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Am J Physiol Heart Circ Physiol 282: H1395-H1403, 2002; doi:10.1152/ajpheart.00683.2001
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Vol. 282, Issue 4, H1395-H1403, April 2002

H2O2 opens mitochondrial KATP channels and inhibits GABA receptors via protein kinase C-epsilon in cardiomyocytes

Hong Yan Zhang, Bradley C. McPherson, Huiping Liu, Timir S. Baman, Peter Rock, and Zhenhai Yao

Department of Anesthesiology, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxygen radicals and protein kinase C (PKC) mediate ischemic preconditioning. Using a cultured chick embryonic cardiomyocyte model of hypoxia and reoxygenation, we found that the oxygen radicals generated by ischemic preconditioning were H2O2. Like preconditioning, H2O2 selectively activated the epsilon -isoform of PKC in the particulate compartment and increased cell viability after 1 h of hypoxia and 3 h of reoxygenation. The glutathione peroxidase ebselen (converting H2O2 to H2O) and the superoxide dismutase inhibitor diethyldithiocarbamic acid abolished the increased H2O2 and the protection of preconditioning. PKC activation with phorbol 12-myristate 13-acetate increased cell survival; the protection of preconditioning was blocked by epsilon V1-2, a selective PKC-epsilon antagonist. Similar to preconditioning, the protection of PKC activation was abolished by mitochondrial KATP channel blockade with 5-hydroxydecanoate or by GABA receptor stimulation with midazolam or diazepam. In addition, PKC, mitochondrial ATP-sensitive K+ (KATP) channels, and GABA receptors had no effects on H2O2 generated by ischemic preconditioning before prolonged hypoxia and reoxygenation. We conclude that H2O2 opens mitochondrial KATP channels and inhibits GABA receptors via activating PKC-epsilon . Through this signal transduction, preconditioning protects ischemic cardiomyocytes.

gamma -aminobutyric acid receptors; preconditioning


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

OXYGEN RADICALS are important intracellular second messengers that mediate cardioprotection (7, 9). Preconditioning and flumazenil generate oxygen radicals and protect cardiomyocytes during ischemia-reperfusion (42). Flumazenil inhibits the GABA receptor complex (20). The GABA receptor antagonist is used clinically for reversal of apnea and loss of consciousness associated with oversedation (2) and might be beneficial in protecting against cerebral ischemia (20). Rapid administration of a GABA complex antagonist produces minimal coronary and left ventricular hemodynamic responses (24). GABA receptor antagonists might have a role in the management of patients with ischemic heart disease if they can mimic preconditioning to protect the heart and thus have cardiac protective properties. The first objective of our study was to examine the role of GABA receptors in the development of preconditioning protection.

GABA receptors have been identified in mitochondria (2, 19, 27, 28). Reactive oxygen species originating from mitochondria, ATP-sensitive K+ (KATP) channels, and protein kinase C (PKC) are second messengers in cardioprotection produced by hypoxic preconditioning, acetylcholine, opioids, and flumazenil (25, 31, 34, 37, 38). A second objective of this study was to examine the role of GABA receptors on preconditioning-induced oxygen radicals before the start of ischemia and to determine the sequence of GABA receptors, PKC, and oxygen radicals in the signal transduction pathway of ischemic preconditioning.

Cardiocyte injury in ischemia-reperfusion models correlates with the amount of free radicals produced (33), especially during the first few minutes of reperfusion (33, 43). Free radicals contribute to myocardial stunning, and the role of these radicals in lethal cell injury is unequivocal (16). Vanden Hoek et al. (33) demonstrated that in cultured cardiomyocytes, hypoxic preconditioning attenuated oxidant stress at reoxygenation. We hypothesize that ischemic preconditioning attenuates oxidant stress at hypoxia and reoxygenation and that GABA receptors play a role in development of this oxidant stress.

