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Am J Physiol Heart Circ Physiol 285: H1420-H1427, 2003. First published June 12, 2003; doi:10.1152/ajpheart.00167.2003
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Myocardial oxygenation and high-energy phosphate levels during KATP channel blockade

Jianyi Zhang, Arthur H. L. From, Kamil Ugurbil, and Robert J. Bache

Departments of Medicine, Radiology, Physiology and the Center for Magnetic Resonance Research, University of Minnesota Health Sciences Center, Minneapolis, Minnesota 55455

Submitted 23 February 2003 ; accepted in final form 26 May 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Inhibition of ATP-sensitive K+ (KATP) channel activity has previously been demonstrated to result in coronary vasoconstriction with decreased myocardial blood flow and loss of phosphocreatine (PCr). This study was performed to determine whether the high-energy phosphate abnormality during KATP channel blockade can be ascribed to oxygen insufficiency. Myocardial blood flow and oxygen extraction were measured in open-chest dogs during KATP channel blockade with intracoronary glibenclamide, whereas high-energy phosphates were examined with 31P magnetic resonance spectroscopy (MRS), and myocardial deoxymyoglobin (Mb-{delta}) was determined with 1H MRS. Glibenclamide resulted in a 20 ± 8% decrease of myocardial blood flow that was associated with a loss of phosphocreatine (PCr) and accumulation of inorganic phosphate. Mb-{delta} was undetectable during basal conditions but increased to 58 ± 5% of total myoglobin during glibenclamide administration. This degree of myoglobin desaturation during glibenclamide was far greater than we previously observed during a similar reduction of blood flow produced by a coronary stenosis (22% of myoglobin deoxygenated during stenosis). The findings suggest that reduction of coronary blood flow with an arterial stenosis was associated with a decrease of myocardial energy demands and that this response to hypoperfusion was inhibited by KATP channel blockade.

blood flow; myoglobin; oxygen saturation

ATP-SENSITIVE POTASSIUM (KATP) channels have the potential to influence energy metabolism in the heart. Opening of KATP channels on vascular smooth muscle cells allows efflux of potassium; the resultant membrane hyperpolarization closes voltage-gated calcium channels to result in vasodilatation with an increase of blood flow (25). KATP channels on coronary arterioles open in response to hypoxia and appear to be critical for metabolic vasoregulation by which coronary blood flow is regulated in response to myocardial metabolic requirements (11, 20). We previously observed that in intact awake dogs, KATP channel blockade with intracoronary glibenclamide resulted in vasoconstriction with a 17–20% decrease in coronary blood flow (4, 5). This decrease in coronary flow was associated with a parallel reduction of myocardial oxygen consumption (MO2) and a significant decrease of systolic wall thickening. These findings suggested that glibenclamide resulted in coronary vasoconstriction with a decrease in myocardial blood flow sufficient to result in ischemic contractile dysfunction. However, an alternative possibility is that glibenclamide caused a primary decrease of MO2 so that the reduction of coronary flow occurred secondary to the decreased oxygen uptake. This possibility is based on studies of KATP channels located on the inner mitochondrial membrane where they have the potential to influence respiration and ATP synthesis (10, 15). If blockade of mitochondrial KATP channels by glibenclamide caused a decrease of MO2, then the consequent reduction of ATP synthesis could lead to decreased contractile performance despite adequate oxygen availability.

1H nuclear magnetic resonance (NMR) spectroscopy can be used to detect myoglobin desaturation. The unpaired electron spin in the heme-Fe(II) complex of deoxymyoglobin (Mb-{delta}) extends over the proximal histidyl N{delta} proton to cause a chemical shift that produces a characteristic resonance on 1H NMR spectroscopy (6). Using isolated perfused rat hearts, Kreutzer and Jue (12) demonstrated that the degree of myoglobin deoxygenation can be quantitated with (NMR) spectroscopy and used to determine intracellular oxygen tension. We have adapted this technique for in vivo use and have demonstrated that decreases of myocardial blood flow produced by graded coronary artery stenoses resulted in an increasing Mb-{delta} signal that was proportional to the decrease in blood flow (2, 27). In the present study, 1H NMR spectroscopic detection of Mb-{delta} was used to assess myoglobin oxygenation during KATP channel blockade with glibenclamide. This allowed evaluation of whether the decrease in blood flow produced by glibenclamide resulted in insufficient myocardial oxygen availability or resulted from a primary decrease in respiration, in which case myoglobin desaturation would not be expected. 31P NMR spectroscopy was combined with 1H NMR to determine whether the reductions of coronary flow produced by glibenclamide resulted in alterations in high-energy phosphates (HEP) similar to those when blood flow is reduced by a coronary stenosis.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Studies were performed in 12 adult mongrel dogs of either sex weighing 20–27 kg. All experimental procedures were approved by the University of Minnesota Animal Care Committee. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985).

