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Am J Physiol Heart Circ Physiol 288: H559-H567, 2005. First published September 30, 2004; doi:10.1152/ajpheart.00845.2004
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Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction

Michael A. Moses,1,2 Patrick D. Addison,1,2 Peter C. Neligan,1,2 Homa Ashrafpour,1 Ning Huang,1 Murtuza Zair,1,3 Alispasha Rassuli,1 Christopher R. Forrest,1,2 Gary J. Grover,4 and Cho Y. Pang1,2,3

1Research Institute, The Hospital for Sick Children; and Departments of 2Surgery and 3Physiology, University of Toronto, Toronto, Ontario, Canada; and 4Bristol-Myers Squibb Pharmaceutical Research Institute, Pennington, New Jersey


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (~300 mmHg) elicited protection against ischemia-reperfusion injury (infarction) in multiple distant skeletal muscles subsequently subjected to 4 h of ischemia and 48 h of reperfusion, but the mechanism was not studied. The aim of this project was to test our hypothesis that mitochondrial ATP-sensitive potassium (KATP) (mKATP) channels play a central role in the trigger and mediator mechanisms of hindlimb remote ischemic preconditioning (IPC) of skeletal muscle against infarction in the pig. We observed in the pig that hindlimb remote IPC reduced the infarct size of latissimus dorsi (LD) muscle flaps (8 x 13 cm) from 45 ± 2% to 22 ± 3% (n = 10; P < 0.05). The nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 (3 mg/kg), abolished the infarct-protective effect of hindlimb remote IPC in LD muscle flaps (n = 10, P < 0.05) when these drugs were injected intravenously at 10 min before remote IPC. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at the end of hindlimb remote IPC also abolished the infarct protection in LD muscle flaps (n = 10; P < 0.05). Furthermore, intravenous injection of the specific mKATPchannel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of ischemia protected the LD muscle flap against infarction to a similar extent as hindlimb remote IPC, and this infarct-protective effect of BMS-191095 was abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before or after intravenous injection of BMS-191095 (n = 10; P < 0.05). The infarct protective effect of BMS-191095 was associated with a higher muscle content of ATP at the end of 4 h of ischemia and a decrease in muscle neutrophilic myeloperoxidase activity at the end of 1.5 h of reperfusion compared with the time-matched control (n = 10, P < 0.05). These observations led us to conclude that mKATPchannels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig, and the opening of mKATP channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.

pig skeletal muscle; remote ischemic preconditioning; trigger and mediator mechanism

MURRY ET AL. (20) reported that ischemic preconditioning (IPC) of dog myocardium with four cycles of 5 min of coronary artery occlusion interspersed with 5 min of reperfusion attenuated myocardial infarction by 75% when the preconditioned myocardium was subsequently subjected to 40 min of sustained ischemia and 4 days of reperfusion. Przyklenk et al. (35) reported that this regional infarct protective effect of IPC extended beyond the preconditioned myocardium. Specifically, they observed that instigation of four cycles of 5 min of occlusion and reperfusion of the circumflex coronary artery in the dog heart not only protected the myocardium perfused by the circumflex artery but also protected the myocardium perfused by the left anterior descending coronary artery. This cardioprotective effect of "intraorgan" IPC may be limited to animal species and model (12, 21, 26). Subsequently, it was reported that a brief ischemia in a distant organ also provided cardioprotection similar to that of regional IPC. For example, a brief occlusion of the mesenteric artery triggered cardioprotection in the rat (6, 17, 32, 38, 52, 54) and the rabbit (44). A brief occlusion of a renal artery in the rabbit (33, 43) and a femoral artery or the infrarenal aorta in the rat (34, 53) also induced cardioprotection against infarction. However, these remote IPC techniques are invasive and thus have limited clinical application. Of special interest to us was the report from Oxman et al. (25) that 10 min of hindlimb ischemia by tourniquet application preconditioned the heart against reperfusion tachyarrhythmia in the rat. This cardioprotective effect of noninvasive remote IPC was subsequently studied in human coronary artery bypass surgery using two cycles of 3 min of ischemia and 2 min of reperfusion in an upper limb, but the result was inconclusive (11). More recently, Pang et al. (27) demonstrated in the pig that three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (i.e., hindlimb remote IPC) protected multiple distant skeletal muscles against infarction. This noninvasive remote IPC has potential clinical application in prophylactic treatment against skeletal muscle ischemia-reperfusion (I/R) injury in autogenous muscle transplantation and vascular and musculoskeletal reconstructive surgery performed under tourniquet or vascular clamp control. Subsequently, other investigators have also reported that four 5-min cycles of occlusion and reperfusion in a hindlimb of the pig induced cardioprotection against infarction (15).

