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Am J Physiol Heart Circ Physiol 278: H305-H312, 2000;
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Vol. 278, Issue 1, H305-H312, January 2000

RAPID COMMUNICATION
Ischemic preconditioning in rats: role of mitochondrial KATP channel in preservation of mitochondrial function

Ryan M. Fryer, Janis T. Eells, Anna K. Hsu, Michele M. Henry, and Garrett J. Gross

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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

5-hydroxydecanoic acid; HMR-1098; adenosine 5'-diphosphate-sensitive potassium channel; mitochondria


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC PRECONDITIONING (IPC) is a phenomenon in which single or multiple brief periods of ischemia have been shown to protect the heart against a more prolonged ischemic insult, the result of which is a marked reduction in myocardial infarct size, severity of stunning, or incidence of cardiac arrhythmias. IPC was first demonstrated in 1986 (25), when Reimer's group made the novel discovery that brief periods of ischemia and reperfusion afforded cardioprotection to a prolonged ischemic insult in the canine model. Since this discovery, extensive studies have demonstrated this cardioprotection in all animals studied and have shown that IPC-like effects exist in cultured cells exposed to hypoxia and metabolic inhibition.

Stimulation of either the adenosine A1 receptor (1) or delta 1-opioid receptor (29) has been shown to induce IPC-like cardioprotection mediated via Gi/o protein activation (30). Intracellular enzyme mediators of IPC-induced cardioprotection include protein kinase C (10, 35), tyrosine kinase (11, 23, 24), and mitogen-activated protein kinase-activated protein kinase 2 (23, 24). Although a number of pharmacological agents and signaling pathways have been proposed to be involved in mediating the cardioprotective effect of IPC, current research suggests that the ATP-sensitive potassium (KATP) channel is an important component of this phenomenon and may serve as the end effector.

Sarcolemmal KATP channel involvement in IPC was first suggested by Gross and Auchampach (14). However, since the discovery of functional KATP channels in the mitochondria (18), investigators have sought to elucidate a potential role for the mitochondrial KATP channel in IPC-induced cardioprotection. Initially, it was hypothesized that the surface or sarcolemmal KATP channels (14) mediated protection observed after IPC; however, more recent evidence suggests that the mitochondrial KATP channel may mediate IPC-induced cardioprotection (12, 21).

It is generally accepted that ischemia-reperfusion (I/R) injury profoundly disrupts mitochondrial energy metabolism, and numerous studies have shown that mitochondria isolated from I/R hearts manifest reduced function, decreased membrane potential, and respiratory impairment (7, 9). Recent evidence suggests that IPC may protect against mitochondrial dysfunction and improve myocardial energy metabolism (19, 26, 34). We hypothesize that activation of the mitochondrial KATP channel during IPC is important in preventing I/R-induced mitochondrial damage. Thus the purpose of these studies was twofold: first, to examine the contribution of the mitochondrial versus sarcolemmal KATP channel in IPC-induced cardioprotection, and second, to investigate the role of the mitochondrial KATP channel in the preservation of mitochondrial bioenergetics in IPC. For these studies we utilized the cardioselective and putative sarcolemmal KATP channel-selective inhibitor HMR-1098 (3, 13, 20), the mitochondrial KATP channel-selective inhibitor 5-hydroxydecanoic acid (5-HD) (28), and the mitochondrial KATP channel-selective agonist diazoxide (21) to examine the role of these two channels in IPC.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

General Surgical Preparation

Male Wistar rats, 350-450 g, were used for all phases of this study. The rats were anesthetized via intraperitoneal administration of Inactin (100 mg/kg), a long-acting barbiturate. A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent ventilator (model CIV-101, Columbus Instruments, Columbus, OH, or model 683, Harvard Apparatus, South Natick, MA). The rats were ventilated with room air supplemented with O2 at 60-65 breaths/min. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5-10 mmH2O. Arterial pH, PCO2, and PO2 were monitored at control, after 15 min of occlusion, and after 60 and 120 min of reperfusion by a blood gas system (AVL 995 pH/Blood Gas Analyzer) and maintained within a normal physiological range (pH 7.35-7.45; PCO2 25-40 mmHg; and PO2 80-110 mmHg) by adjusting the respiratory rate and/or tidal volume. Body temperature was maintained at 38°C with the use of a heating pad, and bicarbonate was administered intravenously as needed to maintain arterial blood pH within normal physiological levels.

