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Am J Physiol Heart Circ Physiol 280: H1882-H1888, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 4, H1882-H1888, April 2001

ATP consumption by uncoupled mitochondria activates sarcolemmal KATP channels in cardiac myocytes

Norihito Sasaki, Toshiaki Sato, Eduardo Marbán, and Brian O'Rourke

Institute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, Maryland 21205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K+ (KATP) current (IK,ATP) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on IK,ATP. In contrast, in the presence of pinacidil, exposure to 100 µM DNP rapidly activated IK,ATP with complex kinetics consisting of a quick rise [time constant of IK,ATP increase (tau ) = 0.13 ± 0.01 min], an early partial recovery (tau  = 0.43 ± 0.04 min), and then a more gradual increase. This DNP-induced activation of IK,ATP was reversible and accompanied by mitochondrial flavoprotein oxidation. The F1F0-ATPase inhibitor oligomycin abolished the DNP-induced activation of IK,ATP. The initial rapid rise in IK,ATP was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau  = 0.66 ± 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau  = 0.20 ± 0.01 min, P < 0.05) and the subsequent declining phase (tau  = 0.88 ± 0.19 min, P < 0.05). From single KATP channel recordings, we excluded a direct effect of DNP on KATP channels. Taken together, these results indicate that rapid changes in F1F0-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed KATP channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal KATP channels can be rationalized by reversal of F1F0-ATPase and the facilitation of coupling by the creatine kinase system.

dinitrophenol; ATP hydrolysis; patch-clamp; ATP-sensitive K+ channels


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MITOCHONDRIAL F1f0-atpase generates ATP from ADP using the proton gradient established by the electron transport chain of the inner membrane. Mitochondrial uncouplers such as 2,4-dinitrophenol (DNP) or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) are chemical protonophores that dissipate the mitochondrial inner membrane potential causing oxidation of mitochondrial matrix NADH (8, 22). Mitochondrial uncoupling can also rapidly induce net ATP hydrolysis as a result of reverse-mode F1F0-ATPase activity (6, 17). Thus mitochondrial ATP consumption is likely to play a significant role in the cellular response to mitochondrial permeability transitions (6, 10), redox oscillations (20), and ischemia (25, 26), conditions that dissipate the mitochondrial inner membrane potential. Exposure to a mitochondrial uncoupler changes cardiac muscle contractility before activation of sarcolemmal ATP-sensitive K+ (KATP) channels (1). In isolated ventricular myocytes, FCCP immediately decreases the cytosolic Ca2+ transient and increases diastolic Ca2+ concentration, suggesting that impaired Ca2+ handling precedes significant activation of sarcolemmal KATP channels (9).

Two factors are likely to be involved in preventing early KATP channel activation in the face of impaired mitochondrial function. First, ATP derived from nearby glycolytic enzymes may provide effective spatiotemporal inhibition of KATP channels (27, 28). Second, the creatine kinase (CK) system is an important buffer of high-energy phosphate (14), maintaining the cytosolic ATP level despite perturbations of mitochondrial energy metabolism (12). On the other hand, the CK system also acts as an ATP shuttle, connecting the sites of energy production to the sites of energy consumption including sarcolemma (14). If the mitochondrial membrane potential collapses and the mitochondria switch from ATP generation to ATP consumption, this change could also be rapidly transmitted back to sarcolemma by the CK system even if the bulk ATP concentration were above that required to inhibit KATP channels.

The present study provides novel evidence for close coupling between mitochondrial energy production and/or consumption and subsarcolemmal energy charge. To assay energetics in the subsarcolemmal space, we converted KATP channels into sensitive reporters of local energy metabolism. Such channels normally require extreme ATP depletion (and/or ADP accumulation) to open (19), but their nucleotide sensitivity can be shifted into the physiological range by exposure to KATP channel agonists such as pinacidil (13). Thus our strategy was to manipulate mitochondrial energetics while measuring drug-primed KATP current (IK,ATP) as an indicator of nucleotide concentrations just under the surface membrane.


