Sarcolemmal KATP Channel Triggers Opioid-Induced Delayed Cardioprotection in the Rat
Recently, the involvement of sarcolemmal KATP (sarcKATP) channels in ischemic and pharmacological preconditioning (IPC and PPC) has been minimized by numerous studies suggesting a primary role for mitochondrial KATP (mitoKATP) channels in early and delayed cardioprotection. Although the mitoKATP channel has clearly been shown to be a distal effector of delayed IPC and PPC, studies implicating it as a trigger of protection in delayed IPC are lacking. Accordingly, we characterized the role of cardiac KATP channels as triggers or distal effectors of delayed cardioprotection induced by opioids in rats, and the data suggest that the sarcKATP channel triggers and that the mitoKATP channel is a distal effector of opioid-induced delayed cardioprotection.
Protection from myocardial infarction via ischemic preconditioning (IPC) has been described to be a biphasic event. The early phase occurs immediately after IPC and lasts 1 to 3 hours1; the late phase of protection is seen 12 to 24 hours after the initial stimulus and lasts up to 72 hours.2,3⇓ We have previously shown that δ opioid agonists induce a delayed cardioprotective effect that appears to be mediated by a burst of reactive oxygen species (ROS).4
In recent investigations, the mitochondrial ATP-dependent K+ (mitoKATP) channel has received considerable attention as being the trigger of early IPC. Studies in the isolated rabbit heart5 and in isolated myocytes6,7⇓ have suggested that this channel contributes to protective signals via a redox-sensitive mechanism.8,9⇓ Similarly, data suggest that the mitoKATP channel could be either a trigger that induces the expression of proteins or a distal effector in delayed IPC. However, little information is available on the triggering role of the sarcolemmal KATP (sarcKATP) channel in delayed IPC.
Therefore, we investigated the role of the sarcKATP channel as a trigger or distal effector of delayed cardioprotection induced by δ opioid agonists. We present evidence suggesting that the sarcKATP channel functions as the trigger and the mitoKATP channel as a distal effector in opioid-induced delayed PC.
Materials and Methods
This study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.
Study Groups and Experimental Protocols
Male Sprague-Dawley rats (250 to 300 g) were obtained from Charles River Laboratories, Wilmington, Mass, and randomly divided into groups and subjected to pretreatment with SNC-121 (0.1 mg/kg, IV), a δ opioid agonist, followed by a 24-hour recovery period. Selective and nonselective KATP channel blockers were administered intravenously either with opioid pretreatment or after the recovery period just before index ischemia. All rats underwent 30 minutes of index ischemia followed by 2 hours of reperfusion.
General Surgical Procedure and Infarct Determination
General surgical procedures and infarct determination were performed as described previously.4 Briefly, rats were anesthetized, vessels cannulated for delivery of drugs and blood pressure measurements, and a tracheotomy performed for artificial ventilation. Subsequently a left thoracotomy was performed at the fifth intercostal space, and a pericardiotomy was performed followed by adjustment of the left atrial appendage to locate the left coronary artery. A ligature was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle. The ends of the suture were threaded through a propylene tube to form a snare. Occlusion was elicited by clamping the snare onto the epicardial surface using a hemostat.
After 2 hours of reperfusion, the coronary artery was again occluded. The area at risk (AAR) was determined by negative staining. The heart was excised, and the left ventricle was separated from the remaining tissue and cut into thin cross-sectional pieces. The normal area and AAR were separated and placed in different vials containing 1% 2,3,5-triphenyltetrazolium chloride (TTC) in 100 mmol/L phosphate buffer (pH 7.4). Tissues were fixed overnight in 10% formaldehyde, and the infarcted tissue was dissected from the AAR using a dissecting microscope. In 9 additional rats (5 treated with SNC only and 4 treated with SNC+HMR-1098), epicardial monophasic action potential duration at 50% repolarization (APD50) was determined by a bipolar electrode (EP Technologies) as previously described.10 The average of 10 consecutive arrhythmia-free readings was used to calculate APD50.
All values are expressed as mean SEM. For the hemodynamic data, left ventricle mass, AAR, infarct size, and APD, statistical significance was determined by performing a one-way ANOVA with Bonferroni’s multiple-comparison test as the post hoc test. Significance was set at P<0.05.
