Changes of the Plasma Atrial Natriuretic Peptide duringMyocardial Infarction in Rats

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Changes of the Plasma Atrial Natriuretic Peptide during Myocardial Infarction in Rats

Dong-Choon Ahn and In-Shik Kim¹

Department of Veterinary Anatomy, College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju 561-756, Korea

(Accepted: February 09, 2012)

Abstract : Atrial natriuretic peptide (ANP) is associated with the variety of disorders of myocardial function. The effect, however, of myocardial infarction (MI) on ANP has not been fully described. Thus, this study investigated the effect of experimental MI, induced by left coronary artery ligation, on ANP secretion. Male Sprague-Dawley rats aged 60 d underwent ligation of the left anterior descending coronary artery to induce MI and were compared with a group that underwent a sham operation. Rats of sham operation had a similar procedure without having the suture tightened around the coronary artery. Animals were sacrificed at 1, 3, 6, 12, and 18 h or 1, 3, 5, 7, 14, and 30 d after the procedure. MI size was assessed by planimetry and perimetry, and plasma ANP levels were determined by radioimmunoassay. Mean infarct size was 39.6-44.5% of the left ventricle after coronary occlusion in experimental groups. No significant differences were observed in infarct size among groups. In contrast, the concentration of plasma ANP was significantly higher at 1, 3, 6, 12, 18, and 24 h after left coronary artery ligation than in sham animals.

This parameter, however, did not differ significantly at 3, 5, 7, 14, and 30 d after ligation compared with sham-ligated controls. These results demonstrate that plasma ANP levels are markedly increased in the early phase of MI in the male rat and can be a useful biomarker for diagnosis in acute MI.

Key words : Myocardial infarction, ANP, rat, coronary artery ligation.

Introduction

The natriuretic peptide family of hormones contributes to the control of body fluid homeostasis and blood pressure regulation through its combined actions on vasculature, kidneys, and adrenal glands (10). The three best-known members of this family are atrial natriuretic peptide (ANP) (9), brain natriuretic peptide (BNP) (24), and C-type natriuretic peptide (CNP) (25). ANP and BNP are produced predominantly by the cardiac atria and ventricles, respectively, in response to increased atrial and ventricular transmural pressure (15,17).

ANP has potent diuretic, natriuretic, and vasorelaxation properties, and is probably involved in the regulation of fluid balance and hemodynamics. It is secreted into plasma at pico- molar concentrations, predominantly from atrial myocytes in response to local wall stretch and increased atrial pressure.

ANP affects the renin-angiotension system by acting on the adrenal glomerulosa (13,16) to inhibit the stimulated secretion of renin and directly inhibit the adrenal secretion of aldosterone. ANP also increases renal blood flow by acting on the glomerular apparatus, increases afferent vessel diameter, de- creases efferent vessel diameter, and increases glomerular sur-

face area by the inhibition of mesangial cell concentration.

These actions increase the glomerular filtration rate, which ultimately results in augmented diuresis and natriuresis (14, 22); increased vascular permeability, facilitating the move- ment of plasma from blood capillaries to the interstitial fluid (32); and relaxation of the vascular smooth muscle, resulting in a reduction in blood pressure (22).

Plasma ANP concentrations are elevated in dogs with mitral regurgitation (11), heart worm disease (27) and congestive heart failure (31), and may be secondary to increased atrial stretch (10). Plasma ANP level was suggested to be an biochemical marker for cardiac decompensation but not left atrial and left ventricular enlargement (11). Recent studies have demon- strated that the cardiac atria are deficient in ANP in Syrian hamsters with cardiomyopathy or rats with myocardial infarction (MI) (5,29,30). However, the effect of MI on ANP has not been fully described and in this study was examined in rats with MI experimentally induced by left coronary artery ligation.

Materials and Methods

Experimental animals

Male Sprague-Dawley rats weighing 293 ± 15.8 g were pur- chased from Dae Han Experimental Animal Co., Ltd. (Chun- gju, Korea). They were maintained under conditions of con- trolled temperature (25^oC) and lighting (14L : 10D). All ani-

1Corresponding author.

E-mail: iskim@jbnu.ac.kr

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mals were fed normal rat chow and had access to water ad libitum. This study was carried out in accordance with the Chonbuk National University Animal Experimental Guide- lines.

