Asian Cardiovasc Thorac Ann 2006;14:239-243
© 2006 Asia Publishing EXchange Ltd
Landiolol Enhances Effect of Ischemic Preconditioning in Isolated Rabbit Hearts
Shigetoshi Mieno, MD,
Hitoshi Horimoto, MD,
Kenji Kishida, MD,
Sachiko Horimoto, MD,
Shinjiro Sasaki, MD
Department of Thoracic and Cardiovascular Surgery Osaka Medical College Osaka, Japan
For reprint information contact: Shigetoshi Mieno, MD Tel: 81 72 683 1221 Fax: 81 72 684 6542 Email: tho050{at}poh.osaka-med.ac.jp, Department of Thoracic and Cardiovascular Surgery, Osaka Medical College, 2-7 Daigakucho, Takatsuki, Osaka 569-8686, Japan
 |
ABSTRACT
|
|---|
The effect of the ultra-short-acting beta blocker, landiolol, on ischemic preconditioning was examined in isolated rabbit hearts. Ischemic preconditioned hearts received 2 episodes of 5 min each of global ischemia and reperfusion. The left anterior descending coronary artery was occluded for 1 hour and reperfused for 1 hour. Left ventricular end-systolic and end-diastolic pressures and infarct size were measured. Seven control hearts had no drug infused. Four groups of 6 hearts each were pretreated with 1 or 3 µM of landiolol or a combination of 1 or 3 µM landiolol and ischemic preconditioning. A further group of 6 hearts had ischemic preconditioning without landiolol. Ischemic preconditioning significantly reduced left ventricular end-diastolic pressure and infarct size compared to the controls. Landiolol alone did not change left ventricular end-diastolic pressure or infarct size, but landiolol 3 µM and ischemic preconditioning decreased left ventricular end-diastolic pressure more than preconditioning alone. These data suggest that pre-ischemic landiolol infusion may enhance the cardioprotective effect of ischemic preconditioning.
|
INTRODUCTION
|
|---|
Ischemic preconditioning (IPC) enhances tolerance to subsequent ischemic injury.1,2 Ischemic preconditioning results in changes such as reduction of infarct size, preservation of ionic homeostasis, and a decrease in ventricular arrhythmias after ischemia-reperfusion. Several researchers, including ourselves, have proposed subcellular mechanisms for the IPC response, including the mitochondrial adenosine triphosphate-sensitive potassium channel, nitric oxide, protein kinase C, opioid receptors, and ß-adrenergic receptors.3–7 Sandhu and colleagues8 reported that ß-receptor stimulation before ischemia reduced infarct size and mimicked IPC effects. An increase in cyclic adenosine monophosphate, a key mediator of the ß-receptor signaling pathway, is essential for the IPC response. Moreover, we have recently shown that IPC fails to protect after myocardial infarction, but the adenylate cyclase agonist forskolin restores the protective effects of IPC.9 This accumulated evidence indicates that ß-receptors are involved in the IPC mechanism.
Recent experiments have shown beneficial effects of blocking ß1-adrenergic receptors in ischemia-reperfusion injury.10 However, we found that ß2-receptors play a pivotal role in the IPC protective response.11 This suggests that ß1 blockade might enhance the cardioprotective effect of IPC, whereas activation of ß2 receptors is associated with the cardioprotective effect of IPC. To assess the role of ß1 blockade in IPC, we used a specific ß1 blocker, landiolol. Landiolol (ONO-1101) is an ultra-short-acting beta blocker with a shorter half-life (4 min) and higher affinity for ß1 receptors (255-fold > ß2 receptors) than other ultra-short-acting beta blockers.12 However, the precise effect of ß1 blockade during the IPC response is unknown. The purpose of this study was to determine the effect of landiolol on IPC in isolated perfused rabbit hearts.
