Cardiovasc Toxicol DOI 10.1007/s12012-015-9323-1
Protective Effect of Hydroxytyrosol Against Cardiac Remodeling After Isoproterenol-Induced Myocardial Infarction in Rat Kais Mnafgui1,2 • Raouf Hajji2 • Fatma Derbali2 • Ines Khlif3 • Ftouma Kraiem4 Hedi Ellefi5 • Abdelfattah Elfeki1 • Noureddine Allouche3 • Neji Gharsallah6
•
Ó Springer Science+Business Media New York 2015
Abstract The present study aimed to investigate the cardioprotective effect of hydroxytyrosol (HT) against isoproterenol-induced myocardial infarction in rats. Male rats were randomly divided into four groups, control, isoproterenol (Isop) and pretreated animals with HT in two different doses (2 and 5 mg/kg) orally for 7 days and intoxicated with isoproterenol (Isop ? HT1) and (Isop ? HT2) groups. Myocardial infarction in rats was induced subcutaneously by isoproterenol (100 mg/kg, s.c.) at an interval of 24 h on 6th and 7th day. On 8th day, electrocardiographic (ECG) pattern, gravimetric and biochemical parameters were assessed. Isoproterenol exhibited changes in ECG pattern, including significant ST-segment elevation and increase in the serum troponin-T level by 317 % as compared to control rats. Moreover, cardiac injury markers (creatine kinase-MB,
lactate dehydrogenase, alanine aminotransferase) underwent a notable rise in serum of infarcted animals. Else, a disturbance in lipids profile and significant increase in lipase and angiotensin-converting enzyme (ACE) activities and heart weight ratio were observed in isoproterenol group. However, pre- and co-treatment with HT (2 and 5 mg/kg) improved the myocardium injury, restored the hemodynamic function and inhibited the ACE activity that prevent cardiac hypertrophy and remodeling. Overall, these findings demonstrated that HT exerted a potent cardioprotective effect against isoproterenol-induced myocardial infarction. Keywords ACE ECG Hydroxytyrosol Myocardial infarction Troponin-T
Introduction Raouf Hajji and Fatma Derbali have contributed equally to this work. & Kais Mnafgui [email protected] 1
Laboratory of Animal Physiology, Faculty of Sciences of Sfax, University of Sfax, P.O. Box 95, 3052 Sfax, Tunisia
2
Department of Internal Medicine, Hospital of Sidi Bouzid, 9100 Sidi Bouzid, Tunisia
3
Laboratory of Chemistry of Natural Products, Faculty of Sciences of Sfax, University of Sfax, B.P. 1171, 3000 Sfax, Tunisia
4
Faculty of Medicine of Monastir, Avenue Avicenne, 5019 Monastir, Tunisia
5
Department of Cardiology, Centre Hospitalier Intercommunal Robert Ballanger, 93600 Boulevard Robert Ballanger, Aulnay-Sous-Bois, France
6
Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, University of Sfax, B.P. 1171, 3000 Sfax, Tunisia
Acute myocardial infarction is an important ischemic heart disease and a major cause of morbidity and mortality worldwide [1]. It is the acute condition of myocardial necrosis that occurs as a result of imbalance between coronary blood supply and myocardial demand. Clinical markers of myocardial necrosis and dysfunction include blood pressure, heart rate and electrocardiographic (ECG) changes and left ventricular (LV) dysfunction accompanied with increased serum levels of cardiac-specific proteins. Cardiac troponins are frequently associated with myocardial infarction and inflammation-related proteins at elevated levels with heart attack [2]. The animal model of isoproterenol-induced myocardial infarction presents an important endorsed technique for studying the effects of several potential cardioprotective bioactive compounds [3]. Isoproterenol [1-(3,4-dihydroxyphenyl)-2-isopropylaminoethanol hydrochloride] is a synthetic catecholamine
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and b-adrenergic agonist used in inframaximal doses to regulate heart function. However, the administration of isoproterenol in surpramaximal doses could induce severe stress in the myocardium through the depletion of the cardiomyocytes energy reserve which is resulting in irreversible cellular injury and ultimately infract like necrosis [3, 4]. Many synthetic drugs are used for the management of heart attack but they are not free from side effects. Hence, several studies have focused on identifying new therapeutic approaches to prevent myocardial infarction. Recently, greater attention has been focused on the phenols as effective bioactive compounds that protect cells or molecules from myocardial damage. In this regard, hydroxytyrosol (HT), a natural phenol present in large amounts in all parts of olive trees (Olea europaea), has been found to be a potent antioxidant and hypocholesterolemic agent in various animal models of disease including dyslipidemia, atherosclerosis and diabetes [4–6]. Polyphenols are excellent cardioprotectors. Hence, we suggested that HT may have a cardioprotective effect. The purpose of the current study was designed to investigate the protective effect of the oral pretreatment of HT on experimentally induced myocardial infarction in Wistar rats.
Materials and Methods Chemicals Lipase kit was purchased from Biolabo reagents France, HT [2-(3,4-dihydroxyphenyl)ethanol] was obtained from Cayman Chemical (Ann Arbor, MI, USA); the purity was C98 %. Isoproterenol hydrochloride powder was obtained from Sigma-Aldrich, St. Louis, USA. Angiotensin-converting enzyme (ACE) kit was purchased from Trinity, UK. The remaining chemicals used were of analytical grade.
subcutaneously at an interval of 24 h for 2 days to induce experimental myocardial infarction [7]. Animals were killed 48 h after the first dose of isoproterenol. Experimental Protocols After acclimatization, the animals were randomly divided into the following groups consisting of eight rats each: Group I: (Control) rats received standard laboratory diet and drinking saline water ad libitum and served as a control; Group II: (Isop) rats received saline water for 7 days and at the 6th day subcutaneously injected with isoproterenol (100 mg/kg, subcutaneously injected, once at an interval of 24 h for two consecutive days); Group III: (Isop ? HT1) rats pretreated with hydroxytyrosol (2 mg/kg bw; gastric gavages, respectively) for 7 days and at the 6th day subcutaneously injected with isoproterenol (100 mg/kg bw) for two consecutive days; Group IV: (Isop ? HT2) rats received HT (5 mg/kg bw) for 7 days and were injected subcutaneously with isoproterenol (100 mg/kg bw) on day 6th and 7th. All rats are fasted overnight but had free access to water at the last administration of the drug. After the 7-days induction, the animals were weighted and killed by decapitation in order to minimize the handling stress, and the trunk blood collected. The serum was prepared by centrifugation (15009g, 15 min, 4 °C), frozen and stored at -20 °C until analysis. Immediately after killing, the heart was excised out, washed with saline and fixed in fixed in a Bouin solution for 24 h and then embedded in paraffin. Sections of 5 lm thickness were stained with hematoxylin–eosin. The slides were photographed with an Olympus U-TU1X-2 camera connected to an Olympus CX41 microscope (Tokyo, Japan).