However, very little is known about the mechanism of noninvasive hindlimb remote IPC. We observed in the pig that hindlimb remote IPC against infarction in various distant muscles was mediated by a humoral but not neuronal pathway, and opioid but not adenosine receptors were involved in the humoral pathway (1). The postreceptor signaling pathway of hindlimb remote IPC has not been studied. We and other investigators (9, 29) observed previously that the specific mitochondrial ATP-sensitive potassium (mKATP) channel opener diazoxide and BMS-191095 increased ischemic tolerance in the pig and dog skeletal muscle, respectively. Therefore, we hypothesized that mKATP channels play a pivotal role in the hindlimb remote IPC of skeletal muscle against infarction. We tested our hypothesis by utilizing pharmacological probes to investigate the role of mKATP channels in the trigger and mediator mechanisms of hindlimb IPC against infarction in distant skeletal muscle in the pig. We also investigated the effect of pharmacological activation of mKATP channels on muscle content of ATP and myeloperoxidase activity during sustained I/R. Results obtained from the present studies support our hypothesis that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental Surgery

Castrated Yorkshire pigs (18.9 ± 1.1 kg) were used. The following experimental surgery and protocols were approved by the Animal Care Committee of The Hospital for Sick Children and were in compliance with the guidelines of the Canadian Council of Animal Care.

The pigs were under general anesthesia during surgery, IPC, sustained muscle ischemia, muscle biopsies, and excision of latissimus dorsi (LD) muscle flaps for assessment of ischemic necrosis (infarction). All these procedures were performed in a temperature-controlled (24°C) surgical room and were described previously (1).

Anesthesia. General anesthesia was induced with intramuscular ketamine (25 mg/kg) and intravenous pentobarbitone sodium (10–15 mg/kg). The pig was intubated with an endotracheal tube of 6.5 mm in diameter and mechanically ventilated with 50% oxygen-50% nitrous oxide to a tidal volume of 15 mg/kg. Body fluid and general anesthesia were maintained by intravenous infusion of saline (2 ml/min) containing pentobarbitone sodium (~0.5 mg·kg–1·min–1). Rectal temperature was monitored and kept between 38°C and 39°C by warming the pig with a heating blanket.

Bilateral LD muscle flap model. An island LD muscle flap (8 x 13 cm) based on the thoracodorsal neurovascular bundle was dissected and raised bilaterally in each pig. The thoracodorsal nerve was transected to mimic the clinical situation in autogenous muscle transplantation. The muscle tendon (1 cm wide) used to support the vascular pedicle of the LD muscle flap was ligated with 2-0 silk sutures. Therefore, blood supply to the island LD muscle flap was derived entirely from the thoracodorsal artery and drained by two thoracodorsal veins. The muscle flap was sutured to its original site with 3-0 Vicryl sutures, and the skin overlying the muscle flap was closed with 3-0 nylon sutures, leaving a small opening in the axilla for access to the vascular bundle for clamping to induce total muscle flap ischemia.

Induction of I/R injury in LD muscle flaps. Two microvascular clamps (2 x 8 mm, Weck) were used to occlude the vascular pedicle of each island LD muscle flap to render it globally ischemic. Absence of yellow fluorescence under ultraviolet light within 10 min after intravenous injection of fluorescein dye (15 mg/kg) confirmed global ischemia in the LD muscle flap. Vascular clamps were removed at the end of 4 h of warm global ischemia. Reperfusion was ascertained by a second intravenous injection of fluorescein dye and by the presence of yellow fluorescein after 5 min of dye injection. Skin wounds were closed with 3-0 nylon sutures, and the pig was allowed to recover and was returned to an animal holding room.

We previously observed that muscle infarction did not occur in 8 x 13 cm pig LD muscle flaps if the muscle flaps were not subjected to an ischemic insult (31). Therefore, a nonischemic control group was not planned in the present studies to avoid unnecessary euthanizing of pigs.