Induction of myocardial infarction

Rats were divided into 11 groups of eight rats for analysis at 1, 3, 6, 12, and 18 h and 1, 3, 5, 7, 14, and 30 d after left coronary artery ligation. Sham operated animals were included in groups analyzed at each time point. Animals were anesthe- tized and maintained under anesthesia with intraperitoneal ket- amine hydrochloride (80 mg/kg) and xylazine (50 mg/kg). A 16-gauge catheter (Abbocath; Abbott Korea Ltd., Seoul, Korea) was inserted into the trachea through the oral cavity and connected to a rodent ventilator (Harvard Apparatus, Hollis- ton, MA, USA). Artificial ventilation (room air, 80-90 strokes/

min, 20 ml·kg-1·stroke-1) was provided throughout the procedure, which was conducted under aseptic conditions. The tho- racic cavity was opened through the left fourth intercostal space, and the heart was exposed in lateral oblique view. Iden- tification of the left main descending coronary artery (LMDA) was assisted by a controlling illuminator. To induce a large MI, a 7-0 polypropylene ligature was placed 0.9-2.1 mm to the left of the LMDA origin. The origin of the LMDA was detected after the auricle of the left atrium was elevated using fine, smooth-tipped forceps. The ligature was placed and tied provisionally for observation of the change in color of an appropriate area of the myocardium. The ligature was then placed and tied around the LMDA at the level of the tip of the auricle, similar to previous reports (1,2,20,26). Proper place- ment of the ligature was confirmed by observing the change in color on a large area of the left ventricle (LV), and the tho- racic wall was closed with layered sutures. In rats that underwent the sham procedure, the suture was not tightened around the coronary artery. The animals were removed from artificial ventilation and placed in a warm cage until recovery, and were maintained on a normal diet.

Sample preparation

Animals were sacrificed by decapitation after cervical dis- location at each time point. The arterial blood was collected for measurement of plasma ANP levels. Hearts were har- vested and fixed in 10% neutral buffered formalin solution for histological examination.

Estimation of myocardial infarction size

Different histological techniques were necessary to detect the extent of necrotic myocardium or scar tissue after coronary artery ligation. For time points ≤3d after ligation, 1-mm heart sections were stained with 1% triphenyl tetrazolium chloride (TTC) (1,2,20,26). For time points > 3d, hearts were fixed and embedded in paraffin blocks. They were sectioned (6µm) at regular intervals of 600 µm for a total of 712 sections and stained with hematoxylin and eosin (H & E). In some cases, sections were stained for collagen using Masson's

trichrome (MT). The MI size for each section was calculated as the ratio of the infarcted segment to the total LV perimeter, averaged between exterior and interior measurements, using the Image Lab program (Leica Q 500 MC, Germany), and the average MI of all sections was expressed as a percentage of total LV perimeter (1,2,20,26).

Plasma preparation

To measure plasma ANP concentration, the arterial blood was collected into pre-chilled tubes containing ethylenedi- aminetetraacetic acid (EDTA, 1 mg/ml of whole blood), phe- nylmethylsulfonyl fluoride (PMSF, 0.4 mM), and soybean trypsin inhibitor (50 mM Na-benzoyl-L-arginine ethyl ester/ml).

Plasma samples were obtained after centrifugation.

Preparation of ¹²⁵I-labeled atrial natriuretic peptide

125I-ANP was prepared as described previously (6). Synthetic ANP (5µg/5 µl in 0.1 M acetic acid; Peninsula Laboratories, Belmont, CA, USA) was added to a vial containing 25µl of 0.5 M phosphate-buffered saline (pH 7.4), followed by the addi- tion of 1 mCi of ¹²⁵I-Na (Amersham International, Bucking- hamshire, UK). Chloramine-T (10µg/10 µl) was added to the reaction vial, mixed gently, and bovine serum albumin (BSA, 60 mg/200µl) was added 30 s later. The reaction mixture was immediately applied to a Sephadex G-25 column (1.0× 24 cm) and ¹²⁵I-ANP was eluted with 0.1 M acetic acid containing 0.3% BSA, 0.3% lysozyme, 0.1% glycine, and 200 KIU/

ml aprotinin. The iodinated ANP was divided and stored at

−70°C until use. Immediately before use, the ¹²⁵I-ANP was re- purified by high-performance liquid chromatography on a reversed-phase Bondapak C18 column (Waters Associates, Milford, MA, USA) with a linear gradient elution (0-60% ace- tonitrile). The specific activity (~2,000 Ci/mM) of ¹²⁵I-ANP was determined by radioimmunoassay (8). An ANP-specific antibody was prepared from New Zealand White rabbits using the carbodiimide method described previously (7).