Animals
Measurement of Blood Pressure by Noninvasive Method
The essays of this study were conducted on adult male Wistar rats, weighting 260 ± 10 g, which were obtained from the local Central Pharmacy, Tunisia. All rats were kept in an environmentally controlled breeding room (temperature: 20 ± 2 °C; humidity: 60 ± 5 %; 12-h dark/light cycle) where they had standard diets and free access to tap water. The experimental protocols were conducted in accordance with the guide for the care and use of laboratory animals issued by the University of Sfax, Tunisia, and approved by the Committee of Animal Ethics.
Twenty-four hours after the second injection of isoproterenol, blood pressures were measured using noninvasive blood pressure Monitor (CODATM Surgical Monitor, USA) which consists of placing a cuff on the animal’s tail to occlude the blood flow. The pressure was raised and then slowly released. The cuff pressure when the pulse signal reappears is intended as the systolic pressure. The cuff pressure when the pulse signal level recovers its initial level is intended as diastolic pressure.
Induction of Experimental Myocardial Infarction
Electrocardiography
Isoproterenol was dissolved in normal saline and injected to rats (100 mg/kg body weight) and injected
Twenty-four hours after the second dose of isoproterenol, rats of all the groups were anesthetized with ketamine
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hydrochloride (100 mg/kg bw) intraperitoneally. Needle electrodes were inserted under the skin of the animals in lead II position. ECG recordings were made using veterinary electrocardiograph (ECG VET 110, Biocare, China).
by 17 and 37 %, respectively, as compared to untreated myocardial infarcted rats. Electrocardiogram Patterns of Normal and Experimental Animals
Biochemical Analysis After killing, the heart was dissected out, immediately washed in ice-cold saline and a homogenate was prepared in 0.1 M Tris–HCl buffer (pH 7.4). Homogenate was centrifuged, and supernatant was used for the assay of marker enzymes. The collected serum was used for the determination of serum activity of lipase and ACE, the cardiac marker enzymes as creatinine kinase-MB (CK-MB), lactate dehydrogenase (LDH), alanine transaminase (ALT) and troponin-I rates were measured in frozen aliquots of serum by standardized enzymatic procedures using commercial kits from (Biolabo, France) on an automatic biochemistry analyzer (Vitalab Flexor E, USA) using commercially available standard enzymatic kits (Biolabo, France). Serum lipids level of triglycerides (TG), total cholesterol (T-Ch), high density lipoprotein-cholesterol (HDL-c) and low density lipoprotein-cholesterol (LDL-c) were measured using the corresponding commercial kits (Biolabo, France) on an automatic biochemistry analyzer (Kenza, France) at the clinic pathological laboratory of Sidi Bouzid Hospital. Serum LDL-cholesterol concentration was determined according to formula: LDL_cholesterol = Total cholesterol_(Triglycerides/5) - (HDL_cholesterol) performed by Friedewal et al. [8].
Control animals showed normal ECG pattern with normal heart rate (375 ± 13.92 bpm). While infarcted rats evidenced a significant (P \ 0.05) increase in heart rate (430 ± 11.65 bpm) which became irregular, remarkable elevation of ST-segment (wave of Paradee) and QT interval along with no identifiable P wave as compared to the normal animals (Figs. 1, 2). However, oral pre- and cotreatment of isoproterenol-induced rats with 2 mg/kg of HT showed acceleration in heart rate (430 ± 12.34 bpm), remaining regular sinus with the appearance of a discrete ST-elevation. Else, an aspect of electrical alternating was observed (alternating between QRS ample and QRS less ample). Though, the HT2 group exhibited sinus rhythm not accelerated relative to healthy control (375 ± 10.31 bpm), with no ST-segment elevation, no alternating electric sinus and rare atrial premature as compared to groups II and III. Effect of Hydroxytyrosol on Hemodynamic Function As shown in Table 2, significant decreases in systolic, diastolic and mean arterial blood pressure were observed in isoproterenol-induced rats as compared to control group (P \ 0.05). The pre- and co-treatment of infarcted rats with HT at 2 and 5 mg/kg significantly restored arterial pressure in a dose-dependent pattern.
Statistical Analysis Data are presented as mean ± standard deviation (SD). Determinations were performed from eight animals per group, and differences were examined by a one-way analysis of variance (ANOVA) followed by the Fisher test (Stat View). *P \ 0.05 was considered statistically significant.
Results Effect of Hydroxytyrosol (HT) on Heart Weight to Body Weight Ration of Experimental Rats As shown in Table 1, there was no significant difference in the body weight between the groups observed. Isoproterenol-induced myocardial infarcted rats exhibited significant increases in heart weight and the heart weight to body weight ration as compared to control rats (P \ 0.05). However, in response to HT1 and HT2, the infarcted rats showed significant (P \ 0.05) decrease in the heart weight
Effect of Hydroxytyrosol on Serum Cardiac Markers Table 3 indicated the variation of CK-MB and LDH both in serum and heart tissues and ALT, troponin-T in serum of control and experimental rats. The rats induced with isoproterenol evidenced a significant (P \ 0.05) increase in the serum levels CK-MB, ALT, LDH and troponin-T by 66, 112, 70 and 317 %, respectively, as compared to control rats associated with significant decrease in myocardium LDH and CK-MB, whereas pre- and co-treatment with HT (5 mg/kg bw) daily for 7 days significantly (P \ 0.05) decreased the levels of serum cardiac markers better than the administrated dose of 2 mg/kg of HT in isoproterenolinduced rats compared to untreated infarcted rats. Histopathological Examination of Cardiac Tissues The histopathological examination of heart tissues showed that control group of rats evidenced a normal myofibrillar structure without any infarction edema and inflammatory
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Cardiovasc Toxicol Table 1 Effect of hydroxytyrosol pre- and co-treatment on heart weight, body weight and heart weight/body weight ratio in isoproterenolinduced myocardial infarction in rats Parameters
Control
Isop
Isop ? HT1
Body weight (g)
269.20 ± 9.83
271.20 ± 10.28
273.80 ± 10.44
Heart weight (g)
0.95 ± 0.10
Heart weight/body weight ratio
1.50 ± 0.09*
0.35 ± 0.02
0.55 ± 0.02*
Isop ? HT2 268.21 ± 8.70
,#
0.96 ± 0.11#,@
,#
0.36 ± 0.03#,@
1.24 ± 0.13*
0.45 ± 0.04*
Values are given as mean ± SD for groups of eight animals each HT1, 2 mg/kg bw of hydroxytyrosol; HT2, 5 mg/kg bw of hydroxytyrosol Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group of rats. @P \ 0.05 significant differences compared to HT1 group
0.5 *
0.45
ST-Segment (mV)
0.4 0.35 0.3 0.25 0.2 0.15
*#
0.1
#@
0.05 0
79 %, respectively, with notable decrease in serum HDL-c by 26 % when compared to normal control animals (Table 4), whereas the administration of HT at 2 and 5 mg/ kg to infarcted rats induced a significant decrease in pancreatic lipase activity by 35 and 42 %, respectively, accompanied with remarkable decrease in T-Ch, TG, LDL-c by (13, 23 and 24 %) and (21, 35 and 39 %), respectively, with considerable increase in HDL-c by 19 and 29 %, respectively, as compared to isoproterenol-induced rats alone (P \ 0.05). Effect of Hydroxytyrosol on Serum ACE Activity
Control
Isop
Isop+HT1
Isop+HT2
Groups
Fig. 1 Effect of hydroxytyrosol on ST-segment elevation (mV) in the ECG (recorded from limb lead II) in normal control, isoproterenol alone injected and treated rats. Values are given as mean ± SD for group of eight rats. Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
Figure 5 showed that the ACE activity in serum of untreated infarcted rats underwent a significant increase by 67 % as compared to normal control animals (P \ 0.05). However, treatment with HT at doses of 2 and 5 mg/kg induced a remarkable decrease of ACE activity by 28 and 36 %, respectively, in serum of isoproterenol-induced myocardial infarction in rats.
