKATP channel early can also act as a trigger of cytoprotection. Neither 5-HD, 2-MPG or NAC had an independent effect of cell viability (see Fig. 2).


Figure 2
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  		Fig. 2 Effects on cell viability of diazoxide for 15 min administered as a "pulse" followed by washout and 15 min drug-free interval. I/R: simulated ischemia/reoxygenation; DZ: diazoxide 30 µM; 5-HD: 5-hydroxydecanoate sodium 50 µM, MPG: 2-mercaptopropionyl glycine 400 µM, NAC: N-acetylcysteine 2 mM. Data are mean±S.E.M. *=P<0.001 vs. I/R.

 
3.2 Effect of diazoxide, 5-HD and free radical scavengers on ROS production
Fig. 3 demonstrates that incubation of cells with diazoxide under normoxic conditions independent of simulated ischaemia/reperfusion, caused significant generation of free radicals as measured by cumulative generation of the fluorescent mitotracker orange from its non-fluorescent precursor. Cells with no intervention generate a basal level of free radicals as seen by the increase in mean fluorescence intensity in channel FL2 to 49.5 arbitrary units (AU). Incubation with diazoxide caused a significant increase in free radical production (diazoxide 65.2 vs. control 49.5 AU, respectively, P<0.001). This increase was abolished by the free radical scavengers 2-MPG and NAC (45.17 and 53 AU, respectively versus diazoxide, 65.2 AU). Similarly, this increase was abolished by concomitant incubation with 5-HD (45.2 AU vs. diazoxide, 65.2 AU). This shows that opening of the KATP channel directly with diazoxide can generate free radical production, the likely source of which is the mitochondrion, and this generation can be inhibited by the KATP channel blocker 5-HD. Fig. 4 expresses the generation of ROS as a function of the generation in the control cell group.


Figure 3
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  		Fig. 3 Increase in fluorescence signal of mitotracker orange probe over 15 min incubation. Control: cells with no intervention; DZ: diazoxide 30 µM; 5-HD: 5-hydroxydecanoate sodium 50 µM, MPG: 2-mercaptopropionyl glycine 400 µM, NAC: N-acetylcysteine 2 mM. Data are mean±S.E.M. *=P<0.001 vs. control cells.

 

Figure 4
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  		Fig. 4 Relative change in ROS production expressed proportionally to control ROS generation. Values are mean±coefficient of variation relative to control mean. *=P<0.001 vs. control cells.

 
3.3 Effect of diazoxide on index of mitochondrial mass and membrane potential
Fig. 5 demonstrates the effect of interventions on mitochondrial membrane potential as measured by JC-1. Incubation with diazoxide, 5-HD and the combination of diazoxide and 5-HD had no significant effect on membrane potential. The protonophore CCCP had a dramatic effect on membrane potential; Fig. 5 shows the effects of 15 min incubation with 100 µM CCCP. Mitochondrial mass as measured by nonyl acridine orange fluorescence showed no significant change with any intervention. However when there was complete collapse of the mitochondrial membrane potential with CCCP at 2 mM, a significant decrease in nonyl acridine orange fluorescence is seen, indicating disruption of mitochondrial membrane lipid integrity and loss of the nonyl~lipid binding (data not shown). These results indicate that in the intact cell, diazoxide causes no change in mitochondrial membrane potential or mass.


Figure 5
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  		Fig. 5 Value of 520 nm light scatter analysis. DZ: diazoxide 30 µM; CCCP: carbonyl cyanide chlorophenylhydrazone 100 µM; 5-HD: 5-hydroxydecanoate sodium 50 µM. *=P<0.001 vs. control cells.

 
3.4 Effect of diazoxide on mitochondrial volume as measured by changes in light scattering
Fig. 6 demonstrates the effect of changes in light scattering by intact cells. Results are derived from the relationship between narrow angle scatter (FS) and wide angle scatter (SS) and expressed as arbitrary units. An increase in this value corresponds with an increase in mitochondrial volume. As can be seen, diazoxide causes a time dependent and significant increase in volume vs. controls. This increase is inhibited by concomitant incubation with 5-HD. The protonophore CCCP at 100 µM caused the most swelling and this was reversible when the compound was washed out. No change was seen in the event flight time value (directly proportional to cell size) with any interaction. These results indicate that incubation with diazoxide causes an increase in matrix volume as measured by increase in forward light scattering with no significant change in overall cell volume and this effect is abrogated by incubation with 5-HD.


