Opening of mitochondrial KATP channels enhances cardioprotection ...

Am J Physiol Heart Circ Physiol 287: H1967–H1976, 2004. First published July 8, 2004; doi:10.1152/ajpheart.00338.2004.

Opening of mitochondrial KATP channels enhances cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation, and respiration Anthony J. Rousou,1 Maria Ericsson,3 Micheline Federman,2 Sidney Levitsky,1 and James D. McCully1 1

Division of Cardiothoracic Surgery, 2Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Harvard Medical School; and 3Electron Microscopy Core Facility, Harvard Medical School, Boston, Massachusetts 02215

Submitted 8 April 2004; accepted in final form 6 July 2004

Rousou, Anthony J., Maria Ericsson, Micheline Federman, Sidney Levitsky, and James D. McCully. Opening of mitochondrial KATP channels enhances cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation, and respiration. Am J Physiol Heart Circ Physiol 287: H1967–H1976, 2004. First published July 8, 2004; doi:10.1152/ajpheart.00338.2004.—Previously, we have shown that the pharmacological opening of the mitochondrial ATP-sensitive K channels with diazoxide (DZX) enhances the cardioprotection afforded by magnesium-supplemented potassium (K/Mg) cardioplegia. To determine the mechanisms involved in the cardioprotection afforded by K/Mg ⫹ DZX cardioplegia, rabbit hearts (n ⫽ 24) were subjected to isolated Langendorff perfusion. Control hearts were perfused for 75 min. Global ischemia (GI) hearts were subjected to 30 min of equilibrium, 30 min of GI, and 15 min of reperfusion. K/Mg and K/Mg ⫹ DZX cardioplegia hearts received either K/Mg or K/Mg ⫹ DZX for 5 min before GI and reperfusion. Tissue was harvested for mitochondrial isolation and transmission electron microscopy (TEM). Mitochondrial structure, area, matrix volume, free calcium, and oxygen consumption were determined. TEM demonstrated that GI mitochondria were damaged and that K/Mg and K/Mg ⫹ DZX preserved mitochondrial structure. TEM and light scattering demonstrated separately that mitochondrial matrix and cristae area and matrix volume were significantly increased after GI and reperfusion with GI ⬎ K/Mg ⫹ DZX ⬎ K/Mg hearts (P ⬍ 0.05 vs. control). Mitochondrial free calcium was significantly increased in GI and K/Mg hearts. K/Mg ⫹ DZX significantly decreased mitochondrial free calcium accumulation (P ⬍ 0.05 vs. GI and K/Mg). State 3 oxygen consumption and respiratory control index in malate (complex I substrate)- and succinate (complex II substrate)energized mitochondria were significantly decreased (P ⬍ 0.05 vs. control) in the GI and K/Mg ⫹ DZX groups. These data indicate that the enhanced cardioprotection afforded by K/Mg ⫹ DZX cardioplegia occurs through the preservation of mitochondrial structure and the significant decrease in mitochondrial free calcium accumulation and mitochondrial state 3 oxygen consumption. myocardium; ischemia; reperfusion injury CARDIOPLEGIA is used as a myoprotective agent for the alleviation of surgically induced ischemia-reperfusion injury incurred during cardiac operative procedures. These solutions allow for the rapid electromechanical arrest of the myocardium through alteration of cellular electrochemical gradients (35). The advantage of cardioplegia arrest is that it provides the surgeon a bloodless field in which to operate. This advantage is, however,

Address for reprint requests and other correspondence: J. D. McCully, Div. of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Rm. 144, Boston, MA 02115 (E-mail: [email protected]). http://www.ajpheart.org

tempered in that the depolarization resulting from cardioplegia also leads to the alteration of ion flux across the sarcolemmal membrane and is associated with increased cytosolic calcium accumulation and significant depletion of cellular high-energy (ATP) reserves (6, 7, 39, 40). Most cardioplegia solutions use high potassium (K), which maintains the heart in a depolarized state. In previous reports (6, 7, 39, 40), we have shown that magnesium-supplemented K cardioplegia (K/Mg) provides superior cardioprotection compared with high-K cardioplegia. In a series of reports, using the isolated, perfused rabbit heart (6, 7, 39, 40) and the in situ blood-perfused sheep and pig (28, 29, 41) heart models, we have shown that K/Mg cardioplegia significantly decreases cytosolic and nuclear calcium accumulation, significantly enhances the preservation and resynthesis of high-energy phosphates, and significantly decreases myocardial ischemia-reperfusion injury compared with high-K cardioplegia (39, 40). Recently, we have demonstrated that the cardioprotection afforded by K/Mg cardioplegia is modulated by mitochondrial ATP-sensitive K (mitoKATP) channels (37, 41). We have also shown that the cardioprotection afforded by K/Mg cardioplegia can be significantly enhanced by the pharmacological opening of mitoKATP channels with diazoxide (DZX) when added to K/Mg cardioplegia (37, 41). The mechanism by which the opening of mitoKATP channels affords enhanced cardioprotection has been suggested to act either by inhibiting the uptake of Ca2⫹ (22) or by modulating mitochondrial matrix volume and optimizing respiration and energetics (11, 12). All of these mechanisms have been shown to be associated with the modulation of myocardial cell death (necrosis and apoptosis) (1, 5, 12, 16, 24). In previous reports by others (24), it has been have shown that the opening of the mitoKATP channels causes a significant increase in matrix volume, which is reversed by the addition of the mitoKATP blocker 5-hydroxydecanoate. It has been hypothesized that mitochondrial volume regulation facilitates efficient energy transfer of high-energy phosphate moieties and inhibits ATP wastage (1, 5, 12, 15, 16, 24). Alternatively, it has been suggested that opening mitoKATP channels alters mitochondrial calcium uptake. It has been shown that the use of DZX selectively opens mitoKATP channels, reducing the rate of mitochondrial Ca2⫹ accumulation in a concentration-dependent manner in both the isolated perfused rat heart (1, 22) and in isolated adult rabbit ventricular myocytes (30). It has been 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.

0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society

H1967

H1968

MITOCHONDRIAL KATP CHANNEL OPENING AND CARDIOPROTECTION

further suggested that opening of the mitoKATP channel exerts cardioprotective effects by modulating mitochondrial oxygen consumption during ischemia-reperfusion injury (5, 26, 31). These effects are controversial, because it has been shown by different groups (5, 24, 31) that the opening of mitoKATP channels with DZX can either increase, decrease, or have no effect on mitochondrial oxygen consumption. All of these studies were performed in either isolated mitochondrial preparations, isolated myocytes, or isolated hearts without depolarizing cardioplegic protection (1, 5, 12, 16, 22, 24, 30). It was therefore unknown whether alterations in mitochondrial matrix volume, mitochondrial calcium accumulation, and mitochondrial oxygen consumption could be altered with K/Mg cardioplegia. It was also unknown whether the pharmacological opening of the mitochondrial KATP channels with DZX when added to K/Mg cardioplegia (K/Mg ⫹ DZX) would affect these mechanisms. The purpose of this investigation was to examine the role of mitochondrial matrix volume, mitochondrial calcium accumulation, and mitochondrial oxygen consumption during normothermic global ischemia (GI) in unprotected hearts and in hearts protected by K/Mg and K/Mg ⫹ DZX cardioplegia. MATERIALS AND METHODS

