Scholarly article on topic 'Studies of Ischemic Preconditioning Mechanisms in Langendorff Rat Heart Model the Impact of Phosphocreatin Kinase and ATP Sensitive Potasium Channels Pharmachological Openers and Blockers on Cardioprotection'

Studies of Ischemic Preconditioning Mechanisms in Langendorff Rat Heart Model the Impact of Phosphocreatin Kinase and ATP Sensitive Potasium Channels Pharmachological Openers and Blockers on Cardioprotection Academic research paper on "Agriculture, forestry, and fisheries"

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{"ischemic preconditioning (IPC)" / "1 / 2-dioctanoyl-sn-glycerol (DOG)" / "pharmachological preconditioning (PP)" / "Phosphocreatin kinase (PCK)" / "KATP channels" / ischemia-reperfusion / Gliblenclamide / Cromakalim / "Chelerytrine. "}

Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Cristian Romeo Revnic, Carmen Ginghina, Adriana Sarah Nica, Silviu Voinea, Alexandru Sonea, et al.

Abstract Ischemic preconditioning (IPC) induced by administration of brief episodes of ischemia-reperfusion represents a protective mechanism of the heart against prolonged episodes of ischemia. Although the mechanism of ischemic preconditioning has been extensively studied, however, until now it is insufficient elucidated. Using a Langendorff rat heart model with 45minutes ischemia followed by 120minute reperfusion we aimed to evaluate the role of Phosphocreatin Kinase-C (PCK) in ischemic rat myocardium and demonstration of its involvment in the path of pharmachological preconditioning (PP) by using PKC activators 1,2-dioctanoyl-sn-glycerol (DOG) and inhibitors chelerythrine (CHE) and evaluation of the role of KATP channels in pharmachological preconditioning (PP) mechanism by administration of a KATP channel opener (Cromakalim) (CRK) or by blocking the opening of KATP channels with glibenclamide(GLB). The activators of PCK(DOG) and of K ATP channel (CRK) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart When activator of PCK DOG + GLB, a KATP channel inhibitor were coperfused before the lethal ischemia, there was an increase in myocardial infarct size, expressed as a percentage of the area at risk, versus control, the cardioprotective effect of DOG being abolished by GLB. The same results were obtained when CHE the inhibitor of PCK was coperfused with CRK the activator of K ATP channel. The effect of CHE and GLB perfused at the beginning of IPC resulted in loss of protection accompanied by a significant increase in infarct size area. The finding that treatment with a DOG PKC activator and CRK activator of K ATP channel gives a similar degree of protection against infarction as that seen after ischemic preconditioning and that this protection can be blocked by CHE a PCK pharmachological inhibitor provides support for the hypothesis that PKC plays a pivotal role in ischemic preconditioning. Our data may have a significance in pharmachological preconditioning (PP) with decrease in infarct size as an end point.

Academic research paper on topic "Studies of Ischemic Preconditioning Mechanisms in Langendorff Rat Heart Model the Impact of Phosphocreatin Kinase and ATP Sensitive Potasium Channels Pharmachological Openers and Blockers on Cardioprotection"

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Agriculture and Agricultural Science

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Agriculture and Agricultural Science Procedia 10 (2016) 299 - 310

5th International Conference "Agriculture for Life, Life for Agriculture'

Studies of Ischemic Preconditioning Mechanisms in Langendorff Rat Heart Model the Impact of Phosphocreatin Kinase and ATP Sensitive Potasium Channels Pharmachological Openers and Blockers on Cardioprotection

Cristian Romeo REVNICa, Carmen GINGHINAb, Adriana SarahNICAb, Silviu VOINEAb, Alexandru SONEAc, Cosmin SONEAc, Catalina PENAd, Bogdan PALTINEANUd, Flory REVNICd*

aAmbroise Pare' Hospital, Pierre et Marie Curie University, Paris VI, France bU.M.F. "CarolDavila", Bucharest, Romania cUniversity of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Marasti Blvd, District 1, Bucharest, Romania d'N.I.G.G. "Ana Aslan ,Biology of Aging Department, 9 Caldarusani Str., 78178, Bucharest, Romania eU.M.F. Tg.Mures, Surgery Department, Tg. Mures

Ischemic preconditioning (IPC) induced by administration of brief episodes of ischemia-reperfusion represents a protective mechanism of the heart against prolonged episodes of ischemia. Although the mechanism of ischemic preconditioning has been extensively studied, however, until now it is insufficient elucidated. Using a Langendorff rat heart model with 45 minutes ischemia followed by 120 minute reperfusion we aimed to evaluate the role of Phosphocreatin Kinase-C (PCK) in ischemic rat myocardium and demonstration of its involvment in the path of pharmachological preconditioning (PP) by using PKC activators 1,2-dioctanoyl-sn-glycerol (DOG) and inhibitors chelerythrine (CHE) and evaluation of the role of KArP channels in pharmachological preconditioning (PP) mechanism by administration of a KATP channel opener (Cromakalim) (CRK) or by blocking the opening of KATP channels with glibenclamide(GLB). The activators of PCK(DOG) and of K ATP channel (CRK) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart When activator of PCK DOG + GLB, a KATP channel inhibitor were coperfused before the lethal ischemia, there was an increase in myocardial infarct size, expressed as a percentage of the area at risk, versus control, the cardioprotective effect of DOG being abolished by GLB. The same results were obtained when CHE the inhibitor of PCK was coperfused with CRK the activator of K ATP channel. The effect of CHE and GLB perfused at the beginning of IPC resulted in loss of protection accompanied by a significant increase in infarct size area. The finding that treatment with a DOG PKC activator and CRK activator of K ATP channel gives a similar degree of protection against infarction as that seen after ischemic preconditioning and that this protection can be blocked by CHE a PCK pharmachological inhibitor provides support for the hypothesis that PKC plays a pivotal role in ischemic preconditioning. Our data may have a significance in pharmachological preconditioning (PP) with decrease in infarct size as an end point.