Clinically, tolerance of skeletal muscle to warm global ischemia is limited to <2.5 h (3, 16, 47). We observed previously in the pig that 8 x 13 cm LD muscle flaps sustained 42–45% infarction when subjected to 4 h of warm ischemia and 48 h of reperfusion (1, 14, 28, 30, 31). Therefore, the same I/R time was used in the present studies.

Noninvasive remote IPC technique. With the pig under general anesthesia, remote IPC was elicited by three cycles of 10 min of occlusion and reperfusion in a hindlimb of the pig by tourniquet application (~300 mmHg). From the result from our previous study, this protocol of hindlimb IPC would reduce the infarct size of 8 x 13 cm LD muscle flaps by ~55% when these muscle flaps were subsequently subjected to 4 h of ischemia and 48 h of reperfusion (1).

Assessment of I/R injury (infarction) in LD muscle flaps. Bilateral LD muscle flaps were excised under general anesthesia. Each 8 x 13 cm LD muscle flap was cut transversely into thirteen 8 x 1 cm segments for assessment of muscle infarction, using the nitroblue tetrazolium dye staining technique as described by us previously (31). Muscle infarction was calculated as the percentage of total muscle flap at risk.

The pig was then euthanized with an overdose of pentobarbitone sodium.

Chemical analysis of muscle samples. Muscle biopsies (0.5 x 0.5 cm) were taken sequentially from the dorsal edge of the LD muscle flap immediately before and at the end of 2 and 4 h of ischemia and 1.5 h of reperfusion. The locations and technique for taking muscle biopsies were described previously (31). All muscle biopsies were immediately rinsed with cold (4°C) isotonic saline, blotted, frozen in liquid nitrogen, and stored at –80°C. The procedures for muscle sample extraction and assay of ATP, protein content, and myeloperoxidase (MPO) activity were described by us previously (1).

Drug Preparation

All chemicals, drugs, and assay kits were purchased from Sigma (Oakville, Ontario, Canada) unless otherwise stated. (3R)-trans-4-[(4-chlorophenyl)-N-(1H-imidazole-2-ylmethyl)dimethyl-2H-1-benzopyran-6-carbonitril monohydrochloride] (BMS-191095, Bristol-Myers Squibb Pharmaceutical) was dissolved (2 mg/kg) in 2 ml of polyethylene glycol 400 (10). Glibenclamide (0.3 or 1.0 mg/kg) was dissolved in a mixture of NaCl (0.9%), ethanol (70%), and NaOH (80 mM) and in a ratio of 35:4:1 in volume, respectively (41). 1-[[5-[2-(5-Chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea sodium salt (HMR-1098, Aventis Pharma) was dissolved (3 mg/kg) in 10 ml of saline (0.9%). 5-Hydroxydecanoate sodium (5 or 10 mg/kg) was dissolved in 10 ml of saline (0.9%). Purified distilled water from a Milli-Q Water System (Bedford, MA) was used for making solutions and buffers. All drug solutions were prepared ~30 min before injection.

Drug Dosage

It was reported that the optimal intravenous dose of the sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 and the specific mKATP channel inhibitor 5-HD were 3 and 10 mg/kg, respectively (4). Other investigators also reported that the effective dose of the nonspecific KATP channel inhibitor glibenclamide and 5-HD for blocking the cardioprotective effect of acute local IPC in laboratory animals were 0.3–1.0 mg/kg (7, 40, 46) and 5 mg/kg (13), respectively. On the other hand, the effective dose of the specific mKATP channel opener BMS-191095 for cardioprotection in the dog was 0.4–3.6 mg/kg (9, 10). From this published information, we chose the following intravenous drug dosages for the present studies: 3 mg/kg HMR-1098, 5–10 mg/kg 5-HD, 0.3–1 mg/kg glibenclamide, and 2 mg/kg BMS-191095.

Experimental Protocol

Protocol 1: Role of mKATP channels in the trigger mechanism of hindlimb remote IPC of skeletal muscle against infarction. Pigs with bilateral LD muscle flaps were assigned to one of eight groups as shown in Fig. 1. All LD muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion. In groups 2–5, remote IPC against infarction in LD muscle flaps was elicited by instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb of the pig by tourniquet application. The selective sKATP channel inhibitor HMR-1098 (3 mg/kg), nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg), or selective mKATP channel inhibitor 5-HD (5 mg/kg) was injected intravenously in pigs of groups 3, 4, and 5, respectively, at 10 min before IPC. HMR-1098, glibenclamide, or 5-HD (the same doses as stated previously) was injected in pigs of groups 6, 7, and 8, respectively, at 10 min before sustained ischemia. Infarction in LD muscle flaps was assessed at the end of 48 h of reperfusion using the nitroblue tetrazolium dye staining technique.



