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Am J Physiol Heart Circ Physiol 280: H649-H657, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 2, H649-H657, February 2001

Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria

Alicia J. Kowaltowski, Subramaniam Seetharaman, Petr Paucek, and Keith D. Garlid

Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There is an emerging consensus that pharmacological opening of the mitochondrial ATP-sensitive K+ (KATP) channel protects the heart against ischemia-reperfusion damage; however, there are widely divergent views on the effects of openers on isolated heart mitochondria. We have examined the effects of diazoxide and pinacidil on the bioenergetic properties of rat heart mitochondria. As expected of hydrophobic compounds, these drugs have toxic, as well as pharmacological, effects on mitochondria. Both drugs inhibit respiration and increase membrane proton permeability as a function of concentration, causing a decrease in mitochondrial membrane potential and a consequent decrease in Ca2+ uptake, but these effects are not caused by opening mitochondrial KATP channels. In pharmacological doses (<50 µM), both drugs open mitochondrial KATP channels, and resulting changes in membrane potential and respiration are minimal. The increased K+ influx associated with mitochondrial KATP channel opening is ~30 nmol · min-1 · mg-1, a very low rate that will depolarize by only 1-2 mV. However, this increase in K+ influx causes a significant increase in matrix volume. The volume increase is sufficient to reverse matrix contraction caused by oxidative phosphorylation and can be observed even when respiration is inhibited and the membrane potential is supported by ATP hydrolysis, conditions expected during ischemia. Thus opening mitochondrial KATP channels has little direct effect on respiration, membrane potential, or Ca2+ uptake but has important effects on matrix and intermembrane space volumes.

myocardial injury; potassium; cardioprotection; ischemia-reperfusion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SINCE 1989, many laboratories have shown that openers of ATP-sensitive K+ (KATP) channels protect the heart against ischemia-reperfusion injury (4, 6, 15, 16, 18). Although attention was initially focused on the sarcolemmal KATP channel, recent evidence strongly suggests that the mitochondrial KATP channel is the receptor for cardioprotective KATP openers (13, 14). An important basis for this hypothesis was the finding in heart that diazoxide is highly selective for mitochondrial KATP channels, having no effect on sarcolemmal KATP channels in the therapeutic dose range; nevertheless, diazoxide was protective in a model of ischemia-reperfusion (13). Moreover, 5-hydroxydecanoate, a mitochondria-specific KATP channel blocker (26), was found to block cardioprotection by KATP channel openers (13, 14).

Since these findings appeared, KATP channel openers have continued to play a major role in the effort to understand the mechanisms of cardioprotection. A major barrier to achieving this goal is the continued uncertainty about the effects of these drugs on mitochondria. Garlid (8) proposed that mitochondrial KATP channels are primarily involved in the volume regulation mechanism and that direct bioenergetic effects of opening mitochondrial KATP channels would be small. Several recent reports appear to contradict this prediction by showing large decreases in mitochondrial membrane potential (Delta Psi ) after treatment with a variety of KATP channel openers (24, 25, 29, 40). Thus, Liu et al. (29), using an indirect assay, reported profound uncoupling of mitochondria in cardiomyocytes after administration of high doses of diazoxide and pinacidil and proposed that this uncoupling mediates cardioprotection by reducing Ca2+ uptake into mitochondria. In apparent support of this hypothesis, Holmuhamedov et al. (24, 25) demonstrated membrane depolarization and inhibition of Ca2+ uptake in the presence of mitochondrial KATP channel openers in isolated rat heart mitochondria. Thus two conflicting hypotheses have been advanced to explain what happens when mitochondrial KATP channels are opened. To determine which of these hypotheses is correct, we investigated the effects of diazoxide and pinacidil on isolated rat heart mitochondria.