Experimental preparation. The dogs were anesthetized with pentobarbital sodium (30–35 mg/kg bolus, followed by 4 mg · kg–1 · h–1 iv), intubated, and ventilated with a respirator with supplemental oxygen to maintain arterial blood gases within the physiological range. A heparin-filled polyvinyl chloride catheter (3.0 mm od) was introduced into the right femoral artery and advanced into the ascending aorta. A left thoracotomy was performed through the fourth intercostal space, and the heart was suspended in a pericardial cradle. A heparin-filled catheter (3.0 mm od) was introduced into the left ventricle (LV) through the apical dimple and secured with a purse-string suture. A similar catheter was inserted into the left atrium through the atrial appendage. The proximal left anterior descending coronary artery (LAD) was dissected free, and a hydraulic occluder constructed of polyvinyl chloride tubing (2.7 mm od) was placed around the artery. A silicone elastomer catheter (0.30 mm id) was placed into the LAD distal to occluder. A similar catheter was placed into the anterior interventricular vein for blood sampling from the region perfused by the LAD. The region of the LV that became cyanotic on inflation of the occluder was determined by visual inspection. A 28-mm diameter magnetic resonance spectroscopy (MRS) surface coil was sutured onto the pericardium overlying the ischemic area. The pericardial cradle was then released and the heart allowed to assume its normal position. The surface coil leads were connected to a balanced tuned circuit, and the animals were placed into the magnet.

NMR spectroscopy: general methods. Measurements were performed in a 40-cm bore, 4.7-Tesla magnet interfaced with a Spectroscopy Imaging Systems (SISCO; Fremont, CA) computer console. The LV pressure signal was used to gate MRS data acquisition to the cardiac cycle, whereas respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions (14, 18, 19). 31P and 1H MRS frequencies were 81 and 200.1 MHz, respectively.

Detection of myoglobin desaturation. The method for 1H MRS detection of the proximal histidyl N-{delta} proton resonance of Mb-{delta} has been described in detail (2, 27). Briefly, a single-pulse collection sequence with a Gaussian pulse (1 ms) was used to selectively excite the N-{delta} proton signal of the proximal histidyl of Mb-{delta}. This frequency-selective pulse provided sufficient water suppression due to the large chemical shift difference between the water resonance and Mb-{delta} (>14 kHz). A short repetition time (35 ms) was used due to the short T1 value of Mb-{delta}. Each spectrum is acquired in 6 min (10,000 free induction decay). Although the short T1 of Mb-{delta} and the fast acquisition prevented gating of data acquisition to the cardiac cycle, signal loss as a result of heart motion was negligible because of the inherently broadline width of the Mb-{delta} peak. Although the Mb-{delta} resonance is temperature sensitive, the chemical shift of this resonance, which appeared at 71–72 parts/million (ppm) (relative to H2O), remained virtually constant during the study protocol. No other resonances were detected within a 10-ppm region. We have previously established that the Mb-{delta} resonance originates from all layers across the LV wall with a slight preference for the mid and inner layers (27).

31P NMR technique. 31P NMR spectra were acquired in late diastole with a pulse repetition time of 6–7 s. This repetition time allowed full relaxation for ATP and inorganic orthophosphate (Pi) resonances, and ~90% relaxation for the phosphocreatinine (PCr) resonance. PCr resonance intensities were corrected for this minor saturation. Radiofrequency transmission and signal detection were performed with a 28-mm diameter surface coil. A capillary containing 15 µl of 3 M phosphonoacetic acid was placed at the coil center to serve as a reference. Details of the method of spatial localization across the LV wall have been previously published (18, 19). Briefly, signal origin was restricted using B0 gradients and adiabatic inversion pulses to an 18 mm x 18 mm column perpendicular to the LV wall. Resonance intensities were quantified using integration routines provided by SISCO software. Chemical shifts were measured relative to PCr, which was assigned a chemical shift of –2.55 ppm relative to 85% phosphoric acid at 0 ppm. Values for PCr and ATP were normalized to those present in the basal state, and the PCr-to-ATP ratio was determined for each voxel. Pi resonances were measured, and the ratio of Pi to PCr was calculated. Mean HEP for the whole LV wall was obtained by averaging the HEP contents in the subepicardial, midwall, and subendocardial voxels.