Discussion cells (Fig. 3a). However, tissues from isoproterenol-treated rats revealed widespread myocardial structure disorder and subendocardial necrosis with loss of transverse striation and obvious leukocyte infiltration (Fig. 3b) as compared to control group, whereas pretreatment with HT (2 and 5 mg/ kg, respectively) exhibited marked improvement in isoproterenol-induced myocardial degeneration and inflammatory cells infiltration (Fig. 3c, d). Effect of Hydroxytyrosol on Serum Lipase Activity and Lipids Profile As shown in Fig. 4, the isoproterenol-induced group underwent a significant (P \ 0.05) increase in serum pancreatic lipase activity by 89 % which led to remarkable rise in serum levels of T-Ch, TG and LDL-c by 30, 67 and
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HT (3,4-dihydroxyphenylethanol) is an amphipathic molecule that is derived from the hydrolysis of oleuropein. Oleuropein hydrolysis takes places during olive maturation, as well as during olive oil storage [9, 10]. This phenolic bioactive compound has been proved to be endowed with several pharmaceutical properties such as antioxidant, anti-inflammatory, antithrombotic, antidiabetic and hypolipidemic effects [11–14]. In the current study, we explored, for the first time, the therapeutic efficacy of HT in rats with myocardial infarction. A subcutaneous injection of a supramaximal dose of isoproterenol was a consequence of electrocardiographic (ECG), biochemical and structural changes in the heart as mimetic to that occurs in patients with myocardial infarction. Changes in ECG pattern are commonly used for the diagnosis of acute
Cardiovasc Toxicol
Fig. 2 Effect of hydroxytyrosol on electrocardiographic (ECG) pattern in normal and experimental rats. ECG pattern of normal control group showing normal cardiograph. ECG pattern of isoproterenol (100 mg/kg)-induced group rats showing pathological changes such as ST-segment elevation. Electrocardiogram pattern
Table 2 Hemodynamic parameters in different experimental groups
Parameters
of HT (2 mg/kg)-treated isoproterenol-induced group rats showing minimized ST-segment elevation with aspect of electrical alternating. ECG pattern of HT (5 mg/kg) pre- and co-treated isoproterenolinduced group rats showing almost normal cardiograph without any elevation in ST-segment
Control
Isop
Isop ? HT1
Isop ? HT2
SAP (mmHg) DAP (mmHg)
128 ± 5.56 83.40 ± 3.04
88.20 ± 4.76* 56 ± 3.80*
104.40 ± 4.03*,# 69.80 ± 4.81*,#
122 ± 5.24#,@ 79.60 ± 4.15#,@
MAP (mmHg)
105.70 ± 4.13
72.12 ± 1.51*
87.10 ± 2.82*,#
100.80 ± 4.57#,@
HR (bpm)
375 ± 13.92
430 ± 11.65*
375 ± 10.31#,@
430 ± 11.65*
SAP systolic arterial blood pressure, DAP diastolic arterial blood pressure, MAP mean arterial blood pressure, HR heart rate Values are given as mean ± SD for group of eight animals each. Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences to diabetic rats treated with HT1
Table 3 Effect of hydroxytyrosol (HT) on cardiac markers in serum and heart tissues
Groups
Control
Isop
Isop ? HT1
Isop ? HT2
Serum ALT (UI/L)
41.4 ± 4.27
88 ± 4.01*
62.40 ± 6.24*,# ,#
54.60 ± 9.65*,# 543.2 ± 65.72#,@
LDH (UI/L)
519.8 ± 37.31
883.2 ± 40.61*
671.6 ± 63.38*
CK-MB (UI/L)
21.70 ± 0.96
36.16 ± 1.21*
25.65 ± 0.71*,#
22.72 ± 1.40#,@
0.57 ± 0.06
2.38 ± 0.33*
,#
0.76 ± 0.13*
0.53 ± 0.05#,@
7.31 ± 0.53
4.28 ± 0.21*
5.67 ± 0.47*,#
6.87 ± 0.32#,@
0.97 ± 0.16*
,#
2.07 ± 0.24#,@
Troponin-T (ng/mL) Heart LDH (lKat/mg) CK-MB (lKat/mg)
2.45 ± 0.14
1.45 ± 0.13*
Alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB) and serum troponin-T level of control and experimental groups of rats. CK-MB and LDH in the heart tissue are expressed in lkat/mg of proteins. Values are given as mean ± SD for group of eight animals each Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls. P \ 0.05 significant differences compared to isoproterenol group @P \ 0.05 significant differences to diabetic rats treated with HT1
#
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Fig. 3 Pathological changes of myocardial tissue (H&E 9500). a Control group showing normal myocardial histology, clear transverse striations and no inflammatory cell infiltration. b Isoproterenol group showing myocardial cells necrosis, separation of cardiac myofibrillar and large
Serum lipase activity (UI/L)
180
*
160 140 *#
120
*#@
100 80 60 40 20 0
Control
Isop
Isop+HT1
Isop+HT2
Groups
Fig. 4 Effect of hydroxytyrosol on serum lipase activity in normal and experimental rats. Values are given as mean ± SD for group of eight rats. Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
myocardial infarction. Rats treated with isoproterenol showed a marked elevation of ST-segment with remarkable decrease in P wave indicative of myocardial ischemia and infarction in accordance with previous study [15]. The alteration of ECG pattern could be related to the consecutive loss of integrity of cell membrane in injured myocardium [16]. The supramaximal dose of isoproterenol also injected increased heart rate (tachycardia) due to involvement of baroreceptor reflex-mediated changes in autonomic nerve activity [17], whereas pre- and co-treatment with HT
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inflammatory cells infiltration. c Isop ? HT1 (2 mg/kg)-treated group showing few inflammatory cell infiltration and improvement of myocardium necrosis. d Isop ? HT2 (5 mg/kg) showing normal myocardial arrangement, clear transverse striations and little few inflammatory cells
(5 mg/kg) significantly reduced the abnormalities observed in the ECG of isoproterenol-induced rats. Accordingly, it has been demonstrated that HT improved the cardiac disturbances enhanced by doxorubicin by significantly reducing the percentage of altered mitochondria also enhancing the mitochondrial electron transport chain and antioxidant defense system [18]. Several previous studies have demonstrated that the myocardium injury induced by isoproterenol in experimental rats is strongly associated with deterioration of hemodynamic and LV contractile function [14–17]. While, HT pre- and co-treatment showed significant improvement in systolic and diastolic arterial pressure (SAP and DAP, respectively) and subsequent increase in mean arterial pressure (MAP), a marker of after load. Serum cardiac troponins and cytosolic enzyme activities such as CK-MB, LDH and AST are very sensitive and specific diagnostic markers of acute myocardial infarction [19, 20]. Our results confirmed previous studies reporting that isoproterenol induced significant increases in serum troponin-T level and CK-MB, LDH and AST activities which reflected the severe damage in myocardium tissues [20, 21]. HT pre- and co-treatment reduced the activity of the marker enzymes in serum of isoproterenol-induced rats. It confirmed that HT could maintain membrane integrity, so restricting the leakage of these enzymes. Previous study clearly evidenced that administration of HT to normal rats showed no toxicity effect in tolerant dose, and there was no significant difference between