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

Materials. Collagenase (type II) was purchased from Worthington (Lakewood, NJ). DNP, FCCP, NaCN, glibenclamide, oligomycin, atractyloside, and 2,4-dinitrofluorobenzene (DNFB) were obtained from Sigma (St. Louis, MO). Pinacidil was purchased from Research Biochemicals International (Natick, MA). Pinacidil, glibenclamide, oligomycin, and atractyloside were dissolved in DMSO before being added into experimental solutions. The final concentration of DMSO was <0.3%.

Cell isolation. New Zealand White rabbits of either sex (1-2 kg) were anesthetized by intravenous injection of pentobarbitone (30 mg/kg) until consciousness was lost. After the absence of a corneal reflex was confirmed, hearts were rapidly removed and mounted on a Langendorff apparatus. Ventricular myocytes were isolated by conventional enzymatic dissociation as described previously (18, 23). Hearts were perfused with a constant flow (12-14 ml/min at 37°C) for 5 min with normal modified Tyrode solution [containing (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH)] and then 5 min of Ca2+-free Tyrode, 20 min of Ca2+-free Tyrode containing collagenase (1 mg/ml), and 5 min of Ca2+-free Tyrode solution sequentially. Cells were then cultured on laminin-coated coverslips in M199 culture medium with 2% fetal bovine serum at 37°C. Experiments were performed 24-48 h after isolation.

Electrophysiology and flavoprotein fluorescence measurement. For whole cell patch-clamp recordings, the internal pipette solution contained (in mM) 120 potassium glutamate, 25 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, and 3 MgATP (pH 7.2 with KOH). The composition of the external solution was the same as the Tyrode solution used for cell isolation. The K+-free external solution (Fig. 4A) was made by omitting K+ from the Tyrode solution. Currents were elicited every 6 s from a holding potential of -80 mV by consecutive steps to -40 mV for 100 ms and then to 0 mV for 380 ms. To quantify IK,ATP, currents were measured 200 ms into the second pulse. Single-channel recordings were performed in high-K+ pipette and bath solutions containing (in mM) 120 potassium glutamate, 125 KCl, 1 MgCl2, 10 EGTA, 10 HEPES, and 10 glucose (pH 7.2 with KOH) in either the cell-attached mode or excised inside-out mode. In some excised patch experiments, 1 mM MgATP was added to the intracellular (bath) solution as indicated. Steady-state single-channel currents were recorded every 5 s for 800 ms at a holding potential of -80 mV. To quantify single KATP channel activation from such recordings, we calculated the product of the channel number (N) and open probability (Po) from each trace recorded.

In some whole cell clamp experiments (Fig. 3, A and B), flavoprotein fluorescence was measured simultaneously with membrane current to monitor mitochondrial redox state. Endogenous flavoprotein fluorescence was excited with a xenon arc lamp with a band-pass filter centered at 480 nm, but only during the 100-ms step to -40 mV to minimize photobleaching. Emitted fluorescence was recorded at 530 nm by a photomultiplier tube and digitized (18, 23). All experiments were performed at room temperature (22-23°C).

Quantitative analysis of IK,ATP activation. To quantify the kinetics of activation of KATP channels induced by DNP in the presence of pinacidil, the rapid rising phase and the subsequent partial recovery phase were fitted by exponential functions (Fig. 5, A and B).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DNP rapidly activates IK,ATP in presence of pinacidil. To test for coupling between mitochondria and basal energetics in the subsarcolemmal space, we measured whole cell IK,ATP during exposure to the mitochondrial inhibitor DNP. Exposure to DNP alone did not induce any acute change, but after a considerable delay, it did slowly activate outward current at 0 mV. This current was identified as IK,ATP because of its sensitivity to glibenclamide inhibition (Fig. 1A). We then checked whether pinacidil-primed channels might be able to report energetic changes to which KATP channels are normally oblivious. Pinacidil (100 µM) alone failed to activate IK,ATP (Fig. 1C). In contrast, in the presence of pinacidil, exposure to DNP rapidly turned on IK,ATP, and this activation was readily reversed by washout of DNP (Fig. 1C). These observations can be rationalized as indicative of DNP-induced changes in local energetics just below the surface membrane, suggesting rapid coupling between mitochondria and sarcolemma.