Animals. New Zealand White male rabbits (n ⫽ 24, 15–20 wk; 3– 4 kg) were obtained from Millbrook Farm (Amherst, MA). All animals were housed individually and provided with laboratory chow and water ad libitum. All experiments were approved by the Beth Israel Deaconess Medical Center Animal Care and Use Committee and the Harvard Medical Area Standing Committee on Animals (institutional animal care and use committee) and conformed to the National Institutes of Health “Guidelines Regulating the Care and Use of Laboratory Animals” (NIH Pub. No. 5377-3, 1996). All research was performed in accordance with the American Physiological Society’s “Guiding Principles in the Care and Use of Animals.” Langendorff perfusion. Langendorff retrograde perfusion was performed as described by Feinberg et al. (8). All rabbits were anesthetized with pentobarbital sodium (Nembutal; 100 mg/kg) and heparin (200 U/kg) intravenously through the marginal ear vein. The heart was excised and placed in a 4°C bath of Krebs-Ringer solution equilibrated with 95% O2-5% CO2 (pH 7.4 at 37°C) in which spontaneous beating ceased within a few seconds. Krebs-Ringer solution contained (in mM) 100 NaCl, 4.7 KCl, 1.1 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 1.7 CaCl2, 11.5 glucose, 4.9 pyruvic acid, and 5.4 fumaric acid. Langendorff retrograde perfusion was performed as previously described (37). In brief, a latex balloon containing a catheter-tip transducer (Millar Instruments; Houston, TX) was inserted into the left ventricle. The volume of the water-filled balloon was determined at a constant physiological end-diastolic pressure in a range of 5 to 10 mmHg by using a calibrated microsyringe during equilibrium, and this balloon volume was maintained for the duration of the experiment. The aorta was cannulated with a metal cannula and the heart was subjected to Langendorff retrograde perfusion at a constant pressure of 75 cmH2O at 37°C. Hearts were paced through the right atrium at 180 ⫾ 3 beats/min throughout the experiment using a Medtronic (Minneapolis, MN) model 5330 stimulator. Hemodynamic variables were acquired by using the PO-NE-MAH digital data-acquisition system (Gould; Valley View, OH) with an Acquire Plus processor board and left ventricular (LV) pressure analysis software (37). Experimental protocol. The experimental protocol is shown in Fig. 1. Hearts were perfused for 30 min to establish equilibrium hemodynamics. Equilibrium was ceased when heart rate, LV end-diastolic pressure (LVEDP), and LV peak developed pressure (LVPDP), which is defined as the difference from the LV systolic pressure to the AJP-Heart Circ Physiol • VOL

Fig. 1. Hearts were perfused for 30 min to establish equilibrium function and paced at 180 beats/min. Control hearts were perfused without global ischemia (GI) at 37°C for 75 min. GI hearts were subjected to 30-min GI and 15-min reperfusion. GI was achieved by cross-clamping the perfusion line. Magnesium-supplemented potassium (K/Mg) hearts received K/Mg cardioplegia (K, 20 mM; Mg, 20 mM in Krebs-Ringer solution) for 5 min before ischemia. K/Mg ⫹ diazoxide (K/Mg ⫹ DZX) hearts received K/Mg cardioplegia with 50 ␮M DZX for 5 min before ischemia.

LVEDP maintained at the same level for three continuous measurement periods timed 5 min apart. Control hearts (n ⫽ 6) were perfused without GI at 37°C for 75 min. GI hearts (n ⫽ 6) were subjected to 30 min of GI and 15 min of reperfusion. GI was achieved by crossclamping the perfusion line. The K/Mg hearts (n ⫽ 6) were perfused with normothermic (37°C) K/Mg cardioplegia (20 mM K⫹, 20 mM Mg2⫹ in Krebs-Ringer solution) for 5 min before ischemia. The K/Mg ⫹ DZX hearts (n ⫽ 6) were perfused with normothermic K/Mg cardioplegia with 50 ␮M DZX for 5 min before ischemia. DZX was dissolved in DMSO (Fisher Scientific; Fair Lawn, NJ) before being added into Krebs solution. The final concentration of DMSO was ⬍0.1%. DMSO was added to control and GI hearts at the same concentration. Tissue samples. At the conclusion of the perfusion protocol, the heart was rapidly removed from the Langendorff apparatus and immediately placed in ice-cold Krebs-Ringer solution. The left ventricle was dissected from the rest of the heart and used for the remainder of the experiment. Wet weight-to-dry weight ratios. LV tissue samples (⬃0.1g) obtained from the apex from all experimental groups were weighed (wet weight), dried at 80°C for 24 h for reweighing (dry weight), and then used for the determination of wet weight-to-dry weight ratios, as previously described (37). Transmission electron microscopy. A second myocardial tissue sample (⬃0.1 g) excised from the LV free wall was fixed for transmission electron microscopy (TEM) with a fixative of 1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% picric acid in 100 mM cacodylate buffer and embedded in resin (41). Thin sections were stained with 1% uranyl acetate and examined with TEM by an independent investigator. Mitochondrial isolation. The left ventricle was used for mitochondrial isolation by differential centrifugation as described by Kowaltowski et al. (24). Briefly, the LV tissue was minced with scissors and washed twice with ice-cold Krebs-Ringer solution. The LV tissue was then placed in ice-cold buffer (buffer A) containing 300 mM sucrose, 10 mM K⫹-HEPES buffer, pH 7.2, and 1 mM K⫹-EGTA, pH 8.0. The minced tissue was homogenized by using a blender-type homogenizer (Sorvall Omnimixer; Sorvall, Bloomington, DE) for 10 –15 s and in a glass-grinding vessel (Thomas; Philadelphia, PA) with a motor-driven pestle for 5–10 s at 4°C. Nagarse (⬃4 mg/20 ml homogenate) was added to the homogenate, which was then incubated on ice for 10 min. The homogenate was then centrifuged (International Equipment; Clinical Centrifuge) for 4 min at 750 g (1,500 rpm). The supernatant was saved, and 1 mg/ml BSA was added. The supernatant was then recentrifuged for 4 min at 750 g, and the pellet was discarded. The resulting supernatant was then centrifuged at 9,000 g (9,500 rpm) for

