Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

Opening of mitochondrial K+ channels increases ischemic ATP levels by preventing hydrolysis

Mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) have been proposed to mediate protection against ischemic injury by increasing high-energy intermediate levels. This study was designed to verify if mitochondria are an important factor in the loss of cardiac ATP associated to ischemia, and determine the possible role of mitoK_ATP in the control of ischemic ATP loss. Langendorff-perfused rat hearts subjected to ischemia were found to have significantly higher ATP contents when pretreated with oligomycin or atractyloside, indicating that mitochondrial ATP hydrolysis contributes toward ischemic ATP depletion. MitoK_ATP opening induced by diazoxide promoted a similar protection against ATP loss. Diazoxide also inhibited ATP hydrolysis in isolated, nonrespiring mitochondria, an effect accompanied by a drop in the membrane potential and Ca²⁺ uptake. In hearts subjected to ischemia followed by reperfusion, myocardial injury was prevented by diazoxide, but not atractyloside or oligomycin, which, unlike diazoxide, decreased reperfusion ATP levels. Our results suggest that mitoK_ATP-mediated protection occurs due to selective inhibition of mitochondrial ATP hydrolysis during ischemia, without affecting ATP synthesis after reperfusion.     

Opening of the mitoKATP channel and decoupling of mitochondrial complex II and III contribute to the suppression of myocardial reperfusion hyperoxygenation

Diazoxide, a mitochondrial ATP-sensitive potassium (mitoK_ATP) channel opener, protects the heart from ischemia–reperfusion injury. Diazoxide also inhibits mitochondrial complex II-dependent respiration in addition to its preconditioning effect. However, there are no prior studies of the role of diazoxide on post-ischemic myocardial oxygenation. In the current study, we determined the effect of diazoxide on the suppression of post-ischemic myocardial tissue hyperoxygenation in vivo, superoxide (O₂ ^??) generation in isolated mitochondria, and impairment of the interaction between complex II and complex III in purified mitochondrial proteins. It was observed that diazoxide totally suppressed the post-ischemic myocardial hyperoxygenation. With succinate but not glutamate/malate as the substrate, diazoxide significantly increased ubisemiquinone-dependent O₂ ^?? generation, which was not blocked by 5-HD and glibenclamide. Using a model system, the super complex of succinate-cytochrome c reductase (SCR) hosting complex II and complex III, we also observed that diazoxide impaired complex II and its interaction with complex III with no effect on complex III. UV–visible spectral analysis revealed that diazoxide decreased succinate-mediated ferricytochrome b reduction in SCR. In conclusion, our results demonstrated that diazoxide suppressed the in vivo post-ischemic myocardial hyperoxygenation through opening the mitoK_ATP channel and ubisemiquinone-dependent O₂ ^?? generation via inhibiting mitochondrial complex II-dependent respiration.     

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.     

MitoK ATP -dependent changes in mitochondrial volume and in complex II activity during ischemic and pharmacological preconditioning of langendorff-perfused rat heart

It has been proposed that activation of the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) is part of signaling pathways triggering the cardioprotection afforded by ischemic preconditioning of the heart. This work was to analyze the mitochondrial function profile of Langendorff-perfused rat hearts during the different phases of various ischemia-reperfusion protocols. Specifically, skinned fibers of ischemic preconditioned hearts exhibit a decline in the succinate-supported respiration and complex II activity during ischemia, followed by a recovery during reperfusion. Meanwhile, the apparent affinity of respiration for ADP (which reflects the matrix volume expansion) is increased during preconditioning stimulus and, to a larger extent, during prolonged ischemia. This evolution pattern is mimicked by diazoxide and abolished by 5-hydroxydecanoate. It is concluded that opening the mitoK_ATP channel mediates the preservation of mitochondrial structure-function via a mitochondrial matrix shrinkage and a reversible inactivation of complex II during prolonged ischemic insult.     

A role of opening of mitochondrial ATP-sensitive potassium channels in the infarct size-limiting effect of ischemic preconditioning via activation of protein kinase C in the canine heart

The opening of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels triggers or mediates the infarct size (IS)-limiting effect of ischemic preconditioning (IP). Because ecto-5′-nucleotidase related to IP is activated by PKC, we tested whether the opening of mitoK_ATP channels activates PKC and contributes to either activation of ecto-5′-nucleotidase or IS-limiting effect. In dogs, IP procedure decreased IS and activated ecto-5′-nucleotidase, both of which were mimicked by transient exposure to either cromakalim or diazoxide, and these effects were blunted by either GF109203X (a PKC inhibitor) or 5-hydroxydecanoate (a mitoK_ATP channel blocker), but not by HMR-1098 (a surface sarcolenmal K_ATP channel blocker). Either cromakalim or diazoxide activated both PKC and ecto-5′-nucleotidase, which was blunted by either GF109203X or 5-hydroxydecanoate, but not by HMR-1098. We concluded that the opening of mitoK_ATP channels contributes to either activation of ecto-5′-nucleotidase or the infarct size-limiting effect via activation of PKC in canine hearts.     

