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Cardiovascular Research 2008 77(2):406-415; doi:10.1016/j.cardiores.2007.08.008
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©European Society of Cardiology 2007. All rights reserved.For permissions please email: journals.permissions@oxfordjournals.org

Inhibited mitochondrial respiration by amobarbital during cardiac ischaemia improves redox state and reduces matrix Ca2+ overload and ROS release{dagger}

Mohammed Aldakkak1, David F. Stowe1,2,3,4,5, Qun Chen6, Edward J. Lesnefsky6,7 and Amadou K.S. Camara1,*

1 Anesthesiology Research Laboratories, Department of Anesthesiology, The Medical College of Wisconsin, Milwaukee, Wisconsin, USA
2 Department of Physiology, The Medical College of Wisconsin, Milwaukee, Wisconsin, USA
3 Cardiovascular Research Center, The Medical College of Wisconsin, Milwaukee, Wisconsin, USA
4 Research Service, Zablocki VA Medical Center, Milwaukee, Wisconsin, USA
5 Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin, USA
6 Department of Medicine, Case Western Reserve University, Division of Cardiology, Cleveland, Ohio, USA
7 Medical Service, Louis Stokes VA Medical Center, Cleveland, Ohio, USA

* Corresponding author. M4280, 8701 Watertown Plank Road, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States. Tel: +1 414 456 5624; fax: +1 414 456 6507. E-mail address: aksc{at}mcw.edu (A.K.S. Camara).

Received 26 March 2007; revised 13 August 2007; accepted 20 August 2007

Time for primary review: 49 days


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 
Aim: Damage to the mitochondrial electron transport chain (ETC) occurs during ischaemia. Blockade of electron flow in the ETC just before ischaemia with the reversible complex I inhibitor amobarbital protects isolated mitochondria against ischaemic damage and preserves oxidative phosphorylation and cytochrome c content. We hypothesized that brief amobarbital perfusion just before ischaemia would improve cardiac recovery and decrease infarct size after ischaemia and reperfusion (IR) by preserving the mitochondrial redox state and reducing mitochondrial superoxide (O2–•) generation, in turn would decrease mitochondrial Ca2+ accumulation (mt[Ca2+]).

Methods: Guinea pig Langendorff-perfused hearts were treated with Krebs Ringer solution (KR; untreated) or amobarbital (2.5 mM) in KR for 1 min immediately before 30 min of no flow, global ischaemia, followed by reperfusion without additional treatment. Cardiac function, mitochondrial NADH, FAD, mt[Ca2+], and O2–• levels were assessed during the 1 min perfusion period and throughout IR.

Results: Amobarbital perfusion alone before ischaemia significantly increased O2–• levels and NADH, without altering FAD, and decreased mt[Ca2+]. During ischaemia, mitochondrial NADH was higher, O2–• levels were lower, and mt[Ca2+] was less elevated in the amobarbital group. On reperfusion O2–• levels and mt[Ca2+] were significantly reduced, NADH-FAD redox state was preserved and cardiac function was markedly improved in the amobarbital group; infarct size was smaller in the amobarbital group compared to the untreated group.

Conclusion: Temporary blockade of mitochondrial complex I activity by amobarbital protects hearts by reducing production of O2–• and mtCa2+ loading during IR injury.

KEYWORDS Mitochondria; Energy metabolism; Free radicals; Ischaemia; Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 
Cardiac mitochondria are an important source of reactive oxygen species (ROS) during ischaemia and reperfusion (IR). ROS contribute to myocardial injury during reperfusion after ischaemia,1 but more recent studies show significant ROS generation occurs also during ischaemia,2 especially during late ischaemia,3,4 when myocardial O2 content is very low.5 ROS increase during 30 min ischaemia3 and irreversible damage occurs at complexes III and IV.6,7 Now that mitochondria are known as a major source of superoxide (O2–•) generation during ischaemia, ROS have been implicated in damage to the electron transport chain (ETC) during ischaemia as well as during reperfusion.8 Ischaemic preconditioning attenuates ROS-induced damage which protects the heart from ischaemic injury in part by blunting the surge in ROS production during late ischaemia.3,9

