Intramitochondrial signaling: interactions among mitoK_ATP, PKCɛ, ROS, and MPT

Abstract

Activation of protein kinase Cɛ (PKCɛ), opening of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP), and increased mitochondrial reactive oxygen species (ROS) are key events in the signaling that underlies cardioprotection. We showed previously that mitoK_ATP is opened by activation of a mitochondrial PKCɛ, designated PKCɛ1, that is closely associated with mitoK_ATP. mitoK_ATP opening then causes an increase in ROS production by complex I of the respiratory chain. This ROS activates a second pool of PKCɛ, designated PKCɛ2, which inhibits the mitochondrial permeability transition (MPT). In the present study, we measured mitoK_ATP-dependent changes in mitochondrial matrix volume to further investigate the relationships among PKCɛ, mitoK_ATP, ROS, and MPT. We present evidence that 1) mitoK_ATP can be opened by H₂O₂ and nitric oxide (NO) and that these effects are mediated by PKCɛ1 and not by direct actions on mitoK_ATP, 2) superoxide has no effect on mitoK_ATP opening, 3) exogenous H₂O₂ or NO also inhibits MPT opening, and both compounds do so independently of mitoK_ATP activity via activation of PKCɛ2, 4) mitoK_ATP opening induced by PKG, phorbol ester, or diazoxide is not mediated by ROS, and 5) mitoK_ATP-generated ROS activates PKCɛ1 and induces phosphorylation-dependent mitoK_ATP opening in vitro and in vivo. Thus mitoK_ATP-dependent mitoK_ATP opening constitutes a positive feedback loop capable of maintaining the channel open after the stimulus is no longer present. This feedback pathway may be responsible for the lasting protective effect of preconditioning, colloquially known as the memory effect.

it is now generally accepted that mitochondria are both essential effectors of cardioprotection and primary targets of cardioprotective signaling. It is widely agreed both that opening of the mitochondrial permeability transition (MPT) is responsible for the pathogenesis of necrotic cell death following ischemia-reperfusion (9, 13) and that opening of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) is essential for cardioprotection (21). The conditions that obtain during reperfusion after prolonged ischemia—high levels of calcium, phosphate, and reactive oxygen species (ROS)—cause opening of the MPT (32). Thus mechanisms of cardioprotection must target mitochondria in such a way as to ensure that MPT opening is inhibited. This notion is supported by the recent findings that mitochondria isolated from preconditioned (2) or postconditioned (3) hearts are more resistant to induction of MPT.

Progress has been made in understanding the intramitochondrial signaling pathway that leads to MPT inhibition and the central roles played in this process by mitoK_ATP, PKCɛ, and ROS. Most signals reach mitochondria from the cytosol, and bradykinin triggers the protected phenotype by activating guanylyl cyclase to produce cGMP, which then activates a cGMP-dependent protein kinase (PKG) (44). PKG was shown to be the last cytosolic step in the signaling pathway by the demonstration that PKG opened mitoK_ATP in isolated mitochondria to the same extent as cromakalim and diazoxide given at concentrations to yield a V_max response (6). PKG interaction with mitochondria causes the signal to be transmitted to a PKCɛ (PKCɛ1) bound to the mitochondrial inner membrane (MIM), which in turn phosphorylates mitoK_ATP and causes it to open (6, 24). The resulting increase in K⁺ influx with attendant matrix alkalinization causes increased ROS production by complex I of the respiratory chain (1). This increase in ROS then activates a second inner membrane PKCɛ (PKCɛ2), which inhibits MPT (7).

A number of questions remain regarding the interactions among PKCɛ, mitoK_ATP, ROS, and MPT. In the present study, we show that H₂O₂, the PKCɛ-activating peptide ψɛRACK (receptor for activated C kinase), and PMA cause mitoK_ATP opening through a PKCɛ that is bound to the inner membrane and that this mitoK_ATP opening depends on phosphorylation. We show for the first time that nitric oxide (NO) and H₂O₂ open mitoK_ATP indirectly, through their activation of PKCɛ, and do not act on mitoK_ATP directly. We find that PKG reacts with and phosphorylates an unknown mitochondrial outer membrane (MOM) protein and that an intact MOM is necessary for transmission of the signal from the cytosolic surface of the MOM to PKCɛ on the inner membrane. Exogenous NO and H₂O₂ are also able to inhibit MPT through their activation of a second PKCɛ, and this occurs independently of mitoK_ATP. Superoxide anion was found not to open mitoK_ATP, and superoxide-dependent mitoK_ATP opening is shown to be due to superoxide dismutation to H₂O₂. Finally, we demonstrate mitoK_ATP-dependent mitoK_ATP opening, which occurs via an increase in mitoK_ATP-dependent ROS, ROS activation of PKCɛ, and persistent, phosphorylation-dependent mitoK_ATP opening. The latter finding provides evidence for the first time of a positive feedback loop within mitochondria that may be responsible for the lasting (memory) effect of preconditioning (15, 47).

METHODS

Langendorff-perfused hearts.

