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CARDIOVASCULAR

6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (Cilostazol), a Phosphodiesterase Type 3 Inhibitor, Reduces Infarct Size via Activation of Mitochondrial Ca²⁺-Activated K⁺ Channels in Rabbit Hearts

Departments of Pharmacology (M.F., H.Ni., T.S., H.Na.) and General Surgery (H.Ni., M.M.), Chiba University Graduate School of Medicine, Chiba, Japan

Received for publication January 4, 2008
Accepted March 31, 2008.

	Abstract

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6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone (cilostazol), a phosphodiesterase type 3 (PDE III) inhibitor, activates cAMP-dependent protein kinase A (PKA). The cAMP/PKA pathway potentiates the opening of mitochondrial Ca²⁺-activated K⁺ (mitoK_Ca) channels and confers cardioprotection. Although cilostazol has been reported to directly activate sarcolemmal large-conductance Ca²⁺-activated K⁺ channels, it remains unclear whether cilostazol modulates the opening of mitoK_Ca channels. Therefore, we tested the possibility that cilostazol opens mitoK_Ca channels and protects hearts against ischemia/reperfusion injury. Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK_Ca channel activity. Infarct size in the isolated perfused rabbit hearts subjected to 30-min global ischemia and 120-min reperfusion was determined by triphenyltetrazolium chloride staining. Cilostazol (1, 3, 10, and 30 µM) oxidized flavoprotein in a concentration-dependent manner. The oxidative effect of cilostazol (10 µM) was antagonized by the mitoK_Ca channel blocker paxilline (2 µM). Activation of PKA by 8-bromoadenosine 3'5'-cyclic monophosphate (0.5 mM) potentiated the cilostazol-induced flavoprotein oxidation. Treatment with cilostazol (10 µM) for 10 min before ischemia significantly reduced the infarct size from 67.2 ± 1.3 (control) to 33.6 ± 5.3% (p < 0.05). This infarct size-limiting effect of cilostazol was abolished by paxilline (60.3 ± 4.9%) but not by the PKA inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester (KT5720) (200 nM, 40.5 ± 3.5%). On the other hand, another PDE III inhibitor, milrinone (10 µM), neither oxidized flavoprotein nor reduced infarct size. Our results suggest that cilostazol exerts a cardioprotective effect via direct activation of mitoK_Ca channels.

Therefore, the goal of this study was to examine the effect of cilostazol on mitoK_Ca channels in cardiomyocytes and determine whether cilostazol can protect the heart from ischemia/reperfusion injury. Our results indicate that cilostazol reduces infarct size via direct activation of mitoK_Ca channels.

	Materials and Methods

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All procedures complied with the Institute of Laboratory Animal Resources (1996) and were approved by the Institutional Animal Care and Use Committee of Chiba University.

Isolation of Rabbit Ventricular Myocytes. Adult rabbit ventricular myocytes were isolated by collagenase digestion, as described previously (Nishida et al., 2008). Hearts were excised from anesthetized (i.v. injection of 30 mg/kg pentobarbital sodium) rabbits (Japanese White) that weighed 2.5 to 3.5 kg and were mounted on a Langendorff apparatus. The heart was perfused with modified HEPES-buffered Tyrode's solution composed of 143 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.33 mM NaH₂PO₄, 0.5 mM MgCl₂, 5.5 mM glucose, and 5 mM HEPES, pH 7.4. The perfusate was bubbled with 100% O₂ and maintained at 36°C. After 5-min perfusion, hearts were perfused without Ca²⁺ for another 10 min, after that the perfusion solution was switched to one containing collagenase (0.25 mg/ml, Wako type I). After 25 to 30 min of digestion, the heart was perfused with the high-K⁺ low-Cl^– (modified KB) solution containing 70 mM KOH, 50 mM L-glutamic acid, 40 mM KCl, 20 mM taurine, 20 mM KH₂PO₄, 3 mM MgCl₂, 10 mM glucose, 1 mM EGTA, and 10 mM HEPES-KOH buffer, pH 7.4. Ventricular tissue was cut into small pieces in the modified KB solution, and the pieces were gently agitated to dissociate cells. Cells were then filtered through nylon mesh. Once isolated, the cells were suspended in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at room temperature until use. The cells used in the present experiments had a regular shape with clear cross-striation.