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  		Fig. 1. Experimental protocol 1. Noninvasive remote ischemic preconditioning (RIPC) against infarction in pig latissimus dorsi (LD) muscle flaps was elicited by instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb of the pig by tourniquet application. HMR-1098, glibenclamide, or 5-hydroxydecanoate (5-HD) was injected intravenously at 10 min before IPC or before 4 h of sustained ischemia. All muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion.

 
Protocol 2: Role of mKATP channels in the mediator mechanism of hindlimb remote IPC of skeletal muscle against infarction. Pigs with bilateral LD muscle flaps were assigned to one of eight groups as shown in Fig. 2. All muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion. In groups 2–6, remote IPC against infarction in LD muscle flaps was elicited by instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb of the pig by tourniquet application. A low dose of the nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-HD (5 mg/kg) was injected intravenously in pigs of groups 3 and 4, respectively, at 10 min after IPC. A higher dose of glibenclamide (1 mg/kg) was injected intravenously in pigs of groups 5 and 6, respectively, at 10 min after IPC. A high dose of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) was injected intravenously in pigs of groups 7 and 8, respectively, at 10 min before 4 h of sustained ischemia. Infarction in LD muscle flaps was assessed at the end of 48 h of reperfusion, using the nitroblue tetrazolium dye staining technique.



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  		Fig. 2. Experimental protocol 2. Noninvasive RIPC against infarction in pig LD muscle flaps was elicited by instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb of the pig by tourniquet application. A high or low dose of glibenclamide or 5-HD was injected intravenously at 10 min before 4 h of sustained ischemia, with or without preceding RIPC. All LD muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion.

 
Protocol 3: Critical time points in opening mKATP channels for protection of skeletal muscle against infarction. Pigs with bilateral island LD muscle flaps were assigned to one of the six groups to receive the following treatments: 1) 4 h of sustained ischemia in LD muscle flaps (ischemic control); 2) hindlimb remote IPC before 4 h of sustained ischemia in LD muscle flaps; 3) intravenous bolus injection of the specific mKATP channel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of sustained ischemia; 4) intravenous bolus injection of the selective mKATP channel inhibitor 5-HD (5 mg/kg) at 10 min before intravenous bolus injection of BMS-191095 (2 mg/kg), and 10 min later followed by 4 h of sustained ischemia in LD muscle flaps; 5) intravenous bolus injection of 5-HD (5 mg/kg) at 10 min after bolus injection of BMS-191095 (2 mg/kg), and 10 min later followed by 4 h of sustained ischemia in LD muscle flaps. The muscle infarction in LD muscle flaps was assessed at 48 h after reperfusion, using the nitroblue tetrazolium dye staining technique.

Protocol 4: Effect of the selective mKATP channel opener BMS-191095 on energy metabolism and neutrophil accumulation in ischemic skeletal muscle. Muscle biopsies (0.5 x 0.5 cm) were taken from bilateral LD muscle flaps immediately before and at the end of 2 and 4 h of sustained ischemia and 1.5 h of reperfusion, with or without (control) intravenous bolus injection of BMS-191095 (2 mg/kg) at 10 min before 4 h of sustained ischemia.

Statistics

All values are expressed as means ± SE. The number of observations and the specific statistical analysis are described in the legends of the figures and table. Statistical significance was set at P ≤ 0.05 for all tests.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of Hindlimb Remote IPC and Drug Treatments on Mean Arterial Blood Pressure of the Pig

In a preliminary study, the mean arterial blood pressure was monitored for 60 min in anesthetized pigs after three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application or after intravenous bolus injection of the selective mKATP channel opener BMS-191095 (2 mg/kg), the selective sKATP channel inhibitor HMR 1018 (3 mg/kg), the selective mKATP channel inhibitor 5-HD (10 mg/kg), or the nonselective KATP channel inhibitor glibenclamide (1 mg/kg). It was observed that neither hindlimb remote IPC nor intravenous drug injection had any significant effect on mean arterial blood pressure in the pig compared with the control (baseline) within each group (Table 1).