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  		Fig. 1.   Dinitrophenol (DNP)-induced activation of ATP-sensitive K+ (KATP) current (IK,ATP) in the presence or absence of pinacidil. Time course of whole cell currents (I) measured at 0 mV during exposure to 100 µM DNP in the absence (A) or presence (C and E) of 100 µM pinacidil. Note that 30 µM glibenclamide (gliben) inhibited DNP-induced current (A). B and D: original current traces recorded at the time indicated by symbols [black-triangle and  (B) and  and star  (D)] in A and C, respectively.

The DNP-induced activation of IK,ATP in the presence of pinacidil had complex kinetics consisting of a rapid rise, an early partial recovery, and then a more gradual increase. When the order was reversed and DNP was applied first, pinacidil activated IK,ATP monotonically, an effect that was reversed on pinacidil washout (Fig. 1E). Brisk activation of IK,ATP (i.e., within seconds of drug application) always required the simultaneous presence of DNP and pinacidil. These phenomena were also observed in experiments using FCCP, another mitochondrial uncoupler, as an alternative to DNP (data not shown). Taken together, these findings can be explained by the combination of two effects: the ATP depletion induced by DNP and the shift of the ATP sensitivity of KATP channels induced by pinacidil (13). In other words, we could detect a mitochondrially mediated rapid decrease of subsarcolemmal ATP by measuring IK,ATP in the presence of pinacidil.

Figure 2 presents, in schematic form, a framework for the interpretation of our experimental findings. Under physiological conditions (Fig. 2, left), ATP synthesized by F1F0-ATPase using the proton gradient is transferred from the mitochondrial matrix to sarcolemma through an adenine nucleotide translocator (ANT) and two types of CK, i.e., cytosolic and mitochondrial CK (14, 24, 29). Several lines of evidence indicate that ANT is functionally coupled with mitochondrial CK (24, 29). The CK system, however, is not essential for the bulk diffusion of ATP between membranes, because many cell types lack the CK system. Nevertheless, in excitable tissues with high metabolic demands (notably neurons, skeletal muscle, and cardiac muscle), the CK system expedites the transport of high-energy metabolites by shuttling creatine phosphate in an energetic chain reaction. In addition, the maintenance of low ADP levels by the CK system is important for preserving a high free energy of ATP hydrolysis at energy-consuming sites (14).


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  		Fig. 2.   Scheme of subcellular energy transport between mitochondria and the sarcolemma. Left: physiological condition; right: mitochondrial uncoupling. Pi required in almost all steps is abbreviated. CK, creatine kinase; mtCK, mitochondrial CK; PCr, phosphocreatine; Cr, creatine; ANT, adenine nucleotide translocator; Q, coenzyme Q; c, cytochrome c; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; DFNB, 2,4-dinitrofluorobenzene.

In contrast, in the case of mitochondrial uncoupling (Fig. 2, right), F1F0-ATPase hydrolyzes ATP and pumps protons out in the reverse mode. Such mitochondrial perturbation may be rapidly transferred to the sarcolemma through the ANT and the CK systems, which can be demonstrated by examining the activation kinetics of IK,ATP.

Fast activation of IK,ATP requires rapid dissipation of mitochondrial potential. To confirm that DNP actually affected mitochondria even when no IK,ATP is elicited, we measured flavoprotein fluorescence (an indicator of matrix redox state) and membrane current simultaneously. Figure 3A demonstrates that exposure to DNP alone rapidly increased flavoprotein oxidation; nevertheless, IK,ATP was activated with a considerable latency. In contrast, Fig. 3B shows that, in the presence of pinacidil, additional application of DNP rapidly increased both IK,ATP and flavoprotein oxidation. DNP-induced mitochondrial oxidation could be observed not only during dialysis in the whole cell patch-clamp configuration but also in untouched cells (18, 22, 23). Thus the lack of rapid IK,ATP activation in the absence of pinacidil is not due to the lack of an uncoupling effect of DNP on mitochondria.


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  		Fig. 3.   Simultaneous measurement of membrane currents and flavoprotein (FP) fluorescence induced by DNP in the absence (A) or presence of pinacidil (Pina; B). C: effects of 2 mM CN- on membrane currents in the presence of 100 µM pinacidil.