Flavoprotein Fluorescence Measurement. To index mitoK_Ca channel activity, the autofluorescence of mitochondrial flavoprotein was measured by modifying the method described by Sato et al. (2005). In brief, the cells were superfused with glucose-free Tyrode's solution containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.33 mM NaH₂PO₄, 0.5 mM MgCl₂, and 5 HEPES, pH 7.4, at room temperature (22°C). Flavoprotein fluorescence was excited at 480 nm (for 200 ms every 10 s) and emitted at 520 nm. Relative fluorescence was calibrated with signals recorded after application of the mitochondrial uncoupler 2,4-dinitrophenol (DNP; 100 µM). Emitted fluorescence was monitored with a cooled charge-coupled device digital camera (C4742-95; Hamamatsu Photonics, Hamamatsu, Japan). The imaging of flavoprotein was analyzed for average pixel intensities of regions of interest drawn to include whole-cell and expressed as a percentage of the DNP-induced maximal oxidation, using an Aquacosmos image-processing system (Hamamatsu Photonics).

Langendorff-Perfused Rabbit Heart. Female white rabbits (Japanese White, 2.5–3.5 kg) were anesthetized with an i.v. injection of 30 mg/kg pentobarbital sodium. The hearts were then dissected out and mounted on a Langendorff apparatus for perfusion with a modified Krebs-Henseleit solution composed of 119 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 24.9 mM NaHCO₃, 10 mM glucose, and gassed with 95% O₂/5% CO₂, pH 7.4 (36°C). Using a roller pump (Cole-Parmer, Vernon Hills, IL), Krebs-Henseleit solution was delivered at a constant rate of 25 to 30 ml/min that established an initial mean coronary artery perfusion pressure of >40 mm Hg. To measure left ventricular pressure, a fluid-filled balloon was inserted via the left atrium and positioned in the left ventricle. The balloon was expanded with distilled water to achieve an initial baseline left ventricular end-diastolic pressure (LVEDP) between 4 and 8 mm Hg. Hemodynamic parameters, i.e., heart rate, left ventricular developed pressure (LVDP; difference between left ventricular end-systolic pressure and LVEDP), and velocity of contraction (+dP/dt), were monitored continuously using a PowerLab data acquisition system (ADInstruments, Castle Hill, Australia).

Ischemia/Reperfusion Protocol. Langendorff-perfused hearts were stabilized and randomly assigned to the study groups. Control (CONT; n = 8): the preparations were subjected to 30 min of normothermic global ischemia followed by 120 min of reperfusion. Global ischemia was achieved by complete interruption of coronary perfusion. CILO (n = 7): preparations were treated with cilostazol (10 µM) for 10 min before the onset of ischemia. CILO + PX (n = 7): preparations were treated with the mitoK_Ca channel blocker paxilline (2 µM) during cilostazol. CILO + KT (n = 7): preparations were treated with the PKA inhibitor KT5720 (200 nM) 5 min before and during cilostazol. In a separate experiment, another PDE III inhibitor, milrinone (10 µM), was administered for 10 min before the onset of ischemia (n = 4). After 2 h of reperfusion, the heart was removed from the Langendorff apparatus and then cut into six to eight transverse slices from apex to base. The slices were incubated for 5 min at 37°C in a 1% solution of 2,3,5-triphenyltetrazolium chloride to visualize infarcts. All slices were weighed and photographed after staining. The area of infarct and that of the left ventricle in each slice were measured by computed planimetry (Aquacosmos; Hamamatsu Photonics). Infarct weight was determined with the following equation: percentage of infarct area x weight of each slice, as described previously (Suzuki et al., 2002), and expressed as a percentage of the total tissue weight.