We show first that the reported dramatic bioenergetic effects of these drugs (24, 25) have nothing to do with mitochondrial KATP channels but, rather, are caused by the intrinsic uncoupling and inhibitory properties of these agents when used at excessive doses. We show that regulated K+ influx via mitochondrial KATP channels is small in magnitude, causing minimal stimulation of respiration and an estimated depolarization of only 1-2 mV. Finally, we show that opening and closing mitochondrial KATP channels causes significant changes in matrix volume, secondary to the entrance of osmotically obligated water accompanying net flux of K+ and anions.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitochondrial isolation. Mitochondria were isolated by differential centrifugation from rat heart (34). Briefly, two or three rat hearts were washed in ice-cold buffer containing 300 mM sucrose, 10 mM K+-HEPES buffer, pH 7.2, and 1 mM K+-EGTA. After the tissue was finely minced, it was incubated for 10 min in the presence of 2-4 mg of nagarse. Excess nagarse was removed by washing in the same buffer supplemented with 1 mg/ml BSA, and the samples were homogenized. The suspension was then centrifuged for 4 min at 600 g. The resulting supernatants were recentrifuged at 9,000 g for 8 min. The mitochondrial pellet was then washed once or twice until a blood-free, compact pellet was obtained. The final pellet was suspended at ~35 mg/ml and kept over ice.

Measurement of Delta Psi . Delta Psi was estimated by measuring the distribution of 1 µM tetraphenylphosphonium (TPP+) using a TPP+-selective electrode prepared in our laboratory. Delta Psi was calculated according to Jensen et al. (27) assuming a matrix volume of 1 µl/mg protein. Alterations in mitochondrial volume due to mitochondrial KATP channel activity, as determined by quantitative light scattering (2), could not alter values of Delta Psi by >4 mV.

Measurement of mitochondrial Ca2+ uptake. Mitochondrial Ca2+ uptake was estimated from fluorescence changes of 0.1 µM Ca2+ green 5N (hexapotassium salt), using an Aminco SLM 8000 fluorescence spectrophotometer at excitation and emission wavelengths of 506 and 531 nm, respectively. Calibration was performed by addition of known quantities of CaCl2.

Measurement of mitochondrial respiration. Respiration was measured using a Yellow Springs Instruments oxygen electrode.

Measurement of mitochondrial volume. Changes in mitochondrial volume, which accompany net salt transport into mitochondria, were followed using a quantitative light-scattering technique (2, 26). This technique is based on the principle that reciprocal absorbance of the mitochondrial suspension, when corrected for the extrapolated absorbance at infinite protein concentration, is linearly related to matrix volume within well-defined regions, as described in detail by Beavis et al. (1) and Garlid and Beavis (10). Note that light scattering measures total particle volume. In the isotonic range, matrix swelling is largely compensated by intermembrane space (IMS) contraction, so that total volume changes are small (1), and the light-scattering traces have correspondingly low signal-to-noise characteristics (see Figs. 5-7).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

K+-independent uncoupling and respiratory inhibition by high doses of diazoxide and pinacidil. Holmuhamedov and co-workers (24, 25) reported that diazoxide and pinacidil cause a decrease in Delta Psi and Ca2+ uptake. We were able to reproduce these results under similar experimental conditions (Figs. 1 and 2). Two characteristics of these experiments suggested to us that these effects are unrelated to mitochondrial KATP channel opening: 1) Mitochondrial KATP channels are not sensitive to K+ channel openers under these conditions, because mitochondria incubated in the absence of ATP and Mg2+ present mitochondrial KATP channels in the open state (2, 14, 26). 2) Diazoxide or pinacidil at >50 µM was necessary to achieve significant decreases in Delta Psi and Ca2+ uptake rates (24, 25). These concentrations are in excess of those necessary to promote mitochondrial KATP channel opening. In rat heart mitochondria, diazoxide opens mitochondrial KATP channels with a half-maximal saturation (K1/2) of 2.3 µM (14) and pinacidil with a K1/2 of 11.7 ± 3.7 µM (n = 4; results not shown).


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  		Fig. 1.   Mitochondrial depolarization induced by high concentrations of diazoxide (A) or pinacidil (B). Rat heart mitochondria (0.5 mg/ml) were incubated in the presence of K+ () or Li+ () salts of Cl- (110 mM), succinate (5 mM), EGTA (100 µM), and MOPS buffer (10 mM), pH 7.2. Mitochondrial membrane potential (Delta Psi ) was estimated in the presence of the diazoxide or pinacidil concentrations shown. Similar results were obtained in 3 independent experiments.



