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.     

Cardioprotection by glucose-insulin-potassium: dependence on KATP channel opening and blood glucose concentration before ischemia

We tested the hypothesis that glucose-insulin-potassium (GIK)-induced protection against myocardial infarction depends on ATP-dependent K(+) (K(ATP)) channel activation and is abolished by hyperglycemia before the ischemia. Dogs were subjected to a 60-min coronary artery occlusion and 3-h reperfusion in the absence or presence of GIK (25% dextrose; 50 IU insulin/l; 80 mM/l KCl infused at 1.5 ml x kg(-1) x h(-1)) beginning 75 min before coronary artery occlusion or 5 min before reperfusion. The role of K(ATP) channels was evaluated by pretreatment with glyburide (0.1 mg/kg). The efficacy of GIK was investigated with increases in blood glucose (BG) concentrations to 300 or 600 mg/dl or experimental diabetes (alloxan/streptozotocin). Infarct size (IS) was 29 +/- 2% of the area at risk in control experiments. GIK decreased (P < 0.05) IS when administered beginning 5 min before reperfusion. This protective action was independent of BG (13 +/- 2 and 12 +/- 2% of area at risk; BG = 80 or 600 mg/dl, respectively) but was abolished in dogs receiving glyburide (30 +/- 4%), hyperglycemia before ischemia (27 +/- 4%), or diabetes (25 +/- 3%). IS was unchanged by GIK when administered before ischemia independent of BG (31 +/- 3, 27 +/- 2, and 35 +/- 3%; BG = 80, 300, and 600 mg/dl, respectively). The insulin component of GIK promotes cardioprotection by K(ATP) channel activation. However, glucose decreases K(ATP) channel activity, and this effect predominates when hyperglycemia is present before ischemia.     

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.     

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.     

Isoflurane-induced preconditioning is attenuated by diabetes

Volatile anesthetics stimulate, but hyperglycemia attenuates, the activity of mitochondrial ATP-regulated K(+) channels. We tested the hypothesis that diabetes mellitus interferes with isoflurane-induced preconditioning. Acutely instrumented, barbiturate-anesthetized dogs were randomly assigned to receive 0, 0.32, or 0.64% end-tidal concentrations of isoflurane in the absence or presence of diabetes (3 wk after administration of alloxan and streptozotocin) in six experimental groups. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size (triphenyltetrazolium staining) was 29 +/- 3% (n = 8) of the left ventricular area at risk in control experiments. Isoflurane reduced infarct size (15 +/- 2 and 13 +/- 1% during 0.32 and 0.64% concentrations; n = 8 and 7 dogs, respectively). Diabetes alone did not alter infarct size (30 +/- 3%; n = 8) but blocked the protective effects of 0.32% (27 +/- 2%; n = 7) and not 0.64% isoflurane (18 +/- 3%; n = 7). Infarct size was directly related to blood glucose concentrations in diabetic dogs, but this relationship was abolished by higher concentrations of isoflurane. The results indicate that blood glucose and end-tidal isoflurane concentrations are important determinants of infarct size during anesthetic-induced preconditioning.     

Is the sarcolemmal or mitochondrial KATP channel activation important in the antiarrhythmic and cardioprotective effects during acute ischemia/reperfusion in the intact anesthetized rabbit model?