Chemicals. Cilostazol was generously donated by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan) and was dissolved in dimethyl sulfoxide (DMSO) as a 10-mM stock solution. Milrinone, paxilline, 8Br-cAMP, and KT5720 were purchased from Sigma-Aldrich (St. Louis, MO) and were dissolved in DMSO before they were added to the experimental solution, and the final concentration of DMSO was 0.1%. DNP was purchased from Wako Pure Chemicals (Osaka, Japan) and dissolved in the perfusate.

Statistical Analysis. Data are expressed as the mean ± S.E.M., and the number of cells or experiments is shown as n. Statistical comparisons were made using Student's t test or analysis of variance combined with Fisher's post hoc test, as appropriate. A value of p < 0.05 was regarded as significant.

	Results

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Effect of Cilostazol on Flavoprotein Oxidation. The effects of cilostazol on mitoK_Ca channels in rabbit ventricular myocytes were investigated by measuring flavoprotein fluorescence. Whereas the mitochondrial redox potential is an indirect way of detecting mitochondrial ion channel activity, this approach enabled us to assay the function of mitoK_Ca channels in intact cells (Sato et al., 2005). As summarized in Fig. 1, cilostazol (10 µM) reversibly oxidized flavoprotein and increased the fluorescence intensity to 19.9 ± 3.8% (n = 5), when the redox signal was calibrated by exposing the cells to DNP at the end of the experiments. In the presence of the mitoK_Ca channel blocker paxilline (2 µM), the oxidative effect of cilostazol (10 µM) was significantly reduced to 6.5 ± 1.7% of the DNP value (n = 5, p < 0.05). On the other hand, another PDE III inhibitor, milrinone, had no significant effect on flavoprotein oxidation (10 µM, 5.0 ± 1.0%, n = 4; 30 µM, 4.8 ± 1.6%, n = 4). As shown in Fig. 2, cilostazol caused a concentration-dependent increase in flavoprotein fluorescence. Furthermore, in the presence of the cell-permeable cAMP analog 8Br-cAMP (0.5 mM), the oxidative effect of cilostazol was augmented. In the absence of cilostazol, 8Br-cAMP alone had no significant effect on flavoprotein fluorescence (data not shown). Taken together, these data indicate that cilostazol activates mitoK_Ca channels, and its effect is augmented by the cAMP/PKA pathway.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Summarized data for percentage of flavoprotein oxidation. CILO (10 µM); PX (2 µM); MIL(10) and MIL(30), milrinone (10 and 30 µM, respectively). Values are expressed as percentage relative to those obtained with DNP. *, p < 0.05.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Concentration-dependent effects of cilostazol on flavoprotein oxidation measured in absence (open circles) and presence (open triangles) of 8Br-cAMP (0.5 mM). Each point constitutes measurements from 2 to 6 cells.

Effect of Cilostazol on Mechanical Function of Isolated Hearts. There were no significant differences in baseline values of hemodynamic parameters among the groups (Table 1). Treatment with cilostazol (10 µM) significantly increased heart rate before ischemia. Representative time courses of changes in left ventricular contractile function during ischemia/reperfusion are shown in Fig. 3. In CONT, LVDP declined and the LVEDP gradually increased during ischemia. After reperfusion, contractile function only partially recovered. Brief cilostazol treatment immediately before ischemia attenuated the elevation of LVEDP during ischemia, and it improved the recovery of LVDP (CILO). Coadministration of paxilline (2 µM) abolished the effects of cilostazol (CILO + PX). In contrast, the PKA inhibitor KT5720 (200 nM) did not abolish the effects of cilostazol (CILO + KT). As summarized in Table. 1, compared with untreated controls, cilostazol treatment resulted in significant improvement of postischemic recovery of contractile function. Paxilline significantly antagonized the cilostazol-mediated improvement in postischemic recovery of contractile function, whereas KT5720 did not inhibit the cardioprotective effect of cilostazol.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Representative time course of changes left ventricular pressure in CONT, cilostazol-treated (CILO), cilostazol plus paxilline-treated (CILO + PX), and cilostazol plus KT5720-treated (CILO + KT) hearts are shown.