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  		Fig. 2.   Inhibition of mitochondrial Ca2+ uptake by diazoxide (A) or pinacidil (B). Rat heart mitochondria (0.5 mg/ml) were incubated in the presence of 20 µM CaCl2 and K+ () or Li+ () salts of Cl- (110 mM), succinate (5 mM), and MOPS buffer (10 mM), pH 7.2. Ca2+ uptake was estimated in the presence of the diazoxide or pinacidil concentrations shown. Similar results were obtained in 3 independent experiments.

The suspicion that the decrease in Delta Psi and Ca2+ uptake is not due to mitochondrial KATP channel opening was confirmed by carrying out parallel experiments in a K+-free, Li+-based medium. Identical effects on Ca2+ uptake and Delta Psi were observed in K+ and Li+ media (Figs. 1 and 2). Since Li+ cannot be transported through mitochondrial KATP channels, the observed effects of high doses of diazoxide and pinacidil are demonstrably unrelated to mitochondrial KATP channel activity.

Mechanisms of toxicity of diazoxide and pinacidil in rat heart mitochondria. Further experiments were conducted to elucidate the mechanism of the effects described in Figs. 1 and 2. At high doses, pinacidil stimulated resting respiration, as reported by Holmuhamedov et al. (24), but diazoxide did not (Fig. 3). Both drugs decreased the maximal respiratory rates of mitochondria treated by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; Fig. 3). These effects are unrelated to mitochondrial KATP channel activity, because they were observed in K+ and Li+ salts (Fig. 3).


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  		Fig. 3.   Mitochondrial respiration in the presence of diazoxide (A) or pinacidil (B). Rat heart mitochondria (0.5 mg/ml) were incubated in K+ (squares) or Li+ (circles) salts of Cl- (110 mM), succinate (5 mM), EGTA (100 µM), and MOPS buffer (10 mM), pH 7.2, in the presence (open symbols) or absence (filled symbols) of 0.5 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Respiration was measured in the presence of the diazoxide or pinacidil concentrations shown. Similar results were obtained in 3 independent experiments.

At doses that reduced Delta Psi and Ca2+ uptake (Figs. 1 and 2), diazoxide did not stimulate resting respiration (Fig. 3). This finding is atypical of agents that are pure inhibitors of electron transport, which normally affect resting respiration before significant changes in Delta Psi occur (42). Accordingly, we investigated this phenomenon further. We assayed nonrespiring mitochondria in K+-acetate medium containing valinomycin, in which a protonophore is required for net salt and water uptake (12) (Fig. 4). The requirement for proton permeation is illustrated by the effects of FCCP, a known protonophore. The results show that diazoxide and pinacidil also increase membrane permeability to protons. The concentration dependence of the protonophoretic actions of the K+ channel openers is interesting. Unlike weak acid protonophores, such as FCCP, weak bases cannot transport protons by a cycling mechanism, because they cannot delocalize the protonic charge. Rather, they form transient multimers that partially span the membrane, thereby creating a series of energy wells along which the protons can jump. This property is responsible for the nonlinear concentration dependences exhibited by diazoxide and pinacidil in Fig. 4. A similar phenomenon and mechanism were observed for uncoupling by bupivacaine (39). Independently of mechanism, a combination of respiratory inhibition and increased proton permeability can readily account for the decrease in Delta Psi observed in Fig. 1. This, in turn, is sufficient to account for reduced Ca2+ uptake (Fig. 2), because Delta Psi is the driving force for mitochondrial Ca2+ uptake (22).


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  		Fig. 4.   Protonophoretic effects of diazoxide and pinacidil. Initial rates of matrix swelling (dW/dt) are plotted vs. concentrations of FCCP (), pinacidil (), or diazoxide (black-triangle). The principle of the assay is that valinomycin-induced swelling due to uptake of K+-acetate and water requires the presence of a protonophore (12). Rat heart mitochondria (0.1 mg/ml) were incubated in K+ salts of acetate (55 mM), HEPES (10 mM, pH 7.2), and EGTA (0.1 mM) in the presence of 2 µM antimycin A. The reaction was initiated by addition of 1 µM valinomycin. Solid curves are regression fits to the following equation: dW/dt = A * [pronotophore]n, where A is absorbance and n = 1, 2, and 3, for FCCP, pinacidil, and diazoxide, respectively. Similar results were obtained in 3 independent experiments.

