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Endocrinology, doi:10.1210/en.2008-0472
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Endocrinology Vol. 149, No. 10 5172-5176
Copyright © 2008 by The Endocrine Society

Early Ghrelin Treatment after Myocardial Infarction Prevents an Increase in Cardiac Sympathetic Tone and Reduces Mortality

Daryl O. Schwenke, Takeshi Tokudome, Ichiro Kishimoto, Takeshi Horio, Mikiyasu Shirai, Patricia A. Cragg and Kenji Kangawa

Department of Physiology (D.O.S., P.A.C.), University of Otago, Dunedin 9054, New Zealand; Department of Biochemistry (T.T., I.K., T.H., K.K.), National Cardiovascular Center Research Institute, Osaka 565-8565, Japan; and Faculty of Health Sciences (M.S.), Hiroshima International University, Hiroshima City, Hiroshima Prefecture 730-0016, Japan

Address all correspondence and requests for reprints to: Ichiro Kishimoto, Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: kishimot{at}ri.ncvc.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Acute myocardial infarction (MI) initiates an increase in cardiac sympathetic nerve activity (CSNA), which ultimately exacerbates chronic cardiac dysfunction. Ghrelin (Ghr), a GH-releasing peptide, is an effective treatment for improving cardiac function in chronic heart failure. Ghr also suppresses renal sympathetic nerve activity (SNA) and, therefore, may have important therapeutic benefits in the early stages of acute MI: by reducing CSNA. In this study we hypothesized that early Ghr administration may prevent an increase in CSNA in the acute phase after MI. CSNA was continuously recorded in urethane-anaesthetized rats before and for 5 h after acute MI (or sham). MI was induced by ligation of the left anterior descending coronary artery. Rats received an injection of either saline or Ghr (150 µg/kg, sc) 1 min, or 2 h, after the infarct. CSNA remained stable during the 5-h recording duration in sham rats. MI induced a maximal 110% increase in SNA, which was prevented in rats that received Ghr 1 min after infarct. When Ghr was injected 2 h after MI (SNA had increased by ~85%), SNA decreased to pre-MI activity. Importantly, early Ghr administration significantly reduced the high mortality rate associated with MI (61% mortality in untreated MI rats cf. ~23% in Ghr-treated MI rats). These results show that early Ghr treatment prevents the increase in CSNA after MI, which may contribute to the improved chances of survival. Whether these early beneficial effects of Ghr also have long-term benefits for improving cardiac function is an area that requires further investigation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MYOCARDIAL infarction (MI) is the most common cause of death in industrialized societies. In most instances, death occurs within the early stage, i.e. the first few hours, after MI. This high morbidity has been strongly associated with an adverse and sustained increase in cardiac sympathetic nerve activity (CSNA), which begins within the first hour of the initial infarction (1). Even for those that survive the immediate infarct, the adverse damage to cardiac tissue impairs the functional capacity of the heart, which is further exacerbated by the sustained increase in sympathetic tone, such that the long-term survival prognosis is bleak.

The peptide hormone ghrelin (Ghr), first discovered in 1999 (2), has improved cardiac function in patients suffering from end-stage chronic heart failure (3). Ghr is a GH-releasing peptide, and so the mechanism by which Ghr improves cardiac function has been linked, at least in part, to the anabolic properties of GH. To date, there is a paucity of studies describing the treatment of myocardial ischemia within the first few hours of onset. Indeed, it is this time period when autonomic modulation of cardiac function is enhanced, in which the opportunity to improve outcome by therapeutic intervention is so great.

Some studies have shown that Ghr is able to centrally suppress renal sympathetic nerve activity (SNA) (4, 5). More recently, we have also shown, using heart-rate spectral analyses, that Ghr treatment appears to attenuate cardiac sympathetic tone within the first week after MI (6). However, it is the initial increase in CSNA within the first hours after MI that significantly contributes to ventricular arrhythmia (1, 7) and, consequently, a high mortality (8). Therefore, in this study we hypothesized, and aimed to show, that the early administration of Ghr immediately after MI would be able to prevent, or at least attenuate, the early increase in CSNA, which could potentially improve early survival prognosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Experiments were conducted on 41 male Sprague Dawley rats (8 wk old; body weight ~280–340 g). All rats were on a 12-h light, 12-h dark cycle at 25 ± 1 C, and provided with food and water ad libitum. All experiments were approved and conducted in accordance with the guidelines stipulated by the Animal Ethics Committee of the University of Otago, New Zealand.

