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Ann Thorac Surg 1996;62:1748-1751
© 1996 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Calcitonin Gene-Related Peptide–Induced Preconditioning Improves Preservation With Cardioplegia

Department of Cardiosurgery, Xiang Ya Hospital, and Department of Pharmacology, Hunan Medical University, Hunan, People's Republic of China

Accepted for publication June 19, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. Our recent work has shown that calcitonin gene-related peptide (CGRP) may play an important role in mediation of ischemic preconditioning. Therefore, we tested the hypothesis that CGRP-induced preconditioning protects against myocardial damage after prolonged cardioplegic arrest in isolated rat hearts.

Methods. Six groups were studied: the control, ischemic preconditioning, and CGRP-pretreated groups for both 4- and 8-hour hypothermic ischemia. All hearts were arrested using St. Thomas Hospital cardioplegia, and then reperfused with normothermic Krebs-Henseleit solution for 60 minutes after the 4- or 8-hour hypothermic ischemic period. Hearts were subjected to two cycles of 5-minute ischemia and 10-minute reperfusion in the ischemic preconditioning group. In the CGRP-pretreated group, Krebs-Henseleit solution containing CGRP (5 x 10^-9 mol/L) was substituted for the ischemic period.

Results. At 30 minutes of reperfusion after 4-hour storage, left ventricular pressure (mm Hg) and its first derivative (dp/dt_max, mm Hg/s) in the control, ischemic preconditioning, and CGRP groups were 65.2 ± 5.93 and 1,170 ± 119, 94.13 ± 4.93 and 1,825 ± 145.83, and 85.47 ± 4.17 and 1,900 ± 123.13, respectively (p < 0.01). After 8-hour storage, left ventricular pressure (mm Hg) and dp/dt_max (mm Hg/s) in the same groups were 51.07 ± 5.83 and 815 ± 107.17, 83.47 ± 6.54 and 1,480 ± 120.91, and 84.8 ± 8.49 and 1,396 ± 126.16 (p < 0.01). Ischemic preconditioning and CGRP-induced preconditioning also significantly reduced the release of myocardial enzymes.

Conclusions. The present studies suggest that ischemic preconditioning protects against ischemia-reperfusion injury even after 8 hours of hypothermic preservation in isolated rat hearts, and that CGRP exerts preconditioning-like cardioprotection.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Ischemic preconditioning was first described by Murry and colleagues [1] in 1986, and this phenomenon has been confirmed in various animals and in humans [2]. Recently, some researchers have begun to investigate the prolonged preservation of isolated hearts by ischemic preconditioning and hypoxic preconditioning [3, 4].

The mechanism responsible for preconditioning has not been fully elucidated. There is an increasing amount of evidence that endogenous myocardial protective substances may play an important role in ischemic preconditioning, and adenosine or catecholamine can substitute for ischemic preconditioning [5].

Calcitonin gene-related peptide (CGRP) is a principal transmitter of sensory nerves and is present in the heart [6]. Recently we found in the isolated rat heart that CGRP_8–37, a selective CGRP receptor antagonist, abolished the cardioprotection of ischemic preconditioning, which suggests that CGRP may be an endogenous myocardial protective substance [7]. The present study was designed to explore the effect of ischemic or CGRP-induced preconditioning on myocardial salvage after prolonged cardioplegic arrest in the isolated rat heart.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).

Isolated Perfused Heart Preparation
Male Wistar rats weighing 180 to 200 g were anesthetized with ether. The hearts were excised rapidly and immersed in cold Krebs-Henseleit buffer solution (4°C). The aorta was mounted on a cannula attached to a perfusion apparatus. Retrograde reperfusion of the heart was started in the Langendorff mode at a constant perfusion pressure (100 cm H₂O) and a constant temperature (37°C) [8]. The perfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer (in mmol/L: NaCl, 119; NaHCO₃, 25.5; KCl, 4.3; KH₂PO₄, 1.2; MgSO₄, 1.2; CaCl₂, 2.5; and glucose, 11.0) gassed with 95% O₂ and 5% CO₂.

An isovolumic balloon catheter, attached to a pressure transducer, was inserted into the left ventricle through the left atrium. The balloon was then inflated with water to maintain a left ventricular end-diastolic pressure of 2 to 3 mm Hg [9]. Both the left ventricular pressure (LVP) and its first derivative (dp/dt_max) were recorded throughout the experiment by inputting signals to a Nihon Kohden polygraph system. Coronary flow (CF) was measured by timed collection of coronary effluent. The epicardial electrocardiogram was recorded using the Nihon Kohden electrocardiographic recorder, by which the heart rate was analyzed.

Measurement of Myocardial Enzymes
The activity of myocardial enzymes, including aspartate transaminase, lactate dehydrogenase, hydroxybutyrate dehydrogenase, and creatine phosphokinase, in the coronary effluent before ischemia and at 45 minutes of reperfusion was measured spectrophotometrically using an enzymatic method (Hitachi Automatic Analyzer 7170A, Japan). Kits for measurement of enzymes were obtained from Long March Chemical Co (Shanghai, People's Republic of China).

