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Am J Physiol Heart Circ Physiol 290: H434-H440, 2006. First published August 26, 2005; doi:10.1152/ajpheart.00763.2005
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Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

David F. Stowe,1,2,3,4,5 Mohammed Aldakkak,1 Amadou K. S. Camara,1 Matthias L. Riess,1 Andre Heinen,1 Srinivasan G. Varadarajan,1 and Ming-Tao Jiang1

Departments of 1Anesthesiology and 2Physiology, Anesthesiology Research Laboratories, 3Cardiovascular Research Center, The Medical College of Wisconsin, Milwaukee; 4Veterans Affairs Medical Center Research Service, Milwaukee; and 5Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin

Submitted 19 July 2005 ; accepted in final form 19 August 2005


   ABSTRACT
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 ABSTRACT
METHODS
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ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BKCa) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BKCa channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BKCa channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2–·), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BKCa channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2–·, or an antagonist of the BKCa channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2–· generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBKCa channels; 2) NS effects are blocked by BKCa antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.

cell signaling; ischemic biology; oxidant stress; energy metabolism; heart; mitochondria; reactive oxygen species; redox balance

DEPRESSED MITOCHONDRIAL BIOENERGETICS with excess reactive oxygen species (ROS) generation and mitochondrial (m)Ca2+ loading are major factors underlying ischemia-reperfusion (I/R) injury. Prophylactic measures to reduce cardiac I/R injury include ischemic preconditioning (IPC), i.e., brief pulses of I/R before longer ischemia, and pharmacological preconditioning (PPC), i.e., cardiac protection elicited some time after a drug is washed out. PPC is theoretically a better approach because it does not require brief ischemia. A few exogenous drugs, e.g., pinacidil and diazoxide, that induce PPC possibly act on a ATP-sensitive K+ (KATP) channel. It would appear likely that the opening of other mK+ channels would also elicit PPC.

The membranes of vascular smooth muscle, neural, and secretory cells contain large or Big conductance (100–300 pS) K+ channels (BKCa) activated by increased intracellular [Ca2+] and by cell membrane depolarization (4). BKCa channel opening allows cytosolic K+ efflux, which promotes cell membrane repolarization; this, in turn, reduces Ca2+ entry by closing voltage-dependent Ca2+ channels (40, 41, 45). Siemen et al. (35) first demonstrated BKCa channels in the inner mitochondrial membrane (IMM) in a glioma cell line. Xu et al. (42) reported 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619)-induced activation of BKCa channels isolated to IMM of guinea pig cardiac myocytes; they also reported that NS protected against global I/R injury in rabbit isolated hearts. Although they continuously infused NS up to the onset of ischemia, this led to the possibility that drugs that open mBKCa, like mKATP channels, also induce cardiac PPC via a memory effect. More recently, others have shown that NS could precondition isolated mice (39) and rat (7) hearts subjected to global ischemia and in situ dog hearts subjected to regional ischemia (34). NS-induced effects were attenuated or blocked by charybdotoxin and iberiotoxin (34) or by paxilline (PX) (7, 39), an antagonist of BKCa channel opening (32, 42).

The mechanism for PPC-induced protection by BKCa channel opening is not known. We hypothesized that Ca2+-sensitive K+ channel opening within the IMM initiates a mitochondrial protective effect evidenced by attenuated changes in several indexes of mitochondrial function during and after ischemia, as well as improved myocardial contractile and vascular function, and reduced infarct size on reperfusion after ischemia. To examine this, we infused and washed out NS before ischemia and examined specifically the effect of NS on attenuating mitochondrial dysfunction during I/R by near continuous measurement of NADH, ROS (superoxide, O2–·), and m[Ca2+] in isolated hearts. We gave PX to verify its effect to antagonize NS. Because protective effects of putative KATP channel openers can be abolished by ROS scavengers (28), we also bracketed NS with a dismutator of O2–·, Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), to assess whether NS-induced mBKCa opening requires O2–·, presumably generated within the mitochondrial respiratory complex, to initiate PPC.