Anesthesia and surgical preparation
Rats were anesthetized with urethane (1.5 g/kg, ip). Adequate anesthesia was confirmed by elimination of the limb withdrawal reflex. Throughout the experiment, body temperature was maintained at 38 C using a rectal thermistor coupled with a thermostatically controlled heating pad. The trachea was cannulated, and the lungs were ventilated with a Harvard rodent ventilator (model 680; Harvard Apparatus, Holliston, MA). The inspirate gas was enriched with O2 (~50% O2), and the ventilator settings were adjusted (tidal volume ~3.5 ml; breathing rate ~80/min) to maintain arterial PCO2 normocapnic. The femoral artery and vein were cannulated for measurement of systemic arterial blood pressure (ABP) and fluid administration (saline at 3 ml/h), respectively. The arterial line contained heparinized saline (50 U/ml).

Recording CSNA
The stellate ganglion was exposed through a left thoracotomy between the first and second rib. The cardiac sympathetic nerve was identified as a branch from the stellate ganglion, dissected free of surrounding connective tissue, sectioned, and the proximal section (containing efferent fibers) was placed on a pair of platinum recording electrodes. The signal was filtered (low cutoff 0.1 kHz; high cutoff 1 kHz;) and amplified, and subsequently passed through an amplitude discriminator (model WD-2, Dagan Corp., Minneapolis, MN) for counting nerve discharge frequency (impulse frequency).

Raw SNA, impulse frequency, and ABP were continuously sampled at 4 KHz, 200 Hz, and 400 Hz, respectively, using a PowerLab data-acquisition system (model 8/S; ADInstruments Pty Ltd., Bella Vista New South Wales, Australia). Heart rate (HR) was derived from the arterial systolic peaks. The raw nerve signal was rectified and integrated (1-sec resetting interval) online, and the integrated nerve signal was displayed in real time.

Experimental protocol
A 7.0-Prolene suture (Ethicon, Inc., Johnson & Johnson, Somerville, NJ) was loosely placed around the left anterior descending (LAD) coronary artery, which was located between the appendage of the left atrium and the base of the pulmonary artery. CSNA, and mean ABP (MABP) and HR were continuously recorded before occlusion of the LAD coronary artery, and for 5 consecutive hours after: 1) no manipulation (sham, n = 7); 2) LAD occlusion (MI, n = 13); and 3) MI with an immediate injection of Ghr (150 µg/kg, sc) (MI plus Ghr, n = 13). We also aimed to assess whether Ghr could reduce sympathetic tone after it had already increased after MI (to simulate those patients that receive delayed therapeutic treatment). Therefore, in this study we also tested a group of rats that received Ghr 2 h after MI (MI plus delayed Ghr, n = 8). Ghr was obtained from the Peptide Institute, Inc. (Osaka, Japan).

Measurement of infarct size
At the completion of each experiment, each rat was euthanized, and the heart was excised and sectioned into 2-mm horizontal slices down the vertical plane. The sections were then stained with 2,3,5-triphenyltetrazolium solution (Sigma-Aldrich Corp., St. Louis, MO) and subsequently fixed in 10% formalin for 20 min. Slices were mounted and photographed. Total infarct size was determined by measuring the area of the infarction for each slice, multiplying the area by the slice thickness, and summing the area of all slices. Infarct size was presented as a percentage of the total left ventricular wall.

