Sarcolemmal and mitochondrial K(ATP) channels and myocardial ischemic preconditioning

Ischemic preconditioning (IPC) is the phenomenon whereby brief periods of ischemia have been shown to protect the myocardium against a sustained ischemic insult. The result of IPC may be manifest as a marked reduction in infarct size, myocardial stunning, or incidence of arrhythmias. While many substances and pathways have been proposed to play a role in the signal transduction mediating the cardioprotective effect of IPC, overwhelming evidence indicates an intimate involvement of the ATP-sensitive potassium channel (K_ATP channel) in this process. Initial hypotheses suggested that the surface or sarcolemmal K_ATP (sarcK_ATP) channel mediated the cardioprotective effects of IPC. However, much research has subsequently supported a major role for the mitochondrial K_ATP channel (mitoK_ATP) as the one involved in IPC-mediated cardioprotection. This review presents evidence to support a role for the sarcK_ATP or the mitoK_ATP channel as either triggers and/or downstream mediators in the phenomenon of IPC.     

Differential role of sarcolemmal and mitochondrial K(ATP) channels in adenosine-enhanced ischemic preconditioning

Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic preconditioning (IPC) by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. The purpose of this study was to determine whether APC is modulated by ATP-sensitive potassium (K(ATP)) channels and to determine whether this modulation occurs before ischemia or during reperfusion. The role of K(ATP) channels before ischemia (I), during reperfusion (R), or during ischemia and reperfusion (IR) was investigated using the nonspecific K(ATP) blocker glibenclamide (Glb), the mitochondrial (mito) K(ATP) channel blocker 5-hydroxydecanoate (5-HD), and the sarcolemmal (sarc) K(ATP) channel blocker HMR-1883 (HMR). Infarct size was significantly increased (P < 0.05) in APC hearts with Glb-I, Glb-R, and 5-HD-I treatment and partially with 5-HD-R. Glb-I and Glb-R treatment significantly decreased APC functional recovery (P < 0.05 vs. APC), whereas 5-HD-I and 5-HD-R had no effect on APC functional recovery. HMR-IR significantly decreased postischemic functional recovery (P < 0.05 vs. APC) but had no effect on infarct size. These data indicate that APC infarct size reduction is modulated by mitoK(ATP) channels primarily during ischemia and suggest that functional recovery is modulated by sarcK(ATP) channels during ischemia and reperfusion.     

Role of mitochondrial and sarcolemmal K(ATP) channels in ischemic preconditioning of the canine heart

We tested whether mitochondrial or sarcolemmal ATP-sensitive K(+) (K(ATP)) channels play a key role in ischemic preconditioning (IP) in canine hearts. In open-chest beagle dogs, the left anterior descending artery was occluded four times for 5 min each with 5-min intervals of reperfusion (IP), occluded for 90 min, and reperfused for 6 h. IP as well as cromakalim and nicorandil (nonspecific K(ATP) channel openers) markedly limited infarct size (6.3 +/- 1.2, 8.9 +/- 1.9, and 7.2 +/- 1.6%, respectively) compared with the control group (40.9 +/- 4.1%). A selective mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate, partially blunted the limitation of infarct size in the animals subjected to IP and those treated with cromakalim and nicorandil (21.6 +/- 3.8, 25.1 +/- 4.6, and 19.8 +/- 5.2%, respectively). A nonspecific K(ATP) channel blocker, glibenclamide, completely abolished the effect of IP (38.5 +/- 6.2%). Intracoronary or intravenous administration of a mitochondria-selective K(ATP) channel opener, diazoxide, at >100 micromol/l could only partially decrease infarct size (19.5 +/- 4.3 and 20.1 +/- 4.4%, respectively). In conclusion, mitochondrial and sarcolemmal K(ATP) channels independently play an important role in the limitation of infarct size by IP in the canine heart.     

