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Effect of Coenzyme Q10 Supplementation on Mitochondrial Function after Myocardial Ischemia Reperfusion
Journal of Surgical Research 102, 221–228 (2002) doi:10.1006/jsre.2001.6324, available online at http://www.idealibrary.com on
Effect of Coenzyme Q 10 Supplementation on Mitochondrial Function after Myocardial Ischemia Reperfusion 1 Juan A. Crestanello, M.D.,* ,2 Nicolai M. Doliba, Ph.D.,† Natalia M. Doliba, M.D.,† Andriy M. Babsky, Ph.D.,† Koki Niborii, M.D.,* Mary D. Osbakken, M.D., Ph.D.,† ,‡ and Glenn J. R. Whitman, M.D., FACS* *Division of Cardiothoracic Surgery, University of Maryland Medical System, Baltimore, Maryland; †Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania; and ‡Aventis Pharmaceuticals, Inc., Bridgewater, New Jersey Submitted for publication June 19, 2001; published online December 13, 2001
Background. Coenzyme Q 10 (CoQ 10) protects myocardium from ischemia-reperfusion (IR) injury as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine during reperfusion. This protection may result from CoQ 10’s bioenergetic effects on the mitochondria, from its antioxidant properties, or both. The purpose of this study was to elucidate the effects of CoQ 10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation. Methods. Isolated hearts (n ⴝ 6/group) from rats pretreated with liposomal CoQ 10 (10 mg/kg iv, CoQ 10), vehicle (liposomal only, Vehicle), or saline (Saline) 30 min before the experiments were subjected to 15 min of equilibration (EQ), 25 min of ischemia (I), and 40 min of reperfusion (RP). Left ventricular-developed pressure (DP) was measured. Mitochondria were isolated at end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP). Mitochondrial respiratory function (State 2, 3, and 4, respiratory control index (RCI, ratio of State 3 to 4), and ADP:O ratio) was measured by polarography using NADH (␣-ketoglutarate, ␣-KG)- or FADH (succinate, SA)dependent substrates. Results. CoQ 10 improved recovery of DP at end-RP (67 ⴞ 11% in CoQ 10 vs 47 ⴞ 5% in Vehicle and 50 ⴞ 11% in Saline, P < 0.05 vs Vehicle and Saline). CoQ 10 did not change preischemic mitochondrial function. IR decreased State 3 and RCI in all groups using either substrate. CoQ 10 had no effect in the mitochondrial 1 Presented at the Surgical Forum, Cardiothoracic Surgery Session, of the 85th Annual Clinical Congress of The American College of Surgeons, San Francisco, CA, October 1999. This work was partially supported by the NIH Grant RO 1-HL39208 (M.D.O.). 2 To whom correspondence should be addressed at Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Fax: (507) 255-7378. E-mail: [email protected].
oxidation of ␣-KG at end-I. CoQ 10 improved State 3 at end-I when SA was used (167 ⴞ 21 in CoQ 10 vs 120 ⴞ 10 in Saline and 111 ⴞ 10 ng-atoms O/min/mg protein in Vehicle, P < 0.05). Using ␣-KG as a substrate, CoQ 10 improved RCI at end-RP (4.2 ⴞ 0.2 in CoQ 10 vs 3.2 ⴞ 0.2 in Saline and 3.0 ⴞ 0.3 in Vehicle, P < 0.05). Using SA, CoQ 10 improved State 3 (181 ⴞ 10 in CoQ 10 vs 142 ⴞ 9 in Saline and 140 ⴞ 12 ng-atoms O/min/mg protein in Vehicle, P < 0.05) and RCI (2.21 ⴞ 0.06 in CoQ 10 vs 1.85 ⴞ 0.11 in Saline and 1.72 ⴞ 0.08 in Vehicle, P < 0.05) at end-RP. Conclusions. The cardioprotective effects of CoQ 10 can be attributed to the preservation of mitochondrial function during reperfusion as evidenced by improved FADH-dependent oxidation. © 2001 Elsevier Science Key Words: mitochondria; rat; heart; ischemia; reperfusion; oxygen consumption; ubiquinone.
INTRODUCTION
Myocardial ischemia reperfusion injury is associated with free oxygen radical generation that causes lipid and protein peroxidation resulting in mechanical and bioenergetic derangements [1–5]. This oxidative injury can be attenuated by treatment with oxygen radical scavengers and with agents that modulate energy use and production [6 –10]. Coenzyme Q 10 (CoQ 10) is a lipid-soluble bezoquinone that has properties that make it a potentially ideal therapeutic agent for preventing ischemia-reperfusion injury [11, 12]. CoQ 10 is a component of the mitochondrial respiratory chain where it transports electrons from the NADH and succinate dehydrogenase to the cytochrome system [13]. It also participates in the coupling of the respiratory chain to oxidative phosphorylation [14 –17]. CoQ 10 is also a powerful antioxidant
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capable of protecting cell structures and membranes from oxidative damage during reperfusion [18 –20]. CoQ 10 has been successfully used to prevent myocardial reperfusion injury [21–23]. Our laboratory has shown that exogenous supplementation with CoQ10 protects myocardium from ischemia-reperfusion injury, as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine (PCr) during reperfusion [24, 25]. We have also shown decreased free oxygen radical generation during reperfusion [26]. However, the effects of CoQ 10 supplementation on mitochondrial function during ischemia-reperfusion have not been clearly defined. The purpose of this study was to elucidate the effect of CoQ 10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation. We hypothesized that CoQ 10 supplementation improves mitochondrial function during reperfusion. GLOSSARY ADP:O ratio
DP end-EQ end-I end-RP EQ I IR PCr RCI RP State 2 State 3 State 4
is the ratio of nanomoles of ADP consumed to nanogram-atoms of oxygen consumed during State 3 left ventricular-developed pressure end-equilibration (minute 15 of the experiment, see Fig. 1) end-ischemia (minute 40 of the experiment, see Fig. 1) end-reperfusion (minute 80 of the experiment, see Fig. 1) equilibration ischemia ischemia-reperfusion phosphocreatine respiratory control index (ratio of State 3 to State 4) reperfusion mitochondrial oxygen consumption with substrate only mitochondrial oxygen consumption stimulated by ADP phosphorylation mitochondrial oxygen consumption after cessation of ADP phosphorylation
METHODS
Pretreatment with CoQ 10 Male adult Sprague-Dawley rats weighing 200 to 250 g (Harlan Sprague Dawley Inc., Indianapolis, IN) were anesthetized with sodium pentobarbital (60 mg/kg ip) and heparinized (heparin sodium 500 IU ip). Rats (n ⫽ 6 per group) were then pretreated either with liposomal coenzyme Q 10 (10 mg/kg iv CoQ 10 group, Eisai Co. Ltd., Tokyo, Japan), vehicle (liposomal only iv, Vehicle group, Eisai Co. Ltd., Tokyo, Japan), or saline (Saline group) 30 min before the physiological intervention.