Statistical analysis
All statistical analyses were conducted using StatView (version 5.01; SAS Institute Inc., Cary, NC). All results are presented as means ± SEM. Two-way ANOVA (repeated measures) was used to test significance for temporal changes in CSNA after LAD occlusion. One-way ANOVA (factorial) was used to test for differences among the groups of rats. Where statistical significance was reached, post hoc analyses were incorporated using the paired or unpaired t test with the Dunnet’s correction for multiple comparisons. The Kaplan-Meier survival analysis was performed to compare survival curves between saline-treated and Ghr-treated rats after LAD occlusion. A P value less than or equal to 0.05 was predetermined as the level of significance for all statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Survival
Eight out of 13 MI rats (61% mortality) died within 6 h after MI, compared with 23% of those MI plus Ghr rats (three out of 13 rats; P = 0.039; Fig. 1Go). Of the eight MI rats that died, three died after the fifth hour. Rats treated with Ghr 2 h after infarction had a mortality of 25% (two of eight rats); one death occurring after 5 h recording. Therefore, we were able to collect 5 h CSNA data from seven rats for each group.


Figure 1
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  		FIG. 1. Kaplan-Meier survival analysis showing a greater mortality rate in untreated MI rats (n = 13) compared with Ghr-treated rats (MI plus Ghr; n = 13) within 6 h after LAD occlusion (P = 0.0399).

 
Arrhythmias
Cardiac arrhythmias were evident within the first few minutes of LAD occlusion for all MI rats (treated with or without Ghr). As illustrated in Fig. 2AGo, arrhythmic episodes often consisted of numerous ectopic beats occurring in succession, followed by the return of a "normal" cardiac rhythm. In untreated MI rats, the arrhythmic episodes often persisted for up to 5 h recording, although the incidence of episodes became less frequent with time (Fig. 2BGo), and there were less ectopic beats per episode. For all MI rats that died within 5 h after MI (n = 5), the cause of death was due to sudden cardiac arrest after an arrhythmic insult. Of the three rats that died after the fifth hour after MI, one died of sudden cardiac arrest after an arrhythmic insult, whereas critical hypotension (MABP ~30 mm Hg) after the fifth hour after MI preempted the death of the remaining two rats.


Figure 2
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  		FIG. 2. "Chart" recording showing a severe example of arrhythmias immediately after LAD occlusion (this rat subsequently died of cardiac arrest ~2 h after MI) (A), and the incidence of arrhythmic episodes at each time interval period (i.e. 0–5 h) after MI (B). {dagger}, Significant difference between MI (n = 7) and MI plus Ghr rats (n = 6) (P < 0.05). Imp, Impulse frequency; LAD, left anterior descending artery.

 
The incidence of arrhythmias within the first hour after MI for MI plus Ghr rats was similar to untreated MI rats, although in the ensuing hours, MI plus Ghr rats had fewer arrhythmic insults, significant by the second to third hour after MI (Fig. 2BGo). Indeed, in all but two of the MI plus Ghr rats, the incidence of arrhythmias had subsided from the third hour after MI. The persistence of arrhythmias in one MI plus Ghr rat ultimately resulted in cardiac arrest and death. The cause of death in the remaining two MI plus Ghr rats that died prematurely was linked to critical hypotension.

CSNA
Typical chart recordings of hemodynamic and CSNA data before and 5 h after either MI or MI plus Ghr are presented in Fig. 3Go. Baseline cardiovascular and CSNA data are presented in Table 1Go. In sham rats (i.e. control), CSNA remained stable for the 5-h recording period. MI rats elicited a significant increase in CSNA (110 ± 27%), which was completely prevented in those rats that also immediately received Ghr after MI (i.e. MI plus Ghr) (Fig. 4Go). Moreover, when Ghr was administered 2 h after MI (when CSNA had increased by 85 ± 23%), CSNA declined to pre-MI activity by the fifth hour of recording. MABP and HR did not significantly change in sham or MI plus Ghr rats. MI rats experienced a mild 12% decrease in MABP ({Delta}MABP 9 ± 3 mm Hg) and a 15% increase in HR ({Delta}HR 55 ± 24 beats/min; not significant). The hypotension was more pronounced in MI plus delayed Ghr rats ({Delta}MABP 14 ± 3 mm Hg), although HR did not increase.