Male Sprague-Dawley rats (200–220 g) were briefly anesthetized with carbon dioxide and immediately decapitated. Hearts were rapidly excised, submerged in iced Krebs buffer, and perfused by an aortic cannula delivering normothermic (37°C) modified Krebs-Henseleit solution containing (in mM) 118 NaCl, 5.9 KCl, 1.75 CaCl₂, 1.2 mM MgSO₄, 0.5 EDTA, 25 NaHCO₃, and 16.7 glucose at pH 7.4. The perfusate was gassed with 95% O₂-5% CO₂, which results in a Po₂ >600 mmHg at the level of the aortic cannula. Hearts were allowed to stabilize for 25 min, after which diazoxide (30 μM) was perfused for 15 min, followed by a 10-min wash. Hearts were then collected for isolation of mitochondria. Control perfusions were for 50 min without interruption (sham-perfused hearts). The experimental protocols used in these studies were performed in compliance with the American Physiological Society's “Guiding Principles in the Care and Use of Animals” and were approved by the Institutional Animal Care and Use Committee at Portland State University.

Preparation of mitochondria and mitoplasts.

Heart mitochondria were isolated by differential centrifugation and further purified in a 26% self-generating Percoll gradient exactly as previously described (6, 8). Mitoplasts were prepared from isolated mitochondria by digitonin treatment (58). Protein phosphatase inhibitors were added to all mitochondrial preparations from Langendorff-perfused hearts.

Matrix volume measurements.

Respiration-driven influx of K⁺, with accompanying anions and water, causes swelling of the mitochondrial matrix (20). Within well-defined limits, changes in matrix volume are linearly related to the reciprocal of the absorbance of the suspension (1/A), corrected for the extrapolated value at infinite protein concentration (1/A∞) (4): V = a + b(1/A − 1/A∞). The conversion parameters a and b are estimated to be −0.1026 and 0.5855, respectively. The absolute values of these parameters are unimportant when normalized differences are considered. Thus data in Figs. 2–8 are summarized as “volume change (%)”, given by 100 × [V(x) − V(ATP)]/[V(0) − V(ATP)], where V(x) is the observed steady-state volume at 120 s under the given experimental condition and V(ATP) and V(0) are observed values in the presence and absence of ATP, respectively. The assay medium composition was (in mM) 120 KCl, 10 HEPES pH 7.2, 10 succinate, 5 inorganic phosphate, 0.5 MgCl₂, and 0.1 EGTA, supplemented with 1 μM rotenone and 0.67 μg/ml oligomycin. Light-scattering changes were followed at 520 nm and 30°C.

Fig. 2.

H₂O₂-induced mitoK_ATP opening is blocked by PKCɛ inhibitors. Shown are the effects of several compounds on mitoK_ATP opening induced by H₂O₂ plotted as volume change (%). Indicated additions to the assay were ATP (0.2 mM), H₂O₂ (2 μM), 5-HD (0.3 mM), glibenclamide (glib, 10 μM), Ro-318220 (Ro, 0.5 μM), ɛV_1-2 (0.5 μM), PKCɛ scrambled peptide (scr, 1 μM), δV_1-1 (0.2 μM), genistein (gen, 5 μM), and cyclosporin A (CsA, 1 μM). Data are means ± SD of at least 4 independent experiments.

Fig. 8.

Phosphorylation-dependent persistence of mitoK_ATP opening in diazoxide-perfused hearts. Shown are the effects of sham perfusion or Dzx perfusion on mitoK_ATP activity in mitoplasts isolated from perfused rat hearts. mitoK_ATP activity is plotted as volume change (%). Indicated additions to the assay were ATP (0.2 mM), Dzx (30 μM), and PP2A (11 ng/ml). Data are means ± SD of at least 3 independent experiments.

It should be noted that mitoK_ATP-dependent K⁺ flux has been validated by five independent measurements: light scattering, direct measurements of K⁺ flux, H⁺ flux, respiration, and H₂O₂ production. Each of these was found to yield quantitatively identical measures of K⁺ flux when calibrated with the K⁺ ionophore valinomycin. In each of these assays, the effect of 30 μM diazoxide, to yield a V_max response, matched the effect of 1 pmol valinomycin/mg protein (1, 8). Moreover, other pharmacological openers at concentrations to yield a V_max response, including cromakalim (8, 21, 22, 25, 52), bimakalim (52), nicorandil (52, 57), pinacidil (38), BMS195095 (23), bepridil (56), sevoflurane (53), as well as activators of PKCɛ (Refs. 7, 24 and present study) yielded results comparable with those of diazoxide.

MPT was assayed exactly as described by Costa et al. (7). MPT opening was synchronized by sequential additions at 20-s intervals of CaCl₂ (100 μM free Ca²⁺), ruthenium red (0.5 μM, to block further Ca²⁺ uptake), and CCCP (250 nM, to synchronize MPT opening; Ref. 50). Mitochondria (0.1 mg/ml) were added to assay medium in the presence of ATP (50 μM). Rates of matrix volume change were obtained by taking the linear term of a second-order polynomial fit of the light scattering trace, calculated over the initial 2 min after MPT induction by CCCP. MPT inhibition was calculated by taking the Ca²⁺-induced swelling rates in the presence and absence of 1 μM cyclosporin A (CsA) as 100% and 0%, respectively.