   METHODS
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Langendorff heart model. The investigation conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, Revised 1996). Approval for this study was obtained from the Medical College of Wisconsin Biomedical Resources Studies Committee. Guinea pig hearts were isolated and prepared as described in detail (2, 6, 17, 18, 29–31) with care to minimize IPC. These were preoxygenation, maintaining respiration after anesthesia with ketamine (50 mg/kg) and immediately perfusing the aortic root with cold solution. Hearts were instrumented with a saline-filled balloon and transducer to measure left ventricular (LV) pressure (LVP) and an aortic flow probe to measure coronary flow (CF). Heart rate (HR) and rhythm were measured via atrial and ventricular electrodes. Hearts were perfused in Langendorff mode at 55 mmHg with modified Krebs-Ringer solution at 37°C. HR and rhythm, myocardial function (isovolumetric LVP), and CF were measured continuously. At 120-min reperfusion, hearts were stained with 2,3,5-triphenyltetrazolium chloride, and infarct size was determined as a percentage of ventricular heart weight (2).

Cardiac measurements. Either NADH, m[Ca2+], or ROS (primarily O2–·) were measured nearly continuously from the LV using one of three excitation and emission fluorescence spectra (2, 6, 17, 18, 29, 31) 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 Amico-Bowman and Photon Technology International). NADH autofluorescence was assessed at 350 nm excitation and 450/390 nm emission wavelengths. Alternatively, hearts were loaded with 6 µM indo 1 AM (load, 30 min; washout, 20 min), and Ca2+ transients were recorded by using the excitation wavelength for NADH with emissions recorded at 390 and 450 nm. After the Ca2+ transients were initially recorded, hearts were perfused for 15 min with 100 µM MnCl2 to quench cytosolic Ca2+ to reveal primarily m[Ca2+] (29). In other hearts, as described earlier (6, 17, 18), dihydroethidium (10 µM, DHE) was loaded for 20 min and washed out for 20 min. The LV wall was excited at 540 nm, and emitted light was recorded at 590 nm to measure a labile fluorescence signal that is primarily a marker of the free radical O2–· (37, 44). DHE enters cells and is oxidized by O2–·, which converts it to a reversible ethidium-like compound that causes a red-shift in the electromagnetic light spectrum (44).

Myocardial fluorescence intensity was recorded in arbitrary fluorescence units over 12 s each minute at a 100 µs/s sampling rate throughout experiments for DHE and over 2.5 s at a 100 µs/s sampling rate during 35 discrete sampling periods throughout each experiment for NADH and m[Ca2+]. For each fluorescence study, none of the drugs alone had any appreciable effect on autofluorescence. m[Ca2+] was corrected for underlying changes in NADH autofluorescence during I/R for each group. Each signal was digitized and recorded at 200 Hz (Power LAB/16sp, Chart and Scope 3.6.3, ADInstruments) on G4 Macintosh computers for later analysis with the use of specifically designed programs (MATLAB, MathWorks and Microsoft Excel) software. All variables were averaged over the 2.5- or 12-s sampling period.

Protocol. Hearts were infused with 3 µM NS for 15 min, ending 25 min before the onset of 30 min of global ischemia. In some hearts, NS was bracketed with 1 µM PX, a blocker of the BKCa channel (32), or 20 µM TB, a dismutator of O2–·. PX or TB was given for 5 min before starting NS, during NS, and for 5 min after stopping NS. Reperfusion lasted 120 min. Additional studies (not shown) demonstrated that PX or TB given alone as a pretreatment, i.e., without NS, compared with drug-free controls had no effect on any of the variables measured.

Statistical analyses. A total of 126 experiments with isolated hearts were divided into 6 groups: control, NS, NS+PX, NS+TB, PX, and TB. NADH was measured in 6–8 hearts/group, ROS in 6–8 hearts/group, and m[Ca2+] in 6–8 hearts/group. LVP and coronary flow were measured in all hearts, so that there were 21 hearts/group for functional data. Infarct size was measured in a blinded manner in 7 hearts of each group. All data are means ± SE. Appropriate comparisons were made among groups that differed by a variable at a given condition or time and within a group over time compared with the initial control data. Statistical differences were measured across groups at specific time points (20, 50, 80, 100, 130, and 240 min). Differences among variables were determined by two-way multiple ANOVA for repeated measures (Statview and CLR ANOVA software; Macintosh computers). If F tests were significant, appropriate post hoc tests (Student-Newman-Keuls or Duncan) were used to compare means. Mean values were considered significant at P (two-tailed) < 0.05.


