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Calcitonin gene-related peptide-mediated cardioprotection of postconditioning in isolated rat hearts
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Regulatory Peptides 147 (2008) 4 – 8 www.elsevier.com/locate/regpep
Calcitonin gene-related peptide-mediated cardioprotection of postconditioning in isolated rat hearts Dai Li, Nian-Sheng Li, Qing-Quan Chen, Ren Guo, Ping-Sheng Xu, Han-Wu Deng, Yuan-Jian Li ⁎ Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, No. 110 Xiang-Ya Road, Changsha 410078, China Received 31 August 2007; received in revised form 3 November 2007; accepted 22 November 2007 Available online 3 December 2007
Abstract Previous studies have demonstrated that endogenous calcitonin gene-related peptide (CGRP) plays an important role in mediation of ischemic preconditioning. In the present study, we tested whether CGRP is also involved in mediation of the protective effects of postconditioning in isolated rat hearts. Sixty minutes of left coronary artery occlusion and followed by 60 min of reperfusion caused a significant decrease in cardiac function and a significant increase in creatine kinase (CK) release and infarct size. Postconditioning with three cycles of 1-min ischemia and 1-min reperfusion produced a marked improvement of cardiac function and decreased CK release and infarct size, concomitantly with an increase in the release of CGRP release in coronary effluent. However, the cardioprotection afforded by postconditioning was abolished by CGRP 8-37 (10− 7 M), a selective CGRP receptor antagonist, or pretreatment with capsaicin (50 mg/kg, s.c.), which depletes transmitters in sensory nerves. Exogenous CGRP (5 × 10− 9 M) administration of CGRP reappeared postconditioning-like cardioprotection in the rats pretreated with capsaicin. These results suggest that the protective effects of ischemic postconditioning are related to stimulation of endogenous CGRP release in rat hearts. © 2007 Elsevier B.V. All rights reserved. Keywords: Calcitonin gene-related peptide; Ischemia-reperfusion; Postconditioning; Rat
1. Introduction A number of studies have indicated that postconditioning affords a potent cardioprotection as that provided by preconditioning [1,2]. It is well known that the protection of preconditioning is triggered by brief cycles of iterative ischemia-reperfusion performed before a long time of coronary artery occlusion [3]. Compared to preconditioning, postconditioning is induced by an intermittent ischemia-reperfusion but is applied after the prolonged ischemic insult [4]. It has been documented that multiple active substance including neurotransmitters and antacoids are involved in mediation of the cardioprotection of preconditioning [5–7]. Recent investigations have showed that some endogenous substances such as nitric oxide, adenosine or acetylcholine are
⁎ Corresponding author. Tel.: +86 731 2355078; fax: +86 731 2355078. E-mail address: [email protected] (Y.-J. Li). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.11.004
also involved in mediation of the protective effect of postconditioning [4]. Calcitonin gene-related peptide (CGRP), the principal transmitter in capsaicin-sensitive sensory nerves, is widely distributed in cardiovascular tissues. We and others have documented that CGRP plays an important role in mediation of ischemia preconditioning [8,9]. In the present study, therefore, we tested whether endogenous CGRP is involved in the protective effects of postconditioning. 2. Materials and methods 2.1. Perfusion of the isolated heart Sprague–Dawley male rats weighing 200–250 g were obtained from the Animal Center of Xiang-Ya School of Medicine. All animals received humane care in compliance with “The Guide for the Care of Use of Laboratory Animals” published by the National Institute of Health (NIH Publication No. 85-23, revised 1996).
D. Li et al. / Regulatory Peptides 147 (2008) 4–8
5
Rats were anesthetized by intraperitoneal administration of sodium pentobarbital (60 mg/kg). The heart was rapidly excised and placed in ice-cold Krebs–Henseleit buffer solution containing NaCl 119.0, NaHCO3 25.5, KCl 4.3, KH PO3 1.2, MgSO3 1.2, CaCl2 2.5, and glucose 11.0 mM. The heart was attached to a Langendorff apparatus via the aorta for retrograde perfusion with Krebs–Henseleit buffer solution. The perfusate was equilibrated with 95% O2 and 5% CO2, maintained at 37 °C and pH 7.4. Perfusion pressure was maintained at 85 cm H2O. A water-filled latex balloon was inserted into the left ventricle via the mitral valve. The left ventricular pressure (LVP) and its first derivative of left ventricular pressure (±dp / dtmax), and heart rate (HR) were continuously monitored by a Power Lab system (ADIntruments Shanghai Trading Co, Ltd). A 4–0 silk suture was passed around the left coronary artery at a point two-thirds of the way between its origin near the pulmonary conus and the cardiac apex and a snare was formed by passing both ends of the suture through a piece of polyethylene tubing. Occlusion of the left coronary artery, by clamping the snare against the surface of the heart, caused an area of epicardial cyanosis with regional hypokinesis. Reperfusion was achieved by releasing the snare and was confirmed by conspicuous hyperaemic blushing of the previously ischemic myocardium. Coronary flow was measured by timed collection of coronary effluent, and coronary effluent was collected for measurement of CGRP level and creatine kinase activity.
were incubated in 1% triphenyl tetrazolium chloride phosphate buffer solution at 37 °C for 20 min to stain the viable myocardium brick red. The samples were then fixed in a 10% formalin solution for 24 h. Sections were traced onto acetate sheets. The area of infarct and risk zone was determined by planimetry of the tracings. Infarct and risk area volumes were expressed as mm3 and infarct size was calculated as a percentage of the risk zone.
2.2. Experimental protocols
CGRP, CGRP 8-37 and capsaicin were purchased from Sigma. Capsaicin was dissolved in a vehicle containing 10% Tween 80, 10% ethanol, and 80% saline. The radioimmunoassay kits for measurement of CGRP were obtained from Dongya Immunity Technology Institution, Beijing, China. Creatine kinase kits were obtained from High-tech Bioengineering Co. Ltd, Beijing, China.