The right carotid artery was cannulated to measure blood pressure and heart rate via a Gould PE50 or Gould PE23 pressure transducer connected to a Grass (model 7) polygraph. The right jugular vein was cannulated for saline, bicarbonate, and drug infusion. A left thoracotomy was performed at the fifth intercostal space, followed by a pericardiotomy and adjustment of the left atrial appendage to reveal the location of the left coronary artery. A ligature (6-0 prolene) 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. Pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat elicited occlusion of the coronary artery and resulted in regional left ventricular ischemia. Epicardial cyanosis and subsequent decrease in blood pressure verified coronary artery occlusion. Reperfusion of the heart was initiated by unclamping the hemostat and loosening the snare and was confirmed by visualizing an epicardial hyperemic response. Heart rate and blood pressure were allowed to stabilize before the following protocols were initiated.

Drugs

Inactin (thiobutabarbital sodium) and 5-HD were purchased from RBI (Natick, MA). 2,3,5-Triphenyltetrazolium chloride (TTC) was purchased from Sigma Chemical (St. Louis, MO). HMR-1098, the sodium salt of HMR-1883, was a generous gift from Hoechst Marion Roussel (Frankfurt, Germany). Inactin and HMR-1098 were dissolved in distilled water. 5-HD was dissolved in saline. Diazoxide was dissolved in 0.1 ml of NaOH and saline. All drugs were dissolved in ~0.9 ml of vehicle for administration at all doses.

Study Groups and Experimental Protocols

Rats were randomly assigned to 1 of 10 groups (Fig. 1). All groups underwent a 30-min coronary artery occlusion and 2 h of reperfusion. 5-HD or HMR-1098 was administered in the presence or absence of IPC. The control group underwent only a 30-min coronary artery occlusion and subsequent 2 h of reperfusion. IPC was elicited by 5 min of coronary artery occlusion and 5 min of reperfusion. The effect of HMR-1098 (3 or 6 mg/kg), a sarcolemmal KATP channel-selective antagonist, was also examined in the absence or presence of IPC. HMR-1098 (3 mg/kg) was administered 15 min before the control protocol. The effects of HMR-1098 on IPC-induced cardioprotection were examined by administering 3 mg/kg HMR-1098 5 or 15 min before IPC or 6 mg/kg HMR-1098 15 min before IPC. Similarly, the effect of pretreatment with 5-HD, the mitochondrial KATP channel-selective antagonist, was examined by administering 10 mg/kg 5-HD. To determine the effect of 5-HD treatment on infarct size in control groups, 5-HD was administered 5 min before 30 min of coronary artery occlusion and 2 h of reperfusion. The effect of the importance of timing in 5-HD treatment on IPC was assessed by administering the drug 5, 10, or 30 min before IPC. Diazoxide (10 mg/kg) was administered 10 min before prolonged ischemia to determine the effects of opening the mitochondrial KATP channel in control animals and was administered 5 min before or 5 min after 5-HD treatment.


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  		Fig. 1.   Protocol bars depicting experiments used to study effects of HMR-1098 (HMR) or 5-hydroxydecanoic acid (5-HD) on ischemic preconditioning (IPC) in intact rat heart. All groups underwent a 30-min coronary artery occlusion and a 2-h reperfusion period. HMR-1098 (3 or 6 mg/kg ) was administered 15 min before control or IPC protocols. 5-HD (10 mg/kg) was administered 5 min before prolonged ischemia or 5, 10, or 30 min before IPC. Diazoxide (DZX, 10 mg/kg) was given 15 min before ischemia and 5-HD was administered 5 min before or after DZX in 2 additional groups.

Determination of Infarct Size

When these protocols were completed, the coronary artery was reoccluded and the area at risk (AAR) determined by negative staining. Patent blue dye was administered via the jugular vein to effectively stain the nonoccluded area of the left ventricle. The rat was euthanized with a 15% KCl solution. The heart was excised, and the left ventricle was removed from the remaining tissue and subsequently cut into six thin cross-sectional pieces. This preparation allowed for the delineation of the normal area (stained blue) versus the AAR, which subsequently remained pink. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 min with a 1% TTC stain in 100 mM phosphate buffer (pH 7.4) at 37°C. TTC is an indicator of viable and nonviable tissue. Tissues were stored in vials of 10% formaldehyde overnight, and the infarcted myocardium was dissected from the AAR under the illumination of a dissecting microscope (Cambridge Instruments). Infarct size (IS) and AAR were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR).

