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Cardiovascular Research 2001 52(2):236-245; doi:10.1016/S0008-6363(01)00395-9
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Mitochondria are the main ATP source for a cytosolic pool controlling the activity of ATP-sensitive K+ channels in mouse cardiac myocytes

Andreas Knopp, Stephan Thierfelder, Bettina Doepner and Klaus Benndorf*

Institut für Physiologie, Abt. Herz-Kreislauf-Physiologie, Friedrich-Schiller-Universität Jena, D-07740 Jena, Germany

* Corresponding author. Tel.: +49-3641-934-351; fax: +49-3641-933-202 kben{at}mti-n.uni-jena.de

Received 16 January 2001; accepted 5 June 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The aim was to identify the major ATP source controlling the activity of sarcolemmal KATP channels in ventricular cardiomyocytes. Methods: KATP-channel current (IKATP) was measured with the patch-clamp technique in either the whole-cell (glycogenolysis blocked by 10 mmol/l EGTA), cell-attached, or inside-out configuration. Results: In the absence of any substrate, IKATP (amplitude 31±4 nA; n=5) appeared spontaneously 520±160 s (n=6) after whole-cell access. This latency was shortened by exposure to anoxia (117±33 s, n=32) and even more by uncoupling (1–10 µmol/l FCCP; 25±3 s; n=13) while the amplitude was unchanged. During metabolic inhibition the latency was remarkably prolonged when the F1F0-ATPase was blocked by oligomycin, suggesting that under those conditions the F1F0-ATPase is the major ATP consumer. Glucose (5.5–20.0 mmol/l) in the bath solution did not affect the amplitude of IKATP but prolonged its latency compared to respective substrate-free conditions. However, IKATP was blocked immediately by mitochondrial substrates. FCCP also induced large IKATP in cell-attached measurements in either the absence or presence of glucose and oligomycin. Conclusions: The activity of KATP channels in cardiomyocytes of mice is controlled by a cytosolic [ATP] pool for which oxidative phosphorylation is the predominant ATP source.

KEYWORDS Energy metabolism; Glycolysis; Hypoxia/anoxia; K-ATP channel; Mitochondria; Oxidative phosphorylation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
In excised membrane patches from cardiac myocytes, sarcolemmal KATP channels open at [ATP] below the range of several tens to hundreds of micromolar [1,2]. In whole myocytes, opening of KATP channels occurs during uncoupling of oxidative phosphorylation [3,4], inhibition of the cytochrome oxidase or removal of oxygen [5,6], even in the presence of glucose [7]. These findings strongly argue for an essential contribution of oxidative phosphorylation to the synthesis of ATP controlling the KATP channels. We showed recently that KATP channels in cell-attached patches of quiescent myocytes may be effectively controlled by the ATP consumption of Na+–K+-ATPases outside the membrane patch [8], indicating that KATP channels are controlled by ATP sources located several micrometers away from the channels. Distances in this order of magnitude may be taken as an upper limit for the mean distance between mitochondria and sarcolemmal KATP channels.

In contrast, a preferential control of the KATP channels by glycolytic ATP has been reported in cardiomyocytes that were permeabilized with saponin and whose cytosolic ATP consumption was artificially enhanced [9,10]. These results support the hypothesis of a functional compartmentation of ATP [11].

Herein, we describe patch-clamp experiments in cardiac myocytes without permeabilization of the membrane and in the key experiments we did not use any artificial ATP-consuming system. Our results in quiescent cardiac myocytes show that sarcolemmal KATP channels are controlled by ATP of a common cytosolic pool to which oxidative phosphorylation provides the main ATP portion whereas glycolysis provides only a subsidiary ATP portion.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Isolation of single mouse myocytes
Mouse ventricular myocytes were prepared at 37°C as described previously [5]. In brief: On a Langendorff apparatus, the heart was perfused for 5 min with a solution that was not especially oxygenized and that contained (mmol/l): 140.0 NaCl, 5.8 KCl, 0.5 KH2PO4, 0.4 Na2HPO4, 0.9 MgSO4, 11.1 glucose and 10.0 HEPES, pH 7.1 (NaOH). The perfusion was continued for 25 min with the same solution supplemented with 10 µmol/l CaCl2 and 150 mg/l collagenase. The ventricles were cut and agitated in KB solution containing (mmol/l): Glutamic acid 50.0, HEPES 20.0, taurine 20.0, glucose 10.0, MgSO4 3.0, EGTA 0.5, KCl 30.0 and KH2PO4 30.0, pH 7.3 (KOH). The cells were stored in KB solution.