Finally, we wanted to determine the nature of oxygen radicals and which isoform of PKC is important in mediating cardioprotection of ischemic preconditioning. The findings of this study demonstrate that in isolated cultured ventricular myocytes, hydrogen peroxide/hydroxyl radicals inhibit mitochondrial GABA receptors by activating the PKC-epsilon isoform. Through this signal transduction pathway, ischemic preconditioning protects cardiocytes during hypoxia and reoxygenation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cardiomyocyte preparation. Ventricular myocytes from 10-day-old chick embryos were prepared according to a method described previously (3). Briefly, hearts were harvested and placed in Hanks' balanced salt solution lacking magnesium and calcium (Life Technologies; Grand Island, NY). Ventricles were minced, and myocytes were dissociated by use of trypsin degradation (0.025%, Life Technologies) repeated four to six times at 37°C with gentle agitation. Isolated cells were then transferred to a solution with trypsin inhibitor for 8 min, filtered through a 100-µm mesh filter, centrifuged for 5 min at 1,200 rpm at 4°C, and finally resuspended in a nutritive medium described previously (25). Resuspended cells were placed in a petri dish in a humidified incubator (5% CO2-95% air at 37°C) for 45 min to promote early adherence of fibroblasts. Nonadherent cells were counted with a hemocytometer, and viability was measured with trypan blue (0.4%). Approximately 1 × 106 cells in nutritive medium were pipetted onto coverslips (25 mm) and incubated for 3-4 days, after which synchronous contractions of the monolayer were noted. Experiments were performed on spontaneously contracting cells at day 3 or 4 after isolation.

Perfusion system. Glass coverslips containing spontaneously beating chick myocytes were placed in a stainless steel, 1-ml, flow-through chamber (Penn Century; Philadelphia, PA). The chamber was sealed with Kynar film (McMaster-Carr; Elmhurst, IL) placed between the coverslip and the metal hypoxic chamber to minimize oxygen exchange between the chamber wall and the perfusate and then mounted on a temperature-controlled platform (37°C) on an inverted microscope. A water-jacketed glass equilibration column mounted above the microscope stage was used to equilibrate the perfusate to known oxygen tensions (PO2). The standard perfusion medium was equilibrated for 1 h before the experiment by bubbling through it a gas mixture of 21% O2-5% CO2-74% N2. A hypoxia solution, composed of balanced salt solution (BSS) containing no glucose with 2-deoxyglucose (20 mmol/l) added to inhibit glycolysis, was bubbled with a gas mixture of 20% CO2 and 80% N2 for 1 h before the experiments. The pH of the perfusion solution was routinely verified [normoxic buffered salt solution (BSS) 7.4, simulated ischemia BSS 6.8]. Stainless steel or low oxygen solubility polymer tubing connected the equilibration column to the flow-through chamber to minimize ambient oxygen transfer into the perfusate. Hypoxic chamber PO2 was routinely monitored by Oxyspot (Medical Systems; Greenvale, NY) under conditions identical to those experiments using an optical phosphorescence quenching method (35).

Cell viability. An inverted microscope, equipped for epifluorescent illumination, included a xenon light source (75 W), a 12-bit digital, cooled CCD camera (Princeton Instruments), a shutter and filter wheel (Sutter), and appropriate excitation and emission filter cubes. The microscope was also equipped with Hoffman-modified phase illumination to accentuate surface topology and facilitate the measurement of contractile motion. Fluorescent cell images were obtained using a magnification of ×10 (Nikon Plan Fluor). Data were acquired and analyzed with Metamorph software (Universal Imaging). Cell viability was quantified with the nuclear stain propidium iodide (PI, 5 µM) (Molecular Probe; Eugene, OR), an exclusion fluorescent dye that binds to chromatin upon loss of membrane integrity (1, 4). PI is not toxic to cells over 8 h, permitting its addition to the perfusate throughout the experiments. At the completion of each experiment, digitonin (300 µM) was added to the perfusate for 1 h. Digitonin disrupts membrane integrity of all cells allowing a PI to enter all cells and thus permitting determination of the maximal value of PI (when all cells' membranes have been disrupted). Loss of viability (cell death) was then expressed as a percentage of the maximum value of PI after 1 h of digitonin exposure (100%).