Isolated Heart Preparation Thoracotomy was performed 30 min after the injections. Hearts were quickly excised and arrested with 4°C Krebs-Henseleit buffer (containing in mM) NaCl, 118; KCl, 4.6; KH2PO 4, 1.17; MgSO4, 1.17; CaCl2, 1.16; NaHCO3, 23; and glucose, 5.3. Hearts were then transferred to a
nonrecirculating Langendorff apparatus and perfused at a constant aortic pressure of 76 mm Hg with Krebs-Henseleit buffer, previously equilibrated with a gas mixture of 95% O2 and 5% CO 2 (pH 7.4, pO2 ⬎ 500 mm Hg, 37°C). They were paced at 360 beats per minute at 2 ⫻ threshold (Grass Instruments, Quincy, MA). An intraventricular latex balloon was inserted into the left ventricle through the mitral valve and attached to a pressure transducer (COBE, Lakewood, CO). Left ventricular end diastolic pressure was set at 6 mm Hg. Developed pressure (peak systolic minus end diastolic pressure), contractility (⫹dP/dt), and compliance (⫺dP/dt) were recorded throughout the experiment. Data were amplified, acquired, and recorded on a Macintosh IIci computer (Apple Computers, Cupertino, CA) using LabView software (National Instruments, Austin, TX). Animals were treated in accordance with the Principles of Laboratory Animal Care (National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985).
Experimental Protocol Hearts in all groups were subjected to 15 min of equilibration (EQ), 25 min of global normothermic ischemia (I), and 40 min of reperfusion (RP) (Fig. 1). Nonflow ischemia was achieved via a stopcock at the level of the aortic root. Pacing was halted during ischemia. Heart temperature was maintained at 37°C throughout the experiment by immersion in a water-jacketed nongassed perfusate bath.
Isolation of Heart Mitochondria Mitochondria were isolated by differential centrifugation according to Kondrashova and Doliba [27]. At end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP), parallel groups of hearts (Saline, Vehicle, and CoQ 10) were chilled, weighed, and homogenized in a glass Potter-Elvehjem homogenizer with a motor driven Teflon pestle. The homogenization medium contained 300 mM sucrose, 10 mM HEPES buffer, 1 mM EDTA, and 0.25% bovine serum albumin (fraction V) at pH 7.4. The nuclear fraction and cellular debris were isolated from a 12% suspension of homogenate by centrifugation for 3 min at 150g and again for 4 min at 330g at 0°C. The mitochondrial fraction was obtained by centrifugation of the supernatant for 17 min at 8000g at 0°C. Each heart generated one mitochondrial preparation. There were 6 hearts in each group.
Measurement of Mitochondrial Oxygen Consumption Mitochondrial respiratory function was measured by the polarographic method of Chance and Williams using a Clark oxygen electrode [28]. The incubation media contained sucrose 250 mM, KCl 50 mM, KH 2PO 4 1 mM, HEPES 5 mM buffer (pH 7.4, 27°C). Mitochondrial samples (approximately 2 mg of protein) were added to 1.5 ml of incubation media containing either an NADH (␣-ketoglutarate, ␣-KG, 2 mM) or a FADH dependent (succinic acid, SA, 2mM) substrate for each measurement. Parameters measured were as follows: State 2, oxygen consumption before the addition of ADP; State 3, oxygen consumption stimulated by ADP (320 nmoles); State 4, oxygen consumption after completion of ADP phosphorylation. The respiratory control index (RCI: ratio of State 3 to State 4) [28, 29], and the ADP:O ratio (ratio between the nanomoles of ADP phosphorylated and the nanomoles of oxygen consumed during State 3) were also determined. Mitochondrial protein concentration in the polarographic chamber was measured at the end of each experiment by the Lowry method [30]; results were used to normalize respiratory rates to mitochondrial protein. Data were expressed as means ⫾ SEM.
Chemicals Chemicals for isolation of heart mitochondria and polarography were obtained from Sigma (St. Louis, MO).
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FIG. 1. Experimental protocol: 30 min after treatment, hearts in the different groups were perfused in a Langendorff apparatus and subjected to 15 min of equilibration (EQ), 25 min of ischemia (Ischemia), and 40 min of reperfusion (Reperfusion). At the end of the equilibration period (end-EQ), at the end of the ischemic period (end-I), and at the end of the reperfusion period (end-RP) parallel groups of hearts (Saline, Vehicle, and CoQ 10) were homogenized for mitochondria (mito) isolation.