These data show that the toxicity of diazoxide and pinacidil may be attributed to uncoupling, due to an intrinsic protonophoretic property of the drugs, and respiratory inhibition, a common feature of all hydrophobic drugs (30). Ca2+ uptake is a useful parameter for toxicity studies, because it is affected by uncoupling and respiratory chain inhibition. Diazoxide at <100 µM caused no depression of Ca2+ uptake, whereas pinacidil began to depress Ca2+ uptake at 50 µM (Fig. 2, inset). The margin of safety for opening mitochondrial KATP channels is therefore exceedingly low for pinacidil, with K1/2 of ~12 µM for opening mitochondrial KATP channels.

Mitochondrial KATP channels regulate mitochondrial volume. We previously showed in rat liver mitochondria that matrix volume changes secondary to the uptake of K+, but not tetraethylammonium (TEA+), salts are mediated by mitochondrial KATP channel openers (14, 26). The traces in Fig. 5 follow the volume changes due to opening and closing mitochondrial KATP channels in rat heart mitochondria. The initial massive uptake of K+ salts and water essentially reverses the losses that occurred during mitochondrial isolation in K+-free medium. K+ loss via the K+/H+ antiporter continues during isolation until free matrix Mg2+ rises to a level sufficient to block the carrier (8). When the contracted mitochondria are now allowed to respire in K+ medium, as in Fig. 5, they rapidly take up K+ salts and water until a steady-state volume is reached, a process requiring ~3-4 min at 25°C (Fig. 5).


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  		Fig. 5.   Effect of diazoxide on mitochondrial volume (Wm). Rat heart mitochondria (0.1 mg/ml) were incubated in K+ salts of Cl- (135 mM), succinate (5 mM), Pi (2.5 mM), and EGTA (100 µM), pH 7.2, in the presence of 0.5 mM MgCl2, 0.5 µg/ml oligomycin, and no further additions (a), 200 µM ATP (b), 200 µM ATP + 30 µM diazoxide (DZX, c), or 200 µM ATP + 30 µM diazoxide + 300 µM 5-hydroxydecanoate (5-HD, d). Mitochondrial volume changes were determined through quantitative light scattering as described previously (10). Similar results were obtained in 3 independent experiments.

In the absence of ATP (Fig. 5, trace a), the mitochondrial KATP channel is open, and mitochondria take up a larger amount of K+ and water to arrive at a higher steady-state volume than that observed in the presence of ATP (trace b), which inhibits mitochondrial KATP channel-dependent K+ uptake. Concomitant addition of diazoxide (trace c) reverses the ATP inhibition due to mitochondrial KATP channel opening. No effect of diazoxide is observed when it is added to mitochondria in the absence of ATP, because mitochondrial KATP channels are already open (14, 26). Finally, the diazoxide effect is reversed by the addition of the mitochondrial KATP channel inhibitor 5-hydroxydecanoate (trace d). Thus, although the mitochondrial KATP channel is not responsible for the large changes in Delta Psi and Ca2+ accumulation observed in Figs. 1 and 2, it does promote significant changes in matrix volume, amounting to 20% of the maximum steady-state water content.

K+ uptake through mitochondrial KATP channels may be an important mechanism to maintain mitochondrial volume under physiological conditions in which Delta Psi falls. In Fig. 6, we show that the volume of mitochondria incubated in the presence of ATP (trace a) is larger than that of mitochondria incubated in the presence of both ATP and ADP (trace b). This volume decrease is caused by a sharp reduction in K+ uptake secondary to the decrease in Delta Psi that normally attends high rates of oxidative phosphorylation. Indeed, it is not observed when oxidative phosphorylation is prevented by oligomycin (trace c). Diazoxide (trace d) prevents the contraction caused by oxidative phosphorylation and reestablishes a steady-state volume similar to that observed in the absence of ADP. This effect of diazoxide is prevented by 5-hydroxydecanoate (trace e).


