Materials and Methods. This study investigated the effects of LAC, RAM and VAL on serum BNP levels in acute and chronic periods after ISO-induced MI in rats.

Results. Serum BNP was found to be significantly increased in the acute MI model, but not in the chronic MI model. RAM and VAL application decreased BNP levels that had been increased after acute MI induction. Additionally, no significant differences were seen in chronic MI+drug groups compared with both intact and chronically infarcted control groups.

Conclusion. The acute MI model, but not the chronic MI model, was associated with increased serum BNP levels. Pretreatment with RAM and VAL, but not LAC, prevented the acute MI-induced increase in serum BNP levels, suggesting that inhibition of the renin-angiotensin system has prophylactic effects in the acute MI model. Therefore, both RAM and VAL may become first-line drugs for the treatment of hypertensive patients who are at high risk for cardiovascular failure.

It is now well recognized that isoproterenol (ISO), a synthetic catecholamine and B-adrenergic agonist, causes severe stress in the myocardium, resulting in infarct-like necrosis of the heart muscles and, in supramaximal doses, MI ³. Among various mechanisms proposed to explain ISO-induced cardiac damage are hypoxia due to myocardial hyperactivity and coronary hypotension ^4, calcium overload ^5, depletion of energy reserves ⁶ and generation of highly cytotoxic free radicals through auto-oxidation of catecholamines ⁷. One of the systems known to be involved in heart failure is the natriuretic peptide system.

The natriuretic peptide system (atrial natriuretic peptide, brain natriuretic peptide (BNP) and C natriuretic peptide) is an important marker of cardiac failure. These peptides are synthesized in atrial or ventricular myocytes in response to wall tension. Several studies have demonstrated the correlation between high BNP levels and mortality in patients with acute coronary syndrome and heart failure. BNP could be used, for instance, as an early diagnostic marker for the differential diagnosis between cardiogenic and non-cardiogenic dyspnea ⁸. Moreover, plasma BNP and NT-proBNP levels are increased in heart failure and aid its diagnosis. These peptide levels provide prognostic information in patients with heart failure and guide the optimization of heart failure therapy ⁹–¹².

For this study, we chose drugs that belong to the most common groups used in hypertension treatment and have the highest prescription rate; lacidipine (LAC), a calcium channel blocker, ramipril (RAM), an angiotensin-converting enzyme inhibitor, and valsartan (VAL), an angiotensin II type 1 receptor blocker. A previous study had determined the cardioprotective activity of LAC, RAM and VAL, which are important medicinal drugs with antihypertensive properties against ISOinduced MI ¹³. However the levels of BNP, an indicator of left ventricular failure, have not been evaluated.

The aim of the present study was to evaluate and compare the preventive role of LAC, RAM and VAL on ventricles in acute and chronic periods after ISO-induced MI in rats by determining the serum levels of BNP.

Experimental animals
A total of 72 male, Sprague-Dawley rats, weighing 180–200 g, were used in the study. The experiments were conducted according to the ethical norms approved by the Ethics Committee of the Experimental Animal Teaching and Researcher Center (No: B.30.2.ATA.0.70/94). Rats were obtained from the Experimental Animal Laboratory of the Pharmacology Department of Ataturk University, Faculty of Medicine and the Medicinal and Experimental Application and Research Center, Erzurum, Turkey. They were kept under standard laboratory conditions in a natural cycle of light and dark. The animals were fed a normal diet and water.

Experimental protocols of isoproterenol induced MI (acute and chronic)
In this study, a total of 72 rats were divided into 12 groups (n = 6 in each group), and the experimental groups are summarized below: Group 1, intact control; Group 2, acute MI control; Group 3, chronic MI control; Group 4, Lacidipine intact control; Group 5, Ramipril intact control; Group 6, Valsartan intact control; Group 7, Acute MI + Lacidipine; Group 8, acute MI + Ramipril; Group 9, acute MI + Valsartan; Group 10, chronic MI + Lacidipine; Group 11, chronic MI + Ramipril; Group 12, chronic MI + Valsartan.

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 to the rats in treatment groups . For induction of both acute and chronic MI models, we used ISO 180 mg/kg (17) subcutaneously. The intact control group (Group 1) was administered isotonic NaCl subcutaneously as the vehicle.