Cardioprotection by K(ATP) channels in wild-type hearts and hearts overexpressing A(1)-adenosine receptors

We studied the role of mitochondrial ATP-sensitive K(+) (K(ATP)) channels in modifying functional responses to 20 min global ischemia and 30 min reperfusion in wild-type mouse hearts and in hearts with approximately 250-fold overexpression of functionally coupled A(1)-adenosine receptors (A(1)ARs). In wild-type hearts, time to onset of contracture (TOC) was 303 +/- 24 s, with a peak contracture of 89 +/- 5 mmHg. Diastolic pressure remained elevated at 52 +/- 6 mmHg after reperfusion, and developed pressure recovered to 40 +/- 6% of preischemia. A(1)AR overexpression markedly prolonged TOC to 517 +/- 84 s, reduced contracture to 64 +/- 6 mmHg, and improved recovery of diastolic (to 9 +/- 4 mmHg) and developed pressure (to 82 +/- 8%). 5-Hydroxydecanoate (5-HD; 100 microM), a mitochondrial K(ATP) blocker, did not alter ischemic contracture in wild-type hearts, but increased diastolic pressure to 69 +/- 8 mmHg and reduced developed pressure to 10 +/- 5% during reperfusion. In transgenic hearts, 5-HD reduced TOC to 348 +/- 18 s, increased postischemic contracture to 53 +/- 4 mmHg, and reduced recovery of developed pressure to 22 +/- 4%. In summary, these data are the first to demonstrate that endogenous activation of K(ATP) channels improves tolerance to ischemia-reperfusion in murine myocardium. This functional protection occurs without modification of ischemic contracture. The data also support a role for mitochondrial K(ATP) channel activation in the pronounced cardioprotection afforded by overexpression of myocardial A(1)ARs.     

Adenosine-induced late preconditioning in mouse hearts: role of p38 MAP kinase and mitochondrial K(ATP) channels

We investigated the role of p38 mitogen-activated protein kinase (MAPK) phosphorylation and opening of the mitochondrial ATP-sensitive K(+) [(K(ATP))(mito)] channel in the adenosine A(1) receptor (A(1)AR)-induced delayed cardioprotective effect in the mouse heart. Adult male mice were treated with vehicle (5% DMSO) or the A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 0.1 mg/kg ip). Twenty-four hours later, hearts were subjected to 30 min of global ischemia and 30 min of reperfusion in the Langendorff mode. Genistein or SB-203580 (1 mg/kg i.p.) given 30 min before CCPA treatment was used to block receptor tyrosine kinase or p38 MAPK phosphorylation, respectively. 5-Hydroxydecanoate (5-HD; 200 microM) was used to block (K(ATP))(mito) channels. CCPA produced marked improvement in left ventricular function, which was partially blocked by SB-203580 and 5-HD and completely abolished with genistein. CCPA caused a reduction in infarct size (12.0 +/- 2.0 vs. 30.3 +/- 3.0% in vehicle), which was blocked by genistein (29.4 +/- 2.3%), SB-203580 (28.3 +/- 2.6%), and 5-HD (33.9 +/- 2.4%). CCPA treatment also caused increased phosphorylation of p38 MAPK during ischemia, which was blocked by genistein, SB-203580, and 5-HD. The results suggest that A(1)AR-triggered delayed cardioprotection is mediated by p38 MAPK phosphorylation. Blockade of cardioprotection with 5-HD concomitant with decrease in p38 MAPK phosphorylation suggests a potential role of (K(ATP))(mito) channel opening in phosphorylation and ensuing the late preconditioning effect of A(1)AR.     