   RESULTS
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Spontaneous HR averaged 244 ± 12 beats/min before ischemia for all groups; this was not statistically different at 120 min reperfusion for all groups (data not shown). Each of the figures shows the marked degree of dysfunction or damage in the untreated control group during and after global ischemia and the beneficial effect of PPC elicited by NS treatment before ischemia. Figure 1A shows that diastolic LVP rose above baseline only slightly during initial reperfusion in the NS group; in all other groups, diastolic LVP was markedly elevated during late ischemia and throughout reperfusion. Figure 1B shows that developed (systolic and diastolic) LVP was improved most on reperfusion in the NS group compared with all other groups. Figure 2A similarly shows that CF was highest in the NS group on reperfusion. PX or TB was perfused for 25 min and washed out before ischemia. Changes in diastolic LVP, developed LVP, and CF were not statistically different in the PX- and TB-alone groups than those from the control group (not shown). These data indicate that NS induces PPC as evidenced by improved myocardial function and that this can be blocked by either PX or TB.



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  		Fig. 1. Effects of 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS or NS-1619) and antagonists on diastolic left ventricular (LV; A) and systolic-diastolic (developed) LV pressure (B). Effects of NS on improving these functional variables during ischemia and reperfusion were abolished by either Big conductance Ca2+-sensitive K+ (BKCa) channel blocker paxilline (PX) or O2–· dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP). PX or TB alone had effects no different from those of drug-free conrols (Con) (data not shown). *P < 0.05 vs. Con, NS+PX, NS+TB; {dagger}P < 0.05 vs. Con.

 


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  		Fig. 2. Effects of NS and antagonists on coronary flow (A) and on mitochondrial [Ca2+] (B). Effect of NS to increase flow on reperfusion was abolished, and effect to reduce mitochondrial [Ca2+] was largely reversed by either BKCa channel blocker PX or O2–· dismutase mimetic TB. PX or TB alone had effects no different from those of drug-free Con (data not shown). *P < 0.05 vs. Con, NS+PX, NS+TB.

 
Figure 2B shows indo 1 fluorescence indicative of m[Ca2+] in four of the six groups during I/R. m[Ca2+] remained lower throughout ischemia and during early reperfusion in the NS group compared with all other groups. In the PX and TB groups (not shown), m[Ca2+] was not significantly different at any time from the control group. This indicates that NS markedly reduced mCa2+ loading both during I/R, an effect that was blocked by either PX or TB. Figure 3, A and B, shows DHE fluorescence, indicating ROS (O2–·) and NADH autofluorescence in four of the six groups before, during, and after ischemia. There were no detectable changes in these values by the drugs given and terminated before ischemia. PX and TB were given and washed out before ischemia. There were no significant differences among control, PX, and TB groups throughout the protocol, so the latter two groups are again not shown for clarity. ROS increased less during I/R in the NS group compared with all other groups and returned to baseline on reperfusion only in the NS group. Concurrently, NADH increased less during early ischemia and decreased less during late reperfusion in the NS group compared with other groups. This indicates better preservation of mitochondrial redox potential and less production of O2–· during I/R after PPC with NS. Bracketing NS with PX or TB antagonized these changes in ROS induced by NS alone. This indicates that both PX and TB greatly reversed the beneficial effect of NS to reduce ROS levels during I/R. Figure 4 shows that percent ventricular infarct size was lowest in the NS-alone group and was not different among the other five groups. This indicates less permanent cardiac cell damage after PPC with NS.



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  		Fig. 3. Effects of NS and antagonists on dihydroethidium (DHE) (O2–·; A) and NADH fluorescence (B). Effects of NS on attenuating changes O2–· and NADH during ischemia and reperfusion were largely reversed by either BKCa channel blocker PX or O2–· dismutase mimetic TB. PX or TB alone had effects no different from those of drug-free Con (data not shown). afu, arbitrary fluorescence units. *P < 0.05 vs. Con, NS+PX, NS+TB; {dagger}P < 0.05 vs. Con.

 


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  		Fig. 4. Effect of NS on ventricular infarct size. Infarct-lowering effect of NS was reversed by either BKCa channel blocker PX or O2–· dismutase mimetic TB. *P < 0.05 vs. Con, NS+PX, PX, TB.