For the acute MI model, on days 29 and 30 of drug treatment, the rats of the ISO acute control (Group 2) and LAC, RAM and VAL (Groups 7, 8 and 9) treatment groups were administered ISO (180 mg/kg) subcutaneously at 24 h intervals.

For the chronic MI model, on days 1 and 2 of drug treatment, the rats in the Chronic MI Control (Group 3) and LAC, RAM and VAL (Groups 10, 11 and 12) treatment groups were administered ISO subcutaneously at 24 h intervals.

LAC, RAM and VAL (Groups 4, 5 and 6) were administered to the non-MI healthy rats (control groups) orally at 24 h intervals for 30 days. On day 31, blood samples (~3 ml) were collected from the hearts of all animals and the samples were allowed to coagulate at room temperature for 30 min to 1 h. Following coagulation, the samples were centrifuged and serum was collected on dry ice prior to storage at -80 ºC. Serum samples were used to measure the levels of rat brain natriuretic peptide (BNP)-32, with an ELISA kit (ERB1201-1, Assaypro LLC, AssayMax ELISA kits) according to the manufacturer's instructions.

Statistical analyses
Data describing serum BNP levels were subjected to one-way ANOVA using SPSS 13.0 software. Differences among groups were assessed using the LSD option, and significance was determined at P<0.05.

Click Here to Zoom

Table 1: Effects of LAC, RAM and VAL on serum BNP levels after acute and chronic MI induction in rats

Isoproterenol-induced MI serves as a standardized model to study the beneficial effects of many drugs, such as nicorandil and amlodipine ^18, on cardiac function ¹⁹. High-dose ISO induces hemodynamic, morphological and functional alterations in the heart and leads to myocardial necrosis ^20,21. The effects of ISO on the heart are mediated through B-1 and B-2 adrenoceptors. Both B-1 and B2 adrenoceptors mediate the positive inotropic and chronotropic effects of B adrenoceptor agonists ²². It has been reported that supramaximal doses of ISO, a B-adrenergic agonist and a well-known inducer of myocardial hypertrophy ^17,23, produce subendocardial myocardial ischemia, hypoxia, necrosis and, finally, fibroblastic hyperplasia. ISO also contributes to decreased myocardial compliance and inhibition of diastolic and systolic function, which are similar to local myocardial infarction-like pathological changes differing from the orthostatic collapse induced by hemorrhagic shock ^17,23. In previous studies, serum BNP levels were shown to increase in ISO-induced MI models ^24-26. We also observed a signifi- cant (P < 0.05) increase in the serum levels of BNP during the acute period (Group 2) after ISO-induction in rats. However, there was no significant alteration in levels during the chronic period (Group 3) compared with the levels observed in the intact control group (Group 1). The release of BNP reflects the alterations in left ventricular potency as a response to B-adrenergic stimulation ²⁷. In our study, the insignificant results we observed in chronic MI groups may be a result of an adaptive response from the heart after ISO induction.

An increasing number of studies have shown that plasma BNP levels predict all-cause mortality and cardiovascular events that include heart failure, myocardial infarction, stroke, atrial fibrillation and cardiovascular death in stable patients with or without known cardiovascular disease. BNP levels also provide information about cardiovascular risk in addition to that provided by traditional risk factors ²⁸. Echocardiography is the gold standard for detection of left ventricular dysfunction, both systolic and diastolic, and echocardiographic evidence of left ventricular dysfunction is a strong and independent risk factor for heart failure and cardiac death ^29-32. However, prospective studies have shown that most incident cases of heart failure had normal echocardiographs at baseline ^30,32, and that B-type natriuretic peptides are superior to echocardiography in the prediction of cardiovascular risk. BNP remained a predictor of heart failure, atrial fibrillation and stroke after adjustment for echocardiographic estimates of left ventricular mass, left atrial diameter and left ventricular systolic function ³³.

Pretreatment with RAM and VAL decreased the level of serum BNP, which had increased as a result of ISOapplication to rats, while LAC application failed to prevent the increase in BNP levels. This could be due to the protective effect of RAM and VAL on the myocardium, which reduces the cardiac damage and thereby inhibits the renin-angiotensin system. RAM is an angiotensin converting enzyme blocker and VAL is an angiotensin receptor type I blocker ³⁴. A previous study reported the protective effects of LAC, RAM and VAL in the acute and chronic MI models ¹³. Previous studies have also demonstrated that inhibiting the renin-angiotensinaldosterone system prevents left ventricular remodeling after MI. For patients with acute MI, a combination of Perindopril and Losartan, which are inhibitors of the renin-angiotensin system, significantly inhibited left ventricular remodeling and improved left ventricular function ³⁵. Our results are in line with the previous data. In our study, a 3 mg/kg dose of LAC failed to prevent the ISO-induced increase in BNP levels. However, high dose amlodipine, a long-lasting calcium channel blocker, has been found to decrease BNP levels in hypertensive patients ³⁵. The ineffectiveness of LAC may be a result of the dose we used.