In mitochondria, complex III is the principal site of O2–• generation during oxidation of complex I substrates.10 Thus, blocking complex I during ischaemia could decrease electron flow to complex III and subsequently reduce O2–• and protect mitochondria from ischaemic injury. Indeed, blocking electron flow from complex I immediately before ischaemia markedly attenuated damage to the ETC as evidenced by decreased ROS production, preserved oxidative phosphorylation, and improved retention of cytochrome c.10–12 These studies focused on protection of mitochondria isolated after ischaemia11 or reperfusion.12

The present study directly monitored effects of amobarbital, a short-acting barbiturate anesthetic that reversibly blocks complex I,12,13 on real-time in situ mitochondrial function in the intact heart in order to evaluate the contribution of mitochondrial-derived cardiac injury during IR. Our aim was to assess dynamic changes in mitochondrial redox state (nicotinamide adenine dinucleotide hydrogen (NADH) and flavin adenine dinucleotide (FAD)), and indicators of mitochondrial damage (O2–•, Ca2+ overload) during and after global ischaemia in intact hearts in the presence or absence of a block in electron transport at complex I during ischaemia. An increase in mitochondrial Ca2+ (mt[Ca2+]), along with excess ROS production, may lead to uncoupling of oxidative phosphorylation or to irreversible mitochondrial damage. We proposed that blockade of complex I with amobarbital during ischaemia in intact hearts would decrease both O2–• production and mt[Ca2+] uptake. This protective effect of amobarbital would be evidenced by a more reduced redox state, better mechanical recovery, and smaller infarct size on reperfusion.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Langendorff heart model
The investigation conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health Publication No. 85-23, Revised 1996). The Medical College of Wisconsin Biomedical Resources Studies Committee approved this study. Guinea pig hearts (n = 59) were isolated and prepared as previously described in detail.3,14–18 Animals were anesthetized with ketamine (50 mg/kg). Following decapitation and thoracotomy, hearts were removed and perfused at 55 mmHg via the aortic root with a Krebs Ringer solution (KR: in mM 138 Na+, 4.5 K+, 1.2 Mg2+, 2.5 Ca2+, 134 Cl–, 15 HCO3–, 1.2 H2PO4–, 11.5 glucose, 2 pyruvate, 16 mannitol, 0.1 probenecid, 0.05 EDTA, and 5 U/L insulin) gassed with 3% CO2, 97% O2 (pH 7.4) at 37°C. A saline-filled balloon and transducer were used to measure left ventricular pressure (LVP). Coronary flow was measured and myocardial O2 extraction was determined.15

2.2 Cardiac mitochondrial measurements
NADH and FAD, mt[Ca2+], or ROS were measured near continuously through the LV free wall using one of four excitation and emission fluorescence spectra3,14–18 in different subsets of hearts. A trifurcated fiberoptic probe (3.8 mm2/bundle) was placed against the LV to excite and record light signals at specific wavelengths using spectrophotofluorometers (SLM Instruments Inc, Urbana, IL; or Photon Technology International, London, Canada). In a subset of hearts, as described earlier,3,16,19 10 µM dihydroethidium (DHE) was loaded for 20 min and the LV free wall was excited at 540 nm, and emitted light was recorded at 590 nm, to measure fluorescence that is primarily a marker of the O2–• radical.4,20 DHE enters cells and is reversibly oxidized by O2–• to a labile product that is similar but not identical to ethidium bromide, and is not dependent on the presence of DNA.20 In other hearts NADH autofluorescence was assessed at 350 nm excitation and 450/390 nm emissions and FAD autofluorescence at 480 nm excitation and 540 nm emission. Alternatively, hearts were loaded with 6 µM indo 1 AM for 30 min and Ca2+ transients were recorded at 350 nm excitation and 390 and 450 nm emissions. After Ca2+ transients were initially observed, hearts were perfused for 15 min with 100 µM MnCl2 to quench the cytosolic indo 1 signal, which permitted measurement of mt[Ca2+].21 mt[Ca2+] was corrected for underlying changes in NADH autofluorescence during IR for each group. Each signal was digitized and recorded at 200 Hz on computers for later analysis using specifically designed computational programs and commercial software.