Statistical Analysis Paired and nonpaired t tests were used for statistical significance. Significance was defined as P ⬍ 0.05.
RESULTS
Mechanical Function Hearts perfused continuously for 80 min preserved 95 ⫾ 2% of their end-equilibration DP demonstrating the stability of our isolated perfused rat heart model. Saline, Vehicle, and CoQ 10 groups had similar developed pressure (DP) at end-equilibration (106 ⫾ 10 mm Hg in Saline, 105 ⫾ 10 mm Hg in Vehicle, 114 ⫾ 9 mm Hg in CoQ 10, P ⫽ NS). At end-reperfusion, CoQ 10 hearts recovered 67 ⫾ 5% of their preischemic DP, whereas Saline and Vehicle hearts recovered 50 ⫾ 11 and 47 ⫾ 5% of their preischemic DP, respectively (P ⬍ 0.05 vs Saline and Vehicle) (Fig. 2). Both ⫹dP/dt and ⫺dP/dt behaved in a similar fashion.
cle had an effect on mitochondrial function at endischemia. CoQ 10 significantly improved State 3 compared to Saline and Vehicle when SA was used as a substrate (P ⬍ 0.05 vs Saline and Vehicle at end-I, Fig. 4B). All other parameters remained unchanged. Mitochondria Respiratory Function at End-Reperfusion Mitochondrial oxygen consumption data from CoQ 10, Vehicle, and Saline at end-RP are shown in Fig. 3 and 4, using ␣-KG or SA as substrate, respectively. Ischemia-reperfusion decreased State 3 and RCI in all groups compared to their respective end-EQ values (P ⬍ 0.05 vs end-EQ). However, the efficiency of oxidative phosphorylation was preserved in all groups as evidenced by unchanged ADP:O ratios at end-RP.
Mitochondria Respiratory Function at End-Equilibration Preischemic mitochondria function is not affected by CoQ 10 administration using either ␣-KG or SA as substrate (Figs. 3 and 4). Mitochondria Respiratory Function at End-Ischemia Using ␣-KG as a substrate, ischemia decreased State 3, RCI, and ADP:O ratios (P ⬍ 0.05 vs end-EQ). State 2 and State 4 remained unchanged. Using SA as a substrate, ischemia decreased State 3, RCI, ADO:O ratios (P ⬍ 0.05 vs end-EQ). State 2 and 4 remained unchanged. Using ␣-KG as a substrate, neither CoQ 10 nor Vehi-
FIG. 2. Recovery of mechanical function at end-reperfusion in Saline, Vehicle, and CoQ 10 hearts expressed as a percentage of the initial left ventricular developed pressure (DP). *P ⬍ 0.05 vs Saline.
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FIG. 3. State 2 respiration (A), State 3 respiration (B), State 4 respiration (C), respiratory control index (D), and ADP:O ratio (E) in Saline, Vehicle, and CoQ 10 mitochondria at end-equilibration (End-EQ), end-Ischemia (end-I), and end-reperfusion (End-RP) using ␣-ketoglutarate as a substrate. State 2 is mitochondrial oxygen consumption before the addition of ADP. State 3 is oxygen consumption stimulated by ADP. State 4 is oxygen consumption after ADP phosphorylation has ceased. RCI is the respiratory control index of Chance (State 3/State 4). ADP/O ratio is the ratio of nanomoles of ADP consumed to nanogram-atoms of oxygen consumed during State 3. Values are mean ⫾ SEM. CoQ 10 supplementation had no effect on end-EQ nor on end-I mitochondrial function. CoQ 10 improved RCI at end-RP (*P ⬍ 0.05 vs Saline and Vehicle). ⌿ P ⬍ 0.05 vs the respective group at end-EQ.
CoQ 10 improved RCI at end-RP using ␣-KG as a substrate (P ⬍ 0.05, Fig. 3D). CoQ 10 also improved State 3 and RCI at end-RP using SA as a substrate, (P ⬍ 0.05, Figs. 4B and 4D).
reperfusion. Data demonstrate that CoQ 10 supplementation improves RCI at end-reperfusion using either a NADH- or a FADH-dependent substrate. CoQ 10 also improves mitochondrial State 3 at end-ischemia and at end-RP when a FADH-dependent substrate is used.
DISCUSSION
This study confirms the protective effects of CoQ 10 on myocardial function after ischemia-reperfusion and provides evidence that CoQ 10 supplementation improves mitochondrial function during ischemia-
Mitochondrial Function at End-Equilibration Preischemic mitochondrial function is not changed by the administration of CoQ 10. Previously, using a different CoQ 10 formulation we were able to show an
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FIG. 4. State 2 respiration (A), State 3 respiration (B), State 4 respiration (C), respiratory control index (D), and ADP:O ratio (E) in Saline, Vehicle, and CoQ 10 mitochondria at end-equilibration (end-EQ), end-ischemia (end-I), and end-reperfusion (end-RP) using succinic acid as a substrate. Legends are as in Fig. 3. Values are mean ⫾ SEM. Using succinic acid as a substrate, CoQ 10 supplementation had no effect on preischemic mitochondrial function. However, CoQ 10 improved State 3 at end-I and State 3 and RCI at end-RP (*P ⬍ 0.05 vs Saline and Vehicle at end-I and at end-RP). ⌿P ⬍ 0.05 vs the respective group at end-EQ.
