The Ceiling Effect of Pharmacological Postconditioning with the Phytoestrogen Genistein Is Reversed by the GSK3 Inhibitor SB 216763 [3-(2,4-Dichlorophenyl)-4(1-methyl-1H- indol-3-yl)-1H-pyrrole-2,5-dione] through Mitochondrial ATP- Dependent Potassium Channel Opening

In the present study, we investigated the efficacy of pharmacological postconditioning induced by 17 -estradiol and the phytoestrogen, genistein, against myocardial infarction induced by increasing durations of coronary artery occlusion (CAO). Anesthetized rabbits underwent either 20-min (protocol A) or 30-min (protocol B) CAO, followed by 4 h of coronary artery reperfusion (CAR). Before CAR, they randomly received an intravenous injection of either vehicle (control), 100 or 1000 g/kg genistein (Geni100 and Geni1000, respectively), or 100 g/kg 17 -estradiol (17 -E100). In protocol A, infarct size was significantly reduced in Geni100 (n 6), Geni1000 (n 6), and 17 -E100 (n 6) versus control (n 9) (6 2, 15 4, and 11 3 versus 35 5%, respectively). In protocol B, none of these drugs reduced infarct size versus control. Western blots demonstrated an increase of Akt phosphorylation in the Geni100 and 17 -E100 hearts submitted to 20-min CAO but not to 30-min CAO. The selective GSK3 inhibitor SB 216763 (0.2 mg/kg) [3-(2,4)-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole2,5-dione] did not exhibit cardioprotection at this dose, but its administration restored the cardioprotective effect of genistein and 17 -estradiol with 30-min CAO. Administration of 5-hydroxydecanoate (5 mg/kg) abolished the cardioprotective effects of Geni100 and 17 -E100 alone with 20-min CAO and also those observed when combined to SB 216763 with 30-min CAO. Thus, pharmacological postconditioning with genistein and 17 -estradiol is limited by a “ceiling effect of protection” along with a loss of Akt phosphorylation. However, this ceiling effect is reversed by concomitant inhibition of GSK3 by SB 216763 through opening of mitochondrial ATP-dependent potassium channels. In 2003, Zhao et al. (2003) introduced for the first time the concept of ischemic postconditioning (PCD). The authors demonstrated that application of brief intermittent episodes of myocardial ischemia-reperfusion at the onset of reperfusion was associated with a significant reduction of infarct size in a canine model. Among the mechanisms involved in the cardioprotection afforded by PCD, a key role of mitochondrial ATP-dependent potassium (KATP) channel (Yang et al., 2004b), phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Tsang et al., 2004; Yang et al., 2004b; Hausenloy et al., 2005; Sivaraman et al., 2007), protein kinase C (Philipp et al., 2006; Zatta et al., 2006), connexin 43 (Heusch et al., 2006), or the inhibition of mitochondrial transition pore permeability (Argaud et al., 2005) has been reported. Besides ischemic PCD, several studies have demonstrated the possibility of inducing pharmacological PCD by administration of drugs such as opioids (Gross et al., 2004; Förster et al., 2007; Obame et al., 2008), bradykinin (Yang et al., 2004a; Penna et This work was supported by the French Agence Nationale pour la Recherche [Grant ANR-06-PHYSIO-Ischermdiol]; and “Fondation de l’Avenir” [Grant ET5-406]. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.152587. ABBREVIATIONS: PCD, postconditioning; KATP, ATP-dependent potassium; PI3K, phosphatidylinositol 3-kinase; SB 216763, 3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; CAO, coronary artery occlusion; 17 -E100, 17 -estradiol (100 g/kg); Geni100, genistein (100 g/kg); Geni1000, genistein (1000 g/kg); CAR, coronary artery reperfusion; 5-HD, 5-hydroxydecanoate. 0022-3565/09/3293-1134–1141$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 3 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 152587/3476786 JPET 329:1134–1141, 2009 Printed in U.S.A. 1134 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from al., 2007), adenosine receptor agonists (Yang et al., 2004a), erythropoietin (Hanlon et al., 2005; Mudalagiri et al., 2008), or isoflurane (Krolikowski et al., 2005; Tessier-Vetzel et al., 2006). In this setting, we previously reported that administration of 17 -estradiol and the phytoestrogen genistein before reperfusion in rabbits also produced pharmacological PCD through activation of the PI3K/Akt pathway and mitochondrial preservation (Tissier et al., 2007). To date, the cellular mechanism of any investigated cardioprotective strategy has received extensive attention, but only few studies focused on the potential reduction of efficacy with increasing durations of ischemia, i.e., the characterization of a “ceiling of protection” (Murry et al., 1986; Gumina et al., 1999; Tissier et al., 2003). Concerning ischemic PCD, to our knowledge, only one study has reported a ceiling effect. Infarct size was reduced significantly by ischemic PCD after ischemic episodes shorter than 45 min but not after 60 min in conscious rats (Tang et al., 2006). This issue has never been investigated with any reported pharmacological PCD strategy. In accordance, the first goal of the present study was to determine whether a ceiling effect exists with pharmacological PCD induced by 17 -estradiol and the phytoestrogen genistein. It is important to emphasize that ischemic and pharmacological cardioprotective strategies do not always exhibit similar patterns of protection as previously demonstrated (Gumina et al., 1999). Then, we investigated whether the ceiling effect with PCD could be related to differential activation of the PI3K/Akt pathway with increasing durations of ischemia. We next studied whether it was possible to pharmacologically reverse the ceiling effect by concomitant administration of the GSK3 inhibitor SB 216763, i.e., targeting a downstream key step that has been demonstrated to be involved in the signaling pathway of