In conclusion, the acute MI model, but not the chronic model, significantly increased the serum BNP level, which is an indicator of left ventricular systolic function. Pretreatment with RAM and VAL, which are inhibitors of the renin-angiotensin system, but not with LAC, was found to prevent acute the MI-induced increase in serum BNP levels. We thus propose that the inhibition of the renin-angiotensin system has prophylactic effects in the acute MI model. Thus, both RAM and VAL may be candidate first-line drugs for the treatment of hypertensive patients who have a high risk of cardiovascular failure.

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

1) Maxwell SR, Lip GY. Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Int J Cardiol 1997;58: 95–117.

2) Hunt PJ, Richards AM, Nicholls MG, Yandle TG, Doughty RN, Espiner EA. Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of cardiac impairment. Clin Endocrinol 1997; 47: 287–96.

3) Wexler BC, Greenberg BP. Protective effects of clofibrate on ısoproterenol-ınduced myocardial-ınfarction in arteriosclerotic and non-arteriosclerotic rats. Atherosclerosis 1978;29:373–95.

4) Yeager JC, Iams SG. The hemodynamics of isoproterenol-induced cardiac failure in the rat. Circ Shock 1981; 8:151–63.

5) Bloom S, Davis Dl. Calcium as mediator of isoproterenol- induced myocardial necrosis. Am J Pathol 1972;69:459–70.

6) Fleckenstein A, Janke J, Döring HJ, Leder O. Myocardial fiber necrosis due to intracellular Ca overload-a new principle in cardiac pathophysiology. Recent Adv Stud Cardiac Struct Metab 1974;4:563–80.

7) Sharma M, Kishore K, Gupta SK, Joshi S, Arya DS. Cardioprotective potential of ocimum sanctum in isoproterenol induced myocardial infarction in rats. Mol Cell Biochem 2001; 225:75–83.

8) Mazzone M, Forte P, Portale G et al. Brain natriuretic peptide and acute coronary syndrome. Minerva Med 2005;96:11-8.

9) Rademaker MT, Richards AM. Cardiac natriuretic peptides for cardiac health. Clin Sci 2005; 108: 23–36.

10) Campbell DJ, Mitchelhill KI, Schlicht SM, Booth RJ. Plasma aminoterminal pro-brain natriuretic peptide: A novel approach to the diagnosis of cardiac dysfunction. J Card Fail 2000; 6: 130–9.

11) Doust JA, Pietrzak E, Dobson A, Glasziou P. How well does B-type natriuretic peptide predict death and cardiac events in patients with heart failure: Systematic review. BMJ 2005; 330: 625.

12) Jourdain P, Jondeau G, Funck F et al. Plasma brain natriuretic peptide-guided therapy to improve outcome in heart failure: The STARSBNP Multicenter Study. J Am Coll Cardiol 2007; 49: 1733– 9.

13) Bayir Y. Sıçanlarda isoprotrenol ile oluşturulan miyokard infarktüsü modelinde dNA hasarı ve oksidatif stres üzerine lasidipin, ramipril ve valsartan'nın etkilerinin incelenmesi. Doktora tezi. Atatürk Üniversitesi, Sağlık Bilimleri Enstitüsü, Tıp Fakültesi, Biyokimya. 2009.

14) Kyselovic J, Krenek P, Wibo M, Godfraind T. Effects of amlodipine and lacidipine on cardiac remodelling and renin production in saltloaded stroke-prone hypertensive rats. Br J Pharmacol 2001;134: 1516–22.

15) Wölkart G, Pang X, Stessel H, Kirchengast M, Brunner F. Chronic endothelin-A receptor antagonism is as protective as angiotensin converting enzyme inhibition against cardiac dysfunction in diabetic rats. Br J Pharmacol 2007;151:1187–97.