Effect of Cilostazol on Myocardial Infarction after Ischemia/Reperfusion. As shown in Fig. 4, triphenyltetrazolium chloride staining of hearts subjected to 30 min of global ischemia followed by 120 min of reperfusion showed an infarction of 67.2 ± 1.3% of the total tissue weight (CONT, n = 8). Treatment with cilostazol (10 µM) significantly reduced infarct size to 33.6 ± 5.3% (CILO, n = 7, p < 0.05 versus CONT). Although paxilline alone did not alter infarct size (63.7 ± 2.1%, n = 4), there was no significant difference between the myocardial infarct size in paxilline and cilostazol-treated hearts (CILO + PX, 60.3 ± 4.9%, n = 7) compared with untreated controls. Treatment with KT5720 alone did not affect infarct size (62.1 ± 4.3%, n = 4), and coadministration of KT5720 (200 nM) did not block the cilostazol-mediated reduction in infarct size (CILO + KT, 40.5 ± 3.5%, n = 7, p < 0.05 versus CONT, N.S. versus CILO + PX). Unlike cilostazol, administration of milrinone (10 µM) before ischemia failed to reduce infarct size (58.2 ± 5.0%, n = 4) compared with the control group. These results indicate that cilostazol treatment before ischemia confers cardioprotection via activation of mitoK_Ca channels, but activation of PKA itself is not a prerequisite for cardioprotection.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Summarized effects of cilostazol on myocardial infarct size. CONT indicates control; CILO, cilostazol; PX, paxilline; and KT, KT5720. Each open circle represents infarct size in an individual heart, and closed circles with error bars are group means ± S.E.M. *, p < 0.05 versus CONT.

	Discussion

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Cilostazol, a PDE III inhibitor, is known to cause an increase in intracellular cAMP levels, which leads to activation of PKA (Kimura et al., 1985). A previous study has shown that cilostazol increases the activity of BK_Ca channels in pituitary GH₃ cells and pheochromocytoma PC12 cells (Wu et al., 2004). Accordingly, one aim of the present study was to investigate whether cilostazol modulates the activity of mitoK_Ca channels in rabbit ventricular cells. We found that cilostazol alone oxidized flavoprotein in a concentration-dependent manner, and the oxidative effect of cilostazol was antagonized by the mitoK_Ca channel blocker paxilline (Figs. 1 and 2). On the other hand, another PDE III inhibitor, milrinone, did not oxidize flavoprotein (Fig. 1). These data indicate that the flavoprotein oxidation is not a class effect of PDE III inhibitor, and they further suggest that cilostazol-induced oxidation in the absence of 8Br-cAMP may not be produced solely by the activation of the cAMP/PKA pathway. We have previously shown that PKA activation itself is not sufficient to open mitoK_Ca channels, but rather it shifts channels into the primed state from which channels can be opened more intensely (Sato et al., 2005). Consistent with this effect, we found that cilostazol-induced flavoprotein oxidation was enhanced in the presence of 8Br-cAMP (Fig. 2). Taken together, these results indicate that cilostazol itself activates mitoK_Ca channels directly, and mitoK_Ca channels can be opened more intensely in response to activation of cAMP/PKA cascade.

Another aim of this work was to test whether cilostazol confers cardioprotection in Langendorff-perfused rabbit hearts. We found that administration of cilostazol for 10 min before ischemia significantly improved the recovery of contractile function (Fig. 3; Table 1) and reduced infarct size after ischemia/reperfusion (Fig. 4). Because the coronary flow rate was maintained constant throughout experiments using a roller pump, cardioprotective effects of cilostazol might not be ascribed to coronary vasodilation in our study. An increased heart rate, which presumably results from an increase in intracellular cAMP levels, was observed during application of cilostazol before ischemia (Table 1). The heart rate increase could be observed even in the presence of paxilline. However, the cardioprotective effects of cilostazol were completely abolished by the mitoK_Ca channel blocker paxilline. Furthermore, the PKA inhibitor KT5720 failed to inhibit the cilostazol-mediated improvement in postischemic recovery of contractile function and reduction in infarct size (Figs. 3 and 4), whereas the cilostazol-induced increase in heart rate was antagonized by KT5720 (Table 1). These results suggest that elevation of cAMP itself is not cardioprotective, and activation of downstream mitoK_Ca channel is involved in the mechanism of cilostazol-induced cardioprotection. This concept is further supported by the finding that milrinone, another PDE III inhibitor, failed to reduce infarct size.