Assessment of Mitochondrial ATP Synthesis

Isolation of cardiac mitochondria. Mitochondrial ATP synthesis was determined in cardiac mitochondria in a parallel series of experiments using the same treatment protocols described for infarct size experiments. Mitochondria were isolated from rat hearts by differential centrifugation as described by Solem and Wallace (32). Mitochondria prepared by this methodology have been shown to be metabolically active with respiratory control ratios of 3.5-5.0 with succinate and 8.0-10.0 with glutamate/malate and corresponding ratios of ADP to oxygen of 1.5-1.7 and 2.5-2.7 (32). Briefly, the atria and right ventricle were trimmed away and the AAR of the left ventricle was dissected, weighed, and minced into 1-mm pieces in 1 ml of isolation buffer (200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 1 mM EGTA, 5 mM MOPS, and 0.1% BSA; pH 7.15 adjusted with KOH). In untreated control hearts a comparable region of ventricle was dissected. The minced tissue was rinsed clear of blood and light debris with cold isolation buffer and transferred to a glass Potter-Elvehjem homogenizing vessel on ice. Immediately after the addition of 2.5 mg of protease in 2.5 ml of isolation buffer, the tissue was gently homogenized on ice with a Teflon pestle. After 30 s of homogenization, 17.5 ml of cold isolation buffer were promptly added to dilute the protease and homogenization continued for an additional 60 s. Exposure to concentrated protease was limited to 30 s to maintain mitochondrial integrity and yield. The tissue suspension was centrifuged (8,000 g for 10 min) to remove the protease. The pellet was resuspended in 3.5 ml of isolation buffer, and an additional 25 ml of isolation buffer were added and the suspension centrifuged (700 g for 10 min) to remove cellular debris. The supernatant containing the mitochondrial fraction was further centrifuged (8,000 g for 10 min), and the pellet was washed twice by resuspension in 3.5 ml of isolation buffer (without EGTA) and centrifuged (8,000 g for 10 min). The final mitochondrial pellet was resuspended to 1 g original tissue wt/ml cold isolation buffer (without EGTA). All procedures were performed at 4°C, and protein concentration was determined using the dye binding method of Bradford (4) with BSA as a standard.

Mitochondrial ATP synthesis. Mitochondria (10 µg mitochondrial protein/ml) were preincubated for 10 min in medium containing 180 mM sucrose, 45 mM KH2PO4, 10 mM Mg-acetate, 1 mM EDTA, 1 mM pyrophosphate, 1 g/l BSA, and 150 µM ADP in a 25°C water bath. ATP synthesis was initiated by the addition of respiratory substrates (1 mM pyruvate + 1 mM malate to drive electron flow through complex I of the electron transport chain). Aliquots of 50 µl were taken from the incubations at 4-min intervals for 12 min, and reactions were stopped by adding the samples to 500 µl of 2.5% (vol/vol) trichloroacetic acid (8). Samples were neutralized with 100 µl of 1 M Tris base. The neutralized supernatant was assayed for ATP by luciferin-luciferase luminometry using a modification of the luminescence method of Strehler (33) and components of the Sigma Bioluminescent Somatic Cell Assay Kit (catalog no. FL-AA, Sigma). Mitochondrial protein concentration was determined using the Bradford assay (4), and the rate of mitochondrial ATP synthesis was calculated and expressed as micromoles of ATP synthesized per minute per milligram of mitochondrial protein.

Tissue ATP concentrations. Segments of the AAR of the left ventricle were rapidly dissected, frozen in liquid nitrogen, and stored at -70°C until analysis. For analysis, frozen tissue samples (20 mg) were homogenized in 500 µl of 2.5% (vol/vol) trichloroacetic acid. After centrifugation, 400-µl aliquots of supernatant were neutralized with 1 M Tris (80 µl) (22). Tissue ATP concentrations were determined by luciferin-luciferase luminometry on the neutralized supernatant (33). Protein concentrations were determined on the solubilized pellet by the Bradford method (4), and the concentration of ATP was expressed as nanomoles of ATP per milligram of tissue protein.

Exclusion Criteria

A total of 83 rats successfully completed the protocols for IS studies, 40 rats completed the protocols for mitochondrial ATP synthesis experiments, and 22 rats completed the protocols for tissue ATP concentrations. Rats were excluded from data analysis if they exhibited severe hypotension (<30 mmHg systolic blood pressure) or if we were unable to maintain adequate blood gas values within a normal physiological range because of metabolic acidosis. Exclusion of animals from the present study was evenly distributed among the protocol groups.