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  		Fig. 6.   Effect of diazoxide on mitochondrial volume during oxidative phosphorylation. Rat heart mitochondria (0.1 mg/ml) were incubated in K+ salts of Cl- (135 mM), succinate (5 mM), Pi (2.5 mM), EGTA (100 µM), pH 7.2, 0.5 mM MgCl2, and 200 µM ATP in the presence of 0.5 µg/ml oligomycin (a), 1 mM ADP (b), 1 mM ADP + 0.5 µg/ml oligomycin (c), 1 mM ADP + 30 µM diazoxide (d), or 1 mM ADP + 30 µM diazoxide + 300 µM 5-hydroxydecanoate (e). Mitochondrial volume changes were determined through quantitative light scattering as described previously (10). Similar results were obtained in 3 independent experiments.

The volume changes shown in Figs. 5 and 6 are dependent on the presence of a Delta Psi , which provides the driving force for K+ uptake. During myocardial ischemia, the respiratory chain cannot pump protons, because no oxygen is available as an electron acceptor. Under these conditions, hydrolysis of cellular ATP by the proton-pumping ATP synthase should be sufficient to drive K+ uptake via mitochondrial KATP channels and diffusion. Experimental support for this hypothesis is provided in Fig. 7. CN- was used to block mitochondrial respiration, and Delta Psi was supported solely by ATP hydrolysis. Under these conditions, the low matrix volume (trace a) was strongly increased by diazoxide (trace b), demonstrating that mitochondrial KATP channel opening was able to induce a functional response in the absence of respiration. In confirmation that the increased volume is due to mitochondrial KATP channel opening, 5-hydroxydecanoate completely prevented the diazoxide effect (trace c).


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  		Fig. 7.   Effect of diazoxide and respiratory inhibition on mitochondrial volume. Rat heart mitochondria (0.1 mg/ml) were incubated in K+ salts of Cl- (135 mM), succinate (10 mM), EGTA (100 µM), and Pi (2.5 mM), pH 7.2, in the presence of 1 mM MgCl2 and 2 mM ATP. KCN (500 µM) was added where indicated. Mitochondrial volume changes were determined in the presence of no further additions (a), 30 µM diazoxide (b), or 30 µM diazoxide + 300 µM 5-hydroxydecanoate (c). Similar results were obtained in 3 independent experiments.

Estimation of K+ flux through mitochondrial KATP channels. We investigated whether a change in Delta Psi could be detected under the conditions in which we observed mitochondrial volume changes attributable to mitochondrial KATP channel activity. We found that mitochondria incubated in K+ medium containing ATP do not undergo significant changes in Delta Psi when treated with low doses (<50 µM) of diazoxide or pinacidil (results not shown). Thus, even under conditions in which diazoxide or pinacidil is regulating mitochondrial KATP channels and mitochondrial volume, changes in Delta Psi are too small to be measured.

We were able to detect a small, reproducible decrease in tetramethylpentadecane (TMPD)/ascorbate-supported respiratory rate in the presence of ATP (Fig. 8). Confirming that this decreased respiration could be attributed to mitochondrial KATP channel closure, it was reversed by diazoxide in a manner sensitive to 5-hydroxydecanoate. No effects of ATP or drugs were seen when mitochondria were incubated in Li+ salts (Fig. 8). The mitochondrial KATP channel-dependent difference in TMPD/ascorbate-supported respiratory rates averaged 12.1 ng atom O · min-1 · mg-1. Considering that two protons are pumped at the level of cytochrome oxidase, this respiratory difference suggests that mitochondrial KATP channels are responsible for a flux of 24.2 nmol K+ · min-1 · mg-1.


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  		Fig. 8.   Mitochondrial respiratory changes on mitochondrial ATP-sensitive K+ channel opening. Rat heart mitochondria (0.2 mg/ml) were incubated in K+ or Li+ salts of Cl- (110 mM), tetramethylpentadecane (200 µM), ascorbate (2 mM), EGTA (100 µM), Pi (5 mM), and MOPS buffer (10 mM), pH 7.2, in the presence of 1 mM MgCl2 and 0.5 µg/ml oligomycin. ATP (200 µM), diazoxide (30 µM), and 5-hydroxydecanoate (200 µM) were present as indicated. Error bars, SD from 3 individual experiments. Comparisons between data were performed using a pairwise Tukey's test conducted by Sigmastat. *P < 0.01.