Cardiovasc Toxicol Table 4 Effect of hydroxytyrosol (HT) on serum lipids profile
Groups
Control
Isop
Isop ? HT1
T-Ch
1.81 ± 0.12
2.35 ± 0.10*
2.04 ± 0.16*,#
1.86 ± 0.05#,@
1.20 ± 0.10*
,#
0.77 ± 0.11#,@
,#
0.92 ± 0.03*,#,@
,#
0.79 ± 0.02#,@
TG
0.72 ± 0.05
LDL-c
0.84 ± 0.06
HDL-c
0.82 ± 0.05
1.50 ± 0.14* 0.61 ± 0.05*
Isop ? HT2
0.92 ± 0.11* 1.13 ± 0.18* 0.72 ± 0.04*
Total cholesterol (T-ch), triglycerides (TG), low density lipoprotein-cholesterol (LDL-c) and high density lipoprotein-cholesterol (HDL-c) in serum. Values are given as mean ± SD for group of eight rats Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. # P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
ACE activity in serum (UI/L)
90 *
80 70
*#
60
#@
50 40 30 20 10 0
Control
Isop
Isop+HT1
Isop+HT2
Groups
Fig. 5 ACE activity in serum of normal and experimental rats. Values are given as mean ± SD for group of eight rats. Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
normal rats treated with HT and untreated normal group in all tested parameters such as heart weight, ratio, cardiac enzymes markers, oxidative stress statute and myocardium histology [18]. Our histopathological findings of the isoproterenol-induced myocardial infarction showed large infarcted zone with edema, necrosis and obvious separation of cardiac myofibrillar and inflammatory cells which explained the ST-segment elevation observed in the ECG findings. The encroachment of the injured myocardium by neutrophils during ischemia could be considered as the major source of free radicals [22]. However, HT pre- and co-treatment (2 and 5 mg/kg, respectively) showed clear protective effects in isoproterenol-treated groups. Furthermore, lipids have a major role in cardiovascular diseases through the development of hyperlipidemia, hence increasing atherosclerosis risks. Else, it could lead to modify the composition, structure and stability of the cellular membranes. In the present study, a significant increase in serum pancreatic lipase activity was observed which led to
considerable rise in serum T-Ch, TG and LDL-c and remarkable decreased in HDL-c in isoproterenol-induced myocardial infarcted rats. These changes in lipids profile could be attributed to cardiac cyclic adenosine monophosphate that enhanced lipid biosynthesis during the hypoxic condition in isoproterenol-induced myocardial infarcted rats [23]. In fact, lipase is secreted from the pancreas, transported to small intestine and hydrolyzes triglycerides nonabsorbable into absorbable monoglycerides and free fatty acids absorbable by small intestine. Hence, inhibition of lipase activity by HT explains the significant reduced uptake of triglycerides from the circulation of infarcted rats which is confirmed in our previous study [24]. Queenthy and John [7] revealed the positive correlation between the risk of developing ischemic heart disease and serum LDL-c level. In this regard, previous investigations have demonstrated that HT can correct dyslipidemia, improve triglyceride metabolism, enhance endothelial function, increase antioxidant potential and decrease low density lipoprotein-cholesterol (LDL-c), which is an important cardiovascular diseases risk factor due to its role in atherosclerosis [25, 26]. Accumulating evidence suggests that the cardiac rennin–angiotensin system (RAS) is activated during the LV remodeling process after acute myocardial infarction [27, 28]. Our data evidenced that serum ACE activity of isoproterenol-induced rats underwent a significant increase accompanied with elevated heart weight ratio (hypertrophy) indicative of ventricular remodeling process. It includes infarct expansion as well as compensatory reactive hypertrophy and dilation of the noninfarcted LV [28]. Preand co-treatment of isoproterenol-induced myocardial infarction in rat at moderate dose of HT caused a significant decrease of serum ACE activity with marked reduce in heart weight ratio indicative of cardioprotective effect preventing the increased risk of infarct expansion and LV remodeling that follows myocardial infarction. Actually, there is evidence from several experimental studies that the cardiac tissue-type rennin–angiotensin system is activated after myocardial infarction and failure [28, 29]. However, ACE inhibition has been revealed to prevent cardiac
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remodeling and to prolong survival in experimental myocardial infarction. This process has been related to the reduced levels of circulating and tissue angiotensin II which is involved in the development of vascular and ventricular remodeling. Hence, ACE inhibitor increases tissue bradykinin accumulation, and the bradykinin has antigrowth effects and reduces vasomotor [30]. In conclusion, the present study provides experimental evidence that oral pre- and co-treatment with HT (2 and 5 mg/kg) evidence cardioprotective effect in isoproterenolinduced myocardial infarction in rats by improving heart weight, lipids profile and cardiac dysfunctional markers as well as demonstrating the beneficial role of HT as ACE inhibitor to prevent cardiac remodeling and failure. Acknowledgments This research was supported by the Tunisian Ministry of Higher Education and Scientific Research and the Tunisian Ministry of Public Health. Authors wish to thank Mrs. Sadok Slema, Mbarek Nasri and Chedly Tmar for their assistance and cooperation. Conflict of interests
The authors declare no conflict of interest.
