View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector JACC Vol. 29, No. 3 693 March 1, 1997:693–701 Infarct Size Limitation by a New Na1-H1 Exchange Inhibitor, Hoe 642: Difference From Preconditioning in the Role of Protein Kinase C TETSUJI MIURA, MD, PHD, TAKASHI OGAWA, MD, KATSUO SUZUKI, MD, MAHIKO GOTO, MD, KAZUAKI SHIMAMOTO, MD, PHD Sapporo, Japan Objectives. This study examined the effect of a new specific Results. In pentobarbital-anesthetized rabbits, 0.3 mg/kg and Na1-H1 exchange inhibitor, Hoe 642, on infarct size and the 0.6 mg/kg of Hoe 642 given before ischemia limited infarct size (as protective role of protein kinase C (PKC) by this agent. In percent area at risk [%IS/AR]) to 42.7 6 4.4% (SEM) and 26.2 6 addition, we assessed the possible alteration of Hoe 642–induced 5.4%, respectively, from the control value of 55.1 6 3.5%. However, cardioprotection by commonly used animal anesthetic drugs. injection of Hoe 642 before reperfusion did not change infarct size Background. Earlier studies on the contribution of Na1-H1 (%IS/AR 49.6 6 4.9%, p 5 0.387; power 0.81 for detecting 50% exchange to ischemic injury were complicated by nonspecific reduction). Infarct size limitation by the preischemic treatment actions of the Na1-H1 exchange inhibitors, and the role of this with Hoe 642 was similarly observed in the rabbits anesthetized exchanger in myocardial infarction in vivo remains unclear. The with ketamine/xylazine. In the polyB-treated rabbits, 0.6 mg/kg of difference in anesthetic agents used in experiments could have Hoe 642 significantly limited infarct size (%IS/AR was 28.3 6 3.8% resulted in discrepant findings regarding cardioprotection of with Hoe 642 and 50.1 6 7.5% without Hoe 642), although some interventions, such as preconditioning and adenosine preconditioning was blocked by the same dose of polyB (%IS/AR triphosphate–sensitive potassium channel openers. was 39.3 6 6.1% with polyB and 11.3 6 2.4% without polyB). Methods. Infarction was induced by 30 min of coronary occlu- Conclusions. Hoe 642 enhanced myocardial tolerance against sion and3hofreperfusion in the rabbit heart. In the first series infarction, and this enhanced tolerance was not influenced by of experiments, rabbits were anesthetized with pentobarbital or anesthetic agents commonly used for infarct size studies. Infarct ketamine/xylazine. Hoe 642 was injected intravenously 10 min size limitation by Hoe 642 was not inhibited by polyB, suggesting before ischemia or 5 min before reperfusion. In the second series that cardioprotection by Na1-H1 exchange inhibition is not PKC of experiments, rabbits received 25 mg/kg body weight of poly- mediated and thus may be unrelated to preconditioning. myxin B (polyB), Hoe 642 plus polyB, preconditioning with 5 min (J Am Coll Cardiol 1997;29:693–701) of ischemia and 5 min of reperfusion plus polyB or precondition- q1997 by the American College of Cardiology ing alone before 30 min of ischemia. In animal models of myocardial infarction, it has been well a toxic level (i.e., “Ca21 overload”) has been suggested to be established that infarct size after coronary artery occlusion is critical for transition from reversible to irreversible injury (6), determined mainly by three factors: the size of ischemic it is still unclear how a Ca21 overload is induced in ischemic myocardial mass, the level of residual blood flow in the cardiomyocytes. A possible mechanism, which recently re- ischemic region (i.e., coronary collateral blood flow) and the ceived much attention, is increased Ca21 entry through the duration of the coronary occlusion (1,2). Clinical findings also Na1-Ca21 exchange, secondary to the accumulation of Na1 in show that these factors critically influence the size of infarct, the myocytes (7,8). The intracellular Na1 level rapidly in- and thus the outcome in patients with acute myocardial creases after the onset of ischemia, to which the extrusion of infarction (3–5). However, the cellular mechanism of ischemic H1 by Na1-H1 exchange, decreased Na1 efflux by Na1-K1 myocyte death is not clearly understood. Although an elevation pump and Na1 influx through Na1 channels may be contrib- of the cytosolic free Ca21 level in myocytes during ischemia to utory. If an intracellular Na1 buildup does indeed trigger the Ca21 overload during ischemia, inhibition of the Na1-H1 exchange would attenuate ischemic myocardial injury. This From the Second Department of Internal Medicine, Sapporo Medical hypothesis was supported by the recent findings that Na1-H1 University School of Medicine, Sapporo, Japan. This study was supported in part exchange inhibitors suppressed the elevation of intracellular by Grant-in-Aid for Scientific Research 90199951 from the Ministry of Educa- 1 tion, Science and Culture, Tokyo, Japan. Na during ischemia and improved the postischemic contrac- Manuscript received July 16, 1996; revised manuscript received October 25, tile function