2210-7843 © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of the University of Agronomic Sciences and Veterinary Medicine Bucharest doi:10.1016/j.aaspro.2016.09.067

Abstract

© 2016 The Authors. Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Peer-reviewunderresponsibilityoftheUniversityof AgronomicSciencesand VeterinaryMedicineBucharest

Keywords: ischemic preconditioning (IPC), 1,2-dioctanoyl-sn-glycerol (DOG), pharmachological preconditioning (PP), Phosphocreatin kinase (PCK), KAtp channels, ischemia-reperfusion, Gliblenclamide, Cromakalim, Chelerytrine.

* Corresponding author. Tel.: +40721-496-792; Fax: +4021-318-28-88.

E-mail address: f_revnic@yahoo.com

1. Introduction

Coronary artery disease represents a global burden on health care resources, and it is the leading cause of morbidity and mortality in the world by 2020 (Go et al., 2013). Repeated short episodes of ischemia and reperfusion have been demonstrated to make myocardium transiently more resistant to deleterious effects of prolonged ischemia and this paradoxical form of myocardial adaptation has been termed as ischemic preconditioning (Braunwald and Kloner, 1982). The occlusion of circumflex artery has produced protection of myocardium supplied by left anterior descending coronary artery and this phenomenon is termed as intracardiac preconditioning (Murry, 1986).

Acute myocardial infarction(AMI), the clinical manifestation of ischemia-reperfusion (IR) injury, is a leading cause of death in the world. (Go et al., 2013). In 1974, Braunwald first proposed the concept that "just because myocardial tissue lies within the distribution of a recently occluded coronary artery does not mean that it is necessary condemned to death" (Braunwald and Kloner, 1982). This statement was at the origin of the concept of "damage control",which aims at limiting cardiac injury by mean of early reperfusion and adjunctive pharmacologic therapy. Although percutaneous coronary interventions and thrombolytic therapies are effective in limiting the duration of ischemia, the re-introduction of blood flow to previously ischemic area causes additional damage, collectively known as reperfusion injury. The first form of conditioning was discovered by Murry and colleagues in 1986, in this study, the authors made what at that time appeared one of the most effective ways to reduce reperfusion injury is ischemic preconditioning (IPC), which is induced by several cycles of brief ischemia and reperfusion bouts prior to the prolonged ischemia protective against a longer ischemic episode. The protection afforded by this so called "preconditioning" against cell death is dramatic and is observable in all species investigated, as well as in tissues other than the myocardium. Although a large number of studies have identified different triggers and mediators of preconditioning, their clinical applicability has been limited.

Preconditioning signalling. The underlying mechanism of IPC is made up of complex signalling pathways, including triggers, mediators and effectors. Generally, autocrine and paracrine signalling induced by IPC first starts with the activation of membrane receptors, including adenosine, bradykinin B2, angiotensin, opioid and endothelin receptors (Downey et al., 2007).

MOLECULAR MECHANISM

Ischemic preconditioning seems to operate via agonist binding to Gi protein-coupled receptors prior to ischemia, which triggers a signaling cascade that protects the heart from mitochondrial permeability transition (MPT). These triggers in our current understanding, induce the formation of a vesicular caveolar signaling platform (signalosome) that contain the enzymes of the pathway and terminates in activation of guanylyl cyclase resulting in increased production of cGMP and subsequent activation of protein kinase G (PKG) (Quinlan et al., 2008). cGMP-activated PKG phosphorylates a protein on the mitochondrial outer membrane (MOM), which then causes the mitochondrial KATP channel (mitoKATP) on the mitochondrial inner membrane to open, leading to increased production of reactive oxygen species (ROS) by the mitochondria. The protective signal from the MOM is transmitted to the mitochondrial inner membrane by a series of intermembrane signalling steps that includes PKC activation. The resulting ROS then activate a second PKC pool which, through another signal transduction pathway termed the mediator pathway, causes inhibition of MPT and reduction in cell death (Costa et al., 2007). The mechanism of PKC activation during ischemic preconditioning is poorly known, a number of potential mediators of PKC activation has been described in experimental studies carried out on myocardium from rat, dog, rabbit and man among these mediators include: Nitric Oxide (NO), Reactive Oxygen Species (ROS), Diacylglycerol (Steinberg, 2015).