H₂O₂ production.

Hydrogen peroxide production was measured by deesterified 2,7-dichlorofluorescein diacetate (DCF-DA) or Amplex Red, exactly as described previously (1).

Chemicals.

Protein kinase G isoform 1α, cGMP, (±)-S-nitroso-N-acetylpenicillamine (SNAP), KT-5823 and the PKCɛ scrambled peptide (negative control for ɛV_1-2) were from Calbiochem (San Diego, CA). PKCɛ-specific peptides antagonist ɛV_1-2 (EAVSLKPT) or agonist ψɛRACK (HDAPIGYD) and PKCδ-specific peptide antagonist δV_1-1 (SFNSYELGSL) were synthesized with a purity >98% by EZBiolab (Westfield, IN) according to published amino acid sequences (14, 27). All other chemicals were from Sigma (St Louis, MO). The PKG1α concentration (25 ng/ml, corresponding to 1.5 × 10⁻¹⁰ M) and activity used in this study were comparable with those used in our previous study and with the concentration present in cells (see Ref. 6 and references therein).

Data analysis.

All data were analyzed by unpaired Student's t-test. P values <0.05 were considered significant.

RESULTS

PKCɛ-dependent regulation of mitoK_ATP in isolated heart mitochondria.

The experiments in Figs. 1 and 2 were designed to confirm that activation of PKCɛ opens mitoK_ATP. Figure 1 contains light scattering traces from heart mitochondria respiring in K⁺ medium. The addition of the PKCɛ activator peptide ψɛRACK or H₂O₂ to mitochondria incubated in the presence of ATP caused identical increases in steady-state matrix volume to an extent comparable to that obtained in the absence of ATP or in the presence of ATP + K_ATP channel opener (not shown). The addition of either 5-hydroxydecanoate (5-HD) or the PKCɛ inhibitor peptide ɛV_1-2 inhibited mitoK_ATP-dependent K⁺ influx and prevented the increase in matrix volume. It should be noted that ψɛRACK and ɛV_1-2 are small peptides (mol wt 888.5 and 845.5, respectively) that readily diffuse across the MOM. In experiments not shown, we estimated that the EC₅₀ for H₂O₂-induced mitoK_ATP opening was 0.4 μM (±0.1 μM; Hill coefficient = 1) and the EC₅₀ for the specific PKCɛ peptide agonist ψɛRACK was 72 nM (±30 nM; Hill coefficient = 1).

Fig. 1.

PKCɛ-mediated mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP) opening. Changes in mitochondrial matrix volume (V) are plotted vs. time. Rat heart mitochondria (0.1 mg/ml) were suspended in assay medium described in methods. H₂O₂ (2 μM) or ψɛ receptor for activated C kinase (RACK) (0.5 μM) was added to medium in the presence of ATP (0.2 mM) ∼1 s after the mitochondria. Other additions to the assay medium were 5-hydroxydecanoate (5-HD, 0.3 mM) and ɛV_1-2 (0.5 μM). Traces are representative of at least 5 independent experiments.

To confirm that the effects of H₂O₂ and ψɛRACK were specific for PKCɛ and mitoK_ATP, we investigated their effects in the presence of various regulators of PKC and mitoK_ATP and also in the presence of the MPT inhibitor CsA. The results obtained from five or more independent experiments are summarized in Fig. 2. H₂O₂-dependent mitoK_ATP opening was inhibited by the mitoK_ATP blockers 5-HD and glibenclamide and by the PKCɛ blockers ɛV_1-2 and Ro-318220. A scrambled peptide with the same amino acid composition as ɛV_1-2 was without effect. CsA had no effect on steady-state volume in the presence of H₂O₂, indicating that its effects were not caused by opening of MPT. Other tested compounds that had no effect on H₂O₂-dependent mitoK_ATP opening included the PKCδ inhibitors δV_1-1 (mol wt 1,117.8), Gö-6983 (not shown), and the tyrosine kinase inhibitor genistein. Identical results were obtained with the same agents when mitoK_ATP was opened by ψɛRACK or PMA (data not shown).

Superoxide does not open mitoK_ATP.

Superoxide anion has been suggested to induce mitoK_ATP opening in cardiomyocytes and perfused hearts (11, 34, 39, 43, 46, 61). Superoxide was generated with hypoxanthine plus xanthine oxidase (XOx). As shown in Fig. 3, 60 mU of XOx opened mitoK_ATP, but this effect was blocked by catalase, indicating that H₂O₂ from spontaneous dismutation of superoxide is the species responsible for the observed effect. We then lowered the XOx concentration to 6 mU, which was not effective in opening mitoK_ATP. However, 6 mU of XOx plus 30 U of superoxide dismutase (SOD), to convert the superoxide anion to H₂O₂, did cause mitoK_ATP opening, and this effect was abolished by subsequent addition of catalase, to remove H₂O₂. The effects of hypoxanthine + XOx + SOD on mitoK_ATP were inhibited by 5-HD or ɛV_1-2 (Fig. 3).

