517920 research-article2014
PRF0010.1177/0267659113517920PerfusionLiao et al.
Original Paper
Agonist of inward rectifier K+ channels enhances the protection of ischemic postconditioning in isolated rat hearts
Perfusion 2014, Vol. 29(4) 321–326 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0267659113517920 prf.sagepub.com
Z Liao, Z Feng and C Long
Abstract Background: Selective inhibition of inward rectifier K+ channels could abolish the protection mediated by ischemic preconditioning, but the roles of these channels in ischemic postconditioning have not been well characterized. Our study aims to evaluate the effect of inward rectifier K+ channels on the protection induced by ischemic postconditioning. Methods: Langendorff-perfused rat hearts (n=8 per group) were split into four groups: postconditioning hearts (IPO group); ischemic postconditioning with BaCl2 hearts (PB group); ischemic postconditioning with zacopride hearts (PZ group); and without ischemic postconditioning (CON group). After suffering 30 minutes of global ischemia, groups IPO, PB and PZ went through 10 seconds of ischemic postconditioning with three different perfusates: respectively, Krebs–Henseleit buffer (IPO group); 20 μmol/L BaCl2 (antagonist of the channel, PB group); 1 μmol/L zacopride (agonist of the channel, PZ group). Results: At the end of reperfusion, the myocardial performance was better preserved in the PZ group than the other three groups. The PB group showed no significant differences from the CON group. Conclusions: Our study has shown that the IK1 channel agonist zacopride is associated with the enhancement of ischemic postconditioning. Keywords cardioprotection; ischemic postconditioning; inward rectifier K+ channels, zacopride; Langendorff
Introduction Ischemia/reperfusion (I/R) injury has been a focus of cardiac protection research for several decades. In 2003, Zhao et al. found brief and repeated episodes of ischemia reperfusion immediately after a period of sustained ischemia reduced reperfusion injury in dog hearts. This phenomenon is called ‘ischemic postconditioning’ (IPO).1 Many ion channels are involved in IPO protection and it is of interest to many scientists that both ischemic precondition (IPC) and IPO, to some extent, share the common channels and pathways.2 It has been well established that the inward rectifier K+ (Ik1) channel plays a major role in modulating the membrane potential and maintaining excitability of cardiomyocytes.3 Activating the Ik1 channel could facilitate intracellular Cl- effluxing and enhanced cell regulatory volume decrease (RVD) is able to prevent cell swelling in IPC.4-6 Moreover, in the early stage of reperfusion, activating the Ik1 channel could shorten action potential duration (APD) and, therefore, prevent intracellular Ca2+ overload. Additionally, selective inhibition of the
IK1 channels could attenuate the protection of IPC. All the above phenomena share the common mechanism mediated by IPO protection.7 However, so far, there is little evidence to indicate the Ik1 channels are responsible for IPO. Department of Cardiopulmonary Bypass, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China Corresponding author: Zhengyi Feng MD Department of Cardiopulmonary Bypass State Key Laboratory of Cardiovascular Disease Fuwai Hospital National Center for Cardiovascular Diseases Chinese Academy of Medical Sciences and Peking Union Medical College 167, Bei li shi Road Beijing 100037 Peoples Republic of China. Email: [email protected]
Downloaded from prf.sagepub.com at JOHNS HOPKINS UNIVERSITY on December 30, 2014
322
Perfusion 29(4)
The purpose of our study was to demonstrate how exactly Ik1 channels affect IPO protection.
Methods The present experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH Publication No. 85-23, revised 1996) and permitted by the Research Commission on Ethics of Fuwai Hospital.
Surgical Procedure Male Sprague–Dawley rats (weighing 250–350 g, n=32) were anesthetized with pentobarbital (i.p. 50 mg/kg). Heparin (i.v.1,000 UI/kg) was injected through the femoral veins. The hearts were rapidly removed and immersed in ice-cold buffer. After cannulating the heart onto a Langendorff perfusion apparatus, KrebsHenseleit bicarbonate buffer (K-H, containing in mmol/L: glucose 11.0, NaCl 118.5, KCl 4.75, MgSO4 1.19, KH2PO4 1.18, NaHCO3 25.0 and CaCl2 1.4 at pH 7.4) was administered. The buffer was bubbled with 95% O2 and 5% CO2 at 37°C and maintained at 100 cmH2O perfusion pressure during the procedure.
