logy 135 (2009) 187–192www.elsevier.com/locate/ijcard

International Journal of Cardio

Delayed anti-arrhythmic effect of nitroglycerin in anesthetized rats:Involvement of CGRP, PKC and mKATP channels☆

Babak Baharvand a,⁎, Mansour Esmaili Dehaj b, Bahram Rasoulian c, Mehrdad Namdari a,Yaghoob Shikhani d, Ali Asghar Kiani d

a Heart Research Center, Faculty of Medicine, Lorestan University of Medical Sciences, Khoramabad, Iranb Department of Physiology, Faculty of Medicine, Yazd University of Medical Sciences, Yazd, Iran

c Department of Physiology, Faculty of Medicine, Lorestan University of Medical Sciences, Khoramabad, Irand Department of Laboratory Sciences, Faculty of Medicine, Lorestan University of Medical Sciences, Khoramabad, Iran

Received 3 August 2007; received in revised form 2 February 2008; accepted 29 March 2008Available online 26 June 2008

Abstract

Delayed anti-infarct and anti-stunning effects of nitroglycerin (NTG) have well been established in some animal models. The main goalsof this study in anesthetized rats were to determine whether NTG has a delayed anti-arrhythmic effect and if so, whether calcitonin gene-related peptide (CGRP), protein kinase C (PKC) and mitochondrial KATP channels (mKATP) are involved in triggering this response. For thispurpose, on day 0, male Wistar rats received NTG (120 μg/kg, iv) with or without pre-administration of PKC inhibitor chelerythrine (CHE),capsaicin (CAP) to deplete CGRP from sensory nerves or mKATP channel blocker 5-hydroxydecaonic acid (5HD). On day 1, their heartswere subjected to 30 min ischemia and 120 min reperfusion. In rats pretreated with NTG, the incidence of ventricular tachycardia andventricular fibrillation and the mortality rate significantly reduced (from 100%, 61% and 18.1% in the control group to 45.4%, 10% and 0%in the NTG group, respectively). Infarct size also reduced from 58±4.7% in the control group to 31±3.7% in the NTG group. These effectswere abolished by CHE, CAP and 5HD, which none of them alone had any effect on infarct size or the incidence of myocardial arrhythmias.These results show that a low dose of NTG has a delayed anti-arrhythmic effect and this effect may share a common mechanism with anti-infarct effects of this drug, involving CGRP release and PKC and mKATP activation.© 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Nitroglycerin; CGRP; PKC; mKATP; Arrhythmia

1. Introduction

Tolerance of the myocardium to a sustained ischemic da-mage after the heart has been subjected to one or more briefperiods of ischemic insults has been defined as ischemicpreconditioning (IPC) [1–3]. Early phase of IPC lasts for 1–2 hafter an ischemic episode(s), while a late phase of protection

☆ This work was supported by a grant from Lorestan University ofMedicalSciences.⁎ Corresponding author. Tel.: +98 661 3222768; fax: +98 661 3231568.E-mail address: drb.baharvand@yahoo.co.in (B. Baharvand).

0167-5273/$ - see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.ijcard.2008.03.048

reappears about 12–24 h after a brief ischemic period(s) andlasts up to 96 h [4,5].

Nitroglycerin, a nitric oxide (NO) releasing agent, canmimic the late phase of IPC in addition to its early phase andprotects the myocardium against both stunning and infarction[5,6]. Because of the sustained nature of the late phase ofpreconditioning (PC) phenomenon [2] and because nitrogly-cerin is a widely used drug in clinical situations with minimaland known adverse effects [7], this issue may have importantclinical consequences. This will be further emphasized by thefact that this late cardioprotective effect of nitroglycerin alsoexists in humans as it has been shown in patients undergoingcoronary angioplasty [8]. It also must be noted that a major

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problem with nitroglycerin is its hemodynamic effects i.e.developing of tolerance some hours after its usage, that doesnot exist in its PC mimetic action [5]. Nevertheless, althoughmany mortally cases induced by ischemic heart events aredue to cardiac arrhythmias [9,10], most of previous studieshave focused on late anti-infarct and anti-stunning effects ofnitroglycerin [5,11–13] and there are only a few evidencesabout the late anti-arrhythmic effects of this drug in conscioussheep [14]. The main purpose of present study was to in-vestigate possible late anti-arrhythmic effects of nitroglycerinin addition to its late anti-infarct effects, in an in-vivo ratmodel of heart ischemia–reperfusion (IR) injury.

