Original Article

Effects of Lacidipine, Ramipril and Valsartan on Serum BNP Levels in Acute and Chronic Periods Following Isoproterenol-Induced Myocardial Infarction in Rats

Lasidipin, Ramipril ve Valsartanın Sıçanlarda Isoprotrenol ile Uyarılan Miyokard Infarktüsü Sonrası Akut ve Kronik Periyotta Serum BNP Seviyeleri Üzerine Etkileri

Yasin Bayir1, Elif Cadirci2, Halis Suleyman2, Zekai Halici2, Mevlut Sait Keles3

1Ataturk University, Faculty of Pharmacy, Department of Biochemistry, Erzurum, Turkey 2Ataturk University, Faculty of Medicine, Department of Pharmacology, Erzurum, Turkey 3Ataturk University, Faculty of Medicine, Department of Biochemistry, Erzurum, Turkey

Correspondence to: Elif Cadirci, Ph.D., Atatürk University, Faculty of Medicine, Department of Pharmacology, 25240, Erzurum, Turkey. Phone: +90.442.2316563, Fax: +90.442.2360968, e-mail: [email protected]

Abstract Özet

Objective. Myocardial infarction (MI) as a result of cardiovas- Amaç. Miyokard infarktüsü (Mİ) hem gelişmiş hem de cular disease is the principal cause of death in both developed gelişmekte olan ülkelerde kardiyovasküler hastalıklar sonucu and developing countries. Brain natriuretic peptide (BNP) is an oluşan ölümün esas sebebidir. Brain natriuretic peptide (BNP) important marker of cardiac failure. Cardioprotective activities kardiyak hasarın önemli bir göstergesidir. Antihipertansif il- of the antihypertensive drugs lacidipine (LAC), ramipril (RAM) açlar olan lasidipin (LAC), ramipril (RAM) ve valsartanın, izo- and valsartan (VAL), against isoproterenol (ISO)-induced MI, proterenol (ISO) ile indüklenen Mİ üzerinde, koruyucu etkileri have been determined. However, the levels of BNP, an indica- belirlenmiştir. Ancak sol ventrikül hasarının bir göstergesi tor of left ventricular failure, have not been evaluated. olan BNP seviyeleri değerlendirilmemiştir.

Materials and Methods. This study investigated the effects Gereç ve Yöntem. Bu çalışmada LAC, RAM ve VAL’ın serum of LAC, RAM and VAL on serum BNP levels in acute and chron- BNP seviyeleri üzerine olan etkileri sıçanlarda ISO ile indük- ic periods after ISO-induced MI in rats. lenen MI’da akut ve kronik periyot da incelenmiştir.

Results. Serum BNP was found to be significantly increased Bulgular. Serum BNP seviyeleri akut MI modelinde ar- in the acute MI model, but not in the chronic MI model. RAM tarken, kronik MI modelinde artmamıştır. RAM ve VAL and VAL application decreased BNP levels that had been in- uygulamasının, sıçanlarda ISO uygulamasıyla oluşan, akut creased after acute MI induction. Additionally, no significant MI’dan sonra artmış olan BNP seviyelerini anlamlı şekilde differences were seen in chronic MI+drug groups compared azalttığı belirlenmiştir. Kronik MI+ ilaç gruplarında da intakt with both intact and chronically infarcted control groups. ve kronik MI’lı kontrol grupları ile karşılaştırıldığında anlamlı bir değişiklik saptanmamıştır. Conclusion. The acute MI model, but not the chronic MI model, was associated with increased serum BNP levels. Pre- Sonuç. Serum BNP seviyelerinin, akut MI modelinde artırdığı, treatment with RAM and VAL, but not LAC, prevented the ancak kronik MI modelinde artmadığı bulunmuştur. RAM ve acute MI-induced increase in serum BNP levels, suggesting VAL ile yapılan ön tedavinin, serum BNP seviyesinde MI’la in- that inhibition of the renin-angiotensin system has prophylac- düklenen artışı önlemesi, ancak LAC’ın önleyememesi, renin- tic effects in the acute MI model. Therefore, both RAM and anjiotensin sisteminin inhibisyonunun akut MI modelinde pro- VAL may become first-line drugs for the treatment of hyper- filaktik etkileri olduğunu göstermektedir. Bizim bulgularımız tensive patients who are at high risk for cardiovascular failure. gösterdi ki, RAM ve VAL kardiyovasküler yetmezlik riski yük- sek olan hipertansiyon hastalarında ilk tercih edilecek ilaçlar haline gelebilir.