When respiring on succinate, mitochondria incubated in K+ and TEA+ media respired at 35.8 ± 2.3 and 30.9 ± 1.7 ng atom O · min-1 · mg-1, respectively (n = 6, P < 0.05). This difference in respiration can be attributed to K+ flux through mitochondrial KATP channels, because TEA+ is not transported by mitochondrial KATP channels but, rather, diffuses across the inner membrane at rates similar to K+ diffusion (2, 14, 26). With the assumption of an H+:O stoichiometry of 6 for succinate-supported respiration, this respiratory difference implies a K+ flux through mitochondrial KATP channels of 29.4 nmol K+ · min-1 · mg-1, which is very similar to the estimate obtained from TMPD-supported respiration (Fig. 8).

How much depolarization can result from a K+ flux of <30 nmol K+ · min-1 · mg-1? An estimate can be made from the relationship between mitochondrial respiration and Delta Psi when respiration is increased with uncoupler. As seen in Fig. 9, Delta Psi falls linearly with respiration until the maximal respiratory rate is reached. The slope of this line in heart mitochondria (the internal resistance of the overall electron transport system) is 0.34 (n = 2). Thus the respiratory differences observed in the presence and absence of mitochondrial KATP channel activity (~5 ng atom O · min-1 · mg-1 for succinate-supported respiration) would result in a Delta Psi decrease of 1-2 mV, which is not detectable using conventional techniques.


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  		Fig. 9.   Relationship between mitochondrial respiration and Delta Psi . Rat heart mitochondria (0.25 mg/ml) were incubated in Li+ salts of Cl- (110 mM), succinate (10 mM), EGTA (100 µM), Pi (5 mM), and HEPES buffer (10 mM), pH 7.2. Mitochondrial respiration and Delta Psi were estimated in the presence of 0-250 nM FCCP. Similar results were obtained in 2 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hypothesis that opening KATP channels protects the heart against ischemia-reperfusion damage was first tested and verified by Grover et al. (18) and subsequently confirmed by many laboratories (4, 6, 15, 20, 41; the cardiopharmacology of KATP channels is reviewed in Ref. 17). The hypothesis that mitochondrial KATP channels are the site of action for the cardioprotective effects of KATP channel openers (14) was subsequently verified by Garlid et al. (13). These results have led to a new focus on the role of mitochondrial KATP channels in ischemia-reperfusion injury. However, little is known about the functional consequences of mitochondrial KATP channel activity in mitochondria and how this activity ultimately leads to protection. Indeed, two mutually exclusive hypotheses have been put forward for the bioenergetic effects of mitochondrial KATP channel opening.

Liu et al. (29) proposed that futile K+ cycling due to opening mitochondrial KATP channels is sufficient to cause significant uncoupling and depolarization. Indeed, massive mitochondrial depolarization secondary to addition of KATP channel openers has now been reported by three different laboratories (24, 25, 29, 36, 37, 40). This depolarization, in turn, is proposed to reduce mitochondrial Ca2+ uptake, thereby preventing Ca2+ overload and opening of the mitochondrial permeability transition (25). These findings, and the attendant hypotheses, clearly have great import for our understanding of the cardioprotective actions of K+ channel openers. For this reason, we have gone to considerable lengths in this study on rat heart mitochondria to investigate these actions. The data in Figs. 1-5 and associated text show clearly that the reported effects of diazoxide and pinacidil on Delta Psi and Ca2+ uptake (24, 25) have nothing to do with mitochondrial KATP channels. Thus the finding that openers cause the same effects in Li+ medium excludes participation of the mitochondrial KATP channel, because this channel does not transport Li+. Indeed, this was the expected result, because the cited studies were conducted under conditions in which the mitochondrial KATP channel is already open and therefore cannot be affected by KATP channel openers (14). We show further that the bioenergetic effects previously attributed to actions at mitochondrial KATP channels are caused instead by the intrinsic uncoupling and inhibitory properties of these agents when used at concentrations far in excess of those required to open mitochondrial KATP channels. Thus diazoxide and pinacidil, like most hydrophobic compounds, exhibit concentration-dependent inhibition of the respiratory chain and also uncouple respiration. It was reported more than 30 years ago that diazoxide at high doses (150 µM) inhibits respiration in rat heart mitochondria due to inhibition of succinate dehydrogenase (38). It is also unlikely that opening mitochondrial KATP channels in vivo leads to significant mitochondrial uncoupling, as suggested by FAD autofluorescence studies in cardiac myocytes (29, 36, 37). Measurements of cardiac efficiency of oxygen utilization (work/oxygen consumption) in the intact heart show that KATP channel openers in pharmacological doses have no effect on efficiency, which excludes significant uncoupling as a consequence of their administration (19, 21).