287 • NOVEMBER 2004 •

www.ajpheart.org


10 min (Sorvall model RC-5B, DuPont; model SA 600 Rotor, refrigerated superspeed centrifuge) at 4°C. The mitochondrial pellet was then resuspended in ice-cold buffer A containing 1 mg/ml BSA and recentrifuged at 9,000 g at 4°C two times. The final pellet was suspended in ⬃3– 4 ml of respiration medium containing (in mM) 250 sucrose, 2 KH2PO4, 10 MgCl2, 20 K⫹-HEPES buffer, pH 7.2, 0.5 K⫹-EGTA, pH 8.0, at 4°C (46). Mitochondrial matrix and cristae area. The mitochondrial matrix and cristae area was determined by TEM (41). In brief, TEM images from 15–20 transverse section photomicrographs taken from six sites from each heart and from six hearts in each group were used to determine mitochondrial cross-sectional area. All TEM photomicrographs were ⫻24,500 magnification. All mitochondrial profiles in all TEM photomicrographs were measured. The minor axis and the major axis of each mitochondrial profile were measured by using Scion Image 4.02 (NIH Image). Mitochondrial cross-sectional area was determined assuming an ellipsoid structure using the formula (L 䡠 W 䡠 ␲/4, where L is length and W is width) for n ⬎500 mitochondria for each experimental group. Mitochondrial matrix volume. Mitochondrial matrix volumes were measured separately by the light-scattering technique, premised on the observation that swollen mitochondria are associated with a decrease in light absorbance (20). The light-scattering technique is sensitive to changes in the intermembrane space and the shape of mitochondria (19, 20). Mitochondrial matrix volume was measured as the decrease in absorbance at 520 nm and the isosbestic point for the mitochondrial cytochromes by allowing for the estimation of volume insensitive to changes in redox state. In brief, isolated mitochondria from control, GI, K/Mg, and K/Mg ⫹ DZX hearts (0.3 mg mitochondrial protein/ ml) were suspended separately in (in mM) 125 sucrose, 65 KCl, 10 HEPES, pH 7.2, and 2 succinate plus 2 ␮M rotenone. The absorbance of isolated mitochondrial suspensions was followed by using an Ultrospec II spectrophotometer (LKB, Cambridge, UK) at 520 nm. Data were collected at 2-min intervals for a total of 20 min. Mitochondrial free calcium. Mitochondrial free calcium ([Ca2⫹]mt) was determined by using an in-house spectrofluoresence system as previously described (2). Mitochondria were isolated from freezeclamped tissue. Mitochondria (1 mg mitochondrial protein) were suspended in 1 ml buffer containing (in mM) 140 NaCl, 5 KCl, 5.6 glucose, and 5 HEPES; pH 7.4, then loaded with 1 ␮M fura-2 AM (Molecular Probes, Eugene, OR) in DMSO (0.1% final concentration) and 0.006% Pluronic F-127 (Molecular Probes), and allowed to incubate for 15 min at 30°C in the dark. Sample blanks received DMSO (0.1% final concentration) as vehicle only. Mitochondria were stirred in a quartz cuvette with a Teflon-coated magnetic stir bar at

H1969

30°C, and fluorescence was recorded at 340 and 380 nm (excitation) and 510 nm (emission) as previously described (2). Calibration was performed as described previously (2, 39). Measurement of mitochondrial respiration. Mitochondrial oxygen consumption was measured by using a Clark-type electrode (Yellow Springs Instruments, Yellow Springs, OH). Before the assay, the instrument was calibrated to 100% saturation in air-saturated respiration media at 30°C. To measure oxygen consumption mediated by complex I of the mitochondrial electron transport chain, glutamate and malate (5 mM of each) were added to the respiration media before the mitochondria. To determine oxygen consumption mediated by complex II, succinate (8 mM) and rotenone (4 ␮M), an inhibitor of complex I, were added to the respiration media in the place of glutamate and malate. State 2 mitochondrial oxygen consumption was measured for 1 min before the addition of ADP to the respiration medium. State 3 mitochondrial oxygen consumption was initiated by the addition of 1 mM ADP and was allowed to occur for 2 min, at which point state 4 mitochondrial oxygen consumption was initiated by blocking the ATP synthase of complex V of the mitochondrial inner membrane with addition of oligomycin (15 ␮M) (1). The respiratory control index (RCI) was calculated as the ratio of the state 3 to state 4 mitochondrial oxygen consumption (25). In a separate set of trials, atractyloside (13 ␮M) was added to inhibit the adenine nucleotide translocase after 2 min of state 3 mitochondrial oxygen consumption, instead of oligomycin (1). Oxygen consumption was calculated from an initial air-saturated [O2] ⫽ 195 nmol/ml (34). Statistical analysis. Statistical analysis was performed by using the SAS (version 6.12) software package (SAS Institute, Cary, NC). The means ⫾ SE for all data were calculated for all variables. Statistical significance was assessed by using repeated-measures ANOVA. Statistical significance was claimed at P ⬍ 0.05. RESULTS

Equilibrium hemodynamics. No significant difference in LVPDP or LVEDP was observed within or between groups at the end of equilibrium (Fig. 2, A and B). Effect of GI, K/Mg cardioplegia, and K/Mg ⫹ DZX cardioplegia on functional recovery. LVPDP in GI hearts was significantly decreased to 66.3 ⫾ 5.4 mmHg (P ⬍ 0.05 vs. control, K/Mg, and K/Mg ⫹ DZX) after 15 min of reperfusion. In the K/Mg and K/Mg ⫹ DZX groups, LVPDP was 104.0 ⫾ 6.1 and 103.7 ⫾ 6.7 mmHg, respectively, after 15 min of reperfusion. LVPDP in K/Mg and K/Mg ⫹ DZX was signifi-

Fig. 2. A: left ventricular (LV) peak developed pressure (LVPDP) during 30-min equilibrium, 30-min GI, and 15-min reperfusion for control, GI, K/Mg, and K/Mg ⫹ DZX hearts. All results are shown as means ⫾ SE for n ⫽ 6 for each group. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. GI. B: LV enddiastolic pressure (LVEDP) during 30-min equilibrium, 30-min GI, and 15-min reperfusion for control, GI, K/Mg, and K/Mg ⫹ DZX hearts. All results are shown as means ⫾ SE for n ⫽ 6 for each group. *P ⬍ 0.05 vs. control.

AJP-Heart Circ Physiol • VOL

287 • NOVEMBER 2004 •

www.ajpheart.org

H1970


Fig. 3. Transmission electron microscopy (TEM) images (⫻24,500) of LV tissue samples after 75-min perfusion for control, 30min GI, and 15-min reperfusion for GI, K/Mg, and K/Mg ⫹ DZX. In control hearts, TEM revealed preserved sarcomere structure with electron-dense intracristae matrix and few small calcium granules. In GI hearts, TEM revealed sarcomere disruption with severely swollen and electron-transparent mitochondria and numerous mitochondrial calcium granules. In K/Mg hearts, TEM demonstrated intact sarcomeres and swollen and electron-dense mitochondria with numerous calcium granules. In K/Mg ⫹ DZX hearts, the sarcomeres were intact; mitochondria were swollen and electron dense; and there were fewer calcium granules within the mitochondria than GI and K/Mg. Arrows, calcium granules; M, mitochondrion; S, sarcomere; N, nucleus.