Keywords: Brain natriuretic peptide, Isoproterenol, Lacidipine, Myocardial Anahtar Kelimeler: Beyin natriuretik peptid, Izoproterenol, Lasidipin, Miyok- infarction, Ramipril, Valsartan ard infarktüsü, Ramipril, Valsartan

The Eurasian Journal of Medicine 44 Bayir et al.

Introduction cles in acute and chronic periods after ISO-induced MI in rats by determining the serum levels of BNP.

yocardial infarction (MI) as a result of cardiovascular Materials and Methods M disease is the principal cause of death in both devel- oped and developing countries. MI is the irreversible necrosis of heart muscle secondary to prolonged ischemia. Drugs and chemicals This usually results from an imbalance in oxygen supply ver- Lacidipine (Lacipil 4 mg tb) from Glaxo-Smith Kline, sus demand. The appearance of cardiac hormones in the cir- Turkey, Ramipril (Delix 10 mg tb) from Aventis, Turkey and culation generally indicates myocardial tissue injury [1]. For Valsartan (Diovan 80 mg tb) from Novartis, Turkey were ob- example, assays of serum hormones such as brain natriuretic tained. Isoproterenol hydrochloride was purchased from Sig- peptide (BNP) are widely performed in the early or late phase ma Chemical (Germany). All other chemicals used were of of suspected ischemic myocardial injury [2]. analytical grade.

It is now well recognized that isoproterenol (ISO), Experimental animals a synthetic catecholamine and B-adrenergic agonist, causes A total of 72 male, Sprague-Dawley rats, weighing severe stress in the myocardium, resulting in infarct-like necro- 180–200 g, were used in the study. The experiments were sis of the heart muscles and, in supramaximal doses, MI [3]. conducted according to the ethical norms approved by the Among various mechanisms proposed to explain ISO-induced Ethics Committee of the Experimental Animal Teaching and cardiac damage are hypoxia due to myocardial hyperactivity Researcher Center (No: B.30.2.ATA.0.70/94). Rats were and coronary hypotension [4], calcium overload [5], depletion obtained from the Experimental Animal Laboratory of the of energy reserves [6] and generation of highly cytotoxic free Pharmacology Department of Ataturk University, Faculty of radicals through auto-oxidation of catecholamines [7]. One Medicine and the Medicinal and Experimental Application of the systems known to be involved in heart failure is the and Research Center, Erzurum, Turkey. They were kept under natriuretic peptide system. standard laboratory conditions in a natural cycle of light and dark. The animals were fed a normal diet and water. The natriuretic peptide system (atrial natriuretic pep- tide, brain natriuretic peptide (BNP) and C natriuretic peptide) Experimental protocols of isoproterenol induced is an important marker of cardiac failure. These peptides are MI (acute and chronic) synthesized in atrial or ventricular myocytes in response to In this study, a total of 72 rats were divided into 12 wall tension. Several studies have demonstrated the correla- groups (n = 6 in each group), and the experimental groups are tion between high BNP levels and mortality in patients with summarized below: Group 1, intact control; Group 2, acute acute coronary syndrome and heart failure. BNP could be MI control; Group 3, chronic MI control; Group 4, Lacidipine used, for instance, as an early diagnostic marker for the dif- intact control; Group 5, Ramipril intact control; Group 6, Val- ferential diagnosis between cardiogenic and non-cardiogenic sartan intact control; Group 7, Acute MI + Lacidipine; Group dyspnea [8]. Moreover, plasma BNP and NT-proBNP levels are 8, acute MI + Ramipril; Group 9, acute MI + Valsartan; Group increased in heart failure and aid its diagnosis. These peptide 10, chronic MI + Lacidipine; Group 11, chronic MI + Ramipril; levels provide prognostic information in patients with heart Group 12, chronic MI + Valsartan. failure and guide the optimization of heart failure therapy [9–12]. LAC 3 mg/kg (14), RAM 3 mg/kg (15) and VAL 30 mg/kg (16) were administered orally once a day for 30 days For this study, we chose drugs that belong to the to the rats in treatment groups . For induction of both acute most common groups used in hypertension treatment and and chronic MI models, we used ISO 180 mg/kg (17) subcuta- have the highest prescription rate; lacidipine (LAC), a calcium neously. The intact control group (Group 1) was administered channel blocker, ramipril (RAM), an angiotensin-converting en- isotonic NaCl subcutaneously as the vehicle. zyme inhibitor, and valsartan (VAL), an angiotensin II type 1 receptor blocker. A previous study had determined the cardio- For the acute MI model, on days 29 and 30 of drug protective activity of LAC, RAM and VAL, which are important treatment, the rats of the ISO acute control (Group 2) and medicinal drugs with antihypertensive properties against ISO- LAC, RAM and VAL (Groups 7, 8 and 9) treatment groups induced MI [13]. However the levels of BNP, an indicator of were administered ISO (180 mg/kg) subcutaneously at 24 h left ventricular failure, have not been evaluated. intervals.