Animals were randomly divided into 5 groups. All animals were subjected to 60 min of left coronary artery occlusion followed by 60 min of reperfusion, except the control group. (1) control group; (2) ischemia-reperfusion injury, the left coronary artery was occluded for 3 min followed by 60 min of reperfusion; (3) ischemic postconditioning (IPC), immediately at the onset of reperfusion, reflow was initiated with 1 min of full coronary flow, followed by 1 min of re-occlusion, repeated for a total of three cycles; (4) IPC plus CGRP 8-37, the heart was perfused with CGRP 8-37 (10− 7 M) for 10 min before ischemia; (5) IPC plus capsaicin, A single dose of capsaicin (50 mg/kg) which depletes transmitters in sensory nerves was administered by s.c. injection, 4 days before the experiment under anesthesia (sodium pentobarbital, 60 mg/kg, i.p.); and (6) IPC plus capsaicin & CGRP, the heart was perfused with CGRP (5 × 10− 9 M) in the phase of repeated perfusion and capsaicin was given according to the same procedure as for the IPC plus capsaicin group. 2.3. Infarct size and risk area At the end of 60 min reperfusion, the left coronary was reoccluded, and 1 ml Evans blue (1%) was injected into the ventricular cavity and was allowed to perfuse the non-ischemia portions of the heart. The entire heart was weighted, rinsed of excess blue dye, trimmed of right ventricular and atria tissue and sliced into 1-mm thick sections from the apex to base. The slices
2.4. Measurement of creatine kinase activity The creatine kinase activity in the coronary effluent from the heart at 5 min of reperfusion was measured spectrophotometrically. 2.5. CGRP assay The whole coronary effluent of perfusate fractions (5 min) was collected before ischemia and during reperfusion, and acetic acid (final concentration 0.2 M) was added. The samples were desalted using SEP-PAK C18 cartridges and lyophilized. CGRP-like immunoreactivity in the perfusate fraction was measured by using a radioimmunoassay kit with antisera raised against rat CGRP, 125 I-labelled CGRP and rat CGRP standard. 2.6. Reagents
2.7. Statistics Data are expressed as means ± SEM. All values were analyzed by using ANOVA and the Newman–Keuls Student's t-test. The significance level was chosen as P b 0.05.
Table 1 The basal values of cardiac function
Control IR IPC +CGRP8-37 +Cap + Cap&CGRP
n LVP (mm Hg)
+dp / dtmax −dp / dtmax HR (mm Hg/s) (mm Hg/s) (beats/ min)
CF (ml/min)
8 9 9 7 7 6
3346 ± 188 3608 ± 317 3312 ± 337 3070 ± 424 2896 ± 322 2954 ± 234
9.5 ± 0.5 9.7 ± 0.6 8.5 ± 0.6 9.0 ± 1.0 6.1 ± 0.5## 6.8 ± 0.4
132.3 ± 9.2 142.7 ± 9.9 131.7 ± 16.2 125.0 ± 11.0 115.9 ± 11.0 119.4 ± 13.7
2259 ± 129 2043 ± 146 1932 ± 210 1978 ± 185 1827 ± 195 1864 ± 231
253 ± 9 231 ± 10 228 ± 18 208 ± 14 223 ± 17 211 ± 14
All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). ## P b 0.01 vs IPC.
6
D. Li et al. / Regulatory Peptides 147 (2008) 4–8
genous CGRP reappeared postconditioning-like cardioprotection, as shown by improving cardiac function and decreasing creatine kinase release in the rats pretreated with capsaicin (Table 2 and Fig. 1).
3. Results 3.1. Cardiac function and creatine kinase activity There was no significant difference in basal values of HR, CF, and LVP ± dp / dtmax among groups. Pretreatment with capsaicin which depletes transmitters in sensory nerves decreased coronary flow of heart (Table 1). Sixty minutes ischemia and 60-min reperfusion caused a marked decrease in cardiac function (CF, LVP, and LVP ± dp / dtmax) and a significant increase in creatine kinase release. Postconditioning significantly improved cardiac function (Table 2) and decreased creatine kinase release in reperfusion period (Fig. 1), these effects which were abolished by CGRP8-37, a selective CGRP receptor antagonist, or by pretreatment with capsaicin. Exo-
3.2. Infarct size As shown in Table 3, there were no changes in risk zone, indicating that the size of the risk area was comparable in all groups. Ischemia-reperfusion caused a 45.0 ± 2.2 % necrosis in the area at risk. However, postconditioning significantly reduced infarct size, an effect which was abolished by CGRP8-37 or by pretreatment with capsaicin. Exogenous CGRP decreased the amplified infarct size in the rats pretreated with capsaicin.