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11. O’Dowd, Y., Driss, F., Dang, P. M., Elbim, C., Gougerot-Pocidalo, M. A., Pasquier, C., et al. (2004). Antioxidant effect of hydroxytyrosol, a polyphenol from olive oil: Scavenging of hydrogen peroxide but not superoxide anion produced by human neutrophils. Biochemical Pharmacology, 68, 2003–2008. 12. Gonza´lez-Correa, J. A., Navas, M. D., Lopez-Villodres, J. A., Trujillo, M., Espartero, J. L., & De La Cruz, J. P. (2008). Neuroprotective effect of hydroxytyrosol and hydroxytyrosol acetate in rat brain slices subjected to hypoxia-reoxygenation. Neuroscience Letters, 446, 143–146. 13. Va´zquez-Velasco, M. Esperanza, Dı´az, L., Lucas, R., Go´mezMartı´nez, S., Bastida, S., Marcos, A., & Sa´nchez-Muniz, F. J. (2011). Effects of hydroxytyrosol-enriched sunflower oil consumption on CVD risk factors. British Journal of Nutrition, 105, 1448–1452. 14. Cabrerizo, S. La, Cruz, D., Pedro, J., Lo´pez-Villodres, J. A., Mun˜oz-Marı´n, J., Guerrero, A., & Gonza´lez-Correa, J. A. (2013). Role of the inhibition of oxidative stress and inflammatory mediators in the neuroprotective effects of hydroxytyrosol in rat brain slices subjected to hypoxia reoxygenation. Journal of Nutritional Biochemistry, 24, 2152–2157. 15. Kannan, M. M., & Quine, S. D. (2011). Ellagic acid ameliorates isoproterenol induced oxidative stress: Evidence from electrocardiological, biochemical and histological study. European Journal of Pharmacology, 659, 45–52. 16. Kannan, M. M., & Quine, D. S. (2013). Ellagic acid inhibits cardiac arrhythmias, hypertrophy and hyperlipidaemia during myocardial infarction in rats. Metabolism, 62, 52–61. 17. Whalen, E. J., & Lewis, S. J. (1999). In vivo evidence that isoproterenol may increase heart rate in the rat by mechanisms in addition to activation of cardiac b1-or b2-adrenoceptors. European Journal of Pharmacology, 382, 207–210. 18. Granados-Principal, S., El-Azem, N., Pamplona, R., RamirezTortosa, C., Pulido-Moran, M., Vera-Ramirez, L., et al. (2014). Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-induced cardiotoxicity in rats with breast cancer. Biochemical Pharmacology, 90, 25–33. 19. O’Brien, P. J., Smith, D. C., Knechtel, T. J., Marchak, M. A., Pruimboom-Brees, I., Brees, D. J., et al. (2006). Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals. Laboratory Animals, 40, 153–171. 20. Evran, B., Karpuzog˘lu, H., Develi, S., Kalaz, E. B., Soluk-Tekkes¸ in, M., Olgac¸, V., & Uysal, M. (2014). Effects of carnosine on prooxidant–antioxidant status in heart tissue, plasma and erythrocytes of rats with isoproterenol-induced myocardial infarction. Pharmacological Reports, 66, 81–86. 21. Radhiga, T., Rajamanickam, C., Sundaresan, A., Ezhumalai, M., & Pugalendi, K. V. (2012). Effect of ursolic acid treatment on apoptosis and DNA damage in isoproterenol induced myocardial infarction. Biochimie, 94, 1135–1142. 22. Kumaran, K. S., & Prince, P. S. M. (2010). Protective effect of caffeic acid on cardiac markers and lipid peroxide metabolism in cardiotoxic rats: an in vivo and in vitro study. MetabolismClinical and Experimental, 59, 1172–1180. 23. Leroith, D. (2007). Dyslipidemia and glucose dysregulation in over weight and obese patients. Clinical Cornerstone, 8, 38–52. 24. Mnafgui, K., Derbali, A., Sayadi, S., Gharsallah, N., Elfeki, A., & Allouche, N. (2014). Anti-obesity and cardioprotective effects of cinnamic acid in high fat diet-induced obese rats. Journal of Food Science and Technology,. doi:10.1007/s13197-014-1488-2. 25. Fki, I., Sahnoun, Z., & Sayadi, S. (2007). Hypocholesterolemic effects of phenolic extracts and purified hydroxytyrosol recovered from olive mill wastewater in rats fed a cholesterol-rich diet. Journal of Agriculture and Food Chemistry, 55, 624–631. 26. Jemai, H. El, Feki, A., & Sayadi, S. (2009). Antidiabetic and antioxidant effects of hydroxytyrosol and oleuropein from olive
Cardiovasc Toxicol leaves in alloxan-diabetic rats. Journal of Agriculture and Food Chemistry, 57, 8798–8804. 27. Schieffer, B., Wirger, A., Meybrunn, M., Seitz, S., Holtz, J., Riede, U. N., & Drexler, H. (1994). Comparative effects of chronic angiotensin-converting enzyme inhibition and angiotensin II type 1 receptor blockade on cardiac remodeling after myocardial infarction in the rat. Circulation, 89, 2273–2282. 28. Borghi, C., Bacchelli, S., Esposti, D. D., & Ambrosioni, E. (2006). Effects of early angiotensin-converting enzyme inhibition in patients with non–ST-elevation acute anterior myocardial infarction. American Heart Journal, 152, 470–477.