pharmacological PCD (Gross et al., 2004, 2007; Pagel et al., 2006). Finally, we investigated whether the opening of mitochondrial KATP channels was involved in the observed cardioprotection (Gross et al., 2007, 2008). Materials and Methods The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). Animal Surgery. As described previously (Tissier et al., 2003; Couvreur et al., 2006), anesthesia was induced in male New Zealand rabbits (2–3 kg) by zolazepam and tiletamine (both 20–30 mg/kg i.v.). Animals were intubated and mechanically ventilated with 100% oxygen. Anesthesia was maintained by pentobarbital (20–30 mg/kg i.v.). A catheter was positioned in the ear marginal artery for arterial pressure measurement (P23ID strain gauge; Statham Instruments, Oxnard, CA). An external electrocardiogram was also recorded. A left thoracotomy was performed at the fourth intercostal space. The pericardium was opened, and a 3/0 Prolene suture was passed beneath a major branch of the left coronary artery. The ends of the ligature were passed through a short segment of propylene tubing to form a snare. A coronary artery occlusion (CAO) was induced by pulling the snare through the tubing. Ischemia was confirmed by the occurrence of ST segment deviation of the electrocardiogram. Reperfusion was induced by releasing the snare. The chest was then closed in layers. Measurement of Risk Area and Infarct Size. After completion of reperfusion, the animals received heparin and sodium pentobarbital (60 mg/kg i.v.). Potassium chloride was then administered intravenously to induce cardiac arrest, and the hearts were excised. The ascending aorta was cannulated and perfused (120 mm Hg) retrogradely with saline followed by Evans blue (5%) (Sigma-Aldrich, St. Louis, MO) after ligation of the previously occluded artery. The left ventricle was cut into six to eight slices, which were weighed and incubated with 1% triphenyltetrazolium chloride (Sigma-Aldrich) in a pH 7.4 buffer at 37°C. Slices were fixed in 10% formaldehyde and then photographed with a digital camera mounted on a stereomicroscope. Using a computerized planimetric program (Scion Image; Scion Corporation, Frederick, MD), the area at risk and the infarcted zones were quantified. The area at risk was identified as the nonblue region and was expressed as a percentage of the left ventricle weight. Infarcted area was identified as the tissue unstained by triphenyltetrazolium chloride and was expressed as a percentage of the area at risk. Experimental Protocol. The animals were included into five different protocols as illustrated in Figs. 1 and 2. To investigate the ceiling effect, rabbits were subjected either to a 20-min (protocol A) or a 30-min (protocol B) period of CAO. Animals received at random an intravenous injection of vehicle (control), 100 g/kg 17 -estradiol (17 -E100), 100 g/kg genistein (Geni100), or 1000 g/kg genistein (Geni1000) (Sigma-Aldrich). These intravenous injections were performed during the last 5 min of ischemia, before reperfusion. Rabbits underwent 4 h of coronary artery reperfusion (CAR). Heart rate and mean arterial pressure were measured before CAO, during CAO (at 5 and 18 min in protocol A and at 5 and 28 min in protocol B), and at 10-min CAR. To perform myocardial sampling for Western blot analysis, additional rabbits underwent the same protocols, but the animals were sacrificed at 10 min of reperfusion. In protocol C, to investigate whether inhibition of GSK3 with SB 216763 (0.2 mg/kg i.v.) could prevent the ceiling effect, rabbits received at random 6 min before the onset of CAR SB 216763 alone, SB 216763 plus 17 -E100, or SB 216763 plus Geni100. Rabbits were submitted to 30-min CAO followed by 4-h CAR. Control animals in protocol B were used as control in protocol C. The dose of SB 216763 was chosen in accordance with the literature. At the dose of 0.2 mg/kg i.v., SB 216763 has no effect on infarct size when administered before reperfusion associated with 30-min CAO in the anesthetized rabbit (Pagel et al., 2006). In addition, the involvement of mitochondrial KATP channels in the cardioprotective effects observed in protocols A and C was investigated using 5-hydroxydecanoate (5-HD; 5 mg/kg). Additional rabbits were submitted to 20-min CAO (protocol D) in the presence of 5-HD alone, 5-HD 17 -E100, or 5HD Geni100. Other additional rabbits were submitted to 30-min CAO (protocol E) in the presence of 5-HD alone, 5-HD SB 216763 17 -E100, or 5-HD SB 216763 Geni100. The administration of 5-HD was performed 5 min before CAO. Western Blots. Myocardial samples were homogenized in ice-cold buffer containing 50 mM Tris-HCl, pH 7.4 at 4°C, 150 mM NaCl, 1 mM EDTA, 5 l/ml protease inhibitor cocktail, 1 mM sodium orthovanadate, 5 mM sodium fluoride, and 1 mM sodium Na2 -glycerol phosphate (Sigma-Aldrich). The tissues were scissor minced, washed with ice-cold NaCl, and homogenized using an Ultra-Turrax and a Teflon Potter homogenizer. The homogenates were centrifuged at 40,000g for 30 min at 4°C. The protein concentration was determined by the BCA Protein Assay Kit (Pierce Chemical, Rockford, IL). Proteins were separated on 10% SDS-polyacrylamide gels using 40 g of proteins per lane. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA). After blocking with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, membranes were incubated for 1 h at room temperature with the following antibodies: 1:1000 mouse monoclonal antiphospho-Akt (at Ser473) (Cell Signaling Technology Inc., Danvers, MA), mouse monoclonal anti-Akt 1:1000 (R&D Systems, Minneapolis, MN). The membranes were then washed three times with Trisbuffered saline/Tween 20 for 10 min and subsequently incubated for 1 h with the appropriate secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Ceiling and Pharmacological Postconditioning 1135 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from