2.2 Electrophysiology
Pipettes of 0.7–3 M{Omega} were pulled from borosilicate glass tubing. Whole-cell and cell-attached patch-clamp experiments were performed at 37°C in an experimental chamber that has been designed for insulation against atmospheric oxygen [5].

In whole-cell measurements, the bath contained extracellular solution consisting of (mmol/l): NaCl 150.0, KCl 5.4, CaCl2 2.5 or 3.6, MgCl2 0.5 and HEPES 10.0, pH 7.4 (NaOH). IKATP was induced by either anoxia or addition of the uncoupler carbonylcyanide(4-trifluoromethoxy) phenyl hydrazone (FCCP, 1 µmol/l). Oligomycin (2.5 µg/ml) or iodoacetic acid (IAA, 1 mmol/l) were added to block the mitochondrial ATPase and glycolysis, respectively. The standard pipette solution contained (mmol/l): 150.0 KCl, 5.0 HEPES, 10 EGTA and 1 CaCl2, pH 7.3 (KOH). In some experiments of FCCP induced IKATP (cf. Table 2), the pipette solution contained additionally 0.6 mmol/l MgATP and compounds necessary for maintaining glycolysis (Glycolytic substrates, GSS; mmol/l): 0.15 D-fructose 1,6-bisphosphate, 0.17 phosphoenolpyruvate, 0.1 ADP, 0.83 KH2PO4 and 1 NAD. The concentrations of D-fructose 1,6-bisphosphate and phosphoenolpyruvate were 2.5-fold and 8.5-fold as high as in rat cardiomyocytes, respectively [12,13]. The concentration of NAD was three-fold as in lymphocytes [14]. The ADP concentration corresponds to values of 50–100 µmol/l reached within the first 10 min of ischemia or hypoxia [15].


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  		Table 2 Latency and maximum amplitude of IKATP induced by 1 µmol/l FCCP at various bath and pipette solutions (conditions A to G). * indicates a significant difference (t-test; P<0.05); – indicates the absence of a significant difference

 
In the whole-cell measurements the holding potential was set to –80 mV. The current amplitude was measured at the end of 40 ms pulses to +40 mV. The pulsing frequency was either 0.5 or 1 Hz. Currents were filtered at a cut-off frequency of 20 kHz (4-pole Bessel). In cell-attached measurements, both bath and pipettes contained extracellular solution. The holding potential was set to 0 mV, i.e. the true holding potential of the membrane patch was the resting potential Vr of the cell. Voltage ramps from –20 mV to +160 mV (duration 1 s) were applied at a frequency of 0.2 Hz. Hence, during the ramp the trans-patch potential was changed from Vr –20 mV to Vr +160 mV. Inside-out patch experiments were conducted at 22°C. Bath and pipettes contained intracellular solution (mmol/l): KCl 140, EGTA 2 and HEPES 5, pH 7.2 (KOH). IKATP was evaluated at –80 mV (pulse duration 700 ms, holding potential 0 mV, pulsing frequency 1 Hz).