cantly decreased vs. control (115.7 ⫾ 6.4 mmHg) (P ⬍ 0.05) after 15 min reperfusion (Fig. 2A). LVEDP in GI hearts was significantly increased (P ⬍ 0.05 vs. control) to 13.3 ⫾ 1.1 mmHg after 15 min reperfusion. LVEDP in control (7.7 ⫾ 0.7 mmHg), K/Mg (7.3 ⫾ 0.4 mmHg), and K/Mg ⫹ DZX (9.2 ⫾ 1.1 mmHg) after 15 min reperfusion was not statistically different (Fig. 2B). TEM. TEM images of LV tissue for control, GI, K/Mg, and K/Mg ⫹ DZX hearts are shown in Fig. 3. TEM for control hearts (perfused 75 min without GI) revealed preserved sarcomere structure with electron-dense matrix and cristae. In GI hearts, TEM revealed sarcomere disruption with severely swollen matrix and cristae and electron transparent mitochondria. In K/Mg hearts, TEM demonstrated intact sarcomeres with swollen matrix and cristae and electron-dense mitochondria. In K/Mg ⫹ DZX hearts, the sarcomeres were intact; the matrix and cristae were swollen, and the mitochondria were electron dense. Mitochondrial matrix and cristae area. Mitochondrial matrix and cristae area as determined by TEM for control, GI, K/Mg, and K/Mg ⫹ DZX hearts is shown in Fig. 4. Mitochondrial matrix and cristae area was significantly increased (P ⬍ 0.05) in GI, K/Mg, and K/Mg ⫹ DZX after 30 min of GI and 15 min of reperfusion compared with control (75-min perfuAJP-Heart Circ Physiol • VOL

sion without GI). An overlap in mitochondrial matrix and cristae area for all samples was observed (Fig. 4). Mitochondrial matrix and cristae area were significantly increased in GI hearts after 30 min of GI and 15 min of reperfusion (P ⬍ 0.05

Fig. 4. Mitochondrial matrix and cristae areas as determined by TEM after 75-min perfusion for control, 30-min GI, and 15-min reperfusion for GI, K/Mg, and K/Mg ⫹ DZX. The distribution of the area measurements is shown in arbitrary units for control (n ⫽ 510 observations), GI (n ⫽ 533 observations), K/Mg (n ⫽ 527 observations), and K/Mg ⫹ DZX (n ⫽ 523 observations).

287 • NOVEMBER 2004 •

www.ajpheart.org


H1971

State 3 (ADP stimulated) mitochondrial oxygen consumption in GI and K/Mg ⫹ DZX groups was significantly decreased for malate (complex I substrate) (Fig. 7A)- and succinate (complex II substrate) (Fig. 7B)-energized mitochondria (P ⬍ 0.05 vs. control and K/Mg). RCI for both malate (complex I substrate)- and succinate (complex II substrate)-energized mitochondria was significantly decreased (P ⬍ 0.05) in GI and K/Mg ⫹ DZX compared with control and K/Mg (Fig. 8, A and B) after 30 min of GI and 15 min of reperfusion. Wet weight-to-dry weight ratios. No significant difference in wet weight-to-dry weight ratios was found within or among control, GI, K/Mg, and K/Mg ⫹ DZX groups after 30 min of GI and 15 min of reperfusion (results not shown). DISCUSSION Fig. 5. Mitochondrial matrix volume as determined by the light-scattering technique shown as %change in 520-nm absorbance (⌬A520 nm) over 20 min for control (after 75 min perfusion) and for GI, K/Mg, and K/Mg ⫹ DZX (after 30-min GI and 15-min reperfusion). All results are shown as means ⫾ SE for n ⫽ 8 for each group. *P ⬍ 0.05 vs. control; #P ⬍ 0.05 vs. GI.

vs. control, K/Mg, and K/Mg ⫹ DZX). K/Mg cardioplegia significantly decreased mitochondrial matrix and cristae area compared with GI (P ⬍ 0.05 vs. GI). The addition of DZX to K/Mg cardioplegia (K/Mg ⫹ DZX) significantly increased mitochondrial matrix and cristae area compared with K/Mg (P ⬍ 0.05 vs. K/Mg), but this increase in volume was significantly less (P ⬍ 0.05) than that observed in GI. Mean mitochondrial matrix and cristae area expressed as arbitrary units (au) was 18,417 ⫾ 143 au (n ⫽ 510) for control; 23,668 ⫾ 319 au (n ⫽ 527) for K/Mg; 27,497 ⫾ 198 au (n ⫽ 523) for K/Mg ⫹ DZX; and 29,733 ⫾ 192 au (n ⫽ 533) for GI. Mitochondrial matrix volume. Mitochondrial matrix volume as determined by the light-scattering technique was significantly increased in GI hearts after 30 min of GI and 15 min of reperfusion (P ⬍ 0.05 vs. control, K/Mg, and K/Mg ⫹ DZX) (Fig. 5). K/Mg and K/Mg ⫹ DZX cardioplegia significantly decreased mitochondrial matrix volume compared with GI (P ⬍ 0.05 vs. GI); however, mitochondrial matrix volume was significantly increased in K/Mg and K/Mg ⫹ DZX (P ⬍ 0.05) compared with control. There was a significant increase (P ⬍ 0.05) in K/Mg ⫹ DZX matrix volume compared with K/Mg. Final percent change in 520-nm absorbance over 20 min (after 75-min perfusion) was 67.19 ⫾ 0.85 for control; 62.95 ⫾ 0.88 for K/Mg; 59.24 ⫾ 1.06 for K/Mg ⫹ DZX; and 55.35 ⫾ for GI. [Ca2⫹]mt. [Ca2⫹]mt was significantly increased (P ⬍ 0.05) after 30 min of GI and 15 min of reperfusion in GI, K/Mg, and K/Mg ⫹ DZX compared with control (Fig. 6). [Ca2⫹]mt in control hearts was 213.8 ⫾ 5.4 nmol/mg mitochondrial protein. No significant difference in [Ca2⫹]mt was observed between GI (617.3 ⫾ 38.8 nmol/mg mitochondrial protein) and K/Mg (608.5 ⫾ 33.4 nmol/mg mitochondrial protein). K/Mg ⫹ DZX significantly decreased [Ca2⫹]mt to 481.7 ⫾ 24.4 nmol/mg mitochondrial protein (P ⬍ 0.05 vs. GI and K/Mg). Mitochondrial oxygen consumption. No significant differences in state 2 (baseline, no ADP available), state 4 (ATP synthase inhibited, oligomycin), or adenine nucleotide transporter (ANT)-inhibited (atractyloside) mitochondrial oxygen consumption were observed within or between groups after 30 min of GI and 15 min of reperfusion in GI, K/Mg, and K/Mg ⫹ DZX compared with control (75-min perfusion without GI) (Table 1). AJP-Heart Circ Physiol • VOL

In previous reports (6, 7, 28, 29, 40), we have shown that GI significantly increases infarct size (27.8 ⫾ 2.4%) and significantly decreases postischemic functional recovery. We have also shown that K/Mg cardioplegia significantly decreases infarct size to 3.7 ⫾ 0.5% (P ⬍ 0.05 vs. GI) and significantly enhances postischemic functional recovery (P ⬍ 0.05 vs. GI; P not significant vs. control) (6, 7, 28, 29, 40). Recently, we (37, 41) have shown that the cardioprotection afforded by K/Mg cardioplegia is modulated by mitoKATP channels and that the addition of DZX when added to K/Mg cardioplegia significantly decreased infarct size to 1.5 ⫾ 0.4% (P ⬍ 0.05 vs. K/Mg; P not significant vs. control). These data suggested that opening of mitoKATP channels provided a mechanism by which the cardioprotection afforded by K/Mg cardioplegia could be enhanced (37, 41). The mechanisms through which the opening of mitoKATP channels afforded enhanced cardioprotection was unknown; however, it has been suggested that either inhibition of mitochondrial Ca2⫹ uptake (22) or the modulation of mitochondrial matrix volume and/or optimization of respiration and energetics may be involved (11, 12). In the present report, our data indicate that mitochondrial matrix and cristae area as determined by TEM, mitochondrial matrix volume as determined by the light-scattering technique,

Fig. 6. Mitochondrial free matrix calcium concentration ([Ca2⫹]mt) (nM/mg mitochondrial protein) for control (after 75-min perfusion) and for GI, K/Mg, and K/Mg ⫹ DZX (after 30-min GI and 15-min reperfusion) as determined by fura-2 epifluorescent analysis. All results are shown as means ⫾ SE for n ⫽ 6 for each group. *P ⬍ 0.05 vs. control. **P ⬍ 0.05 vs. GI. The calibration curve for [Ca2⫹] at 0, 0.17, 0.34, 0.68, and 1.36 ␮M is shown in the inset.