Inhibition of oxygen consumption in skeletal muscle-derived mitochondria by pinacidil,diazoxide, and glibenclamide,but not by 5-hydroxydecanoate

Cell intermediary metabolism and energy production succeeds by means of mitochondria, whose activity is in relation to transmembrane potential and/or free radical production. Adenosine triphosphate (ATP)-dependent potassium channels (K_ATP) in several cell types have shown to couple cell metabolism to membrane potential and ATP production. In this study, we explore whether oxygen consumption in isolated skeletal-muscle mitochondria differs in the presence of distinct respiration substrates and whether these changes are affected by K_ATP-channel inhibitors such as glibenclamide, 5-Hydroxydecanoate (5-HD), and K_ATP channel activators (pinacidil and diazoxide). Results demonstrate a concentration-dependent diminution of respiration rate by glibenclamide (0.5–20 μM), pinacidil (1–50 μM), and diazoxide (50–200 μM), but no significant differences were found when the selective mitochondrial K_ATP-channel inhibitor (5-HD, 10–500 μM) was used. These results suggest that these K_ATP-channel agonists and antagonists exert an effect on mitochondrial respiration and that they could be acting on mito-K_ATP or other respiratory-chain components.     

Reversible Cyclosporin A-sensitive Mitochondrial Depolarization Occurs within Minutes of Stroke Onset in Mouse Somatosensory Cortex in Vivo: A TWO-PHOTON IMAGING STUDY*

Neuronal structure and function are rapidly damaged during global ischemia but can in part recover during reperfusion. Despite apparent recovery in the hours/days following an ischemic episode, delayed cell death can be initiated, making it important to understand how initial ischemic events affect potential mediators of apoptosis. Mitochondrial dysfunction and the opening of the mitochondrial permeability transition pore (mPTP) are proposed to link ischemic ionic imbalance to mitochondrially mediated cell death pathways. Using two-photon microscopy, we monitored mitochondrial transmembrane potential (Δψ_m) in vivo within the somatosensory cortex during ischemia and reperfusion in a mouse global ischemia model. Our results indicated a synchronous loss of Δψ_m within 1–3 min of ischemic onset that was linked to within seconds of plasma membrane potential (Δψ_p) depolarization. Δψ_m recovered rapidly upon reperfusion, and no delayed depolarization was observed over 2 h. Cyclosporin A treatment largely blocked Δψ_m collapse during ischemia, suggesting a role for the mPTP. Blocking Δψ_m depolarization did not affect structural damage to dendrites, indicating that the opening of the mPTP and damage to dendrites are separable pathways that are activated during Δψ_p depolarization. Our findings using in vivo imaging suggest that mitochondrial dysfunction and specifically the activation of the mPTP are early reversible events during brain ischemia that could trigger delayed cell death.     

Adenine nucleotide translocase mediates the K(ATP)-channel-openers-induced proton and potassium flux to the mitochondrial matrix

K_ATP channel openers have been shown to protect ischemic-reperfused myocardium by mimicking ischemic preconditioning, although their mechanisms of action have not been fully clarified. In this study we investigated the influence of the adenine nucleotide translocase (ANT) inhibitors–carboxyatractyloside (CAT) and bongkrekic acid (BA)–on the diazoxide- and pinacidil-induced uncoupling of isolated rat heart mitochondria respiring on pyruvate and malate (6 + 6 mM). We found that both CAT (1.3 M) and BA (20 M) markedly reduced the uncoupling of mitochondrial oxidative phosphorylation induced by the K_ATP channel openers. Thus, the uncoupling effect of diazoxide and pinacidil is evident only when ANT is not fixed by inhibitors in neither the C- nor the M-conformation. Moreover, the uncoupling effect of diazoxide and pinacidil was diminished in the presence of ADP or ATP, indicating a competition of K_ATP channel openers with adenine nucleotides. CAT also abolished K⁺-dependent mitochondrial respiratory changes. Thus ANT could also be involved in the regulation of K_ATP-channel-openers-induced K⁺ flux through the inner mitochondrial membrane.     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

The human mitochondrial KATP channel is modulated by calcium and nitric oxide: a patch-clamp approach

ATP-sensitive potassium channels of the inner mitochondrial membrane (mtK_ATP) are blocked by ATP. They are suggested to be involved in protective mechanisms such as ischemic preconditioning (IPC). Here we identify this channel type for the first time in a human cell line (Jurkat cells). Vesicles of the inner mitochondrial membrane (mitoplasts) were prepared by hypoosmotic shock. Single-channel currents were measured by means of the patch-clamp technique. We identified an outward-rectifying channel with a slope conductance of 15 and 82 pS at negative and positive potentials, respectively. The block by 5-hydroxydecanoic acid and inhibition by ATP characterize this channel as the mtK_ATP channel. ATP also increased the frequency of events within the burst. This effect was modulated by the Ca²⁺-bath concentration. We also show that the human mtK_ATP channel is a direct target for nitric oxide that blocked the channel activity. Although the molecular structure of this channel type is still unknown, its characterization as an outward-rectifying channel and modulation by calcium ions and nitric oxide may help to elucidate its functional significance, which possibly implicates a role in cell survival after IPC.     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.     

