El . L'
`EX2263
`1 1lly & Co. v. Teva Pharms. lnt'l GMBH
`IPR2018-01422, -01423, 01424, -01425,
`-01426, -01427
`
`ELSEVIER
`
`Regulatory Peptides 82 (1999) 53-57
`
`REGULATORY
`
`PEPTIDES
`
`Evidence for calcitonin gene-related peptide-mediated ischemic
`preconditioning in the rat heart
`
`Rong Lu, Yuan-Jian Li*, Han-Wu Deng
`
`Department of Pharmacology, Hunan Medical University, Changsha, Hunan 410078, People's Republic of China
`
`Received 13 January 1999; received in revised form 7 April 1999; accepted 9 April 1999
`
`Abstract
`
`Previous studies have suggested that calcitonin gene-related peptide (CGRP) may play an important role in the mediation of ischemic
`preconditioning. In the present study, we examined the release ofCGRP during ischemic preconditioning and the effect of preconditioning
`frequency on this effect in the isolated rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant cardiac
`dysfunction and an increase in the release of creatine kinase (CK) during reperfusion. Preconditioning with one, two or three cycles of
`5-min ischemia and 5-min reperfusion caused a marked improvement of cardiac function and a decrease in the release of CK, and there
`was no difference in the degree of improvement among groups. The protective effects of ischemic preconditioning were abolished by the
`CGRP receptor antagonist CGRP8 _37 . A single preconditioning cycle induced a significant increase in the release ofCGRP in the coronary
`effluent. In the hearts treated with two or three preconditioning cycles, the level of CGRP was highest in the first cycle, and was gradually
`decreased with increasing number of cycles of preconditioning. These results suggest that the protective effects of ischemic
`preconditioning are mediated by endogenous CGRP in the isolated rat heart. © 1999 Elsevier Science B.V. All rights reserved.
`
`Keywords: Ischemic preconditioning; Calcitonin gene-related peptide (CGRP); CGRP8 _37 ; Heart; Rat
`
`1. Introduction
`
`Ischemic preconditioning is termed as the protective
`adaptive mechanism produced by brief periods of myocar(cid:173)
`dial ischemic trigger, which provides the great powerful
`form of protection against myocardial necrosis from a
`subsequent prolonged period of ischemia and reperfusion
`[1]. The mechanism responsible for the protective effects
`of ischemic preconditioning is not fully understood. A
`number of studies have suggested that endogenous chemi(cid:173)
`cal substances such as adenosine, catecholamines, brady(cid:173)
`kinin, prostaglands, and nitric oxide are involved in the
`cardioprotection of ischemic preconditioning [2].
`There is substantial evidence to suggest that neurogenic
`mechanisms are involved in protective effects on the
`
`*Corresponding author. Tel.: + 86-731-447-4411, ext. 2704; fax:
`+ 86-731-447-1339.
`
`ischemic myocardium, and peptidergic and sympathetic
`nerves have been shown to participate in the mediation of
`ischemic preconditioning [3-5]. Calcitonin gene-related
`peptide (CGRP), a principal transmitter in capsaicin sensi(cid:173)
`tive sensory nerves, is widely distributed in cardiovascular
`tissues [6]. Recently, we and others have shown endogen(cid:173)
`ous CGRP may play an important role in the mediation of
`ischemic preconditioning in the rat [7,8].
`Since previous studies, by using the CGRP receptor
`antagonist or blocker of capsaicin sensitive sensory nerves,
`have suggested that CGRP may be
`involved in the
`protective effects afforded by ischemic preconditioning
`[7,9], in the present study, we examined the release of
`CGRP during ischemic preconditioning in the isolated rat
`heart. With reference to protection against the ischemic
`myocardium, there is evidence that one cycle of precondi(cid:173)
`tioning is as
`'efficacious' as multiple cycles [10,11].
`According to involvement of endogenous chemical medi-
`
`0167-0115/99/$- see front matter © 1999 Elsevier Science B.V All rights reserved.
`PI!: S0167-01!5(99)00039-7
`
`

`

`54
`
`R. Lu eta/. I Regulatory Peptides 82 (1999) 53-57
`
`ators in the protective effects of ischemic preconditioning,
`we postulate that the release of endogenous substances
`stimulated by a single preconditioning cycle may be
`sufficient to afford maximum protection. Therefore, we
`also explored the effect of preconditioning frequency on
`CGRP release.