 

   DISCUSSION
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Our results show that NS effectively preconditions in situ cardiac mitochondria as evidenced in intact hearts by improved indexes of mitochondrial function, not only during reperfusion but also during ischemia. This was accompanied by markedly improved mechanical function and flow and reduced infarct size. Importantly, O2–· was required to initiate preconditioning by NS, which suggests a protective effect by NS specifically on BKCa channels located in cardiac cell mitochondria. These conclusions are based on our findings that the protective effects of brief treatments with NS were almost completely reversed by bracketing NS with the O2–· dismutator TB or the BKCa blocker PX. Our study supports the work of others (7, 34, 39) who show that mBKCa channel opening, like mKATP channel opening, can induce PPC. Moreover, the present study suggests a common-link, i.e., K+ influx into the mitochondrial matrix with altered mitochondrial function and obligatory O2–· generation in the protective mechanism. Overall, these results demonstrate the marked cardioprotective effects after preconditioning with NS and strongly implicate mBKCa channel opening and generation of O2–·, or its products or reactants, as initiators of mitochondrial as well as myocardial cell preconditioning.

The improvement in mitochondrial function by NS was evidenced by a reduced level of O2–·, reduced m[Ca2+], and improved redox state (more normal NADH) during I/R. Each protective effect of NS was blocked by TB; this suggests that initial formation of O2–· is required to trigger mBKCa channel activation. Similar O2–·-requiring effects have been observed for the putative mKATP channel opener diazoxide and for volatile anesthetics (17, 18, 28). Although the mitochondrial preconditioning effect of NS appears to require mBKCa channel opening and enhanced formation of O2–·, apparently these factors do not mediate preconditioning because the drugs are washed out before ischemia. Triggering of the memory effect by ROS is possibly mediated via downstream cytosolic phosphorylation and dephosphorylation pathways, with a feedback effect on mitochondrial receptors regulating mitochondrial bioenergetics. It also follows that mitochondrial preconditioning could be initiated by matrix K+ influx via any one or more mK+ channels.

NS infusion with or without PX or TB before ischemia did not demonstrate any detectable effects on myocardial function or mitochondrial indexes in our isolated heart model. This may be due to undetectable changes in mitochondrial redox state, m[Ca2+], and ROS levels induced by the low concentration of NS that we used. Higher concentrations of NS depressed contractility. However, our preliminary investigations in isolated mitochondria from guinea pig myocytes showed that NS-enhanced state 2 and state 4 respiration in a concentration-dependent manner and that low NS concentrations (5–30 µM), while not inducing any depolarization of the IMM, markedly increased the H2O2 release rate; these effects were fully reversible by PX (15, 15a). Similar effects could be elicited by low concentrations of valinomycin, a K+ ionophore, or by increasing buffer Ca2+. Together, these data strongly support the existence of cardiac-cell mBKCa channels and furnish a mechanistic link between mBKCa channel opening and ROS-induced initiation of mitochondrial and myocardial preconditioning.

Changes in mitochondrial redox state, ROS, and Ca2+ with I/R injury.

There is much evidence that ROS play an important role in triggering cardioprotection, but how or which ROS initiate or mediate mBKCa opening is unknown. O2–· bursts occur during reperfusion when excess [O2] is supplied, but O2–· is also formed in low O2 states, i.e., during ischemia, in isolated hearts (17, 18, 29), and during simulated ischemia in isolated cardiomyocytes when electron flow through the respiratory chain is impeded (5). The putative mKATP channel opener diazoxide (13) mimicked IPC on reducing infarct size, and ROS scavengers N-acetylcysteine (11) and N-mercapto-propionyl-glycine (28) blocked the preconditioning effect of diazoxide; this suggested that mKATP channel opening causes ROS formation (22). A mild ROS stress is believed to trigger protection by activating protein kinases (8). But ROS have also been shown to activate the sarcolemmal KATP channel by modulating KATP channel binding sites because this effect is blocked by glibenclamide or by ROS scavengers (36). Those studies (21) suggested that ROS produced during IPC may afford cardioprotection on reperfusion directly or via a feed-forward mechanism for KATP channel-induced ROS production.

The cause and effect relationship and timing of mCa2+ loading, and changes in mitochondrial membrane potential ({Delta}{psi}m), NADH, and ROS during cardiac I/R injury are not clear (3). However, studies in the intact heart model from Stowe's laboratory (17, 18, 24, 25, 29, 31) show a close association of redox changes, ROS produced, and mCa2+ influx during I/R. NADH and ROS were markedly changed not only during reperfusion but also during ischemia. Ischemia-induced increases in NADH (31), ROS (18), and cytosolic [Ca2+] (2) and m[Ca2+] (30) returned closer to normal values on reperfusion after PPC. These effects were reversed by ROS scavengers or by blocking sarcolemmal KATP and/or mKATP opening with glibenclamide or 5-hydroxydecanoate (17, 26, 30, 31). Preconditioning also led to reduced ROS generation and improved ATP synthesis in isolated mitochondria (25). All these studies suggest that PPC is largely mediated by mK+ channel opening and that improved mitochondrial bioenergetics during ischemia is early evidence of improved myocardial function and reduced tissue damage on reperfusion.