Myocardial blood flow measurements. Myocardial blood flow was measured with 15-µm diameter microspheres labeled with gamma-emitting radionuclides (51Cr, 85Sr, 95Nb, or 46Sc). Microsphere suspension containing 2 x 106 microspheres was injected through the left atrial catheter, whereas a reference sample of arterial blood was withdrawn from the aortic catheter at a rate 15 ml/min. Myocardial and blood reference specimen radioactivity was used to compute blood flow as milliliter per gram of myocardium per minute (27).

Myocyte oxygenation. Myocyte oxygenation was estimated from the myoglobin oxygen saturation-PO2 relationship described in Eq. 1.

(1)
In Eq. 1, Mb-O2 and Mb-{delta} are the fractional contents of oxymyoglobin and deoxymyoglobin, respectively, PO2 is the intramyocyte partial pressure of O2, and [PO2]50 is the partial pressure of O2 at which myoglobin is half saturated with O2 (2.38 mmHg at 37°C) (21). Therefore, if the integral of the Mb-{delta} resonance measured during total coronary occlusion performed at the conclusion of each study represents the total myoglobin content (in the Mb-{delta} form), then the resonance integrals determined during other experimental conditions can be normalized relative to this value to result in Mb-{delta} expressed as a fractional value of the total myoglobin content. The fractional content of Mb-O2 can be calculated from the relationship Mb-O2 = (1 – Mb-{delta}). The fractional contents of Mb-O2 and Mb-{delta} and the temperature-corrected [PO2]50 value can then be employed to calculate intracellular PO2 using Eq. 1. Because of continuing collateral blood flow, we assumed that the Mb-{delta} resonance measured during total coronary occlusion represented 95% of the total myoglobin and normalized the other Mb-{delta} resonances in relation to that value. We assumed that the fractional Mb-O2 content in the basal state (i.e., a time when no Mb-{delta} resonance was detected) was ~90%. This follows from calculations using Eq. 1; at 37°C calculated intramyocyte PO2 would be 21 and 46 mmHg for Mb-O2 saturations of 90 and 95%, respectively. We assumed a fractional Mb-O2 content of ~90% during basal conditions because this value is consistent with the known oxygen gradient between the capillary and intracellular myoglobin and the observed values for coronary venous PO2 (9).

Study protocol. Aortic and LV pressures were measured with transducers positioned at midchest level and recorded on an eight-channel direct-writing recorder (Coulbourne Instrument; Lehigh Valley, PA). LV pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic measurements and 31P and 1H MRS spectra were first obtained under basal conditions. Midway through the 20-min data acquisition period, a microsphere injection was performed for determination of myocardial blood flow. After completion of baseline measurements, adenosine receptor blockade was produced by administration of 8-phenyltheophylline (5 mg/kg iv). This dose has been previously shown to produce >90% inhibition of the coronary vasodilator response to adenosine while causing minimal phosphodiesterase inhibition (22). At the start of 10 min after administration of 8-phenyltheophylline, glibenclamide was infused at a rate of 20 µg · kg–1 · min–1 through the coronary artery catheter. After 10 min was allowed to achieve steadystate conditions, 31P and 1H spectra were obtained again, and a microsphere injection was performed. After completion of data acquisition, the glibenclamide infusion was discontinued, and 20 min were allowed to return to baseline conditions. Finally, to provide a calibration signal for the Mb-{delta} resonance, the LAD was totally occluded by inflating the hydraulic occluder, and all measurements were repeated.

Data analysis. Hemodynamic data were measured from the chart recordings. 31P and 1H NMR spectra were analyzed as described above. Myocardial blood flow in three transmural layers from epicardium to endocardium was determined from the microsphere measurements. Data were analyzed with one-way analysis of variance for repeated measures. Multiple-regression analysis (Statistica) was used for comparison of the regression equations obtained from historical data with similar data from the present animals. A value of P < 0.05 was considered significant. When a significant result was found, individual comparisons were made using the method of Scheffé.