Proteinkinase-C (PKC) is comprised of a family of signal-regulated enzymes that play pleiotropic roles in the control of many physiological and pathological responses (Garlid et al., 2008). Experiments conducted on rat myocardium showed that there are several isoforms of PKC which have been divided into three categories: classic, new and unusual, the difference between them being on the need of the presence of different activators (Kawamura et al., 1998). The classic PKC group contains PKC-a which requires for activation the presence of Ca2+, phosphatidylserine, DOG; the new PKC group contains PKC 8 and PKC-e, their activation is done in the presence of DOG and phosphatidylserine. The group of atypical PKC includes PKC-^ which requires for activation only the presence of phosphatidylserine (Nishizuka, 1992).

ISCHEMIC PRECONDITIONING AND KATP CHANNELS

The main functions of K ATP are concerned, it seem to be as responders to cardiac stress (Wang et al., 2015) and shortening the duration of the cardiac action potential should decrease myocyte Ca++ influx and force of contraction, and, consequently, the requirement for ATP which is in short supply in hypertrophic cardiac myocytes (Kane et al., 2015).

A number of studies have evaluated the role of KAtp channels in ischemic preconditioning mechanism; there were described two types of KATP channels involved: sarcoplasmic (sarc-KATP ) si mitochondrial (mito-KATP).

Cardiomyocite ischemia involves sarc-KATP opening, leading to an increased influx of K+ and shortening the action potential with increased risk of arrhythmias (Kane et al., 2005).

Informations regarding IPC has been derived mainly from the use of K channels openers (KCOs) such as nicorandil, diazoxside, pinacidil and Cromakalim, which can mimic IPC and that interact primarly with mito-KATP channels rather than sarcolemal (sarc-KATP ) channels and from the use of blockers such as gliblenclamide and 5 hydroxidecanoic acid (5HD) which diminish the beneficial effects of short ischemic events on cardiac tissue.

Therefore, a considerable attention has been focused on the opening of the mitochondial (mito-KATP) channel as a primary regulatory event in mitocondrial cardioprotective signaling in ischemic preconditioning (IPC) (Dolinsky and Dyck, 2006). Cardioprotective effect of IPC is mediated by (mito-KATP) opening, mitochondrial Calcium overloading and increase mitochondrial reactive species (ROS) generation leading to further PCK activation. The cascade of cardioprotective events can be initiated by the binding of (adenosine, opioids, acetylcholine, bradykinin) ligands to G- coupled receptors and subsequent activation of calcium flux, TPK (tyrosine protein kinase), the P3IK/Akt pathways and (mito-KATP) modulation (Revnic et al., 2009). Regulation of ion channel through activation of kinases such as protein kinase A (PKA) and PKC is an important mechanism that regulates a wide variety of cellular functions. The phosphorylation by PKA and PKC on serine and threonine residue is known to alter channel properties by modifying the kinetics and/or number of channels present on plasma membrane, including KATP channels. Classical KATP consist of inward rectifier Kir6.2 subunits and sulfonylurea receptor subunits (SUR1 or SUR2). The SUR is a member of the ATP-binding cassette (ABC) family of proteins and acts as a regulatory subunit, conferring ADP sensitivity and the distinctive pharmacological characteristics on the KATP channel complex. On the other hand, the Kir6.x subunit forms the pore of the channel and mediates the defining ATP dependent inhibition of KATP channels In addition to being regulated by various nucleotides, KATP channels are modulated by hormones, noradrenaline, intracellular signals such as G proteins (Gs), phosphatidylinositol-4,5 phosphate (PIP2) that modulate KATP channel activity (Szwczyk and Wojtczak, 2002).

OBJECTIVES:

Using a Langendorff rat heart model with 45 minutes ischemia followed by 120 minute reperfusion we aimed to evaluate:

1. The effect of ischemic preconditioning (IPC) (2x5 minutes ischemia alternated with 5 minutes reperfusion) on infarct size as an end point, because is a. robust indicator of preconditioning-induced protection;

2. The effect of pharmachological inhibitor of PCK chelerythrine (CHE) and pharmachological inhibitor of KATP channel Gliblenclamide (GLB) perfused before ischemic preconditioning (IPC) protocol;

3. The role of Phosphocreatin Kinase (PCK) in ischemic rat myocardium and demonstration of the involvment of PCK in the path of pharmachological preconditioning (PP) by using PKC activators (DOG) and inhibitors chelerythrine (CHE) and evaluation of the role of KATP channels in pharmachological preconditioning (PP)

mechanism by administration of an KAtp channel opener (Cromakalim) (CRK) or by inhibiting the opening of KAtp channels with glibenclamide (GLB.);

4. To test the hypothesis whether pharmachological activators of PCK and 1,2-dioctanoyl-sn-glycerol (DOG), a diacylglycerol analogue and KAtp channels (Cromakalim (CRK) perfused in rat heart before lethal ischemia could mimic the ischemic preconditioning with limiting the infarct size as an end point;

5. The effect of coperfusion of DAG pharmachological activator of CPK + Gliblenclamide (GLB) pharmachological inhibitor of K ATP channel, administrated in rat heart before lethal ischemia, on infarct size;

6. The effect of coperfusion of pharmachological activator of KAip channel Cromakalim (CRK)+ Chelerythrine (CHE) the pharmachological inhibitor of PCK administrated in rat heart before lethal ischemia, on infarct size.

2. Materials and Methods

Animals

50 White male Wistar rats aged 12 months old (330+/-30 g body weight) were used in our study. All animals were fed a standard diet, housed under the same conditions, and received humane care in accordance with The Guidance on the Operation of the Animals (Scientific Procedures, 1986).