Ca2+-sensitive K+ channels and role of ROS.

KCa channels are widely distributed on surface membranes in smooth muscle and neurons where they modulate action potential repolarization to regulate arterial tone and neurotransmitter release, respectively. They are traditonally classified into three groups (small, intermediate, and large), based on their conductance levels. Pharmacological manipulation of these channels alters redox potential in smooth muscle (40). The BKCa channel is sensitive to charybdotoxin and is activated by both voltage and Ca2+ (27, 35). PKC appears to regulate activity of these channels (45).

Sieman et al. (35) first documented BKCa channels in IMM of glial cells. Xu et al. (42) recently reported BKCa channels are present in mitoplasts prepared from cardiac myocytes and suggested that opening of mBKCa channels to enhance matrix K+ influx is an important factor in mitigating I/R injury in a manner similar to mKATP channels. These investigators proposed further that the function of mBKCa channels might be to improve efficiency of mitochondrial energy production. They showed that activation of the mBKCa channel with 30 µM NS, which was given up at the onset of ischemia, reduced infarct size by half in rabbit hearts; these effects were similar to mKATP activation by diazoxide (42). They also identified a 55-kDa BK segment from the IMM of guinea pig heart and a 80-kDa BK-{alpha} subunit from liver IMM; both molecular weight sizes are much smaller than other previously reported BKCa weights, i.e., ~120 kDa. mBKCa channels appear to have properties similar to those of BKCa channels in the sarcolemma of vascular smooth muscle cells (42).

Xu et al. (42) first showed a link between mBKCa channel opening and protection against cardiac I/R injury. Others (7, 34, 39) demonstrated recently that NS could induce a cardiac preconditioning state. We observed additionally that putative mBKCa channel opening leads to a mitochondrial preconditioned state as shown by better preservation of mitochondrial function not only during reperfusion but also during ischemia. We also showed that ROS are involved. However, it has been unclear how the opening of mK+ channels alters mitochondrial energetics and functions to mediate myocardial preconditioning. In theory, the opening of any mK+ channel might act as an energy-dissipating channel that subsequently leads to preserved mitochondrial function and reduced I/R injury. For example, it was shown recently that activation of BKCa channels led to decreased {Delta}{psi}m in isolated pulmonary (20) and coronary artery (43) smooth muscle cells. Moreover, Sato et al. (33) recently reported that NS reversibly increased flavoprotein oxidation of cardiac myocytes in a concentration-dependent fashion, which indicates a direct effect of NS on mitochondrial electron transport chain activity. However, we could not detect any change in tissue NADH fluorescence during NS exposure in the isolated heart. The reason for this discrepancy may be the fact that Sato et al. (33) used substrate-depleted myocytes to minimize the production of electron donors and so to amplify the small flavoprotein signal due to the opening of mKCa and/or mKATP channels. Furthermore, they demonstrated that NS induced a slight depolarization of the IMM and attenuated a ouabain-induced increase in m[Ca2+]; all these effects were blocked by PX. Thus the present findings and those above suggest a direct effect of mBKCa channel opening on mitochondrial bioenergetics.

Analogous to the mBKCa channel, it is similarly unclear how mKATP channel opening might lead to cardiac preconditioning (1, 9, 19). Several proposals about mKATP channel opening may help to explain how mBKCa channel opening could mediate PPC. mKATP opening may largely depolarize {Delta}{psi}m to cause uncoupling with accelerated respiration (1, 16). Subsequently, ischemia would then reduce the driving force for Ca2+ influx through the Ca2+ uniporter; this would attenuate mCa2+ overload (10, 23) so that energized mitochondria on reperfusion would perform more efficiently. The putative mKATP opener diazoxide was reported to reduce matrix Ca2+ uptake by depolarizing {Delta}{psi}m and so to decrease the driving force for mCa2+ entry (1, 16). Others (12, 13, 19), however, propose that the physiological role of mKATP channels is the control of mitochondrial matrix volume rather than dissipation of {Delta}{psi}m and uncoupling. It was postulated that matrix swelling by K+ uptake is caused by concomitant uptake of anions (mostly Cl–) and water by osmotic forces. Concomitant mK+/H+ exchange would then only partially dissipate the proton gradient ({Delta}µH) by increasing matrix acidity and so only slightly decrease {Delta}{psi}m. In turn, mitochondrial matrix swelling to counteract matrix contraction during ischemia might optimize mitochondrial function, because partial uncoupling was seen to improve efficiency of oxidative phosphorylation (14). In either scenerio, mBKCa opening, like mKATP opening, may suppress mCa2+ overload and ROS production and thereby improve mitochondrial function during I/R injury.