Experimental Protocol After a 20 minutes stabilization period of perfusion with K-H buffer, all the hearts were subjected to 30 minutes of global ischemia (24°C). The hearts were randomly assigned to the following groups (Figure 1): control hearts (CON group, n=8), the hearts were re-perfused with K-H buffer continuously for 60 minutes; ischemic postconditioning hearts (IPO group, n=8), six cycles of 10 seconds of reperfusion with K-H buffer followed by 10 seconds re-occlusion, then continuous K-H buffer reperfusion was performed for 60 minutes; ischemic postconditioning with BaCl2 hearts (PB group, n=8), six cycles of 10 seconds of reperfusion with K-H buffer containing 20 μmol/L BaCl2 followed by 10 seconds reocclusion then continuous K-H buffer without BaCl2 reperfusion was performed for 60 minutes; ischemic postconditioning with zacopride hearts (PZ group, n=8), six cycles of 10 seconds of reperfusion with K-H buffer containing 1 μmol/L zacopride followed by 10 seconds re-occlusion then continuous K-H buffer without zacopride re-perfusion for 60 minutes.
Cardiac function and coronary flow (CF) measurements A water-filled balloon was connected to a pressure transducer (AD Instruments, Colorado Springs, CO)
Figure 1. Experimental protocol. CON: CON group (control); IPO: IPO group (six cycles of 10 seconds of reperfusion with K-H buffer followed by 10 seconds ischemia immediately after the continuous ischemia period); PZ: PZ group (six cycles of 10 seconds of reperfusion with K-H buffer containing 1 μmol/L zacopride followed by 10 seconds of global ischemia immediately after the continuous ischemia period; PB: PB group (six cycles of 10 seconds of reperfusion with K-H buffer containing 20μmol/L BaCl2 followed by 10 seconds of ischemia immediately after the continuous ischemia).
and inserted into the left ventricle through the mitral valve. Heart rate (HR), left ventricular developed pressure (LVDP) and maximum positive and negative derivative of left ventricular pressure (±dp/dtmax) were recorded with a monitor (Chart V; AD Instruments) for analysis. Coronary flow (CF) was evaluated by measuring the volume of coronary effluent which was returned from the coronary sinus and expressed in milliliters per minute. Cardiodynamic data and CF were recorded at different time points (the end of stabilization period (20 min, baseline), 5, 10, 30 and 60 min after reperfusion).
Determination of cTnI, MDA and SOD I/R injury was evaluated by troponin I (cTnI) release in the coronary effluent at baseline and after 60 min of reperfusion (Troponin I ELISA. TPI Kit, Kamiya Biochemical Company, Seattle, Washington, USA). Results were recorded in nanograms per milliliter. The malondialdehyde (MDA) activity was compared by measuring the concentration of MDA in the coronary effluent collected at baseline and after 60 min of reperfusion. Assays were conducted using a Lipid Peroxidation Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. MDA values are given as nanomoles per milliliter of coronary effluent. The activity of the total superoxide dismutase (T-SOD) was measured by using an assay kit (Nanjing Jiancheng Bioengineering Institute), according to the
Downloaded from prf.sagepub.com at JOHNS HOPKINS UNIVERSITY on December 30, 2014
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Liao et al.
Figure 2. (A) cTnI release in the coronary effluent of the hearts at baseline and 60 min of reperfusion. PZ hearts had significantly reduced cTnI release compared with IPO hearts. Values shown are the mean±SD of eight assessments; (B) The effect of zacopride and BaCl2 on the levels of MDA concentration; and (C) T-SOD activity of coronary effluent in rat Langendorff model. ΔP
PRF0010.1177/0267659113517920PerfusionLiao et al.