The principle of retrograde myocardial perfusion in Langendorff system:

Experimental studies using isolated and perfused rat myocardium in Langendorff retrograde perfusion system are commonly performed in research laboratories to study the physiological, pharmacological, morphological and biochemical aspects of ischemia reperfusion injury as well as to evaluate the ventricular performance, the metabolic parameters and the coronary flow parameters in different experimental settings.

The system was first imagined and created by Oscar Langerdorff in 1897 inquiries were made starting from Cyon Eilas Carl Ludwig Institute of Physiology in Leipzig Germany in 1866, the originally proposed Langerdorff method suffering small changes over the time (Skrzypiec-Spring et al., 2007).

The principle of the method is as follows: isolated myocardium is perfused by retrograde flow from the aorta in which a cannula system is inserted to vehiculate the oxygenated Langerdorff reperfusion infusion liquid.

Myocardium is perfused in diastole when aortic valve closure occurs and infusion liquid enters the coronary system, later it leaves the coronary sinus, arrives into the right atrium then passes the right ventricle and leaves the heart through the pulmonary artery.

The advantage of isolated rat myocardial perfusion study in Langerdorff system for experimental models compared with ischemia-reperfusion studies performed on living rats is that the extracardiac mechanisms of neurohormonal controlol are abolished and we can better study some of the myocardial properties.

Using this system has been demonstrated the role of various factors upon the electrical activity and myocardial contractile function: temperature, oxygen, calcium ions, adrenaline, acetylcholine and the evaluation of myocardial reperfusion lesions and the preparation for cardiac transplantation (Skrzypiec-Spring et al., 2007).

Description of retrograde perfusion system Langerdorff

For this study, we used Langendorff apparatus ML870B2 (production AD Instruments) and time recording software PowerLab systems for monitoring and analyzing the following parameters: heart rate, ECG, coronary flow, left ventricular developed pressure, left ventricular systolic pressure, end diastolic left ventricular pressure and temperature. For all these measurements there are sensors connected to CPU PowerLab/8SP.

Ensuring a constant coronary flow perfusion is made using the Minipuls™ 3 pump and the pump control unit (STH Pump Controller); the system used by us having the advantage over the classical system that glass containers for ensuring the coronary perfusion were eliminated, there is a better control of temperature during reperfusion with different solutions can be made because the tank is divided in different compartments.

Isolated heart surgical preparation for Langendorffperfusion system

Rats were anesthetized with sodium pentobarbital (55 mg/kg intraperitonally) and given heparin sodium (300 IU). Hearts were excised, arrested in ice-cold buffer, and mounted on a constant-pressure (80 mm Hg) Langendorff perfusion system. They were perfused with a modified Krebs-Henseleit bicarbonate buffer containing the following

chemicals (in mmol/L): NaCl 118.5, NaHC03 25.0, KC1 4.8, MgS04 1.2, KH2P04 1.2, CaCl2 1.7, and glucose 12.0.The temperature-controlled value of 37°C was continuously monitored by a thermoprobe inserted into the right ventricle. Infused buffer was freshly prepared and filtered using 0.45 nm hydrophobic microfiltration membrane based on a polyether sulfone polymer (Sartorius AG, Gottingen).

A latex, fluid-filled, isovolumic balloon was introduced into the left ventricle through the left atrial appendage and inflated to give a preload of 8 to 10 mm Hg. The device was previously set to a constant flow rate (9.7 ± 0.5 ml/min) the perfused liquid having a concentration of 95% O2, 5% CO2, pH 7.4. Left ventricular developed pressure, heart rate, and coronary flow were registered at regular intervals. A surgical needle it has been used which was passed under the left main coronary artery, and the ends of the thread were passed through a small plastic tube to form a snare. Regional ischemia was induced by tightening the snare, and reperfusion was started by releasing the ends of the thread.

Treatment Protocols

The experimental protocols are presented in Table 1. All treatment solutions were initially dissolved in dimethyl sulfoxide (DMSO) and finally in Krebs-Henseleit solution so that final concentration of DMSO did not exceed 0.04% in order not to interfere with cardiac physiology: 1,2-dioctanoil-sn-glycerol (DOG) - PKC activator -solution 30 ^mol/L; cheleritrine (CHE) - PKC inhibitor solution 30^mol/L; cromakalim (CRK) - KAtp -opener-solution 7 ^mol/L; glibenclamide (GLIB) - KAtp -blocker solution 10^ mol/L.

Table 1. Scheme oftreatments individualized by groups.