The aim of the present study was to evaluate and For the chronic MI model, on days 1 and 2 of drug compare the preventive role of LAC, RAM and VAL on ventri- treatment, the rats in the Chronic MI Control (Group 3) and

EAJM: 41, April 2009 45 Effects of Lacidipine, Ramipril and Valsartan on Serum BNP Levels

LAC, RAM and VAL (Groups 10, 11 and 12) treatment groups Discussion were administered ISO subcutaneously at 24 h intervals.

LAC, RAM and VAL (Groups 4, 5 and 6) were admin- This study investigated effects of both acute and istered to the non-MI healthy rats (control groups) orally at chronic MI on serum BNP levels. Additionally, the protective 24 h intervals for 30 days. On day 31, blood samples (~3 ml) effects of LAC, VAL, and RAM in the MI model were deter- were collected from the hearts of all animals and the samples mined by evaluating their inhibitory effects on the increase of were allowed to coagulate at room temperature for 30 min BNP levels. to 1 h. Following coagulation, the samples were centrifuged and serum was collected on dry ice prior to storage at -80 °C. Isoproterenol-induced MI serves as a standardized Serum samples were used to measure the levels of rat brain model to study the beneficial effects of many drugs, such natriuretic peptide (BNP)-32, with an ELISA kit (ERB1201-1, as nicorandil and amlodipine [18], on cardiac function [19]. Assaypro LLC, AssayMax ELISA kits) according to the manu- High-dose ISO induces hemodynamic, morphological and facturer’s instructions. functional alterations in the heart and leads to myocardial necrosis [20,21]. The effects of ISO on the heart are mediated Statistical analyses through B-1 and B-2 adrenoceptors. Both B-1 and B2 adreno- Data describing serum BNP levels were subjected ceptors mediate the positive inotropic and chronotropic ef- to one-way ANOVA using SPSS 13.0 software. Differences fects of B adrenoceptor agonists [22]. It has been reported among groups were assessed using the LSD option, and sig- that supramaximal doses of ISO, a B-adrenergic agonist and a nificance was determined at P<0.05. well-known inducer of myocardial hypertrophy [17,23], pro- duce subendocardial myocardial ischemia, hypoxia, necrosis Results and, finally, fibroblastic hyperplasia. ISO also contributes to decreased myocardial compliance and inhibition of diastolic and systolic function, which are similar to local myocardial Levels of serum BNP were found to be significantly infarction-like pathological changes differing from the ortho- increased in the acute MI model; however, those in the chron- static collapse induced by hemorrhagic shock [17,23]. In pre- ic MI model were not altered compared with the intact con- vious studies, serum BNP levels were shown to increase in trol group. Pretreatment with three antihypertensive agents ISO-induced MI models [24-26]. We also observed a signifi- (LAC, RAM and VAL) without MI induction did not alter the cant (P < 0.05) increase in the serum levels of BNP during the BNP levels in intact rats. However, RAM and VAL application acute period (Group 2) after ISO-induction in rats. However, decreased BNP levels that had increased after the acute MI there was no significant alteration in levels during the chronic induction. No significant differences were observed in chronic period (Group 3) compared with the levels observed in the MI+drug groups compared with either intact or chronically intact control group (Group 1). The release of BNP reflects infarcted control groups (Table 1). the alterations in left ventricular potency as a response to