Measurement of oxygen radicals. Oxygen radicals were assessed using the probe 2,7-dichlorofluorescin (DCFH). The membrane-permeable diacetate form of the dye DCFH (DCFH-DA) was added to the perfusate at a final concentration of 5 µM. Within the cell, esterases cleave the acetate groups on DCFH-DA, thus trapping the reduced probe (DCFH) intracellularly. Intracellular oxygen radicals lead to the oxidation of DCFH, yielding the fluorescent product DCF. The probe DCFH in cardiomyocytes is readily oxidized by H2O2 or hydroxyl radical but is relatively insensitive to superoxide. Fluorescence was measured using an excitation wavelength of 480 nm, dichroic 505-nm long pass, and emitter bandpass of 535 nm (Chroma Technology) with neutral density filters to attenuate the excitation light intensity. Fluorescence intensity was assessed in clusters of several cells identified as regions of interest. Background fluorescence was identified as an area without cells or with minimal cellular fluorescence. Intensity values are reported as percentage of initial values after the background value was subtracted.

PKC enzyme assay. Enzyme activity of total PKC, PKC-epsilon , and PKC-delta isoforms was measured by a method described previously (17, 26). Briefly, 5 million cells for each experiment were collected in a sample buffer containing 50 mmol/l Tris · HCl (pH 7.5), 5 mmol/l EDTA, 10 mmol/l EGTA, 10 mmol/l benzamidine, 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.3% beta -mercaptoethanol (Sigma; St. Louis, MO). The collection was centrifuged at 45,000 g for 30 min and separated into a cytosolic fraction and particulate fraction. The particulate pellet was dissolved ultrasonically in the sample buffer. Protein concentration was determined with the Bradford method (5). Each fraction of 50-100 µg was assayed for total PKC, PKC-epsilon , and PKC-delta activity using a kit (Amersham Pharmacia; Piscataway, NJ). For PKC-epsilon and PKC-delta assays, the protein was immunoprecipitated overnight by PKC-epsilon and PKC-delta monoclonal antibody (BD Transduction Lab) in immunoprecipitation buffer (pH 7.4) (150 mM NaCl, 50 mM Tris, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 16 µg/ml benzamidine-HCl, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A; Sigma) with protein A/G beads (Santa Cruz Biotechnologies). In addition, PKC-epsilon - or delta -specific substrate (ERMRPRKRQGSVRRRV) (BioMol; Plymouth Meeting, PA) was used for phosphorylation reaction with [32P]ATP (Amersham Pharmacia; Piscataway, NJ).

Chemicals. Midazolam was purchased from Roche Pharma (Humacao, PR), and diazepam was purchased from Elkins-Sinn (Cherry Hill, NJ). PKC-epsilon antagonist (epsilon V1-2) was purchased from Calbiochem-Novabiochem (San Diego, CA). Phorbol 12-myristate 13-acetate (PMA), 5-hydroxydecanoate (5-HD), ebselen, and diethyldithiocarbamic acid (DDC) were purchased from Sigma. All agents were dissolved in BSS buffer before administration. PI and DCFH-DA were purchased from Molecular Probes (Eugene, OR). Rottlerin was purchased from BioMol (Plymouth Meeting, PA) and dissolved in a 1:5 cocktail of ethanol-saline.

Doses of PMA, midazolam, diazepam, epsilon V1-2, 5-HD, ebselen, and DDC were chosen on the basis of our previous studies (37, 42) and preliminary results in which these agents were shown to almost completely abolish the beneficial effects of ischemic preconditioning.

Statistical analysis. Data are expressed as means ± SE. Differences between groups for cell death and oxygen radical production were compared using a two-factor analysis of variance (ANOVA) with repeated measures and Fisher's least significant difference test. Differences between groups were considered significant when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of preconditioning on cell death, contraction, and oxidant stress. In this model of hypoxia and reoxygenation, preconditioning with 10 min of hypoxia markedly reduced cell death. The pattern and extent of cell death were similar to that previously reported (25, 42). After 3 h of reoxygenation, cell death was 58.2 ± 7.3% in controls (n = 8) and 15.8 ± 2.3% in preconditioned cells (n = 8, P < 0.05) (Fig. 1B). At the end of 3 h of reoxygenation, visible contractile activity was noticed in 14 of 16 preconditioned cells (87.5%) and 4 of 18 in controls (22.2%, P < 0.05). Oxidant stress during hypoxia and reoxygenation was lower in preconditioned cells (Fig. 1A). Therefore, preconditioning markedly attenuated oxidant stress during hypoxia and reoxygenation and conferred protection from cell death and contractile dysfunction.


