Intramitochondrial signaling: interactions among mitoKATP, PKCepsilon, ROS, and MPT

Abstract

Activation of protein kinase Cepsilon (PKCepsilon), 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 PKCepsilon, designated PKCepsilon1, 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 PKCepsilon, designated PKCepsilon2, 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 PKCepsilon, mitoK(ATP), ROS, and MPT. We present evidence that 1) mitoK(ATP) can be opened by H(2)O(2) and nitric oxide (NO) and that these effects are mediated by PKCepsilon1 and not by direct actions on mitoK(ATP), 2) superoxide has no effect on mitoK(ATP) opening, 3) exogenous H(2)O(2) or NO also inhibits MPT opening, and both compounds do so independently of mitoK(ATP) activity via activation of PKCepsilon2, 4) mitoK(ATP) opening induced by PKG, phorbol ester, or diazoxide is not mediated by ROS, and 5) mitoK(ATP)-generated ROS activates PKCepsilon1 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.

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.

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. 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.

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.

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.

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.

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.

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.

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.

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. 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).