Original Paper
Agonist of inward rectifier K+ channels enhances the protection of ischemic postconditioning in isolated rat hearts
Perfusion 2014, Vol. 29(4) 321–326 © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0267659113517920 prf.sagepub.com
Z Liao, Z Feng and C Long
Abstract Background: Selective inhibition of inward rectifier K+ channels could abolish the protection mediated by ischemic preconditioning, but the roles of these channels in ischemic postconditioning have not been well characterized. Our study aims to evaluate the effect of inward rectifier K+ channels on the protection induced by ischemic postconditioning. Methods: Langendorff-perfused rat hearts (n=8 per group) were split into four groups: postconditioning hearts (IPO group); ischemic postconditioning with BaCl2 hearts (PB group); ischemic postconditioning with zacopride hearts (PZ group); and without ischemic postconditioning (CON group). After suffering 30 minutes of global ischemia, groups IPO, PB and PZ went through 10 seconds of ischemic postconditioning with three different perfusates: respectively, Krebs–Henseleit buffer (IPO group); 20 μmol/L BaCl2 (antagonist of the channel, PB group); 1 μmol/L zacopride (agonist of the channel, PZ group). Results: At the end of reperfusion, the myocardial performance was better preserved in the PZ group than the other three groups. The PB group showed no significant differences from the CON group. Conclusions: Our study has shown that the IK1 channel agonist zacopride is associated with the enhancement of ischemic postconditioning. Keywords cardioprotection; ischemic postconditioning; inward rectifier K+ channels, zacopride; Langendorff
Introduction Ischemia/reperfusion (I/R) injury has been a focus of cardiac protection research for several decades. In 2003, Zhao et al. found brief and repeated episodes of ischemia reperfusion immediately after a period of sustained ischemia reduced reperfusion injury in dog hearts. This phenomenon is called ‘ischemic postconditioning’ (IPO).1 Many ion channels are involved in IPO protection and it is of interest to many scientists that both ischemic precondition (IPC) and IPO, to some extent, share the common channels and pathways.2 It has been well established that the inward rectifier K+ (Ik1) channel plays a major role in modulating the membrane potential and maintaining excitability of cardiomyocytes.3 Activating the Ik1 channel could facilitate intracellular Cl- effluxing and enhanced cell regulatory volume decrease (RVD) is able to prevent cell swelling in IPC.4-6 Moreover, in the early stage of reperfusion, activating the Ik1 channel could shorten action potential duration (APD) and, therefore, prevent intracellular Ca2+ overload. Additionally, selective inhibition of the
IK1 channels could attenuate the protection of IPC. All the above phenomena share the common mechanism mediated by IPO protection.7 However, so far, there is little evidence to indicate the Ik1 channels are responsible for IPO. Department of Cardiopulmonary Bypass, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China Corresponding author: Zhengyi Feng MD Department of Cardiopulmonary Bypass State Key Laboratory of Cardiovascular Disease Fuwai Hospital National Center for Cardiovascular Diseases Chinese Academy of Medical Sciences and Peking Union Medical College 167, Bei li shi Road Beijing 100037 Peoples Republic of China. Email: [email protected]
Downloaded from prf.sagepub.com at JOHNS HOPKINS UNIVERSITY on December 30, 2014
322
Perfusion 29(4)
The purpose of our study was to demonstrate how exactly Ik1 channels affect IPO protection.
Methods The present experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH Publication No. 85-23, revised 1996) and permitted by the Research Commission on Ethics of Fuwai Hospital.
Surgical Procedure Male Sprague–Dawley rats (weighing 250–350 g, n=32) were anesthetized with pentobarbital (i.p. 50 mg/kg). Heparin (i.v.1,000 UI/kg) was injected through the femoral veins. The hearts were rapidly removed and immersed in ice-cold buffer. After cannulating the heart onto a Langendorff perfusion apparatus, KrebsHenseleit bicarbonate buffer (K-H, containing in mmol/L: glucose 11.0, NaCl 118.5, KCl 4.75, MgSO4 1.19, KH2PO4 1.18, NaHCO3 25.0 and CaCl2 1.4 at pH 7.4) was administered. The buffer was bubbled with 95% O2 and 5% CO2 at 37°C and maintained at 100 cmH2O perfusion pressure during the procedure.