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  		Fig. 1.   A: effects of different protocols on 2,7-dichlorofuorescin (DCFH) oxidation, an index of oxygen radical generation. In control cells (n = 10), intensity of 2,7'-dichlorofluorescein (DCF) fluorescence increased slightly over 1 h (open circles). Preconditioning (PC) with 10 min of hypoxia and 10 min of reoxygenation produced oxygen radicals before the prolonged ischemia (n = 8). During prolonged hypoxia and reoxygenation, oxygen radical production was markedly lower in PC-treated cells compared with controls. GABA receptor agonist midazolam (100 µmol/l), given during baseline period, did not affect the increased oxygen radicals produced by PC before hypoxia (n = 7). B: PC reduced cardiocyte death from hypoxia and reoxygenation. Midazolam (100 µmol/l) alone had no effects on cell death and oxidant stress, but it abolished protection (n = 6) and attenuated oxidant stress of PC at ischemia-reperfusion (filled triangles in A, n = 7). A lower dose of midazolam (10 µmol/l) had no effects on cell death and oxidant stress by itself, either on the protection or attenuated oxidant stress of PC. PI, propidum iodide. * P < 0.05 compared with corresponding point in controls.

GABA receptors, PKC, and mitochondrial KATP channels. The effects of preconditioning on reduced cell death and attenuated oxidant stress at hypoxia and reoxygenation were lost with the presence of GABA receptor agonists midazolam (100 µmol/l, 51.0 ± 6.5%, n = 6) or diazepam (100 µmol/l, 57.1 ± 8.1%, n = 7) versus controls (58.2 ± 7.3%, n = 8, P > 0.05). In contrast, a lower dose of midazolam or diazepam (10 µmol/l) had no effect on these beneficial effects of preconditioning. Midazolam or diazepam at a concentration of 100 µmol/l had no effect on cell death and oxidant stress compared with controls. Loss of GABA receptor activity is important in the cardioprotection of ischemic preconditioning.

The beneficial effects of ischemic preconditioning on reduced cell death and oxidant stress were lost in the presence of the specific PKC-epsilon inhibitor (18) epsilon V1-2 at 10 µmol/l (52.3 ± 3.4%, n = 8) or the selective mitochondrial KATP channel blocker 5-HD at 100 µmol/l (47.6 ± 6.4%, n = 7 vs. 58.2 ± 7.3%, n = 8 in controls, P > 0.05). epsilon V1-2 (10 µmol/l) and 5-HD (100 µmol/l) alone had no effects on cell death compared with ischemic controls (Figs. 3 and 4). Furthermore, rottlerin (3 µmol/l), which selectively antagonizes PKC-delta at the dose <3 µmol/l, did not block the protection of preconditioning in our model.


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  		Fig. 2.   GABA receptor agonist diazepam (100 µmol/l) was given during the baseline period. It had no effects on cell death (B, n = 4) or on oxygen radical production (n = 3) by itself. It did not affect the increased oxygen radicals produced by PC before hypoxia (A, n = 6). However, it abolished protection (B, n = 7) and attenuated oxidant stress of PC at prolonged hypoxia and reoxygenation (A, n = 6). A lower dose of diazepam (10 µmol/l) had no effect on cell death and oxidant stress by itself, either on the protection or attenuated oxidant stress of PC. *P < 0.05 compared with corresponding point in controls.