Most of the whole-cell experiments were done with a discontinuous single-electrode voltage clamp (dSEVC) amplifier (SEC 05L/H, npi Electronics, Germany). The switching frequency was 40–50 kHz. Intervals for recording of voltage and current injection were equally long. In part of the whole-cell experiments, a conventional patch clamp amplifier was used (Axopatch 200B, Axon Instruments, Foster City, USA). The series resistance was compensated for by about 80%. The amplitude of the measured currents was independent of the used amplifier. Cell-attached and excised-patch experiments were carried out with an Axopatch 200A amplifier at 5 kHz band width.

Recording and analysis of the data were performed with the ISO2 software (MFK, Niedernhausen, Germany). All traces were recorded with a sampling rate of 10 kHz (12-bit resolution).

2.3 Oxygen measurement
In part of the whole-cell recordings, the oxygen tension (pO2) near the cell was continuously measured as described previously [5]. In brief: We added to the bath solution 0.4 g/l of the phosphorescence dye Pd-meso-tetra (4-carboxyphenyl) porphin (PTP). pO2 was determined by evaluating the phosphorescence life time of PTP [5,16].

Anoxic bath solution was prepared by leaving it flow through an oxygen-permeable Silastic silicon tubing wound within a brass tube that was gassed with pure nitrogen [5]. The bath was kept free of oxygen by insulation with ultra pure argon.

2.4 Chemicals
Collagenase was obtained from Sigma (type I), Worthington Biochemical Corporation, or Biochrom. PTP was purchased from Medical Systems Corp. (Greenvale, NY). Ascorbate oxidase, phosphoenol pyruvate, oligomycin, iodoacetic acid (IAA), and FCCP were obtained from Sigma. D-fructose 1,6-bisphosphate was purchased from Fluka. All other chemicals were standard.

2.5 Statement
The investigation conforms with the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.6 Statistics
Statistical data are given as mean±SEM. Statistical significance was tested with the Student’s t-test (P<0.05).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
A myocardial cell produces ATP along three series of reactions (Fig. 1). We utilized various experimental interventions to determine which source of ATP predominantly regulates the KATP channels in the presence of external glucose. Effects on amplitude and time course of IKATP were evaluated. In whole-cell experiments, glycogenolysis was supposed to be inhibited by 10 mmol/l EGTA in the pipette (see textbooks of biochemistry). The efficacy of the dialysis with EGTA could be inferred from the rapid cessation of contraction after gaining access to the cell interior.


Figure 1
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  		Fig. 1 Scheme of cellular ATP production. (1) ATP production by glycolytic degradation of glucose to pyruvate in the cytosol. (2) Mitochondrial ATP synthesis in the citrate cycle via GTP. (3) Oxidative ATP production in the inner mitochondrial membrane via cytochromes, F1F0-ATPase, and ATP/ADP translocase. The two mitochondrial membranes are symbolized by stippled lines. Main pathways for ATP production are indicated by bold lines and arrows. The rectangular boxes indicate the experimental interventions: FCCP, anoxia and oligomycin were used for uncoupling and to block cytochrome oxidase and F1F0-ATPase, respectively. IAA was used to block glycolysis. Pyruvate, acetate, and lactate were used as mitochondrial and glucose as glycolytic substrate.

 
3.1 Mitochondrial substrates are highly effective in blocking spontaneous IKATP
When voltage clamping a myocyte in the absence of any inhibiting agent of the metabolism, IKATP developed spontaneously, most likely due to dialysis of ATP and other substrates via the pipette [17]. The insets in Fig. 2 show typical current traces of IKATP in either the absence of glucose (A), the presence of glucose (B), or the absence of glucose at blocked glycolysis (C). Control currents (1) are dominated by the voltage-dependent K+ current [18]. After a latency IKATP appeared (2). Glucose did not affect the amplitude of IKATP but significantly prolonged the latency compared to substrate-free conditions. Conclusively, at the time when IKATP appeared the degradation of bath glucose in glycolysis and mitochondria did not provide sufficient ATP to reduce IKATP. Glycolysis was presumably blocked by cell dialysis during the relatively long recording interval. Hence, the prolongation of the latency by bath glucose may have been the result of stimulated ATP production before glycolysis was blocked. When glycolysis was blocked by adding IAA to the substrate-free bath solution, the latency of IKATP was similar to that in the absence of IAA. This result shows that under our experimental conditions (dialysis of the cell with high EGTA) glycogenolysis did not provide substrate for the ATP synthesis.