Statistical Analysis of Data

All values are expressed as means ± SE. ANOVA with a Newman-Keuls post hoc test was used to determine whether any significant differences existed among groups for hemodynamics, IS, and AAR. Significant differences were determined at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamics

Table 1 summarizes heart rate (HR), mean arterial blood pressure (MAP), and rate-pressure product (RPP) in all groups as determined at baseline, after 15 min of coronary artery occlusion, and after 120 min of reperfusion. Blood pressure in the protocols was maintained at baseline values after the saline, 5-HD, or HMR-1098 treatment was administered. No significant differences were found at baseline or after 15 min of coronary artery occlusion for all parameters. RPP and MAP after 2 h of reperfusion were not significantly different for most groups versus control; however, for the group pretreated with 5-HD (10 mg/kg) 5 min before diazoxide (10 mg/kg) administered 10 min before I/R, RPP was significantly different from control (P < 0.05). In addition, because of the high HR for control animals at 2 h of reperfusion, there existed a significant difference between control and the other study groups for this parameter.

                              
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  		Table 1.   Hemodynamics

Infarct Size and Area at Risk

There were no significant differences for any group versus control for left ventricle weight (g) or AAR (data not shown). Control animals exhibited an IS/AAR of 56 ± 1%. IPC elicited by 5 min of ischemia and 5 min of reperfusion significantly reduced infarct size (7 ± 1%). The effects of HMR-1098 pretreatment and 5-HD pretreatment are shown in Figs. 2 and 3, respectively. HMR-1098 and 5-HD in the absence of IPC did not significantly change IS/AAR (56 ± 4% and 63 ± 4%, respectively). HMR-1098 (3 mg/kg) administered 5 or 15 min before IPC did not significantly attenuate cardioprotection (14 ± 4% or 19 ± 5%, respectively). Similarly, HMR-1098 (6 mg/kg) administered 15 min before IPC did not significantly attenuate cardioprotection induced by IPC (9 ± 1%). 5-HD pretreatment 5 min before IPC partially abolished cardioprotection (40 ± 1%). However, when pretreatment was extended to either 10 or 30 min before IPC, 5-HD did not attenuate IPC (14 ± 7% or 9 ± 3%, respectively). Diazoxide (10 mg/kg) administered 15 min before I/R (Fig. 4) significantly reduced IS/AAR (36.2 ± 4.8%). However, when 5-HD (10 mg/kg) was administered either 5 min before or 5 min after diazoxide, cardioprotection was completely abolished (56.4 ± 3.4% or 51.8 ± 2.8%, respectively).


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  		Fig. 2.   Infarct size expressed as a percentage of the area at risk (IS/AAR) in rat hearts subjected to prolonged ischemia, IPC, HMR-1098, or IPC in presence of HMR-1098. HMR-1098 was administered at 3 [HMR(3)]or 6 mg/kg [HMR(6)]. Drug treatment in control hearts (Con) did not affect infarct size in absence of a preconditioning stimulus. Similarly, HMR-1098 did not significantly affect IS/AAR in presence of IPC. * P < 0.05 vs. control; dagger  P < 0.05 vs. IPC.



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  		Fig. 3.   IS/AAR in rat hearts subjected to prolonged ischemia, IPC, 5-HD, or IPC in presence of 5-HD. 5-HD exhibited a time-dependent partial attenuation of cardioprotection when administered either 5, 10, or 30 min before IPC. 5-HD in absence of IPC did not affect IS/AAR vs. control. Cardioprotection was attenuated by 5-min, but not by 10- or 30-min, pretreatment with 5-HD. * P < 0.05 vs. control; dagger   P < 0.05 vs. IPC.



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  		Fig. 4.   IS/AAR in rat hearts subjected to control, DZX, or DZX administered before or after 5-HD treatment. DZX reduced infarct size, and this cardioprotection was abolished with 5-HD administration either 5 min before or 5 min after DZX treatment. DZX(10), 10 mg/kg DZX; 5-HD(10), 10 mg/kg 5-HD. * P < 0.05 vs. control.