Delayed cardioprotective effects of nitroglycerin are me-diated by calcitonin gene-related peptide (CGRP) — a prin-ciple transmitter in capsaicin-sensitive sensory nerves, proteinkinase C (PKC) [13,15,16].We investigate in the present studywhether anti-infarct and anti-arrhythmic effects of nitrogly-cerin share a commonmechanism. In this regard PKC inhibitorchelerythrine (CHE), mitochondrial KATP (mKATP) channelblocker 5-hydroxydecaonate (5HD), and capsaicin (CAP)which depletes sensory nerves from CGRP were used.

2. Methods

2.1. Animals

Male Wistar rats weighing 250–300 g were used. The in-vestigation was approved by the Animal Ethics Committee ofSchool of Medical Sciences in Lorestan University (MedicalSciences).

2.2. Surgical method

Animals were anesthetized with sodium pentobarbital(50 mg/kg, ip) and the anesthetic was administered as neededthroughout the protocol. The neck was opened with a ventralmidline incision and a thoracotomy was performed. The ratswere mechanically ventilated with room air via a smallanimal ventilator (Harvard apparatus, South Natica, MA) at70–80 breaths per minute. Body temperature was maintainedat 37±0.5 °C. A standard limb lead II electrocardiogram(ECG) was continuously monitored and recorded throughoutthe experiment, using a computerized data acquisitionsystem. Catheters flashed with heparinized saline wereinserted into the left carotid artery and right jugular vein tomonitor blood pressure and administration of drugs. A leftthoracotomy was performed in the fourth intercostal spaceand pericardium was opened to expose the heart. A 5-0 silksuture was passed the left main descending coronary artery(LDA). The ends were threaded through a small polyethy-lene tube to form a snare. Any animal in which thisprocedure produced dysrhythmias or sustained fall in meanarterial pressure to less than 50 mm Hg was excluded fromthe study. For induction of ischemia, following stabilizationperiod of 20 min the snare was tightened and held in placewith another tube. Myocardial ischemia was confirmed by

regional cyanosis, ST elevation in the ECG and a drop inblood pressure. Following 30 min ischemia, reperfusion wasestablished by releasing the snare, confirmed by the visibleobservation of hyperemia over the surface.

2.3. Experimental protocol

Animals were randomly assigned to 9 groups. On day 1,all hearts were subjected to 30 min of ischemia and 120 minof reperfusion. On day 0, they were pharmacologically pre-conditioned with NTG and/or treated with PKC inhibitor,(CHE), mKATP channel blocker, (5HD) or (CAP) as follows:

Group 1 (control, n=13), rats received no treatment.Group 2 (NTG, n=11), rats received only NTG [120 μgkg− 1, iv (2 μg kg− 1 min− 1 iv over 1 h)] [13,17].Group 3 (CHE+NTG, n=11), rats received CHE (5 mgkg− 1, iv), 10 min before NTG infusion.Group 4 (5HD+NTG, n=10), rats received 5HD (10 mgkg− 1, iv) 10 min before NTG infusion.Group 5 (CAP+NTG, n=11), rats were pretreated withCAP (50 mg kg− 1, s.c) for 4 days and received NTG 24 hbefore the ischemia (on day 1).Group 6 (VCP+NTG, n=12), rats obtained the vehiclefor CAP (s.c) for 4 days and received NTG 24 h before theischemia (on day 1).Group 7 (CHE, n=10) rats obtained only CHE as ingroup 3.Group 8 (5HD, n=12) rats obtained only 5HD as ingroup 4.Group 9 (CAP, n=11), rats were pretreated with CAP asin group 5.

2.4. Measurement of infarct size and risk zone

After 120 min of reperfusion, the LDA was reoccludedand 2 mL of 2% solution of Evans blue dye was infused intothe jugular vein catheter to distinct the ischemic from non-ischemic areas. The hearts then were excised and frozen for24 h at − 20 °C. Afterwards, the hearts were cut into 2 mm-thick transverse slices and stained by incubation for 20 minin 1% triphenyltetrazolium chloride (TTC) in sodium phos-phate buffer (pH 7.4). The slices were then soaked in 10%formalin to enhance the contrast of the stain. Upon removalfrom the formalin, the slices were pressed between two glassplates to form a uniform 2-mm thickness. The infarcted andrisk zone regions were traced through the glass. The in-farcted and risk zone areas were determined by planimetry ofthe tracings. Infarct size is expressed as a percentage of areaat risk.