Figure 3C demonstrates that, in the presence of pinacidil, exposure to 2 mM CN- (an electron transport chain inhibitor) did not induce rapid activation of IK,ATP. Furthermore, the DNP-induced rapid activation of IK,ATP was not inhibited by CN-, although CN- could inhibit the oxidative effect of DNP on mitochondria (data not shown). These results indicate that acute activation of IK,ATP in the presence of pinacidil requires rapid dissipation of mitochondrial inner membrane potential, which is induced by uncouplers but not by CN-.

DNP-induced IK,ATP activation is inhibited by oligomycin. Mitochondrial uncouplers have been reported to induce ATP hydrolysis caused by reverse-mode F1F0-ATPase activity (6, 17). To determine whether mitochondrial ATP hydrolysis was involved in the DNP-induced rapid activation of IK,ATP, the effect of the F1F0 inhibitor oligomycin (cf. Fig. 2) was examined (4, 6, 11, 17). These experiments were performed under external K+-free conditions to eliminate the potential for indirect inhibitory effects of oligomycin on the Na+-K+ pump (21); however, similar results were obtained in the presence of external K+. Figure 4A shows that the increase in IK,ATP by DNP in the presence of pinacidil was inhibited by additional exposure to oligomycin. In addition, pretreatment with oligomycin abolished the DNP-induced activation of IK,ATP (data not shown). These findings indicate that DNP-induced activation of IK,ATP is mediated by F1F0-ATPase, probably because mitochondrial uncoupling not only interrupts ATP synthesis but also accelerates ATP hydrolysis, thus rapidly depleting subsarcolemmal ATP.


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  		Fig. 4.   A: effects of mitochondrial modulator on DNP-induced rapid activation of IK,ATP. Oligomycin (30 µM) inhibited DNP-induced activation of IK,ATP in the presence of pinacidil. B and C: effects of 300 µM atractyloside (B) and 100 µM DNFB (C) on DNP-induced rapid activation of IK,ATP. D: effects of second exposure to DNP on IK,ATP in the presence of pinacidil.

The first step of the interaction between mitochondrial matrix ATPase activity and the cytosol is the translocation of ATP and ADP across the inner membrane by ANT (cf. Fig. 2) (6). We tested the effects of the ANT inhibitor atractyloside (6, 15) on the DNP-induced acute activation of IK,ATP. Figure 4B demonstrates that the application of atractyloside alone induced a small transient increase of IK,ATP but significantly attenuated the fast rising phase of DNP-induced activation. Although 300 µM atractyloside might not be able to inhibit ANT completely because of its poor cell membrane permeability, these data indicate that, in the presence of pinacidil, small perturbations of intracellular ATP can be detected by measuring IK,ATP, revealing the existence of cross-talk between the mitochondrial inner membrane and the subsarcolemma.

To define the contribution of the CK system (cf. Fig. 2) to the DNP-induced activation of IK,ATP, the effects of DNFB were examined (5). DNFB was reported to inhibit both cytoplasmic and mitochondrial CK equally in rabbit isolated mitochondria (30) (cf. Fig. 2). DNFB did not activate IK,ATP within 10 min in the absence of pinacidil. In contrast, in the presence of pinacidil, subsequent exposure to DNFB modestly but consistently increased IK,ATP (n = 6). Additional exposure to DNP induced rapid activation of IK,ATP, followed by a gradual decrease (Fig. 4C). Although the effect of DNFB may have been complicated by local ATP production by glycolytic enzymes or by incomplete inhibition of CK, it is important to note that the rising and the declining phases of IK,ATP were blunted by coapplication of DNFB. This finding suggests that the CK system contributes to energy transport between mitochondria and sarcolemma during mitochondrial uncoupling. Interestingly, second exposures to DNP in the presence of pinacidil also revealed slowing of the rapid rise of DNP-induced IK,ATP activation in a manner reminiscent of atractyloside (Fig. 4D).