Mitochondrial ATP Synthesis

The rate of mitochondrial ATP synthesis was determined in cardiac mitochondria in a parallel series of experiments using hearts from untreated animals and hearts from animals subjected to the same treatment protocols described for IS experiments. As shown in Figs. 5-7, the rate of mitochondrial ATP synthesis in hearts subjected to 30 min of ischemia followed by 2 h of reperfusion (0.67 ± 0. 06 µmol ATP · min-1 · mg mitochondrial protein-1) was reduced to 32% of the rate measured in untreated rat hearts (2.12 ± 0. 30 µmol ATP · min-1 · mg-1). IPC significantly improved ATP synthesis in the AAR to 1.86 ± 0.18 µmol ATP · min-1 · mg-1 (89% of the rate measured in untreated rat hearts) versus I/R hearts (0.67 ± 0.06 µmol ATP · min-1 · mg-1). 5-HD, in the absence of IPC, did not alter the rate of ATP synthesis versus I/R (0.45 ± 0.06 µmol ATP · min-1 · mg-1). HMR-1098 (6 mg/kg) and 5-HD (10 mg/kg), in the absence of IPC (Figs. 5 and 6, respectively), did not alter the rate of ATP synthesis versus I/R (0.68 ± 0.13 and 0.45 ± 0.06 µmol ATP · min-1 · mg-1, respectively). Similarly, HMR-1098 (6 mg/kg) administered 15 min before IPC (Fig. 5) did not alter the rate of ATP synthesis (2.01 ± 0.03 µmol ATP · min-1 · mg-1) relative to IPC (1.86 ± 0.18 µmol ATP · min-1 · mg-1). However, when 5-HD, was administered to animals 5 min before IPC (Fig. 6), the rate of ATP synthesis (1.18 ± 0.15 µmol ATP · min-1 · mg-1) was significantly attenuated relative to the rate measured in IPC hearts. Diazoxide (10 mg/kg) administered 10 min before I/R (Fig. 7) had no effect on the rate of ATP synthesis relative to I/R alone.


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  		Fig. 5.   Rate of mitochondrial ATP synthesis (expressed as µmol · min-1 · mg mitochondrial protein-1) in mitochondria isolated from untreated control hearts and from AAR of hearts subjected to prolonged ischemia, IPC, HMR-1098, or IPC in presence of HMR 1098. HMR-1098 (6 mg/kg) was administered 15 min before ischemia or IPC protocols. Rate of ATP synthesis was significantly reduced by ischemia-reperfusion (I/R). IPC prevented the reduction in ATP synthesis produced by I/R. HMR-1098, in absence of IPC, did not affect the rate of ATP synthesis vs. I/R alone. Similarly, HMR-1098 did not alter the rate of ATP synthesis relative to I/R in presence of IPC. ¥ P < 0.05 vs. untreated control; * P < 0.05 vs. I/R; dagger  P < 0.05 vs. IPC.



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  		Fig. 6.   Rate of mitochondrial ATP synthesis in mitochondria isolated from AAR of hearts subjected to prolonged ischemia, IPC, 5-HD, or IPC in presence of 5-HD. 5-HD (10 mg/kg) was administered 5 min before ischemia or IPC protocols. Rate of ATP synthesis in mitochondria isolated from hearts subjected to IPC was significantly greater than that measured in mitochondria isolated from hearts subjected to I/R. 5-HD, in absence of IPC, did not affect the rate of ATP synthesis vs. I/R. However, when administered before IPC, 5-HD significantly attenuated the protection of ATP synthesis conferred by IPC. * P < 0.05 vs. I/R; dagger   P < 0.05 vs. IPC.



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  		Fig. 7.   Rate of mitochondrial ATP synthesis in mitochondria isolated from AAR of hearts subjected to prolonged ischemia, IPC, or DZX. DZX (10 mg/kg) was administered 10 min before ischemia. Rate of ATP synthesis in mitochondria isolated from hearts subjected to IPC was significantly greater than that measured in mitochondria isolated from hearts subjected to I/R. DZX did not alter the rate of ATP synthesis vs. I/R. * P < 0.05 vs. I/R; dagger   P < 0.05 vs. IPC.