Table 2 Effect of IPC on cardiac function n
Reperfusion (min) 5
10
20
30
60
LVP(mm Hg) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
128.9 ± 9.5 58.1 ± 8.2 ⁎ ⁎ 84.5 ± 11.0+ 46.5 ± 6.6## 61.1 ± 3.5 77.3 ± 4.3$
130.4 ± 9.1 59.8 ± 7.8⁎⁎ 85.2 ± 12.4+ 43.6 ± 6.7## 58.6 ± 2.9# 75.7 ± 5.8$
130.4 ± 9.6 60.0 ± 7.7⁎⁎ 82.9 ± 10.9+ 47.4 ± 4.0## 57.3 ± 3.6# 71.0 ± 8.2$
128.1 ± 9.4 54.5 ± 6.7 ⁎ ⁎ 79.8 ± 9.1++ 47.4 ± 4.1## 55.4 ± 3.2# 70.4 ± 2.0$
127.3 ± 9.9 48.4 ± 5.6⁎⁎ 78.2 ± 7.2+ 39.3 ± 5.0## 51.1 ± 2.4## 65.8 ± 4.1$
+ dp / dtmax (mm Hg/s) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
3299 ± 192 1309 ± 166⁎⁎ 1903 ± 258+ 873 ± 211## 1553 ± 189## 1764 ± 155$
3180 ± 189 1356 ± 172⁎⁎ 1970 ± 197 904 ± 193## 1429 ± 181## 1773 ± 192$
3289 ± 218 1311 ± 165⁎⁎ 2027 ± 216++ 978 ± 194## 1362 ± 178## 1718 ± 139$
3281 ± 201 1220 ± 128⁎⁎ 1942 ± 209++ 1025 ± 205## 1354 ± 189## 1652 ± 213$
3233 ± 240 1146 ± 156⁎⁎ 1924 ± 223++ 985 ± 195## 1286 ± 192## 1623 ± 152$
− dp / dtmax (mm Hg/s) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
2250 ± 149 736 ± 117⁎⁎ 1296 ± 116++ 683 ± 132## 881 ± 94## 1037 ± 96$
2233 ± 132 687 ± 74⁎⁎ 1127 ± 98+ 655 ± 126## 877 ± 82## 986 ± 64$
2218 ± 127 695 ± 86⁎⁎ 1150 ± ±111++ 669 ± 93## 817 ± 75## 994 ± 84$
2227 ± 120 648 ± 75⁎⁎ 1084 ± 89++ 691 ± 83## 790 ± 69## 951 ± 79$
2210 ± 135 553 ± 73⁎⁎ 1043 ± 92++ 656 ± 78## 753 ± 67## 917 ± 75$
HR (beats/min) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
248 ± 12 218 ± 15 191 ± 18 172 ± 11 174 ± 14 187 ± 9
246 ± 10 179 ± 18 201 ± 18 171 ± 5 185 ± 14 176 ± 13
248 ± 11 187 ± 22 212 ± 16 181 ± 4 186 ± 14 183 ± 12
246 ± 11 183 ± 22 214 ± 16 167 ± 11 184 ± 13 174 ± 17
247 ± 12 165 ± 20 202 ± 12 168 ± 8 182 ± 11 177 ± 11
CF(ml/min) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
9.4 ± 0.5 4.0 ± 0.3⁎⁎ 5.8 ± 0.6+ 3.9 ± 0.4## 2.3 ± 0.2## 4.6 ± 0.4$
9.4 ± 0.5 3.8 ± 0.3⁎⁎ 5.7 ± 0.6+ 3.8 ± 0.4## 2.2 ± 0.1## 4.2 ± 0.2$$
9.2 ± 0.5 3.4 ± 0.3⁎⁎ 5.5 ± 0.5+ 3.7 ± 0.6## 2.2 ± 0.2## 4.1 ± 0.5$$
9.3 ± 0.4 3.2 ± 0.3⁎⁎ 5.4 ± 0.5+ 3.6 ± 0.6## 2.1 ± 0.1## 3.9 ± 0.2$
9.2 ± 0.4 2.7 ± 0.3⁎⁎ 5.3 ± 0.5+ 3.4 ± 0.6## 2.0 ± 0.1## 3.7 ± 0.3$
All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). ⁎⁎P b 0.01 vs control; +P b 0.05, ++P b 0.01 vs IR; #P b 0.05, ##P b 0.01 vs IPC; $P b 0.05, $$P b 0.01 vs + Cap.
D. Li et al. / Regulatory Peptides 147 (2008) 4–8
Fig. 1. Effect of IPC on creatine kinase activity. All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). n = 6–9. ⁎⁎P b 0.01 vs control; ++P b 0.01 vs IR; ##P b 0.01 vs IPC; $P b 0.05 vs + Cap.
3.3. Release of CGRP As shown in Fig. 2, the content of CGRP-like immunoreactivity in coronary effluent was significantly increased during reperfusion compared with in pre-ischemia period. CGRP content of coronary effluent in the postconditioning group was higher than that of the ischemia-reperfusion group. However, the increased release of CGRP by postconditioning was abolished by pretreatment with capsaicin. 4. Discussion The cardioprotection afforded by postconditioning exhibits improvement of cardiac function, reduction of infarct size and CK release and limitation of tissue edema, which has been demonstrated in various models of ischemia-reperfusion [4,10]. However, the mechanisms responsible for the beneficial effect of postconditioning on the ischemic myocardium are not fully understood. Recently, it has been reported that the protection of postconditioning was triggered by endogenous active substances such as nitric oxide, adenosine or acetylcholine [11,12]. It has been demonstrated that the heart is innervated by both autonomic and capsaicin-sensitive sensory nerves, and it has been suggested that capsaicin-sensitive sensory nerves may Table 3 Effect of IPC on infarct size and risk area
IR IPC +CGRP8-37 +Cap +Cap&CGRP
n Heart wet weight (g)
Area at risk Infarct size (mm3) (mm3)
Infarct size/ risk area (%)
9 9 7 7 6
375.0 ± 14.4 377.0 ± 11.8 347.6 ± 17.8 370.0 ± 18.6 362.1 ± 10.8
45.0 ± 2.2 27.8 ± 2.7++ 41.0 ± 1.9## 41.1 ± 2.8## 33.1 ± 3.2$
0.86 ± 0.02 0.82 ± 0.02 0.85 ± 0.03 0.83 ± 0.03 0.83 ± 0.04
168.3 ± 9.3 104.7 ± 10.6++ 142.1 ± 9.1## 152.4 ± 11.2## 119.7 ± 10.2$
All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). ++ P b 0.01 vs IR; ##P b 0.01 vs IPC; $P b 0.05 vs + Cap.
7
Fig. 2. Effect of IPC on release of CGRP. All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). n = 7–9. ⁎⁎P b 0.01 vs IR of pre-ischemia period; ++P b 0.01 vs IR of reperfusion period; #P b 0.05, ##P b 0.01 vs IR and IPC, respectively.