Diabetes and hyperglycemia impair activation of mitochondrial K(ATP) channels

Hyperglycemia is an important predictor of cardiovascular mortality in patients with diabetes. We investigated the hypothesis that diabetes or acute hyperglycemia attenuates the reduction of myocardial infarct size produced by activation of mitochondrial ATP-regulated potassium (K(ATP)) channels. Acutely instrumented barbiturate-anesthetized dogs were subjected to a 60-min period of coronary artery occlusion and 3 h of reperfusion. Myocardial infarct size (triphenyltetrazolium chloride staining) was 25 +/- 1, 28 +/- 3, and 25 +/- 1% of the area at risk (AAR) for infarction in control, diabetic (3 wk after streptozotocin-alloxan), and hyperglycemic (15% intravenous dextrose) dogs, respectively. Diazoxide (2.5 mg/kg iv) significantly decreased infarct size (10 +/- 1% of AAR, P < 0.05) but did not produce protection in the presence of diabetes (28 +/- 5%) or moderate hyperglycemia (blood glucose 310 +/- 10 mg/dl; 23 +/- 2%). The dose of diazoxide and the degree of hyperglycemia were interactive. Profound (blood glucose 574 +/- 23 mg/dl) but not moderate hyperglycemia blocked the effects of high-dose (5.0 mg/kg) diazoxide [26 +/- 3, 15 +/- 3 (P < 0.05), and 11 +/- 2% (P < 0.05), respectively]. There were no differences in systemic hemodynamics, AAR, or coronary collateral blood flow (by radioactive microspheres) between groups. The results indicate that diabetes or hyperglycemia impairs activation of mitochondrial K(ATP) channels.     

Ketamine abolishes ischemic preconditioning through inhibition of K(ATP) channels in rabbit hearts

Although ketamine inhibits ATP-sensitive K (K(ATP)) channels in rat ventricular myocytes and abolishes the cardioprotective effect of ischemic preconditioning in isolated rat hearts and in rabbits in in vivo, no studies to date specifically address the precise mechanism of this prevention of ischemic preconditioning by ketamine. This study investigated the mechanism of the blockade of ischemic preconditioning by ketamine in rabbit ventricular myocytes using patch-clamp techniques and in rabbit heart slices model for simulated ischemia and preconditioning. In cell-attached and inside-out patches, ketamine inhibited sarcolemmal K(ATP) channel activities in a concentration-dependent manner. Ketamine decreased the burst duration and increased the interburst duration without a change in the single-channel conductance. In the heart slice model of preconditioning, heart slices preconditioned with a single 5-min anoxia, pinacidil, or diazoxide, followed by 15-min reoxygenation, were protected against subsequent 30-min anoxia and 1-h reoxygenation, and the cardioprotection was blocked by the concomitant presence of ketamine. These data are consistent with the notion that inhibition of sarcolemmal or mitochondrial K(ATP) channels may contribute, at least in part, to the mechanism of the blockade of ischemic preconditioning by ketamine.     

Ischemic preconditioning in rats: role of mitochondrial K(ATP) channel in preservation of mitochondrial function

We examined the role of the sarcolemmal and mitochondrial K(ATP) channels in a rat model of ischemic preconditioning (IPC). Infarct size was expressed as a percentage of the area at risk (IS/AAR). IPC significantly reduced infarct size (7 +/- 1%) versus control (56 +/- 1%). The sarcolemmal K(ATP) channel-selective antagonist HMR-1098 administered before IPC did not significantly attenuate cardioprotection. However, pretreatment with the mitochondrial K(ATP) channel-selective antagonist 5-hydroxydecanoic acid (5-HD) 5 min before IPC partially abolished cardioprotection (40 +/- 1%). Diazoxide (10 mg/kg iv) also reduced IS/AAR (36.2 +/- 4.8%), but this effect was abolished by 5-HD. As an index of mitochondrial bioenergetic function, the rate of ATP synthesis in the AAR was examined. Untreated animals synthesized ATP at 2.12 +/- 0.30 micromol x min(-1) x mg mitochondrial protein(-1). Rats subjected to ischemia-reperfusion synthesized ATP at 0.67 +/- 0.06 micromol x min(-1) x mg mitochondrial protein(-1). IPC significantly increased ATP synthesis to 1.86 +/- 0.23 micromol x min(-1) x mg mitochondrial protein(-1). However, when 5-HD was administered before IPC, the preservation of ATP synthesis was attenuated (1.18 +/- 0.15 micromol x min(-1) x mg mitochondrial protein(-1)). These data are consistent with the notion that inhibition of mitochondrial K(ATP) channels attenuates IPC by reducing IPC-induced protection of mitochondrial function.     