improvement in myocardial ATP and PCr levels measured by 31P NMR spectroscopy [24]. However, mitochondrial function at end-EQ is not affected by CoQ 10 supplementation in this experimental model. Mitochondrial Function at End-Ischemia Twenty-five minutes of ischemia results in significant derangements in mitochondrial function. State 3, RCI, and ADP:O ratios are decreased while States 4 and 2 have a tendency to increase. These findings are consistent with progressive uncoupling, decrease in the
phosphorylation capability, and loss of aerobic efficiency of the mitochondria [34, 35]. CoQ 10 supplementation had no effects on the mitochondrial oxidation of NADH-dependent substrates at end-ischemia. However, when a FADH-dependent substrate is used, State 3 is improved at end-ischemia, which suggests an improvement in mitochondrial phosphorylation capability. Mitochondrial Function at End-Reperfusion CoQ 10 supplementation improves mitochondrial ability to oxidize FADH-dependent substrates and mito-
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chondrial coupling during reperfusion, as seen by preservation of State 3 and RCI in CoQ 10 hearts. CoQ 10 supplementation has no significant effect on the oxidation of NADH-dependent substrates during reperfusion. However, it improves mitochondrial coupling as seen by improved RCI. The improvement in mitochondrial function at endreperfusion is responsible, at least in part, for the improvement in myocardial tolerance to ischemiareperfusion seen in CoQ 10 hearts. This improvement in mitochondrial function may be the result of CoQ 10 induced intrinsic bioenergetic properties in the mitochondria, of its action as an antioxidant, or the ability of CoQ 10 to replete intracellular CoQ 10 levels, which are depleted during ischemia-reperfusion. In the mitochondria, CoQ 10 is a component of the respiratory electron transfer chain. It transports electrons among NADH dehydrogenase, succinate dehydrogenase, and the cytochrome b-c 1 [13–15]. It also participates in the proton motive Q cycle transferring protons across the inner-mitochondrial membrane coupling respiration to oxidative phosphorylation [13–15]. Battino et al. [17] and Lenaz et al. [14, 16] have shown that CoQ 10 concentration in mitochondrial membrane is physiologically not saturated for maximal electron transfer. They showed that the addition of exogenous CoQ 10 increases the rate of electron transfer in the respiratory chain, thus improving the efficiency of oxidative phosphorylation and mitochondrial coupling. This set of experiments clearly showed an improvement in mitochondrial coupling and State 3 at end-RP, which agrees with previous results from our laboratory showing an improvement on myocardial phosphocreatine and ATP levels at end-RP in CoQ 10-supplemented hearts [24]. However, no effect on mitochondrial function was demonstrated prior to ischemia. The association of these two findings (no improvement at end-EQ and improvement at end-RP) suggests that (1) an intrinsic improvement in mitochondrial function is not the main mechanism for CoQ 10’s effects and (2) the main mechanisms responsible for its protective effects are either its antioxidant properties or the supplementation of depleted mitochondrial CoQ 10 stores. Myocardial CoQ 10 levels are decreased during ischemia reperfusion [31]. Exogenously administered CoQ 10 is nonspecifically incorporated into cell and mitochondrial membranes [32]. We have previously shown that iv supplementation with liposomal CoQ 10 increases myocardial CoQ 10 levels by 38% [25]. Maulick et al. and others have shown that CoQ 10 supplementation not only increases preischemic CoQ 10 levels but also increases postischemic CoQ 10 levels, improving the total antioxidant reserve of the heart during reperfusion [33]. Improvement of mitochondrial CoQ 10 concentration during reperfusion may be
partially responsible for the better mitochondrial function seen at end-RP. Mitochondrial free oxygen radical generation is increased during reperfusion [36]. Specific segments of the respiratory chain (the reduced flavin mononucleotide of NADH dehydrogenase in complex I and the ubisemiquinone associated with cytochrome b-c 1 segment of complex III) are primarily responsible for its generation [36 –38]. These segments are also the ones preferentially damaged by oxygen radicals [39]. The loss of activity of complex I and III results in decreased State 3 and RCI during RP [40 – 42]. CoQ 10, in its fully reduced form (ubiquinol), is a powerful antioxidant preventing lipid peroxidation during reperfusion. There are two mechanisms for CoQ 10 (ubiquinol) antioxidant function: (1) CoQ 10 provides hydrogen equivalents to reduce peroxyl and/or alkoxyl radicals preventing both the initiation and the propagation of lipid peroxidation; and (2) CoQ 10 also regenerates the reduced form of vitamin E, another powerful antioxidant, from the tocopheroxyl radical produced by oxygen radicals [18]. Ubiquinol is also continuously regenerated from the semiquinone radical by complex III (ubiquinol cytochrome c reductase complex) providing an endless supply of antioxidant closely associated to the main sites of oxygen radical generation in the inner-mitochondrial membrane (complexes I and III) [18 –20]. Our laboratory has previously demonstrated that these antioxidant properties result in decreased total myocardial free oxygen radical production during reperfusion as measured by lucigenin enhanced chemiluminescence [26]. Oxygen radicals also induce changes in mitochondrial membrane permeability that result in collapse of the proton motive force, inhibition of oxidative phosphorylation, ATP hydrolysis, and cell death [43, 44]. Greenberg et al. have shown that by preventing membrane oxidation, CoQ 10 stabilizes cell membranes and prevents membrane damage during reperfusion [12]. Although these results are encouraging, it is premature to suggest, based on our data and on those available in the literature, a dose of CoQ 10 for human use in the setting of cardiac surgery. The human data show that it is possible to effectively use either short-term parenteral supplementation or long-term enteral supplementation with CoQ 10 to decrease oxidative stress during reperfusion [45, 46]. In conclusion, this study confirms that short-term parenteral supplementation with liposolube CoQ 10 provides cardioprotection against ischemia-reperfusion injury. This cardioprotective effect is the result of improving mitochondrial function during ischemiareperfusion by repleting mitochondrial CoQ 10 stores potentiating its antioxidant and bioenergetic properties.
