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1Division 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
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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myocardium; ischemia; reperfusion injury
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 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.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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 cross-clamping 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 µM 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.
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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 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 x24,500 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·W·
/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 µM 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 freeze-clamped 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 µM 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 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 µM), 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 µM) (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 µM) 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.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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, 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.
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 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 oxygen 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 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 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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 B.-q. Zhu, U. Simonis, G. Cecchini, H.-z. Zhou, L. Li, J. R. Teerlink, and J. S. Karliner Comparison of Pyrroloquinoline Quinone and/or Metoprolol on Myocardial Infarct Size and Mitochondrial Damage in a Rat Model of Ischemia/Reperfusion Injury Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2006; 11(2): 119 - 128. [Abstract] [PDF]
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L. Wang, C. Kinnear, J. M. Hammel, W. Zhu, Z. Hua, W. Mi, and C. A. Caldarone Preservation of Mitochondrial Structure and Function After Cardioplegic Arrest in the Neonate Using a Selective Mitochondrial K(ATP) Channel Opener. Ann. Thorac. Surg., May 1, 2006; 81(5): 1817 - 1823. [Abstract] [Full Text] [PDF]
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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.