|
MATERIALS AND METHODS
|
|---|
All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, and published by the National Academy Press, revised 1996. Thirty-seven adult male New Zealand rabbits, weighing 6–7 kg, were anesthetized with sodium pentobarbital (30 mg·kg–1) and anticoagulated with sodium heparin (200 U·kg–1) via an ear vein. Once the corneal reflex was abolished, the rabbits were placed in the supine position and the chest was entered through a bilateral thoracotomy. The heart was rapidly excised and placed in an ice-bath (4°C) of Krebs-Henseleit solution (Na+ 135 mM, K+ 4.7 mM, Ca2+ 1.7 mM, PO4– 1.1 mM, Mg2+ 1.2 mM, HCO3– 25 mM, glucose 11.5 mM, pyruvate 4.9 mM, fumarate 5.4 mM). The aorta was cannulated, and the heart was suspended within a heated glass chamber. The aorta was perfused with oxygenated (95% O2 + 5% CO2) Krebs-Henseleit solution at 37°C and 75 mm Hg root pressure, and the pulmonary artery was incised. The heart was allowed to equilibrate for 30 min while the left atrium was excised and a water-filled balloon was placed through the mitral valve into the left ventricle. The initial left ventricular end-diastolic pressure (LVEDP) was set at 5–10 mm Hg by water inflation, and the volume remained constant throughout the experiment. Left ventricular end-systolic pressure (LVESP) and LVEDP were measured from the balloon tracing. The heart was paced at 150 beats·min–1 with an asynchronous pacemaker. Ischemia was induced by ligating the left anterior descending coronary artery (LAD) close to its origin with 3/0 silk suture. After 1 hour of ischemia, reperfusion was achieved by releasing the ligature and briefly massaging the LAD with a moistened cotton swab. After 1 hour of reperfusion, the heart was removed from the perfusion apparatus, religated, and 3 mL of 0.4% disodium indigotin-disulfonate (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan) was infused through the aortic cannula. The right ventricle was excised, and the left ventricle was sectioned horizontally at 2-mm intervals into 5 or 6 slices. The slices were incubated with triphenyltetrazolium chloride (Sigma Chemical Co., St. Louis, MO, USA) at 37°C, pH 7.4, for 15 min. The unstained area was scanned (SigmaScan Pro; SPSS, Inc., Chicago, IL, USA) and the infarcted area was labeled. The infarcted area (IA) was expressed as a percentage of the total area at risk. The overall infarcted area was computed as a weighted average of the slices by weight.
The experimental protocol is shown in Figure 1
. Seven hearts served as a control group without any treatment prior to LAD ischemia and reperfusion. In another 6 hearts, IPC was accomplished with two 5-min episodes of complete aortic occlusion followed by 5 min of reperfusion prior to LAD ischemia. Four groups of 6 hearts each were pretreated with 1 or 3 µM of landiolol (Ono Pharmaceutical Co., Ltd., Osaka, Japan) or a combination of 1 or 3 µM of landiolol and IPC prior to LAD ischemia. To evaluate the effect of landiolol on cardiac pressure, LVEDP and LVESP were measured 5 min after 1 µM (n = 6) and 3 µM (n = 6) of landiolol infusion.
View larger version (31K):
[in this window]
[in a new window]
|
Figure 1. Experimental protocol: landiolol was infused during pretreatment and ischemic preconditioning (IPC), but not during regional ischemia or reperfusion, landiolol was infused from –25 to 0 min and IPC consisting of 2 cycles of 5-min global ischemia and reperfusion, regional ischemia of the left anterior descending coronary artery (LAD) occurred from 0 to 60 min, and reperfusion from 60 to 120 min.
|
|
All results are expressed as mean ± standard error. LVEDP and LVESP data were analyzed using two-way repeated measures analysis of variance. Infarct size was analyzed using one-way analysis of variance. The effect of landiolol was analyzed using the paired two-tailed t test. Statistical analyses were performed using GraphPad Prism version 4 (GraphPad Software Inc., San Diego, CA, USA). A p-value < 0.05 was considered significant.