Figure 2
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  		Fig. 2 Pyruvate added to the bath solution rapidly removes spontaneous IKATP. Here, and in the following figures, diamonds indicate the current at the end of a test pulse to +40 mV (late current) and filled squares indicate the holding current. The insets show original recordings elicited by voltage steps from –80 to 40 mV. The level of zero current is indicated by a dashed line. (A) Glucose-free bath solution. The illustrated time course starts 200 s after whole-cell access. Short pulses of pyruvate removed IKATP. (B) Bath solution with 5.5 mmol/l glucose. Pyruvate removed IKATP as effective as without glucose. (C) At blocked glycolysis (1 mmol/l IAA) and without glucose in the bath, short pulses of pyruvate blocked IKATP.

 
Next the influence of the mitochondrial substrates pyruvate, acetate and lactate on the spontaneous IKATP was tested. Washing pyruvate (5–10 mmol/l) on and off allowed repeated inhibition and induction of IKATP, respectively, under all conditions tested (Fig. 2): in the presence of glucose (n=14), in the absence of glucose (n=4), and at blocked glycolysis without glucose (n=6). However, after rigor contracture had once started, pyruvate had no effect (Fig. 2C). Pyruvate did not stop the cell shortening, indicating that it cannot overcome the ATP consumption caused by rigor contracture itself [19]. In the absence of glucose, pyruvate exerted the same effect at concentrations between 0.1 and 10 mmol/l (n=14). Similar effects were also observed with 10 mmol/l acetate (n=4) and 5 mmol/l lactate (n=5). In the continuous presence of pyruvate and IAA, IKATP did not appear (600–1100 s; n=7).

In order to prove that pyruvate acts exclusively as mitochondrial substrate, we blocked mitochondria by anoxia (Fig. 3A) or 1 µmol/l FCCP (not shown). Shortly after development of IKATP, application of pyruvate failed to abolish IKATP in 12 out of 12 cells with anoxia and in 14 out of 14 cells with FCCP. In the case of anoxia, subsequent reoxygenation immediately inhibited IKATP, confirming intact mitochondrial function (Fig. 3A). We also excluded that pyruvate has any direct or indirect effects on KATP channels in inside-out patches. These experiments were performed in patches containing 10–15 channels. In the presence of 10 mmol/l pyruvate at the extracellular side of the membrane (pipette solution), 10 mmol/l pyruvate applied and removed at the intracellular membrane side did not influence IKATP (n=3), neither at –80 mV (Fig. 3B) nor at +80 mV (not shown).


Figure 3
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  		Fig. 3 Pyruvate has no effect on KATP channels apart from being mitochondrial substrate. (A) Whole-cell experiment. Same symbols as in Fig. 2. Pyruvate did not remove IKATP when mitochondria were blocked by anoxia. Subsequent reoxygenation removed IKATP. (B) Inside-out patch experiment. Test Pulses of 700 ms duration were elicited from a holding potential of 0 mV to the test potential of –80 mV at a frequency of 1 Hz. The pipette solution contained 10 mmol/l pyruvate. Supplements of the bath solution are indicated. Removal of 5 mmol/l ATP generated an inwardly directed IKATP of about 70 nA. The presence or absence of 10 mmol/l pyruvate in the bath solution had no influence on IKATP.

 
3.2 IKATP develops when mitochondria are blocked by anoxia or FCCP, even in the presence of glycolytic substrate
When blocking mitochondria by either anoxia or the uncoupler FCCP, an essential control of KATP channels by glycolytic ATP should be clearly revealed because both interventions activate glycolysis [13,20]. We started anoxia or FCCP application within 30 s after whole-cell access in order to minimize the wash-out of cytoplasmic substrates.