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Effect of Coenzyme Q 10 Supplementation on Mitochondrial Function after Myocardial Ischemia Reperfusion 1 Juan A. Crestanello, M.D.,* ,2 Nicolai M. Doliba, Ph.D.,† Natalia M. Doliba, M.D.,† Andriy M. Babsky, Ph.D.,† Koki Niborii, M.D.,* Mary D. Osbakken, M.D., Ph.D.,† ,‡ and Glenn J. R. Whitman, M.D., FACS* *Division of Cardiothoracic Surgery, University of Maryland Medical System, Baltimore, Maryland; †Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania; and ‡Aventis Pharmaceuticals, Inc., Bridgewater, New Jersey Submitted for publication June 19, 2001; published online December 13, 2001
Background. Coenzyme Q 10 (CoQ 10) protects myocardium from ischemia-reperfusion (IR) injury as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine during reperfusion. This protection may result from CoQ 10’s bioenergetic effects on the mitochondria, from its antioxidant properties, or both. The purpose of this study was to elucidate the effects of CoQ 10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation. Methods. Isolated hearts (n ⴝ 6/group) from rats pretreated with liposomal CoQ 10 (10 mg/kg iv, CoQ 10), vehicle (liposomal only, Vehicle), or saline (Saline) 30 min before the experiments were subjected to 15 min of equilibration (EQ), 25 min of ischemia (I), and 40 min of reperfusion (RP). Left ventricular-developed pressure (DP) was measured. Mitochondria were isolated at end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP). Mitochondrial respiratory function (State 2, 3, and 4, respiratory control index (RCI, ratio of State 3 to 4), and ADP:O ratio) was measured by polarography using NADH (␣-ketoglutarate, ␣-KG)- or FADH (succinate, SA)dependent substrates. Results. CoQ 10 improved recovery of DP at end-RP (67 ⴞ 11% in CoQ 10 vs 47 ⴞ 5% in Vehicle and 50 ⴞ 11% in Saline, P < 0.05 vs Vehicle and Saline). CoQ 10 did not change preischemic mitochondrial function. IR decreased State 3 and RCI in all groups using either substrate. CoQ 10 had no effect in the mitochondrial 1 Presented at the Surgical Forum, Cardiothoracic Surgery Session, of the 85th Annual Clinical Congress of The American College of Surgeons, San Francisco, CA, October 1999. This work was partially supported by the NIH Grant RO 1-HL39208 (M.D.O.). 2 To whom correspondence should be addressed at Mayo Clinic, 200 First St. SW, Rochester, MN 55905. Fax: (507) 255-7378. E-mail: [email protected].
oxidation of ␣-KG at end-I. CoQ 10 improved State 3 at end-I when SA was used (167 ⴞ 21 in CoQ 10 vs 120 ⴞ 10 in Saline and 111 ⴞ 10 ng-atoms O/min/mg protein in Vehicle, P < 0.05). Using ␣-KG as a substrate, CoQ 10 improved RCI at end-RP (4.2 ⴞ 0.2 in CoQ 10 vs 3.2 ⴞ 0.2 in Saline and 3.0 ⴞ 0.3 in Vehicle, P < 0.05). Using SA, CoQ 10 improved State 3 (181 ⴞ 10 in CoQ 10 vs 142 ⴞ 9 in Saline and 140 ⴞ 12 ng-atoms O/min/mg protein in Vehicle, P < 0.05) and RCI (2.21 ⴞ 0.06 in CoQ 10 vs 1.85 ⴞ 0.11 in Saline and 1.72 ⴞ 0.08 in Vehicle, P < 0.05) at end-RP. Conclusions. The cardioprotective effects of CoQ 10 can be attributed to the preservation of mitochondrial function during reperfusion as evidenced by improved FADH-dependent oxidation. © 2001 Elsevier Science Key Words: mitochondria; rat; heart; ischemia; reperfusion; oxygen consumption; ubiquinone.
INTRODUCTION
Myocardial ischemia reperfusion injury is associated with free oxygen radical generation that causes lipid and protein peroxidation resulting in mechanical and bioenergetic derangements [1–5]. This oxidative injury can be attenuated by treatment with oxygen radical scavengers and with agents that modulate energy use and production [6 –10]. Coenzyme Q 10 (CoQ 10) is a lipid-soluble bezoquinone that has properties that make it a potentially ideal therapeutic agent for preventing ischemia-reperfusion injury [11, 12]. CoQ 10 is a component of the mitochondrial respiratory chain where it transports electrons from the NADH and succinate dehydrogenase to the cytochrome system [13]. It also participates in the coupling of the respiratory chain to oxidative phosphorylation [14 –17]. CoQ 10 is also a powerful antioxidant
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capable of protecting cell structures and membranes from oxidative damage during reperfusion [18 –20]. CoQ 10 has been successfully used to prevent myocardial reperfusion injury [21–23]. Our laboratory has shown that exogenous supplementation with CoQ10 protects myocardium from ischemia-reperfusion injury, as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine (PCr) during reperfusion [24, 25]. We have also shown decreased free oxygen radical generation during reperfusion [26]. However, the effects of CoQ 10 supplementation on mitochondrial function during ischemia-reperfusion have not been clearly defined. The purpose of this study was to elucidate the effect of CoQ 10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation. We hypothesized that CoQ 10 supplementation improves mitochondrial function during reperfusion. GLOSSARY ADP:O ratio
DP end-EQ end-I end-RP EQ I IR PCr RCI RP State 2 State 3 State 4
is the ratio of nanomoles of ADP consumed to nanogram-atoms of oxygen consumed during State 3 left ventricular-developed pressure end-equilibration (minute 15 of the experiment, see Fig. 1) end-ischemia (minute 40 of the experiment, see Fig. 1) end-reperfusion (minute 80 of the experiment, see Fig. 1) equilibration ischemia ischemia-reperfusion phosphocreatine respiratory control index (ratio of State 3 to State 4) reperfusion mitochondrial oxygen consumption with substrate only mitochondrial oxygen consumption stimulated by ADP phosphorylation mitochondrial oxygen consumption after cessation of ADP phosphorylation
METHODS
Pretreatment with CoQ 10 Male adult Sprague-Dawley rats weighing 200 to 250 g (Harlan Sprague Dawley Inc., Indianapolis, IN) were anesthetized with sodium pentobarbital (60 mg/kg ip) and heparinized (heparin sodium 500 IU ip). Rats (n ⫽ 6 per group) were then pretreated either with liposomal coenzyme Q 10 (10 mg/kg iv CoQ 10 group, Eisai Co. Ltd., Tokyo, Japan), vehicle (liposomal only iv, Vehicle group, Eisai Co. Ltd., Tokyo, Japan), or saline (Saline group) 30 min before the physiological intervention.