Fig. 3.

H₂O₂, but not superoxide, opens mitoK_ATP via PKCɛ. Shown are the effects of xanthine oxidase (XOx) + hypoxanthine on mitoK_ATP activity, plotted as volume change (%). Rat heart mitochondria (0.1 mg/ml) were suspended in assay medium containing ATP (0.2 mM) and hypoxanthine (hypo, 0.2 μM). XOx (6 or 60 U/ml) was added to medium ∼1 s after mitochondria. As indicated, medium also contained catalase (cata, 10 U/ml), superoxide dismutase (SOD, 30 U), 5-HD (0.3 mM), and ɛV_1-2 (0.5 μM). Data are means ± SD of at least 4 independent experiments.

In experiments not shown, we measured H₂O₂ production under these experimental conditions, using Amplex Red or deesterified carboxy-DCF (1) in the absence of mitochondria. Addition of 60 mU of XOx produced ∼30 times more H₂O₂ by spontaneous dismutation than addition of 6 mU, which led to barely detectable levels of H₂O₂. Addition of SOD to medium containing 6 mU of XOx resulted in a 60 ± 10% increase in H₂O₂ detected by both fluorescent probes, and addition of catalase resulted in barely detectable levels of H₂O₂. 5-HD and ɛV_1-2 did not affect H₂O₂ production under these in vitro experimental conditions. We conclude from these studies that superoxide anion does not activate mitoK_ATP directly, but rather through its dismutation products.

NO opens mitoK_ATP via PKCɛ.

We next examined the effects of NO, which has been suggested to induce mitoK_ATP opening in cardiomyocytes and perfused hearts (55, 64, 65). Using mitochondrial membranes incubated in KCl medium, Dahm et al. (10) found that 1 mM SNAP generates 1 μM NO within 3 min, at an approximate rate of 0.3 μM NO/min. As shown in Fig. 4, SNAP reversed the ATP inhibition of mitoK_ATP with an apparent K_m of 2 mM, corresponding to ∼2 μM NO. SNAP inhibits mitochondrial swelling at concentrations above 50 mM, probably due to inhibition of cytochrome-c oxidase (12, 51). Therefore, we used 10 mM SNAP in our studies. SNAP-induced mitoK_ATP opening in mitochondria was inhibited by 5-HD, N-(2-mercaptopropionyl)glycine (MPG) (not shown), and ɛV_1-2, but not by catalase (Fig. 4). SNAP-induced mitoK_ATP opening in mitoplasts was blocked by MPG and protein phosphatase 2A (PP2A). From these results, we conclude that NO opens mitoK_ATP indirectly, through activation of PKCɛ.

Fig. 4.

Nitric oxide (NO) opens mitoK_ATP via PKCɛ. Shown are the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on mitoK_ATP activity, plotted as volume change (%). Rat heart mitochondria (0.1 mg/ml) were suspended in assay medium containing ATP (0.2 mM). SNAP (10 mM) was added 2 min before mitochondria to allow generation of NO. As indicated, medium also contained 5-HD (0.3 mM), ɛV_1-2 (0.5 μM), and catalase (10 U/ml). Columns on right demonstrate the results of experiments with mitoplasts lacking the mitochondrial outer membrane (MOM). N-(2-mercaptopropionyl)glycine (MPG, 0.3 mM) and protein phosphatase 2A (PP2A, 11 ng/ml) blocked mitoK_ATP opening by SNAP in mitoplasts. Data are means ± SD of at least 4 independent experiments.

Role of mitochondrial outer membrane in PKG- and PKCɛ-dependent mitoK_ATP opening.

PKCɛ copurifies and coreconstitutes with mitoK_ATP in a fully functional manner (24), and we refer to this mitoK_ATP-associated enzyme as PKCɛ1 (7). PKCɛ1 is retained in heart mitoplasts and appears to be tightly bound to the inner membrane (7). The experiments summarized in Fig. 5 were performed in intact mitochondria and in mitoplasts lacking the MOM. PMA, which can readily diffuse across the MOM to activate PKCɛ1, opened mitoK_ATP in both intact mitochondria and mitoplasts (Fig. 5). Exogenous PKG + cGMP opens mitoK_ATP in a PKCɛ-dependent process (6, 7). PKG, which cannot cross the MOM, opens mitoK_ATP in intact mitochondria but not in mitoplasts. Thus PKG-dependent mitoK_ATP opening requires an intact MOM. The Ser/Thr phosphatase PP2A, which cannot cross the MOM, had no effect on PMA-induced mitoK_ATP opening in intact mitochondria, but it negated the effects of PMA in mitoplasts, indicating that mitoK_ATP is opened by PKCɛ-dependent phosphorylation at the level of the inner membrane. PP2A blocked the effect of PKG in intact mitochondria, showing that PKG-dependent mitoK_ATP opening depends on phosphorylation of a MOM protein necessary for signal transmission to mitoK_ATP.

Fig. 5.