The relative contributions of cardiomyocyte sarcolemmal ATP-sensitive K(+) (K(ATP)) and mitochondrial K(ATP) channels in the cardioprotection and antiarrhythmic activity induced by K(ATP) channel openers remain obscure, though the mitochondrial K(ATP) channels have been proposed to be involved as a subcellular mediator in cardioprotection afforded by ischemic preconditioning. In the present study, we sought to investigate the effects of administration of ATP-sensitive K(+) channel (K(ATP)) openers (nicorandil and minoxidil), a specific mitochondrial K(ATP) channel blocker (5-hydroxydecanoate (5-HD)) and a specific sarcolemmal K(ATP) channel blocker (HMR 1883; (1-[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl-3-methylthiourea) prior to coronary occlusion as well as prior to post-ischemic reperfusion on survival rate, ischemia-induced and reperfusion-induced arrhythmias and myocardial infarct size in anesthetized albino rabbits. The thorax was opened in the left 4th intercostal space and after pericardiotomy the heart was exposed. In Group I (n=88), occlusion of the left main coronary artery and hence, myocardial ischemia-induced arrhythmias was achieved by tightening a previously placed loose silk ligature for 30 min. In Group II (n=206), arrhythmias were induced by reperfusion following a 20-min ligation of the left main coronary artery. Both in Group I and Group II, intravenous (i.v.) administration of nicorandil (0.47 mg/kg), minoxidil (0.5 mg/kg), HMR 1883 (3 mg/kg)/nicorandil and HMR 1883 (3 mg/kg)/minoxidil before coronary artery occlusion increased survival rate (86%, 75%, 75% and 86% vs. 55% in the control subgroup in Group I; 75%, 67%, 67% and 75% vs. 46% in the control subgroup in Group II), significantly decreased the incidence and severity of life-threatening arrhythmias. In Group II, i.v. administration of nicorandil and minoxidil before coronary artery occlusion significantly decreased myocardial infarct size. However, i.v. administration of nicorandil or minoxidil before reperfusion did neither increase survival rate nor confer any antiarrhythmic or cardioprotective effects. The antiarrhythmic and cardioprotective effects of both nicorandil and minoxidil were abolished by pretreating the rabbits with 5-HD (5 mg/kg, i.v. bolus), a selective mitochondrial K(ATP) channel blocker but not by HMR 1883 (3 mg/kg). In the present study, higher levels of malondialdehyde (MDA) and lower levels of reduced glutathione (GSH) and superoxide dismutase (SOD) in necrotic zone of myocardium in all the 16 subgroups in Group II suggest little anti-free radical property of nicorandil and minoxidil. We conclude that intervention by intravenous administration of nicorandil and minoxidil (through the selective activation of mitochondrial K(ATP) channels) increased survival rate and exhibited antiarrhythmic and cardioprotective effects during coronary occlusion and reperfusion in anesthetized rabbits when administered prior to coronary occlusion. The cardiomyocyte mitochondrial K(ATP) channel may be a pharmacologically modulable target of cardioprotection and antiarrhythmic activity.     

Role of K(ATP)(+) channels in regulation of systemic, pulmonary, and coronary vasomotor tone in exercising swine

The role of ATP-sensitive K(+) (K(ATP)(+)) channels in vasomotor tone regulation during metabolic stimulation is incompletely understood. Consequently, we studied the contribution of K(ATP)(+) channels to vasomotor tone regulation in the systemic, pulmonary, and coronary vascular bed in nine treadmill-exercising swine. Exercise up to 85% of maximum heart rate increased body O(2) consumption fourfold, accommodated by a doubling of both cardiac output and body O(2) extraction. Mean aortic pressure was unchanged, implying that systemic vascular conductance (SVC) also doubled, whereas pulmonary artery pressure increased almost in parallel with cardiac output, so that pulmonary vascular conductance (PVC) increased only 25 +/- 9% (both P < 0.05). Myocardial O(2) consumption tripled during exercise, which was paralleled by an equivalent increase in O(2) supply so that coronary venous PO(2) was maintained. Selective K(ATP)(+) channel blockade with glibenclamide (3 mg/kg iv), decreased SVC by 29 +/- 4% at rest and by 10 +/- 2% at 5 km/h (both P < 0.05), whereas PVC was unchanged. Glibenclamide decreased coronary vascular conductance and hence myocardial O(2) delivery, necessitating an increase in O(2) extraction from 76 +/- 2% to 86 +/- 2% at rest and from 79 +/- 2% to 83 +/- 1% at 5 km/h. Consequently, coronary venous PO(2) decreased from 25 +/- 1 to 17 +/- 1 mmHg at rest and from 23 +/- 1 to 20 +/- 1 mmHg at 5 km/h (all values are P < 0.05). In conclusion, K(ATP)(+) channels dilate the systemic and coronary, but not the pulmonary, resistance vessels at rest and during exercise in swine. However, opening of K(ATP)(+) channels is not mandatory for the exercise-induced systemic and coronary vasodilation.     

Mitochondrial ATP dependent potassium channels mediate non-ischemic preconditioning by tachycardia in dogs

Brief episodes of tachycardia without myocardial ischemia prior to a coronary occlusion decrease myocardial infarct size in dogs. This non-ischemic preconditioning is mediated by adenosine. Because ischemic preconditioning is mediated through ATP dependent potassium channels, particularly the mitochondrial ones, we studied whether non-ischemic preconditioning is also mediated through these channels. In anesthetized dogs heart rate was kept constant at 120 cycles/min and aortic pressure changes were damped. Myocardial infarction was induced by occlusion of the anterior descending coronary artery for 60 min and reperfusion for 270 min. In a control group the infarct size (necrotic volume/risk region volume × 100) was 15.8 ± 1.5%. Preconditioning with five periods of tachycardia, 5 min in duration each at 213 cycles/min with intervening periods of 5 min of basal heart rate at 120 cycles/min, reduced the infarct size by 45.6% (p < 0.05) with respect to the control group. This effect was completely reverted by the blockade of ATP dependent potassium channels with glibenclamide or 5 hydroxydecanoate (a specific blocker of mitochondrial ATP dependent potassium channels) prior to preconditioning. These effects were not due to differences in collateral flow, risk region size or hemodynamic variables between the groups. These results show that mitochondrial ATP dependent potassium channels mediate non-ischemic preconditioning by tachycardia in dogs.     