CONTROL (Group A) Stabilization 20' 15' perfusion Ischemia 45' Reperfusion 120'

CHK - 7 I^mol/L (Group B) Stabilization 20' CHK - 5' 10'perfusion Ischemia 45' Reperfusion 120'

GLB -10 |^mol/L (Group C) Stabilization 20' GLY- 15' Ischemia45' Reperfusion 120'

DOG-30 |^mol/L (Grpup D) Stabilization 20' DOG 5' 10'perfusion Ischemia 45' Reperfusion 120'

CHE - 30 |^mol/L (Group E) Stabilization 20' CHE 15' Ischemia 45' Reperfusion 120'

DOG30 |^mol/L + GLB 10 ^mol/L Stabilization 20' DOG + GLY- 15' Ischemia 45' Reperfusion 120'

(Group F)

CHE + CRK (Group G) Stabilization 20' CHE+ CHK -15' Ischemia 45' Reperfusion 120'

Precond (Group H) Stabilization 20' Isch-5' Isch-5' Reperf-10' Ischemia 45' Reperfusion 120'

CHE + Precond (Group I) Stabilization 20' CHE-5' Isch-5' Reperf-10' Ischemia 45' Reperfusion 120'

GLB + Precond (Group J) Stabilization 20' GLY-5' Isch-5' Reperf-10' Ischemia 45' Reperfusion 120'

Experimental groups

After mounting the isolated heart in the Langerdorff retrograde perfusion system, perfusion of hearts was

performed with Krebs Henseleit solution for 20 minutes with the stabilization of the hemodynamic parameters (heart

rate, coronary flow, left ventricular systolic pressure), that period is called the stabilization period.

The hearts were randomly assigned to 1 of 10 treatment groups:

1. (Group A) Control hearts (n=5) were perfused with 0.02% dimethyl sulfoxide for 20 minutes during stabilization, followed by 15 minutes Krebs Henseleit solution, before 45 minutes of regional ischemia and 120 minutes of reperfusion.

2. Group B hearts (n=5), received 5minutes perfusion with Cromakalim (CRK) 7 ^mol/L after 20 minutes stabilization, followed by 10 minutes infusion with Krebs Henseleit solution before 45 minutes of regional ischemia and 120 minutes reperfusion.

3. Group C hearts (n=5), received infusion solution with Glibenclamide (Glib) 10 ^mol/L for 15 minutes, before 45 minutes regional ischemia and 120 minutes reperfusion.

4. Group D hearts (n=5), infusion with 1.2 dioctanoil-sn-glycerol (DOG) 30 ^ mol/L for 5 min followed by perfusion with Krebs Henseleit solution for 10 min (group D), before 45 minutes regional ischemia and 120 minutes reperfusion.

5. Group E hearts (n=5), infusion with chelerythrine (CHE) 30 ^ mol/L solution for 15 min before 45 minutes regional ischemia and 120 minutes reperfusion.

6. Group F hearts (n=5) coperfused with (DOG) 30 ^ mol/L and 10 ^mol/L for 15 minutes before 45 minutes regional ischemia and 120 minutes reperfusion.

7. Group G hearts (n=5) coperfused with combined solutions of (CHE) 30^ mol/L and CRK 7 ^mol/L concentrations for 15 minutes before 45 minutes regional ischemia and 120 minutes reperfusion.

8. Group H hearts (n=5). Hearts were preconditioned (IPC) for 2x5 min global ischaemia, alternated with 5 minutes reperfusion before 45 minutes regional ischemia and 120 minutes reperfusion.

9. Group I hearts (n=5) underwent the IP protocol in the presence of 30 ^mol/L (CHE). The drug was added to the perfusate 20 minutes before starting the preconditioning protocol and was present throughout this protocol.

10. Group J hearts (n=5). Underwent the IPC protocol in the presence of 10^mol/L (Glib) which was added to the perfusate 20 minutes before starting the preconditioning protocol and was present throughout this protocol. After mounting the isolated heart in the Langerdorff retrograde perfusion system, perfusion of hearts was

performed with Krebs Henseleit solution for 20 minutes with the stabilization of the hemodynamic parameters (heart rate, coronary flow, left ventricular systolic pressure), that period is called the stabilization period.

Infarct Size Measurement

At the end of the reperfusion period the snare was tightened to in order to reocclude the coronary artery, and a saline solution of 0.12% Evans blue was infused slowly by way of the aorta. This procedure delineated the nonischemic zone of the myocardium as a dark blue area. After 1 to 4 hours at 20°C, the hearts were sliced into 1-mm-thick transverse sections and incubated in triphenyltetrazolium chloride solution (1% in phosphate buffer, pH 7.4) at 37°C for 10 to 15 minutes. The tissue slices were then fixed in 10% formalin. At the end of this procedure, in the risk zone the viable tissue was stained red and the infarcted tissue appeared pale. The slices were drawn onto acetate sheets. To determine myocardial area on sections computerized planimetry has been used, each section was analyzed separately by drawing the area of infarction using an electronic tablet (Genius G-PEN RS M609X, 9x6 Multimedia Tablet) connected to the computer measured as percentage of infarcted tissue within the volume of myocardium at risk.

Statistical analysis

For data processing we used SPSS 14.0 and Microsoft Excel in Office 2003. The results were expressed as mean value ± standard deviation, for statistical significance testing we used ANOVA and Fisher test, p < 0.05.

3. Results and Discussions

Hemodynamic Data

Baseline data relating to physiological parameters of cardiac function (Figure 3, Table 5) and coronary flow rates before regional ischemia where similar in all experimental groups. During regional ischemia, coronary flow and left ventricular developed pressure decreased to a similar extent in all groups. An increase in coronary flow during the first minutes of reperfusion was indicative of successful reflow, but coronary flow subsequently declined in all groups during the following 120 minute reperfusion period (Figure 1, Table 3). During reperfusion, left ventricular developed pressure recovered gradually, though never reaching stabilization values (Figure 2, Table 4).