2.5. Classification of ischemic arrhythmias

Ischemia-induced ventricular arrhythmias were determinedin accordance with the Lambeth conventions [18]: prematureventricular beat (VEB) as a discrete and identifiable premature

Table 1Heart rate (HR) and mean arterial blood (MBP) pressure during 30 mincoronary occlusion and 120 min reperfusion in anesthetized rats.

Baseline Ischemia Reperfusion

Groups HR MBP HR MBP HR MBP

CON 356±10 104±4.1 377±16 82±3.2 a 362±12 99±5NTG 343±12 107±6.3 351±14 84±4.6 a 346±11 93±4.1CHE+NTG 378±13 98±4.2 365±13 78±3.3 a 373±9 92±3.85HD+NTG 358±9 110±5.6 361±17 85±4.9 a 344±13 102±4.3CAP+NTG 384±12 103±6.7 368±14 80±3.5 a 376±10 94±3.8VCP+NTG 397±10 112±6.1 382±15 87±5 a 362±13 105±4.4CHE 345±14 106±5.5 356±11 79±3.8 a 369±11 97±5.15HD 392±11 99±6.4 403±16 76±4.6 a 387±15 93±3.9CAP 367±12 105±7.3 363±12 82±3.5 a 375±12 96±4.2

Data are expressed as mean±SEM.CON = control; NTG = Nitroglycerin; 5HD = 5-hydroxydecanoic acid;CHE = chelerythrine; CAP = capsaicin; VCP = vehicle for capsaicin.a Significantly (Pb0.05) different from baseline.

Fig. 1. Episodes of VEBs (A) and incidence of VT (B) and VF(C) in controland the eight treatment groups during 30 min ischemia. CON = Control;NTG = Nitroglycerin; 5HD = 5-hydroxydecanoic acid; CHE = Cheler-ythrine; CAP =Capsaicin; VCP =Vehicle for capsaicin. ⁎Pb0.01, #Pb0.001vs. CON.

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QRS complex (premature with respect to the P wave), ven-tricular tachycardia (VT) as a run of four or more VEB at a ratefaster than the resting sinus rate and ventricular fibrillation(VF) as a signal for which individual QRS deflection canno longer distinguished from one another. Complex forms(bigeminy and salvos) were added to VEB count and notanalyzed separately. The incidence of VT and VF weredetermined in each experimental group. Also the VEB countin the 30 min recording period and the total duration of VTand VF were determined in each rat. The arrhythmia scorewas determined using the previously published scale [14]:0 = sinus rhythm, 1 = VEB, 2 = ventricular tachycardia, 3 =VT, 4 = VF, and 5 = irreversible VF.

2.6. Measurement of plasma creatine kinase (CK) activity

At the end of reperfusion, blood samples were taken andplasma was collected and stored at − 40 °C. CK activity wasmeasured by a standard kit, using an autoanalyzer and ex-pressed as unit per liter (U/L).

2.7. Materials

Capsaicin, chelerythrine, 5HD, TTC and Evans blue werepurchased from the Sigma (St. Louis, Mo., USA). NTG waspurchased from Tehran Pharmaceutical Factory (Tehran,Iran) and was diluted in 0.9% saline. Capsaicin was dissolvedin a vehicle containing 10% Tween 80, 10% ethanol, and80% saline. CK kit was provided by Parsazmoon Company(Tehran, Iran).

2.8. Statistics

Data were expressed as means±SEM, except for inci-dence of VT, VF and mortality rate. Heart rate, mean bloodpressure, infarct size and CK activity were analyzed withone-way ANOVA followed by Tukey's post hoc test. The

number of arrhythmia episodes and arrhythmia durationhad a non-Gaussian distribution; therefore, were comparedusing a Mann–Whitney U-test. A Fisher's exact test was

Table 2VT and VF duration (s) in control and the eight treatment groups during30 min ischemia.

Groups VT duration VF duration

CON 74.8±12 16.1±6NTG 8.09±3.2 a 4CHE+NTG 66.9±14.9 15.6±6.45HD+NTG 58.8±14 15.2±7.6CAP+NTG 68.9±13 12.1±4.5VCP+NTG 10.36±3.1 a 9CHE 68.2±15.3 16±5.25HD 52.5±9.8 9.4±3.9CAP 76.2±15 12.3±4.6

Data are expressed as mean±SEM.CON = control; NTG = Nitroglycerin; 5HD = 5-hydroxydecanoic acid;CHE = chelerythrine; CAP = capsaicin; VCP = vehicle for capsaicin.a Significantly (Pb0.05) different from baseline.