Cardiac Tissue ATP Concentrations

Tissue concentrations of ATP were measured in untreated control hearts and in the AAR of hearts subjected to the same treatment protocols described for IS experiments. ATP concentrations in untreated control hearts were 3.8 ± 0.4 nmol ATP/mg tissue protein (n = 4). In the AAR of hearts exposed to I/R, ATP concentrations were reduced to 1.1 ± 0.2 nmol ATP/mg tissue protein (n = 4). ATP concentrations in the AAR of IPC hearts (1.1 ± 0.1 nmol ATP/mg tissue protein, n = 4) were not significantly different from the concentrations measured in the AAR of hearts subjected to I/R. Our preliminary findings (n = 2 for each treatment group) show no differences in ATP concentrations relative to either I/R or IPC in any drug treatment group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These experiments demonstrate that inhibition of the mitochondrial, but not sarcolemmal, KATP channel attenuates cardioprotection induced by IPC. We suggest that IPC-induced cardioprotection is mediated, at least in part, via activation of the mitochondrial KATP channel in the in vivo rat heart because the mitochondrial KATP channel-selective inhibitor 5-HD attenuated cardioprotection in a time-dependent manner. Similarly, we were able to antagonize the cardioprotection induced via diazoxide-induced opening of the mitochondrial KATP channel with 5-HD administration.

Because IS/AAR was not affected by treatment with HMR-1098 at 3 or 6 mg/kg, these experiments suggest that the sarcolemmal KATP channel is not the main effector of cardioprotection or may play only a minimal, supplementary role relative to the mitochondrial KATP channel. 5-HD exhibited a time-dependent inhibition of the mitochondrial KATP channel. We demonstrated that IPC-induced cardioprotection could be markedly attenuated with 5 min of 5-HD pretreatment; however, when pretreatment was extended to either 10 or 30 min before IPC, 5-HD did not abolish cardioprotection. These results may be a consequence of rapid 5-HD metabolism in Wistar rats and suggest that the use of 5-HD as a selective inhibitor of the mitochondrial KATP channel in the in vivo rat myocardium requires time considerations similar to those previously shown in our laboratory for glibenclamide (31).

Our results also demonstrate that IPC protects mitochondrial oxidative phosphorylation. In contrast to the profound reduction in mitochondrial ATP synthesis observed after ischemia and reperfusion, mitochondria isolated from the AAR of the hearts of preconditioned rats exhibited rates of ATP synthesis that were virtually identical to ATP synthesis rates measured in untreated rat hearts. These findings are consistent with studies showing that IPC reduces superoxide production and prevents the impairment of state 3 mitochondrial respiration induced by ischemia and reperfusion (26). Our studies also extend the evidence of mitochondrial protection to mitochondrial oxidative phosphorylation. Although the rates of mitochondrial ATP production were improved in IPC hearts relative to I/R, tissue ATP concentrations in the AAR were not different between the two treatment groups, suggesting that cellular ATP utilization after IPC is greatly stimulated. Moreover, there was a remarkable degree of correlation between the degree of mitochondrial protection conferred by IPC and the reduction of IS observed in preconditioned hearts, suggesting that mitochondria may play an important role in the cardioprotection produced by IPC.

Importantly, our findings provide the first evidence linking mitochondrial KATP channel activation to cardioprotection at the level of mitochondrial function. The mitochondrial KATP channel-selective antagonist 5-HD significantly attenuated the protective effect of IPC on mitochondrial ATP synthesis. 5-HD pretreatment 5 min before IPC decreased the rate of ATP synthesis to 63% of the rate measured in IPC hearts. In contrast, the putative sarcolemmal KATP channel-selective inhibitor HMR-1098 had no effect on the protective effect of IPC on mitochondrial ATP synthesis. Again, there was a remarkable correlation between the effect of 5-HD pretreatment on mitochondrial ATP synthesis and the attenuation of cardioprotection as assessed by IS observed in 5-HD pretreated hearts. By assessing both IS as an index of cardiac injury and ATP synthesis as an index of mitochondrial function, the present studies provide a link among IPC, mitochondrial KATP channel activation, and improved mitochondrial function in the intact rat heart.

Although diazoxide treatment reduced IS, it had no effect on the rate of ATP synthesis relative to I/R. It is possible that the cardioprotective action of diazoxide is unrelated to any action on oxidative phosphorylation. Alternatively, it is possible that diazoxide, by virtue of its long biological half-life, continues to affect mitochondrial function in isolated mitochondria. Direct activation of the mitochondrial KATP channel by diazoxide in isolated mitochondria has been shown to depolarize mitochondria and reduce the driving force for ATP synthesis (17).