play a role in modulation of cardiac function through local reflex mechanisms [13]. CGRP, the major transmitter in capsaicin-sensitive sensory nerves, is distributed in cardiovascular tissues. CGRP, besides regulating vascular smooth muscle tone, possesses a protection of the ischemic myocardium. Recently, we and others have documented that CGRP is involved in mediation of cardioprotection afforded by ischemic or pharmacological preconditioning [14,15], and CGRP is thought as an endogenous myocardial protective substance. The release of CGRP is regulated by multiple factors. Myocardial ischemia, even a brief ischemia period of 5 min, causes a significant increase of CGRP release in isolated guinea pig heart [13]. Studies in clinics have shown that the plasma concentration of CGRP is markedly elevated in patient with and without early reperfusion after acute myocardial infarction [13,16]. Our recent work has shown that a single preconditioning episode of 5-min ischemia caused a marked increase in the release of CGRP in the isolated rat heart [17]. It is likely that the protection afforded by postconditioning is related to stimulation of CGRP release, because the release of endogenous CGRP could be regulated by transient ischemia which is similar to brief episodes provided by postconditioning. The results of the present study revealed postconditioning improved cardiac function and decreased infarct size, concomitantly with an increase in the release of CGRP in coronary effluent, and the protective effects of postconditioning were abolished by CGRP8-37, an antagonist of CGRP receptor, or pretreatment with capsaicin, which depletes CGRP in sensory nerves. Exogenous CGRP reappeared postconditioning-like cardioprotection in the rat pretreated with capsaicin. These results support the hypothesis that endogenous CGRP may play an important role in mediation of the cardioprotection of postconditioning in rats. Capsaicin-sensitive sensory nerves contain a number of peptides, including CGRP, substance P and neurokinin A [13]. Besides CGPR, substance P and neurokinin A could also mediate the effect of capsaicin. Among these peptides, CGRP,
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D. Li et al. / Regulatory Peptides 147 (2008) 4–8
endogenous or exogenous, has been shown to possess a beneficial effect of the myocardium [18,19]. This study is consistent with our previous observation that brief ischemia evokes CGRP release and the cardioprotection afforded by preconditioni1ng or postconditioning is abolished by CGRP 837, an antagonist of CGRP receptor [17]. These findings allow us to speculate that CGRP may play an important role in mediation of the protection provided by postconditioning. TNF-α, as an autocrine cytokine, has been shown to be released from the ischemic heart and to be involved in myocardial dysfunction in ischemia and reperfusion [20]. It has been shown that an increase in the level of TNF-α is localized in cardiomyocytes from human myocardial tissues following acute ischemia, and pretreatment with neutralising anti-TNF-a antibody reduces myocardial damage during ischemia [21]. Our previous study showed that CGRP-mediated preconditioning is related to the inhibition of myocardial TNF-α production [22]. Recently, it was reported that postconditioning attenuated cardiomyocyte apoptosis, mediated by reducing TNF-α release [23]. The mechanism responsible for the inhibition of TNF-α production by CGRP remains unclear. There is evidence that endogenous mediators, including neurotransmitters, bind to specific receptors and then activate the endogenous protective mechanisms via complex signal pathways associated with the activation of protein kinase C (PKC) [24,25]. Previous studies have shown that PKC inhibition abolished CGRP-induced preconditioning [8]. It is probable that the protective effect of CGRP-mediated postconditioning is related to inhibition of TNF-α production via activation of PKC. However, further work is needed before drawing a definitive conclusion on this matter. In conclusion, the present study suggests that endogenous CGRP is involved in the mediation of cardioprotection afforded by postconditioning in rats. Acknowledgements This work was supported by grants from the National Nature Science Foundation (No. 30430740) and the National Basic Research Program (973 Program, No. 2007CB512000), China. References [1] Thibault H, Piot C, Ovize M. Postconditioning in man. Heart Fail Rev 2007;12:245–8. [2] Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME, Kerendi F, Guyton RA, Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 2004;62:74–85. [3] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–36. [4] Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten-Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:H579–88. [5] Yao Z, Gross GJ. A comparison of adenosine-induced cardioprotection and ischemic preconditioning in dogs. Efficacy, time course, and role of KATP channels. Circulation 1994;89:1229–36.
[6] Parratt JR. Protection of the heart by ischaemic preconditioning: mechanisms and possibilities for pharmacological exploitation. Trends Pharmacol Sci 1994;15:19–25. [7] Chien GL, Van Winkle DM. Naloxone blockade of myocardial ischemic preconditioning is stereoselective. J Mol Cell Cardiol 1996;28:1895–900. [8] Li YJ, Li YJ, Peng CF, Li NS, You JM, Deng HW. Calcitonin gene-related peptide protects against endothelial cell damage due to oxidised lowdensity lipoprotein. Med Sci Res 1995;23:253–4. [9] Pajdo R, Brzozowski T, Konturek PC, Kwiecien S, Konturek SJ, Sliwowski Z, Pawlik M, Ptak A, Drozdowicz D, Hahn EG. Ischemic preconditioning, the most effective gastroprotective intervention: involvement of prostaglandins, nitric oxide, adenosine and sensory nerves. Eur J Pharmacol 2001;427:263–76. [10] Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of “modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3-kinase-Akt pathway. Circ Res 2004;95:230–2. [11] Kin H, Zatta AJ, Lofye MT, Amerson BS, Halkos ME, Kerendi F, Zhao ZQ, Guyton RA, Headrick JP, Vinten-Johansen J. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosine. Cardiovasc Res 2005;67:124–33. [12] Zhang JS, Tan YR, Xiang Y, Luo ZQ, Qin XQ. Regulatory peptides modulate adhesion of polymorphonuclear leukocytes to bronchial epithelial cells through regulation of interleukins, ICAM-1 and NFkappaB/IkappaB. Acta Biochim Biophys Sin (Shanghai) 2006;38:119–28. [13] Franco-Cereceda A. Calcitonin gene-related peptide and tachykinins in relation to local sensory control of cardiac contractility and coronary vascular tone. Acta Physiol Scand Suppl 1988;569:1–63. [14] Zhu HQ, Jiang JL, Lu R, Zhang XH, Deng HW, Li YJ. The protective effects of monophosphoryl lipid A on the ischemic myocardium and endothelium in rats. Cardiovasc Drugs Ther 2003;17:311–8. [15] Chai W, Mehrotra S, Jan Danser AH, Schoemaker RG. The role of calcitonin gene-related peptide (CGRP) in ischemic preconditioning in isolated rat hearts. Eur J Pharmacol 2006;531:246–53. [16] Li G, Chen S, Lu E, Li Y. Ischemic preconditioning improves preservation with cold blood cardioplegia in valve replacement patients. Eur J Cardiothorac Surg 1999;15:653–7. [17] Lu R, Li YJ, Deng HW. Evidence for calcitonin gene-related peptidemediated ischemic preconditioning in the rat heart. Regul Pept 1999;82:53–7. [18] Li YJ, Xiao ZS, Peng CF, Deng HW. Calcitonin gene-related peptideinduced preconditioning protects against ischemia-reperfusion injury in isolated rat hearts. Eur J Pharmacol 1996;311:163–7. [19] Zhou FW, Li YJ, Lu R, Deng HW. Protection of calcitonin gene-related peptide-mediated preconditioning against coronary endothelial dysfunction induced by reperfusion in the isolated rat heart. Life Sci 1999;64:1091–7. [20] Gurevitch J, Frolkis I, Yuhas Y, Lifschitz-Mercer B, Berger E, Paz Y, Matsa M, Kramer A, Mohr R. Anti-tumor necrosis factor-alpha improves myocardial recovery after ischemia and reperfusion. J Am Coll Cardiol 1997;30:1554–61. [21] Bozkurt B, Kribbs SB, Clubb Jr FJ, Michael LH, Didenko VV, Hornsby PJ, Seta Y, Oral H, Spinale FG, Mann DL. Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats. Circulation 1998;97:1382–91. [22] He SY, Deng HW, Li YJ. Monophosphoryl lipid A-induced delayed preconditioning is mediated by calcitonin gene-related peptide. Eur J Pharmacol 2001;420:143–9. [23] Sung CP, Arleth AJ, Aiyar N, Bhatnagar PK, Lysko PG, Feuerstein G. CGRP stimulates the adhesion of leukocytes to vascular endothelial cells. Peptides 1992;13:429–34. [24] Vinten-Johansen J. Postconditioning: a mechanical maneuver that triggers biological and molecular cardioprotective responses to reperfusion. Heart Fail Rev in press. [25] Zhou ZH, Peng J, Ye F, Li NS, Deng HW, Li YJ. Delayed cardioprotection induced by nitroglycerin is mediated by alpha-calcitonin gene-related peptide. Naunyn Schmiedebergs Arch Pharmacol 2002;365:253–9.
Regulatory Peptides 147 (2008) 4 – 8 www.elsevier.com/locate/regpep
Calcitonin gene-related peptide-mediated cardioprotection of postconditioning in isolated rat hearts Dai Li, Nian-Sheng Li, Qing-Quan Chen, Ren Guo, Ping-Sheng Xu, Han-Wu Deng, Yuan-Jian Li ⁎ Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, No. 110 Xiang-Ya Road, Changsha 410078, China Received 31 August 2007; received in revised form 3 November 2007; accepted 22 November 2007 Available online 3 December 2007
Abstract Previous studies have demonstrated that endogenous calcitonin gene-related peptide (CGRP) plays an important role in mediation of ischemic preconditioning. In the present study, we tested whether CGRP is also involved in mediation of the protective effects of postconditioning in isolated rat hearts. Sixty minutes of left coronary artery occlusion and followed by 60 min of reperfusion caused a significant decrease in cardiac function and a significant increase in creatine kinase (CK) release and infarct size. Postconditioning with three cycles of 1-min ischemia and 1-min reperfusion produced a marked improvement of cardiac function and decreased CK release and infarct size, concomitantly with an increase in the release of CGRP release in coronary effluent. However, the cardioprotection afforded by postconditioning was abolished by CGRP 8-37 (10− 7 M), a selective CGRP receptor antagonist, or pretreatment with capsaicin (50 mg/kg, s.c.), which depletes transmitters in sensory nerves. Exogenous CGRP (5 × 10− 9 M) administration of CGRP reappeared postconditioning-like cardioprotection in the rats pretreated with capsaicin. These results suggest that the protective effects of ischemic postconditioning are related to stimulation of endogenous CGRP release in rat hearts. © 2007 Elsevier B.V. All rights reserved. Keywords: Calcitonin gene-related peptide; Ischemia-reperfusion; Postconditioning; Rat
1. Introduction A number of studies have indicated that postconditioning affords a potent cardioprotection as that provided by preconditioning [1,2]. It is well known that the protection of preconditioning is triggered by brief cycles of iterative ischemia-reperfusion performed before a long time of coronary artery occlusion [3]. Compared to preconditioning, postconditioning is induced by an intermittent ischemia-reperfusion but is applied after the prolonged ischemic insult [4]. It has been documented that multiple active substance including neurotransmitters and antacoids are involved in mediation of the cardioprotection of preconditioning [5–7]. Recent investigations have showed that some endogenous substances such as nitric oxide, adenosine or acetylcholine are
⁎ Corresponding author. Tel.: +86 731 2355078; fax: +86 731 2355078. E-mail address: [email protected] (Y.-J. Li). 0167-0115/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2007.11.004
also involved in mediation of the protective effect of postconditioning [4]. Calcitonin gene-related peptide (CGRP), the principal transmitter in capsaicin-sensitive sensory nerves, is widely distributed in cardiovascular tissues. We and others have documented that CGRP plays an important role in mediation of ischemia preconditioning [8,9]. In the present study, therefore, we tested whether endogenous CGRP is involved in the protective effects of postconditioning. 2. Materials and methods 2.1. Perfusion of the isolated heart Sprague–Dawley male rats weighing 200–250 g were obtained from the Animal Center of Xiang-Ya School of Medicine. All animals received humane care in compliance with “The Guide for the Care of Use of Laboratory Animals” published by the National Institute of Health (NIH Publication No. 85-23, revised 1996).