In anoxia experiments, the latency was defined as the time interval between the drop of pO2 below 0.2 mmHg, the level of half maximum activity of the cytochrome oxidase, and the onset of IKATP [5]. Fig. 4A shows the time courses of pO2, holding current, and late current of a typical anoxia experiment in the presence of external glucose. An extra current developed after a latency of 6 min which is IKATP [21]. Reoxygenation removed IKATP and the scenario was repeated. A following longer interval of anoxia generated the typical IKATP transient [5]. The maximum amplitude of IKATP was similar in the absence and presence of glucose. However, glucose prolonged the latency (Table 1). This effect of glucose was independent of the glucose concentration (5.5, 10, or 20 mmol/l). When glycolysis was blocked by 1 mmol/l IAA, anoxia produced IKATP and reoxygenation removed the current as rapid as in the absence of IAA (Fig. 4B). The effective removal of IKATP by reoxygenation implies that it is the ATP produced by the reactivated mitochondria that has blocked the KATP channels.


Figure 4
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  		Fig. 4 Effect of anoxia and reoxygenation on IKATP. The open squares represent pO2, the other symbols correspond to Fig. 2. (A) Anoxia evoked IKATP in the presence of 5.5 mmol/l glucose. Reoxygenation removed IKATP. (B) Anoxia-induced IKATP at blocked glycolysis (1 mmol/l IAA) and in the absence of glycolytic substrate in the bath solution. Under both conditions IKATP was immediately abolished by reoxygenation, indicative that KATP channels are effectively blocked by mitochondrial ATP. The transient pO2 increase after 260 s in B started at a pO2 much larger than 0.2 mmHg and was thus without relevance for the experiment.

 

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  		Table 1 Amplitude and latency of spontaneous and anoxia induced IKATP

 
2-deoxyglucose (DG) is phosphorylated by the cytosolic hexokinase to 2-deoxyglucose 6-phosphate, which is nonmetabolizable, utilizing ATP. When 10 mmol/l DG was added 3 min before starting substrate-free anoxia, the latency until the appearance of IKATP was extremely shortened (Table 1). This dramatic effect of DG shows that the glucose uptake and hexokinase activity in our cells was intact.

Table 2 summarizes all results obtained with FCCP and various supplements in the bath and the pipette solution. With standard pipette solution, 1 µmol/l FCCP generated large IKATP in the absence (condition A) and presence of glucose (condition B; cf. Fig. 5A). In analogy to anoxia, the mean maximum amplitude was unchanged whereas the latency between drug application and appearance of IKATP was longer in the presence of glucose. In the case of anoxia- and FCCP-induced IKATP, glucose may have prolonged the latency by an increased ATP production before (aerobic metabolism) and during mitochondrial blockade (anaerobic metabolism). However, the similarity of the amplitudes in the absence and presence of glucose indicates that stimulation of glycolysis by glucose has no relevant effect on the number of KATP channels opened by anoxia or FCCP.


Figure 5
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  		Fig. 5 FCCP-induced uncoupling in the presence of glucose. Same symbols as in Fig. 2. (A) IKATP appeared after a latency of 80 s. Shortly after the peak current (45 nA), a series of test pulses to voltages between –60 and +40 mV was elicited from a holding potential of –120 mV. Rigor did not develop during the experiment. (B) FCCP-induced IKATP in the additional presence of oligomycin. The latency (440 s) was much longer whereas the amplitude (39 nA) was similar compared to the experiment in A.