Isolated Heart Preparation Thoracotomy was performed 30 min after the injections. Hearts were quickly excised and arrested with 4°C Krebs-Henseleit buffer (containing in mM) NaCl, 118; KCl, 4.6; KH2PO 4, 1.17; MgSO4, 1.17; CaCl2, 1.16; NaHCO3, 23; and glucose, 5.3. Hearts were then transferred to a
nonrecirculating Langendorff apparatus and perfused at a constant aortic pressure of 76 mm Hg with Krebs-Henseleit buffer, previously equilibrated with a gas mixture of 95% O2 and 5% CO 2 (pH 7.4, pO2 ⬎ 500 mm Hg, 37°C). They were paced at 360 beats per minute at 2 ⫻ threshold (Grass Instruments, Quincy, MA). An intraventricular latex balloon was inserted into the left ventricle through the mitral valve and attached to a pressure transducer (COBE, Lakewood, CO). Left ventricular end diastolic pressure was set at 6 mm Hg. Developed pressure (peak systolic minus end diastolic pressure), contractility (⫹dP/dt), and compliance (⫺dP/dt) were recorded throughout the experiment. Data were amplified, acquired, and recorded on a Macintosh IIci computer (Apple Computers, Cupertino, CA) using LabView software (National Instruments, Austin, TX). Animals were treated in accordance with the Principles of Laboratory Animal Care (National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985).
Experimental Protocol Hearts in all groups were subjected to 15 min of equilibration (EQ), 25 min of global normothermic ischemia (I), and 40 min of reperfusion (RP) (Fig. 1). Nonflow ischemia was achieved via a stopcock at the level of the aortic root. Pacing was halted during ischemia. Heart temperature was maintained at 37°C throughout the experiment by immersion in a water-jacketed nongassed perfusate bath.
Isolation of Heart Mitochondria Mitochondria were isolated by differential centrifugation according to Kondrashova and Doliba [27]. At end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP), parallel groups of hearts (Saline, Vehicle, and CoQ 10) were chilled, weighed, and homogenized in a glass Potter-Elvehjem homogenizer with a motor driven Teflon pestle. The homogenization medium contained 300 mM sucrose, 10 mM HEPES buffer, 1 mM EDTA, and 0.25% bovine serum albumin (fraction V) at pH 7.4. The nuclear fraction and cellular debris were isolated from a 12% suspension of homogenate by centrifugation for 3 min at 150g and again for 4 min at 330g at 0°C. The mitochondrial fraction was obtained by centrifugation of the supernatant for 17 min at 8000g at 0°C. Each heart generated one mitochondrial preparation. There were 6 hearts in each group.
Measurement of Mitochondrial Oxygen Consumption Mitochondrial respiratory function was measured by the polarographic method of Chance and Williams using a Clark oxygen electrode [28]. The incubation media contained sucrose 250 mM, KCl 50 mM, KH 2PO 4 1 mM, HEPES 5 mM buffer (pH 7.4, 27°C). Mitochondrial samples (approximately 2 mg of protein) were added to 1.5 ml of incubation media containing either an NADH (␣-ketoglutarate, ␣-KG, 2 mM) or a FADH dependent (succinic acid, SA, 2mM) substrate for each measurement. Parameters measured were as follows: State 2, oxygen consumption before the addition of ADP; State 3, oxygen consumption stimulated by ADP (320 nmoles); State 4, oxygen consumption after completion of ADP phosphorylation. The respiratory control index (RCI: ratio of State 3 to State 4) [28, 29], and the ADP:O ratio (ratio between the nanomoles of ADP phosphorylated and the nanomoles of oxygen consumed during State 3) were also determined. Mitochondrial protein concentration in the polarographic chamber was measured at the end of each experiment by the Lowry method [30]; results were used to normalize respiratory rates to mitochondrial protein. Data were expressed as means ⫾ SEM.
Chemicals Chemicals for isolation of heart mitochondria and polarography were obtained from Sigma (St. Louis, MO).
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FIG. 1. Experimental protocol: 30 min after treatment, hearts in the different groups were perfused in a Langendorff apparatus and subjected to 15 min of equilibration (EQ), 25 min of ischemia (Ischemia), and 40 min of reperfusion (Reperfusion). At the end of the equilibration period (end-EQ), at the end of the ischemic period (end-I), and at the end of the reperfusion period (end-RP) parallel groups of hearts (Saline, Vehicle, and CoQ 10) were homogenized for mitochondria (mito) isolation.