   RESULTS
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DISCUSSION
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Hemodynamic and myocardial blood flow data. Hemodynamic measurements during each experimental condition are shown in Table 1. Infusion of glibenclamide caused no significant change in heart rate, mean aortic pressure, or LV systolic pressure but increased LV end-diastolic pressure from 3 ± 1 to 8 ± 1 mmHg (P < 0.05). Occlusion of the LAD was associated with significant reductions of mean aortic and LV systolic pressures, whereas LV end-diastolic pressure was similar to that during the glibenclamide infusion.


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  		Table 1. Hemodynamic data

 

As shown in Table 2, glibenclamide in the presence of adenosine receptor blockade resulted in a 20 ± 8% decrease of mean myocardial blood flow. The reduction of blood flow produced by glibenclamide was more prominent in the subepicardium than in the subendocardium, resulting in a significant increase in the subendocardial-to-subepicardial ratio from 1.11 ± 0.07 to 1.42 ± 0.13 (P < 0.05). Infusion of glibenclamide into the LAD resulted in no significant change of blood flow in the remote myocardial region. During total occlusion of the LAD, blood flow in the anterior LV wall fell to 3 ± 1% of the baseline value (P < 0.01). The reduction of myocardial blood flow during total occlusion was most marked in the subendocardium, with a decrease of the subendocardial-to-subepicardial flow ratio to 0.07 ± 0.04.


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  		Table 2. Myocardial blood flow

 

Myocardial Mb-{delta} levels. Typical 31P and 1H NMR spectra from one heart are shown in Figs. 1 and 2, respectively. The mean normalized Mb-{delta} data are in Table 3. During infusion of glibenclamide a resonance corresponding to Mb-{delta} was seen in the 1H NMR spectra; the resonance area was 58 ± 5% of the Mb-{delta} resonance detected during total coronary occlusion. To compare the Mb-{delta} changes during glibenclamide infusion with changes that occurred when coronary flow was limited by an arterial stenosis, data from the present animals were plotted with data previously obtained when flow was limited by a coronary stenosis (27). As shown in Fig. 3, for similar levels of blood flow reduction, glibenclamide produced a greater degree of myoglobin desaturation than did the stenosis (P < 0.05).



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  		Fig. 1. Typical 31P nuclear magnetic resonance (NMR) spectra showing myocardial high-energy phosphate (HEP) and Pi resonances from one heart under baseline conditions (A) and during infusion of glibenclamide (20 µg · kg–1 · min–1)(B). PCr, phosphocreatine. ppm, Parts/million.

 


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  		Fig. 2. Interleaved 1H NMR spectra obtained from the same heart as in Fig. 1, showing myocardial deoxymyoglobin (Mb-{delta}) resonances under baseline conditions (A), during infusion of glibenclamide (20 µg · kg–1 · min–1) (B), and during total left descending coronary artery occlusion (C).

 

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  		Table 3. Myocardial high-energy phosphate level and oxygenation data

 


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  		Fig. 3. Fractional myoglobin saturation (A) and calculated myocyte PO2 (B) plotted against normalized myocardial blood flow (MBF) during glibenclamide administration. Data are superimposed on a previously published plot obtained when myocardial blood flow was decreased with a coronary stenosis (27).

 

Myocardial HEP levels. Spectra obtained during baseline conditions demonstrated prominent resonances corresponding to PCr and the three phosphates of ATP, whereas Pi was below the limit of detectability. Infusion of glibenclamide was associated with a significant decrease of PCr but no significant change of ATP (Table 3), resulting in a decrease of PCr/ATP from 2.42 ± 0.11 during baseline conditions to 1.66 ± 0.08 during glibenclamide (P < 0.05). These changes were associated with appearance of a resonance corresponding to Pi ({Delta}Pi/PCr = 0.61 ± 0.03). When the glibenclamide infusion was discontinued, the spectra returned to the baseline state (data not shown). Total LAD occlusion at the end of the experiment was associated with a marked decrease of PCr and PCr/ATP, a moderate reduction of ATP, and a large Pi resonance. To determine whether the HEP changes during glibenclamide infusion were different from changes that occurred when coronary flow was limited by an arterial stenosis, PCr measurements from the present animals were plotted with similar data previously obtained when flow was limited by a coronary stenosis (27). As shown in Fig. 4, reductions of myocardial PO2 produced by a coronary stenosis and by glibenclamide resulted in essentially identical reductions of normalized PCr (P = not significant).