 
Under both anoxia or uncoupling, the mitochondrial F1F0-ATPase reverses, becoming an important consumer of ATP [22]. In order to test for an influence of the F1F0-ATPase on the development of IKATP, this enzyme was blocked by oligomycin that was applied 5–10 min before gaining whole-cell access. In the presence of 10 mmol/l glucose, FCCP induced a large and transient IKATP after a much longer latency (Fig. 5B) than in the absence of oligomycin whereas the amplitude was not affected (cf. Table 2, condition B versus D). The same result was obtained in the absence of glucose (condition A versus C). In the presence of oligomycin glucose had no effect on the mean maximum amplitude and the mean latency of FCCP-induced IKATP (condition C versus D). The pronounced oligomycin-induced prolongation of the latency of IKATP confirms that the F1F0-ATPase essentially consumes ATP after FCCP application [22]. On the other hand, the presence of oligomycin did not reveal a glucose-induced reduction of the IKATP amplitude. Hence, glycolysis did not keep a relevant number of KATP channels closed even when the mitochondrial F1F0-ATPase was blocked and ATP consumption was low.

In whole-cell experiments with long latencies there is a risk that glycolysis is blocked by dialysis of intermediate products or activators of the glycolytic pathway via the pipette solution. To address this question, we added to the pipette solution the substrates of the two ATP producing steps of the glycolytic pathway, D-fructose 1,6-bisphosphate and phosphoenolpyruvate, as well as NAD, ADP and PO43–. Glucose, oligomycin, and FCCP were added to the bath solution as described above. With oligomycin, the additives in the pipette solution had no significant effect on the latency and maximum amplitude (Table 2, condition G versus D). Even when 0.6 mmol/l ATP was additionally supplemented to the pipette solution, latency and maximum amplitude of IKATP were not significantly affected, neither in the presence (condition E versus D) nor in the absence of oligomycin (condition F versus B). The result was that the dialysis of our myocytes with glycolytic substrates ultimately did not enable glycolytic ATP to keep the KATP channels closed and that also the dialysis of ATP itself did not suffice to close the channels. These results strongly suggest that in the experiments with anoxia and FCCP and with control pipette solution, cell dialysis was not relevant for generating IKATP.

3.3 IKATP in cell-attached measurements
In order to further confirm that IKATP observed in the whole-cell experiments was not induced by an inhibited glycolysis due to dialysis via the pipette, we performed cell-attached experiments. We expected IKATP to appear after a longer latency compared to respective whole-cell experiments because additional substrate for glycolysis would be provided by glycogenolysis, which should remain intact under cell-attached conditions. Experiments were conducted with FCCP (1 µmol/l) only, FCCP plus glucose (5.5 mmol/l), and FCCP plus glucose plus oligomycin. Fig. 6A, top shows current traces before (lower trace) and after the development of IKATP (upper trace). The corresponding current differences (shown below) were taken to be caused by IKATP because of their characteristic inward rectification. Fig. 6B shows three typical time courses of IKATP in cell-attached patches. FCCP alone generated IKATP after a latency of 195 s. In the presence of glucose, IKATP appeared after 255 s. After additionally inhibiting the F1F0-ATPase by pre-incubation with oligomycin, IKATP developed after the longer latency of 720 s. The respective mean values were 275±70 s (n=4), 245±12 s (n=5), 1860±830 s (n=6), respectively. In the presence of oligomycin, two other cells did not develop IKATP within 3000 and 3600 s. As expected, the latencies were generally longer than measured under respective whole-cell conditions. The main result of the cell-attached experiments, however, is that IKATP could be induced under all tested conditions. This result is in line with those of the whole-cell measurements. Hence, glycolytic ATP production by utilization of external glucose does not suffice to keep KATP channels closed, not even when the massive ATP consumption by the F1F0-ATPase is blocked.


Figure 6
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  		Fig. 6 FCCP-induced IKATP in cell-attached patches. (A). Top: Current traces during voltage ramps from Vr –20 mV to Vr +160 mV measured before (lower trace) and after development of IKATP (upper trace). Bottom: The current difference of the traces in the top diagram shows strong inward rectification, typical for IKATP. (B) Time course of IKATP (mean current difference) measured between Vr +60 mV and Vr +70 mV. Experimental conditions are indicated above the current peaks. [glucose]=5.5 mmol/l; oligomycin was applied 5 min before starting the experiment.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We identified the source of the ATP controlling the sarcolemmal KATP channels in mouse cardiomyocytes by measuring amplitude and latency of IKATP under various conditions. These conditions included the block of metabolic reactions and the application of substrates specific for the type of ATP production.