`
`2. Materials and methods
`
`2.1. Perfusion of the isolated heart
`
`Male Sprague-Dawley rats weighing 180-220 g, were
`anesthetized with sodium pentobarbital (60 mg/kg, i.p.).
`The hearts were excised, rapidly placed in iced Krebs(cid:173)
`Henseleit buffer solution ( 4°C), mounted, and perfused on
`a modified Langendorf[ apparatus at a constant pressure of
`80 em H20 with Krebs-Henseleit solution (mmol/1: NaCl,
`1.2;
`25.0; KCl, 4.7; KH 2P04 ,
`119.0; NaHC0 3 ,
`MgS0 4.7H20, 1.2; CaC1 2 , 2.5; and glucose, 11.0). The
`perfusate solution was saturated with 95% 0 2 and 5% C0 2
`(37°C, pH 7.4).
`A water-filled latex balloon was inserted into the left
`ventricle through the left artiotomy, and the volume was
`adjusted to achieve a stable left ventricular end-diastolic
`pressure of 5 to 6 mmHg during initial equilibration and
`maintained throughout the experiments. The heart rate
`(HR.), left ventricular pressure (LVP) and its first derivative
`(LV dp/dtmaJ were recorded by a polyphysiological
`recorder. Coronary flow (CF) was measured by timed
`collection of coronary effluent.
`
`2.2. Radioimmunoassay
`
`The whole coronary effluent of perfusate fraction (5
`min) were collected before and after brief ischemia respec(cid:173)
`tively, and acetic acid (final concentration 0.2 M) was
`added. The samples were desalted using sep-pak C 18
`cartridge, lyophilized and stored at - 20°C until assay
`[12].
`Kits for measurement of CGRP were obtained from
`Dongya Immunity Technology Institution (P.R. China).
`The perfusated fraction samples were redissolved in appro(cid:173)
`priate buffer. CGRP-like immunoreactivity (CGRP-LI) of
`perfusated fraction was measured using antisera raised
`against rat CGRP, 125I-labelled CGRP and rat CGRP
`standard.
`
`2.3. Creatine kinase measurement
`
`Myocardial injury was monitored by assaying creatine
`kinase (CK). The activity of CK in the coronary effluent
`from the heart at 5 min of reperfusion was assayed
`spectrophotometrically.
`
`2.4. Experimental protocols
`
`All hearts had an initial stabilization period for 15-20
`min. The experiment was divided into seven groups. The
`control group was perfused with Krebs-Henseleit solution
`throughout
`the experiment. The ischemia~reperfusion
`group experienced 30-min global ischemia and 40-min
`reperfusion. For ischemic preconditioning, hearts were
`subjected to one, two or three cycles of 5-min global
`ischemia and 5-min reperfusion, respectively, before 30-
`min ischemia and 40-min reperfusion. In the case of
`CGRP8_37 , hearts were perfused with CGRP8_37 (10- 7 M)
`for 20 min before 30-min ischemia and 40-min reperfu(cid:173)
`sion. For the studies on the effects of CGRP8_37 on the
`protective effects of ischemic preconditioning, hearts were
`perfused with CGRP8_37 for 5 min, and then preconditioned
`with three cycles of 5-min global ischemia and 5-min
`reperfusion in the presence of CGRP8_37 before 30-min
`ischemia and 40-min reperfusion.
`
`2.5. Statistics
`
`All values are expressed as means±S.E.M. The data
`from the content of CGRP was evaluated by paired t-test.
`The data from cardiac function were determined by
`ANOVA and the Newman-Keuls test. The level of signifi(cid:173)
`cance was chosen as P < 0.05.
`
`3. Results
`
`The basal values ofLVP, LV dp/dtmax• HR. and CF in all
`groups are listed in Table 1. There was no difference in
`LVP, LV dp/dtmax• HR. and CF among groups.
`Table 2 summarizes the percentage recovery of cardiac
`function compared with matched baseline for all groups.
`Thirty minutes of global ischemia and 40 min of reperfu(cid:173)
`sion caused a significant decline in cardiac function (LVP,
`LV dp I dtmax' and CF) and an increase in the release of CK
`(Fig. 1 ). Ischemic preconditioning induced by one, two or
`three cycles of 5-min ischemia and 5-min reperfusion
`enhanced the improvement of cardiac function and reduced
`the release of CK, and there was no difference in the
`degree of improvement among groups. The content of
`CGRP in the coronary effluent during ischemic precondi(cid:173)
`tioning was significantly increased in a single precondi(cid:173)
`tioning cycle. In the hearts treated with two or three
`preconditioning cycles, the release of CGRP was highest in
`the first cycle, and gradually decreased with increasing
`frequency of preconditioning. A slight increase in the
`release of CGRP was shown in the third preconditioning
`cycle, and there was no significant difference compared
`with pre-preconditioning (Fig. 2).