D. Li et al. / Regulatory Peptides 147 (2008) 4–8
5
Rats were anesthetized by intraperitoneal administration of sodium pentobarbital (60 mg/kg). The heart was rapidly excised and placed in ice-cold Krebs–Henseleit buffer solution containing NaCl 119.0, NaHCO3 25.5, KCl 4.3, KH PO3 1.2, MgSO3 1.2, CaCl2 2.5, and glucose 11.0 mM. The heart was attached to a Langendorff apparatus via the aorta for retrograde perfusion with Krebs–Henseleit buffer solution. The perfusate was equilibrated with 95% O2 and 5% CO2, maintained at 37 °C and pH 7.4. Perfusion pressure was maintained at 85 cm H2O. A water-filled latex balloon was inserted into the left ventricle via the mitral valve. The left ventricular pressure (LVP) and its first derivative of left ventricular pressure (±dp / dtmax), and heart rate (HR) were continuously monitored by a Power Lab system (ADIntruments Shanghai Trading Co, Ltd). A 4–0 silk suture was passed around the left coronary artery at a point two-thirds of the way between its origin near the pulmonary conus and the cardiac apex and a snare was formed by passing both ends of the suture through a piece of polyethylene tubing. Occlusion of the left coronary artery, by clamping the snare against the surface of the heart, caused an area of epicardial cyanosis with regional hypokinesis. Reperfusion was achieved by releasing the snare and was confirmed by conspicuous hyperaemic blushing of the previously ischemic myocardium. Coronary flow was measured by timed collection of coronary effluent, and coronary effluent was collected for measurement of CGRP level and creatine kinase activity.
were incubated in 1% triphenyl tetrazolium chloride phosphate buffer solution at 37 °C for 20 min to stain the viable myocardium brick red. The samples were then fixed in a 10% formalin solution for 24 h. Sections were traced onto acetate sheets. The area of infarct and risk zone was determined by planimetry of the tracings. Infarct and risk area volumes were expressed as mm3 and infarct size was calculated as a percentage of the risk zone.
2.2. Experimental protocols
CGRP, CGRP 8-37 and capsaicin were purchased from Sigma. Capsaicin was dissolved in a vehicle containing 10% Tween 80, 10% ethanol, and 80% saline. The radioimmunoassay kits for measurement of CGRP were obtained from Dongya Immunity Technology Institution, Beijing, China. Creatine kinase kits were obtained from High-tech Bioengineering Co. Ltd, Beijing, China.
Animals were randomly divided into 5 groups. All animals were subjected to 60 min of left coronary artery occlusion followed by 60 min of reperfusion, except the control group. (1) control group; (2) ischemia-reperfusion injury, the left coronary artery was occluded for 3 min followed by 60 min of reperfusion; (3) ischemic postconditioning (IPC), immediately at the onset of reperfusion, reflow was initiated with 1 min of full coronary flow, followed by 1 min of re-occlusion, repeated for a total of three cycles; (4) IPC plus CGRP 8-37, the heart was perfused with CGRP 8-37 (10− 7 M) for 10 min before ischemia; (5) IPC plus capsaicin, A single dose of capsaicin (50 mg/kg) which depletes transmitters in sensory nerves was administered by s.c. injection, 4 days before the experiment under anesthesia (sodium pentobarbital, 60 mg/kg, i.p.); and (6) IPC plus capsaicin & CGRP, the heart was perfused with CGRP (5 × 10− 9 M) in the phase of repeated perfusion and capsaicin was given according to the same procedure as for the IPC plus capsaicin group. 2.3. Infarct size and risk area At the end of 60 min reperfusion, the left coronary was reoccluded, and 1 ml Evans blue (1%) was injected into the ventricular cavity and was allowed to perfuse the non-ischemia portions of the heart. The entire heart was weighted, rinsed of excess blue dye, trimmed of right ventricular and atria tissue and sliced into 1-mm thick sections from the apex to base. The slices
2.4. Measurement of creatine kinase activity The creatine kinase activity in the coronary effluent from the heart at 5 min of reperfusion was measured spectrophotometrically. 2.5. CGRP assay The whole coronary effluent of perfusate fractions (5 min) was collected before ischemia and during reperfusion, and acetic acid (final concentration 0.2 M) was added. The samples were desalted using SEP-PAK C18 cartridges and lyophilized. CGRP-like immunoreactivity in the perfusate fraction was measured by using a radioimmunoassay kit with antisera raised against rat CGRP, 125 I-labelled CGRP and rat CGRP standard. 2.6. Reagents
2.7. Statistics Data are expressed as means ± SEM. All values were analyzed by using ANOVA and the Newman–Keuls Student's t-test. The significance level was chosen as P b 0.05.
Table 1 The basal values of cardiac function
Control IR IPC +CGRP8-37 +Cap + Cap&CGRP
n LVP (mm Hg)
+dp / dtmax −dp / dtmax HR (mm Hg/s) (mm Hg/s) (beats/ min)
CF (ml/min)
8 9 9 7 7 6
3346 ± 188 3608 ± 317 3312 ± 337 3070 ± 424 2896 ± 322 2954 ± 234
9.5 ± 0.5 9.7 ± 0.6 8.5 ± 0.6 9.0 ± 1.0 6.1 ± 0.5## 6.8 ± 0.4
132.3 ± 9.2 142.7 ± 9.9 131.7 ± 16.2 125.0 ± 11.0 115.9 ± 11.0 119.4 ± 13.7
2259 ± 129 2043 ± 146 1932 ± 210 1978 ± 185 1827 ± 195 1864 ± 231
253 ± 9 231 ± 10 228 ± 18 208 ± 14 223 ± 17 211 ± 14
All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). ## P b 0.01 vs IPC.
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D. Li et al. / Regulatory Peptides 147 (2008) 4–8
genous CGRP reappeared postconditioning-like cardioprotection, as shown by improving cardiac function and decreasing creatine kinase release in the rats pretreated with capsaicin (Table 2 and Fig. 1).
3. Results 3.1. Cardiac function and creatine kinase activity There was no significant difference in basal values of HR, CF, and LVP ± dp / dtmax among groups. Pretreatment with capsaicin which depletes transmitters in sensory nerves decreased coronary flow of heart (Table 1). Sixty minutes ischemia and 60-min reperfusion caused a marked decrease in cardiac function (CF, LVP, and LVP ± dp / dtmax) and a significant increase in creatine kinase release. Postconditioning significantly improved cardiac function (Table 2) and decreased creatine kinase release in reperfusion period (Fig. 1), these effects which were abolished by CGRP8-37, a selective CGRP receptor antagonist, or by pretreatment with capsaicin. Exo-
3.2. Infarct size As shown in Table 3, there were no changes in risk zone, indicating that the size of the risk area was comparable in all groups. Ischemia-reperfusion caused a 45.0 ± 2.2 % necrosis in the area at risk. However, postconditioning significantly reduced infarct size, an effect which was abolished by CGRP8-37 or by pretreatment with capsaicin. Exogenous CGRP decreased the amplified infarct size in the rats pretreated with capsaicin.