To evaluate quantitatively the kinetics of activation of KATP channels induced by DNP in the presence of pinacidil, the time courses of IK,ATP during the rapid rise and subsequent partial recovery were fitted by single exponential curves. Figure 5A shows representative data for the rising phase of DNP-induced IK,ATP, whereas Fig. 5B shows the declining phase during the first exposure to DNP with (from Fig. 4C) or without (from Fig. 4D) concomitant DNFB. In the other groups, e.g., second exposure to DNP or first exposure to DNP with atractyloside, the early decline in IK,ATP was not observed. Figure 5C summarizes the data for the kinetics of the rising phase, indicating that the rate of rise is considerably blunted during second exposures to DNP (P < 0.01), with DNP + atractyloside (P < 0.01), and in the DNFB-treated group (P < 0.05). In addition, the declining phase of IK,ATP in the DNFB group was also slowed, as shown in Fig. 5D (P < 0.05). These results indicate that mitochondrial uncoupling induced-ATP hydrolysis by F1F0-ATPase is partially inactivated after a rapid activation and that CK inhibition slows the kinetics of DNP-induced IK,ATP activation, probably because under such conditions interactions between mitochondria and sarcolemma depend solely on bulk diffusion through the cytoplasm (cf. Fig. 2). Furthermore, there is a difference in the time course of DNP-induced IK,ATP activation between the first and second application, indicating cellular memory similar to that of preconditioning phenomena (24, 25). This effect of multiple exposures and the ANT inhibitor may reflect endogenous inactivation of the F1F0-ATPase activity as a result of mitochondrial ADP accumulation (8).


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  		Fig. 5.   Quantitative analysis for the kinetics of DNP-induced IK,ATP. The initial rising phase (A) and the subsequent declining phase (B) were fitted by single-exponential curves. C and D: summarized data for tau  during the rising (C) and declining phases (D). DNP(1) and DNP(2) indicate first and second exposures to DNP, respectively; ATR, atractyloside; tau , time constant of IK,ATP increase. *P < 0.05 vs. DNP(1), ** P < 0.01 vs. DNP(1).

DNP-induced activation of single KATP channels in presence of pinacidil. To eliminate the influence of intracellular dialysis by the whole cell patch electrode, we measured single KATP channel currents in the cell-attached mode during application of DNP in the presence of pinacidil. Figure 6 shows that the Po of KATP channels was not affected by exposure to pinacidil alone (n = 18), but additional application of DNP dramatically increased Po. This DNP-induced activation of single-channel current was inhibited by the additional application of oligomycin. In only two cells, exposure to pinacidil alone induced activation of KATP channels in the cell-attached mode. In the majority of cells (n = 15), DNP activated KATP channels within 3 min after exposure to DNP in the presence of pinacidil, whereas in only three cells did channel activation by DNP take more than 3 min. In contrast, application of DNP alone did not activate any single-channel current in the cell-attached mode within 5 min (n = 6).


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  		Fig. 6.   Effects of DNP on the single KATP channels in cell-attached mode. Top: time course of the channel number (N) multiplied by the open probability (Po) during exposure to drugs indicated by bars. Bottom: consecutive 5 traces recorded at the time (a, b, and c in top) are shown. Single-channel activity was measured as inward current at -80 mV for 800 ms (1 kHz). The opening of 3 channels was observed at maximum.

To exclude the possibility that DNP-induced activation of IK,ATP is caused by a direct effect of DNP on the channels, we recorded single-channel current in inside-out excised patches during application of DNP in the presence of pinacidil. Exposure to DNP did not enhance the activity of KATP channels (Fig. 7, 1 mM intracellular [MgATP]). The single-channel currents were also not enhanced by DNP in bath solutions containing either 0 or 3 mM intracellular [MgATP] (data not shown). These results indicate that DNP-induced IK,ATP activation is not mediated by direct effects of DNP on KATP channels. In addition, we confirmed that 30 µM oligomycin had minimal effects on KATP in the excised inside-out patch (data not shown).


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  		Fig. 7.   Effects of DNP on single KATP channel current in excised inside-out mode. Time course of N × Po is indicated, and the opening of 2 channels was observed at maximum. Currents were measured at -80 mV for 800 ms (1 kHz).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that mitochondrial uncoupling can induce rapid activation of IK,ATP in the presence of pinacidil. This activation is due to ATP hydrolysis by F1F0-ATPase, as confirmed by the sensitivity to oligomycin. In the absence of pinacidil, DNP does not have an acute effect on IK,ATP, indicating that, although the mitochondria are inhibited, the ATP depletion is not severe enough to cross the threshold for activation of sarcolemmal KATP channels. Other studies confirm that under normal conditions, mitochondrial inhibitors produce a slow effect on IK,ATP. Leyssens et al. (17) reported that in rat myocytes exposed to FCCP, there was a rapid decrease in intracellular ATP, as indicated by an increase in free Mg2+, but no rapid activation of IK,ATP. Regarding this point, Alekseev et al. (2) reported that DNP alone directly activated IK,ATP in guinea pig ventricular myocytes. However, the DNP-induced activation of IK,ATP observed in the present study differs with respect to the requirement for pinacidil and the very rapid time course of IK,ATP development. By decreasing the sensitivity of sarcolemmal KATP channels to ATP inhibition using pinacidil, we were able to reveal dynamic early changes in energy balance within the cell during mitochondrial uncoupling.