EAJM: 41, April 2009 46 Bayir et al.

B-adrenergic stimulation [27]. In our study, the insignificant tem. RAM is an angiotensin converting enzyme blocker and results we observed in chronic MI groups may be a result of VAL is an angiotensin receptor type I blocker [34]. A previous an adaptive response from the heart after ISO induction. study reported the protective effects of LAC, RAM and VAL in the acute and chronic MI models [13]. Previous studies An increasing number of studies have shown that have also demonstrated that inhibiting the renin-angiotensin- plasma BNP levels predict all-cause mortality and cardiovas- aldosterone system prevents left ventricular remodeling after cular events that include heart failure, myocardial infarction, MI. For patients with acute MI, a combination of Perindopril stroke, atrial fibrillation and cardiovascular death in stable and Losartan, which are inhibitors of the renin-angiotensin patients with or without known cardiovascular disease. BNP system, significantly inhibited left ventricular remodeling and levels also provide information about cardiovascular risk in ad- improved left ventricular function [35]. Our results are in line dition to that provided by traditional risk factors [28]. Echocar- with the previous data. In our study, a 3 mg/kg dose of LAC diography is the gold standard for detection of left ventricular failed to prevent the ISO-induced increase in BNP levels. How- dysfunction, both systolic and diastolic, and echocardiograph- ever, high dose amlodipine, a long-lasting calcium channel ic evidence of left ventricular dysfunction is a strong and blocker, has been found to decrease BNP levels in hyperten- independent risk factor for heart failure and cardiac death sive patients [35]. The ineffectiveness of LAC may be a result [29-32]. However, prospective studies have shown that most of the dose we used. incident cases of heart failure had normal echocardiographs at baseline (30, 32), and that B-type natriuretic peptides are In conclusion, the acute MI model, but not the superior to echocardiography in the prediction of cardiovas- chronic model, significantly increased the serum BNP level, cular risk. BNP remained a predictor of heart failure, atrial which is an indicator of left ventricular systolic function. Pre- fibrillation and stroke after adjustment for echocardiographic treatment with RAM and VAL, which are inhibitors of the estimates of left ventricular mass, left atrial diameter and left renin-angiotensin system, but not with LAC, was found to ventricular systolic function [33]. prevent acute the MI-induced increase in serum BNP levels. We thus propose that the inhibition of the renin-angiotensin Pretreatment with RAM and VAL decreased the system has prophylactic effects in the acute MI model. Thus, level of serum BNP, which had increased as a result of ISO- both RAM and VAL may be candidate first-line drugs for the application to rats, while LAC application failed to prevent the treatment of hypertensive patients who have a high risk of increase in BNP levels. This could be due to the protective ef- cardiovascular failure. fect of RAM and VAL on the myocardium, which reduces the cardiac damage and thereby inhibits the renin-angiotensin sys-

Conflict interest statement The authors declare that they have no conflict of interest to the publication of this article.

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