Figure 6
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  		Fig. 6 Change in JC-1 mean fluorescence intensity in FL2 detector (orange signal) vs. time. Data are mean±S.E.M. *=P<0.001 vs. control cells.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
5 Summary
 References
 
4.1 Cytoprotection by diazoxide: role of mitochondrial KATP
We have previously demonstrated that diazoxide is protective in a human cardiac derived cell line of simulated ischaemia and reoxygenation [20]. We have now shown that the protective effects of diazoxide can be abolished by two chemically distinct free radical scavengers, 2-MPG and NAC. This is in keeping with others who have shown the ability of both NAC [15] and 2-MPG [16], to abolish diazoxide mediated cardioprotection in ex-vivo models, alluding to a role for free radicals in the observed protection. To our knowledge, this is the first evidence directly demonstrating the ability of diazoxide to generate free radicals and the ability of 5-hydroxydecanoate and free radical scavengers to abolish this effect.

4.2 Generation of free radicals
In our experimental system we have demonstrated the ability of diazoxide to directly cause the generation of ROS in an isolated cardiocytes. Other triggers of preconditioning such as hypoxic incubation [14] and acetylcholine [9] have been shown to cause free radical generation in cardiocytes but only indirect evidence exists as to the role of free radical generation in diazoxide mediated cardioprotection [15,16]. Using the probe reduced mitotracker orange, which is a non-specific marker of free radical generation, we were able to detect free radicals that did not effect generation of ethidium from dihydroethidium, which may otherwise have remained undetected in other model systems. Of note, we were not able to measure any significant generation of ethidium from dihydroethidium in this model, similarly we found a variety of reduced fluorescein analogues unreliable due to product leakage in this cell model. This probe is most sensitive to the superoxide moiety, suggesting that if superoxide was being generated, the kinetics of its dismutation by intracellular enzyme systems was such that the kinetics of reaction with the probe were unfavourable.

As yet there is no consenus as to the mechanism(s) of free radical generation by diazoxide. One potential explanation may be "functional uncoupling" of electron transport and oxidative phosphorylation [23] which could generate superoxide radical which would be rapidly dismuted by mitochondrial superoxide dismutase to hydrogen peroxide with further reactions generating hydroxyl radicals. However we accept other possibilities exist, such as modification of ion transporters or channels in the inner mitochondrial membrane which are responsible for the generation of free radicals. However in the absence of other documented intracellular effects of diazoxide we propose the hypothesis of functional uncoupling to be responsible for this free radical release from mitochondria. We acknowledge that there may be other effects of diazoxide in isolated mitochondria. This was recently observed in a study in using saponin-permeabilized cardiomyocytes incubated with diazoxide in which a reduction in mitochondrial basal and maximal respiration was seen [24].

Furthermore it is also unclear how free radicals may confer protection, but by activation of protein kinase C [25], and stimulation of subsequent signaling cascades, they could trigger hyperphosphorylation of small heat shock proteins [26], actin or both, with a concomitant increase in the ability of the cell to withstand osmotic and subsequent free radical-induced stresses during hypoxia and reoxygenation.