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  		Table 1. Effect of hindlimb IPC and intravenous bolus injection of BM-191095, HMR-1018, 5-HD, or glibenclamide on mean arterial blood pressure

 
Role of mKATP Channels in Trigger Mechanism of Hindlimb Remote IPC of Skeletal Muscle Against Infarction

Remote IPC elicited by three cycles of 10 min of occlusion and reperfusion in a hindlimb of the pig reduced the infarct size of LD muscle flaps by 50% (P < 0.05) compared with the ischemic control when these LD muscles were subsequently subjected to 4 h of ischemia and 48 h of reperfusion (Fig. 3). Intravenous bolus injection of the selective sKATP channel inhibitor HMR-1098 (3 mg/kg) at 10 min before remote IPC did not have any significant effect on the infarct protective effect of hindlimb remote IPC because the infarct size of LD muscle flaps in this treatment group was similar to that of LD muscle flaps undergoing hindlimb remote IPC without any drug treatment (Fig. 3). On the other hand, intravenous bolus injection of the nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-HD (5 mg/kg) at 10 min before hindlimb remote IPC completely abolished the infarct protective effect of remote IPC because the infarct size of these two treatment groups was similar to that of the ischemic control. In addition, intravenous injection of HMR-1098 (3 mg/kg), glibenclamide (0.3 mg/kg), or 5-HD (5 mg/kg) at 10 min before 4 h of sustained ischemia did not affect the infarct size of LD muscle flaps compared with the ischemic control (Fig. 3). Taken together, these observations indicate that mKATP, but not sKATP channels, are involved in the trigger mechanism of hindlimb remote IPC of skeletal muscle against infarction.



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  		Fig. 3. Role of mitochondrial ATP-sensitive potassium (mKATP) channels in the trigger mechanism of hindlimb remote RIPC of skeletal muscle against infarction. The specific sarcolemmal (sKATP) channel inhibitor HMR-1018 (HMR, 3 mg/kg), the nonspecific KATP channel inhibitor glibenclamide (Glib, 0.3 mg/kg), or the specific mKATP channel inhibitor 5-HD (5 mg/kg) was injected intravenously at 10 min before RIPC or sustained ischemia. Control and treatment LD muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion. Values are means ± SE; n = 5 pigs (10 muscle flaps per group). *Means are similar, and these means are significantly (P < 0.05) different from the rest of the means without an asterisk (one-way ANOVA and Newman-Keuls multiple comparison test).

 
Role of mKATP Channels in Mediator Mechanism of Hindlimb Remote IPC of Skeletal Muscle Against Infarction

Intravenous bolus injection of the nonspecific KATP channel inhibitor glibenclamide or the specific mKATP channel inhibitor 5-HD at the end of hindlimb IPC abolished the protective effect of hindlimb IPC against distant muscle infarction in a dose-dependent manner. Specifically, intravenous bolus injection of a low dose of glibenclamide (0.3 mg/kg) or 5-HD (5 mg/kg) at the end of hindlimb remote IPC abolished the infarct protective effect of remote IPC in LD muscle flaps by 48% and 59%, respectively, but this partial blockade of infarct protection was not statistically significant. However, intravenous bolus injection of a higher dose of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) significantly (P < 0.05) and completely blocked the infarct protective effect of hindlimb remote IPC against infarction in LD muscle flaps. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at 10 min before 4 h of sustained ischemia followed by 48 h of reperfusion did not have any effect on infarct size compared with the ischemic control (Fig. 4). Collectively, these observations indicate that mKATP channels also play a central role in the mediator mechanism of hindlimb remote IPC of distant skeletal muscle against infarction in the pig.



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  		Fig. 4. Role of mKATP channels in the mediator mechanism of hindlimb RIPC of skeletal muscle against infarction. Nonspecific KATP channel inhibitor Glib (0.3 or 1.0 mg/kg) or the specific mKATP channel inhibitor 5-HD (5 or 10 mg/kg) was injected intravenously at the end of hindlimb RIPC or 10 min before sustained ischemia. All LD muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion. Values are means ± SE; n = 5 pigs (10 LD muscle flaps per group). *Means significantly different (P < 0.05) from means without an asterisk or dagger. {dagger}Means similar to means with or without an asterisk (one-way ANOVA and Newman-Keuls multiple comparison test).