Statistical Analysis Paired and nonpaired t tests were used for statistical significance. Significance was defined as P ⬍ 0.05.
RESULTS
Mechanical Function Hearts perfused continuously for 80 min preserved 95 ⫾ 2% of their end-equilibration DP demonstrating the stability of our isolated perfused rat heart model. Saline, Vehicle, and CoQ 10 groups had similar developed pressure (DP) at end-equilibration (106 ⫾ 10 mm Hg in Saline, 105 ⫾ 10 mm Hg in Vehicle, 114 ⫾ 9 mm Hg in CoQ 10, P ⫽ NS). At end-reperfusion, CoQ 10 hearts recovered 67 ⫾ 5% of their preischemic DP, whereas Saline and Vehicle hearts recovered 50 ⫾ 11 and 47 ⫾ 5% of their preischemic DP, respectively (P ⬍ 0.05 vs Saline and Vehicle) (Fig. 2). Both ⫹dP/dt and ⫺dP/dt behaved in a similar fashion.
cle had an effect on mitochondrial function at endischemia. CoQ 10 significantly improved State 3 compared to Saline and Vehicle when SA was used as a substrate (P ⬍ 0.05 vs Saline and Vehicle at end-I, Fig. 4B). All other parameters remained unchanged. Mitochondria Respiratory Function at End-Reperfusion Mitochondrial oxygen consumption data from CoQ 10, Vehicle, and Saline at end-RP are shown in Fig. 3 and 4, using ␣-KG or SA as substrate, respectively. Ischemia-reperfusion decreased State 3 and RCI in all groups compared to their respective end-EQ values (P ⬍ 0.05 vs end-EQ). However, the efficiency of oxidative phosphorylation was preserved in all groups as evidenced by unchanged ADP:O ratios at end-RP.
Mitochondria Respiratory Function at End-Equilibration Preischemic mitochondria function is not affected by CoQ 10 administration using either ␣-KG or SA as substrate (Figs. 3 and 4). Mitochondria Respiratory Function at End-Ischemia Using ␣-KG as a substrate, ischemia decreased State 3, RCI, and ADP:O ratios (P ⬍ 0.05 vs end-EQ). State 2 and State 4 remained unchanged. Using SA as a substrate, ischemia decreased State 3, RCI, ADO:O ratios (P ⬍ 0.05 vs end-EQ). State 2 and 4 remained unchanged. Using ␣-KG as a substrate, neither CoQ 10 nor Vehi-
FIG. 2. Recovery of mechanical function at end-reperfusion in Saline, Vehicle, and CoQ 10 hearts expressed as a percentage of the initial left ventricular developed pressure (DP). *P ⬍ 0.05 vs Saline.
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FIG. 3. State 2 respiration (A), State 3 respiration (B), State 4 respiration (C), respiratory control index (D), and ADP:O ratio (E) in Saline, Vehicle, and CoQ 10 mitochondria at end-equilibration (End-EQ), end-Ischemia (end-I), and end-reperfusion (End-RP) using ␣-ketoglutarate as a substrate. State 2 is mitochondrial oxygen consumption before the addition of ADP. State 3 is oxygen consumption stimulated by ADP. State 4 is oxygen consumption after ADP phosphorylation has ceased. RCI is the respiratory control index of Chance (State 3/State 4). ADP/O ratio is the ratio of nanomoles of ADP consumed to nanogram-atoms of oxygen consumed during State 3. Values are mean ⫾ SEM. CoQ 10 supplementation had no effect on end-EQ nor on end-I mitochondrial function. CoQ 10 improved RCI at end-RP (*P ⬍ 0.05 vs Saline and Vehicle). ⌿ P ⬍ 0.05 vs the respective group at end-EQ.
CoQ 10 improved RCI at end-RP using ␣-KG as a substrate (P ⬍ 0.05, Fig. 3D). CoQ 10 also improved State 3 and RCI at end-RP using SA as a substrate, (P ⬍ 0.05, Figs. 4B and 4D).
reperfusion. Data demonstrate that CoQ 10 supplementation improves RCI at end-reperfusion using either a NADH- or a FADH-dependent substrate. CoQ 10 also improves mitochondrial State 3 at end-ischemia and at end-RP when a FADH-dependent substrate is used.
DISCUSSION
This study confirms the protective effects of CoQ 10 on myocardial function after ischemia-reperfusion and provides evidence that CoQ 10 supplementation improves mitochondrial function during ischemia-
Mitochondrial Function at End-Equilibration Preischemic mitochondrial function is not changed by the administration of CoQ 10. Previously, using a different CoQ 10 formulation we were able to show an
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FIG. 4. State 2 respiration (A), State 3 respiration (B), State 4 respiration (C), respiratory control index (D), and ADP:O ratio (E) in Saline, Vehicle, and CoQ 10 mitochondria at end-equilibration (end-EQ), end-ischemia (end-I), and end-reperfusion (end-RP) using succinic acid as a substrate. Legends are as in Fig. 3. Values are mean ⫾ SEM. Using succinic acid as a substrate, CoQ 10 supplementation had no effect on preischemic mitochondrial function. However, CoQ 10 improved State 3 at end-I and State 3 and RCI at end-RP (*P ⬍ 0.05 vs Saline and Vehicle at end-I and at end-RP). ⌿P ⬍ 0.05 vs the respective group at end-EQ.