287 • NOVEMBER 2004 •

www.ajpheart.org

H1972


Table 1. Mitochondrial oxygen consumption Complex I

Complex II

Group

State 2

State 4

ANT Inhibited

State 2

State 4

ANT Inhibited

Control GI K/Mg K/Mg ⫹ DZX

11.7⫾0.8 11.7⫾0.4 10.7⫾0.4 9.8⫾0.5

12.6⫾1.2 12.4⫾0.6 11.4⫾0.3 11.9⫾0.4

13.2⫾1.2 12.3⫾0.4 11.0⫾0.5 11.6⫾0.4

43.2⫾1.7 45.5⫾0.9 47.7⫾0.7 43.4⫾1.1

66.4⫾1.4 71.9⫾1.3 65.3⫾1.8 71.0⫾1.6

56.8⫾1.8 59.9⫾0.8 58.2⫾1.3 58.6⫾1.6

Values are means ⫾ SE (in nmol O2䡠min⫺1䡠mg mitochondrial protein⫺1); n ⫽ 6 animals/group. Isolated mitochondrial oxygen consumption was measured after 75-min perfusion in control hearts and 30-min global ischemia (GI), and 15-min reperfusion in GI, magnesium-supplemented potassium (K/Mg), and K/Mg ⫹ diazoxide (DZX) hearts.

and mitochondrial structure, free calcium accumulation, and oxygen consumption are altered after ischemia-reperfusion. Our results also show that the use of depolarizing (K/Mg, K/Mg ⫹ DZX) cardioplegia modulates these effects and is associated with enhanced cardioprotection (6, 7, 28, 29, 37, 39, 40, 41). The importance of the mitochondrion in providing for cellular homeostasis has been previously documented (1, 5, 7, 12, 15, 16, 22, 24). Under control (nonstressed) conditions, the energy generated by the electron transport chain allows for the active transport of hydrogen ions across the inner mitochondrial membrane, out of the matrix to the intermembrane space, creating an electrochemical gradient (3). This gradient allows for the energy that drives the release of ATP from ATP synthase and export to the cytosol via a series of complexes, which include the ATP/ADP translocator (ANT), mitochondrial creatine kinase (Mi-CK), and the voltage-dependent anion channel (VDAC) (3). Mi-CK bridges the space between the outer and the inner membranes and is in close contact with the VDAC and ANT, allowing for low-conductance transfer of high-energy phosphates. The binding of Mi-CK to the VDAC confers a low permeability to nucleotides and requires a narrow intermembrane distance (1, 5, 12, 15, 16, 24). It has been proposed that GI alters mitochondrial matrix volume and increases intermembrane distance between VDAC, ANT, and Mi-CK, thus compromising efficient energy transfer during reperfusion (5, 11, 12). Our results, as determined by TEM, indicate that after 30 min of GI and 15 min of reperfusion, the matrix and cristae of mitochondria are severely swollen and electron transparent with the greatest matrix and cristae areas (P ⬍ 0.05) being

observed in GI hearts compared with control hearts. These results are in agreement with our results obtained by the light-scattering technique and with those of Ozcan et al. (32), who have previously shown that 88% of mitochondria were swollen with increased intermembrane space and swollen cristae and disrupted matrix after 20 min anoxia-reoxygenation. Our results also indicate that [Ca2⫹]mt accumulation is significantly increased (P ⬍ 0.05) in GI hearts compared with control hearts. This would agree with our previous reports (6, 7) in which we have shown by using sequential fura-2 epifluorescence that there is a significant increase in cytosolic calcium during GI and that increased cytosolic calcium accumulation is associated with a significant increase in [Ca2⫹]mt accumulation. Previous studies (10) have indicated that increased [Ca2⫹]mt accumulation increases mitochondrial volume. Under homeostatic conditions, the mitochondrial inner membrane (cristae), containing the electron transport chain, expels protons to the cytosol, creating a charge gradient that provides the passive energy for Ca2⫹ influx by the Ca2⫹ uniporter. In GI, increased [Ca2⫹]mt accumulation destabilizes the inner mitochondrial membrane and causes the inner membrane pore to open and permit further cation movement (futile calcium cycling), an energy-dependent process requiring ATP to transport calcium against the electrochemical gradient out of the mitochondrion (10). The collapse of the mitochondrial inner transmembrane potential accompanies the uncoupling of the respiratory chain. As calcium enters the mitochondria, water follows, leading to mitochondrial matrix swelling and eventually mitochondrial rupture and myocyte cell death (36). These events are in agreement with our data.

Fig. 7. A: mitochondrial (1 mg mitochondrial protein/ml buffer) state 3 oxygen consumption in malate (complex I substrate)-energized mitochondria (nM 䡠 min⫺1 䡠 mg mitochondrial protein⫺1) after 75-min perfusion for control, 30min GI, and 15-min reperfusion for GI, K/Mg, and K/Mg ⫹ DZX. All results are shown as means ⫾ SE for n ⫽ 6 animals for each group. *P ⬍ 0.05 vs. control and K/Mg. B: mitochondrial (1 mg mitochondrial protein/ml buffer) state 3 oxygen consumption in succinate (complex II substrate)-energized mitochondria (nM 䡠 min⫺1 䡠 mg mitochondrial protein⫺1) after 75-min perfusion for control, 30-min GI, and 15-min reperfusion for GI, K/Mg, and K/Mg ⫹ DZX. *P ⬍ 0.05 vs. control and K/Mg.


287 • NOVEMBER 2004 •

www.ajpheart.org


H1973

Fig. 8. A: respiratory control index (RCI) in malate (complex I substrate)-energized mitochondria after 75-min perfusion for control, 30-min GI, and 15-min reperfusion for GI, K/Mg, and K/Mg ⫹ DZX. All results are shown as means ⫾ SE for n ⫽ 6 animals for each group. *P ⬍ 0.05 vs. control and K/Mg. B: RCI in succinate (complex II substrate)energized mitochondria after 75-min perfusion for control, 30-min GI, and 15-min reperfusion for GI, K/Mg, and K/Mg ⫹ DZX. *P ⬍ 0.05 vs. control and K/Mg.