七氟醚预处理对大鼠海马脑片缺氧无糖损伤的保护作用:线粒体KATP通道的作用

利用离体海马脑片缺氧无糖(oxygen-glucose deprivation,OGD)损伤模型,探讨七氟醚预处理对神经细胞的保护作用及该作用与线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channels,mitoKATPchannels)的关系,随机将脑片用2%、4%、6%七氟醚,以及6%七氟醚复合mitoKATP通道阻滞剂5-羟基奎酸盐(5-hydroxydecanoic acid,5-HD)预处理30 min,观察OGD损伤14 min复氧1 h期间顺向群峰电位(orthodromic population spike,OPS)的变化,并应用透射电镜观察细胞超微结构的改变.结果表明,与单纯OGD组相比,七氟醚预处理可使海马脑片OPS消失时间明显延长(P＜0.01),使OPS明显恢复,其中4%、6%七氟醚组的恢复率均为71.4%(P＜0.05 vs OGD),相应恢复程度为(61.0±42.3)%和(78.7±21.1)%(P＜0.01),而且6%七氟醚的保护作用可被5-HD取消.OGD组的海马CA1区锥体细胞明显水肿,核膜皱缩、破裂,染色质聚集,线粒体肿胀畸形,嵴断裂或消失,而4%和6%七氟醚组仅见海马CA1区锥体细胞轻度水肿,核膜皱缩不明显,染色质均匀,线粒体轻度肿胀.结果提示,七氟醚预处理对大鼠海马脑片OGD损伤有一定的保护作用,且七氟醚对神经细胞的保护作用与激活mitoKATP通道有关.     

Myocardial preconditioning factors evoke mesenteric ischemic tolerance via opioid receptors and K(ATP) channels

We have shown that a reverse-phase concentrate generated from the effluent of preconditioned (PC) rabbit hearts evokes a cardioprotective effect in virgin acceptor hearts. With the use of a model of sustained (1 h) simulated ischemia in isolated, spontaneously contracting rabbit jejunum, our current aims were to 1) determine whether protective factor(s) released from PC hearts can improve ischemic tolerance in noncardiac tissue; and 2) obtain preliminary insight into the mediator(s) involved in triggering and eliciting this remote protection. Recovery of contractile force following reoxygenation (our index of ischemic tolerance) was enhanced in jejunal segments pretreated with concentrate generated from PC hearts (33 +/- 3% of baseline, P < 0.01) versus segments that received no concentrate (21 +/- 2%) and segments treated with concentrate from normoxic hearts (16 +/- 3%; P < 0.01). Protection achieved with PC concentrate was attenuated by coadministration of naloxone or glibenclamide, thereby implicating the involvement of opioids and ATP-sensitive potassium channels. Moreover, evaluation of purified subfractions of the crude PC concentrate identified a specific bioactive fraction that may participate in triggering the improved jejunal ischemic tolerance.     

Pharmacological modulation of K(ATP) channels

Pharmacological modulation of ATP-sensitive K+ (K(ATP)) channels is used in the treatment of a number of clinical conditions, including type 2 diabetes and angina. The sulphonylureas and related drugs, which are used to treat type 2 diabetes, stimulate insulin secretion by closing K(ATP) channels in pancreatic beta-cells. Agents used to treat angina, by contrast, act by opening K(ATP) channels in vascular smooth and cardiac muscle. Both the therapeutic K(ATP) channel inhibitors and the K(ATP) channel openers target the sulphonylurea receptor (SUR) subunit of the K(ATP) channel, which exists in several isoforms expressed in different tissues (SUR1 in pancreatic beta-cells, SUR2A in cardiac muscle and SUR2B in vascular smooth muscle). The tissue-specific action of drugs that target the K(ATP) channel is attributed to the properties of these different SUR subtypes. In this review, we discuss the molecular basis of tissue-specific drug action, and its implications for clinical practice.     