Cardioprotection by multiple preconditioning cycles does not require mitochondrial K(ATP) channels in pigs

To test whether cardioprotection induced by ischemic preconditioning depends on the opening of mitochondrial ATP-sensitive K(+) (K(ATP)) channels, the effect of channel blockade was studied in barbital-anesthetized open-chest pigs subjected to 30 min of complete occlusion of the left anterior descending coronary artery and 3 h of reflow. Preconditioning was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. 5-Hydroxydecanoate (5 mg/kg iv) was injected 15 min before preconditioning or pharmacological preconditioning induced by diazoxide (3.5 mg/kg, 1 ml/min iv). Infarct size (percentage of the area at risk) after 30 min of ischemia was 35.1 +/- 9.9% (n = 7). Preconditioning markedly limited myocardial infarct size (2.7 +/- 1.6%, n = 7), and 5-hydroxydecanoate did not abolish protection (2.4 +/- 0.9%, n = 8). Diazoxide infusion also significantly limited infarct size (14.6 +/- 7.4%, n = 7), and 5-hydroxydecanoate blocked this effect (30.8 +/- 8.0%, n = 7). Thus the opening of mitochondrial K(ATP) channels is cardioprotective in pigs, but these data do not support the hypothesis that opening of mitochondrial K(ATP) channels is required for the endogenous protection afforded by preconditioning.     

Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.     

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.     

Delayed cardioprotection afforded by the glycogen synthase kinase 3 inhibitor SB-216763 occurs via a KATP- and MPTP-dependent mechanism at reperfusion

Previous studies in our laboratory suggest that an acute inhibition of glycogen synthase kinase 3 (GSK3) by SB-216763 (SB21) is cardioprotective when administered just before reperfusion. However, it is unknown whether the GSK inhibitor SB21 administered 24 h before ischemia is cardioprotective and whether the mechanism involves ATP-sensitive potassium (K(ATP)) channels and the mitochondrial permeability transition pore (MPTP). Male Sprague-Dawley rats were administered the GSK inhibitor SB21 (0.6 mg/kg) or vehicle 24 h before ischemia. Subsequently, the rats were acutely anesthetized with Inactin and underwent 30 min of ischemia and 2 h of reperfusion followed by infarct size determination. Subsets of rats received either the sarcolemmal K(ATP) channel blocker HMR-1098 (6 mg/kg), the mitochondrial K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD; 10 mg/kg), or the MPTP opener atractyloside (5 mg/kg) either 5 min before SB21 administration or 5 min before reperfusion 24 h later. The infarct size was reduced in SB21 compared with vehicle (44 +/- 2% vs. 61 +/- 2%, respectively; P < 0.01). 5-HD administered either before SB21 treatment or 5 min before reperfusion the following day abrogated SB21-induced protection (54 +/- 4% and 61 +/- 2%, respectively). HMR-1098 did not affect the SB21-induced infarct size reduction when administered before the SB21 treatment (43 +/- 1%); however, HMR-1098 partially abrogated the SB21-induced infarct size reduction when administered just before reperfusion 24 h later (52 +/- 1%). The MPTP opening either before SB21 administration or 5 min before reperfusion abrogated the infarct size reduction produced by SB21 (61 +/- 2% and 62 +/- 2%, respectively). Hence, GSK inhibition reduces infarct size when given 24 h before the administration via the opening K(ATP) channels and MPTP closure.     

MCC-134, a blocker of mitochondrial and opener of sarcolemmal ATP-sensitive K+ channels, abrogates cardioprotective effects of chronic hypoxia