4.3 Changes in mitochondrial membrane potential, mass and volume
4.3.1 Membrane potential
There are limited data on the effects of diazoxide, 5-hydroxydecanoate or simulated ischemic preconditioning on the mitochondrial membrane potential of cardiac mitochondria either in isolation or, more particularly, in situ in the intact cell. We have measured the effect of a variety of interventions in situ using the ratiometric cationic fluorophore, JC-1. Normally this compound exists as a monomer with peak emission at 520 nm (green). However when the compound enters the negatively charged mitochondrial matrix, it forms oligomers or "J-complexes" with a shift in emission to 560 nm (orange). This shift in fluorescence can readily be measured by flow cytometry. A number of workers have found an excellent correlation between the ratiometric analysis of JC-1 spectra and more direct measurements of mitochondrial membrane potential such as the tetraphenylphosphonium electrode [27–30]. Comparative studies have been performed using other markers of membrane potential such as Rhodamine 123 and DiOC6(3) which show that other factors such as loading conditions and binding to non-mitochondrial organelles and proteins can compromise the accuracy of Rhodamine 123 and DiOC6(3) [31]. With adequate compensation and correct cell loading, the ratiometric signature allows cell by cell analysis of mean mitochondrial transmembrane potential in situ. More recent work by DiLisa et al. has indicated that the two forms of the compound may have different sensitivities to mitochondrial membrane potential, with the monomer being more sensitive to potentials below 140 mV [32]. We found no significant changes in membrane potential with the cardioprotective concentration of diazoxide (30 µM) or concentrations of diazoxide up to 300 µM in this model (data not shown). This is in contrast to analysis of the effects of higher concentrations of potassium channel openers on membrane potential of isolated liver mitochondria measured by the TPP electrode method [33], but it seems more likely that the opening of the mitochondrial KATP channel will result in change in volume rather than change in membrane potential. We appreciate that there are potential secondary effects of high concentrations of carbocyanine dyes such as JC-1 on mitochondrial metabolism but feel due to the rapidity of the assays used and the fact that we were able to demonstrate reversible depolarization of mitochondrial membrane potential with recovery following CCCP incubation that effects in our model were not significant.

We appreciate that our data contrasts with work published by Holmuhamedov et al. which showed in their model that high concentrations of potassium channel openers were able to depolarise isolated mitochondria in suspension and isolated rat neonatal cardiomyocytes grown on cover slips. It may be that in their model differing measurement environments as well as the use of potassium channel openers at higher concentrations than used in our study may have allowed for non-specific effects to occur. In must also be remembered that in our study we examined membrane potential of mitochondria in situ in the intact cell. Using isolated mitochondria we have seen similar results to those of Holmuhamedov and colleagues (data not shown).

4.3.2 Mitochondrial mass
We employed the agent nonyl acridine orange in order to evaluate mitochondrial mass. This compound binds stoichiometrically to cardiolipin, a membrane lipid found exclusively in the mitochondrial inner membrane and fluoresces at 520 nm. The relative fluorescence intensity generated per cell has been shown to be directly proportional to mitochondrial mass [34–37]. We found that none of the interventions generating cardioprotection altered mitochondrial mass.

4.3.3 Changes in matrix volume
The effect of changes in mitochondrial matrix on light scattering is complex [38]. A number of studies have correlated an increase in matrix volume measured by direct techniques involving cell disruption with a decrease in light scattering measured at 520 nm in cells [39–41]. The ability of flow cytometric techniques to measure this parameter in cells [42] allows rapid measurements of relative changes of matrix volume to be performed in situ without disruption of the cell. We observed a relative increase in forward scatter signal in keeping with a smaller increase in side scatter signal during interventions known to cause mitochondrial swelling such as CCCP administration [43] and this correlated with the transition from thin, filamentous mitochondria to larger, punctate mitochondria when cells loaded with JC-1 were viewed under the fluorescent microscope (data not shown). By virtue of the fact that the Bryte HS flow Cytometer collects light at all angles >1.5° in the side scatter detector, the relative changes in the ratios of forward to side scatter will differ from that of other flow cytometers that collect only signals >85° for side scatter analysis [42]. To account for this, we used off-line discriminant function analysis to express the measured change in light scatter with a major component provided by the increase in forward scatter, equivalent to the decrease in absorbance seen in other systems [42]. Opening of the mitoKATP channel causes influx of the K+ anion accompanied by various cations, the overall effect being a change in volume of the matrix, not a change in membrane potential that could be reliably measured (estimated to be about 3%) [17]. This change in volume can be followed in real time by analysis of change in the pattern of light scatter signatures of cells in suspension [38], and has been shown to increase the rate of ATP synthesis and a variety of enzyme activities in the mitochondria of isolated hepatocytes [41], but little is known of the effect of this process in the cardiomyocyte in situ. Others have measured the effects of mitochondrial swelling on light scatter parameters in intact cells using flow cytometry and found comparable results in different cell lines [42]. This technique lends itself to rapid and repeated measures of cells in suspension, and has a high degree of reproducibility. We chose CCCP 100 µM as a control due to the fact that the rapid contraction of the matrix is followed by rapid secondary swelling of such a magnitude that can be observed by fluorescence microscopy. If one is to extrapolate the relationship between measured changes in matrix volume and light scattering, the change in light scattering can be used as a semi-quantitative index of change in matrix volume provided all other parameters are uniform [41,42].