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  		Fig. 3.   Specific protein kinase C-epsilon inhibitor epsilon V1-2 (10 µmol/l), administered during baseline period, had no effects on cell death (B, n = 4) or on oxygen radical production (n = 4) by itself. It did not affect the increased oxygen radicals produced by PC before hypoxia (A, n = 6). However, it abolished protection (B, n = 7) and attenuated oxidant stress of PC at prolonged hypoxia and reoxygenation (A, n = 6). *P < 0.05 compared with corresponding point in controls.



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  		Fig. 4.   Selective mitochondrial ATP-sensitive K+ (KATP) channel blocker 5-hydroxydecanoate (5-HD, 100 µmol/l), administered during baseline period, had no effects on cell death (B, n = 4) and on oxygen radical production (n = 4) by itself. It did not affect the increased oxygen radicals produced by PC before hypoxia (A, n = 6). However, it abolished protection (B, n = 7) and attenuated oxidant stress of PC at prolonged hypoxia and reoxygenation (A, n = 6). *P < 0.05 compared with corresponding point in controls.

Preconditioning generates H2O2, which regulates GABA receptors and mitochondrial KATP channels through PKC-epsilon isoform. Preconditioning increased DCFH oxidation, an index of oxygen radical production, before prolonged hypoxia (Figs. 1A-4A). These results were consistent with our previous finding (42). These oxygen radicals and cardioprotection were abolished by DDC, an inhibitor for superoxide dismutase (catalyzing O2 to H2O2) or ebselen, a glutathione peroxidase that converts H2O2 to H2O (Fig. 5). Like preconditioning, H2O2 (5 µmol/l) also increased DCFH oxidation and reduced cardiocyte death (Fig. 5).


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  		Fig. 5.   A: H2O2 (5 µmol/l) was infused for 10 min instead of 10 min of PC ischemia. PC and H2O2 markedly increased DCFH oxidation to DCF (1,106 ± 69, n = 8 and 1,890 ± 233, n = 5, respectively) compared with baseline controls (Control, 251 ± 23, n = 6, arbitrary units). PC-produced oxygen radicals were abolished by ebselen, a glutathione peroxidase that converts H2O2 to H2O. The precursor of H2O2 is superoxide (O2·). Superoxide dismutase (SOD) is an enzyme in the cytosol that catalyzes the conversion of O2· to H2O2. Diethyldithiocarbamic acid (DDC), a cytosol Cu,Zn-SOD inhibitor, attenuated oxygen radical production of PC. These data indicate that oxygen radicals produced by PC are mainly H2O2 radicals and that SOD and glutathione peroxidase have important roles in their generation and degradation. B: protection of PC was abolished by DDC (DDC + PC, n = 8) or ebselen (ebselen + PC, n = 6). H2O2 (5 µmol/l) mimicked PC to reduce cardiocyte death. *P < 0.05.

Midazolam, diazepam, epsilon V1-2, or 5-HD had no effect on the oxygen radicals generated by preconditioning (Fig. 1A-4A). In addition, a higher dose of 5-HD (1 mmol/l, n = 5) had no effect on preconditioning-generated oxygen radicals. These results suggest that ischemic preconditioning-induced oxygen radicals are not PKC, mitochondrial KATP channels, or GABA receptor mediated.

Preconditioning and H2O2 (5 µmol/l) markedly increased the enzyme activity of the PKC-epsilon isoform in the particulate fraction but had no effects on the enzyme activity of total PKC and PKC-delta isoform compared with controls. In the cytosolic fraction, no difference was observed in enzyme activity of total PKC, -delta , or -epsilon isoforms (Fig. 6).


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  		Fig. 6.   Preconditioning and H2O2 (5 µmol/l) selectively increased enzyme activity of the PKC-epsilon isoform in the particulate fraction but had no effect on enzyme activity of total PKC or the delta -isoform compared with controls (B). Cytosol enzyme activity of total PKC, the epsilon -isoform, and the delta -isoform were not different among control, preconditioned, and H2O2-treated cardiocytes (A). *P < 0.05.