Table 2 Effect of IPC on cardiac function n
Reperfusion (min) 5
10
20
30
60
LVP(mm Hg) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
128.9 ± 9.5 58.1 ± 8.2 ⁎ ⁎ 84.5 ± 11.0+ 46.5 ± 6.6## 61.1 ± 3.5 77.3 ± 4.3$
130.4 ± 9.1 59.8 ± 7.8⁎⁎ 85.2 ± 12.4+ 43.6 ± 6.7## 58.6 ± 2.9# 75.7 ± 5.8$
130.4 ± 9.6 60.0 ± 7.7⁎⁎ 82.9 ± 10.9+ 47.4 ± 4.0## 57.3 ± 3.6# 71.0 ± 8.2$
128.1 ± 9.4 54.5 ± 6.7 ⁎ ⁎ 79.8 ± 9.1++ 47.4 ± 4.1## 55.4 ± 3.2# 70.4 ± 2.0$
127.3 ± 9.9 48.4 ± 5.6⁎⁎ 78.2 ± 7.2+ 39.3 ± 5.0## 51.1 ± 2.4## 65.8 ± 4.1$
+ dp / dtmax (mm Hg/s) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
3299 ± 192 1309 ± 166⁎⁎ 1903 ± 258+ 873 ± 211## 1553 ± 189## 1764 ± 155$
3180 ± 189 1356 ± 172⁎⁎ 1970 ± 197 904 ± 193## 1429 ± 181## 1773 ± 192$
3289 ± 218 1311 ± 165⁎⁎ 2027 ± 216++ 978 ± 194## 1362 ± 178## 1718 ± 139$
3281 ± 201 1220 ± 128⁎⁎ 1942 ± 209++ 1025 ± 205## 1354 ± 189## 1652 ± 213$
3233 ± 240 1146 ± 156⁎⁎ 1924 ± 223++ 985 ± 195## 1286 ± 192## 1623 ± 152$
− dp / dtmax (mm Hg/s) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
2250 ± 149 736 ± 117⁎⁎ 1296 ± 116++ 683 ± 132## 881 ± 94## 1037 ± 96$
2233 ± 132 687 ± 74⁎⁎ 1127 ± 98+ 655 ± 126## 877 ± 82## 986 ± 64$
2218 ± 127 695 ± 86⁎⁎ 1150 ± ±111++ 669 ± 93## 817 ± 75## 994 ± 84$
2227 ± 120 648 ± 75⁎⁎ 1084 ± 89++ 691 ± 83## 790 ± 69## 951 ± 79$
2210 ± 135 553 ± 73⁎⁎ 1043 ± 92++ 656 ± 78## 753 ± 67## 917 ± 75$
HR (beats/min) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
248 ± 12 218 ± 15 191 ± 18 172 ± 11 174 ± 14 187 ± 9
246 ± 10 179 ± 18 201 ± 18 171 ± 5 185 ± 14 176 ± 13
248 ± 11 187 ± 22 212 ± 16 181 ± 4 186 ± 14 183 ± 12
246 ± 11 183 ± 22 214 ± 16 167 ± 11 184 ± 13 174 ± 17
247 ± 12 165 ± 20 202 ± 12 168 ± 8 182 ± 11 177 ± 11
CF(ml/min) Control IR IPC +CGRP8-37 +Cap +Cap&CGRP
8 9 9 7 7 6
9.4 ± 0.5 4.0 ± 0.3⁎⁎ 5.8 ± 0.6+ 3.9 ± 0.4## 2.3 ± 0.2## 4.6 ± 0.4$
9.4 ± 0.5 3.8 ± 0.3⁎⁎ 5.7 ± 0.6+ 3.8 ± 0.4## 2.2 ± 0.1## 4.2 ± 0.2$$
9.2 ± 0.5 3.4 ± 0.3⁎⁎ 5.5 ± 0.5+ 3.7 ± 0.6## 2.2 ± 0.2## 4.1 ± 0.5$$
9.3 ± 0.4 3.2 ± 0.3⁎⁎ 5.4 ± 0.5+ 3.6 ± 0.6## 2.1 ± 0.1## 3.9 ± 0.2$
9.2 ± 0.4 2.7 ± 0.3⁎⁎ 5.3 ± 0.5+ 3.4 ± 0.6## 2.0 ± 0.1## 3.7 ± 0.3$
All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). ⁎⁎P b 0.01 vs control; +P b 0.05, ++P b 0.01 vs IR; #P b 0.05, ##P b 0.01 vs IPC; $P b 0.05, $$P b 0.01 vs + Cap.
D. Li et al. / Regulatory Peptides 147 (2008) 4–8
Fig. 1. Effect of IPC on creatine kinase activity. All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). n = 6–9. ⁎⁎P b 0.01 vs control; ++P b 0.01 vs IR; ##P b 0.01 vs IPC; $P b 0.05 vs + Cap.