We have shown a significant change in the light scatter characteristics of our cells at 520–540 nm that cannot be accounted for by change in cell volume or size or any other measurable parameter. We are satisfied therefore, that this indicates a change in matrix volume.

Recent work by Garlid et al. [44], showed an increase in mitochondrial volume with incubation of low concentrations of mitochondrial KATP channel openers in isolated rat mitochondria.

Our study however is the first study to demonstrate that low doses of mitochondrial KATP channel openers are able to induce mitochondrial swelling and hence increase mitochondrial volume in the intact human cardiac derived cell.


   5 Summary
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 5 Summary
References
 
In this study we have shown that treatment with the mitoKATP channel opener diazoxide is able to act as a trigger of preconditioning as well as being cardioprotective in our cell model. This is the first study to demonstrate that this protection involves the generation of free radicals by the mitochondria in tandem with a change in matrix volume both of which can be abolished by incubation with 5-HD and specific free radical scavengers, NAC and MPG. The effect observed is independent of a change in mitochondrial membrane potential as measured by a ratiometric fluorescent probe in situ. We believe we have demonstrated a common mechanism by which the mitoKATP channel openers and certain triggers of preconditioning effect protection.

Time for primary review 32 days.


   Acknowledgements
 
We are grateful to the British Heart Foundation for continued support.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 5 Summary
 References
 