in isolated whole hearts and papillary muscle 1996, accepted November 5, 1996. preparations (8–16). However, the agents used in earlier Address for correspondence: Dr. Tetsuji Miura, Second Department of 1 1 Internal Medicine, Sapporo Medical University School of Medicine, South-1, studies for inhibiting Na -H exchange (i.e., amiloride and its West-16, Chuo-ku, Sapporo, 060 Japan. E-mail: [email protected]. derivatives) are not necessarily specific to this antiporter, and q1997 by the American College of Cardiology 0735-1097/97/$17.00 Published by Elsevier Science Inc. PII S0735-1097(96)00522-0 694 MIURA ET AL. JACC Vol. 29, No. 3 INFARCT SIZE LIMITATION BY HOE 642 March 1, 1997:693–701 istration of this agent before ischemia and that before reper- Abbreviations and Acronyms fusion was examined. In the second series of experiments, ANOVA 5 analysis of variance involvement of PKC in the cardioprotective mechanism of Hoe EIPA 5 ethylisopropyl amiloride 642 was examined by an inhibitor of PKC, polymyxin B %IS/AR 5 infarct size as percent area at risk (polyB). KATP 5 ATP-sensitive potassium channel MIA 5 methylisobutyl amiloride NHE-1 5 Na1-H1 exchange type 1 PC 5 preconditioned Methods PKC 5 protein kinase C This study conformed to the guidelines of Sapporo Medical polyB 5 polymyxin B University on animal usage. TTC 5 triphenyltetrazolium Experiment 1: Effect of Hoe 642 on Myocardial Infarct Size act on Na1-Ca21 exchange as well as Ca21 and Na1 channels Surgical preparation. Male albino rabbits (Japanese (17). white) weighing 2.1 to 2.5 kg were anesthetized with pentobar- Recently, infarct size limitation by a Na1-H1 exchange inhib- bital sodium in Protocol 1 or with ketamine/xylazine in Protocol itor was reported in swine models of infarction (18,19). However, 2. Pentobarbital sodium was administered as a slow bolus it remains unclear whether this protection is independent of injection of 40 mg/kg body weight, and an additional small experimental conditions, particularly in animal species and with bolus (;4 mg/kg) was given when necessary. A ketamine/ anesthetic agents. These problems are not negligible for assessing xylazine injection (a mixture of 10 mg/ml of ketamine and 5 the clinical applicability of Na1-H1 exchange inhibitors, and mg/ml of xylazine) was first given as a 1.0- to 1.5-ml bolus to actual differences in the infarct size-limiting effect with animal introduce anesthesia and was then continuously infused at species and anesthetic agents can be exemplified by adenosine ;0.06 to 0.10 ml/min to maintain anesthesia at a sufficient level trisphosphate–sensitive potassium channel (KATP) openers and to abolish the corneal reflex. The rabbit was tracheostomized ischemic preconditioning (20–25). In the experiments using pen- and ventilated with a Harvard rodent respirator using oxygen tobarbital, KATP openers protected the heart from infarction in supplement. The tidal volume was 15 ml, and the ventilation dogs (20) but not in rabbits (21). However, infarct size limitation rate and oxygen flow were adjusted to maintain arterial blood byaKATP opener was detected in the rabbit when ketamine/ gasses within the physiologic range. Fluid-filled catheters were xylazine was used as the anesthetic drug (24). In contrast, inserted into the carotid artery and jugular vein for monitoring preconditioning against infarction was more clearly observed in arterial pressure and infusing drugs, respectively. The chest pentobarbital-anesthetized rabbits compared with those anesthe- was opened through a left thoracotomy, the heart was exposed tized by ketamine/xylazine (23,25). and a 4-0 silk thread was passed around the marginal branch of Another intriguing question is the role of PKC in the the left circumflex coronary artery using a tapered needle. The mechanism of cardioprotection by Na1-H1 exchange inhibi- silk thread was threaded through a small vinyl tube to make a tors. Preconditioning with brief transient ischemia enhances coronary snare. Precordial electrocardiography was monitored myocardial resistance against infarction more than any phar- using bipolar leads corresponding to a CC5 lead. The rabbit macologic agent, and the involvement of PKC has been was then heparinized with 1,000 U of heparin, and 10 min was supported by a number of recent studies, including ours allowed for stabilization. According to the experimental pro- (26–29). Inhibition of Na1-H1 exchange would reduce Ca21 tocol (as outlined later), the coronary artery was occluded by entry through Na1-Ca21 exchange, which may suppress acti- the coronary snare for 30 min and then reperfused. After 3 h vation of calcium-dependent PKC. However, this does not of reperfusion, 2,000 U of heparin was intravenously adminis- necessarily exclude the possibility that inhibition of Na1-H1 tered, and the heart was excised and processed for postmortem might result in the enhanced production of a PKC-activating analysis of infarct.