Similar to preconditioning, PKC activation with PMA (0.2 µmol/l) markedly reduced cardiocyte death (16.3 ± 4.5%, n = 6) compared with controls (42.7 ± 5.5%, n = 6, P < 0.05) (Fig. 7). Midazolam (100 µmol/l) or the selective mitochondrial KATP channel antagonist 5-HD (100 µmol/l), which were given during the prolonged hypoxia and reoxygenation period, had no effect on cell death by itself but it abolished the protection of PMA (Fig. 7).


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  		Fig. 7.   Effects of different protocols on cell death expressed as a percentage of PI uptake. Treatment with phorbol 12-myristate 13-acetate (PMA, 0.2 µM) during hypoxia and reoxygenation reduced cell death. Treatment with midazolam (MIDA; 100 µmol/l) or 5-HD (100 µmol/l), a selective mitochondrial KATP channel antagonist, abolished the protective effect of PMA (PMA + MIDA, PMA + 5-HD). Midazolam or 5-HD was infused during hypoxia and reoxygenation. *P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In isolated cardiomyocytes, preconditioning increased cell survival and improved recovery of spontaneous contraction after hypoxia and reoxygenation. Large amounts of free radicals, assessed by DCFH oxidation, were detected during hypoxia and reoxygenation. Preconditioning attenuated this oxidant stress. GABA receptor agonists midazolam or diazepam abolished protection and restored oxidant stress. Thus preconditioning protects cardiocytes by attenuating oxidant stress through a GABA receptor-related mechanism.

GABA receptors and ischemic preconditioning. Ischemic preconditioning reduced cardiomyocyte death from hypoxia and reoxygenation. These results are consistent with our previous findings (38, 42) and those of others (21, 34). The GABA receptor antagonist flumazenil and preconditioning produced a similar protection (37, 42). Kochs et al. (20) showed that flumazenil protected against cerebral ischemic damage. The protection of preconditioning was abolished by GABA receptor stimulation with midazolam or diazepam. These observations suggest that loss of GABA receptor activity contributes to the development of preconditioning.

Hypoxia and reoxygenation generated a large amount of oxygen radicals that were attenuated by preconditioning (Fig. 1). Preconditioning decreases superoxide levels. Elevated endogenous manganese-superoxide dismutase (SOD) activity and increased SOD expression were found in rat cardiomyocytes 24 h after preconditioning ischemia (41). However, results of Turrens and co-workers (32) showed no changes of SOD, catalase, or glutathione peroxidase activities in preconditioned hearts. Controversy still exists regarding the role of endogenous antioxidant enzymes in preconditioning. Preconditioning preserves mitochondrial function in rat hearts and mitochondria from preconditioned hearts generate less superoxide radicals (36). Studies also suggest nonmitochondrial sources of free radicals are important in reperfusion injury (18). Antioxidants given during reperfusion protect cardiocytes to a similar degree as preconditioning (25, 33). The attenuated oxidant stress during the simulated ischemic period by preconditioning could slow down the depletion of endogenous antioxidants of cardiocytes, which would preserve the cells' ability to reduce oxidant stress at reoxygenation and result in increased survival. Thus a greater level of endogenous antioxidants as a result of preconditioning could be as important a mechanism of preconditioning as the reduced oxidant stress at reoxygenation for the cardioprotection of preconditioning. Free radicals, generated during both hypoxia and reoxygenation, contribute to the pathogenesis of cardiocyte damage. The mechanism by which free radicals damage cardiocytes is not established. The burst of free radical production at reoxygenation is transient and only lasts 15 min in our system. However, cell death constantly increases over the whole reoxygenation period. Free radicals, superoxide in particular, induce apoptosis (40). Therefore, apoptosis may play a role in progressive cell death during reoxygenation.