|
RESULTS
|
|---|
Infusion of landiolol per se tended to decrease LVESP but it did not reduce LVEDP compared to the pretreatment value (Table 1). Changes in LVEDP during the experiment are shown in Figure 2. Baseline LVEDP was preserved during pretreatment and LAD ischemia in all groups. Ischemic preconditioning reduced LVEDP compared to controls ( p = 0.06). Landiolol 3 µM + IPC reduced LVEDP after reperfusion compared to IPC alone ( p = 0.03) or controls ( p < 0.01). Landiolol 3 µM + IPC also reduced LVEDP compared to landiolol 3 µM ( p = 0.01), landiolol 1 µM ( p < 0.01), and landiolol 1 µM + IPC ( p < 0.01). Landiolol 1 µM + IPC did not reduce LVEDP compared to IPC alone ( p = 0.19), landiolol 1 µM ( p = 0.35), or controls ( p = 0.70). Landiolol 1 µM did not affect LVEDP compared to controls ( p = 0.14) or IPC alone ( p = 0.68). Changes in LVESP during the experiment are shown in Figure 3. Regional LAD ischemia decreased LVESP in all groups, and it did not recover to the pretreatment value in any group. None of the treatments had a significant effect on LVESP: IPC vs. controls, p = 0.35; landiolol 3 µM + IPC vs. IPC, p = 0.33; landiolol 3 µM + IPC vs. controls, p = 0.95; landiolol 3 µM + IPC vs. landiolol 3 µM, p = 0.62; landiolol 3 µM + IPC vs. landiolol 1 µM or 1 µM landiolol + IPC, p = 0.83. Infusion of 1 µM of landiolol before ischemia with IPC did not change LVESP vs. IPC ( p = 0.47), landiolol 1 µM ( p = 0.95), or controls ( p = 0.88). Landiolol 1 µM alone did not change LVESP compared to controls ( p = 0.74) or IPC ( p = 0.45).
View larger version (23K):
[in this window]
[in a new window]
|
Figure 2. Time-course of changes in left ventricular end-diastolic pressure, left ventricular end diastolic pressure in the Lan3µM + IPC group was significantly lower than the other groups. IPC = ischemic preconditioning, Lan1µM = pretreatment with 1 µM of landiolol, Lan3µM = pretreatment with 3 µM of landiolol.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Figure 3. Time-course of changes in left ventricular end-systolic pressure during the experiment, there were no significant difference between the groups. Abbreviations as in Figure 2 .
|
|
Infarct size is shown in Figure 4. Control hearts had an IA of 48% ± 4%. Ischemic preconditioning reduced IA to 25% ± 3% ( p < 0.01 vs. controls). Landiolol 3 µM + IPC and 1 µM + IPC also reduced IA to 17% ± 3% and 22% ± 3%, respectively ( p < 0.01 vs. controls for both doses). No significant difference in IA was observed between IPC and landiolol 3 µM + IPC ( p = 0.11). Landiolol 3 µM alone did not reduce IA ( p = 0.8093 vs. controls). Landiolol 3 µM + IPC reduced IA compared to 3 µM ( p < 0.01) and 1 µM alone ( p < 0.01). No significant difference was found between 1 and 3 µM of landiolol.
View larger version (12K):
[in this window]
[in a new window]
|
Figure 4. Left ventricular infarct size (% of area at risk), significant differences versus control, IPC, IPC + Lan1µM, Lan3µM, and IPC + Lan3µM are designated by *, **, ##, and #, respectively. Abbreviations as in Figure 2.
|
|
|
DISCUSSION
|
|---|
These results demonstrate that pre-ischemia infusion of landiolol enhances the cardioprotective effect of IPC after reperfusion. Ischemic preconditioning reduces infarct size and arrhythmic events, and improves left ventricular function. Recent reports suggest that ß-adrenergic receptor stimulation is involved in the mechanism of IPC. During short periods of ischemia, IPC causes release of catecholamines, resulting in increased catecholamine concentration in the myocardium. In agreement with these results, this study demonstrated that IPC reduced LVEDP and infarct size. Pre-ischemic ß-adrenergic stimulation by isoproterenol administration also reduces infarct size.7 Lochner and colleagues13 demonstrated that IPC increases cyclic adenosine monophosphate concentration, a downstream effect of ß-receptor stimulation during the short period of preconditioning, suggesting a mechanism for the IPC-mediated effects. Adenylate cyclase activity is also increased in preconditioned hearts.14 This accumulated evidence suggests that ß-receptor activation during IPC activates the ß-adrenergic signaling pathway, and promotes the cardioprotective effect after reperfusion.
Landiolol is a highly cardioselective agent with a 255-fold higher affinity for ß1-adrenergic receptors than ß2-receptors.15 In comparison with other beta blockers, esmolol, widely used as an ultra-short-acting beta blocker, has a 33-fold greater affinity for ß1-receptors than ß2-receptors, while propranolol, a non-selective beta blocker, possesses a 0.68-fold greater affinity for ß1-receptors. It is known that the ß1-receptors are 60%–80% of the total ß-receptors in myocardium. The 2 types of receptor have different systems to regulate downstream signaling.16 For example, ß1-receptor stimulation induces apoptotic effects through Gs proteins, while ß2-receptor stimulation shows anti-apoptotic effects due to the dual coupling systems through Gs and Gi proteins. The dual coupling system is thought to contribute to cell survival in various types of injury including ischemia. Although further studies are needed to investigate to what extent the ß2-receptor signaling pathway is activated in this model, it is possible that landiolol specifically blocks ß1-receptors, leading to enhanced or selective activation of the ß2-receptor signaling pathway.