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  		Fig. 4. PCr during administration of glibenclamide normalized to the control value plotted against calculated myocyte PO2. Data are superimposed on a previously published plot obtained when myocardial blood flow was decreased with a coronary stenosis (2).

 

MO2. MO2 data from six animals are shown in Table 4. During glibenclamide infusion, the partial pressure of oxygen in coronary venous blood decreased by 13% (P < 0.05), mean myocardial blood flow decreased by 22% (P < 0.05), and MO2 decreased by 12% (P < 0.05).


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  		Table 4. Myocardial oxygen consumption data

 


   DISCUSSION
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 METHODS
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 DISCUSSION
DISCLOSURES
 REFERENCES
 
In a previous study using 1H MRS, we found that no Mb-{delta} was detectable in in vivo canine myocardium during basal conditions or with elevated workstates produced by catecholamine infusion despite the observation that HEP levels fell during very high work states (26). This suggested that even at high work states oxygen availability was nonlimiting and indicated that HEP changes that occur at very high workloads cannot be ascribed to oxygen limitation. In a recent study, we examined the relationships among myocardial blood flow, myocyte PO2, and HEP levels in in vivo myocardium under conditions where oxygen limitation does restrain the rate of oxidative phosphorylation (27). Using graded coronary artery stenoses, we found that reductions of blood flow that resulted in intramyocyte PO2 values <5 mmHg were associated with detectable loss of PCr. PCr was decreased to 80% of the control value when intramyocyte PO2 was reduced to 4 mmHg; further reductions of intracellular PO2 lead to precipitous decreases of PCr (27).

In the present study intracoronary glibenclamide resulted in a 20 ± 8% decrease of blood flow in the myocardial region perfused by the LAD. Similarly, in chronically instrumented dogs, we previously observed that glibenclamide caused a 17–20% decrease in coronary blood flow at rest, and during treadmill exercise that was associated with a decrease of both regional systolic wall thickening (4) and coronary venous oxygen tension, so that for any level of oxygen consumption, oxygen extraction was increased (5). This suggested that glibenclamide caused a decrease in oxygen availability relative to oxygen utilization rather than a primary decrease of mitochondrial respiration. This interpretation is supported by the present findings in which the decrease in myocardial blood flow produced by glibenclamide was associated with a prominent Mb-{delta} resonance that was 58 ± 5% of that observed during total coronary occlusion (when myoglobin is assumed to be fully desaturated). The degree of myoglobin desaturation during glibenclamide administration was substantially greater than when a similar decrease of coronary flow was produced by a stenosis (27). We had previously observed that the degree of myoglobin desaturation was linearly and proportionately related to the degree of flow reduction produced by the stenosis. With the use of data from that study, a 20% decrease in coronary flow resulted in a Mb-{delta} resonance equal to 18% of that during total coronary occlusion (27), far less than during the glibenclamide-induced decrease in flow in the present study. There are several possible explanations for the greater degree of myoglobin desaturation during the coronary flow reduction produced by glibenclamide. First, the transmural distribution of perfusion is different for a flow reduction produced by a stenosis compared with glibenclamide. When coronary flow is limited by a stenosis, the hypoperfusion is most severe in the subendocardium with a decrease of the subendocardial-to-subepicardial flow ratio (27). In contrast, glibenclamide tended to cause a greater decrease of blood flow in the subepicardium. The degree of myoglobin desaturation is likely to be most marked in regions with the most severe hypoperfusion. Thus for similar reductions of mean blood flow, myoglobin deoxygenation would likely be most severe in the subendocardium when flow is limited by a stenosis but more severe in the subepicardium during glibenclamide, which decreased flow preferentially in the outer myocardial layers. However, the 1H NMR technique used in the present study detects Mb-{delta} from all layers across the LV wall with a somewhat higher contribution from the mid and inner layers of the LV and less sensitivity in the subepicardium where the flow reduction was most marked during glibenclamide administration (27). Therefore, differences in the transmural pattern of hypoperfusion produced by glibenclamide versus a stenosis cannot explain the greater than expected myoglobin deoxygenation observed during glibenclamide administration.