Chemical preconditioning with 3-nitropropionic acid in hearts: role of mitochondrial K(ATP) channel

We investigated the cardioprotective effect of 3-nitropropionic acid (3-NPA), an inhibitior of mitochondrial succinate dehydrogenase, and we wanted to show whether this protection is mediated by of opening mitochondrial ATP-sensitive potassium (K(ATP)) channels. Adult rabbits were treated with either 3-NPA (3 mg/kg iv) or saline (n = 6 rabbits/group). After 30 min (for early phase) or 24 h (for late phase) of the treatment, the animals were subjected to 30 min of ischemia and 3 h of reperfusion (ischemia-reperfusion). 5-Hydroxydecanoate (5-HD, 5 mg/kg iv),the mitochondrial K(ATP) channel blocker, was administered 10 min before ischemia-reperfusion in the saline- and 3-NPA-treated rabbits. 3-NPA caused a decrease in the infarct size from 27.8 +/- 4.2% in the saline group to 16.5 +/- 1.0% in the 3-NPA-treated rabbits during early phase and from 30.4 +/- 4.2% in the saline group to 17.6 +/- 1.05 in the 3-NPA group during delayed phase (P < 0.05, % of risk area). The anti-infarct effect of 3-NPA was blocked by 5-HD as shown by an increase in infarct size to 33 +/- 2.7% (early phase) and 31 +/- 2.4% (delayed phase) (P < 0.05 vs. 3-NPA groups). 5-HD had no proischemic effect in control animals. Also, 3-NPA had no effect on systemic hemodynamics. We conclude that 3-NPA induces long-lasting anti-ischemic effects via opening of mitochondrial K(ATP) channels.     

Dual roles of mitochondrial K(ATP) channels in diazoxide-mediated protection in isolated rabbit hearts

Whether the mitochondrial ATP-dependent potassium (mK(ATP)) channel is the trigger or the mediator of cardioprotection is controversial. We investigated the critical time sequences of mK(ATP) channel opening for cardioprotection in isolated rabbit hearts. Pretreatment with diazoxide (100 microM), a selective mK(ATP) channel opener, for 5 min followed by 10 min washout before the 30-min ischemia and 2-h reperfusion significantly reduced infarct size (9 +/- 3 vs. 35 +/- 3% in control), indicating a role of mK(ATP) channels as a trigger of protection. The protection was blocked by coadministration of the L-type Ca(2+) channel blockers nifedipine (100 nM) or 5-hydroxydecanoic acid (5-HD; 50 microM) or by the protein kinase C (PKC) inhibitor chelerythrine (5 microM). The protection of diazoxide was not blocked by 50 microM 5-HD but was blocked by 200 microM 5-HD or 10 microM glybenclamide administrated 5 min before and throughout the 30 min of ischemia, indicating a role of mK(ATP) opening as a mediator of protection. Giving diazoxide throughout the 30 min of ischemia also protected the heart, and the protection was not blocked by chelerythrine. Nifedipine did not affect the ability of diazoxide to open mK(ATP) channels assessed by mitochondrial redox state. In electrically stimulated rabbit ventricular myocytes, diazoxide significantly increased Ca(2+) transient but had no effect on L-type Ca(2+) currents. Our results suggest that opening of mK(ATP) channels can trigger cardioprotection. The trigger phase may be induced by elevation of intracellular Ca(2+) and activation of PKC. During the lethal ischemia, mK(ATP) channel opening mediates the protection, independent of PKC, by yet unknown mechanisms.     

Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial K(ATP) channel-dependent mechanism