These findings not only emphasize the necessity to recognize the difference between toxic and pharmacological effects of K+ channel openers but also provide a means to distinguish between them. In the present study, toxic effects of diazoxide are observed beginning at 100 µM, and toxic effects of pinacidil are observed beginning at 50 µM. At lower doses, these agents have no effect on Ca2+ uptake, which would be reduced by respiratory inhibition or uncoupling (Fig. 2).

Because it increases futile K+ cycling, mitochondrial KATP channel opening is expected to result in a decrease in Delta Psi . However, the magnitude of the depolarization will depend entirely on the magnitude of the added K+ flux, which we have estimated to be between 24 and 30 nmol K+ · min-1 · mg-1 in rat heart mitochondria (Fig. 8 and data in text). This amount of K+ flux would reduce Delta Psi by 1-2 mV, as derived from the relationship between respiratory rates and Delta Psi (Fig. 9). Thus, in isolated rat heart mitochondria, mitochondrial KATP channel opening causes a minimal effect on Delta Psi , Ca2+ uptake, and respiration.

On the basis of these results, we believe that the primary function of mitochondrial KATP channels is to participate in regulation of mitochondrial volume (8). Mitochondrial volume homeostasis, which is essential for maintaining vesicular integrity in the face of high and variable inner membrane traffic of ions and water, is provided largely by regulation of K+ transport (7, 8). K+ enters the matrix electrophoretically via diffusion and via mitochondrial KATP channels, when open. Diffusive K+ uptake is exponential with voltage (11) in mitochondria and, therefore, highly sensitive to changes in Delta Psi . K+ uptake is followed immediately by uptake of phosphate (Pi) on the electroneutral phosphate/OH- exchange carrier. Accordingly, net K+ uptake is always accompanied by anions and osmotically obligated water (8). Excess K+ leaves the matrix via the K+/H+ antiporter. It is important to recall that the K+/H+ antiporter is allosterically regulated to sense changes in matrix volume itself (7, 8); consequently, an increase in K+ uptake will necessarily cause a volume increase until K+/H+ exchange comes back into balance, as seen in the traces of Fig. 5. Thus opening mitochondrial KATP channels will shift the balance between K+ uniport and K+/H+ antiport, causing transient swelling and a higher steady-state volume for as long as mitochondrial KATP channels remain open. Such a "regulated interplay" between K+ uniport and K+/H+ antiport was first postulated by Brierley (3). A fundamental physiological question about mitochondrial KATP channels is, why do mitochondria require a second pathway for K+ uptake? To begin to address this question, we designed the experiments of Figs. 5-7 to mimic three different physiological states of the cardiomyocyte.

The conditions of Fig. 5, i.e., state 4 respiration with high Delta Psi , mimic the resting, low-work state of the cardiomyocyte. When diazoxide is added to the perfused heart in this state, the immediate response is a moderate increase in mitochondrial production of reactive oxygen species (ROS) (43). ROS, in turn, function as a second messenger signaling cardioprotection during ischemic preconditioning (5) and diazoxide preconditioning (43). The mechanism by which opening mitochondrial KATP channels induces increased mitochondrial ROS production is unknown but may be rationalized by the following hypothesis. As can be seen in Fig. 5, opening mitochondrial KATP channels under comparable conditions increases matrix water content by ~20%, secondary to uptake of K+ and Pi from the medium. In the cardiomyocyte, where the ratio of matrix water to cytosol water is ~1:4, this flux would cause a significant depletion of cytosolic Pi, which is already low in the resting state. Significant Pi redistribution will raise the cellular phosphorylation potential, thereby slowing respiration and increasing the rate of ROS production from complex III of the electron transport chain.