We examined the effect of MCC-134, a novel inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels and activator of sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels, on cardioprotection conferred by adaptation to chronic hypoxia. Adult male Wistar rats were exposed to intermittent hypobaric hypoxia (7000 m, 8 h/day, 5-6 weeks) and susceptibility of their hearts to ventricular arrhythmias and myocardial infarction was evaluated in anesthetized open-chest animals subjected to 20-min coronary artery occlusion and 3-h reperfusion on the day after the last hypoxic exposure. MCC-134 was administered intravenously 10 min before ischemia and 5 min before reperfusion in a total dose of 0.3 mg/kg or 3 mg/kg divided into two equal boluses. The infarct size (tetrazolium staining) was reduced from 59.2+/-4.4 % of the area at risk in normoxic controls to 43.2+/-3.3 % in the chronically hypoxic group. Chronic hypoxia decreased the reperfusion arrhythmia score from 2.4+/-0.5 in normoxic animals to 0.7+/-0.5. Both doses of MCC-134 completely abolished the antiarrhythmic protection (score 2.4+/-0.7 and 2.5+/-0.5, respectively) but only the high dose blocked the infarct size-limiting effect of chronic hypoxia (54.2+/-3.7 %). MCC-134 had no effect in the normoxic group. These results support the view that the opening of mitoKATP channels but not sarcKATP channels plays a crucial role in the mechanism by which chronic hypoxia improves cardiac tolerance to ischemia/reperfusion injury.     

K(ATP)(+) channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation

The role of ATP-sensitive K(+) (K(ATP)(+)) channels, nitric oxide, and adenosine in coronary exercise hyperemia was investigated. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus and instrumented with a flow transducer on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Experiments were conducted at rest and during graded treadmill exercise with and without combined inhibition of K(ATP)(+) channels (glibenclamide, 1 mg/kg iv), nitric oxide synthesis (N(omega)-nitro-L-arginine, 35 mg/kg iv), and adenosine receptors (8-phenyltheophylline, 3 mg/kg iv). During control exercise, myocardial oxygen consumption increased ~2.9-fold, coronary blood flow increased ~2.6-fold, and coronary venous oxygen tension decreased from 19.9 +/- 0.4 to 13.7 +/- 0.6 mmHg. Triple blockade did not significantly change the myocardial oxygen consumption or coronary blood flow response during exercise but lowered the resting coronary venous oxygen tension to 10.0 +/- 0.4 mmHg and during exercise to 6.2 +/- 0.5 mmHg. Cardiac adenosine levels did not increase sufficiently to overcome the adenosine receptor blockade. These results indicate that combined inhibition of K(ATP)(+) channels, nitric oxide synthesis, and adenosine receptors lowers the balance between total oxygen supply and consumption at rest but that these factors are not required for local metabolic coronary vasodilation during exercise.     

Effect of combined K(ATP) channel activation and Na(+)/H(+) exchange inhibition on infarct size in rabbits

We tested if combining treatment with cariporide, an Na(+)/H(+) exchange inhibitor, and diazoxide, a mitochondrial ATP-sensitive K(+) (K(ATP)) channel opener, would reduce myocardial infarct size (IS) to a greater extent than either intervention alone. Four groups of rabbits were studied (n = 10 each): cariporide (0.3 mg/kg), diazoxide (10 mg/kg), both drugs, and saline control, given 15 min before a 30-min coronary artery occlusion and 3 h reperfusion. IS in controls comprised 47 +/- 6% of the risk region. Cariporide reduced IS by 55% compared with control (21 +/- 3%), but diazoxide did not significantly reduce IS compared with controls (37 +/- 6%). Combined treatment resulted in an IS of 18 +/- 5%. Also we determined that diazoxide did not potentiate a subthreshold dose of cariporide nor did a mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD), prevent cariporide from reducing IS. Thus cariporide reduced necrosis by >50% in this model, both in the presence and absence of K(ATP) channel blockade. There was no significant difference in IS reduction between the group receiving cariporide alone and the group receiving combined treatment. Because the effect of cariporide was not blocked by 5-HD, it is unlikely that K(ATP) channels play a role as an end effector in cariporide's mechanism.     

Remote preconditioning reduces ischemic injury in the explanted heart by a KATP channel-dependent mechanism