GABA receptor stimulation with midazolam or diazepam abolished preconditioning and restored oxidant stress. These data indicate that loss of GABA receptor activity is important in preconditioning protection (reduced oxidant stress and cell death). The mechanism by which preconditioning inhibits GABA receptors hence attenuating oxidant stress is not established. GABA receptor complexes have been identified in mitochondria and have important function in regulating ion channels (2, 19), although the relationship of these ion channels to mitochondrial KATP channels needs to be defined. The dose of midazolam and diazepam used in this study is higher than expected, which suggest the GABA blockade with these compounds is at an intracellular site (such as mitochondria) rather than at the sarcolemmal membrane. Mitochondrial KATP channel opening attenuates oxidant stress at reoxygenation (33). In this study, blockade of mitochondrial KATP channels with 5-HD abolished the effects of preconditioning. Recently, Zhang and Yao (42) have showed that 5-HD did not affect GABA receptor antagonist flumazenil-generated oxygen radicals but abolished the protection of flumazenil. This study suggested that mitochondrial KATP channel opening was a downstream event of oxygen radicals and GABA complex. It seems reasonable to conclude that preconditioning leads to loss of mitochondrial GABA complex activity with resultant opening of mitochondrial KATP channels.

Role of GABA receptors on oxygen radicals before prolonged hypoxia. Biological oxidants initiate intracellular signal transduction (25, 31). Preconditioning generated oxygen radicals before the prolonged hypoxia. Others (9, 34) have also reported that hypoxic preconditioning increased oxygen radicals in a similar myocyte model. Oxygen radicals are second messengers of preconditioning (7). Oxygen radicals also mediate the cardioprotection induced by transient hypoxia, acetylcholine, flumazenil, and opioids (9, 25, 34, 37).

GABA receptors, although not established in chick cardiomyocytes, have been found in mitochondria (2, 19, 28). Inhibition of these receptors affects various ion channels (19), has antistress activity (28), and regulates neurosteroidgenesis (27). Surprisingly, the GABA receptor agonist midazolam or diazepam had no effects on oxygen radicals produced by preconditioning before prolonged ischemia. Therefore, loss of GABA receptor activity seems to be a downstream event of oxygen radicals.

The oxygen radicals generated by preconditioning are mainly H2O2 because DCFH is more readily oxidized by H2O2 and hydroxyl radicals than by superoxide radicals (8, 35). In addition, the radicals were abolished by ebselen, a glutathione peroxidase that converts H2O2 to H2O (8). The precursor of H2O2 is superoxide (O2·). SOD is an enzyme in the cytosol that catalyzes this reaction. DDC, a cytosol Cu,Zn-SOD inhibitor, attenuated the production of oxygen radicals. Thus preconditioning-generated oxygen radicals are H2O2 present in cytosol. Oxygen radicals generated by hypoxic preconditioning originate from mitochondria (34). Likely, free electrons (radicals) generated in mitochondria were transported to cytosol and converted to H2O2. More importantly, ebselen or DDC, which abolished preconditioning-generated H2O2 before prolonged hypoxia, blocked the beneficial effects of preconditioning. Thus H2O2 is important in mediating preconditioning. Yoshida and co-workers (39) showed that overexpression of glutathione peroxidase in mice increased myocardial tolerance to ischemia-reperfusion injury. Overexpression of glutathione peroxidase in mice results in increased antioxidant levels thus reducing free radical-related cardiac injury during ischemia-reperfusion. In the present study, ebselen was only given transiently before the prolonged hypoxia to prevent preconditioning-generated oxygen radicals and their downstream signal transduction. Ebselen was not present during prolonged hypoxia and reoxygenation and thus it had no antioxidant effects.

How preconditioning produces H2O2 is not clear. A previous study by Vanden Hoek and colleagues (34) suggested that preconditioning generated superoxide inside mitochondria. Superoxide migrates into cytosol via anion channels. In cytosol, Cu,Zn-SOD converts superoxide to H2O2 and hydroxyl radicals.