The differences in body weight and myocardial mass between the studied groups were not statistically significant. Analyzing the evolution of LVDP and expressing the result as a percentage of the initial value, we found an improvement of LVDP recovery at 30 minutes of reperfusion (86 ± 4% in the group that received ischemic preconditioning (group H) compared with controls (group A) 42 ± 3%, p<0019) and a significant recovery of LVDP at 60 minutes of reperfusion (98 ± 5% in the group with ischemic preconditioning and 46 ± 3% in the control group, p<0.010).

The investigation of the involvement of PKC in the mechanism of ischemic preconditioning was done using the administration of PKC pharmachological activators (DOG) and that PKC inhibitors (chelerythrine).

When we compared the group with ischemic preconditioning (group H), there is a significant reduction of LVDP values at 30 minutes of reperfusion, at 60 minutes of reperfusion in the group treated with chelyrethrine (CHE) (group E) (47 ± 3% and 73 ± 5%, p<0.05), LVDP recovery at 30 and 60 minutes of reperfusion in the group that received a PKC activator (DOG) (group E) (61 ± 4% and 68 ± 3%) was not important when compared with the results from the group receiving simple preconditioning (group H), but is important when compared with the control group (group A) (42±3% respectively 46±3%, p<0,05).

Infarct Size Data

The risk zone volume was similar in all experimental groups, at 0.5 cm3. Infarct size is represented as the percentage of tetrazolium-negative tissue in the ischemic risk zone. As expected, IPC significantly reduced the amount of infracted tissue in the risk zone (Group H) heart compared with control (Group A) hearts (26 ± 2.8% vs. 44 ± 4.6%; p < 0.05).

Comparing the final size of myocardial infarction in the studied groups, there is a statistically significant reduction in final infarct size in hearts from ischemic preconditioning group (group H) compared with the control group (group A) (26 ± 2.8% vs. 44 ± 4.6%, p < 0.05), and the group treated with DOG activator of PCK (group D) compared with the control (group A) (20 ± 1.4% vs. 44 ± 4.6%, p<0.01).

Group B treated only with (CHK) the pharmachological activator of K+atp channel showed a reduction in final size of myocardial infarction compared with control (group A) (36 ± 3.5% vs. 44 ± 4.6%, p = 0.15).

In group C treated with GLB an inhibitor of K+atp channel there was an increase in infarct size vs. Control (Group C) (47 ± 3.6%) vs. (44 ± 4.6% (Group A), p=0.12

In Group E treated with CHE as inhibitor of PKC there was an increase in infarct size above Control(A) values (Group E) (49 ± 4.2%) vs. (44 ± 4.6%), (p = 0.12).

When DOG was coperfused with GLB (Group F) the IPC was abolished as the infarct size data have shown (Group F) (41 ± 4.7%) vs. (44 ± 4.6%) (GroupA).

When CHE was coperfused with CRK (Group G), there was no cardioprotective effect, accompanied by an increase in infarct size (Group G) (48 ± 3.8%) vs. (44 ± 4.6%) (p = 0.12).

When CHE (Group I) and GLB (Group J) were perfused before the ICP protocol, there was a loss of the cardioprotective effect of ischemic preconditioning with an increase in infarct size,the values in both groups did not exceed the Control.

CHE (Group I) (42 ± 4.1%) vs. (44 ± 0.6% (Group A) and GLB (Group J) (41 ± 2.7%) vs. (44 ± 4.6%) (Group A)

Table 2. Features ofexperimental groups.

Group No.rats B.W. (g) Heart weight (g)

A 5 302±6 1.17±0.05

B 5 300±2 1.26±0.04

c 5 310±8 1.27±0.03

D 5 296±5 1.16±0.03

E 5 298±7 1.19±0.04

F 5 304±5 1.26±0.04

G 5 312±6 1.29±0.06

H 5 303±4 1.25±0.05

I 5 294±7 1.18±0.03

J 5 297±3 1.19±0.06

stabilization reperfusion reperfusion reperfuzie

(mmHg)

Fig. 1. Pressure of the coronary perfusate (CPP) evolution in the studied groups

Table 3. PCP results.

roup PCP stabilization (mm Hg) PCP treatament (mm Hg) PCP initial reperfusion (mm Hg)/ PCP 60' reperfusion (mm Hg)/ PCP 120' reperfusion (mm Hg)

A 49.3±9.5 47.3±4.5 38.4±6.7 40.7±5.8 43.1±3.7

B 45.8±3.7 36.5±5.4 39.7±6.4 37.4±4.6 41.6±5.2

c 40.5±5.5 44.3±8.3 46.4±7.6 41.9±6.4 39.3±7.4

D 44.7±5.0 46.8±4.2 42.2±3.7 39.2±3.2 37.2±6.3

E 43.8±3.5 42.3±5.4 39.7±3.2 37.3±4.2 34.7±4.9

F 52.3±1.5 47.3±5.8 44.7±4.1 41.1±4.2 38.3±4.8

G 49.0±2.6 51.3±4.3 54.3±3.7 48.3±4.7 46.9±8.3

H 46.5±6.4 49.4±6.7 47.3±8.5 45.2±7.5 44.1±9.3

I 50.3±4.3 48.3±4.4 52.4±7.4 55.2±5.3 52.4±4.6

J 47.8±3.2 45.3±6.4 42.1±8.3 44.6±5.3 46.4±7.3

110 100 90 80 70 60 50 40 30

—♦—A

1--- —____— C

__ ^ E

—i—G

—♦—J

1 1 1 1

Initial LVDP (mmHg)

LVDP after treatment (mmHg)

LVDP at the beginning of reperfusion.