2.3 Protocol
The study consisted of three groups, untreated (KR perfusion only; n = 20), amobarbital (n = 20), and time control (n = 15). Each group contained subsets of hearts to independently measure and record either NADH plus FAD, mt[Ca2+], or O2–•. All hearts underwent a stabilization period followed by loading and washout of unbound dye to specifically measure either mt[Ca2+] or O2–•. NADH and FAD were assessed under the same protocol as used in the fluorescence dye-loaded hearts. All hearts undergoing ischaemia were perfused for only 1 min immediately before the onset of no flow, global ischaemia with either KR alone (untreated) or KR containing amobarbital 2.5 mM. Amobarbital itself did not alter fluorescence characteristics or spectra of any dye. This concentration of amobarbital was shown to provide optimal protection of myocardial and mitochondrial function (improved oxidative phosphorylation) against IR injury.11 To confirm this we examined the dose dependent effects of amobarbital and its washout on contractility, O2 extraction, and NADH without ischaemia (n = 4).

This protocol was designed to maximize amobarbital treatment effect only during ischaemia and to minimize blockade of complex I during aerobic perfusion before and after ischaemia. After ischaemia each heart was reperfused with KR solution without additional treatment. At the end of each experiment hearts were removed and atria were discarded; ventricles were cut into thin transverse sections of approximately 3 mm thick and immersed in 0.1% 2,3,5-triphenyltetrazolium chloride for measurement of infarct size.14

2.4 Data analysis
Measurements for each group were compared at baseline, during drug treatment, at 21 min of protocol time (15 min ischaemia), 36 min (30 min ischaemia), 41 min (5 min reperfusion), 66 min (30 min reperfusion), 96 min (60 min reperfusion), and 156 min (120 min reperfusion). All data are expressed as mean ± SEM. Values for NADH, FAD and O2–• are expressed in arbitrary fluorescence units (afu) and mt[Ca2+] is given in nM. Between group and within group comparisons were done by two-way analysis of variance to determine significance. Differences between means were considered significant when P < 0.05 (two-tailed).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Funding
 References
 
Baseline functional values were not significantly different among groups. Figure 1A and B show the timeline for systolic–diastolic LVP (developed LVP) and diastolic LVP (diaLVP), respectively, at baseline, before, during and after ischaemia. Amobarbital given just before ischaemia arrested all hearts. Blockade of electron transport during ischaemia with amobarbital resulted in a significantly smaller increase in diaLVP during late ischaemia and reperfusion than in untreated hearts. On early reperfusion (5 min) developed LVP was significantly higher and diaLVP was significantly lower after amobarbital. This also demonstrates that amobarbital was washed out immediately on reperfusion and that protection by amobarbital is attributed primarily to its presence during ischaemia. At late reperfusion, developed LVP and diaLVP approached baseline values in the amobarbital but not in the untreated group.


Figure 1
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  		Figure 1 Left ventricular developed pressure (systolic–diastolic LVP in mmHg; (A)), and diastolic left ventricular pressure (diaLVP in mmHg; (B)) before, during, and after 30 min no flow, global ischaemia for untreated (KR; n = 12) and 2.5 mM amobarbital (n = 12) groups. Time control experiments (n = 10) without concomitant ischaemia are also shown. Arrow indicates 1 min amobarbital perfusion immediately before ischaemia. {dagger}P < 0.05, 1 min perfusion before ischaemia, during ischaemia and reperfusion vs. baseline values; *P < 0.05 amobarbital vs. untreated (KR).

 
Figure 2A and B show changes in contractility (dLVP/dtmax) and relaxation (dLVP/dtmin) at baseline, before, during and after ischaemia. Both dLVP/dtmax and dLVP/dtmin returned to higher levels during reperfusion after amobarbital treatment.


Figure 2
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  		Figure 2 Rates of contractility dLVP/dtmax (A) and relaxation dLVP/dtmin, (B) before, during, and after 30 min no flow, global ischaemia for untreated (KR; n = 12) and amobarbital (n = 12) groups. Time control experiments (n = 10) are also shown. Arrow indicates 1 min amobarbital perfusion immediately before ischaemia. See Figure 1 for statistics.

 
Figure 3A and B show changes in coronary flow and O2 extraction (pAO2–pVO2) at baseline, before, during and after ischaemia. Coronary flow was higher after amobarbital throughout reperfusion. O2 extraction was similar in the two groups during initial reperfusion, but was significantly lower during 5 to 30 min reperfusion in the untreated group. Amobarbital perfusion just before ischaemia decreased O2 extraction by 82% (inset), which confirmed the marked inhibition of electron transport. The immediate return of contractility and O2 extraction on reperfusion again indicated complete washout of amobarbital.