  1. Murry C.E, Jennings R.B, Reimer K.A. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation (1986) 74:1124–1136.[Abstract/Free Full Text]
  2. Baxter G.F, Yellon D.M. Delayed myocardial protection following ischaemic preconditioning. Basic Res. Cardiol. (1996) 91(53–56):8300–8428.
  3. Sumeray M, Yellon D. Myocardial preconditioning. What have we learned? Eur. Heart J. (1997) 18:A8–A14.[ISI][Medline]
  4. Yellon D.M, Baxter G.F. A "second window of protection" or delayed preconditioning phenomenon: future horizons for myocardial protection? J. Mol. Cell Cardiol. (1995) 27:1023–1034.[CrossRef][ISI][Medline]
  5. Grover G, Garlid K. ATP-sensitive potassium channels: A review of their cardioprotective pharmacology. J. Mol. Cell Cardiol. (2000) 32:677–695.[CrossRef][ISI][Medline]
  6. Grover G.J. Pharmacology of ATP-sensitive potassium channel (KATP) openers in models of myocardial ischemia and reperfusion. Can. J. Physiol. Pharmacol. (1997) 75:309–315.[CrossRef][ISI][Medline]
  7. McPherson A.B, Yao Z. Morphine mimics preconditioning via free radical signals and mitochondrial KATP in myocytes. Circulation (2001) 103:290–295.[Abstract/Free Full Text]
  8. Liang B.T, Gross G.J. Direct preconditioning of cardiac myocytes via opioid receptors and KATP channels. Circ. Res. (1999) 84:1396–1400.[Abstract/Free Full Text]
  9. Yao Z, Tong J, Tan X, Li C, Shao Z, Kim W.C, Vanden Hoek T.L, Becker L.B, Head C.A, Schumacker P.T. Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes. Am. J. Physiol. (1999) 277:H2504–H2509.[ISI][Medline]
  10. Garlid K.D, Paucek P, Yarov-Yarovoy V, Murray H.N, Darbenzio R.B.A.J.D.A, Lodge N.J, Smith M.A, Grover G.J. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ. Res. (1997) 81:1072–1082.[Abstract/Free Full Text]
  11. Tritto I, D'Andrea D, Eramo N, Scognamiglio A, De Simone C, Violante A, Esposito A, Chiariello M, Ambrosio G. Oxygen radicals can induce preconditioning in rabbit hearts. Circ. Res. (1997) 80:743–748.[Abstract/Free Full Text]
  12. Baines C.P, Goto M, Downey J.M. Oxygen radicals released during ischemic preconditioning contribute to cardioprotection in the rabbit myocardium. J. Mol. Cell Cardiol. (1997) 29:207–216.[CrossRef][ISI][Medline]
  13. Zhou X, Zhai X, Ashraf M. Direct evidence that initial oxidative stress triggered by preconditioning contributes to second window of protection by endogenous antioxidant enzyme in myocytes. Circulation (1996) 93:1177–1184.[Abstract/Free Full Text]
  14. Vanden Hoek T.L, Becker L.B, Shao Z, Li C, Schumacker P.T. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J. Biol. Chem. (1998) 273:18092–18098.[Abstract/Free Full Text]
  15. Forbes A, Steenbergen C, London R, Murphy E. The cardioprotective effect of diazoxide involves signalling by reactive oxygen species. J. Mol. Cell Cardiol. (abstract) (1999) 31:A33.
  16. Yang X, Pain T, Heusch G, Cohen M, Downey J. Diazoxide protects the heart by release of free radicals. J. Mol. Cell Cardiol. (abstract) (2000) 32:A49.
  17. Garlid K.D. Cation transport in mitochondria — the potassium cycle. Biochim. Biophys. Acta (1996) 1275:123–126.[Medline]
  18. Dawson T.L, Gores G.J, Nieminen A.L, Herman B, Lemasters J.J. Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am. J. Physiol. (1993) 264:C961–C967.[ISI][Medline]
  19. Turrens J.F, Alexandre A, Lehninger A.L. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch. Biochem. Biophys. (1985) 237:408–414.[CrossRef][ISI][Medline]
  20. Carroll R, Yellon D.M. Delayed cardioprotection in a human cardiomyocyte-derived cell line: the role of adenosine, p38 MAP kinase and mitochondrial KATP. Basic Res. Cardiol. (2000) 95:243–249.[CrossRef][ISI][Medline]
  21. Carroll R, Gant A, Yellon D. Delayed preconditioning and diazoxide-mediated cytoprotection occur independently of a change of mitochondrial membrane potential in a human cardiac cell line. Br. J. Pharm. 2000:C14.
  22. Esumi K, Nishida M, Shaw D, Smith T.W, Marsh J.D. NADH measurements in adult rat myocytes during simulated ischemia. Am. J. Physiol. (1991) 260:H1743–H1752.[ISI][Medline]
  23. Marber M.S. Ischemic preconditioning in isolated cells. Circ. Res. (2000) 86:926–931.[Free Full Text]
  24. Ovide-Bordeaux S, Ventura-Clapier R, Veksler V. Do modulators of the mitochondrial KATP channel change the function of mitochondria in situ. J. Biol. Chem. (2000) 275:37291–37295.[Abstract/Free Full Text]