`As has been reported previously [1 0], the protective
`
`

`

`R. Lu eta/. I Regulatory Peptides 82 (1999) 53-57
`
`55
`
`Table I
`Basal values of cardiac function
`
`n
`
`LVP
`(mmHg)
`
`LV dp/dlmox
`(mmHg s- 1
`)
`
`HR
`(beats min- 1
`
`)
`
`CF
`(ml min- 1
`
`)
`
`8.0±1.2
`8.1 ±0.5
`8.3±0.6
`8.1 ±0.3
`8.0±0.4
`8.1±0.6
`8.1 ±0.3
`
`Control
`1/R'
`+ CGRPS-37
`+PC!'
`+PC2'
`+PC3'
`+ PC3 and CGRP8 _37
`• 1/R, ischemia-reperfusion; PC!, one preconditioning cycle; PC2, two preconditioning cycles; PC3, three preconditioning cycles.
`
`2499±62
`2484±65
`2646±175
`2512±87
`2436±92
`2543±90
`2421±86
`
`305±6
`304±7
`296±4
`311±8
`317±9
`300± 15
`295±3
`
`7
`7
`4
`7
`8
`8
`5
`
`77.1±6.0
`75.6± 1.2
`81.6±6.9
`78.3±3.5
`71.6±4.7
`74.0±7.9
`80.8±3.1
`
`Table 2
`Percentage recovery of cardiac function of all groups during reperfusion
`
`LVP (% of baseline)
`Control
`1/R'
`+ CGRPS-37
`+PC!'
`+PC2'
`+PC3'
`+ PC3 and CGRP8 _37
`
`LV dp/dtmox (%of baseline)
`Control
`1/R
`+ CGRPS-37
`+PC!
`+PC2
`+PC3
`+ PC3 and CGRP8 _37
`
`HR (% of baseline)
`Control
`1/R
`+CGRPs-37
`+PC!
`+PC2
`+PC3
`+ PC3 and CGRP8 _37
`
`n
`
`7
`7
`4
`7
`8
`8
`5
`
`7
`7
`4
`7
`8
`8
`5
`
`7
`7
`4
`7
`8
`8
`5
`
`Reperfusion (min)
`
`5
`
`10
`
`20
`
`30
`
`40
`
`99.6± 1.3
`36.8±5.8++
`24.7±3.2
`70.5±10.8
`69.1±10.1
`67.0±9.4
`28.2±1.6#
`
`100.0±0.8
`28.4±5.1 ++
`18.1 ±4.4
`58.6± 13.0
`58.5±8.7
`51.8±9.3
`21.0±0.9
`
`101.2±1.5
`76.5±8.0
`60.0±2.5
`79.9±7.4
`92.5±4.0
`87.4±8.5
`67.2±9.2
`
`97.3± 1.9
`39.3±6.3 ++
`31.0±7.5
`78.8±6.5*
`77.7±7.2**
`76.1 ± 10.6**
`38.8±5.8##
`
`98.5±2.0
`30.8±4.9++
`25.0±5.6
`68.1 ±8.8*
`68.3±6.8*
`67.8± 10.5**
`32.3±4.3##
`
`101.3± 1.3
`85.8±3.4
`76.5±5.9
`87.4±4.6
`91.5±3.7
`90.3±7.8
`87.5±0.9
`
`92.3±3.2
`51.6±6.2 ++
`48.7±5.5
`80.3±5.3*
`84.1±6.7*
`87.3± 10.6*
`50.2±6.9#
`
`95.8±2.3
`44.3±5.7++
`39.4±4.4
`77.2±7.0**
`83.3±6.0**
`79.1±10.1**
`43.9±6.3#
`
`101.5 ± 1.5
`86.7±3.3
`81.9±4.6
`89.3±4.1
`87.7±4.0
`100.1±3.5
`82.7± 1.6
`
`92.3±3.9
`44.5±6.7++
`50.6±7.8
`73.8±5.3*
`81.0±5.8*
`90.4± 11.9**
`60.2±9.1
`
`92.8±3.1
`42.3±5.9++
`42.4±6.4
`77.1±3.9**
`81.6±5.2**
`85.4± 10.5**
`55.6±9.7
`
`101.4± 1.8
`88.1 ±3.6
`86.7±3.1
`84.6±3.4
`90.8±3.8
`97.4±2.2
`81.6± 1.5
`
`90.6±2.9
`45.7±6.6++
`55.7±8.8
`68.8±5.3
`76.2±6.2*