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Protective Effect of Hydroxytyrosol Against Cardiac Remodeling After Isoproterenol-Induced Myocardial Infarction in Rat Kais Mnafgui1,2 • Raouf Hajji2 • Fatma Derbali2 • Ines Khlif3 • Ftouma Kraiem4 Hedi Ellefi5 • Abdelfattah Elfeki1 • Noureddine Allouche3 • Neji Gharsallah6
•
Ó Springer Science+Business Media New York 2015
Abstract The present study aimed to investigate the cardioprotective effect of hydroxytyrosol (HT) against isoproterenol-induced myocardial infarction in rats. Male rats were randomly divided into four groups, control, isoproterenol (Isop) and pretreated animals with HT in two different doses (2 and 5 mg/kg) orally for 7 days and intoxicated with isoproterenol (Isop ? HT1) and (Isop ? HT2) groups. Myocardial infarction in rats was induced subcutaneously by isoproterenol (100 mg/kg, s.c.) at an interval of 24 h on 6th and 7th day. On 8th day, electrocardiographic (ECG) pattern, gravimetric and biochemical parameters were assessed. Isoproterenol exhibited changes in ECG pattern, including significant ST-segment elevation and increase in the serum troponin-T level by 317 % as compared to control rats. Moreover, cardiac injury markers (creatine kinase-MB,
lactate dehydrogenase, alanine aminotransferase) underwent a notable rise in serum of infarcted animals. Else, a disturbance in lipids profile and significant increase in lipase and angiotensin-converting enzyme (ACE) activities and heart weight ratio were observed in isoproterenol group. However, pre- and co-treatment with HT (2 and 5 mg/kg) improved the myocardium injury, restored the hemodynamic function and inhibited the ACE activity that prevent cardiac hypertrophy and remodeling. Overall, these findings demonstrated that HT exerted a potent cardioprotective effect against isoproterenol-induced myocardial infarction. Keywords ACE ECG Hydroxytyrosol Myocardial infarction Troponin-T
Introduction Raouf Hajji and Fatma Derbali have contributed equally to this work. & Kais Mnafgui [email protected] 1
Laboratory of Animal Physiology, Faculty of Sciences of Sfax, University of Sfax, P.O. Box 95, 3052 Sfax, Tunisia
2
Department of Internal Medicine, Hospital of Sidi Bouzid, 9100 Sidi Bouzid, Tunisia
3
Laboratory of Chemistry of Natural Products, Faculty of Sciences of Sfax, University of Sfax, B.P. 1171, 3000 Sfax, Tunisia
4
Faculty of Medicine of Monastir, Avenue Avicenne, 5019 Monastir, Tunisia
5
Department of Cardiology, Centre Hospitalier Intercommunal Robert Ballanger, 93600 Boulevard Robert Ballanger, Aulnay-Sous-Bois, France
6
Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, University of Sfax, B.P. 1171, 3000 Sfax, Tunisia
Acute myocardial infarction is an important ischemic heart disease and a major cause of morbidity and mortality worldwide [1]. It is the acute condition of myocardial necrosis that occurs as a result of imbalance between coronary blood supply and myocardial demand. Clinical markers of myocardial necrosis and dysfunction include blood pressure, heart rate and electrocardiographic (ECG) changes and left ventricular (LV) dysfunction accompanied with increased serum levels of cardiac-specific proteins. Cardiac troponins are frequently associated with myocardial infarction and inflammation-related proteins at elevated levels with heart attack [2]. The animal model of isoproterenol-induced myocardial infarction presents an important endorsed technique for studying the effects of several potential cardioprotective bioactive compounds [3]. Isoproterenol [1-(3,4-dihydroxyphenyl)-2-isopropylaminoethanol hydrochloride] is a synthetic catecholamine
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Cardiovasc Toxicol
and b-adrenergic agonist used in inframaximal doses to regulate heart function. However, the administration of isoproterenol in surpramaximal doses could induce severe stress in the myocardium through the depletion of the cardiomyocytes energy reserve which is resulting in irreversible cellular injury and ultimately infract like necrosis [3, 4]. Many synthetic drugs are used for the management of heart attack but they are not free from side effects. Hence, several studies have focused on identifying new therapeutic approaches to prevent myocardial infarction. Recently, greater attention has been focused on the phenols as effective bioactive compounds that protect cells or molecules from myocardial damage. In this regard, hydroxytyrosol (HT), a natural phenol present in large amounts in all parts of olive trees (Olea europaea), has been found to be a potent antioxidant and hypocholesterolemic agent in various animal models of disease including dyslipidemia, atherosclerosis and diabetes [4–6]. Polyphenols are excellent cardioprotectors. Hence, we suggested that HT may have a cardioprotective effect. The purpose of the current study was designed to investigate the protective effect of the oral pretreatment of HT on experimentally induced myocardial infarction in Wistar rats.
Materials and Methods Chemicals Lipase kit was purchased from Biolabo reagents France, HT [2-(3,4-dihydroxyphenyl)ethanol] was obtained from Cayman Chemical (Ann Arbor, MI, USA); the purity was C98 %. Isoproterenol hydrochloride powder was obtained from Sigma-Aldrich, St. Louis, USA. Angiotensin-converting enzyme (ACE) kit was purchased from Trinity, UK. The remaining chemicals used were of analytical grade.
subcutaneously at an interval of 24 h for 2 days to induce experimental myocardial infarction [7]. Animals were killed 48 h after the first dose of isoproterenol. Experimental Protocols After acclimatization, the animals were randomly divided into the following groups consisting of eight rats each: Group I: (Control) rats received standard laboratory diet and drinking saline water ad libitum and served as a control; Group II: (Isop) rats received saline water for 7 days and at the 6th day subcutaneously injected with isoproterenol (100 mg/kg, subcutaneously injected, once at an interval of 24 h for two consecutive days); Group III: (Isop ? HT1) rats pretreated with hydroxytyrosol (2 mg/kg bw; gastric gavages, respectively) for 7 days and at the 6th day subcutaneously injected with isoproterenol (100 mg/kg bw) for two consecutive days; Group IV: (Isop ? HT2) rats received HT (5 mg/kg bw) for 7 days and were injected subcutaneously with isoproterenol (100 mg/kg bw) on day 6th and 7th. All rats are fasted overnight but had free access to water at the last administration of the drug. After the 7-days induction, the animals were weighted and killed by decapitation in order to minimize the handling stress, and the trunk blood collected. The serum was prepared by centrifugation (15009g, 15 min, 4 °C), frozen and stored at -20 °C until analysis. Immediately after killing, the heart was excised out, washed with saline and fixed in fixed in a Bouin solution for 24 h and then embedded in paraffin. Sections of 5 lm thickness were stained with hematoxylin–eosin. The slides were photographed with an Olympus U-TU1X-2 camera connected to an Olympus CX41 microscope (Tokyo, Japan).
Animals
Measurement of Blood Pressure by Noninvasive Method
The essays of this study were conducted on adult male Wistar rats, weighting 260 ± 10 g, which were obtained from the local Central Pharmacy, Tunisia. All rats were kept in an environmentally controlled breeding room (temperature: 20 ± 2 °C; humidity: 60 ± 5 %; 12-h dark/light cycle) where they had standard diets and free access to tap water. The experimental protocols were conducted in accordance with the guide for the care and use of laboratory animals issued by the University of Sfax, Tunisia, and approved by the Committee of Animal Ethics.
Twenty-four hours after the second injection of isoproterenol, blood pressures were measured using noninvasive blood pressure Monitor (CODATM Surgical Monitor, USA) which consists of placing a cuff on the animal’s tail to occlude the blood flow. The pressure was raised and then slowly released. The cuff pressure when the pulse signal reappears is intended as the systolic pressure. The cuff pressure when the pulse signal level recovers its initial level is intended as diastolic pressure.