 
Critical Time Points in Opening mKATP Channels for Protection of Skeletal Muscle Against Infarction

The infarct size of LD muscle flaps preconditioned with an intravenous bolus injection of the specific mKATP channel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of sustained ischemia followed by 48 h of reperfusion was 22 ± 2% (Fig. 5). This infarct size was significantly (P < 0.05) smaller than that of the ischemic control LD muscle flaps (40 ± 2%) but was similar to that of the LD muscle flaps undergoing hindlimb remote IPC before 4 h of sustained ischemia (23 ± 4%). However, intravenous bolus injection of the selective mKATP channel opener 5-HD (5 mg/kg) at 10 min before or after intravenous bolus injection of BMS-191095 completely abolished the infarct protective effect of BMS-191095 because the LD muscle infarct size in these two groups was similar to that of the ischemic control (Fig. 5). These observations indicate that the specific mKATP channel opener BMS-191095 mimicked the infarct protective effect of hindlimb remote IPC in LD muscle flaps in the pig. More importantly, these observations also indicate that, to achieve infarct protection, mKATP channels have to remain open for more than 10 min after the triggering effect, thus supporting the above observation that mKATP channels play a key role in the mediator mechanism of hindlimb remote IPC of skeletal muscle against infarction.



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  		Fig. 5. Critical time points in opening mKATP channels for protection of skeletal muscle against infarction. RIPC was elicited with three cycles of 10 min of occlusion and reperfusion in a hindlimb of the pig. The specific mKATP channel inhibitor 5-HD (5 mg/kg) was injected intravenously in a bolus at 10 min before or after intravenous bolus injection of BMS-191095 (2 mg/kg). All LD muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion. Values are means ± SE; n = 5 pigs (10 muscle flaps per group). *Means are similar, but these means are significantly (P < 0.05) different from the rest of the means (one-way ANOVA and Newman-Keuls multiple comparison test).

 
In protocol 1, we observed that intravenous bolus injection of 5-HD (5 mg/kg) did not have any effect on the infarct size of LD muscle flaps subjected to 4 h of ischemia and 48 h of reperfusion, compared with the ischemic control (Fig. 3). Therefore, this group of LD muscle flaps was not repeated in the present study to avoid unnecessary euthanizing of pigs.

Effect of BMS-191095 on ATP Content and Neutrophilic MPO Activity in Ischemic Skeletal Muscle

In this study, the treatment group received an intravenous bolus injection of BMS-191095 (2 mg/kg) at 10 min before sustained ischemia. There was no difference in the muscle content of ATP in control and treatment LD muscle flaps before 4 h of sustained ischemia (Fig. 6). The muscle content of ATP decreased in the control and treatment group during the 4 h of sustained ischemia. The muscle content of ATP was higher in the treatment group compared with the time-matched control at the end of 4 h of ischemia (16.8 ± 3.1 vs. 7.2 ± 2.4 µmol/g protein) and 1.5 h of reperfusion (19.6 ± 3.1 vs. 9.6 ± 1.7 µmol/g protein) (Fig. 6).



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  		Fig. 6. Muscle content of ATP during 4 h of ischemia and 1.5 h of reperfusion in control LD muscle flaps and in LD muscle flaps preconditioned with intravenous bolus injection of the specific mKATP channel opener BMS-191095 (2 mg/kg) at 10 min before sustained ischemia. Values are means ± SE; n = 5 pigs. *Means are significantly different from the time-matched control (two-way ANOVA and t-test).

 
The neutrophilic MPO activity was similar between the control and treatment LD muscle flaps before and at the end of 2 and 4 h of sustained ischemia (Fig. 7). However, the MPO activity was higher in the treatment group (3.0 ± 0.5 U/g wet muscle wt) compared with the time-matched control (0.8 ± 0.2 U/g wet muscle wt) at the end of 1.5 h of reperfusion.