Our results also demonstrate that state 3 mitochondrial oxygen consumption in malate (complex I substrate)- and succinate (complex II substrate)-energized mitochondria was significantly decreased (P ⬍ 0.05) in GI compared with control hearts, which would compromise nucleotide triphosphate (␤NTP) repletion during reperfusion and compromise myocardial cell viability and postischemic functional recovery. It should be noted that, whereas the effects of [Ca2⫹]mt may not be evident in isolated mitochondrial isolations due to methodological manipulations, these data are in agreement with our previous findings obtained by serial 31P NMR spectroscopy, demonstrating that ␤NTP and phosphocreatine (PCr) levels are significantly decreased to 36 ⫾ 3% of control after 30 min of normothermic GI and remain significantly decreased at 15 min of reperfusion (to 43 ⫾ 3% of control ␤NTP levels) (40). K/Mg cardioplegia. Our results demonstrate that the use of K/Mg cardioplegia preserves mitochondrial structure and is associated with a significant increase in mitochondrial matrix volume (P ⬍ 0.05 vs. control), but this increase is significantly less (P ⬍ 0.05) than that observed in GI and K/Mg ⫹ DZX hearts. K/Mg cardioplegia did not reduce [Ca2⫹]mt accumulation, but significantly preserved state 3 oxygen consumption in malate (complex I substrate)- and succinate (complex II substrate)-energized mitochondria (P ⬍ 0.05 vs. GI; P not significant vs. control). These data are in agreement with our previous results obtained by serial 31P NMR spectroscopy, which demonstrated that the reduction in ␤NTP levels during GI is significantly attenuated with K/Mg cardioplegia (P ⬍ 0.05 vs. GI) and that the recovery of ␤NTP was significantly enhanced (40). Preservation of state 3 oxygen consumption in malate (complex I substrate)- and succinate (complex II substrate)-energized mitochondria would significantly enhance the resynthesis of ␤NTP and would allow for the significant decrease in myocardial infarction (apoptosis and necrosis) and allow for enhanced postischemic functional recovery. K/Mg ⫹ DZX cardioplegia. Garlid and colleagues (12–16) have hypothesized that the opening of the mitoKATP channels plays a key role in affording cardioprotection. The mitoKATP channels are stimulated (opened) by nucleotide diphosphates hypothesized to be accumulated as a consequence of elevated or ischemia-induced metabolic demands with the consequent elevation in the rate of metabolism resulting in the fall of intracellular ATP (18). Under normal conditions, the mitoKATP AJP-Heart Circ Physiol • VOL

channels are inhibited (closed). This inhibition occurs by free inhibited ATP concentration ([ATP]i) and inhibited Mg2⫹ATP concentration at levels ⬎1 mM and is responsive to changes in [ATP]i produced by glycolysis but not by increases through application of exogenous ATP (18). Inhibition of mitoKATP channels by ATP has an absolute requirement for Mg2⫹ or Ca2⫹; however, neither Mg2⫹ nor Ca2⫹ can provide for inhibition independently (33). We have previously shown (6, 40) using serial 31P NMR spectroscopy that the decrease in ␤NTP during K/Mg cardioplegia would be insufficient to open mitoKATP channels until late ischemia when [Ca2⫹]mt levels are significantly increased. These events would delay the opening of the mitoKATP channels. Support for this hypothesis comes from recent experiments in which we have shown that the early or enhanced opening of mitoKATP channels using DZX added to K/Mg cardioplegia significantly decreases infarct size (29, 37, 41). Our results indicate that the pharmacological opening of the mitoKATP channels with DZX when added to K/Mg (K/Mg ⫹ DZX) cardioplegia preserved mitochondrial structure and significantly increased mitochondrial matrix and cristae area and matrix volume compared with control and K/Mg. This finding is contrary to that of Das et al. (4), who have suggested that DZX has no effect on mitochondrial matrix volume and may reflect the effects of depolarizing K/Mg cardioplegia, which would alter mitochondrial K⫹. Our results are in agreement with Dos Santos et al. (5), who have demonstrated that mitochondrial matrix volume increases in response to opening mitoKATP channels with DZX and have suggested that this mechanism allows for the ANT, Mi-CK, and VDAC to remain in close contact with each other, allowing for the maintenance of efficient energy transfer during reperfusion. Effects of opening mitoKATP channels on mitochondrial oxygen consumption are controversial. Ozcan et al. (32) have shown that DZX, when present during anoxia-reoxygenation, diminished structural injury such that 67% of mitochondria remained intact and preserved ADP-stimulated respiration and ATP synthesis. Other laboratories have shown contradictory results indicating decreased (26, 31), increased (5), and unaltered oxygen consumption (24) after the opening of the mitoKATP channels. Our results demonstrate that after 30 min of normothermic GI and 15 min of reperfusion, K/Mg ⫹ DZX cardioplegia significantly decreased (P ⬍ 0.05) state 3 mitochondrial oxy-

287 • NOVEMBER 2004 •

www.ajpheart.org

H1974


gen consumption (complex I and complex II) compared with control and K/Mg. These results would agree with those of Lim et al. (26) and Ovide-Bordeaux et al. (31), who have shown that DZX significantly decreased mitochondrial oxygen consumption. However, our results are in contrast to that of Dos Santos et al. (5), who have shown an increase in malate (complex I substrate)-energized mitochondrial oxygen consumption with DZX in the permeabilized rat heart preparation after 30 min of zero-flow ischemia and 15 min of reperfusion. In previous reports (19, 26, 31), the effects of DZX have been suggested to be limited to modulation of succinate (complex II substrate)-energized mitochondrial oxygen consumption. In our experiments, we have not investigated the effects of DZX alone, because we have previously shown (29, 37, 41) that the use of DZX alone does not provide for improved postischemic functional recovery and is therefore clinically irrelevant as a cardioprotective agent when used alone. It is most likely that the effects of DZX on state 3 mitochondrial oxygen consumption in malate (complex I substrate )- and succinate (complex II substrate)-energized mitochondria represent effects associated with depolarized arrest and with the addition of DZX to depolarizing K/Mg cardioplegia (K/Mg ⫹ DZX). An alternative explanation is that because succinate (complex II substrate)-energized mitochondrial oxygen consumption rates are greater than malate (complex I substrate)energized mitochondrial oxygen consumption rates, it is easier to detect a difference in succinate (complex II substrate)energized mitochondrial oxygen consumption. It is also possible that differences in metabolic rate, heart rate, and myosin heavy-chain isoforms in the rat compared with the rabbit may have resulted in differences that limited the detection of discrepancies in state 3 malate (complex I substrate)-energized mitochondrial oxygen consumption (17). The mechanism by which depolarizing cardioplegia with and without DZX effects matrix volume and function may be related to the presence of Mg in high-K cardioplegia. Garlid and Paucek (14) have speculated that in GI, there is a lack of oxygen available to the mitochondria, oxidative phosphorylation is inhibited, and thus protons are not expelled from the matrix. The mitochondria become depolarized, K entry is inhibited, and proton flow at complex V is reversed. Because K entry is being inhibited, water does not enter and leads to matrix contraction. As the matrix contracts, the intermembrane space expands leading to dissociation of the ANT, Mi-CK, and VDAC (14). This separation prevents efficient energy transfer and allows for ATP to enter the matrix and be hydrolyzed at complex V, leading to its depletion (11, 14). As cytosolic calcium is increased during ischemia, calcium enters the mitochondria via the potential-dependent Ca2⫹ uniporter, H2O accumulates, and severe mitochondrial swelling occurs. This process would result in futile calcium cycling with the loss of ATP (7, 40). The significant decrease in high-energy phosphates (␤NTP) and PCr depletion during GI would exceed the requirements of the myocardium during reperfusion and necrosis, and apoptosis would occur (6, 40). Complicating this process is the loss of matrix volume regulation due to reduction of free Mg and the inhibition of the K⫹/H⫹ antiporter increasing matrix volume, as well as allowing for futile K⫹ cycling AJP-Heart Circ Physiol • VOL