LVDP 30' of reperfusion.

LVDP 60' of reperfusion

Fig. 2. LVDP evolution in the studied groups and the experimental stages

Table 4. LVDP results (p<0,05).

Group LVDP initial (mmHg) LVDP after treatment (mm Hg) LVDP at the begining of reperfusion (mm Hg) LVDP 60' ofreperfusion (mm Hg) LVDP 120' of reperfusion (mm Hg)

A 110±4 109±5 78±1 46±6 51±6

B 110±7 103±6 80±2 70±6 71±8

c 111±9 108±8 90±0,5 56±7 64±7

D 107±7 103±6 89±1 66±6 73±7

E 105±8 101±5 86±1 50±5 77±9

F 109±5 102±4 88±0.5 55±4 63±4

G 107±2 91±7 86±1 53±4 59±4

H 102±7 95±3 87±6 88±3 100±8

I 108±2 113±8 102±6 97±4 93±7

J 104±5 99±5 113±8 94±5 102±4

treatments reperfusion reperfusion reperfusion

Fig. 3. Heart rate evolution in the studied groups

Table 5. Heart rate results.

Groups Stabilization Bpm Special treatments Bpm Initial reperfusion Bpm 60' reperfusion Bpm 120' reperfusion Bpm

A 250±3 252±4 256±8 254±4 256±5

B 257±2 258±6 255±2 256±7 254±8

C 256±7 259±5 257±4 256±3 255±3

D 249±4 258±7 255±6 254±2 254±2

E 255±8 257±3 258±6 254±3 256±8

F 248±7 258±8 257±5 254±6 255±5

G 254±4 256±8 254±4 255±7 256+3

H 255±7 258±5 257±4 256±9 254±7

I 258±5 254±4 255±8 254±6 256±8

J 256±4 258±6 258±7 257±4 255±6

< 50 £ 40

< 30 w 20 E 10

Fig. 4. Evaluation of infarct size determined from risk area (%)

Our data on ischemic (IPC) of the rat myocardium consisting in 2x5 min global ischemia, alternated with 5 min reperfusion before the lethal ischemia, and reperfusion pointed out: a significant reduction in infarct size following IPC protocol (Group H) versus control (Group A) (26 ± 2.8% vs. 44 ± 4.6%) p < 0.05.This value was used as a reference for the infarct size data obtained with mimetics of pharmachological preconditioning (PP) such as diacylglycerol analogue DOG activator of PCK and cromakalim activator of K ATP channels

Conversely, KATP openers mimicked preconditioning, reducing the size of the infracted area, and KATP inhibitors abolished this effect.

Activation of kinases, including PKC_p38 MAPK, PKG, and tyrosine kinase(s) (Revnic et al., 2009) is part of downstream signaling. As already mentioned, KATP channels are targets for various kinases, including PKC, PKA, and PKG, and are activated by reactive oxygen species (ROS) (H202) (Revnic et al., 2009).

Indeed, pharmacological evidence soon identified KATP activation as a major element in the mechanism of preconditioning, since inhibitors abolished the effect (Revnic et al., 2009).

The ATP-sensitive potassium channel (KATP) openers (e.g. chromakalim) were originally developed for the treatment of hypertension due to their potent peripheral vasodilating properties. The cardioprotective effects of KATP openers are not correlated with enhanced sarcolemmal potassium currents. Grover and Sleph (1995) have been hypothesized to involve an intracellular mechanism. Several studies have reached the conclusion that activation of mitochondrial Kchannels reduces or prevents the generation of ROS.

The group treated only with (CRK) (group B) the opener of K+Atp sarcolemal and mitocondrial channel induced cardioprotection by reduction infarct size compared with Control (group A) (36 ± 3.5% vs. 44 ± 4.6% (p = 0.15.) Our results are in accordance with the literature data (Speechly-Dick et al., 1994).

Cromakalim is reported to mimic the effects of adenosine that make rat ventricular myocytes insensitive to alpha-adrenergic agonists by a G(i) protein-dependent mechanism (Grover and Sleph, 1995).

K ATP channel blocker (GLB) abolished this effect (Group C) (n=5) (47 ± 3.6%) vs. (44 ± 4.6%) p=0.12.

The pharmachological activator of CPK(DOG) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart at a value above that achieved with IPC protocol i.e (Group D) (20 ± 1.4% vs. 44 ± 4.6%) (Group A) p<0.01.

The protection was blocked by CPK inhibitor (CHE), these data provides support for the hypothesis that PCK plays a crucial role in ischemic preconditioning (Speechly-Dick et al., 1994).

During ischemia, myocardial sarcolemmal ATP-dependent potassium (KATP) channels, which are normally closed by high ATP concentration, open when ATP generation decreases favoring K+ efflux. This reduces action potential duration (APD) decreasing the time of Ca2+ influx and Ca2+ overload. This behavior suggested that they might be involved in the protection against stunning and arrhythmias and in the mechanism of ischemic preconditioning.