Statistical analysis

Significant differences between the means of different groups were determined by analysis of variance (ANOVA), followed by Duncan's test. Significant differences among the mean values of sham ligation and coronary artery ligation rats within each group were determined by a Student’s t-tests. P values < 0.05 were considered statistically significant.

Results

All the animals had a quick and uneventful recovery follow- ing surgery. MI was evaluated by histochemical staining with TTC and MT. In normal rat myocardium, a small amount of collagen was found in the interstitial and perivascular spaces.

After MI, collagen accumulation was first seen at the MI site on day 3 and became more advanced thereafter. Fig 2 illustrates substantial collagen accumulation at an MI site in a myofibroblast (MyoFb) layer surrounding necrotic myocar-

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dium at 7 d after coronary artery ligation. Collagen accumu- lated continually over time and completely replaced necrotic

myocytes on day 30. Collagen deposition was also observed in non-infarcted myocardium on days 7, 14, and 30 after MI, as well as in the pericardium of sham-operated rats and rats with infarcted hearts. Collagen volume in these animals was less than in those with MI (Figs 1-2). Coronary artery ligation resulted in MIs encompassing 39.6-44.5% of the heart wall circumference. No significant difference was observed in infarct size among groups (Fig 3)

The time course of changes in ANP levels after coronary artery or sham ligation is shown in Fig 4 and 5. The sham- operated control animals showed the same values as obtained in normal, unoperated rats in our laboratory (data not shown).

ANP levels were significantly higher at 1, 3, 6, 12, 18 and 24 h after coronary artery ligation than in sham animals (Fig 4);

however, no difference was observed in this parameter at 3, 5, 7, 14, and 30 d after coronary artery ligation in comparison with sham ligation rats (Fig 5).

Fig 1. Evan’s blue-triphenyltetrazolium chloride (TTC) staining illustrates myocardial infarction size 18 h after ligation of the left main descending coronary artery. White; the nfarction areas, Red;

the non-infarcted areas.

Fig 3. Myocardial infarction size at 1, 3, 5, 7, 14 and 30 d after coronary artery ligation. Values are means ± SEM.

Fig 2. Representative light micrographs of sham-operated rats (a) and myocardial infarct rats at different time points from 1 day (b), 3 days (c), 7 days (d), 14 days (e) and 30 days (f). All figures were of the same magnification (× 50). Masson’s Trichrome stain.

Fig 4. Immunoreactive atrial natriuretic peptide (ANP) concentrations at 1, 3, 6, 12 and 18 h after coronary artery ligation. Values are means ± SEM. Significantly different from sham: ^*P < 0.05.

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Discussion

ANP levels are increased in patients with congestive heart failure (3,23) and in Syrian hamsters with congestive cardiomyopathy (5). Vollmar et al. (31) showed that plasma ANP levels were increased in the dog with renal failure, congestive heart failure, and hyperadrenocorticism. Dogs with chronic renal failure had twice the ANP concentrations of control dogs, while those dogs suffering from congestive heart failure had a 6-fold increase in plasma ANP levels compared to healthy dogs (31). In this study, the concentration of plasma ANP was significantly higher at 1, 3, 6, 12, 18, and 24 h after left coronary artery ligation than in sham animals. This parameter, however, did not differ significantly at 3, 5, 7, 14, and 30 d after ligation compared with sham-ligated controls. The present study is the only study to date that has evaluated serial changes in plasma ANP levels after an MI during a time course correlating with that of chronic heart failure.

This study observed a marked elevation of circulating ANP in the early phase of MI caused by coronary ligation in rats.

ANP levels were highest in rats with the largest MI and the most severe cardiac dysfunction. This is the demonstration of increased ANP concentration in this type of experimental heart failure. Moreover, these data show that plasma ANP increased in parallel with both MI size and signs of heart failure in rats. Rector et al. (21) reported decreased atrial ANP content in rats with heart infarction due to coronary artery ligation. They suggested that reduced atrial ANP could partly explain the impaired ability of such rats to excrete a saline load (12), resulting in the sodium retention often present in clinical heart failure. However, the researchers did not measure plasma ANP levels. The high plasma ANP levels in rats with experimental heart failure observed in the present study support the hypothesis that reduced atrial ANP content and increased circulating ANP levels are due simply to the increased release of ANP from the overloaded atria. In fact, oth- ers have reported decreased atrial ANP concentrations when

plasma ANP concentrations were high and vice versa (18).