There is considerable evidence that activation of PKA before ischemia can protect hearts against ischemia/reperfusion injury (Lochner et al., 1999; Tong et al., 2005). We have demonstrated that adrenomedullin treatment before ischemia reduces infarct size via PKA-mediated activation of mitoK_Ca channels (Nishida et al., 2008). However, unlike cilostazol, milrinone failed to reduce infarct size. The precise reason for this discrepancy in our experimental results is not known. It has been reported that in rabbit cardiomyocytes, milrinone inhibits not only PDE III but also PDE IV and causes greater elevations in intracellular cAMP and calcium than cilostazol (Shakur et al., 2002). Thus, it seems that the optimal level of intracellular cAMP and calcium may be required to provide cardioprotection.

The mechanism by which opening of mitoK_Ca channels could protect the hearts remains to be elucidated. We previously reported that opening of mitoK_Ca channels attenuates the mitochondrial Ca²⁺ overload with accompanying depolarization of the mitochondrial membrane (Sato et al., 2005). A recent report proposed that opening of mitoK_Ca channels generates reactive oxygen species, thereby initiating pharmacological preconditioning (Stowe et al., 2006). However, there is a need to determine whether such a mechanism could offer explanations for our findings, because cilostazol has been shown to suppress reactive oxygen species production (Park et al., 2006). Cilostazol has been shown to activate endogenous nitric-oxide (NO) synthase via a cAMP/PKA-dependent mechanism (Hashimoto et al., 2006). Furthermore, it has been reported that cilostazol protects rat heart against ischemia/reperfusion injury, which seems to be mediated by NO production (Manickavasagam et al., 2007). However, N-nitro-L-arginine methyl ester, a NO synthase inhibitor, did not affect the cilostazol-mediated reduction in infarct size (38.0 ± 2.7%, n = 3; our unpublished observations). Further studies are needed to clarify the possible role of NO in regulating mitoK_Ca channels and in conferring cardioprotection by cilostazol.

In conclusion, our results have demonstrated for the first time that cilostazol activates mitoK_Ca channels and reduces infarct size in rabbit hearts. The therapeutic clinical concentration of cilostazol was reported to be 2 µM (Akiyama et al., 1985). Because in preliminary experiments 3 µM cilostazol could not exert a prominent effect on infarct size, a relatively higher concentration (10 µM) of cilostazol may be needed for the cardioprotective action. However, even in clinically effective doses, noradrenaline that has been released from sympathetic nerve endings in response to myocardial ischemia (Remme, 1998) may augment the cilostazol-induced opening of mitoK_Ca channels through a cAMP/PKA-dependent pathway.

	Acknowledgements

 
We thank M. Tamagawa and I. Sakashita for excellent technical and secretarial assistance.

	Footnotes

 
This study was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science, K. Watanabe Research Foundation, and the Vehicle Racing Commemoration Foundation.

M.F. and H.Ni. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.136218.

ABBREVIATIONS: cilostazol, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)quinolinone; PDE III, phosphodiesterase type 3; PKA, protein kinase A; mitoK_ca, mitochondrial Ca²⁺-activated K⁺; BK_Ca, sarcolemmal large-conductance Ca²⁺-activated K⁺; DNP, 2,4-dinitrophenol; LVEDP, left ventricular end-diastolic pressure; +dP/dt, positive change in pressure over time; LVDP, left ventricular developed pressure; CONT, control; CILO, cilostazol; KT5720, (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2', 1'-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester; KT, KT5720; PX, paxilline; DMSO, dimethyl sulfoxide; 8Br-cAMP, 8-bromoadenosine 3'5'-cyclic monophosphate; NS1619, 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one; NO, nitric oxide.

Address correspondence to: Dr. Haruaki Nakaya, Department of Pharmacology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail: nakaya{at}faculty.chiba-u.jp

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