  25. Takashi E, Wang Y, Ashraf M. Activation of mitochondrial K (ATP) channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway (see comments). Circ. Res. (1999) 85:1146–1153.[Abstract/Free Full Text]
  26. Dana A, Skarli M, Papakrivopoulou J, Yellon D.M. Adenosine A(1) receptor induced delayed preconditioning in rabbits: induction of p38 mitogen-activated protein kinase activation and Hsp27 phosphorylation via a tyrosine kinase- and protein kinase C-dependent mechanism. Circ. Res. (2000) 86:989–997.[Abstract/Free Full Text]
  27. Reers M, Smith T.W, Chen L.B. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry (1991) 30:4480–4486.[CrossRef][ISI][Medline]
  28. Smiley S.T, Reers M, Mottola-Hartshorn C, Lin M, Chen A, Smith T.W, Steele G.D Jr., Chen L.B. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. U.S.A. (1991) 88:3671–3675.[Abstract/Free Full Text]
  29. Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. (1993) 197:40–45.[CrossRef][ISI][Medline]
  30. Reers M, Smiley S.T, Mottola-Hartshorn C, Chen A, Lin M, Chen L.B. Mitochondrial membrane potential monitored by JC-1 dye. Methods Enzymol. (1995) 260:406–417.[ISI][Medline]
  31. Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. (1997) 411:77–82.[CrossRef][ISI][Medline]
  32. Di Lisa F, Blank P.S, Colonna R, Gambassi G, Silverman H.S, Stern M.D, Hansford R.G. Mitochondrial membrane potential in single living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition. J. Physiol. (Lond). (1995) 486:1–13.[Abstract/Free Full Text]
  33. Holmuhamedov E.L, Jovanovic S, Dzeja P.P, Jovanovic A, Terzic A. Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function. Am. J. Physiol. (1998) 275:H1567–H1576.[ISI][Medline]
  34. Cossarizza A, Cooper E.L, Quaglino D, Salvioli S, Kalachnikova G, Franceschi C. Mitochondrial mass and membrane potential in coelomocytes from the earthworm Eisenia foetida: studies with fluorescent probes in single intact cells. Biochem. Biophys. Res. Commun. (1995) 214:503–510.[CrossRef][ISI][Medline]
  35. Mancini M, Anderson B.O, Caldwell E, Sedghinasab M, Paty P.B, Hockenbery D.M. Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line. J. Cell Biol. (1997) 138:449–469.[Abstract/Free Full Text]
  36. Metivier D, Dallaporta B, Zamzami N, Larochette N, Susin S.A, Marzo I, Kroemer G. Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes. Immunol. Lett. (1998) 61:157–163.[CrossRef][ISI][Medline]
  37. Virag L, Salzman A.L, Szabo C. Poly(ADP-ribose) synthetase activation mediates mitochondrial injury during oxidant-induced cell death. J. Immunol. (1998) 161:3753–3759.[Abstract/Free Full Text]
  38. Beavis A.D, Brannan R.D, Garlid K.D. Swelling and contraction of the mitochondrial matrix I. A structural interpretation of the relationship between light scattering and matrix volume. J. Biol. Chem. (1985) 260:13424–13433.[Abstract/Free Full Text]
  39. Garlid K.D, Beavis A.D. Swelling and contraction of the mitochondrial matrix II. Quantitative application of the light scattering technique to solute transport across the inner membrane. J. Biol. Chem. (1985) 260:13434–13441.[Abstract/Free Full Text]
  40. Quinlan P, Thomas A, Armston A, Halestrap A. Measurement of the intramitochondrial volume in hepatocytes without cell disruption and its elevation by hormones and valinomycin. Biochem. J. (1983) 214:395–404.[ISI][Medline]
  41. Halestrap A. The regulation of the matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism. Biochemica et Biophysica Acta (1989) 973:355–382.[Medline]
  42. Macouillard-Poulletier de G, Belaud-Rotureau M.A, Voisin P, Leducq N, Belloc F, Canioni P, Diolez P. Flow cytometric analysis of mitochondrial activity in situ: application to acetylceramide-induced mitochondrial swelling and apoptosis. Cytometry (1998) 33:333–339.[CrossRef][ISI][Medline]
  43. Minamikawa T, Williams D.A, Bowser D.N, Nagley P. Mitochondrial permeability transition and swelling can occur reversibly without inducing cell death in intact human cells. Exp. Cell Res. (1999) 246:26–37.[CrossRef][ISI][Medline]
  44. Kowaltowski A.J, Seetharaman S, Paucek P, Garlid K.D. Bioenergetic consequences of opening the ATP sensitive K+ channel in heart mitochondria. Am. J. Physiol. (2001) 280:H649–H657.[ISI]

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