Local and remote ischemic preconditioning (IPC) reduce ischemia-reperfusion (I/R) injury and preserve cardiac function. In this study, we tested the hypothesis that remote preconditioning is memorized by the explanted heart and yields protection from subsequent I/R injury and that the underlying mechanism involves sarcolemmal and mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Male Wistar rats (300-350 g) were randomized to a control (n = 10), a remote IPC (n = 10), and a local IPC group (n = 10). Remote IPC was induced by four cycles of 5 min of limb ischemia, followed by 5 min of reperfusion. Local IPC was induced by four cycles of 2 min of regional myocardial ischemia, followed by 3 min of reperfusion. The heart was excised within 5 min after the final cycle of preconditioning, mounted in a perfused Langendorff preparation for 40 min of stabilization, and subjected to 45 min of sustained ischemia by occluding the left coronary artery and 120 min of reperfusion. I/R injury was assessed as infarct size by triphenyltetrazolium staining. The influence of sarcolemmal and mitochondrial K(ATP) channels on remote preconditioning was assessed by the addition of glibenclamide (10 microM, a nonselective K(ATP) blocker), 5-hydroxydecanoic acid (5-HD; 100 microM, a mitochondrial K(ATP) blocker), and HMR-1098 (30 microM, a sarcolemmal K(ATP) blocker) to the Langendorff preparation before I/R. The role of mitochondrial K(ATP) channels as an effector mechanism for memorizing remote preconditioning was further studied by the effect of the specific mitochondrial K(ATP) activator diaxozide (10 mg/kg) on myocardial infarct size. Remote preconditioning reduced I/R injury in the explanted heart (0.17 +/- 0.03 vs. 0.39 +/- 0.05, P < 0.05) and improved left ventricular function during reperfusion compared with control (P < 0.05). Similar effects were obtained with diazoxide. Remote preconditioning was abolished by the addition of 5-HD and glibenclamide but not by HMR-1098. In conclusion, the protective effect of remote preconditioning is memorized in the explanted heart by a mechanism that involves mitochondrial K(ATP) channels.     

Delayed cardioprotection by isoflurane: role of K(ATP) channels

Isoflurane mimics the cardioprotective effect of acute ischemic preconditioning with an acute memory phase. We determined whether isoflurane can induce delayed cardioprotection, the involvement of ATP-sensitive potassium (K(ATP)) channels, and cellular location of the channels. Neonatal New Zealand White rabbits at 7-10 days of age (n = 5-16/group) were exposed to 1% isoflurane-100% oxygen for 2 h. Hearts exposed 2 h to 100% oxygen served as untreated controls. Twenty-four hours later resistance to myocardial ischemia was determined using an isolated perfused heart model. Isoflurane significantly reduced infarct size/area at risk (means +/- SD) by 50% (10 +/- 5%) versus untreated controls (20 +/- 6%). Isoflurane increased recovery of preischemic left ventricular developed pressure by 28% (69 +/- 4%) versus untreated controls (54 +/- 6%). The mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5-HD) completely (55 +/- 3%) and the sarcolemmal K(ATP) channel blocker HMR 1098 partially (62 +/- 3%) attenuated the cardioprotective effects of isoflurane. The combination of 5-HD and HMR-1098 completely abolished the cardioprotective effect of isoflurane (56 +/- 5%). We conclude that both mitochondrial and sarcolemmal K(ATP) channels contribute to isoflurane-induced delayed cardioprotection.     

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.     

Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation

We previously demonstrated a role for voltage-dependent K(+) (K(V)) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H(2)O(2), as responses were attenuated by the K(V) channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that K(V) channels participate in coronary reactive hyperemia and examined the role of K(V) channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 +/- 5% and inhibited hyperemic volume 32 +/- 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 +/- 6% block at 9 microg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 microM correolide, a selective K(V)1 antagonist (76 +/- 7% and 47 +/- 2% block, respectively, at 1 microM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 +/- 6% block at 10 microg/min) and by correolide in vitro (29 +/- 4% block at 1 microM). K(V) current in smooth muscle cells was inhibited by 4-AP (IC(50) 1.1 +/- 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for K(V)1 family members was detected in coronary arteries. Our data indicate that K(V) channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.     

Signal mechanism activated by erythropoietin preconditioning and remote renal preconditioning-induced cardioprotection

It has been recently reported that release of erythropoietin could mediate the cardioprotective effects of remote renal preconditioning. However, the mechanism of erythropoietin-mediated cardioprotection in remote preconditioning is still unexplored. Therefore, the present study was designed to investigate the possible signal transduction pathway of erythropoietin-mediated cardioprotection in remote preconditioning in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion followed by 5 min reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Remote renal preconditioning and erythropoietin preconditioning (5,000 IUkg(-1), i.p.) attenuated ischemia-reperfusion-induced myocardial injury and produced cardioprotective effects. However, administration of diethyldithiocarbamic acid (150 mg kg(-1) i.p.), a selective NFkB inhibitor, and glibenclamide (5 mg kg(-1) i.p.), a selective K(ATP) channel blocker, attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning. However, administration of minoxidil (1 mg kg(-1) i.v.), a selective K(ATP) channel opener, restored the attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning in diethyldithiocarbamic acid pretreated rats. These results suggest that K(ATP) channel is a downstream mediator of NFkB activation in remote preconditioning and erythropoietin preconditioning. Therefore, it may be concluded that erythropoietin preconditioning and remote renal preconditioning trigger similar signaling mechanisms for cardioprotection, i.e., NFkB activation followed by opening of K(ATP) channels.