Figure 3
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  		FIG. 3. Chart recordings showing "typical" changes in hemodynamic variables, and CSNA-integrated signal of the raw trace and burst frequency [impulse frequency (Imp) per second] in a MI rat (top trace) and MI plus Ghr rat (bottom trace) 5 h after MI.

 

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  		TABLE 1. Baseline (before MI) CSNA (integrated signal and impulse frequency/sec), MABP, and HR of four groups of rats: control (n = 7); MI (n = 7); MI rats immediately treated with Ghr (150 µg/kg, iv) (MI plus Ghr, n = 7); and rats that received Ghr 2 h after the (MI MI plus delayed Ghr, n = 7)

 

Figure 4
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  		FIG. 4. Transient responses in CSNA (percent increase in CSNA of integrated area of the raw nerve signal), MABP (mm Hg), and HR (beats per minute) in sham rats (n = 7) and three groups of MI rats: untreated (MI; n = 7); Ghr treated immediately after MI (MI plus Ghr; n = 7); and Ghr treated 2 h after MI (MI plus delayed Ghr; n = 7). *, Significantly different from before MI (time "0") (P < 0.05).

 
Infarct size
The size of the infarct of the left ventricular wall did not significantly differ among MI (40.0 ± 2.8%), MI plus Ghr (38.3 ± 1.6%), and MI plus delayed Ghr rats (42.4 ± 3.3%).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary findings of this study highlight the important benefits of early Ghr intervention in preventing the adverse increase in CSNA after acute MI, as well as improving early survival prognosis. Although the therapeutic effects of Ghr have largely been attributed to the release of GH (3, 9), evidence is accumulating that supports a direct cardioprotective effect of Ghr through the central modulation of SNA (4, 5, 6).

In this study we reported that MI initiated an increase in CSNA, which is consistent with previous anesthetized animal models (10, 11, 12). More recently, Jardine et al. (1) described a transient increase in CSNA in conscious sheep: significant by the second hour after MI, which was sustained for at least 7 d. We also observed a rapid and sustained increase in CSNA, although, one limitation of this study is that we could only record CSNA for 5 h in an anesthetized, open-chest, rat model.

The mechanisms for the increase in CSNA after MI remain to be elucidated fully, although altered neural reflexes (e.g. from baroreceptors and chemoreceptors), increased levels of hormones (e.g. angiotensin II), and changes in central mechanisms that may amplify the responses to these inputs have been implicated (13). Although the increase in CSNA after MI appears to have immediate benefits, providing inotropic support to the heart to maintain cardiac output, this enhanced sympathetic tone is associated with an increased risk of ventricular arrhythmias (1), a leading cause for sudden heart failure and death (8). Indeed, we noted in our study that ventricular arrhythmias often preceded, or even instigated, cardiac failure and sudden death.

Early Ghr treatment after MI prevented the adverse increase in CSNA within the first 5 h of recording, and reduced the incidence of arrhythmias. Further research is required to reveal the exact mechanism(s) by which Ghr suppresses CSNA after MI, although studies have shown that the receptor for Ghr [GH secretagogue receptor (GHS-R)] is located in the main cardiovascular control centers in neurons of the nucleus tractus solitarius (NTS) (5), and that the central administration of Ghr directly attenuates renal SNA (4, 5).

We have also demonstrated the existence of GHS-R in the infarcted myocardium (6). Costaining with acetylcholine esterase suggests that the GHS-R is localized in the vagal nerve terminals in the heart, which send afferent projections to the NTS, so that Ghr may act to enhance vagal tone and thereby decrease SNA.

An enhanced vagal tone has also been reported to centrally augment baroreflex control of CSNA (14). Yet, our results imply that Ghr may have reduced baroreflex sensitivity, at least in MI plus delayed Ghr rats, because we observed that CSNA was not elevated above baseline (5 h after MI), despite a 17% decrease in MABP. Matsumura et al. (4) similarly reported that Ghr (iv) reduced MABP without changing renal SNA, but further showed that centrally administered Ghr did decrease SNA, HR, and MABP, and enhance baroreflex sensitivity. They reasoned that iv Ghr has a direct peripheral vasodilatory effect as well as a direct central sympathoinhibitory effect.