Figure 3
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  		Figure 3 Time course of changes in coronary flow and O2 extraction (pAO2–pVO2), before, during, and after 30 min no flow, global ischaemia for untreated (KR; n = 12) and amobarbital (n = 12) groups. Time control experiments (n = 10) are also shown. Arrow indicates 1 min amobarbital perfusion immediately before ischaemia. The inset details the effect of amobarbital perfusion before ischaemia on O2 extraction; white and hatched bars represent baseline and 1 min of treatment, respectively. See Figure 1 for statistics.

 
Perfusion of a range of amobarbital concentrations (0.5–4 mM) resulted in a dose dependent depression of LVP and %O2 extraction that was rapidly reversed on washout. At 2.5 mM, LVP was 81±2, 0±0 and 78±3 mmHg before, during and after amobarbital) and %O2 extraction was 79.1±3.5%, 10%±2.0%, and 74.3±4.2% before, during, and after amobarbital, respectively.

To assess mitochondrial function along with contractile and metabolic function, NADH and FAD, O2–•, and mt[Ca2+] were monitored in different subsets of hearts undergoing the same protocol as noted for cardiac function. Each of the four baseline fluorescence signals was not different among the groups. Amobarbital administered for 1 min and washed out without ischaemia reversibly increased NADH in a dose dependent manner (Table 1) without altering FAD (data not shown), up to a peak increase in NADH at 2.5–3 mM. Amorbabital also decreased mt[Ca2+] (data not shown) in a dose dependent manner with a peak reduction at 2.5–3 mM. Figure 4A and B show changes in NADH and FAD at baseline, and before, during and after ischaemia. NADH (Figure 4A) increased while FAD (Figure 4B) correspondingly decreased during ischaemia. On reperfusion NADH and FAD returned to baseline levels in the amobarbital group (indicative of a more reduced redox state). In the untreated hearts, NADH and FAD changed more on reperfusion (more oxidized state). The insets of Figure 4A and B show baseline values of NADH (55±1 afu) and FAD (61±1 afu); one min perfusion of amobarbital just before ischaemia increased NADH (64±1 afu) but did not alter FAD (61.5±1.5 afu). This increase in NADH confirmed in situ blockade of complex I by amobarbital, whereas the lack of change in FAD demonstrated that reducing equivalents continued to pass through complex II.


Figure 4
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  		Figure 4 Time course of changes in NADH and FAD autofluorescence before, during, and after 30 min no flow, global ischaemia for untreated (n = 6) and amobarbital (n = 6) groups. Time control experiments (n = 5) without concomitant ischaemia are also shown. Arrow indicates 1 min amobarbital perfusion immediately before ischaemia. The insets display the effect of amobarbital perfusion before ischaemia on NADH and FAD; white and hatched bars represent baseline and 1 min of treatment, respectively. See Figure 1 for statistics.

 


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  		Table 1 Dose-dependent increase in NADH (a.f.u.) before, during one min perfusion with amobarbital, and after 1 min of drug washout

 
O2–• production increased in both groups during early ischaemia (Figure 5A); however, during the last 5 min of ischaemia, O2–• generation did not increase in the amobarbital group compared to the untreated group. Within 5 min of reperfusion O2–• production decreased to baseline in the amobarbital group despite resumption of aerobic metabolism, but remained significantly elevated in the untreated group during the first 20 min of reperfusion. In Figure 5A (inset) baseline O2–• production was not significantly different between groups. Amobarbital perfusion slightly but significantly increased O2–• (3.44±0.06 afu) production over baseline (3.30±0.04 afu). The small increase in O2–• again reflects a direct effect of amobarbital to block complex I. In comparison, O2–• production during early and late ischaemia rose to 3.74±0.14 afu and 4.38±0.16 afu, respectively in the untreated group. Baseline mt[Ca2+] was similar between groups, at 159±6 nM. Amobarbital alone decreased mt[Ca2+] to 128±5 nM (Figure 5B inset). During ischaemia the increase in mt[Ca2+] was smaller in the amobarbital group, especially during late ischaemia (Figure 5B). The greater increase in mt[Ca2+] was associated with increased diaLVP in the untreated group. On reperfusion, mt[Ca2+] returned to baseline in the amobarbital group within 5 min, but remained significantly elevated in the untreated group during the initial 30-40 min of reperfusion.