In excised patches it is well documented that the [ATP] of half maximum inhibition of KATP channels (IC50) is 25–100 µmol/l [23]. Although the IC50 value may be higher under various conditions [2,24,25], a drop of the [ATP] to below several hundred µmol/l is always required for a relevant channel opening [3]. Based on these data, we interpret our results in terms of a drop of [ATP] to micromolar levels as the key determinant for the opening of KATP channels.

This interpretation is also supported by the observation that rigor contracture appeared shortly after IKATP reached its maximum amplitude. Rigor contracture, that has been shown to appear at 0.1–100 µmol/l MgATP [26,19], further decreases the cytosolic [ATP] [27]. Also the observed run down of whole-cell IKATP suggests a drop of [MgATP] to very low levels [28]. Our results suggest a common cytosolic ATP pool controlling sarcolemmal KATP channels with an only subsidiary input from substrate-level but a dominating input from oxidative phosphorylation.

4.1 The effect of glucose on whole-cell IKATP
In whole-cell experiments there was dialysis of the cells via the pipette solution: EGTA blocked the contraction by reducing the cytosolic [Ca2+] which should also be responsible for blocking glycogenolysis. However, an influence of dialysis on IKATP by depletion of cytoplasmic components could be excluded in as much as similar results were obtained when glycolytic substrates and ATP were added to the pipette solution (Table 2).

All amplitudes of the whole-cell IKATP were similar, no matter if the current was induced by cell dialyses, FCCP, or anoxia and if glucose was present in the bath solution or not. However, the latencies of IKATP were prolonged in the presence compared to the absence of glucose. One possible reason for the delayed appearance of IKATP may have been a higher level of creatine phosphate and ATP generated from the bath glucose before metabolic inhibition. In the case of anoxia and FCCP another reason may be a decelerated drop of cellular [ATP] caused by an increased glycolytic flux during metabolic inhibition [13,20]. At prolonged mitochondrial inhibition however, glycolysis did not provide enough ATP to keep the KATP channels closed, even when external glucose was added as glycolytic substrate.

Uncoupling stimulates mitochondrial respiration [20]. Hence, during uncoupling the [NADH] cannot increase and thus reduce the activity of glyceraldehyde-3-phosphate dehydrogenase as it is the case during anoxia. Therefore, FCCP presumably activates glycolysis even more than does anoxia. Nevertheless, FCCP generated IKATP after a shorter latency than anoxia in both the absence and presence of glucose. FCCP most likely causes a more rapid decline of the mitochondrial proton gradient, causing the F1F0-ATPase to consume ATP by running in the reverse direction [22]. Thus the cytosolic [ATP] should decrease faster under FCCP. The observed prolongation of the latency by the blocker of the F1F0-ATPase, oligomycin, strengthens this interpretation.

Reoxygenation blocked anoxia-induced IKATP even in the absence of glucose and when glycolysis was blocked by IAA (Fig. 4B). Therefore, mitochondrial ATP must have closed the KATP channels. It is also noticeable that the reoxygenated cell has enough substrates — other than glucose — for sufficient mitochondrial ATP production. This interpretation fits with the observation that substrate deprivation causing opening of KATP channels needs a much longer duration of cell dialysis with the pipette solution. Together, glycolysis probably delays the drop of cellular ATP during mitochondrial inhibition, but it is clear that it does not supply enough ATP to keep a significant portion of KATP channels closed.

In the presence of glucose, FCCP induced an IKATP with a much slower run down than under all other conditions (Fig. 5A) and rigor contracture appeared much later. This finding may be the result of an FCCP-induced stimulation of glycolysis [20] maintaining a basic ATP level that prevented rigor and attenuated channel run down. However, a slowed run down and a delayed rigor contracture was not observed when oligomycin was present in addition to FCCP and glucose (Fig. 5B), even when glycolytic substrates were added to the pipette solution (not shown). Further experiments are necessary to explain this phenomenon.