`87.3± 12.8**
`57.7±7.0
`
`92.3±3.6
`43.3±6.4 ++
`46.8±7.1
`73.2±3.7*
`78.8±5.6**
`83.4±10.1**
`51.4±6.8"'
`
`100.7±2.0
`91.1±5.8
`82.5±7.3
`94.5±4.2
`95.3±3.4
`91.6±5.0
`80.0±2.7
`
`CF (% of baseline)
`98.2±0.9
`7
`Control
`98.0±1.7
`88.8±3.1
`97.0±3.0
`92.1±3.2
`54.9±4.6++
`53.1±6.8++
`50.3±2.6++
`55.9±5.8++
`52.9±3.8++
`7
`1/R
`50.9±10.5
`50.7±10.2
`4
`49.0± 11.3
`50.4±9.3
`52.5± 11.0
`+CGRPs-37
`92.1 ± 11.1 **
`90.9±6.8**
`94.1±5.7**
`7
`+PC!
`94.5±9.7**
`88.3± 12.6**
`81.5±4.7*
`94.2±6.1**
`94.0±6.0**
`92.8±5.5**
`87.5±5.3**
`8
`+PC2
`97.8±6.5**
`96.4±6.1 **
`89.7±6.6**
`81.3±7.0**
`8
`+PC3
`71.7±5.6*
`45.9±5.0##
`49.2± 1.8##
`53.4±2.8##
`52.4±3.4##
`+ PC3 and CGRP8 _37
`50.6±3.4 #
`5
`• 1/R, ischemia-reperfusion; PC!, one preconditioning cycle; PC2, two preconditioning cycles; PC3, three preconditioning cycles. + P < 0.05,
`+ + P < 0.0 I vs. control; * P < 0.05, * * P < 0.0 I vs. 1/R; "'P < 0.05, .... P < 0.01 vs. PC3.
`
`effects of ischemic preconditioning were abolished by
`CGRP8_37 (Table 2, Fig. 3). CGRP8_37 treatment alone had
`no effect on ischemia-reperfusion injury.
`
`4. Discussion
`
`The results of this study confirmed previous observa-
`
`

`

`56
`
`2500
`
`~
`~ 2000
`
`.E' c: ·e 15oo
`3
`.§.
`"' 1000
`~
`
`"' ~ 500
`
`~
`(.)
`
`R. Lu et al. I Regulatory Peptides 82 (1999) 53-57
`
`2500
`
`~
`~ :;!000
`
`w
`·e 15oo
`3
`E
`-; 1000
`"' ...
`"' 'E
`
`5oo
`
`~
`(.)
`
`CON
`
`1/R
`
`+PC1
`
`+PC2
`
`+PC3
`
`CON
`
`1/R
`
`+CGRP,_,, +PC3
`
`+PC3&CGRP8_,7
`
`I. Effect of ischemic preconditioning on release of CK during
`Fig.
`reperfusion. CON, control; 1/R, ischemia-reperfusion; PC!, one pre(cid:173)
`conditioning cycle; PC2, two preconditioning cycles; PC3, three pre(cid:173)
`conditioning cycles. Values are means:!:S.E.M., n = 4-8. **P < 0.01 vs.
`control; + P < 0.05 vs. 1/R.
`
`Fig. 3. Effect of CGRP8 _37 on the reduction of CK release by ischemic
`preconditioning. CON, control; 1/R, ischemia-reperfusion; PC3, three
`preconditioning cycles. Values are means:!:S.E.M., n = 4-8. **P < 0.01
`VS. CON; + p < 0.05 vs. 1/R; # p < 0.05 vs. PC3.
`
`C' 100
`~
`...
`80
`" .::: ...
`,e. -= ~
`= 8
`~
`u
`
`60
`
`40
`
`20
`
`0
`
`A: one cycle
`
`**
`
`CON
`
`I
`
`C'
`~
`...
`.::: ...
`"
`5 -=
`~ = 0 ...