Although a direct link between mitochondrial KATP channel activation and myocardial protection remains to be established, several known consequences of mitochondrial KATP activation are likely to improve mitochondrial function following I/R. Activation of the mitochondrial KATP channel results in K+ influx, expansion of mitochondrial matrix volume, and a reduction of the inner mitochondrial membrane potential established by the proton pump (12, 17). Regulation of matrix volume is an essential element in the regulation of mitochondrial energy production, and matrix expansion, secondary to mitochondrial KATP opening, has been postulated to activate electron transport and stimulate mitochondrial metabolism (15). Our findings of improved rates of ATP synthesis in mitochondria isolated from preconditioned hearts are consistent with this mechanism. Mitochondrial membrane depolarization produced by K+ influx would also be expected to reduce the driving force for Ca2+ influx through the Ca2+ uniport, thus attenuating mitochondrial Ca2+ overload (7, 9). Recent studies by Holmuhamedov et al. (17) have shown that preloaded mitochondria release Ca2+ in response to activation by a KATP channel opener. This suggests that a cell in which Ca2+ overload is already present may be protected by a KATP opener or by activation of the channel following IPC. Mitochondrial Ca2+ overload has been closely correlated with mitochondrial and myocardial damage via a variety of mechanisms, including damage to complex I of the electron transport chain (16), increased production of reactive oxygen species (16), and activation of the mitochondrial permeability transition leading to cell death (5, 6, 27).

Selective inhibitors of the sarcolemmal and mitochondrial KATP channels have recently been developed. Hoechst Marion Roussel recently synthesized the cardioselective drug HMR-1883 and the accompanying sodium salt HMR-1098 (3, 13). Similarly, Marban's group suggested in 1998 that HMR-1883 and HMR-1098 are sarcolemma-selective inhibitors of the KATP channel in rabbit myocytes (unpublished data). In the same laboratory, T. Sato, N. Sasaki, J. Seharasegon, B. O'Rourke, and E. Marban's group has demonstrated that 5-HD is selective for the mitochondrial KATP channel in rabbit cardiomyocytes (28). The effect of 5-HD and HMR 1098 in rat cardiomyocytes, however, has not yet been determined, and the possibility of species differences in 5-HD channel binding and modulation remains a possibility.

Terzic's group (17) demonstrated rapid mitochondrial depolarization on activation of the KATP channel with pinacidil. This decreased mitochondrial membrane potential, decreased the rate of ATP-synthesis, and resulted in a compensatory increase in the rate of mitochondrial respiration. Importantly, opening the mitochondrial KATP channel had dramatic effects on Ca2+ loading in the mitochondria. Mitochondria accumulate and retain Ca2+ in the matrix, a function critical to intracellular Ca2+ homeostasis. Pinacidil addition induced a rapid release of Ca2+ from these preloaded mitochondria. This response may be of physiological importance because ischemic cells have been shown to demonstrate increased cytosolic Ca2+. Because from increased cytosolic Ca2+ we would predict increased mitochondrial Ca2+, and because opening of the KATP channel can increase Ca2+ efflux from the mitochondria, mitochondrial KATP channel opening may be a potential mechanism of cardioprotection from ischemia-induced Ca2+ overload.

Indeed, opening of the mitochondrial KATP channel may exert cardioprotection to the myocardium. Garlid and Grover's group (12) first demonstrated in the isolated rat heart subjected to global ischemia that the mitochondrial KATP channel-selective agonist diazoxide improved postischemic functional recovery in a glibenclamide reversible manner. In addition, 5-HD completely abolished this induced cardioprotection. More recent evidence also suggests a role for the mitochondrial KATP channel in cardioprotection in humans. Preliminary evidence presented by Bell et al. (2) demonstrated a role for the mitochondrial KATP channel in both IPC- and opioid-induced cardioprotection in human atrial trabeculae.

In summary, aforementioned studies in combination with the current studies from our laboratory validate a role for the mitochondrial KATP channel in a rat model of IPC. These conclusions are supported by the demonstration that the mitochondrial KATP channel-selective antagonist 5-HD, but not the sarcolemmal KATP channel-selective antagonist HMR-1098, could abolish IPC-induced cardioprotection in the in vivo rat model. Antagonists of both the sarcolemmal and mitochondrial KATP channels provide powerful tools for the study of these two channels; however, further studies are necessary to more completely characterize the pharmacological actions of these inhibitors in rat cardiomyocytes.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-08311.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. J. Gross, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (ggross{at}mcw.edu)

Received 6 July 1999; accepted in final form 10 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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