Because sarcolemmal KATP channels do not open immediately in response to mitochondrial uncouplers (1), systems must be present that are able to blunt the ATP hydrolytic effect of the mitochondria. One such system could be glycolysis. Weiss and Lamp (27, 28) proposed that glycolytic enzymes localized to the sarcolemma may preferentially produce ATP to inhibit KATP channel opening. This could serve as a local damper on the effect of mitochondrial ATP consumption provided that glycolytic substrates (either exogenous or endogenous) are available. Another possibility is that sufficient stores of phosphocreatine are available to buffer the fall in cytosolic ATP, a mechanism relying on the presence of an active CK system. The role of the CK equilibrium as a transporter of energy equivalents between energy-producing and energy-consuming sites (ATP shuttle; see Refs. 3 and 14) should actually exacerbate subsarcolemmal ATP depletion. Maintenance of ATP transport to distant sites depends on a large positive ATP concentration gradient at the energy-producing membrane, which would become negative if the mitochondria began to hydrolyze ATP. This, in fact, could be a crucial link between the mitochondria and the rapid activation of sarcolemmal KATP channels.

A pronounced rapid transient of IK,ATP is evident at the onset of DNP exposure in the presence of pinacidil (e.g., Figs. 1C and 3). This finding is reminiscent of the results of Leyssens et al. (17), who observed a similar transient in intracellular [Mg2+] that was blunted by oligomycin when myocytes were exposed to an uncoupler. The transient is likely to be due to an early burst of ATP breakdown by F1F0-ATPase that is somehow self-limiting. This is supported by blunting of this phase by atractyloside and by the attenuation of the transient phase when pinacidil is applied after DNP (Fig. 1E) or when DNP is applied for the second time (Fig. 4D). Although the mechanism of self-limitation remains to be determined, local depletion of the nucleotide pool or the binding of an endogenous ATPase inhibitor protein (11) are plausible explanations.

Clearly, the conversion of mitochondria from energy-producing to energy-consuming organelles will substantially impact the response of the myocyte to metabolic stress. This mechanism is likely to be active under conditions known to dissipate the mitochondrial inner membrane potential, such as during mitochondrial permeability transitions (6), ischemia (26), or spontaneous redox oscillations (22). Indeed, in the case of redox oscillations in cardiac cells, we have observed close coupling between mitochondrial redox transitions, mitochondrial membrane potential dissipation, and sarcolemmal IK,ATP activation (20). Further investigation into the contribution of mitochondrial energy consumption to the cellular pathophysiology of metabolic inhibition, apoptosis (7, 16), and ischemia will be facilitated by the approaches described in the present study.


    ACKNOWLEDGEMENTS

This study was supported by the National Heart, Lung, and Blood Institute Grants ROI HL-54598 to (to B. O'Rourke) and R37 HL-36957 (to E. Marbán), a Japan Heart Foundation and Bayer Yakuhin Research Grant Abroad (to N. Sasaki), and a Banyu Fellowship in Lipid Metabolism and Atherosclerosis (to T. Sato). E. Marbán holds the Dr. Michel Mirowski Professorship of Cardiology of the Johns Hopkins University.


    FOOTNOTES

Present address of T. Sato: Dept. of Physiology, Oita Medical Univ., Idaigaoka, Hasama, Oita, 879-5593 Japan

Address for reprint requests and other correspondence: E. Marbán, Director, Institute of Molecular Cardiobiology, Johns Hopkins Univ., 720 Rutland Ave., Ross 844, Baltimore, MD 21205 (E-mail: marban{at}jhmi.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 1 March 2000; accepted in final form 3 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(4):H1882-H1888
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