The MOM is essential for PKG-induced, but not PKCɛ-induced, mitoK_ATP activity. Shown are the effects of PKG or PMA, and PP2A, on the matrix volume of heart mitochondria or mitoplasts. Data are plotted as volume change (%). Indicated additions to the assay were PKG (25 ng/ml), PMA (0.2 μM), and PP2A (11 ng/ml). Data are means ± SD of at least 4 independent experiments.

ROS are not involved in mitoK_ATP opening by diazoxide, PMA, or PKG.

The data in Fig. 6 show that ROS are not involved in mitoK_ATP opening by diazoxide or by PKCɛ-dependent opening mediated by PMA or PKG. 5-HD, which acts directly on the channel, inhibited the effects of all three mitoK_ATP agonists. The PKCɛ inhibitor chelerythrine inhibited the effect of PMA, and the PKG-specific inhibitor KT-5823 inhibited the effect of PKG + cGMP. However, the ROS scavenger MPG had no effect on mitoK_ATP opening by any of these mechanisms.

Fig. 6.

mitoK_ATP opening by diazoxide, PKG, or PMA does not require reactive oxygen species (ROS). Shown are the effects of MPG on diazoxide (Dzx)-, PMA-, or PKG/cGMP-induced mitoK_ATP opening, plotted as volume change (%). Rat heart mitochondria (0.1 mg/ml) were suspended in assay medium as described in methods. Additions to the assay were ATP (0.2 mM), Dzx (30 μM), PKG (25 ng/ml), cGMP (10 μM), PMA (0.2 μM), 5-HD (0.3 mM), chelerythrine (chel, 0.1 μM), MPG (0.3 mM), and KT-5823 (KT, 0.5 μM). Data are means ± SD of at least 4 independent experiments.

mitoK_ATP-induced ROS-generation activates PKCɛ and maintains mitoK_ATP in the open state.

mitoK_ATP opening in isolated heart mitochondria causes an increase in ROS production (1). As shown in Figs. 1–3, H₂O₂ induces mitoK_ATP opening via activation of PKCɛ1 (24). These results led us to hypothesize that the ROS produced by mitoK_ATP opening could activate mitochondrial PKCɛ1 and maintain mitoK_ATP in the open state for prolonged periods. To test this hypothesis, we performed experiments on mitoplasts. They were preincubated in 150-μl aliquots of assay medium with ATP and diazoxide (30 μM), diluted 10-fold in sucrose buffer containing 0.5% fatty acid-free BSA, and centrifuged at 15,000 g for 30 s. The pellet was resuspended and added to assay medium in the presence of ATP. After this treatment, the mitoplasts lost their sensitivity to ATP (Fig. 7). Moreover, they became insensitive to diazoxide, suggesting that the channel was already open. This was confirmed by the findings that PP2A + ATP restored the closed state and the sensitivity to diazoxide (Fig. 7). This was presumably due to dephosphorylation of mitoK_ATP. We interpret these results to mean that preincubation with ATP + diazoxide opened mitoK_ATP, leading to increased ROS that activated mitochondrial PKCɛ1, which in turn phosphorylated mitoK_ATP, leading to a sustained open state. This interpretation is supported by the further finding that preincubation of mitoplasts with ATP, diazoxide, and ɛV_1-2, to inhibit PKCɛ1, resulted in a normal response of mitoK_ATP to ATP and diazoxide in the assay medium (Fig. 7). A similar effect was observed when mitoplasts were preincubated with ATP, diazoxide, and the ROS scavenger MPG (Fig. 7), which restored the normal response to ATP and diazoxide in the assay medium (Fig. 7).

Fig. 7.

Phosphorylation-dependent mitoK_ATP-induced mitoK_ATP opening in vitro. Shown are the effects of preincubating heart mitoplasts (0.3 mg) with ATP (0.2 mM) + Dzx (30 μM), plotted as volume change (%). Preincubations were carried out in assay medium containing phosphatase inhibitors and, where indicated, ɛV_1-2 (0.5 μM) or MPG (0.3 mM, 0.5%) at 30°C. After 3 min, the mitoplasts were washed and diluted 100-fold into the assay medium in order to avoid effects of Dzx, ɛV_1-2, or MPG during the assay. Indicated additions to the assay were ATP (0.2 mM), Dzx (30 μM), and PP2A (11 ng/ml). Data are means ± SD of at least 3 independent experiments.

Persistence of open state in mitochondria from diazoxide-treated hearts.

We next investigated whether a similar effect could be observed in mitoplasts prepared from diazoxide-perfused hearts. Figure 8 shows that mitoplasts from sham-perfused hearts displayed normal sensitivity to diazoxide and PP2A and, further, that the diazoxide response was not modified by PP2A. However, mitoplasts from hearts perfused with diazoxide displayed an open mitoK_ATP, and ATP and diazoxide had no effect unless the diazoxide-dependent phosphorylation was removed by PP2A. These findings agree with those in Fig. 7. The data in Figs. 7 and 8 support the hypothesis that mitoK_ATP activity induced by a K_ATP channel agonist or by preconditioning will maintain mitoK_ATP in an open state, even after the K_ATP channel opener is washed away (19, 20).

NO inhibits MPT via PKCɛ.