4.2 The effect of mitochondrial substrate on whole-cell IKATP
The tested substrates of oxidative phosphorylation pyruvate, acetate, and lactate effectively suppressed spontaneous IKATP as induced by dialysis with the pipette solution (Fig. 2). In guinea-pig myocytes, extracellular pyruvate (0.1–10 mmol/l) was reported to close KATP channels via coupling with the sarcolemmal mono-carboxylate transporter when IKATP was induced by either cyanide or 2,4-dinitrophenol [29]. In contrast, we found no effect of pyruvate when mitochondria were blocked by FCCP or anoxia (Fig. 3A). Moreover, we showed in inside out patches that 10 mmol/l pyruvate at the extracellular side, with or without transmembrane gradient, did not reduce IKATP (Fig. 3B). For mouse myocytes we, therefore, exclude a significant influence of the mono-carboxylate transport on KATP channels and conclude that pyruvate acted exclusively as mitochondrial substrate.

4.3 Cell-attached measurements
We repeated some of our whole-cell experiments with FCCP under cell-attached conditions because then all components of glycolysis should be unaffected. The relatively long latencies observed under these conditions may have been caused by an enhanced glycolytic ATP production from substrate provided by glycogenolysis. Under cell-attached conditions, glycogenolysis is supposed to be intact because the cytosolic [Ca2+] is not buffered by EGTA and phosphorylasekinase is not inhibited. However, external glucose did not prevent KATP channels from opening when mitochondria were blocked by FCCP. Even when reducing the ATP consumption by oligomycin, in most of the myocytes IKATP appeared during the recording time.

4.4 Implications for the ATP production under physiological conditions
Evidence has been presented that in a contracting cell ATP produced by mitochondria is predominantly utilized for contraction [9,10]. Consistently, Weiss and Lamp [9,10] reported that at high ATP utilization, glycolytic ATP production is more effective whereas mitochondrial ATP production is ineffective in regulating IKATP. At low ATP utilization glycolysis and oxidative phoshorylation were reported to be similarly effective.

In our quiescent cardiomyoctes, the utilization of ATP was low. Therefore, even a weak glycolytic rate may have decelerated the ATP drop in the cell. However, the KATP channels were not kept closed by glycolysis alone under all conditions tested. In contrast, mitochondrial ATP production had a large effect on IKATP.

The key experiments of Weiss and Lamp [9,10] were performed in cell-attached patches on saponin-permeabilized myocytes in the presence of an artificial ATP-consuming system. Methodical differences may explain the controversial findings: (1) An underestimation of mitochondrial ATP could have been caused by a nonuniform distribution of the artificial ATP consuming system as well as by saponin, which may have affected mitochondria to such an extent that their ability to produce ATP was reduced. (2) An overestimation of glycolytic ATP production may have been caused by relatively high concentrations of glycolytic substrates (concentrations of D-fructose 1,6-bisphosphate and phosphoenolpyruvate were about 10 fold higher as in our experiments and 200–1000 fold higher than under physiological conditions).

In a beating heart the rate of ATP production and utilization is supposed to be in an oscillating steady state at a much higher rate than in quiescent cardiomyoctes. In this situation ATP is produced by the use of external substrates as glucose, fatty acids, and lactate. The cellular content of glycogen is presumably constant until the ATP demand changes. This situation is similar to the conditions in our whole-cell measurements where glycogenolysis was blocked. With regard to the inefficiency of glycolysis in our quiescent cardiomyocte, in a contracting cell glycolysis would have to be extremely upregulated to gain control of the activity of KATP channels. This is not likely. We therefore conclude, that mitochondria control KATP channels in a beating cell as well.

Time for primary review 22 days.


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

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