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(Circulation Research. 2002;91:186.)
© 2002 American Heart Association, Inc.

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Sarcolemmal K_ATP Channel Triggers Opioid-Induced Delayed Cardioprotection in the Rat

Hemal H. Patel, Anna K. Hsu, Jason N. Peart, Garrett J. Gross

From the Medical College of Wisconsin, Department of Pharmacology and Toxicology, Milwaukee, Wis.

Correspondence to Garrett J. Gross, PhD, Medical College of Wisconsin, Department of Pharmacology and Toxicology, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail ggross{at}mcw.edu

Abstract

Recently, the involvement of sarcolemmal K_ATP (sarcK_ATP) channels in ischemic and pharmacological preconditioning (IPC and PPC) has been minimized by numerous studies suggesting a primary role for mitochondrial K_ATP (mitoK_ATP) channels in early and delayed cardioprotection. Although the mitoK_ATP 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 K_ATP channels as triggers or distal effectors of delayed cardioprotection induced by opioids in rats, and the data suggest that the sarcK_ATP channel triggers and that the mitoK_ATP channel is a distal effector of opioid-induced delayed cardioprotection.

Key Words: delayed preconditioning • opioids • trigger • sarcolemmal K_ATP • mitochondrial K_ATP

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 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⁺ (mitoK_ATP) channel has received considerable attention as being the trigger of early IPC. Studies in the isolated rabbit heart⁵ and in isolated myocytes^6,7 have suggested that this channel contributes to protective signals via a redox-sensitive mechanism.^8,9 Similarly, data suggest that the mitoK_ATP 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 K_ATP (sarcK_ATP) channel in delayed IPC.

Therefore, we investigated the role of the sarcK_ATP channel as a trigger or distal effector of delayed cardioprotection induced by opioid agonists. We present evidence suggesting that the sarcK_ATP channel functions as the trigger and the mitoK_ATP 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 opioid agonist, followed by a 24-hour recovery period. Selective and nonselective K_ATP 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.

Statistical Measurements
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 K_ATP channel blocker, HMR-1098 (6 mg/kg), a selective sarcK_ATP channel blocker, or 5-hydroxydecanoic acid (5-HD; 10 mg/kg), a selective mitoK_ATP channel blocker, before undergoing a 24-hour recovery period.

None of the K_ATP 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).

View larger version (33K): [in this window] [in a new window]   Figure 1. SNC-induced delayed protection in the presence of various KATP channel blockers. Glibenclamide (Glb), HMR-1098 (HMR), or 5-hydroxydecanoic acid (5-HD) were given simultaneously with SNC 24 hours before infarction. The graph represents infarct size expressed as a percentage of the area at risk (IS/AAR, %, mean±SEM). The protective effects of SNC were attenuated by concomitant administration of glibenclamide or HMR-1098. *P<0.001 vs Control; +P<0.001 vs SNC. Numbers in parentheses represent group size.

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 sarcK_ATP channel is a trigger and the mitoK_ATP channel is a distal effector of SNC-induced delayed cardioprotection.

View larger version (34K): [in this window] [in a new window]   Figure 2. Early administered KATP channel blockers 24 hours after SNC administration. Control rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Experimental rats were pretreated with SNC-121 (0.1 mg/kg) and allowed to recover for 24 hours. Glibenclamide (Glb; 3 mg/kg), HMR-1098 (HMR; 6 mg/kg), and 5-hydroxydecanoic acid (5-HD; 10 mg/kg) were given 20, 10, and 5 minutes before ischemia, respectively. The graph represents infarct size expressed as a percentage of the area at risk weight (IS/AAR, %, mean±SEM). The protective effect of SNC was attenuated by glibenclamide and 5-HD but not HMR-1098. *P<0.001 vs Control; +P<0.001 vs SNC. Numbers in parentheses represent group size.

MitoK_ATP 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 mitoK_ATP being the trigger in delayed cardioprotection. However, it appears that modulation of mitoK_ATP 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 mitoK_ATP channel in delayed cardioprotection, one as a trigger and one as a distal effector.

Recently, it was reported that superoxide can activate mitoK_ATP channels in reconstituted mitochondria in lipid bilayers.¹⁴ In addition, evidence suggests that free radicals are also involved in the regulation of sarcK_ATP channel opening.¹⁵ It is not known how opening the sarcK_ATP channel produces a delayed cardioprotective effect in rats; however, it is feasible that ROS are involved.

Opening of the sarcK_ATP 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 sarcK_ATP channel activity to trigger delayed cardioprotection. It has been suggested that there is crosstalk between the mitoK_ATP and sarcK_ATP channels in that ATP consumption by mitochondria activates sarcK_ATP channels.²⁰ It is possible that opioids interact with mitochondria and modify sarcK_ATP channel activity by modulation of redox-sensitive or metabolic mechanisms that trigger delayed cardioprotection. Interestingly, it was shown, in a sarcK_ATP channel knockout mouse, that IPC was absent.²¹ These data and the present results suggest that the sarcK_ATP channel needs to be further evaluated as a possible trigger in other models of delayed cardioprotection.

Acknowledgments

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.).

Received May 15, 2002; revision received June 26, 2002; accepted June 27, 2002.