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used to compare the incidence of VT, VF and mortality rates.P b 0.05 was considered statistically significant.

3. Results

3.1. Hemodynamic parameters

Table 1 summarizes the hemodynamic parameters. Therewere no significant differences at baseline values for heartrate and mean arterial blood pressure among the experi-mental groups. Ischemia caused a significant reduction inblood pressure without any effect on heart rate. Blood pres-sure nearly restored to baseline level during reperfusion.

3.2. Arrhythmias during ischemia

Fig. 1(A–GSC) shows the number of VEBs episodes andthe incidence of VT and VF during 30 min ischemia. Thesevariables were significantly reduced in rats treated with NTG(144±25, 45.4% and 10% in the NTG group vs. 346±53,100% and 61% in the control group for VEBs, VT and VF

Fig. 2. Mortality rate (A) and arrhythmia score (B) in control and theeight treatment groups. CON = Control; NTG = Nitroglycerin; 5HD =5-hydroxydecanoic acid; CHE = Chelerythrine; CAP = Capsaicin; VCP =Vehicle for capsaicin. ⁎Pb0.01, #Pb0.001 vs. CON.

respectively). Administration of CHE and 5HD prior to NTGinfusion and pretreatment with capsaicin for 4 days abo-lished the NTG effect on ischemia-induced arrhythmias.However, vehicle for capsaicin did not change the NTGeffects. Compared with the control group no significant dif-ferences were detected in the VEBs number and the in-cidence of VT and VF in the CHE, 5HD and CAP groups.

There was no death in the NTG and NTG + VCP groupsbut the mortality rate was between 17 and 27% in the othergroups (Fig. 2A). Arrhythmia score decreased from 2.8±0.2in the control group to 1.6±0.2 in the NTG group (Fig. 2B).

Table 2 represents that rats pretreated with NTG ex-perienced not only a significant reduction in the VT percent,but also a significant reduction in the duration of VT. Thiseffect of NTG was abolished by CHE, 5HD and CAP pre-treatment. NTG administration markedly reduced the dura-tion of VT but there was no significant difference in theduration of VF between the NTG and control groups.

Fig. 3. Infarct size following 30 min ischemia and 120 min reperfusion incontrol and the eight treatment groups during 30min ischemia. CON=Control;NTG = Nitroglycerin; 5HD = 5-hydroxydecanoic acid; CHE = Chelerythrine;CAP = Capsaicin; VCP = Vehicle for capsaicin. ⁎Pb0.01, #Pb0.001 vs.CON.

Fig. 4. Plasma CK activity following 30 min ischemia and 120 min reper-fusion in control and the eight treatment groups at the end of reperfusion.CON = Control; NTG = Nitroglycerin; 5HD = 5-hydroxydecanoic acid;CHE = Chelerythrine; CAP = Capsaicin; VCP = Vehicle for capsaicin.⁎Pb0.01 vs. CON.

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3.3. Infarct size

Fig. 3 represents infarct size as a percentage of area atrisk. NTG caused a marked reduction in infarct size (58±4.7% in the control group compared with 31±3.7% in theNTG group). Intravenous administration of CHE and 5HDabolished this NTG effect. The anti-infarct effect of NTGwas eliminated after pretreatment with capsaicin to depletesensory nerves from this transmitter. Capsaicin vehicle didnot have any effect on delayed cardioprotection of NTG (28±3.5%). CHE, 5HD and capsaicin alone did not influenceinfarct size.

3.4. Plasma CK activity

At the end of reperfusion, plasma CK activity was mea-sured (Fig. 4). Creatin kinase activity reduced from 1985±102 U/L in the control group to 715±49 and 702±42 U/L inthe NTG and VCP + NTG groups respectively (p b0.01).Prior administration of CHE, 5HD and capsaicin alone hadno effect on plasma CK activity. Likewise, the vehicle ofcapsaicin did not have any effect on decreased CK activityinduced by NTG.

4. Discussion

The present study in anesthetized rats, demonstrates thatintravenous administration of a relatively low dose of NTG,24 h before a sustained ischemic insult, considerably protectsthe heart against subsequent IR injury as it has been shownby reduced infarct size and lower incidence of variousdysrhythmias such as VT and VF. These effects of NTG mayshare a common mechanism, involving CGRP release andPKC and mKATP channels activation.