Auchampach (1994) first found that preconditioning protection is abolished by KATP channel inhibitors and mimicked by KATP channel openers, suggesting the involvement of this channel in preconditioning.

The infarct size area at risk was increased above Contol when GLB (group C) or CHE (group E) were perfused before the lethal ischemia (Group C) (47 ± 3.6% vs. 44 ± 4.6% (Group A) and (Group E) (49 ± 4.2% vs. 44 ± 4.6% ) (GroupA), (p = 0.12).

Also, there was loss of cardiprotective effect of ischemic preconditioning when CHE or GLB was administrated before the ischemic preconditioning protocol:

(Group I) CHE (Group I) (42 ± 4.1%) vs.(44 ± 4.6%) (GroupA);

GLB (Group J) (41 ± 2.7%) vs. (44 ± 4.6%) (GroupA) protocol.

It is well established that glibenclamide can also block sarcolemmal KATP channels in a number of other cell types, including vascular smooth muscle cells, cardiac myocytes, and vascular endothelium, as well as the KATP channels situated on the inner membrane of mitochondria.

Our data pointed out that Glibenclamide (GLB) abolished the cardioprotective effect of ischemic preconditioning (IP), presumably by inhibiting mitochondrial K ATP channel opening in myocytes (Group C and Group J). Gliblenclamide, but not glimepiride or gliclazide, has the ability to abolish the cardioprotective effect elicited by IPC from its antagonistic actions on the ATP-dependent potassium channels within cardiac mitochondria, which are recognized to be pivotal to IPC-induced cardioprotection (Derek et al., 2012).

In this context, one of the most potent mechanisms of protection against myocardial ischemia/reperfusion injury is ischemic preconditioning (Murry et al., 1986).

A substantial body of evidence implicates mitochondrial KATP channel opening as playing a central role in the acquisition of this protection (Murry et al., 1986; Yellon et al., 1998). Although it is not clearly established whether mitochondrial KATP channel opening plays a trigger role (proximal event) or acts as a distal effector of protection, glibenclamide has been shown to attenuate this preconditioning response in animal studies (Yellon et al., 1998).

There are also data from human studies in which preconditioning has been examined with surrogate end points such as ST-segment deviation during repeated intracoronary balloon inflations that support the notion that glibenclamide blunts the preconditioning response.

Several studies have reached the conclusion that activation of mitochondrial K_ channels reduces or prevents the generation of ROS (Tomai et al., 1994).

When DOG was coperfused with GLB (Group F) before the lethal ischemia, the loss of protection was accompanied by an increase in infarct size aria at risk (GroupF) (41 ± 4.7%) vs. (44 ± 4.6%) (Group A), the values were below Control.

On the other heand, when CRK was coperfused with CHE (Group G), before the lethal ischemia, there was no cardioprotective effect, with an increase in infarct size (Group G) (48±3.8%) vs. (44±4.6%) (Group A), p = 0.12. CHE abolished the cardioprotective effect of CRK accompanied by an increase in infarct size above the Control values.

4. Conclusions

- Ischemic preconditioning is an endogenous cardioprotective mechanism in which accessory nerve pathways are not involved, this fact being demonstrated by the effect of ischemic preconditioning on isolated rat heart.

- Infarct size as a golden standard has been reduced to a better degree than IPC when pharmachological activator (DOG) of PKC has been perfused before lethal ischemia.

- K ATP channel opener Cromakalim (CRK) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart, compared to control group.

- The protective mechanism of K+ channels opener seem to be the result of their capacity to activate KATP channels from ischemic myocardium, they seem to possess a unique anti-ischemic mechanism which cannot be

explained by improving the oxygen supply or reduction of its consumption, and the anti-ischemic effect is produced by a direct cardioprotective action.

- The hypothesis is sustained by the observation that pre-treatments of dogs with GLB reduce the benefit of cromakalim (CRK) upon K+channels.

- Ischemia/reperfusion modifies functional parameters in isolated heart, and the action of factors which act upon K+ channels (either blocking or deblocking them) is expressed by modification of these functional parameters.

- When activator of PCK (DOG) + (GLB), an Katp channel inhibitor were coperfused before the lethal ischemia, there was an increase in myocardial infarct size, expressed as a percentage of the area at risk, versus control, the cardioprotective effect of DOG being abolished by GLB. The same results were obtained when (CHE) the inhibitor ofPCK was coperfused with (CRK) the activator ofKATP channel.

- The effect of (CHE) and (GLB) perfused at the beginning of IPC resulted in loss of protection accompanied by a significant increase in infarct size area.

- The findings of this study that the activators of PCK (DOG) and of K ATP channel (CRK) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart and that this protection can be blocked by (CHE) a PCK pharmachological inhibitor provides support for the hypothesis that PKC activation is a pivotal stage in the pathway of intracellular signalling of preconditioning, playing a role in cell regulation.

- Protection against myocardial ischemia-reperfusion injury is a promising strategy for ameliorating the consequences of coronary disease for individual and societal health.

- The usage of pharmacological agents in ischemic conditioning may provide a more benign approach for eliciting cardioprotection in the clinical setting.

- Further studies are needed in order to complete our understanding of the mechanisms underlying myocardial adaptation and pharmachological conditioning can be used to find new therapeutic agents with novel mechanisms of action to supplement the current treatment options for patients with ischemic heart disease.

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