We previously demonstrated in rats that noninvasive delayed limb ischemic preconditioning (LIPC) induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb per day for three days confers the same cardioprotective effect as local ischemic preconditioning of the heart, but the mechanism has not been studied in depth. The aim of this project was to test the hypothesis that delayed LIPC enhances myocardial antioxidative ability during ischemia-reperfusion by a mitochondrial K(ATP) channel (mito K(ATP))-dependent mechanism. Rats were randomized to five groups: ischemia-reperfusion (IR)-control group, myocardial ischemic preconditioning (MIPC) group, LIPC group, IR-5HD group and LIPC-5HD group. The MIPC group underwent local ischemic preconditioning induced by three cycles of 5-min occlusion and 5-min reperfusion of the left anterior descending coronary arteries. The LIPC and LIPC-5HD groups underwent LIPC induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb using a modified blood pressure aerocyst per day for three days. All rats were subjected to myocardial ischemia-reperfusion injury. The IR-5HD and LIPC-5HD groups received the mito K(ATP) channel blocker 5-hydroxydecanoate Na (5-HD) before and during the myocardial ischemia-reperfusion injury. Compared with the IR-control group, both the LIPC and MIPC groups showed an amelioration of ventricular arrhythmia, reduced myocardial infarct size, increased activities of total superoxide dismutase, manganese-superoxide dismutase (Mn-SOD) and glutathione peroxidase, increased expression of Mn-SOD mRNA and decreased xanthine oxidase activity and malondialdehyde concentration. These beneficial effects of LIPC were prevented by 5-HD. In conclusion, delayed LIPC offers similar cardioprotection as local IPC. These results support the hypothesis that the activation of mito K(ATP) channels enhances myocardial antioxidative ability during ischemia-reperfusion, thereby contributing, at least in part, to the anti-arrhythmic and anti-infarct effects of delayed LIPC.     

Differential role of K(ATP) channels in late preconditioning against myocardial stunning and infarction in rabbits

The role of ATP-sensitive potassium (K(ATP)) channels in the late phase of ischemic preconditioning (PC) remains unclear. Furthermore, it is unknown whether K(ATP) channels serve as end effectors both for late PC against infarction and against stunning. Thus, in phase I of this study, conscious rabbits underwent a 30-min coronary occlusion (O) followed by 72 h of reperfusion (R) with or without ischemic PC (6 4-min O/4-min R cycles) 24 h earlier. Late PC reduced infarct size approximately 46% versus controls. The K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD), given 5 min before the 30-min O, abrogated the infarct-sparing effect of late PC but did not alter infarct size in non-PC rabbits. In phase II, rabbits underwent six 4-min O/4-min R cycles for 3 consecutive days (days 1, 2, and 3). In controls, the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 46% on day 2 and 54% on day 3 compared with day 1, indicating a late PC effect against myocardial stunning. Neither 5-HD nor glibenclamide, given on day 2, abrogated late PC. The K(ATP) channel opener diazoxide, given on day 1, attenuated stunning, and this effect was completely blocked by 5-HD. Thus the same dose of 5-HD that blocked the antistunning effect of diazoxide failed to block the antistunning effects of late PC. Furthermore, when diazoxide was administered in PC rabbits on day 2, myocardial stunning was further attenuated, indicating that diazoxide and late PC have additive anti-stunning effects. We conclude that K(ATP) channels play an essential role in late PC against infarction but not in late PC against stunning, revealing an important pathogenetic difference between these two forms of cardioprotection.     

Oxidative stress inhibits vascular K(ATP) channels by S-glutathionylation

The K(ATP) channel is an important player in vascular tone regulation. Its opening and closure lead to vasodilation and vasoconstriction, respectively. Such functions may be disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrated that S-glutathionylation was a modulation mechanism underlying oxidant-mediated vascular K(ATP) channel regulation. An exposure of isolated mesenteric rings to hydrogen peroxide (H(2)O(2)) impaired the K(ATP) channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, H(2)O(2) or diamide caused a strong inhibition of the vascular K(ATP) channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH). Similar channel inhibition was seen with oxidized glutathione (GSSG) and thiol-modulating reagents. The oxidant-mediated channel inhibition was reversed by the reducing agent dithiothreitol (DTT) and the specific deglutathionylation reagent glutaredoxin-1 (Grx1). Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester (BioGEE) showed incorporation of GSH to the Kir6.1 subunit in the presence of H(2)O(2). These results suggest that S-glutathionylation is an important mechanism for the vascular K(ATP) channel modulation in oxidative stress.     

ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.     