Experimental Protocols
Before any experimental treatment with CGRP or ischemic preconditioning, we measured contractile function, CF, and activities of myocardial enzymes to establish baseline values. Hearts in all groups received a 2-minute infusion of St. Thomas cardioplegic solution (4°C) though a sidearm of the cannula. The St. Thomas cardioplegic solution had the following composition (in mol/L): NaCl, 110; KCl, 16; MgCl₂, 16; CaCl₂, 1.2; and NaHCO₃, 10. The hearts were immersed in cardioplegic solution maintained at 4°C for either 4 or 8 hours, and then were reperfused with Krebs-Henseleit solution for 60 minutes (37°C). In the control group, hearts were allowed to equilibrate for 45 minutes and then were treated as described previously. In the ischemic preconditioning group, hearts were equilibrated for 20 minutes and then were subjected to two cycles of 5 minutes of normothermic ischemia and 10 minutes of reperfusion before treatment with St. Thomas cardioplegia solution. In the CGRP-treated group, hearts were equilibrated for 20 minutes and then subjected to two cycles of 5-minute treatment with CGRP (5 x 10^-9 mol/L) (Sigma Chemical Co, St. Louis, MO) and 10 minutes of CGRP-free Krebs-Henseleit solution before cardioplegic arrest.

Statistical Analysis
All values are expressed as mean ± standard error of the mean. One-way analysis of variance was done first to test for any differences among groups. If significant differences were established, a Tukey test was performed. The level of significance was p less than 0.05.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Effect of Ischemic Preconditioning
There were no significant differences in the basic values of LVP and dp/dt_max, CF, myocardial enzymes among the groups. After hypothermic ischemia for 4 or 8 hours, a decline in cardiac function (LVP and dp/dt_max) and CF and an increase in the release of myocardial enzymes were shown during reperfusion (Table 1). The decreases in the hearts subjected to an 8-hour ischemic period were significantly greater than those with 4-hour ischemia (p < 0.05).

Calcitonin gene-related peptide caused only a slight increase in contractile function (LVP in the absence or presence of CGRP was 72.3 ± 2.7 and 81 ± 2.3 mm Hg, respectively; p < 0.05, n = 12) and vasodilator response (CF in the absence or presence of CGRP was 11.7 ± 0.63 and 13.9 ± 0.49 mL/min, respectively; p < 0.05, n = 12). After the preparation was perfused with CGRP-free Krebs-Henseleit solution for 5 to 10 minutes, the augmented responses to CGRP returned to control levels.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Previous investigations have shown that in isolated, perfused rabbit hearts, ischemic preconditioning can improve preservation with crystalloid cardioplegia [3]. Others have reported that in the isolated rat heart, hypoxic preconditioning enhances functional recovery after prolonged cardioplegic arrest [4]. In the present study, ischemic preconditioning in isolated rat hearts also improved the recovery of cardiac contractile function and reduced the release of myocardial enzymes, even after 8 hours of hypothermic ischemia. Evidence presented here and in past studies suggests that preconditioning has protective effects against ischemia-reperfusion injury in the model of hypothermic storage.

Growing evidence suggests that the release of endogenous protective substances is involved in ischemic preconditioning [5]. Some chemical mediators such as adenosine, nitric oxide, kinins, and prostaglandins have been shown to participate in the cardioprotection of ischemic preconditioning [10]. In particular, extensive research has been performed on mediators released from nerves, and many neurotransmitters such as acetylcholine and norepinephrine are considered to be included in the mechanisms of ischemic preconditioning [11, 12].

It has been demonstrated that capsaicin-sensitive sensory nerves are present in the hearts of animals and humans [6]. Calcitonin gene-related peptide, a principal transmitter in cardiac sensory nerves, possesses numerous physiologic properties, several of which are thought to be beneficial to the ischemic myocardium [13]. Studies have shown that the release of CGRP from cardiac sensory nerves is regulated by various factors. Myocardial ischemia, even for a brief period of 5 minutes, can cause a substantial increase in the release of CGRP in isolated guinea pig hearts [14]. Recently, work in our laboratory showed that CGRP receptor antagonist CGRP_8–37 abolished preconditioning-induced protection in the isolated rat heart [7]. These results suggest that endogenous CGRP may play an important role in mediation of ischemic preconditioning.

A major finding in this study is that CGRP exerts preconditioning-like cardioprotection: It enhanced the recovery of cardiac function, improved CF, and reduced the release of myocardial enzymes. The present study confirms previous observations that pharmacologic preconditioning can mimic ischemic preconditioning and extends these observations to provide evidence that CGRP-induced preconditioning improves myocardial salvage after prolonged hypothermic cardioplegic arrest.

The mechanisms responsible for the cardioprotection of CGRP-induced preconditioning are not clear. Several studies have shown that myocardial protective substances, endogenous or exogenous, offer cardioprotection because of activation of the protein kinase C pathway [5]. It has been shown in adult mammalian ventricular cardiomyocytes that CGRP increases the activation of protein kinase C [15]. Recently, our studies showed that the protective effect of CGRP-induced preconditioning on myocardial damage due to endothelin-1 was negated by H7, an inhibitor of protein kinase C [16]. These results suggest that the cardioprotection of CGRP-induced preconditioning may be due to activation of protein kinase C.

In conclusion, the present study suggests that ischemic preconditioning can improve preservation after prolonged cardioplegic arrest in isolated rat hearts and that CGRP can induce a preconditioning-like cardioprotection.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
This work was supported by a grant from the State Education Commission of the People's Republic of China.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Li, Department of Pharmacology, Hunan Medical University, Changsha, Hunan 410078, People's Republic of China.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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