and energy utilization, in agreement with our present and previous observations (6, 7, 40). K/Mg cardioplegia maintains matrix Mg and decreases free Mg levels during ischemia-reperfusion (53). The maintenance of Mg stores and the reduction of free Mg levels during ischemia-reperfusion would allow for the modulation of mitochondrial matrix volume. The preservation of matrix Mg would also allow for sufficient Mg to complex all of the ADP and ATP molecules and thus enhance the resynthesis of ␤NTP and PCr depletion during GI (40). The beneficial effects of K/Mg cardioplegia are tempered, however, due to the increase in [Ca2⫹]mt via the Ca2⫹ uniporter (7). As the calcium enters, H2O follows, leading to mitochondrial swelling. However, in this case, because high-energy phosphates are preserved, [Ca2⫹]mt is more easily expelled via an Na⫹/Ca2⫹ exchanger and the mitochondrial volume is only slightly increased. This allows for proper interaction among the ANT, Mi-CK, and VDAC, thereby preventing ATP hydrolysis. However, the ATP utilized in an attempt to maintain calcium homeostasis limits energy reserves somewhat, and some infarct does occur due to this limitation. The preservation of Mg stores and the reduction of Mgf levels would allow for inhibition of the K⫹/H⫹ antiporter and modulation of mitochondrial swelling (14). In addition, the preservation of Mg stores would maintain cytochrome oxidase structure and enhance cytochrome oxidase activity (6, 38). We (6) have previously shown that after ischemia-reperfusion, cytochrome oxidase activity [maximal velocity (Vmax); in mM cytochrome c oxidized/min], which is vital for the production of high-energy phosphates, is significantly decreased in GI hearts. Previous reports have demonstrated that the depletion of Mg decreases cytochrome oxidase activity to 40% that of the native enzyme (27). The use of K/Mg cardioplegia allows for the preservation of cytochrome oxidase activity (Vmax) (6). We speculate that the early opening of the mitoKATP channels before GI allows for K⫹ entry into the mitochondrial matrix leading to matrix swelling. This early swelling prevents matrix contraction that would otherwise occur during ischemia, allowing for the ANT, Mi-CK, and VDAC to remain in close contact with each other and the maintenance of efficient energy transfer and prevention of ATP depletion (1, 5, 12, 14, 15, 16, 24). The early opening of the mitoKATP channels would also inhibit [Ca2⫹]mt accumulation. K⫹ entry into the mitochondrial through the open mitoKATP channels would result in depolarization of the mitochondrion. The loss of membrane potential would inhibit the potential-dependent calcium uniporter and prevent excessive calcium accumulation and the resultant severe mitochondrial matrix and cristae swelling (21, 22, 30). It has been suggested that mitochondrial membrane depolarization caused by K⫹ entry through the opening of mitoKATP channels reduces Ca2⫹ entry through the mitochondrial calcium uniporter and thus decreases [Ca2⫹]mt accumulation during GI (20). Subsequently, these events are believed to result in ATP preservation and to enhance cell recovery (9). The preservation of high-energy stores would reduce the requirement for high-energy resynthesis during reperfusion and reduce energy flux through the electron transport with resultant and reactive oxygen species generation. These events would putatively allow for the limitation of infarct size. Support for these putative mechanisms comes from studies (9) indicating

287 • NOVEMBER 2004 •

www.ajpheart.org


that increased [Ca2⫹]mt accumulation destabilizes the mitochondrial inner membrane, causing the inner membrane pores to open, which permits further movement of cations across the mitochondrial membrane. The opening of these pores renders the mitochondrion incapable of synthesizing ATP and has been suggested to be a key event in the process leading to myocardial cell death. Our results are also in agreement with those of Holmuhamedov et al. (21, 22), who have previously shown that the opening of mitoKATP channels in isolated mitochondrial preparations made them more resistant to calcium entry, and with Belisle and Kowaltowski (1), who have previously shown that opening mitoKATP channels with DZX decreased isolated rat heart mitochondrial calcium accumulation. Whereas the mechanism for this cardioprotective action remains to be fully elucidated, our data indicate that the enhanced cardioprotection afforded by the pharmacological opening of the mitoKATP channels when added to depolarizing K/Mg cardioplegia is associated with the preservation of mitochondrial structure and the significant decrease in mitochondrial calcium accumulation and mitochondrial state 3 oxygen consumption. Our results also suggest that these mechanisms are not exclusive.

14. 15.

16.

17. 18.

19.

20.

21.

22.

ACKNOWLEDGMENTS This study was supported by National Heart, Lung, and Blood Institute Grant HL-29077.

23.

24. REFERENCES 1. Belisle E and Kowaltowski AJ. Opening of mitochondrial K⫹ channels increases ischemic ATP levels by preventing hydrolysis. J Bioenerg Biomembr 34: 285–298, 2002. 2. Chen DP, Jimenez E, Ataka K, Levitsky S, and Feinberg H. Fura 2 determination of [Ca2⫹]i in isolated perfused heart using R wave-gated electromechanical shutters. J Appl Physiol 76: 1394 –1399, 1994. 3. Cross RL. Turning the ATP motor. Nature 427: 407– 408, 2004. 4. Das M, Parker JE, and Halestrap AP. Matrix volume measurements challenge the existence of diazoxide/glibenclamide-sensitive KATP channels in rat mitochondria. J Physiol 547:893–902. 5. Dos Santos P, Kowaltowski AJ, Laclau MN, Seetharaman S, Paucek P, Boudina S, Thambo JB, Tariosse L, and Garlid KD. Mechanisms by which opening of the mitochondrial ATP-sensitive K⫹ channel protects the ischemic heart. Am J Physiol Heart Circ Physiol 283: H284 –H295, 2002. 6. Faulk EA, McCully JD, Hadlow NC, Tsukube T, Krukenkamp IB, Federman M, and Levitsky S. Magnesium cardioplegia enhances mRNA levels and the maximal velocity of cytochrome oxidase I in the senescent myocardium during global ischemia. Circulation 92: 405– 412, 1995. 7. Faulk EA, McCully JD, Tsukube T, Hadlow NC, Krukenkamp IB, and Levitsky S. Myocardial mitochondrial calcium accumulation modulates nuclear calcium accumulation and DNA fragmentation. Ann Thorac Surg 60: 338 –344, 1995. 8. Feinberg H, Levitsky S, and Lee SL. The quiescent heart: excitability, compliance, and vascular resistance. Am J Physiol Heart Circ Physiol 251: H1085–H1089, 1986. 9. Ferrari R. The role of mitochondria in ischemic heart disease. J Cardiovasc Pharmacol 28, Suppl 1: S1–S10, 1996. 10. Frolkis VV, Frolkis RA, Mkhitarian LS, Shevchuk VG, Fraifeld VE, Vakulenko LG, and Syrovy I. Contractile function and Ca2⫹ transport system of myocardium in ageing. Gerontology 34: 64 –74, 1988. 11. Garlid KD. On the mechanism of regulation of the mitochondrial K⫹/H⫹ exchanger. J Biol Chem 255: 11273–11279, 1980. 12. Garlid KD. Opening mitochondrial KATP in the heart–what happens, and what does not happen. Basic Res Cardiol 95: 275–279, 2000. 13. Garlid KD, Dos Santos P, Xie ZJ, Costa ADT, and Paucek P. Mitochondrial potassium transport: the role of the mitochondrial ATP-sensitive


25.