From this background and a companion study from Stowe's laboratory (15, 15a), we propose that mBKCa opening increases matrix K+, which in turn increases matrix H+ by K+/H+ exchange; this stabilizes the {Delta}{psi}m in the face of increased respiration, conditions that can enhance the generation of O2–· (38). Although an increase in respiration would tend to depolarize {Delta}{psi}m and retard O2–· generation at mitochondrial respiratory complexes, it is possible that K+/H+ exchange stabilizes {Delta}{psi}m so that excess electrons in the respiratory chain can leak out to combine with O2 to enhance generation of mO2–· and other ROS, which then initiates the cytosolic phosphorylation cascade that mediates PC. Evidence that the protective effect of NS is initially induced by O2–· is indicated by reversal of the protective effect of NS in the presence of TB, which rapidly dismutates O2–· to H2O2; TB alone did not lead to preconditioning. ROS-like O2–· or ·OH, or nonradical reactants like H2O2 or ONOO– (in the presence or absence of NO·) may actually trigger the preconditioning effects, but an excess O2–· appears necessary to initiate the response. Opening of mBKCa channels, like mKATP channels, or any factor that enhances matrix K+ flux (e.g., valinomycin, low m[ATP], or increased m[Ca2+]), could act similarly to modulate ROS levels.

Summary and limitations.

Mitochondrial BKCa opening by NS given only before ischemia initiates a mitochondrial and myocardial preconditioning effect as shown by smaller changes in the redox state (NADH), decreased ROS formation, and reduced mCa2+ loading during ischemia, as well as by the reduced level of ROS, restored NADH level, reduced mCa2+ loading, improved metabolic and functional improvements, and reduced infarct size on reperfusion. These protective effects were effectively blocked by dismutation of O2–· with TB.

PX blocked protection by NS, so this suggests that NS exerts its primary effect on BKCa channels. However, our study does not allow a firm conclusion that NS acts solely or primarily on mBKCa channels. For example, an effect of NS to open sarcolemmal BKCa channels in coronary vascular smooth muscle might lead to the improved coronary flow on reperfusion. Evidence that NS actually opens the mBKCa channel to elicit preconditioning is also dependent on the specificity of PX to block this channel; the effects of NS, however, are not blocked by 5-hydroxydecanoate, a mKATP channel blocker (7). Because mitochondria-induced ROS is required for preconditioning, this suggests the channel exists in mitochondria. However, in the isolated heart, we could not detect any change in mitochondrial bioenergetics or ROS generation during the administration of the drug. Finally, it is likely that some factors that induce preconditioning, like ROS or Ca2+ loading, are the same factors that cause I/R damage. There are likely feed-forward or feed-backward mechanisms between mBKCa channel opening and ROS that may be difficult to separate. Cytosolic mediators also likely play a role in mitochondrial preconditioning. The individual stages of triggering, activation, and end effect must be well delineated in future studies to divulge a comprehensive mechanism for preconditioning.


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This study was supported by grants from the National Institutes of Health RO3-AG-022171 (to D. F. Stowe and M.T. Jiang) and KO1-HL-73246 (to A. K. S. Camara and D. F. Stowe); the Foundation for Anesthesia Education and Research (to S. G. Varadarajan and D. F. Stowe); and the American Heart Association 035568Z (to D. F. Stowe).


   ACKNOWLEDGMENTS
 
The authors thank the following for assistance with these studies: Anita Tredeau, Jim Heisner, Leo Kevin, Samhita S. Rhodes, Richard Carlson Jr., and Steve Contney. This work has been published in part in abstract form: Jiang MT et al., Biophys J 86, Suppl: 465A, 2004; Stowe DF et al., FASEB J 18: 670.4, 2004; and Stowe DF et al., FASEB J 19: 386.26, 2005.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. F. Stowe, M4280, 8701 Watertown Plank Rd., Medical College of Wisconsin, Milwaukee, WI 53226 (e-mail: dfstowe{at}mcw.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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