Figure 5
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  		Figure 5 Time course of changes in superoxide (O2–•) levels before, during, and after 30 min no flow, global ischaemia for untreated (n = 8) and amobarbital (n = 8) groups (A). Time course of changes in mt[Ca2+] before, during, and after 30 min no flow, global ischaemia for untreated (n = 6) and amobarbital (n = 6) groups (B). Time control experiments (n = 5; for each variable) without concomitant ischaemia are also shown. Arrow indicates 1 min amobarbital perfusion immediately before ischaemia. The insets display the effect of amobarbital perfusion before ischaemia on superoxide (O2–•) production and mt[Ca2+]; white and hatched bars represent baseline and 1 min of treatment, respectively. See Figure 1 for statistics.

 
Figure 6 shows ventricular infarct size as a percentage of total ventricular weight after 120 min reperfusion; percent infarction was significantly smaller in the amobarbital group. Time control experiments (no ischaemia) show that cardiac function and fluorescence indicators for O2–•, NADH, FAD, and mt[Ca2+] were stable during continuous perfusion (Figures 1–5).


Figure 6
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  		Figure 6 shows infarct size as a percentage of total ventricular weight measured after 120 min reperfusion. *P < 0.05 amobarbital (n = 20) vs. control (n = 20).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Funding
 References
 
This is the first study to demonstrate in the intact heart that reversible blockade of electron transport at complex I protects against IR injury by direct in situ modulation of mitochondrial bioenergetics. One min perfusion of amobarbital immediately before ischaemia improved recovery of contractility, diastolic relaxation and coronary flow, and reduced infarct size on reperfusion. This improved recovery was associated with a higher mitochondrial redox state, reduced O2–• production, and decreased mtCa2+ accumulation. This unique approach to myocyte protection, complex I inhibition, is different22,23 from a preconditioning (memory) mechanism whereby targeted uncoupling of respiration24,25 and or activation of mitochondrial K+ channels9,26 appear to underlie the mitochondrial protective mechanism.

Direct evidence for blockade of electron flow at complex I during amobarbital treatment was shown by an increase in NADH (dose dependently) without a change in FAD, a small but significant increase in O2–• production, a decrease in mt[Ca2+], and indirectly by the marked decrease in O2 extraction. Blockade of complex I prevents oxidation of NADH, and an increase in NADH with attenuated electron flow can result in enhanced O2–• generation.27 The finding that FAD (an indicator of oxidation through complexes II, III, and IV) did not change with exposure to this concentration of amobarbital, confirms the high specificity for inhibition of complex I in the intact heart. A study in cultured cells also showed that amobarbital reversibly increases NADH content.28

The observed block at complex I is consistent with studies that show amobarbital (less than 3.0 mM) inhibits glutamate respiration without affecting succinate respiration.29 The reduction in mt[Ca2+] may occur in part due to transient blockade of complex I which could lead to a slight mitochondrial depolarization because of reduced proton motive force, since electrons are blocked from entering at complex I.

A major source of mitochondrial O2–• is the ubisemiquinone radical formed during the Q cycle at the Qo site of complex III.30,31 We postulated that blocking complex I during ischaemia would decrease electron flow into complex III, reduce electron leak, and thereby O2–• generation at this site, which would protect the heart during and after ischaemia. Permanent blockade of ETC during ischaemia with rotenone protects the ETC distal to complex I from ischaemic damage.6 Unfortunately, as an irreversible inhibitor, rotenone is not suitable when return of oxidative metabolism is required on reperfusion. Amobarbital, a short acting barbiturate, reversibly blocks complex I at the same site as rotenone,12,13 and protects isolated mitochondria against damage during ischaemia that is carried forward into early reperfusion.6,11 The protective effects of amobarbital observed earlier,6,11,12 would be expected to improve mitochondrial bioenergetics, as well as to reduce cardiac dysfunction and infarct size. The present study strongly supports the notion that ischaemia-induced dysfunction of electron transport6,7,11 is a key mechanism of myocardial IR injury as evidenced by decreased in situ production of ROS, a decrease in mt[Ca2+], and cardiac protection during IR after amobarbital.