improvement in myocardial ATP and PCr levels measured by 31P NMR spectroscopy [24]. However, mitochondrial function at end-EQ is not affected by CoQ 10 supplementation in this experimental model. Mitochondrial Function at End-Ischemia Twenty-five minutes of ischemia results in significant derangements in mitochondrial function. State 3, RCI, and ADP:O ratios are decreased while States 4 and 2 have a tendency to increase. These findings are consistent with progressive uncoupling, decrease in the
phosphorylation capability, and loss of aerobic efficiency of the mitochondria [34, 35]. CoQ 10 supplementation had no effects on the mitochondrial oxidation of NADH-dependent substrates at end-ischemia. However, when a FADH-dependent substrate is used, State 3 is improved at end-ischemia, which suggests an improvement in mitochondrial phosphorylation capability. Mitochondrial Function at End-Reperfusion CoQ 10 supplementation improves mitochondrial ability to oxidize FADH-dependent substrates and mito-
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chondrial coupling during reperfusion, as seen by preservation of State 3 and RCI in CoQ 10 hearts. CoQ 10 supplementation has no significant effect on the oxidation of NADH-dependent substrates during reperfusion. However, it improves mitochondrial coupling as seen by improved RCI. The improvement in mitochondrial function at endreperfusion is responsible, at least in part, for the improvement in myocardial tolerance to ischemiareperfusion seen in CoQ 10 hearts. This improvement in mitochondrial function may be the result of CoQ 10 induced intrinsic bioenergetic properties in the mitochondria, of its action as an antioxidant, or the ability of CoQ 10 to replete intracellular CoQ 10 levels, which are depleted during ischemia-reperfusion. In the mitochondria, CoQ 10 is a component of the respiratory electron transfer chain. It transports electrons among NADH dehydrogenase, succinate dehydrogenase, and the cytochrome b-c 1 [13–15]. It also participates in the proton motive Q cycle transferring protons across the inner-mitochondrial membrane coupling respiration to oxidative phosphorylation [13–15]. Battino et al. [17] and Lenaz et al. [14, 16] have shown that CoQ 10 concentration in mitochondrial membrane is physiologically not saturated for maximal electron transfer. They showed that the addition of exogenous CoQ 10 increases the rate of electron transfer in the respiratory chain, thus improving the efficiency of oxidative phosphorylation and mitochondrial coupling. This set of experiments clearly showed an improvement in mitochondrial coupling and State 3 at end-RP, which agrees with previous results from our laboratory showing an improvement on myocardial phosphocreatine and ATP levels at end-RP in CoQ 10-supplemented hearts [24]. However, no effect on mitochondrial function was demonstrated prior to ischemia. The association of these two findings (no improvement at end-EQ and improvement at end-RP) suggests that (1) an intrinsic improvement in mitochondrial function is not the main mechanism for CoQ 10’s effects and (2) the main mechanisms responsible for its protective effects are either its antioxidant properties or the supplementation of depleted mitochondrial CoQ 10 stores. Myocardial CoQ 10 levels are decreased during ischemia reperfusion [31]. Exogenously administered CoQ 10 is nonspecifically incorporated into cell and mitochondrial membranes [32]. We have previously shown that iv supplementation with liposomal CoQ 10 increases myocardial CoQ 10 levels by 38% [25]. Maulick et al. and others have shown that CoQ 10 supplementation not only increases preischemic CoQ 10 levels but also increases postischemic CoQ 10 levels, improving the total antioxidant reserve of the heart during reperfusion [33]. Improvement of mitochondrial CoQ 10 concentration during reperfusion may be
partially responsible for the better mitochondrial function seen at end-RP. Mitochondrial free oxygen radical generation is increased during reperfusion [36]. Specific segments of the respiratory chain (the reduced flavin mononucleotide of NADH dehydrogenase in complex I and the ubisemiquinone associated with cytochrome b-c 1 segment of complex III) are primarily responsible for its generation [36 –38]. These segments are also the ones preferentially damaged by oxygen radicals [39]. The loss of activity of complex I and III results in decreased State 3 and RCI during RP [40 – 42]. CoQ 10, in its fully reduced form (ubiquinol), is a powerful antioxidant preventing lipid peroxidation during reperfusion. There are two mechanisms for CoQ 10 (ubiquinol) antioxidant function: (1) CoQ 10 provides hydrogen equivalents to reduce peroxyl and/or alkoxyl radicals preventing both the initiation and the propagation of lipid peroxidation; and (2) CoQ 10 also regenerates the reduced form of vitamin E, another powerful antioxidant, from the tocopheroxyl radical produced by oxygen radicals [18]. Ubiquinol is also continuously regenerated from the semiquinone radical by complex III (ubiquinol cytochrome c reductase complex) providing an endless supply of antioxidant closely associated to the main sites of oxygen radical generation in the inner-mitochondrial membrane (complexes I and III) [18 –20]. Our laboratory has previously demonstrated that these antioxidant properties result in decreased total myocardial free oxygen radical production during reperfusion as measured by lucigenin enhanced chemiluminescence [26]. Oxygen radicals also induce changes in mitochondrial membrane permeability that result in collapse of the proton motive force, inhibition of oxidative phosphorylation, ATP hydrolysis, and cell death [43, 44]. Greenberg et al. have shown that by preventing membrane oxidation, CoQ 10 stabilizes cell membranes and prevents membrane damage during reperfusion [12]. Although these results are encouraging, it is premature to suggest, based on our data and on those available in the literature, a dose of CoQ 10 for human use in the setting of cardiac surgery. The human data show that it is possible to effectively use either short-term parenteral supplementation or long-term enteral supplementation with CoQ 10 to decrease oxidative stress during reperfusion [45, 46]. In conclusion, this study confirms that short-term parenteral supplementation with liposolube CoQ 10 provides cardioprotection against ischemia-reperfusion injury. This cardioprotective effect is the result of improving mitochondrial function during ischemiareperfusion by repleting mitochondrial CoQ 10 stores potentiating its antioxidant and bioenergetic properties.
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