Induction of Experimental Myocardial Infarction
Electrocardiography
Isoproterenol was dissolved in normal saline and injected to rats (100 mg/kg body weight) and injected
Twenty-four hours after the second dose of isoproterenol, rats of all the groups were anesthetized with ketamine
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Cardiovasc Toxicol
hydrochloride (100 mg/kg bw) intraperitoneally. Needle electrodes were inserted under the skin of the animals in lead II position. ECG recordings were made using veterinary electrocardiograph (ECG VET 110, Biocare, China).
by 17 and 37 %, respectively, as compared to untreated myocardial infarcted rats. Electrocardiogram Patterns of Normal and Experimental Animals
Biochemical Analysis After killing, the heart was dissected out, immediately washed in ice-cold saline and a homogenate was prepared in 0.1 M Tris–HCl buffer (pH 7.4). Homogenate was centrifuged, and supernatant was used for the assay of marker enzymes. The collected serum was used for the determination of serum activity of lipase and ACE, the cardiac marker enzymes as creatinine kinase-MB (CK-MB), lactate dehydrogenase (LDH), alanine transaminase (ALT) and troponin-I rates were measured in frozen aliquots of serum by standardized enzymatic procedures using commercial kits from (Biolabo, France) on an automatic biochemistry analyzer (Vitalab Flexor E, USA) using commercially available standard enzymatic kits (Biolabo, France). Serum lipids level of triglycerides (TG), total cholesterol (T-Ch), high density lipoprotein-cholesterol (HDL-c) and low density lipoprotein-cholesterol (LDL-c) were measured using the corresponding commercial kits (Biolabo, France) on an automatic biochemistry analyzer (Kenza, France) at the clinic pathological laboratory of Sidi Bouzid Hospital. Serum LDL-cholesterol concentration was determined according to formula: LDL_cholesterol = Total cholesterol_(Triglycerides/5) - (HDL_cholesterol) performed by Friedewal et al. [8].
Control animals showed normal ECG pattern with normal heart rate (375 ± 13.92 bpm). While infarcted rats evidenced a significant (P \ 0.05) increase in heart rate (430 ± 11.65 bpm) which became irregular, remarkable elevation of ST-segment (wave of Paradee) and QT interval along with no identifiable P wave as compared to the normal animals (Figs. 1, 2). However, oral pre- and cotreatment of isoproterenol-induced rats with 2 mg/kg of HT showed acceleration in heart rate (430 ± 12.34 bpm), remaining regular sinus with the appearance of a discrete ST-elevation. Else, an aspect of electrical alternating was observed (alternating between QRS ample and QRS less ample). Though, the HT2 group exhibited sinus rhythm not accelerated relative to healthy control (375 ± 10.31 bpm), with no ST-segment elevation, no alternating electric sinus and rare atrial premature as compared to groups II and III. Effect of Hydroxytyrosol on Hemodynamic Function As shown in Table 2, significant decreases in systolic, diastolic and mean arterial blood pressure were observed in isoproterenol-induced rats as compared to control group (P \ 0.05). The pre- and co-treatment of infarcted rats with HT at 2 and 5 mg/kg significantly restored arterial pressure in a dose-dependent pattern.
Statistical Analysis Data are presented as mean ± standard deviation (SD). Determinations were performed from eight animals per group, and differences were examined by a one-way analysis of variance (ANOVA) followed by the Fisher test (Stat View). *P \ 0.05 was considered statistically significant.
Results Effect of Hydroxytyrosol (HT) on Heart Weight to Body Weight Ration of Experimental Rats As shown in Table 1, there was no significant difference in the body weight between the groups observed. Isoproterenol-induced myocardial infarcted rats exhibited significant increases in heart weight and the heart weight to body weight ration as compared to control rats (P \ 0.05). However, in response to HT1 and HT2, the infarcted rats showed significant (P \ 0.05) decrease in the heart weight
Effect of Hydroxytyrosol on Serum Cardiac Markers Table 3 indicated the variation of CK-MB and LDH both in serum and heart tissues and ALT, troponin-T in serum of control and experimental rats. The rats induced with isoproterenol evidenced a significant (P \ 0.05) increase in the serum levels CK-MB, ALT, LDH and troponin-T by 66, 112, 70 and 317 %, respectively, as compared to control rats associated with significant decrease in myocardium LDH and CK-MB, whereas pre- and co-treatment with HT (5 mg/kg bw) daily for 7 days significantly (P \ 0.05) decreased the levels of serum cardiac markers better than the administrated dose of 2 mg/kg of HT in isoproterenolinduced rats compared to untreated infarcted rats. Histopathological Examination of Cardiac Tissues The histopathological examination of heart tissues showed that control group of rats evidenced a normal myofibrillar structure without any infarction edema and inflammatory
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Cardiovasc Toxicol Table 1 Effect of hydroxytyrosol pre- and co-treatment on heart weight, body weight and heart weight/body weight ratio in isoproterenolinduced myocardial infarction in rats Parameters
Control
Isop
Isop ? HT1
Body weight (g)
269.20 ± 9.83
271.20 ± 10.28
273.80 ± 10.44
Heart weight (g)
0.95 ± 0.10
Heart weight/body weight ratio
1.50 ± 0.09*
0.35 ± 0.02
0.55 ± 0.02*
Isop ? HT2 268.21 ± 8.70
,#
0.96 ± 0.11#,@
,#
0.36 ± 0.03#,@
1.24 ± 0.13*
0.45 ± 0.04*
Values are given as mean ± SD for groups of eight animals each HT1, 2 mg/kg bw of hydroxytyrosol; HT2, 5 mg/kg bw of hydroxytyrosol Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group of rats. @P \ 0.05 significant differences compared to HT1 group
0.5 *
0.45
ST-Segment (mV)
0.4 0.35 0.3 0.25 0.2 0.15
*#
0.1
#@
0.05 0
79 %, respectively, with notable decrease in serum HDL-c by 26 % when compared to normal control animals (Table 4), whereas the administration of HT at 2 and 5 mg/ kg to infarcted rats induced a significant decrease in pancreatic lipase activity by 35 and 42 %, respectively, accompanied with remarkable decrease in T-Ch, TG, LDL-c by (13, 23 and 24 %) and (21, 35 and 39 %), respectively, with considerable increase in HDL-c by 19 and 29 %, respectively, as compared to isoproterenol-induced rats alone (P \ 0.05). Effect of Hydroxytyrosol on Serum ACE Activity
Control
Isop
Isop+HT1
Isop+HT2
Groups
Fig. 1 Effect of hydroxytyrosol on ST-segment elevation (mV) in the ECG (recorded from limb lead II) in normal control, isoproterenol alone injected and treated rats. Values are given as mean ± SD for group of eight rats. Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
Figure 5 showed that the ACE activity in serum of untreated infarcted rats underwent a significant increase by 67 % as compared to normal control animals (P \ 0.05). However, treatment with HT at doses of 2 and 5 mg/kg induced a remarkable decrease of ACE activity by 28 and 36 %, respectively, in serum of isoproterenol-induced myocardial infarction in rats.