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  		Fig. 7. Muscle myeloperoxidase activity during 4 h of ischemia and 1.5 h of reperfusion in control LD muscle flaps and in LD muscle flaps preconditioned with intravenous bolus injection of the specific mKATP channel opener BMS-191095 (2 mg/kg) at 10 min before sustained ischemia. Values are means ± SE; n = 5 pigs. *Means are significantly (P < 0.05) different from the time-matched control (two-way ANOVA and t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Important Findings From Present Studies

In the present studies, we used pharmacological probes to demonstrate for the first time that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig. The infarct protective effect induced by the specific mKATP channel opener BMS-191095 was associated with muscle energy preservation during sustained ischemia and a decrease in neutrophil accumulation during reperfusion.

Role of mKATP Channels in Trigger Mechanism of Hindlimb Remote IPC of Skeletal Muscle Against Infarction

Experimental evidence available thus far indicates that mKATP channels play a central role in acute local or remote IPC of myocardium against infarction. For example, it was observed that the selective mKATP channel inhibitor 5-HD abolished the cardioprotective effect of acute local IPC when administered briefly before IPC in the rat (4, 5, 19, 22, 39, 45), rabbit (13, 26), and dog (2). 5-HD treatment also abolished the cardioprotective effect of the specific mKATP channel opener diazoxide when administered briefly before and during diazoxide infusion in the rabbit heart (48, 51). Furthermore, 5-HD also abolished the cardioprotective effect of acute remote IPC elicited by a 10-min occlusion of a renal artery in the rabbit (33). On the other hand, it was reported that an intravenous bolus injection of 5-HD (5 or 10 mg/kg) at 15 min before acute local IPC failed to attenuate the cardioprotective effect of IPC in the pig. These investigators suggested that mKATP channels are less important in the cardioprotection afforded by IPC in the pig (42). However, it is possible that a shorter 5-HD treatment time before IPC and/or a higher dose of 5-HD could have attenuated the cardioprotective effect of acute local IPC in the pig (4, 41).

Here, we demonstrated for the first time that mKATP channels also play a key role in the trigger mechanism of hindlimb remote IPC of skeletal muscle against infarction. Specifically, intravenous bolus injection of the nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-HD (5 mg/kg) at 10 min before hindlimb remote IPC completely abolished the infarct protective effect of IPC in LD muscle flaps in the pig. In addition, this infarct protective effect of hindlimb remote IPC was not affected by intravenous injection of the specific sKATP channel inhibitor HMR-1098 (3 mg/kg) at 10 min before IPC (Fig. 3). Furthermore, intravenous bolus injection of the selective mKATP channel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of sustained ischemia in pig LD muscle flaps protected the muscle flaps from infarction to a similar extent as hindlimb remote IPC, and this infarct protective effect of BMS-191095 was completely abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before intravenous injection of BMS-191095 (Fig. 5). Taken together, we have demonstrated that mKATP channels play a central role in the trigger mechanism of hindlimb remote IPC of skeletal muscle against infarction in the pig.

Role of mKATP Channels in Mediator Mechanism of Hindlimb Remote IPC of Skeletal Muscle Against Infarction

The role of mKATP channels in the mediation of acute local IPC of myocardium against infarction is unclear at the present time. For example, it was reported that the cardioprotective effect of IPC in isolated rabbit heart could not be abolished by infusion of 5-HD (200 µM) starting after IPC and throughout sustained ischemia, thus suggesting that mKATP channels were not involved in the mediator mechanism of IPC (26). However, a higher dose of 5-HD may be required in this model to inhibit the primed mKATP channels after IPC (48, 51). It was also reported that intravenous bolus injection of glibenclamide (1.5 mg/kg) after IPC plus glibenclamide infusion (150 µg·kg–1·min–1) throughout sustained ischemia failed to attenuate the cardioprotective effect of local IPC in the pig (41). Again, the results from this study may not be conclusive because the dose of glibenclamide used in this study was so high that it also significantly increased the infarct size of the ischemic control group. On the other hand, there were reports to indicate that mKATP channels most likely play a key role in the mediator mechanism of local IPC of myocardium against infarction. Specifically, it was reported that the cardioprotective effect of local IPC was abolished when 5-HD was administered 1) during and 5 min after IPC in the dog (2); 2) after preconditioning ischemia in the rat (5) and rabbit (24); and 3) at 16 min after IPC in the rat (22). The myocardial protective effect of diazoxide was also abolished when 5-HD was administered at 20 min after intravenous bolus injection of diazoxide in the rabbit (23) and at 5–10 min after 10 min of diazoxide infusion in the rabbit heart (48, 51).