Sildenafil (Viagra) induces powerful cardioprotective effect via opening of mitochondrial K(ATP) channels in rabbits

Sildenafil citrate (Viagra) is the pharmacological agent used to treat erectile dysfunction in men. Because this drug has a vasodilatory effect, we hypothesized that such an action may induce a preconditioning-like cardioprotective effect via opening of mitochondrial ATP-sensitive K (K(ATP)) channels. Rabbits were treated with sildenafil citrate (0.7 mg/kg iv) either 30 min (acute phase) or 24 h (delayed phase) before 30 min of ischemia and 3 h of reperfusion. Mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg iv) was given 10 min before ischemia-reperfusion. Infarct size was measured by tetrazolium staining. Sildenafil caused reduction in arterial blood pressure within 2 min of treatment, which returned to nearly baseline levels 3 min later. The infarct size (% risk area, means +/- SE) reduced from 33.8 +/- 1.7 in control rabbits to 10.8 +/- 0.9 during the acute phase (68% reduction, P < 0.05) and 19.9 +/- 2.0 during the delayed phase (41% reduction, P < 0.05). 5-HD abolished protection with an increase in infarct size to 35.6 +/- 0.4% and 36.8 +/- 1.6% during the acute and delayed phase, respectively (P < 0.05). Similar acute and delayed cardioprotective effects were observed when sildenafil was administered orally. Systemic hemodynamics also decreased after oral administration of the drug. However, these changes were mild and occurred slowly. For the first time, we demonstrate that sildenafil induces acute and delayed protective effects against ischemia-reperfusion injury, which are mediated by opening of mitochondrial K(ATP) channels.     

Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

We examined whether the mitochondrial ATP-sensitive K channel (K(ATP)) is an effector downstream of protein kinase C-epsilon (PKC-epsilon) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 +/- 6.8% of the left ventricle to 20.3 +/- 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 +/- 4% of the total to 60 +/- 4%, whereas no translocation was observed for PKC-delta and PKC-alpha. In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial K(ATP) opener, infused 10 min before ischemia limited infarct size to 5.2 +/- 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial K(ATP) opening by PKC, the present results suggest that mitochondrial K(ATP)-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.     

Protection against ischemia-induced ventricular arrhythmias and myocardial dysfunction conferred by preconditioning in the rat heart: involvement of mitochondrial K(ATP) channels and reactive oxygen species

Ischemic preconditioning (I-PC) induced by brief episodes of ischemia and reperfusion (I/R) protects the heart against sustained I/R. Although activation of mitochondrial K(ATP) channels (mitoK(ATP)) interacting with reactive oxygen species (ROS) has been proposed as a key event in this process, their role in the antiarrhythmic effect is not clear. This study was designed: 1) to investigate the involvement of mito K(ATP) opening in the effect of I-PC (1 cycle of I/R, 5 min each) on ventricular arrhythmias during test ischemia (TI, 30-min LAD coronary artery occlusion) in Langendorff-perfused rat hearts and subsequent postischemic contractile dysfunction, and 2) to characterize potential mechanisms of protection conferred by I-PC and pharmacological PC induced by mito K(ATP) opener diazoxide (DZX), with particular regards to the modulation of ROS generation. Lipid peroxidation (an indicator of increased ROS production) was determined by measurement of myocardial concentration of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) in non-ischemic controls, non-preconditioned and preconditioned hearts exposed to TI, I-PC alone, as well as after pretreatment with DZX, mito K(ATP) blocker 5-hydroxydecanoate (5-HD) and antioxidant N-acetylcysteine (NAC). Total number of ventricular premature beats (VPB) that occurred in the control hearts (518+/-71) was significantly (P<0.05) reduced by I-PC (195+/-40), NAC (290+/-56) and DZX (168+/-22). I-PC and NAC suppressed an increase in CD and TBARS caused by ischemia indicating lower production of ROS. On the other hand, I-PC and DZX themselves moderately enhanced ROS generation, prior to TI. Bracketing of I-PC with 5-HD suppressed both, ROS production during PC and its cardioprotective effect. In conclusion, potential mechanisms of protection conferred by mito K(ATP) opening in the rat heart might involve a temporal increase in ROS production in the preconditioning phase triggering changes in the pro/antioxidant balance in the myocardium and attenuating ROS production during subsequent prolonged ischemia.