Evidences for an ATP-sensitive potassium channel (KATP) in muscle and fat body mitochondria of insect

In the present study, we describe the existence of mitochondrial ATP-dependent K⁺ channel (mitoK_ATP) in two different insect tissues, fat body and muscle of cockroach Gromphadorhina coquereliana. We found that pharmacological substances known to modulate potassium channel activity influenced mitochondrial resting respiration. In isolated mitochondria oxygen consumption increased by about 13% in the presence of potassium channel openers (KCOs) such as diazoxide and pinacidil. The opening of mitoK_ATP was reversed by glibenclamide (potassium channel blocker) and 1 mM ATP. Immunological studies with antibodies raised against the Kir6.1 and SUR1 subunits of the mammalian ATP-sensitive potassium channel, indicated the existence of mitoK_ATP in insect mitochondria. MitoK_ATP activation by KCOs resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of mitochondrial ATP-sensitive potassium channel in insects.     

Protective effects of sarpogrelate, a 5-HT2A antagonist, against postischemic myocardial dysfunction in guinea-pig hearts

The protective effects of sarpogrelate (SG), a 5-HT_2A antagonist, were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca²⁺ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored using an nitric oxide (NO) electrode, fluorometry and ³¹P-NMR. The recovery of LVDP from ischemia by reperfusion was 30.1% in the control, while the treatment with SG (5×10^-7 M) in pre- and post-ischemia hearts produced a gradual increase to 73.1 and 53.6%, respectively. At the final stage of ischemia, the intracellular concentration of Ca²⁺ ([Ca²⁺_i) and release of NO increased with no twitching and remained at a high steady level. The addition of SG increased the transient NO signal (T_NO) level at the end of ischemia compared with the control, but [Ca²⁺]_i during ischemia decreased. Meanwhile, mitochondrial Ca²⁺ uptake on acidification or Ca²⁺ content changes of the perfusate was suppressed by pre-treatment with SG or the K_ATP channel opener diazoxide, but not the K_ATP channel blocker 5-HD. The myocardial NO elevated with 5-HT in normal Langendorff hearts was suppressed by the treatment with SG. Therefore, the existence of the 5HT_2A receptor in a Langendorff heart was anticipated. By in vitro EPR, SG was found to directly quench the hydroxy radical. Thus, these findings suggested that the 5-HT_2A receptor induced in ischemia–reperfusion plays an important role in the mitochondrial K_ATP channel of hearts in close relation with NO and active oxygen radicals.     

Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.     

Adenosine A1 and A3 receptor agonists reduce hypoxic injury through the involvement of P38 MAPK

Activation of either the A₁ adenosine receptor (A₁R) or the A₃ adenosine receptor (A₃R), by their specific agonists CCPA and Cl-IB-MECA, respectively, protects cardiac cells in culture against ischemic injury. Yet the full protective mechanism remains unclear. In this study, we therefore examined the involvement of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) phosphorylation in this protective intracellular signaling mechanism. Furthermore, we investigated whether p38 MAPK phosphorylation occurs upstream or downstream from the opening of mitochondrial ATP-sensitive potassium (K_ATP) channels. The role of p38 MAPK activation in the intracellular signaling process was studied in cultured cardiomyocytes subjected to hypoxia, that were pretreated with CCPA or Cl-IB-MECA or diazoxide (a mitochondrial K_ATP channel opener) with and without SB203580 (a specific inhibitor of phosphorylated p38 MAPK). Cardiomyocytes were also pretreated with anisomycin (p38 MAPK activator) with and without 5-hydroxy decanoic acid (5HD) (a mitochondrial K_ATP channel blocker). SB203580 together with the CCPA, Cl-IB-MECA or diazoxide abrogated the protection against hypoxia as shown by the level of ATP, lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Anisomycin protected the cardiomyocytes against ischemic injury and this protection was abrogated by SB203580 but not by 5HD. Conclusions Activation of A₁R or A₃R by CCPA or Cl-IB-MECA, respectively, protects cardiomyocytes from hypoxia via phosphorylation of p38 MAPK, which is located downstream from the mitochondrial K_ATP channel opening. Elucidating the signaling pathway by which adenosine receptor agonists protect cardiomyocytes from hypoxic damage, will facilitate the development of anti ischemic drugs.     