Experimental Protocol After a 20 minutes stabilization period of perfusion with K-H buffer, all the hearts were subjected to 30 minutes of global ischemia (24°C). The hearts were randomly assigned to the following groups (Figure 1): control hearts (CON group, n=8), the hearts were re-perfused with K-H buffer continuously for 60 minutes; ischemic postconditioning hearts (IPO group, n=8), six cycles of 10 seconds of reperfusion with K-H buffer followed by 10 seconds re-occlusion, then continuous K-H buffer reperfusion was performed for 60 minutes; ischemic postconditioning with BaCl2 hearts (PB group, n=8), six cycles of 10 seconds of reperfusion with K-H buffer containing 20 μmol/L BaCl2 followed by 10 seconds reocclusion then continuous K-H buffer without BaCl2 reperfusion was performed for 60 minutes; ischemic postconditioning with zacopride hearts (PZ group, n=8), six cycles of 10 seconds of reperfusion with K-H buffer containing 1 μmol/L zacopride followed by 10 seconds re-occlusion then continuous K-H buffer without zacopride re-perfusion for 60 minutes.
Cardiac function and coronary flow (CF) measurements A water-filled balloon was connected to a pressure transducer (AD Instruments, Colorado Springs, CO)
Figure 1. Experimental protocol. CON: CON group (control); IPO: IPO group (six cycles of 10 seconds of reperfusion with K-H buffer followed by 10 seconds ischemia immediately after the continuous ischemia period); PZ: PZ group (six cycles of 10 seconds of reperfusion with K-H buffer containing 1 μmol/L zacopride followed by 10 seconds of global ischemia immediately after the continuous ischemia period; PB: PB group (six cycles of 10 seconds of reperfusion with K-H buffer containing 20μmol/L BaCl2 followed by 10 seconds of ischemia immediately after the continuous ischemia).
and inserted into the left ventricle through the mitral valve. Heart rate (HR), left ventricular developed pressure (LVDP) and maximum positive and negative derivative of left ventricular pressure (±dp/dtmax) were recorded with a monitor (Chart V; AD Instruments) for analysis. Coronary flow (CF) was evaluated by measuring the volume of coronary effluent which was returned from the coronary sinus and expressed in milliliters per minute. Cardiodynamic data and CF were recorded at different time points (the end of stabilization period (20 min, baseline), 5, 10, 30 and 60 min after reperfusion).
Determination of cTnI, MDA and SOD I/R injury was evaluated by troponin I (cTnI) release in the coronary effluent at baseline and after 60 min of reperfusion (Troponin I ELISA. TPI Kit, Kamiya Biochemical Company, Seattle, Washington, USA). Results were recorded in nanograms per milliliter. The malondialdehyde (MDA) activity was compared by measuring the concentration of MDA in the coronary effluent collected at baseline and after 60 min of reperfusion. Assays were conducted using a Lipid Peroxidation Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. MDA values are given as nanomoles per milliliter of coronary effluent. The activity of the total superoxide dismutase (T-SOD) was measured by using an assay kit (Nanjing Jiancheng Bioengineering Institute), according to the
Downloaded from prf.sagepub.com at JOHNS HOPKINS UNIVERSITY on December 30, 2014
323
Liao et al.
Figure 2. (A) cTnI release in the coronary effluent of the hearts at baseline and 60 min of reperfusion. PZ hearts had significantly reduced cTnI release compared with IPO hearts. Values shown are the mean±SD of eight assessments; (B) The effect of zacopride and BaCl2 on the levels of MDA concentration; and (C) T-SOD activity of coronary effluent in rat Langendorff model. ΔP
