Redox regulation of the mitochondrial K(ATP) channel in cardioprotection

Abstract

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK(ATP) regulation. mK(ATP) activity was measured by both osmotic swelling and Tl(+) flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK(ATP) in cardioprotection by nitroxyl. Key findings were as follows: (i) mK(ATP) was activated by O(2)(-) and H(2)O(2) but not other peroxides. (ii) mK(ATP) was inhibited by NADPH. (iii) mK(ATP) was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK(ATP)-dependent manner. Overall, these results suggest that the mK(ATP) channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK(ATP) may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.

Figure 1. Selective mK_ATP Activation by ROS

mK_ATP activity was measured by osmotic swelling as detailed in the methods. Controls (Na⁺ based media) are in Figure S1. (A): mK_ATP is activated by H₂O₂ but not by t-BuOOH or LOOH. Diazoxide (DZX) opening of mK_ATP was used as a positive control. (B): mK_ATP activation by O₂^•- generated by the X/XO system. Glyburide (Glyb) and 5-hydroxydecanoate (5-HD) are mK_ATP antagonists. (C): Dose response of mK_ATP activation by X/XO in K⁺ (black circles) or Na⁺ (gray triangles) based media. *p<0.05 vs. ATP alone. #p<0.05 vs. ATP + H₂O₂ or X/XO. Experimental conditions are listed below the X axis.

Figure 2. Inhibition of DZX-Activated mK_ATP by Reductants

mK_ATP activity was measured by osmotic swelling as detailed in the methods. Data are shown for the baseline condition (open bars) or maximal swelling in the presence of both ATP and DZX (filled bars). Reductants were present in the media before mitochondrial addition, at the concentrations indicated. *p<0.05 vs. the appropriate control (bar marked Ctrl.) in the absence of reductant.

Figure 3. mK_ATP Activation by S-nitrosothiols and Mitochondrial-Associated NOS

mK_ATP activity was measured by osmotic swelling as detailed in the methods. Controls (Na⁺ based media) are in Figure S2. (A): mK_ATP was activated by S-nitrosoacetylpenacillamine (SNAP) and S-nitrosoglutathione (GSNO), at the indicated doses. (B): Dose-response of mK_ATP activation by SNAP in K⁺ (black circles) or Na⁺ (gray triangles) based media. *p<0.05 vs. ATP alone. #p<0.05 vs. ATP plus SNAP. (C): mK_ATP activation by NOS modulators. L- or D-Arginine, L-nitroarginine methyl ester (NAME), Glyb and 5-HD were present at the concentrations indicated. *p<0.05 vs. *p<0.05 vs. ATP alone. #p<0.05 vs. ATP plus L-arginine.

Figure 4. LA-NO₂ Opens mK_ATP and Inhibits Complex II

(A): mK_ATP activity was measured by osmotic swelling as detailed in the methods. LA-NO₂, native linoleate (LA), Glyb, and 5-HD were present at the indicated concentrations. *p<0.05 vs. ATP alone. #p<0.05 vs. ATP plus LA-NO₂. (B): Complex II activity in the presence of LA-NO₂ was determined as detailed in the methods. Values are expressed as percentage of control complex II rate (128 ± 26 nmols DCPIP ^. min^{-1 .} mg protein^-1).

Figure 5. Nitroxyl Inhibits Complex II, Opens mK_ATP and is Cardioprotective

(A): Complex II activity was measured, following nitroxyl exposure of mitochondria, as described in the methods. Values are expressed as percentage of control complex II rate (108 ± 8 nmols DCPIP ^. min^{-1 .} mg protein^-1). (B): Nitroxyl activation of mK_ATP was monitored using a novel Tl⁺ flux assay as described in the methods. Data show the magnitude of change in intra-mitochondrial Tl⁺ based fluorescence following Tl⁺ addition, relative to control. 5-HD, Glyb, the nitroxyl donor Angeli's salt (AS) and decomposed AS (AS^DC) were present at the indicated concentrations. *p<0.05 vs. ATP alone. #p<0.05 vs. ATP plus 5 μM AS. (C): Nitroxyl protects against cardiomyocyte IR injury. Cell viability was measured via Trypan blue exclusion at the end of reoxygenation, as described in the methods, and expressed as percentage of control (normoxic) cell viability. 5-HD, Glyb, Angeli's salt (AS), decomposed AS (AS^DC), the PKG inhibitor KT-5823 (KT) or the soluble guanylate cyclase inhibitor ODQ were present at the indicated concentrations. *p<0.05 vs. IR alone. #p<0.05 vs. IR plus 5 μM AS.

Figure 6. Schematic Showing Redox Regulation of mK_ATP

Nitroxyl (HNO), RSNO, and LA-NO₂ activate the channel, possibly via thiols on the channel itself or on complex II of the respiratory chain. The ability of low molecular weight thiols (RSH) to inhibit the channel may also be mediated via thiols on the channel or on complex II. In contrast, the effects of NADPH are likely no mediated via thiols. The ability of NO^• to activate the channel may be mediated via the generation of secondary RNS (e.g. RSNO, LA-NO₂, HNO), which can activate the channel via PKG-independent mechanisms, or via classical NO^• protein kinase signaling. ROS (in particular H₂O₂) can also activate the channel, via mechanisms that may include thiol modification or protein kinase signaling. The nature of the interaction between complex II and the subunits of the mK_ATP channel itself remains to be elucidated.