Therefore, in this study it is possible that peripheral Ghr administration prevented the increase in CSNA in the acute phase after MI, at least in part, by suppressing SNA directly at the level of the NTS (which would also prevent a baroreflex increase in SNA in response to the vasodilatory effects of Ghr), and indirectly through activation of cardiac vagal afferent nerves. Of course Ghr, which can cross the blood-brain barrier (15), has diverse effects both peripherally and within the central nervous system, and, thus, it is likely that Ghr could modulate CSNA at sites other than the NTS. This is an area of research that warrants further investigation.

In this study, untreated MI rats had a survival rate of only 39%, compared with a 77% survival rate for MI rats treated with Ghr (MI plus Ghr). The observed difference between the two groups of MI rats is likely linked to the fact that Ghr prevented an increase in CSNA in treated rats. Indeed, it may be reasonable to suggest that this benefit of early Ghr treatment contributes, at least in part, to the improved survival prognosis of MI plus Ghr rats (survival of 77%), especially given that arrhythmia-related deaths were less prevalent in MI plus Ghr rats compared with MI rats.

In reality, it may not be possible for all MI patients to receive immediate treatment (i.e. within minutes), and because neurohumoral changes often precede the development of clinically recognizable symptoms of acute heart failure (13), treatment may be delayed by several hours. Yet, it is within this time interval that CSNA has already begun to increase (1). In this study we were able to demonstrate that a 2-h delayed treatment of Ghr was able to reduce the MI-induced increase in CSNA. Furthermore, the survival rate of these rats (75%) was improved compared with untreated rats.

Collectively, the results of this study appear to indicate that early Ghr treatment, at least within the first hours after MI, may improve early survival prognosis, providing clinicians with critical time for implementing supplementary therapeutic measures. Furthermore, this benefit of Ghr is likely associated with the prevention or attenuation of an enhanced cardiac sympathetic drive. Whether these early beneficial effects of Ghr also have long-term benefits for improving cardiac function, and ultimately long-term survival, is an important area that urgently requires further investigation.


   Footnotes
 
This study was supported by the Department of Physiology, University of Otago, New Zealand, and, in part, by the research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation of Japan, and the Takeda Scientific Foundation.

Disclosure Summary: The authors have nothing to disclose. There are no conflicts of interest concerning the material in this study.

First Published Online July 3, 2008

Abbreviations: ABP, Arterial blood pressure; CSNA, cardiac sympathetic nerve activity; Ghr, ghrelin; GHS-R, GH secretagogue receptor; HR, heart rate; LAD, left anterior descending; MABP, mean arterial blood pressure; MI, myocardial infarction; NTS, nucleus tractus solitarius; SNA, sympathetic nerve activity.

Received April 3, 2008.

Accepted for publication June 23, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jardine DL, Charles CJ, Ashton RK, Bennett SI, Whitehead M, Frampton CM, Nicholls MG 2005 Increased cardiac sympathetic nerve activity following acute myocardial infarction in a sheep model. J Physiol 565(Pt 1):325–333
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  5. Lin Y, Matsumura K, Fukuhara M, Kagiyama S, Fujii K, Iida M 2004 Ghrelin acts at the nucleus of the solitary tract to decrease arterial pressure in rats. Hypertension 43:977–982[Abstract/Free Full Text]
  6. Soeki T, Kishimoto I, Schwenke DO, Tokudome T, Horio T, Yoshida M, Hosoda H, Kangawa K 2008 Ghrelin suppresses cardiac sympathetic activity and prevents early left ventricular remodeling in rats with myocardial infarction. Am J Physiol Heart Circ Physiol 294:H426–H432
  7. Keating MT, Sanguinetti MC 2001 Molecular and cellular mechanisms of cardiac arrhythmias. Cell 104:569–580[CrossRef][Medline]
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