Results and Discussion
Rats were simultaneously pretreated with SNC and glibenclamide (3 mg/kg), a nonselective KATP channel blocker, HMR-1098 (6 mg/kg), a selective sarcKATP channel blocker, or 5-hydroxydecanoic acid (5-HD; 10 mg/kg), a selective mitoKATP channel blocker, before undergoing a 24-hour recovery period.
None of the KATP channel blockers given alone resulted in an infarct different from control; however, the delayed cardioprotective effect of SNC (29±2% versus 58±1%, P<0.001) was attenuated by simultaneous treatment with glibenclamide (46±4%, P<0.001) or HMR-1098 (51±5%, P<0.001) but not with 5-HD (35±3%) (Figure 1). In addition, SNC shortened APD50 from 61±9 to 31±4 ms (P<0.01) (n=5); however, SNC did not shorten APD50 in the presence of HMR-1098 (47±2 to 49±2 ms, n=4).
Treatment with glibenclamide (3 mg/kg) or 5-HD (10 mg/kg), given 20 and 5 minutes before index ischemia, attenuated the protective effects of SNC pretreatment on the following day (52±4% and 50±5% versus 29±2%, P<0.001, respectively, Figure 2). However, HMR-1098 had no effect on the protective effects of SNC. These results suggest that the sarcKATP channel is a trigger and the mitoKATP channel is a distal effector of SNC-induced delayed cardioprotection.
MitoKATP channels have been implicated as triggers of delayed cardioprotection. Takashi el al11 showed that administration of diazoxide, 24 hours before prolonged ischemia, protected hearts from damage. This protection was abrogated by simultaneous administration of 5-HD on day 1. Additional studies with diazoxide in rabbits12 also suggested the possibility of mitoKATP being the trigger in delayed cardioprotection. However, it appears that modulation of mitoKATP and its involvement as a trigger of delayed cardioprotection may be dependent on the conditioning stimulus. Hoag et al13 reported that heat stress induced a delayed cardioprotective effect in which infarct size was reduced 24 hours later in rabbits. Administration of glibenclamide and 5-HD after the recovery period before ischemia abolished this protective effect. Interestingly, administration of glibenclamide or 5-HD before heat stress did not abolish delayed cardioprotection. Taken together, these data suggest a possible dual role for the mitoKATP channel in delayed cardioprotection, one as a trigger and one as a distal effector.
Recently, it was reported that superoxide can activate mitoKATP channels in reconstituted mitochondria in lipid bilayers.14 In addition, evidence suggests that free radicals are also involved in the regulation of sarcKATP channel opening.15 It is not known how opening the sarcKATP channel produces a delayed cardioprotective effect in rats; however, it is feasible that ROS are involved.
Opening of the sarcKATP channel has been associated with shortening of the APD,16 hyperpolarization of the cell, decreased calcium entry, and preserved ATP production.17–19⇓⇓ Mechanistically, our data suggest that SNC may be facilitating the trigger phase via shortening of APD because HMR-1098 blocked the APD shortening and cardioprotective effect of SNC. We have shown in previous studies4 that the trigger phase of early IPC or pharmacological preconditioning (PPC) is also linked to the generation of an early burst of ROS, the source and timing of which are presently unknown. The possibility exists that ROS may modulate sarcKATP channel activity to trigger delayed cardioprotection. It has been suggested that there is crosstalk between the mitoKATP and sarcKATP channels in that ATP consumption by mitochondria activates sarcKATP channels.20 It is possible that opioids interact with mitochondria and modify sarcKATP channel activity by modulation of redox-sensitive or metabolic mechanisms that trigger delayed cardioprotection. Interestingly, it was shown, in a sarcKATP channel knockout mouse, that IPC was absent.21 These data and the present results suggest that the sarcKATP channel needs to be further evaluated as a possible trigger in other models of delayed cardioprotection.
This work was supported by National Heart, Lung, and Blood Institute grant HL-08311 (G.J.G.) and by a predoctoral fellowship from the American Heart Association (H.H.P.).
Original received May 15, 2002; revision received June 26, 2002; accepted June 27, 2002.
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