H₂O₂ inhibits MPT opening by oxidizing thiols in PKCɛ and causing its activation (7). We reasoned that NO should have a similar effect. The results in Fig. 9 show that the NO donor SNAP inhibited MPT opening in isolated heart mitochondria to the same extent as diazoxide. This result agrees with that reported by Brookes et al. (5), who found that 0.7 μM NO inhibited MPT in liver mitochondria. Figure 9 also shows that inhibition of mitoK_ATP by 5-HD had no effect on SNAP inhibition of MPT, and that ɛV_1-2 completely abolished the protective effects of SNAP. No further inhibition was observed when mitochondria were incubated with 5-HD and ɛV_1-2 at the same time. These effects of NO on MPT are similar to those previously observed with H₂O₂ (7). We conclude, as before, that NO is activating a second PKCɛ, PKCɛ2, that regulates MPT (7). The added NO acts directly on PKCɛ2, thereby bypassing mitoK_ATP; hence, 5-HD has no effect.

Fig. 9.

NO inhibits mitochondrial permeability transition (MPT) via PKCɛ and independently of mitoK_ATP. Shown are the effects of Dzx or the NO donor SNAP on MPT-induced swelling, expressed as MPT inhibition (%). Synchronized MPT opening in rat heart mitochondria (0.1 mg/ml) was elicited by Ca²⁺ and the uncoupler CCCP, as described in methods. Rates of matrix swelling in the presence and absence of 1 μM CsA were taken as 0% and 100%, respectively. SNAP (10 mM) was added 2 min before mitochondria to allow generation of NO. 5-HD (0.3 mM) and ɛV_1-2 (0.5 μM) were added immediately before mitochondria. Data are means ± SD of at least 3 independent experiments.

Inhibition of GSK-3β has no effect on MPT in isolated mitochondria.

It has been proposed that GSK-3β, a negative modulator of preconditioning (62), is “immediately proximal to the permeability transition pore complex” (29). Given that mitoK_ATP opening by a variety of mechanisms has been shown to inhibit MPT in isolated mitochondria (Ref. 7 and present study), it should be possible to demonstrate inhibition of MPT under similar conditions by inhibiting GSK-3β. However, the inhibitor SB-415286 had no effect on MPT opening in isolated mitochondria (not shown), indicating that the GSK-3β that interferes with cardioprotection is extramitochondrial.

DISCUSSION

The interactions among mitoK_ATP, PKCɛ, ROS, and MPT constitute a well-regulated intramitochondrial signaling pathway. The diagram in Fig. 10 summarizes several years of work on this pathway (1, 6–8, 18, 24), including the present findings. The primary function of the pathway is to inhibit MPT opening, which is widely considered to be the cause of cell death after ischemia-reperfusion (3, 9, 13). The sequence begins with mitoK_ATP opening, which may occur by three distinct mechanisms: direct, by administration of a K_ATP channel opener (KCO) (18); indirect, by activation of PKCɛ1 (24); and physiological, by cytosolic signaling kinases such as PKG (6). Each of these methods has its counterpart in cardioprotection of the perfused heart. Thus diazoxide and other KCOs are protective (21), PKCɛ activation is protective (42, 66), and PKG activation, arising for example from perfusion of the heart with bradykinin, is protective (44).

Fig. 10.

The intramitochondrial signaling pathways. There are 3 distinct ways of opening mitoK_ATP and initiating the signaling sequence described. 1) Direct mitoK_ATP opening by K_ATP channel openers (KCO) has been demonstrated in mitochondria and in liposomes containing reconstituted mitoK_ATP (22). 2) Indirect mitoK_ATP opening by activation of PKCɛ1 was demonstrated by the effects of the PKCɛ-specific peptide agonist ψɛRACK, PMA, H₂O₂, and NO. That these agents were acting via PKCɛ1 was verified by the finding that the PKCɛ-specific binding antagonist ɛV_1-2 blocked all 4 modes of PKCɛ activation of mitoK_ATP but did not block mitoK_ATP opening by diazoxide (Ref. 24 and present study). PKCɛ1 effect requires phosphorylation, perhaps of mitoK_ATP itself. Thus, when given access in mitoplasts to the mitochondrial inner membrane (MIM), PP2A prevented mitoK_ATP-dependent swelling induced by PKCɛ agonists. 3) Physiological mitoK_ATP opening by signals arriving at the MOM from the cytosol, such as PKG (6). PKG + cGMP open mitoK_ATP by phosphorylating a MOM receptor (labeled R1). Thus PKG-dependent mitoK_ATP opening is blocked either if PP2A is added to the assay or if the MOM is removed. Phosphorylation of R1 leads by an unknown mechanism to activation of PKCɛ1 and opening of mitoK_ATP. Once mitoK_ATP is opened, the increase in K⁺ uptake leads to increased matrix volume (ΔV), which is the basis of the light scattering assay for mitoK_ATP activity (8). More K⁺ than phosphate will be taken up into the matrix, because the cytosolic concentration of K⁺ is much higher than that of phosphate. This imbalance leads to matrix alkalinization (8). Matrix alkalinization, in turn, inhibits complex I, leading to increased production of superoxide and its products, H₂O₂ and hydroxyl anion radical (1). The increase in ROS plays 2 roles. It activates a second PKCɛ, PKCɛ2, that then inhibits the MPT in a phosphorylation-dependent reaction (7). We hypothesize that this effect is the primary means by which preconditioning and ischemic postconditioning prevent cardiac cell death. The increase in ROS also activates PKCɛ1, which is bypassed when KCOs are administered to the heart, to cause a persistent phosphorylation-dependent open state of mitoK_ATP (present study). We hypothesize that this positive feedback loop for mitoK_ATP opening is the mechanism of memory, which is seen with all preconditioning triggers (15, 47).