References

1. Kuzuya T, Hoshida S, Yamashita N, Fuji H, Oe H, Hori M, Kamada T, Tada M. Delayed effects of sublethal ischemia on the acquisition of tolerance to ischemia. Circ Res. 1993; 72: 1293–1299.[Abstract/Free Full Text]

2. Marber MS, Latchman DS, Walker JM, Yellon DM. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress is associated with resistance to myocardial infarction. Circulation. 1993; 88: 1264–1272.[Abstract/Free Full Text]

3. Baxter GF, Goma FM, Yellon DM. Characterisation of the infarct-limiting effect of delayed preconditioning: time course and dose-dependency studies in rabbit myocardium. Basic Res Cardiol. 1997; 92: 159–167.[CrossRef][Medline] [Order article via Infotrieve]

4. Patel HH, Hsu A, Moore J, Gross GJ. BW373U86, a opioid agonist, partially mediates delayed cardioprotection via a free radical mechanism that is independent of opioid receptor stimulation. J Mol Cell Cardiol. 2001; 33: 1455–1465.[CrossRef][Medline] [Order article via Infotrieve]

5. Pain T, Yang X-M, Critz SD, Yue Y, Nakano A, Liu GS, Heusch G, Cohen MV, Downey JM. Opening of mitochondrial K_ATP channels triggers the preconditioned state by generating free radicals. Circ Res. 2000; 87: 460–466.[Abstract/Free Full Text]

6. Forbes RA, Steenbergen C, Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res. 2001; 88: 802–809.[Abstract/Free Full Text]

7. Carroll R, Gant VA, Yellon DM. Mitochondrial K_ATP channel opening protects a human atrial-derived cell line by a mechanism involving free radical generation. Cardiovasc Res. 2001; 51: 691–700.[Abstract/Free Full Text]

8. Becker LB, Vanden Hoek TL, Shao Z-H, Li C-Q, Schumacker PT. Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am J Physiol. 1999; 277: H2240–H2246.[Medline] [Order article via Infotrieve]

9. Vanden Hoek TL, Becker LB, Shao Z, Li C, Schumacker PT. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J Biol Chem. 1998; 273: 18092–18098.[Abstract/Free Full Text]

10. Schultz JJ, Kwok WM, Hsu AK, Gross GJ. Terikalant, an inward rectifier potassium channel blocker, does not abolish the cardioprotection induced by ischemic preconditioning in the rat. J Mol Cell Cardiol. 1998; 30: 1817–1825.[CrossRef][Medline] [Order article via Infotrieve]

11. Takashi E, Wang Y, Ashraf M. Activation of mitochondrial K_ATP channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res. 1999; 85: 1146–1153.[Abstract/Free Full Text]

12. Ockaili R, Emani VR, Okubo S, Brown M, Krottapalli K, Kukreja RC. Opening of mitochondrial K_ATP channel induces early and delayed cardioprotective effect: role of nitric oxide. Am J Physiol. 1999; 277: H2425–H2434.[Medline] [Order article via Infotrieve]

13. Hoag JB, Qian Y-Z, Nayeem MA, D’Angelo M, Kukreja RC. ATP-sensitive potassium channel mediates delayed ischemic protection by heat stress in rabbit heart. Am J Physiol. 1997; 273: H2458–H2464.[Medline] [Order article via Infotrieve]

14. Zhang DX, Chen Y-F, Campbell WB, Zou A-P, Gross GJ, Li P-L. Characteristics and superoxide-induced activation of reconstituted myocardial mitochondrial ATP-sensitive potassium channels. Circ Res. 2001; 89: 1177–1183.[Abstract/Free Full Text]

15. Ichinari K, Kakei M, Matsuoka T, Tanaka H. Direct activation of the ATP-sensitive potassium channel by oxygen-derived free radicals in guinea-pig ventricular cells: its potentiation by MgADP. J Mol Cell Cardiol. 1996; 28: 1867–1877.[CrossRef][Medline] [Order article via Infotrieve]

16. Cole W, McPherson C, Sontag D. ATP-regulated K⁺ channels protect the myocardium against ischemia/reperfusion damage. Circ Res. 1991; 69: 571–581.[Abstract/Free Full Text]

17. Schulz R, Rose J, Heusch G. Involvement of activation of ATP-dependent potassium channels in ischemic preconditioning in swine. Am J Physiol. 1994; 267: H1341–H1352.[Medline] [Order article via Infotrieve]

18. Tan H, Mazon P, Verberne H, Sleeswijk M, Coronel A, Opthof T, Janse M. Ischemic preconditioning delays ischaemia-induced cellular electrical uncoupling in rabbit myocardium by activation of ATP-sensitive potassium channels. Cardiovasc Res. 1993; 27: 644–651.[Abstract/Free Full Text]

19. Yao Z, Gross GJ. Activation of ATP-sensitive potassium channels lowers the threshold for ischemic preconditioning. Am J Physiol. 1994; 267: H1888–H1894.[Medline] [Order article via Infotrieve]

20. Sasaki N, Sato T, Marbán E, O’Rourke B. ATP consumption by uncoupled mitochondria activates sarcolemmal K_ATP channels in cardiac myocytes. Am J Physiol. 2001; 280: H1882–H1888.

21. Suzuki M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Seino S, Marbán E, Nakaya H. Role of sarcolemmal K_ATP channels in cardioprotection against ischemia/reperfusion injury in mice. J Clin Invest. 2002; 109: 509–516.[CrossRef][Medline] [Order article via Infotrieve]

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This Article Abstract Full Text (PDF) All Versions of this Article: 91/3/186    most recent 01.RES.0000029085.69891.F2v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Patel, H. H. Articles by Gross, G. J. Search for Related Content PubMed PubMed Citation Articles by Patel, H. H. Articles by Gross, G. J. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information Heart Attack Related Collections Ischemic biology - basic studies Ion channels/membrane transport