`~
`
`u
`
`100
`
`8: two cycles
`
`80
`
`60
`
`40
`
`20
`0
`
`**
`
`CON
`
`I
`
`n
`
`0
`
`60
`
`C' 100 C: three cycles
`'l:l ...
`80
`**
`"
`t
`5
`c
`~ 40
`= 0 ...
`20
`~-
`!;!)
`u
`
`0
`
`n m
`CON
`Fig. 2. The content of CGRP in the coronary effluent. CON, pre(cid:173)
`preconditioning; I, the first cycle; II, the second cycle; III, the third cycle.
`Values are means±S.E.M., n = 6. *P < 0.05, **P < 0.01 vs. CON.
`
`I
`
`tions that in the isolated rat heart, the protective effects of
`ischemic preconditioning are abolished by CGRP8_37 [7].
`Our previous work has shown that brief perfusion of
`capsaicin or calcitonin gene-related peptide (CGRP) pro(cid:173)
`vides a preconditioning-like cardioprotection [9]. Others
`have reported that pretreatment with capsaicin to deplete
`CGRP abolishes the protective effects of pacing-induced
`preconditioning in the rat [13]. In the present study, we
`found that the release of CGRP was significantly increased
`during ischemic preconditioning in the isolated rat heart, in
`further support of the hypothesis that the cardioprotective
`effects of ischemic preconditioning are mediated by endog(cid:173)
`enous CGRP in the rat heart.
`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 [6]. Studies in clinics have shown
`is markedly
`that the plasma concentration of CGRP
`elevated in patients with and without early reperfusion
`after acute myocardial infarction [14]. In the present study,
`a single preconditioning episode of 5-min ischemia caused
`a marked increase in the release of CGRP in the isolated
`rat heart. Recently, it has been found that pretreatment
`with capsaicin aggravates myocardial infarction in the
`porcine heart, and one postulates the elevated level of
`CGRP during ischemia probably constitutes a compensat(cid:173)
`ory response [15]. The predominance of findings, ours as
`well as others, suggest that CGRP is an endogenous
`myocardial protective substance.
`The effect of preconditioning frequency on cardioprotec(cid:173)
`in a variety of animals
`tion has been examined
`[10,11,16,17]. The present results are consistent with
`previous observations which one cycle is as 'efficacious'
`as multiple cycles of preconditioning in rat hearts [I 0]. As
`mentioned above, endogenous myocardial protective sub(cid:173)
`stances play a pivotal role in the protection afforded by
`ischemic preconditioning. In the present study, we tested
`the effect of preconditioning frequency on CGRP release.
`The results revealed that a single preconditioning episode
`
`

`

`R. Lu et al. I Regulatory Peptides 82 (1999) 53-57
`
`57
`
`of 5-min ischemia caused a significant increase in the
`release of CORP, and the release of CORP was decreased
`with increasing number of cycles of ischemic precondition(cid:173)
`ing. It is likely that the release of CORP stimulated by a
`single cycle is sufficient to afford effective protection. It
`has been reported that ischemic or pharmacological pre(cid:173)
`conditioning protects against myocardial injury by trig(cid:173)
`gering signal transduction pathways [2]. As discussed in
`more detail below, CORP-mediated ischemic precondition(cid:173)
`ing is related to activation of protein kinase C (PKC)
`[ 18, 19]. A possible explanation of our findings is that the
`effects of ischemic preconditioning will not increase with
`increasing frequency once endogenous protective mecha(cid:173)
`nisms have been triggered.
`CORP, besides regulating vascular smooth tone, has a
`direct protective effect on myocytes and endothelial cells,
`which has been demonstrated by previous studies in
`cultured myocytes and endothelial cells [20,21]. The
`mechanism responsible for the protective effects of CORP
`is unknown. A number of studies have suggested that
`ischemic preconditioning stimulates the release of endog(cid:173)
`enous active substances, with a subsequent triggering of
`signal transduction pathways, resulting in cardioprotection.
`There is evidence that CORP increases PKC activity and
`activates kATP channel [18,22]. Previous investigations
`is
`have
`shown
`that CORP-induced preconditioning
`abolished by the PKC inhibitor H-7 [19], suggesting that
`the protective effects of CORP are related to activation of
`PKC.
`In summary, the present results suggest that the protec(cid:173)
`tive effects of ischemic preconditioning are mediated by
`endogenous CGRP in the isolated rat heart.
`
`Acknowledgements
`
`This study was supported by a grant from the Ministry
`of Education, China.
`
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`
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