3.3. Release of CGRP As shown in Fig. 2, the content of CGRP-like immunoreactivity in coronary effluent was significantly increased during reperfusion compared with in pre-ischemia period. CGRP content of coronary effluent in the postconditioning group was higher than that of the ischemia-reperfusion group. However, the increased release of CGRP by postconditioning was abolished by pretreatment with capsaicin. 4. Discussion The cardioprotection afforded by postconditioning exhibits improvement of cardiac function, reduction of infarct size and CK release and limitation of tissue edema, which has been demonstrated in various models of ischemia-reperfusion [4,10]. However, the mechanisms responsible for the beneficial effect of postconditioning on the ischemic myocardium are not fully understood. Recently, it has been reported that the protection of postconditioning was triggered by endogenous active substances such as nitric oxide, adenosine or acetylcholine [11,12]. It has been demonstrated that the heart is innervated by both autonomic and capsaicin-sensitive sensory nerves, and it has been suggested that capsaicin-sensitive sensory nerves may Table 3 Effect of IPC on infarct size and risk area
IR IPC +CGRP8-37 +Cap +Cap&CGRP
n Heart wet weight (g)
Area at risk Infarct size (mm3) (mm3)
Infarct size/ risk area (%)
9 9 7 7 6
375.0 ± 14.4 377.0 ± 11.8 347.6 ± 17.8 370.0 ± 18.6 362.1 ± 10.8
45.0 ± 2.2 27.8 ± 2.7++ 41.0 ± 1.9## 41.1 ± 2.8## 33.1 ± 3.2$
0.86 ± 0.02 0.82 ± 0.02 0.85 ± 0.03 0.83 ± 0.03 0.83 ± 0.04
168.3 ± 9.3 104.7 ± 10.6++ 142.1 ± 9.1## 152.4 ± 11.2## 119.7 ± 10.2$
All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). ++ P b 0.01 vs IR; ##P b 0.01 vs IPC; $P b 0.05 vs + Cap.
7
Fig. 2. Effect of IPC on release of CGRP. All values were expressed as mean ± SEM. IR: ischemia-reperfusion; IPC: ischemic postconditioning; Cap: rats were pretreated with capsaicin (50 mg/kg). n = 7–9. ⁎⁎P b 0.01 vs IR of pre-ischemia period; ++P b 0.01 vs IR of reperfusion period; #P b 0.05, ##P b 0.01 vs IR and IPC, respectively.
play a role in modulation of cardiac function through local reflex mechanisms [13]. CGRP, the major transmitter in capsaicin-sensitive sensory nerves, is distributed in cardiovascular tissues. CGRP, besides regulating vascular smooth muscle tone, possesses a protection of the ischemic myocardium. Recently, we and others have documented that CGRP is involved in mediation of cardioprotection afforded by ischemic or pharmacological preconditioning [14,15], and CGRP is thought as an endogenous myocardial protective substance. The release of CGRP is regulated by multiple factors. Myocardial ischemia, even a brief ischemia period of 5 min, causes a significant increase of CGRP release in isolated guinea pig heart [13]. Studies in clinics have shown that the plasma concentration of CGRP is markedly elevated in patient with and without early reperfusion after acute myocardial infarction [13,16]. Our recent work has shown that a single preconditioning episode of 5-min ischemia caused a marked increase in the release of CGRP in the isolated rat heart [17]. It is likely that the protection afforded by postconditioning is related to stimulation of CGRP release, because the release of endogenous CGRP could be regulated by transient ischemia which is similar to brief episodes provided by postconditioning. The results of the present study revealed postconditioning improved cardiac function and decreased infarct size, concomitantly with an increase in the release of CGRP in coronary effluent, and the protective effects of postconditioning were abolished by CGRP8-37, an antagonist of CGRP receptor, or pretreatment with capsaicin, which depletes CGRP in sensory nerves. Exogenous CGRP reappeared postconditioning-like cardioprotection in the rat pretreated with capsaicin. These results support the hypothesis that endogenous CGRP may play an important role in mediation of the cardioprotection of postconditioning in rats. Capsaicin-sensitive sensory nerves contain a number of peptides, including CGRP, substance P and neurokinin A [13]. Besides CGPR, substance P and neurokinin A could also mediate the effect of capsaicin. Among these peptides, CGRP,
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endogenous or exogenous, has been shown to possess a beneficial effect of the myocardium [18,19]. This study is consistent with our previous observation that brief ischemia evokes CGRP release and the cardioprotection afforded by preconditioni1ng or postconditioning is abolished by CGRP 837, an antagonist of CGRP receptor [17]. These findings allow us to speculate that CGRP may play an important role in mediation of the protection provided by postconditioning. TNF-α, as an autocrine cytokine, has been shown to be released from the ischemic heart and to be involved in myocardial dysfunction in ischemia and reperfusion [20]. It has been shown that an increase in the level of TNF-α is localized in cardiomyocytes from human myocardial tissues following acute ischemia, and pretreatment with neutralising anti-TNF-a antibody reduces myocardial damage during ischemia [21]. Our previous study showed that CGRP-mediated preconditioning is related to the inhibition of myocardial TNF-α production [22]. Recently, it was reported that postconditioning attenuated cardiomyocyte apoptosis, mediated by reducing TNF-α release [23]. The mechanism responsible for the inhibition of TNF-α production by CGRP remains unclear. There is evidence that endogenous mediators, including neurotransmitters, bind to specific receptors and then activate the endogenous protective mechanisms via complex signal pathways associated with the activation of protein kinase C (PKC) [24,25]. Previous studies have shown that PKC inhibition abolished CGRP-induced preconditioning [8]. It is probable that the protective effect of CGRP-mediated postconditioning is related to inhibition of TNF-α production via activation of PKC. However, further work is needed before drawing a definitive conclusion on this matter. In conclusion, the present study suggests that endogenous CGRP is involved in the mediation of cardioprotection afforded by postconditioning in rats. Acknowledgements This work was supported by grants from the National Nature Science Foundation (No. 30430740) and the National Basic Research Program (973 Program, No. 2007CB512000), China. References [1] Thibault H, Piot C, Ovize M. Postconditioning in man. Heart Fail Rev 2007;12:245–8. [2] Kin H, Zhao ZQ, Sun HY, Wang NP, Corvera JS, Halkos ME, Kerendi F, Guyton RA, Vinten-Johansen J. Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion. Cardiovasc Res 2004;62:74–85. [3] Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124–36. [4] Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, Vinten-Johansen J. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol 2003;285:H579–88. [5] Yao Z, Gross GJ. A comparison of adenosine-induced cardioprotection and ischemic preconditioning in dogs. Efficacy, time course, and role of KATP channels. Circulation 1994;89:1229–36.
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