16) Yu CM, Wing-Hon Lai K, Li PS, Lam KY, Leung JC, Lai KN. Normalization of renal aquaporin-2 water channel expression by fosinopril, valsartan, and combination therapy in congestive heart failure: a new mechanism of action. J Mol Cell Cardiol 2004;36:445–53.

17) Rona G, Chappel CI, Balazs T, Gaudry R. An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. AMA Arch Pathol 1959;67:443–55.

18) Sathish V, Ebenezar KK, Devaki T. Synergistic effect of Nicorandil and Amlodipine on tissue defense system during experimental myocardial infarction in rats. Mol Cell Biochem 2003;243: 133–8.

19) Jayalakshmi R, Thirupurasundari CJ, Devaraj SN. Pretreatment with alcoholic extract of Crataegus oxycantha (AEC) activates mitochondrial protection during isoproterenolinduced myocardial infarction in rats. Mol Cell Biochem 2006;292: 59–67.

20) Whealthy AM, Thandroyen FT, Opie LH. Catecholamine induced myocardial cell damage: catecholamines or adrenochrome. J Mol Cell Cardiol 1985;17: 349–59.

21) Rajadurai M, Prince P. Preventive effect of naringin on isoproterenol-induced cardiotoxicity in Wistar rats: an in vivo and in vitro study. Toxicology 2007;232: 216–25.

22) Brodde OE. Beta 1- and beta 2-adrenoceptors in the human heart: properties, function, and alterations in chronic heart failure. Pharmacol Rev 1991;43: 203–42.

23) Qi YF, Shi YR, Bu DF, Pang YZ, Tang CS. Changes of adrenomedullin and receptor activity modifying protein 2 (RAMP2) in myocardium and aorta in rats with isoproterenol-induced myocardial ischemia. Peptides 2003;24: 463–8

24) Grimm D, Elsner D, Schunkert H et al.Development of heart failure following isoproterenol administration in the rat: role of the renin-angiotensin system. Cardiovasc Res 1998;37:91–100.

25) Yang J, Zhao D, Chang YZ et al. Protective effect of adrenomedul in (13–52) on isoproterenolinduced myocardial in jury in rats. Chin Pharmacol Bull 1996;12: 530–3.

26) Pan CS, Jin SJ, Cao CQ et al. The myocardial response to adrenomedullin involves increased cAMP generation as well as augmented Akt phosphorylation. Peptides 2007;28:900-9.

27) Jiang W, Cai DY, Pan CS et al. Changes in production and metabolism of brain natriuretic peptide in rats with myocardial necrosis. Eur J Pharmacol 2005;507: 153-62.

28) Campbell DJ. Can measurement of B-type natriuretic peptide levels improve cardiovascular disease prevention? Clin Exp Pharmacol Physiol 2008;35:442-6.

29) Redfield MM, Jacobsen SJ, Burnett JC Jr, Mahoney DW, Bailey KR, Rodeheffer RJ. Burden of systolic and diastolic ventricular dysfunction in the community: Appreciating the scope of the heart failure epidemic. JAMA 2003; 289: 194–202.

30) Aurigemma GP, Gottdiener JS, Shemanski L, Gardin J, Kitzman D. Predictive value of systolic and diastolic function for incident congestive heart failure in the elderly: The cardiovascular health study. J. Am. Coll. Cardiol. 2001; 37: 1042–8.

31) Bella JN, Palmieri V, Roman MJ et al. Mitral ratio of peak early to late diastolic filling velocity as a predictor of mortality in middle-aged and elderly adults: The Strong Heart Study. Circulation 2002; 105: 1928–33.

32) Gottdiener JS, Arnold AM, Aurigemma GP et al. Predictors of congestive heart failure in the elderly: The Cardiovascular Health Study. J Am Coll Cardiol 2000; 35: 1628–37.

33) Wang TJ, Larson MG, Levy D et al. Plasma natriuretic peptide levels and the risk of cardiovascular events and death. N Engl J Med 2004;350: 655–63.

34) Süzer Ö. Süzer Farmakoloji. İstanbul, Klinisyen Tıp Kitabevleri, 2005.

35) Uno H, Ishikawa J, Hoshide S et al. Effects of strict blood pressure control by a long-acting calcium channel blocker on brain natriuretic peptide and urinary albumin excretion rate in Japanese hypertensive patients. Hypertens Res 2008;31:887-96.