A second possible difference between hypoperfusion caused by a coronary stenosis compared with glibenclamide relates to microheterogeneity of blood flow. In studies of tissue oxygenation during progressive decreases of blood flow in the hindlimbs of pigs, Vallet et al. (23) found that glibenclamide caused reductions of oxygen uptake and in the level of cytochrome aa3 oxygenation during moderate decreases of blood flow that did not cause these changes in control legs. They proposed that glibenclamide interfered with capillary recruitment as blood flow and pressure were decreased, resulting in small areas of ischemia in parallel with other areas that were adequately perfused. This microheterogeneity resulted in more prominent metabolic markers of ischemia when blood flow was reduced in limbs treated with glibenclamide. This explanation is supported by the known importance of smooth muscle KATP channels in mediating vasodilation of resistance vessels as perfusion pressure is decreased (11). Because the 1H NMR spectra represents mean myoglobin deoxygenation for the entire volume sensed, the present data do not allow determination of whether microheterogeneity could have contributed to the greater Mb-{delta} signal obtained during glibenclamide administration.

A third possible explanation for greater myoglobin deoxygenation when blood flow was reduced by glibenclamide than by a stenosis is related to changes in myocardial oxygen demands during hypoperfusion. As originally described by Gregg (7), MO2 can be influenced by coronary flow and perfusion pressure. A coronary pressure or flow-related decrease in energy demands would allow the heart to accommodate (at least in part) to the decreased oxygen availability during hypoperfusion. This modulation of oxygen consumption might result from signals during stenosis-induced hypoperfusion that decrease contractile activity (and therefore energy demands and oxygen consumption), or that directly inhibit mitochondrial respiration. Although the final rate of mitochondrial respiration might be similar (and determined by the rate of oxygen delivery by the limited blood flow), cytosolic oxygen tension would be higher if energy production or energy utilization were downregulated by signals sensitive to hypoperfusion rather than by the lowest level of oxygen tension that can sustain mitochondrial respiration.

The present data suggest that KATP channel blockade interferes with this mechanism. In our previous study (27) in which flow was limited by a stenosis, a 20% reduction of coronary flow resulted in an intracellular PO2 of 11 Torr. In contrast, in the present study, the 20% decrease of coronary blood flow produced by glibenclamide was associated with an intracellular PO2 of 1.72 Torr. The higher intracellular PO2 when coronary flow was reduced by a stenosis implies a decrease in oxygen demands so that respiration was not limited exclusively by oxygen availability in the cytosol. Several mechanisms might modulate oxygen demands during hypoperfusion. The pressure in the coronary arterial system has been proposed to distend the ventricle, thereby augmenting contractility and oxygen consumption by causing an increase of sarcomere length or ventricular stiffness (1, 24). Consequently, the decreased coronary pressure that occurs when a stenosis limits blood flow can act to reduce myocardial contractile force and energy utilization. The decrease in flow produced by glibenclamide occurs secondary to constriction of arterioles so that pressure in the coronary arteries is maintained (11); if coronary pressure is the principal mechanism for the Gregg effect, then contractile performance and oxygen demands might not be decreased when flow is reduced with glibenclamide, thereby resulting in a greater perfusion-metabolism mismatch with greater myoglobin deoxygenation.

Marban and associates (16) have proposed an alternate mechanism by which myocardial energy demands may be coupled to coronary flow. They observed that in the range of 60–160 mmHg, decreases of coronary perfusion pressure in isolated ferret hearts resulted in preload-independent decreases of the calcium transient. They speculated that this represented a protective mechanism that minimizes energy demand during low-flow ischemia. Although the intracellular signaling pathway responsible for this change in calcium kinetics is unclear, Lewandowski et al. (13) have reported that metabolic responses in the mitochondrial oxidative pathways also respond to signals other than changes in oxygen delivery and tissue blood flow. These authors reported that in open-chest swine, myocardium perfused with a coronary stenosis preferentially oxidized short-chain fatty acids over endogenous long-chain fatty acids. Surprisingly, this occurred in both the hypoperfused subendocardium as well as the subepicardium where blood flow was maintained near normal, indicating that signals other than oxygen availability regulate fatty acid utilization during hypoperfusion. The finding that in our experimental model cytosolic PO2 was higher when myocardial blood flow was limited by a stenosis than by glibenclamide suggests that inhibition of KATP channel activity can interfere with the modulation of oxygen consumption that normally occurs during myocardial hypoperfusion.