We have previously shown that IPC fails to provide cardioprotective effects in hearts in which ß2-receptor expression is decreased after myocardial infarction, and that gene transfer of ß2-receptors enhances the cardioprotective effects of IPC, resulting in preservation of left ventricular pressure after reperfusion.9,11 In addition, the adenylate cyclase agonist forskolin restores the refractory effect of IPC in the heart after myocardial infarction. The present results, therefore, imply that pre-ischemic infusion of landiolol in combination with IPC may allow IPC to activate ß2-receptor signaling in the absence of ß1-receptor signaling pathways. Acute myocardial ischemia rapidly induces catecholamine release from nerve terminals. Ischemic preconditioning caused by repetitive and reversible ischemic stress protects myocardium from subsequent prolonged ischemia. However, there may not be an increase in catecholamines without IPC, which may explain why landiolol infusion alone failed to show cardioprotective effects after reperfusion. In addition, because landiolol has only a 4-min half-life in plasma, our results are not related to the direct effect of landiolol on LVEDP at 1 hour after reperfusion.12 It is generally accepted that IPC exhibits a cardioprotective effect after repetitive and/or more than 5 min of ischemia, and that this effect continues for 2–3 hour after reperfusion.17 Therefore, a pre-ischemic infusion of landiolol is likely to enhance the cardioprotective effect of IPC, resulting in inhibition of the increase in LVEDP during reperfusion.
Landiolol may have its own cardioprotective effects against ischemia-reperfusion injury. Yasuda and colleagues18 reported that landiolol has the potential to enhance post-ischemic cardiac function after warm cardioplegic arrest in isolated rat heart. Kurosawa and colleagues10 demonstrated that infusion of landiolol for 10 min before global ischemia preserved left ventricular pressure after reperfusion. As we saw no protective effect of landiolol alone, our results are not in accordance with previous reports. We chose the concentrations of landiolol in this study based on previous experiments using esmolol. Although these dosages showed some ß-blocking activity (Table 1), they may not be sufficient to exert a cardioprotective effect in this experimental protocol. Thus, the difference in drug effects between this study and others may be due to the experimental protocol and species.
It is known that beta blockers, including landiolol, have direct effects on the myocardial membrane, antiperoxidative activity, antiradical actions, sarcoplasmic reticular function, and cardiac gene expression, independent of ß-blocking activity. We previously found that pre-ischemic treatment with the nitric oxide donor nipradilol exhibits cardioprotection mainly due to the antioxidative effects of this agent.4 In addition, the nonselective beta blocker propranolol inhibits the activity of reduced nicotinamide adenine dinucleotide oxidase, the effect of which is to slow mitochondrial respiration, thereby preserving adenosine triphosphate stores.19 It is unclear to what extent the cardioprotective effect of landiolol is due to additional effects such as those with nipradilol and propranolol, and thus further investigation is needed.
Recent studies have advanced the development of potent cardioprotective agents as an alternative to IPC. As repeated short periods of ischemia are needed to accomplish IPC, it is not relevant to all patients with serious ischemic heart disease and complications of other organs. However, IPC activates several signaling pathways simultaneously, and consequently provides a greater cardioprotective effect than currently available pharmacological agents. In this context, clarifying the mechanism responsible for IPC will contribute to the development of the ideal cardioprotective agent for preconditioning. It was concluded from these results that infusion of landiolol in combination with IPC reduced LVEDP after ischemia-reperfusion, but did not significantly affect LVESP or infarct size. This suggests that landiolol enhanced the cardioprotective effect of IPC. The mechanism by which landiolol exerts these effects may be through selective regulation of adrenergic receptor-mediated signals in response to IPC.
|
ACKNOWLEDGMENTS
|
|---|
We thank R. Clements, PhD, for helpful advice and editing of the manuscript. Landiolol was kindly provided by Ono Pharmaceutical Company, Osaka, Japan.
|
REFERENCES
|
|---|
- Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–36.[Abstract/Free Full Text]
- Liu Y, Downey JM. Ischemic preconditioning protects against infarction in rat heart. Am J Physiol 1992;263(4 Pt 2):H1107–12.