Direct mitoK_ATP opening by KCOs has been described previously (22). KCOs act on the regulatory sulfonylurea receptors (SUR) of K_ATP channels. Pinacidil, cromakalim, and nicorandil are effective openers of cardiac K_ATP through their action on SUR2A, but ineffective on the pancreatic β-cell K_ATP, which uses SUR1. Conversely, diazoxide is an effective opener of β-cell K_ATP but ineffective on the cardiac channel (40). Interestingly, all KCOs we have examined, including those listed above, open mitoK_ATP (8, 21–23, 25, 38, 45, 52).

Indirect mitoK_ATP opening by activation of PKCɛ1 was demonstrated by the effects of the PKCɛ-specific peptide agonist ψɛRACK (Figs. 1 and 2) and the PKCɛ agonists H₂O₂ (Figs. 1–3), NO (Fig. 4), and PMA (Figs. 5 and 6). That these agents were acting via PKCɛ1 was verified by the finding that the PKCɛ-specific binding antagonist ɛV_1-2 blocked all four modes of PKCɛ activation of mitoK_ATP but did not block mitoK_ATP opening by diazoxide (Ref. 24 and present study). Importantly, superoxide cannot activate PKCɛ1 to open mitoK_ATP, as shown in Fig. 3. Jaburek et al. (24) observed similar effects of ψɛRACK and ɛV_1-2 in liposomes reconstituted with partially purified mitoK_ATP and PKCɛ1. The PKCδ-specific peptide antagonist δV_1-1, or a scrambled analog of ɛV_1-2, had no effect on H₂O₂-dependent mitoK_ATP opening, and we conclude that this effect is mediated specifically by an intrinsic mitochondrial PKCɛ. PKCɛ1 effect requires phosphorylation, perhaps of mitoK_ATP itself. Thus, when given access in mitoplasts to the MIM, PP2A prevented mitoK_ATP-dependent swelling induced by PKCɛ agonists (Fig. 4).

PKCɛ requires anionic phospholipids for activity and is activated physiologically by one of two second messengers—diacylglycerol (or phorbol ester) and a sulfhydryl oxidizing agent, such as H₂O₂ (60) or NO (present study). Addition of PMA or H₂O₂ has been shown to open up one of the two zinc fingers in PKCɛ (30, 37). The phospholipid requirement is met by cardiolipin, which is abundant in mitochondria and enhances PKCɛ activity three- to fourfold compared with phosphatidylserine (36). ψɛRACK, PMA, H₂O₂, and NO each open mitoK_ATP. These agents cause conformational changes that expose the substrate domain on PKCɛ and cause its binding to its RACK (54). ψɛRACK is a PKCɛ-specific peptide agonist that acts by regulating intramolecular PKCɛ binding, and ɛV_1-2 is a PKCɛ-specific peptide antagonist that acts by preventing protein-protein interactions between PKCɛ and its binding protein, RACK (27, 54, 59). Murriel and Mochly-Rosen (42) found that ψɛRACK protected cardiac cells from ischemic damage, whereas ɛV_1-2 caused a loss of protection.

Physiological mitoK_ATP opening is mediated by cytosolic signaling kinases, such as PKG, that act on the MOM. The data in Figs. 5 and 6 show that PKG + cGMP induce mitoK_ATP opening that is blocked by the specific PKG inhibitor KT-5823, the mitoK_ATP inhibitor 5-HD, and the PKC inhibitor chelerythrine. The latter finding shows that activation of PKCɛ (PKCɛ1 in Fig. 10) is an essential step in PKG-dependent mitoK_ATP opening (6). Activation of PKCɛ1 by this mechanism is not prevented by MPG (Fig. 6); therefore it does not involve ROS. In mitochondria with an intact MOM, PKG-dependent mitoK_ATP opening is blocked by PP2A (Fig. 5). In mitoplasts with the MOM disrupted, PKG is no longer able to induce mitoK_ATP opening (Fig. 5). These findings show that the MOM is required for transmission of cytosolic signals to mitoK_ATP and that PKG phosphorylates a MOM receptor protein (R1 in Fig. 10), whose molecular identity is not yet known. The mechanism of signal transmission from MOM to PKCɛ1 on the inner membrane is also not known but may involve a pseudo-RACK mechanism.

Once mitoK_ATP is opened, the increase in K⁺ uptake leads to increased matrix volume (ΔV in Fig. 10), which is the basis of the light scattering assay for mitoK_ATP activity (8). The cytosolic concentration difference between K⁺ and phosphate means that more K⁺ than phosphate will be taken up, leading to matrix alkalinization (8). Matrix alkalinization, in turn, inhibits complex I, leading to increased production of superoxide and its products, H₂O₂ and hydroxyl anion radical (1).