ROS can be beneficial or harmful to the cell. Modest increases in ROS act as a signaling stimulus in ischaemic and pharmacologic preconditioning.3,9 In contrast, in pathophysiologic settings, robust production of ROS leads to cytotoxicity, as in IR injury.32,33 In the present study, O2–• generated during the 1 min amobarbital perfusion was evidently not injurious to mitochondria. It is unlikely that this small amount of O2–• generated by amobarbital protected against IR injury through a classical preconditioning mechanism because ischaemia was instituted immediately in the presence of the drug. During sustained ischaemia, amobarbital blockade of complex I prevented the surge in O2–• production observed in the untreated group during late ischaemia. Our study is in line with the observation that blocking electron flow from complex I to complex III during ischaemia reduces ROS generation from mitochondria isolated after reperfusion.12

Overload of mtCa2+ is a key event in developing IR injury. mt[Ca2+] overload after ischaemia can contribute to ETC damage,34,35 mitochondrial membrane depolarization,36 uncoupling of oxidative phosphorylation, and impaired ATP synthesis.37 These events impair the ability of mitochondria to resist Ca2+-induced opening of the mitochondrial permeability transition pore (MPTP).38 A reduced mt[Ca2+] load (Figure 5B) during and after ischaemia would be expected to minimize the probability of MPTP opening and impede onset of cell death.37,39 Reduced mt[Ca2+] in the amobarbital group may also attenuate the ATP hydrolysis induced by reversal of ATP synthase, resulting in better utilization of NADH with improved contractile recovery and reduced infarct size. ROS production can also trigger MPTP opening and impair complex III and IV activities.37 MPTP with subsequent cytochrome c release not only triggers apoptosis but also inhibits respiration, leading to a further increase in O2–•.40 This interdependent relationship of ROS and mt[Ca2+] on mitochondrial ETC and MPTP does not establish if excess ROS production or mtCa2+ overload is the primary deleterious event in ischaemia. It is possible that amobarbital indirectly decreased ROS production during ischaemia by abating the rise in mtCa2+ uptake. On the other hand, ROS can also alter Ca2+ homeostasis. Favero et al.41 showed that H2O2 modifies the thiol residues of the ryanodine receptor and stimulates Ca2+ release in skeletal muscle SR. Nonetheless, excess ROS generation and mt[Ca2+] overload work in concert to impair mitochondrial function and augment cell death. Amobarbital attenuated both the excess production of ROS and mt[Ca2+] overload, probably by targeting the ROS component of this vicious cycle to reduce mitochondrial damage during ischaemia,11 which eventually attenuated the cardiac injury evident on reperfusion. Note the similar increases in ROS and mt[Ca2+] in the treated and untreated groups early in ischaemia are consistent with our previous studies,3,21 which suggests that the increases in these variables during late ischaemia but not early ischaemia contribute to the severity of the ischaemia and reperfusion injury.

We and others showed that as the supply of O2 diminishes during ischaemia, electron flux through the ETC falters, NADH accumulates,17 and oxidative phosphorylation rapidly declines.42–44 In the present study the NADH fluorescence signal likely represents the average NADH per cell and the number of viable cells underlying the fiberoptic probe. The marked and irreversible decline in NADH during late ischaemia and reperfusion in the untreated group could represent greater cell death45 or increased volume of irreversibly oxidized and energy-depleted mitochondria.17 It is noteworthy that in the untreated group the decline in NADH (more oxidized redox state) during late ischaemia occurred simultaneously as O2–• production increased, which is likely a further manifestation of mitochondrial injury. In contrast, after amobarbital, the increase in NADH during ischaemia was sustained and the O2–• level was lower. This suggests a greater number of salvageable cells in the amobarbital group and better preservation of mitochondrial redox state (higher NADH content). During late ischaemia there was a significant increase in mt[Ca2+], and on reperfusion mt[Ca2+] remained significantly elevated over baseline in the untreated group. Reductions in O2–• production and mt[Ca2+] overload during later ischaemia after amobarbital, despite elevated NADH, supports the notion that amobarbital blocks complex I, reduces ROS generation, and reduces the driving force for mtCa2+ loading.