The Intracellular Localization and Function of the ATP-Sensitive K+ Channel Subunit Kir6.1

Our aim was to determine the subcellular localization and functional roles of the K_ATP channel subunit Kir6.1 in intracellular membranes. Specifically, we focused on the potential role of Kir6.1 as a subunit of the mitochondrial ATP-sensitive K⁺ channel. Cell imaging showed that a major proportion of heterologously expressed Kir6.1-GFP and endogenously expressed Kir6.1 was distributed in the endoplasmic reticulum with little in the mitochondria or plasma membrane. We used pharmacological and molecular tools to investigate the functional significance of this distribution. The K_ATP channel opener diazoxide increased reactive oxygen species production, and glibenclamide abolished this effect. However, in cells lacking Kir6.1 or expressing siRNA or dominant negative constructs of Kir6.1, the same effect was seen. Ca²⁺ handling was examined in the muscle cell line C2C12. Transfection of the dominant negative constructs of Kir6.1 significantly reduced the amplitude and rate of rise of [Ca²⁺] _c transients elicited by ATP. This study suggests that Kir6.1 is located in the endoplasmic reticulum and plays a role in modifying Ca²⁺ release from intracellular stores.     

Quinine Inhibits Mitochondrial ATP-regulated Potassium Channel from Bovine Heart

The mitochondrial ATP-regulated potassium (mitoK_ATP) channel has been suggested as trigger and effector in myocardial ischemic preconditioning. However, molecular and pharmacological properties of the mitoK_ATP channel remain unclear. In the present study, single-channel activity was measured after reconstitution of the inner mitochondrial membrane from bovine ventricular myocardium into bilayer lipid membrane. After incorporation, a potassium-selective current was recorded with mean conductance of 103 ± 9 pS in symmetrical 150 mM KCl. Single-channel activity of this reconstituted protein showed properties of the mitoK_ATP channel: it was blocked by 500 μM ATP/Mg, activated by the potassium-channel opener diazoxide at 30 μM, inhibited by 50 μM glibenclamide or 150 μM 5-hydroxydecanoic acid, and was not affected by the plasma membrane ATP-regulated potassium-channel blocker HMR1098 at 100 μM. We observed that the mitoK_ATP channel was blocked by quinine in the micromolar concentration range. The inhibition by quinine was additionally verified with the use of ⁸⁶Rb⁺ flux experiments and submitochondrial particles. Quinine inhibited binding of the sulfonylurea derivative [³H]glibenclamide to the inner mitochondrial membrane. We conclude that quinine inhibits the cardiac mitoK_ATP channel by acting on the mitochondrial sulfonylurea receptor.(P. Bednarczyk and A. Kicińska) These authors contributed equally to this work.This revised version was published online in August 2005 with a corrected cover date.     

Modeling Cardiac Action Potential Shortening Driven by Oxidative Stress-Induced Mitochondrial Oscillations in Guinea Pig Cardiomyocytes

Ischemia-induced shortening of the cardiac action potential and its heterogeneous recovery upon reperfusion are thought to set the stage for reentrant arrhythmias and sudden cardiac death. We have recently reported that the collapse of mitochondrial membrane potential (ΔΨ_m) through a mechanism triggered by reactive oxygen species (ROS), coupled to the opening of sarcolemmal ATP-sensitive potassium (K_ATP) channels, contributes to electrical dysfunction during ischemia-reperfusion. Here we present a computational model of excitation-contraction coupling linked to mitochondrial bioenergetics that incorporates mitochondrial ROS-induced ROS release with coupling between the mitochondrial energy state and electrical excitability mediated by the sarcolemmal K_ATP current (I_K,ATP). Whole-cell model simulations demonstrate that increasing the fraction of oxygen diverted from the respiratory chain to ROS production triggers limit-cycle oscillations of ΔΨ_m, redox potential, and mitochondrial respiration through the activation of a ROS-sensitive inner membrane anion channel. The periods of transient mitochondrial uncoupling decrease the cytosolic ATP/ADP ratio and activate I_K,ATP, consequently shortening the cellular action potential duration and ultimately suppressing electrical excitability. The model simulates emergent behavior observed in cardiomyocytes subjected to metabolic stress and provides a new tool for examining how alterations in mitochondrial oxidative phosphorylation will impact the electrophysiological, contractile, and Ca²⁺ handling properties of the cardiac cell. Moreover, the model is an important step toward building multiscale models that will permit investigation of the role of spatiotemporal heterogeneity of mitochondrial metabolism in the mechanisms of arrhythmogenesis and contractile dysfunction in cardiac muscle.