There is substantial evidence that myocardial KATP channels act as metabolic sensors that are critical for maintenance of cellular homeostasis during cardiac stress. Thus Zingman et al. (28) reported that mice deficient in the Kir6.2 subunit of the KATP channel had decreased endurance during treadmill exercise, impaired calcium handling during catecholamine stimulation, and decreased survival under extreme {beta}-adrenergic stress. The present findings suggest that KATP channels may also act to inhibit respiration of myocardial myocytes during hypoperfusion. Decreases of PO2 result in increased levels of cytosolic free [ADP] with a fall in PCr/ATP; Zingman et al. (28) found that deletion of Kir6.2 caused no major shift in the bulk adenine nucleotide content of the murine heart. Similarly, in our animals the relationship between intracellular PO2 and PCr/ATP was essentially identical whether the decreased PO2 was caused by a coronary stenosis or by glibenclamide, suggesting that KATP channel blockade did not interfere with phosphoryl transfer within the myocyte.

In a previous study by Duncker et al. (4), they observed that intracoronary glibenclamide caused a 19% average decrease in coronary blood flow in resting awake dogs, but did not impair the normal increase in blood flow during exercise, resulting in a parallel downward shift in the relationship between coronary flow and the rate-pressure product. The coronary vasodilation in response to exercise after KATP channel blockade with glibenclamide was in part mediated by increased adenosine production in the hypoperfused myocardium, because the addition of adenosine receptor blockade with 8-phenyltheophylline markedly blunted the increase in coronary flow during exercise in animals receiving glibenclamide (5). (When K+ channels were intact, 8-phenyltheophylline had no effect on coronary flow, indicating that adenosine does not normally contribute to metabolic coronary vasodilation in the normal heart.) Samaha et al. (20) also obtained results indicating increased importance of adenosine after KATP channel blockade. Using openchest dogs, they observed that glibenclamide caused coronary vasoconstriction with a 15% decrease of MO2. Despite this very modest decrease of MO2, glibenclamide caused a shift from lactate consumption to lactate production, a decrease in PCr/ATP, an increase in free Pi, and intracellular acidosis, consistent with tissue ischemia. In that study the initial reduction of coronary flow produced by glibenclamide was sometimes followed by flow oscillations with marked reductions of blood flow alternating with increased coronary flow. In animals that developed coronary flow oscillations in response to glibenclamide, the subsequent addition of adenosine receptor blockade (8-phenyltheophylline) inhibited the periodic increases of flow and in some animals caused near cessation of coronary flow followed by hypotension and death. In the present study adenosine receptor blockade was produced with 8-phenyltheophylline before the administration of glibenclamide to prevent the coronary flow oscillations described by Sammaha et al. (20). The finding that inhibition of KATP channels with glibenclamide decreased coronary blood flow implies that KATP channels in coronary vascular smooth muscle are tonically active during basal conditions and that this activity is necessary to maintain coronary blood flow sufficient to meet myocardial metabolic requirements.

Limitations. The methylxanthines that exert adenosine receptor-blocking activity also produce some degree of phosphodiesterase inhibition. Inhibition of PDE2, which is principally responsible for catabolism of cAMP, would have the potential to increase myocardial contractile performance and thereby increase metabolic demands. 8-Phenyltheophylline was used in the present study because it exerts very low levels of phosphodiesterase inhibition (22). In support of this, 8-phenyltheophylline caused no increase of heart rate or first derivative of LV pressure, suggesting that the dose used in the present study did not cause significant phosphodiesterase inhibition.


   DISCLOSURES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-50470, HL-61353, HL-33600, HL-20598, HL-21872, HL-58067, HL-58840, HL-67828, and HL-71970.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Zhang, Univ. of MN Health Science Center, Mayo Mail Code 508, 420 Delaware St. SE, Minneapolis, MN 55455 (E-mail: zhang047{at}umn.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.


   REFERENCES
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 ABSTRACT
 METHODS
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 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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