The role of mKATP channels in the mediation of remote IPC has not been studied in any tissue or organ. Here, we have presented convincing evidence to demonstrate for the first time that mKATP channels play a key role in the mediator mechanism of hindlimb remote IPC of skeletal muscle against infarction in the pig. Specifically, intravenous bolus injection of glibenclamide or 5-HD at the end of three cycles of 10-min hindlimb remote IPC attenuated the muscle infarction of LD muscle flaps in a dose-dependent manner, and a high dose of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) completely abolished the infarct protective effect of remote IPC (Fig. 4). This high dose of glibenclamide or 5-HD did not significantly aggravate infarction in the ischemic control muscle flaps. In addition, intravenous bolus injection of 5-HD (5 mg/kg) at 10 min after intravenous bolus injection of the selective mKATP opener BMS-191095 (3 mg/kg) also abolished the infarct protective effect of BMS-191095 (Fig. 5). At the present time, it is not known whether the cellular and molecular mechanisms by which mKATP channels serve as a trigger and mediator in hindlimb remote IPC are similar to those described for local IPC (8, 18, 55).

Effect of BMS-191095 on ATP Content and Neutrophilic MPO Activity in Ischemic Skeletal Muscle

We reported previously that hindlimb remote IPC maintained a higher content of ATP in LD muscle flaps in the pig at the end of 4 h of sustained ischemia compared with the time-matched ischemic control (1). Here, we demonstrated that intravenous bolus injection of the selective mKATP channel opener BMS-191095 at 10 min before 4 h of sustained ischemia in LD muscle flaps in the pig also maintained a higher muscle ATP content at the end of 4 h of ischemia compared with the control, but the mechanism is not known (Fig. 6). There is the possibility that opening the mKATP channels in the LD muscle as a result of hindlimb remote IPC reduces the rate of ATP hydrolysis (37) or mitochondrial ATPase activity (49, 50), thereby decreasing the rate of ATP depletion during sustained ischemia. It is unlikely that this energy-sparing effect was related to opening sKATP channels to cause action potential shortening and reduce contractility because the LD muscle flaps used in the present studies were surgically denervated and were noncontractile.

We also observed previously that hindlimb remote IPC reduced neutrophil accumulation as indicated by the neutrophilic MPO activity within 1.5 h of reperfusion in pig LD muscle flaps subjected to 4 h of ischemia (1). Here, we observed that intravenous bolus injection of BMS-191095 at 10 min before 4 h of sustained ischemia in LD muscle flaps in the pig also attenuated neutrophil accumulation at 1.5 h of reperfusion, but the mechanism is not known (Fig. 7). We speculate that the infarct protective effect of hindlimb remote IPC mediated by opening mKATP channels decreases ischemic injury and inflammation in LD muscle flaps, and this in turn decreases local neutrophil adhesion and accumulation, thus reducing cellular injury at reperfusion.

In summary, we demonstrated for the first time that mKATP channels play a pivotal role in the trigger and mediator mechanisms in hindlimb remote IPC of skeletal muscle against infarction in the pig. In addition, we also demonstrated in the pig LD muscle flaps that the infarct protective effect of the specific mKATP channel opener BMS-191095 is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion to the similar extent as hindlimb remote IPC in pig LD muscle flaps. Hindlimb remote IPC and intravenous BMS-191095 treatment are noninvasive and have no hypotensive side effect. Therefore, these treatment modalities warrant further investigation for their clinical application as a prophylactic treatment for global protection of skeletal muscle against infarction in autogenous skeletal muscle transplantation and vascular and musculoskeletal reconstructive surgery, which requires a long period of vascular occlusion during the operation and occasionally disposes skeletal muscle to unpredictable thrombosis and vasospasm resulting in prolonged and/or repeated ischemic insult.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research project was supported by an operating grant from the Canadian Institutes of Health Research Grant MOP 62925. P. Addison and M. Moses were supported by a postdoctoral fellowship from the Wharton Endowment Fund.


   ACKNOWLEDGMENTS
 
The authors thank D. McIntyre for performing the word processing of this manuscript. BMS-191095 and HMR-1098 were donated by Bristol-Myers Squibb Pharmaceutical and Aventis Pharma, respectively.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Y. Pang, The Hospital for Sick Children, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: pang{at}sickkids.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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