26.

27. 28.

29.

30.

31.

32.

33.

34.

35.

H1975

K⫹ channel in cardiac function and cardioprotection. Biochim Biophys Acta 1606: 1–21, 2003. Garlid KD and Paucek P. Mitochondrial potassium transport: the K⫹ cycle. Biochim Biophys Acta 1606: 23– 41, 2003. Garlid KD, Paucek P, Yarov-Yarovoy V, and Sun X, Schindler PA. The mitochondrial KATP channel as a receptor for potassium channel openers. J Biol Chem 271: 8796 – 8799, 1996. Garlid KD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D’Alonzo AJ, Lodge NJ, Smith MA, and Grover GJ. Cardioprotective effect of diazoxide and its interactions with mitochondrial ATP-sensitive K⫹ channels: Possible mechanism of cardioprotection. Circ Res 81: 1072–1082, 1997. Gross DR. Animals in Cardiovascular Research (1st ed.). Boston, MA: Martinus Nijhoff, 1985. Grover GJ. Pharmacology of ATP-sensitive potassium channel (KATP) openers in models of myocardial ischemia and reperfusion. Can J Physiol Pharmacol 75: 309 –315, 1997. Halestrap AP. The regulation of matrix volume of mammalian mitochondria in vivo and in vitro and its role in the control of mitochondrial metabolism. Biochim Biophys Acta 973: 355–382, 1989. Halestrap AP, Kerr PM, Javadov S, and Suleiman S. The mitochondrial permeability transition: role in ischemia/reperfusion injury. Sepsis 2: 313–325, 1998. Holmuhamedov EL, Jovanovic S, Dzeja PP, Jovanovic A, and Terzic A. Mitochondrial ATP-sensitive K⫹ channels modulate cardiac mitochondrial function. Am J Physiol Heart Circ Physiol 275: H1567–H1576, 1998. Holmuhamedov EL, Wang L, and Terzic A. ATP-sensitive K⫹ channel openers prevent Ca2⫹ overload in rat cardiac mitochondria. J Physiol 519: 347–360, 1999. Jung DW and Brierley GP. Matrix free Mg2⫹ and the regulation of mitochondrial volume. Am J Physiol Cell Physiol 277: C1194 –C1201, 1999. Kowaltowski AJ, Seetharaman S, Paucek P, and Garlid KD. Bioenergetic consequences of opening of the ATP-sensitive K⫹ channel of heart mitochondria. Am J Physiol Heart Circ Physiol 280: H649 –H657, 2001. Lee SH, Doliba NM, Osbakken MD, Oz M, and Mancini D. Improvement of myocardial mitochondrial function following hemodyanamic support with left ventricular assist devices in patients with heart failure. J Thorac Cardiovasc Surg 116: 344 –349, 1998. Lim KHH, Javadov SA, Das M, Clarke SJ, Suleiman MS, and Halestrap AP. The effects of ischaemic preconditioning, diazoxide and 5-hydroxydecanoate on rat heart mitochondrial volume and respiration. J Physiol 545: 961–974, 2002. Lin J, Pan LP, and Chan SI. The subunit location of magnesium in cytochrome c oxidase. J Biol Chem 268: 22210 –22214, 1996. McCully JD, Levitsky S. Mitochondrial ATP-sensitive potassium channels in surgical cardioprotection. Arch Biochem Biophys 420: 237–245, 2003. McCully JD, Wakiyama H, Cowan DB, Federman M, and Levitsky S. Diazoxide amelioration of myocardial injury and mitochondrial damage during cardiac surgery. Ann Thorac Surg 74: 2138 –2146, 2002. Murata M, Akao M, O’Rourke B, and Marban E. Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca2⫹ overload during simulated ischemia and reperfusion. Circ Res 89: 891– 898, 2001. Ovide-Bordeaux S, Ventura-Clapier R, and Veksler V. Do modulators of the mitochondrial KATP channel change the function of mitochondria in situ? J Biol Biochem 275: 37291–37295, 2000. Ozcan C, Holmuhamedov EL, Jahangir A, Terzic A. Diazoxide protects mitochondria from anoxic injury: implication for myopreservation. J Thorac Cardiovasc Surg 121: 298 –306, 2001. Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis G, and Garlid KD. Reconstitution and partial purification of the glibenclamidesensitive, ATP-dependent K⫹ channel from rat liver and beef heart mitochondria. J Biol Chem 267: 26062–26069, 1992. Stadler J, Bentz BG, Harbrecht BG, Di Silvio M, Curran RD, Billiar TR, Hoffman RA, and Simmons RL. Tumor necrosis factor alpha inhibits hepatocyte mitochondrial respiration. Ann Surg 216: 539 – 46, 1992. Sternbergh WC, Brunsting LA, Abd-Elfattah AS, and Wechsler AS. Basal metabolic energy requirements of polarized and depolarized arrest in rat heart. Am J Physiol Heart Circ Physiol 256: H846 –H851, 1989.

287 • NOVEMBER 2004 •

www.ajpheart.org

H1976


36. Suleiman MS, Halestrap AP, and Griffiths EJ. Mitochondria: a target for myocardial protection. Pharmacol Ther 89: 29 – 46, 2001. 37. Toyoda Y, Levitsky S, and McCully JD. Opening of mitochondrial ATP-sensitive potassium channels enhances cardioplegic protection. Ann Thorac Surg 71: 1281–1289, 2001. 38. Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H, Shinazawa-Itoh K, Nakashima R, Yaono R, and Yoshikawa S. Structures of the metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A. Science 269: 1069 –1074, 1995. 39. Tsukube T, McCully JD, Faulk E, Federman M, LoCicero J III, Krukenkamp IB, and Levitsky S. Magnesium cardioplegia reduces


cytosolic and nuclear calcium and DNA fragmentation in the senescent myocardium. Ann Thorac Surg 58: 1005–1011, 1994. 40. Tsukube T, McCully JD, Metz RM, Cook CU, and Levtisky S. Amelioration of ischemic calcium overload correlates with high energy phosphates in the senescent myocardium. Am J Physiol Heart Circ Physiol 42: H418 –H427, 1997. 41. Wakiyama H, Cowan DB, Toyoda Y, Federman M, Levitsky S, and McCully JD. Selective opening of mitochondrial ATP-sensitive potassium channels during cardiopulmonary bypass decreases apoptosis and necrosis in a model of acute myocardial infarction. Eur J Cardiothorac Surg 21: 424 – 433, 2002.

287 • NOVEMBER 2004 •

www.ajpheart.org