In two related studies, we showed that perfusion with cold KR solution increased ROS generation19,21 and NADH,21 likely due to slowed electron flow from upstream complexes, which may be analogous to blocking electron flow with amobarbital. During and after ischaemia, cardiac function was better preserved in cold perfused hearts than in warm perfused hearts, as supported by less ROS production, a higher redox state (NADH), and less mt[Ca2+] overload.21 These studies provide further evidence that ischaemic injury may be initiated in the mitochondrion due to increased O2–• production from the distal ETC, and that preventing electrons from reaching these sites better preserves respiratory chain activity and attenuates O2–• production. Therefore, hypothermia, in addition to preserving the myocardial content of high-energy phosphates,46,47 may also protect via temperature-sensitive blockade of electron transport during cold ischaemia.21

Cardiac protection mediated by blocking electron transport during ischaemia is also reflected in the improved coronary flow and higher O2 extraction in the amobarbital group on reperfusion. Mitochondrial protection during ischaemia could protect against contracture and swelling of endothelial and vascular smooth muscle cells as a means to maintain normal coronary flow.48 In previous preliminary experiments we showed that forcefully increasing coronary flow to unprotected hearts with compromised contractility does not improve cardiac function (unpublished data). Thus, it is possible that blocking electron transport in endothelial and vascular smooth muscle cells also leads to decreased oxidative injury during IR as a mechanism underlying improved coronary flow. It is interesting that the higher O2–• release on reperfusion was accompanied by less O2 extraction in the untreated group; this also suggests impaired respiration in the absence of protection by amobarbital. Others have reported that increased ROS generation and subsequent mitochondrial and cellular injury during IR can be associated with differences in tissue pO2.49

4.1 Summary, conclusions and limitations
Reversible interruption of mitochondrial oxidative metabolism during ischaemia paradoxically protected the myocardium from mitochondrial-derived ROS production and Ca2+ overload. Inhibition of electron transport during ischaemia minimized O2–• production and mt[Ca2+] overload during late ischaemia and early reperfusion. Blocking electron transport during ischaemia in our related studies11,12 and in this study provides a preemptive intervention against ischaemic damage to mitochondria that allows reperfusion of myocardium in the setting of preserved mitochondrial function.

A pharmacologic approach targeting the mitochondrion has substantial potential to provide a novel mechanism of myocardial protection prior to cardiac surgery or during early onset ischaemia. A rapidly reversible inhibitor of the proximal ETC could be delivered either regionally via coronary arteriotomy during ‘off pump’ coronary artery bypass surgery, or globally to the heart if cardiopulmonary bypass is initiated. Functional mitochondrial integrity, determined by less ROS generation, reduced mt[Ca2+] uptake, and preserved redox state during IR, could then be supported during ischaemia in order to reduce cardiac injury and facilitate contractile recovery.

In future isolated heart studies we will compare amobarbital with other cardiac depressant drugs that protect by mechanisms not related to inhibition of mitochondrial function. In vivo animal studies of amobarbital's protective effects during regional cardiac ischaemia would be important pre-clinically. A potential clinical limitation is that amobarbital-induced attenuation of electron flow as a clinical treatment after acute myocardial infarction may not be feasible.


   Funding
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
References
 
HL 073246-01 NIH (Dr. Camara), 2PO1AG15885 NIH (Drs. Lesnefsky and Chen), National Institutes of Health, Bethesda, MD, 0355608Z (Dr. Stowe), 0425307B (Dr. Chen), AHA Dallas, TX, and Office of Research and Development, Medical Research Service, Department of Veterans Affairs (Drs. Stowe, Chen and Lesnefsky).


   Acknowledgements
 
The authors thank Anita Tredeau and Steve Contney for administrative assistance.

Conflict of interest: none declared.


   Notes
 
{dagger} This work was published in part in abstract form: Aldakkak et al. Circulation 112:S.II,286, 2005. Back

This article was published online by Elsevier on 23 August, 2007. Back


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Funding
 References
 

  1. Turrens JF, Beconi M, Barilla J, Chavez UB, McCord JM. Mitochondrial generation of oxygen radicals during reoxygenation of ischemic tissues. Free Radic Res Commun (1991) 12-13:681–689. Pt 2.
  2. Becker LB, vanden Hoek TL, Shao ZH, Li CQ, Schumacker PT. Generation of superoxide in cardiomyocytes during ischaemia before reperfusion. Am J Physiol (1999) 277:H2240–H2246.[Web of Science][Medline]
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