The increase in ROS now activates a second mitochondrial PKCɛ (PKCɛ2 in Fig. 10). We showed previously (7) that activation of PKCɛ2 inhibits the MPT in a phosphorylation-dependent reaction. The evidence for two distinct mitochondrial PKCɛ, one acting on mitoK_ATP and the other on MPT, is given in Ref. 7. H₂O₂ activates PKCɛ2 and inhibits MPT (7). The results in Fig. 9 show that NO, but not superoxide, also inhibits MPT in a PKCɛ-dependent manner. Thus the redundant modes of cardioprotective mitoK_ATP opening lead by these pathways to inhibition of MPT, and presumably to reduction of cell death after ischemia-reperfusion injury (3, 9, 13).

The mitoK_ATP-dependent increase in ROS plays an additional role in cardioprotection. It should be noted in Fig. 10 that PKCɛ1 is bypassed when KCOs are administered to the heart; however, as shown in Figs. 7 and 8, PKCɛ1 is soon activated by mitoK_ATP-dependent ROS, leading to a persistent phosphorylation-dependent open state of mitoK_ATP. These data define a new, positive feedback loop for mitoK_ATP opening, whose existence, which has been suggested by a number of authors (29, 31, 39), means that mitoK_ATP may be either upstream or downstream of PKCɛ, depending on the triggering stimulus. We suggest that feedback phosphorylation of mitoK_ATP is the mechanism of memory, which is seen with all preconditioning triggers (15, 47). Thus cardioprotective stimuli can be washed away from the system, and the perfused heart remains protected from a major ischemic assault due to phosphorylation of mitoK_ATP. We infer, but have not demonstrated, that mitoK_ATP opening is eventually reversed by an endogenous phosphatase (PP2A in Fig. 10) within the intermembrane space. For example, PP2A has been found in mitochondria, where it is activated by proapoptotic factors (35).

The model in Fig. 10 and the findings reported here help to clarify and extend results of previous studies. Several reports have correlated mitoK_ATP opening, ROS, and PKCɛ activity, but none in isolated mitochondria. Jiang et al. (26) observed PKC and 5-HD regulation of the human cardiac mitoK_ATP in lipid bilayers. Garg and Hu (17) showed that PKCɛ modulates mitoK_ATP activity in cardiomyocytes and COS-7 cells. Penna et al. (49) demonstrated that postconditioning protection involves a redox-sensitive mechanism and persistent activation of mitoK_ATP and PKC. Our results are fully consistent with these studies. Sasaki et al. (55) suggested that NO may open mitoK_ATP directly; however, mitoK_ATP opening by NO is blocked by ɛV_1-2 (Fig. 4), indicating that NO opens mitoK_ATP indirectly through PKCɛ1. Several authors have shown that exogenous and endogenous NO are cardioprotective and have attributed this effect to MPT inhibition (5, 28, 33, 63). Brookes et al. (5) showed that NO inhibited MPT and cytochrome c release in isolated liver mitochondria. Here, we confirm that NO inhibits MPT in heart mitochondria and show that this effect is independent of mitoK_ATP activity and occurs via activation of PKCɛ2 (Fig. 9). Forbes et al. (16) and Pain et al. (47) found that N-acetylcysteine or MPG reversed the protective effect of diazoxide in perfused hearts. Our data suggest that block of protection occurred because mitoK_ATP-dependent ROS was scavenged and unavailable to activate PKCɛ2 and inhibit MPT. Lebuffe et al. (39) found that PMA-induced protection was blocked by 5-HD and that this block was reversed by coadministration of H₂O₂ and NO. This is also consistent with the model of Fig. 10 in that H₂O₂ and NO can bypass the blocked mitoK_ATP and act directly on PKCɛ2, thereby inhibiting MPT and protecting the heart. Some effects of mitoK_ATP-dependent ROS signaling appear to result from a second messenger effect of the ROS on extramitochondrial pathways. Thus diazoxide and other cardioprotective signals cause phosphorylation of GSK-3β in cardiomyocytes and isolated hearts (29, 41, 48, 62); however, inhibition of GSK-3β has no effect on MPT opening in isolated mitochondria (present study), suggesting that the GSK isoform that interferes with cardioprotection resides outside of mitochondria.

Limitations.

In these experiments, mitochondria were respiring on the nonphysiological substrate succinate. However, we showed previously (1) that mitoK_ATP activity is also observed when mitochondria respire on pyruvate/malate. K⁺ flux via mitoK_ATP depends on membrane potential and does not appear to be influenced directly by the mechanism of producing this driving force, so we do not anticipate different behavior in vivo. The results of Fig. 8, in which mitoK_ATP was opened ex vivo by diazoxide, are at least consistent with this view. Also, for practical reasons, this study was based solely on light scattering measurements. As described in methods, this assay has been validated quantitatively by five independent techniques.

GRANTS

This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-067842 and HL-35673.