Thus, rats with experimental heart failure are not deficient in circulating ANP. On the contrary, plasma ANP levels rise in parallel with the increased severity of cardiac failure. Similar to patients with severe congestive heart failure, who do not respond to high plasma ANP levels with natriuresis and diuresis (27), rats with experimental heart failure appear resistant to endogenous ANP. Whether this is due to target-organ insensi- tivity, alterations in receptor functions, or reduced renal blood flow is worthy of further exploration.

Increased atrial stretch is considered the major stimulus for ANP release (4,19). Various experimental models have been used to investigate the role of ANP in heart failure. Chimos- key et al. (5) reported that hamsters with familial cardiomyopathy and congestive heart failure showed a decreased atrial ANP content, but plasma levels were not measured. Metzler et al. (17) found that, in conscious dogs, an acute increase in cardiac filling pressure caused by constriction of the ascending aorta resulted in enhanced release of ANP. Those data are in agreement with the present results, suggesting that increased atrial filling pressure releases ANP into circulation in congestive heart failure (3). We did not measure atrial filling pressures in this study. Using the same heart failure model reported here, Pfeffer et al. (20) found a direct relationship between infarct size and ventricular performance. After coronary artery ligation, the impairment of LV function was directly related to the loss of myocardium.

The above-reported results are also consistent with evidence from human studies showing increased plasma ANP concentration in patients with congestive heart failure. Plasma ANP levels are in good correlation both with the severity of heart failure, as assessed by New York Heart Association (NYHA) functional class (28), and with the increase in cardiac filling pressures measured directly (3). A direct correlation of plasma ANP concentration with atrial stretch has also been demon- strated in cardiac patients undergoing elective coronary bypass surgery. Thus, the measurement of plasma ANP may prove to be a sensitive indicator of cardiac status in patients with cardiac dysfunction (28). The heart failure model is a useful tool to study the role of ANP in the pathophysiology of congestive heart failure due to MI, and ANP may be a useful biomarker in the diagnosis of acute MI.

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Fig 5. Immunoreactive atrial natriuretic peptide (ANP) concentrations at 1, 3, 5, 7, 14 and 30 d after coronary artery ligation.

Values are means ± SEM. Significantly different from sham:

*P < 0.05

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흰쥐 심근경색 모델에서 혈장 Atrial Natriuretic Peptide의 변화

안동춘·김인식¹

전북대학교 생체안전성연구소, 수의과대학 해부학교실

요 약 : Atrial natriuretic peptide (ANP)은 여러 가지 다양한 심근질환과 연관성이 있는 것으로 알려져 있으나 심근경 색에서 ANP의 변화에 대해서는 명확하게 밝혀져 있지 않다. 따라서 본 연구에서는 흰쥐 심근경색 모델을 활용하여 혈장내 ANP의 변화를 살펴보아 심근경색에서 ANP의 역할을 규명해보고자 하였다. 수컷 흰쥐 60일령에서 왼심장동 맥을 결찰 하여 심근경색 모델을 만들었고 개흉하여 sham 대조군을 삼았다. 각각의 실험군과 대조군을 수술 후 1, 3, 6, 12, 18시간과 1, 3, 5, 7, 14 및 30일에 희생시켜 실험에 사용하였다. 심근경색의 크기는 planimetry와 perimetry법 을 적용하여 측정하였고 혈장내 ANP 농도는 방사면역측정법을 적용하여 측정하였다. 왼심장동맥을 결찰한 실험군에 서 평균 심근경색의 크기는 왼심실의 39.6-44.5%이었고 유의성 있는 차이는 없었다. 혈장내 ANP 농도는 심근경색 후 1, 3, 6, 12, 18 및 24시간에 대조군에 비하여 명확하게 증가하였으나 3, 5, 7, 14 및 30일령에서 ANP 농도는 대조 군에 비하여 유의성 있는 차이를 나타내지 않았다. 이러한 결과는 수컷 흰쥐에서 혈장내 ANP 농도는 심근경색 초기 에 명확하게 증가함을 입증하였고 급성심근경색의 진단을 위한 생체표지인자로 이용할 수 있는 가능성을 제시해주고 있다.

주요어 : 심근경색, ANP, 흰쥐, 심장동맥결찰