H2O2 activates the epsilon -isoform of PKC and mitochondrial KATP channels. Preconditioning and H2O2 selectively activated the epsilon -isoform of PKC in the particulate fraction without changing its activity in cytosol (Fig. 8). Activity of total PKC and its delta -isoform in both compartments was not affected by preconditioning nor H2O2. The protective effect of preconditioning was abolished by a specific PKC-epsilon inhibitor epsilon V1-2 but not by the selective PKC-delta antagonist rottlerin (3 µmol/l). In a recent study from our laboratory, Zhang et al. (23) showed that 1 µmol/l rottlerin abolished the protection of opioid (23). Numerous studies (13, 14, 30) support the hypothesis that PKC is a central mediator of preconditioning. In intact rats, inhibition of PKC could partially or completely abolish the effects of preconditioning, depending on the strength of preconditioning stimulus (14). Specific isoforms of PKC are important, and translocation to mitochondrial membrane of specific isoforms of PKC (delta  and epsilon ) is responsible for preconditioning in rats and rabbits (17, 26). The fact that rottlerin (3 µmol/l) did not significantly affect the protection of preconditioning suggests that PKC-epsilon is the key isoform in mediating preconditioning in chick embryonic cardiomyocytes.


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  		Fig. 8.   Signal transduction pathway of ischemic preconditioning.

PKC enhances the ability of diazoxide to open mitochondrial KATP channels in rabbit cardiac myocytes (29). Preconditioning protection was abolished by the selective mitochondrial KATP channel blocker 5-HD (100 µmol/l) (12). This dose or a higher dose (1 mmol/l) of 5-HD did not affect the increased levels of H2O2/hydroxyl radicals before hypoxia. Our previous results showed that 1 mmol/l of 5-HD did not affect flumazenil-produced oxygen radicals (37, 42). It seems that opened mitochondrial KATP channels serves as a downstream event of H2O2 in this model. It has been recently shown that in adult perfused rat hearts, mitochondrial KATP channel activator diazoxide (50 µmol/l) increased DCFH oxidation and that this effect of diazoxide was blocked by 5-HD (100 µmol/l) (10). This discrepancy could be species or age related (adult rat cardiomyocytes versus embryonic chick cardiomyocytes). Nevertheless, mitochondrial KATP channel opening mediates the protection associated with preconditioning, acetylcholine, opioids, and adenosine in vivo and in vitro (21-22, 25, 38). PKC and mitochondrial KATP are linked in mediating preconditioning (11, 30). Liang (21) demonstrated that PKC activation is a component of signal transduction of preconditioning and adenosine. The protection of PKC activation with PMA was blocked by 5-HD (Fig. 7). Thus activation of the channels are involved in downstream signal transduction of PKC-epsilon to mediate the protection of preconditioning.

The present results and our previous study (37, 42) suggest activation of mitochondrial KATP channels is a downstream event of oxygen radicals and PKC-epsilon in mediating cardioprotection of hypoxic preconditioning and flumazenil. However, Forbes et al. (10) recently demonstrated that diazoxide, a selective mitochondrial KATP channel opener, generates oxygen radicals and protects ischemic rat hearts. Acetylcholine, bradykinin, phenylephrine, and opioids generate oxygen radicals that were blocked by selective mitochondrial KATP channel antagonist 5-HD (6, 25, 38). These data indicate that the mitochondrial KATP channel is upstream of oxygen radicals in mediating cardioprotection of these agents. Cohen et al. (6) found that adenosine-induced cardioprotection was independent of oxygen radicals. Taken together, although various pharmacological agents, hypoxic and ischemic preconditioning, all can protect hearts against ischemia-reperfusion injury, they protect cardiomyocytes through different intracellular signal transduction pathways.

In conclusion, in isolated chick cardiomyocytes, preconditioning protection is associated with an attenuated oxidant stress. H2O2 produced by transient preconditioning ischemia results in loss of mitochondrial GABA receptor activity and opening of mitochondrial KATP channels via activation of PKC-epsilon isoform. Preconditioning exerts its protection during hypoxia and reoxygenation through this signal transduction pathway (Fig. 8).


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-03881, HL-70324, and HL-70325.


    FOOTNOTES

10.1152/ajpheart.00683.2001

Address for reprint requests and other correspondence: Z. Yao, Dept. of Anesthesiology, Univ. of North Carolina at Chapel Hill, 223 Burnett-Womack Bldg., CB7010, Chapel Hill, NC 27599-7010 (E-mail: zyao{at}aims.unc.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.

Received 1 August 2001; accepted in final form 20 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 282(4):H1395-H1403
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