The most important cause of mortality in the course ofcardiac surgery and myocardial infarction are ventricular ar-rhythmias such as VT and VF [19,20]. Since current anti-

arrhythmic drugs are proarrhythmic and their effects disap-pears along the time, finding effective and safe preventivemodes against ischemia or reperfusion-induced arrhythmiascould have special clinical relevance [9,20]. In this regard,the above mentioned late anti-arrhythmia effects of NTG arethe new and the most important aspects of our results;because the previous study in sheep has proved the late anti-arrhythmic effects of this drug mostly in the early reper-fusion time [14]. In the present study, the late PC mimeticaction of NTG against ischemia-induced dysrrhythmias wasalso documented in the course of 30 min ischemia. It shouldbe noted that there are no considerable reperfusion-inducedarrhythmias in this rat model of heart IR injury, even in theabsence of any preventive or therapeutic mode as it has beenpreviously reported [20,21].

Several metabolites released during the brief IR cycles hasbeen suggested to preserve the heart against prolongedischemic injury through the expression of some cytoprotectiveproteins [22,23]. One of the possible agents involved in thetrigger of the late phase of preconditioning is NO [14]. It hasbeenwell documented that NO via a reactionwith free radicalsand formation of peroxynitrite will enhance expression ofiNOS and increases the plasma NO [24,25]. NO will increaseCGRP release via a CGMP dependent pathway. CGRP is aprinciple transmitter of sensory nerves in human and animalhearts and have been documented to play an important role inthe mediation of both phase of IPC [12]. Using capsaicin todeplete sensory nerves from CGRP, we showed that theinduction of late anti-infarct and anti-arrhythmic effects ofNTG is CGRP dependent. Zhou et al. also observed a similaranti-infarct effect in the same animal model [17]. CGRP couldlead to PKC translocation [15,26] so we hypothesized thatNTG may trigger the late phase through PKC activation andthe late phase will be abolished using PKC inhibiter, CHE. Ourresults show that anti-arrhythmic and anti-infarct effects ofNTG disappear in the presence of CHE. Banerjee et al. alsorevealed that CHE infusion previous to NTG injection blocksthe late anti-stunning effects of this drug in the consciousnessrabbit hearts [13]. Though, CHE is not able to distinguishdifferent PKC isoforms, it seems that depending on thespecies, different PKC isoforms translocate during PCphenomenon, among them PKCε is the most important [27].Therefore, further researches are necessary to determine whichisoform(s) translocates.

The opening of mKATP channels is facilitated by PKC[28] and it has been suggested that the cardioprotective ef-fects of IPC is mediated by mKATP channels [3,28,29]. Ad-ministration of 5HD as a mKATP channel blocker in our studyshowed that the above mentioned effects of NTG are me-diated by the activation of mKATP channels.

There are not only several animal studies about the delayedcardioprotective effects of NTG, but also a few elegant humanstudies about this late PC mimetic action of NTG. Forexample, NTG infusion 24 h before angioplasty reduced STsegment elevation and anginal pain and improved cardiacfunction during the course of 2 min ischemia induced by

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balloon inflation [8]. Similarly, NTG enhanced tolerance toexercise in coronary patients [30]. These studiesmay lead us tointroduce a new indication for NTG administration in patientswith stable angina, beyond current indications for sympto-maticmanagement. This new indication could be the reductionof possible future acute ischemic injuries with once daily lowdose administration of this drug in known cases of coronaryartery diseases. Of course, this would be possible if tolerancedoes not develop in humans to the delayed cardioprotectiveeffect of this drug as it does not develop in rabbits after 30 daysadministration of NTG through a skin patch [5]. Our resultsabout anti-arrhythmic effects of this drug may further en-courage the use of NTG with this possible new indication, ifthese anti-arrhythmic effects also exist in humans.

In summary, a relatively low dose administration of NTG24 h prior to a prolonged ischemic insult reduces infarct sizeand incidence of arrhythmia in anesthetized rats. These pre-ventive aspects of NTG are mediated through CGRP releaseand the activation of PKC and mKATP channels. The detailsof cellular and molecular mechanisms underlying this pro-tection and whether similar anti-arrhythmic aspects are pre-sent in humans remain to be investigated.

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