- Nakai Y, Horimoto H, Mieno S, Sasaki S. Mitochondrial ATP-sensitive potassium channel plays a dominant role in ischemic preconditioning of rabbit heart. Eur Surg Res 2001;33:57–63.[Medline]
- Suematsu Y, Ohtsuka T, Horimoto H, Maeda K, Nakai Y, Mieno S, et al. Long-term treatment with nipradilol, a nitric oxide-releasing beta-adrenergic blocker, enhances postischemic recovery and limits infarct size. Ann Thorac Surg 2002;73:173–9.[Abstract/Free Full Text]
- Asimakis GK, Inners-McBride K, Conti VR, Yang CJ. Transient beta adrenergic stimulation can precondition the rat heart against postischaemic contractile dysfunction. Cardiovasc Res 1994;28:1726–34.[Abstract/Free Full Text]
- Schultz JE, Rose E, Yao Z, Gross GJ. Evidence for involvement of opioid receptors in ischemic preconditioning in rat hearts. Am J Physiol 1995;268(5 Pt 2):H2157–61.
- Yabe K, Ishishita H, Tanonaka K, Takeo S. Pharmacologic preconditioning induced by beta-adrenergic stimulation is mediated by activation of protein kinase C. J Cardiovasc Pharmacol 1998;32:962–8.[Medline]
- Sandhu R, Thomas U, Diaz RJ, Wilson GJ. Effect of ischemic preconditioning of the myocardium on cAMP. Circ Res 1996;78:137–47.[Abstract/Free Full Text]
- Mieno S, Horimoto H, Watanabe F, Nakai Y, Furuya E, Sasaki S. Potent adenylate cyclase agonist forskolin restores myoprotective effects of ischemic preconditioning in rat hearts after myocardial infarction. Ann Thorac Surg 2002;74:1213–8.[Abstract/Free Full Text]
- Kurosawa S, Kanaya N, Niiyama Y, Nakayama M, Fujita S, Namiki A. Landiolol, esmolol and propranolol protect from ischemia/reperfusion injury in isolated guinea pig hearts. Can J Anaesth 2003;50:489–94.[Medline]
- Mieno S, Horimoto H, Sawa Y, Watanabe F, Furuya E, Horimoto S, et al. Activation of beta2-adrenergic receptor plays a pivotal role in generating ischemic preconditioning in rat hearts. Scand Cardiovasc J 2005;39;313–9.[Medline]
- Iguchi S, Iwamura H, Nishizaki M, Hayashi A, Senokuchi K, Kobayashi K, et al. Development of a highly cardioselective ultra short-acting beta-blocker, ONO-1101. Chem Pharm Bull (Tokyo) 1992;40:1462–9.[Medline]
- Lochner A, Genade S, Tromp E, Podzuweit T, Moolman JA. Ischemic preconditioning and the beta-adrenergic signal transduction pathway. Circulation 1999;100:958–66.[Abstract/Free Full Text]
- Okruhlicova L, Ravingerova T, Pancza D, Tribulova N, Styk J, Stetka R. Activation of adenylate cyclase system in the preconditioned rat heart. Physiol Res 2000;49:251–9.[Medline]
- Sugiyama A, Takahara A, Hashimoto K. Electrophysiologic, cardiohemodynamic and beta-blocking actions of a new ultra-short-acting beta-blocker, ONO-1101, assessed by the in vivo canine model in comparison with esmolol. J Cardiovasc Pharmacol 1999;34:70–7.[Medline]
- Xiao RP, Cheng H, Zhou YY, Kuschel M, Lakatta EG. Recent advances in cardiac beta(2)-adrenergic signal transduction. Circ Res 1999;85:1092–100.[Abstract/Free Full Text]
- Kloner RA, Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications: part 2. Circulation 2001;104:3158–67.[Abstract/Free Full Text]
- Yasuda T, Kamiya H, Tanaka Y, Watanabe G. Ultra-short-acting cardioselective beta-blockade attenuates postischemic cardiac dysfunction in the isolated rat heart. Eur J Cardiothorac Surg 2001;19:647–52.[Abstract/Free Full Text]
- Sakurada A, Voss DO, Brandao D, Campello AP. Effects of propranolol on heart muscle mitochondria. Biochem Pharmacol 1972;21:535–40.[Medline]
TOP
ABSTRACT
INTRODUCTION