The conditions of Fig. 6, i.e., state 3 respiration with low Delta Psi , mimic the high-work state of the cardiomyocyte during positive inotropy. The low Delta Psi will severely reduce diffusive K+ uptake: a 30-mV decrease in Delta Psi will reduce K+ uptake by ~50% (11). The resulting imbalance between K+ uptake and efflux will cause matrix contraction, which is reversed by opening mitochondrial KATP channels, as shown in Fig. 6. The role of mitochondrial KATP channels in positive inotropy is described by the following working hypothesis: Excessive matrix contraction will cause reciprocal expansion of the IMS, which includes the intercristal spaces and the space between the inner and outer membranes. IMS swelling will perturb the architecture of this important compartment and disrupt the structure and function of intermembrane enzymes, including mitochondrial creatine kinase. Metabolic channeling through mitochondrial creatine kinase is required during high-work states in heart (33-35). We hypothesize that it is necessary during positive inotropy to open mitochondrial KATP channels to compensate for the lower driving force that occurs during high electron flows (32). This mechanism would preserve the architecture of the IMS and, therefore, preserve efficient energy transfers between mitochondria and cytosol when these are most needed. In this regard, we consider it significant that mitochondrial KATP channel opening reverses the contraction caused by high rates of ATP synthesis (Fig. 6). In this physiological setting, opening mitochondrial KATP channels serves to maintain constant volume, by adding a parallel K+ conductance pathway to compensate for the lower driving force for K+ uptake. It is also clear that excessive mitochondrial contraction is deleterious for electron transport, which is exquisitely sensitive to matrix volume over a very narrow range (23). Maintenance of matrix volume, secondary to physiological mitochondrial KATP channel opening, may prevent respiratory inhibition due to matrix contraction that would otherwise occur during high rates of ATP synthesis.

The conditions of Fig. 7, i.e., no respiration with low Delta Psi , mimic the anoxic state of the cardiomyocyte during ischemia. Ischemia is also associated with mitochondrial depolarization, resulting in matrix contraction, perturbation of IMS architecture, and accelerated ATP hydrolysis due to disruption of mitochondrial creatine kinase. Indeed, the loss of functional coupling between mitochondrial creatine kinase and the adenine nucleotide translocase is one of the earliest detectable alterations in ischemic hearts (28). Nakano et al. (31) suggested that mitochondrial KATP channels may be an end effector of cardiac protection by preconditioning. A possible mechanism is that opening mitochondrial KATP channels helps preserve IMS architecture with consequent slowing of ATP hydrolysis and preservation of the ability to use creatine efficiently as substrate on reperfusion. Here, we consider it significant that mitochondrial KATP channel opening reverses the contraction caused by cyanide (Fig. 7).

In summary, we propose that the primary consequence of opening mitochondrial KATP channels is a modest increase in K+, Pi, and water uptake by mitochondria. The hypotheses describe the different roles of mitochondrial KATP channels in three different states of the cardiomyocyte and suggest that regulation of mitochondrial KATP channels has important bioenergetic consequences in each.


    ACKNOWLEDGEMENTS

We thank Craig Semrad and Jarmila Pauckova for excellent technical assistance.


    FOOTNOTES

This research was supported in part by National Institute of General Medical Sciences Grant GM-55324 (to K. D. Garlid) and American Heart Association Scientist Development Grant 963 0004N (to P. Paucek).

Present address of A. J. Kowaltowski: Dept. de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.

Address for reprint requests and other correspondence: K. D. Garlid, Dept. of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 N.W. Walker Rd., Beaverton, OR 97006-8921 (E-mail: garlid{at}bmb.ogi.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11 July 2000; accepted in final form 6 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(2):H649-H657
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