Discussion cells (Fig. 3a). However, tissues from isoproterenol-treated rats revealed widespread myocardial structure disorder and subendocardial necrosis with loss of transverse striation and obvious leukocyte infiltration (Fig. 3b) as compared to control group, whereas pretreatment with HT (2 and 5 mg/ kg, respectively) exhibited marked improvement in isoproterenol-induced myocardial degeneration and inflammatory cells infiltration (Fig. 3c, d). Effect of Hydroxytyrosol on Serum Lipase Activity and Lipids Profile As shown in Fig. 4, the isoproterenol-induced group underwent a significant (P \ 0.05) increase in serum pancreatic lipase activity by 89 % which led to remarkable rise in serum levels of T-Ch, TG and LDL-c by 30, 67 and
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HT (3,4-dihydroxyphenylethanol) is an amphipathic molecule that is derived from the hydrolysis of oleuropein. Oleuropein hydrolysis takes places during olive maturation, as well as during olive oil storage [9, 10]. This phenolic bioactive compound has been proved to be endowed with several pharmaceutical properties such as antioxidant, anti-inflammatory, antithrombotic, antidiabetic and hypolipidemic effects [11–14]. In the current study, we explored, for the first time, the therapeutic efficacy of HT in rats with myocardial infarction. A subcutaneous injection of a supramaximal dose of isoproterenol was a consequence of electrocardiographic (ECG), biochemical and structural changes in the heart as mimetic to that occurs in patients with myocardial infarction. Changes in ECG pattern are commonly used for the diagnosis of acute
Cardiovasc Toxicol
Fig. 2 Effect of hydroxytyrosol on electrocardiographic (ECG) pattern in normal and experimental rats. ECG pattern of normal control group showing normal cardiograph. ECG pattern of isoproterenol (100 mg/kg)-induced group rats showing pathological changes such as ST-segment elevation. Electrocardiogram pattern
Table 2 Hemodynamic parameters in different experimental groups
Parameters
of HT (2 mg/kg)-treated isoproterenol-induced group rats showing minimized ST-segment elevation with aspect of electrical alternating. ECG pattern of HT (5 mg/kg) pre- and co-treated isoproterenolinduced group rats showing almost normal cardiograph without any elevation in ST-segment
Control
Isop
Isop ? HT1
Isop ? HT2
SAP (mmHg) DAP (mmHg)
128 ± 5.56 83.40 ± 3.04
88.20 ± 4.76* 56 ± 3.80*
104.40 ± 4.03*,# 69.80 ± 4.81*,#
122 ± 5.24#,@ 79.60 ± 4.15#,@
MAP (mmHg)
105.70 ± 4.13
72.12 ± 1.51*
87.10 ± 2.82*,#
100.80 ± 4.57#,@
HR (bpm)
375 ± 13.92
430 ± 11.65*
375 ± 10.31#,@
430 ± 11.65*
SAP systolic arterial blood pressure, DAP diastolic arterial blood pressure, MAP mean arterial blood pressure, HR heart rate Values are given as mean ± SD for group of eight animals each. Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences to diabetic rats treated with HT1
Table 3 Effect of hydroxytyrosol (HT) on cardiac markers in serum and heart tissues
Groups
Control
Isop
Isop ? HT1
Isop ? HT2
Serum ALT (UI/L)
41.4 ± 4.27
88 ± 4.01*
62.40 ± 6.24*,# ,#
54.60 ± 9.65*,# 543.2 ± 65.72#,@
LDH (UI/L)
519.8 ± 37.31
883.2 ± 40.61*
671.6 ± 63.38*
CK-MB (UI/L)
21.70 ± 0.96
36.16 ± 1.21*
25.65 ± 0.71*,#
22.72 ± 1.40#,@
0.57 ± 0.06
2.38 ± 0.33*
,#
0.76 ± 0.13*
0.53 ± 0.05#,@
7.31 ± 0.53
4.28 ± 0.21*
5.67 ± 0.47*,#
6.87 ± 0.32#,@
0.97 ± 0.16*
,#
2.07 ± 0.24#,@
Troponin-T (ng/mL) Heart LDH (lKat/mg) CK-MB (lKat/mg)
2.45 ± 0.14
1.45 ± 0.13*
Alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB) and serum troponin-T level of control and experimental groups of rats. CK-MB and LDH in the heart tissue are expressed in lkat/mg of proteins. Values are given as mean ± SD for group of eight animals each Values are statistically presented as follows: *P \ 0.05 significant differences compared to controls. P \ 0.05 significant differences compared to isoproterenol group @P \ 0.05 significant differences to diabetic rats treated with HT1
#
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Cardiovasc Toxicol
Fig. 3 Pathological changes of myocardial tissue (H&E 9500). a Control group showing normal myocardial histology, clear transverse striations and no inflammatory cell infiltration. b Isoproterenol group showing myocardial cells necrosis, separation of cardiac myofibrillar and large
Serum lipase activity (UI/L)
180
*
160 140 *#
120
*#@
100 80 60 40 20 0
Control
Isop
Isop+HT1
Isop+HT2
Groups
Fig. 4 Effect of hydroxytyrosol on serum lipase activity in normal and experimental rats. Values are given as mean ± SD for group of eight rats. Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
myocardial infarction. Rats treated with isoproterenol showed a marked elevation of ST-segment with remarkable decrease in P wave indicative of myocardial ischemia and infarction in accordance with previous study [15]. The alteration of ECG pattern could be related to the consecutive loss of integrity of cell membrane in injured myocardium [16]. The supramaximal dose of isoproterenol also injected increased heart rate (tachycardia) due to involvement of baroreceptor reflex-mediated changes in autonomic nerve activity [17], whereas pre- and co-treatment with HT
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inflammatory cells infiltration. c Isop ? HT1 (2 mg/kg)-treated group showing few inflammatory cell infiltration and improvement of myocardium necrosis. d Isop ? HT2 (5 mg/kg) showing normal myocardial arrangement, clear transverse striations and little few inflammatory cells
(5 mg/kg) significantly reduced the abnormalities observed in the ECG of isoproterenol-induced rats. Accordingly, it has been demonstrated that HT improved the cardiac disturbances enhanced by doxorubicin by significantly reducing the percentage of altered mitochondria also enhancing the mitochondrial electron transport chain and antioxidant defense system [18]. Several previous studies have demonstrated that the myocardium injury induced by isoproterenol in experimental rats is strongly associated with deterioration of hemodynamic and LV contractile function [14–17]. While, HT pre- and co-treatment showed significant improvement in systolic and diastolic arterial pressure (SAP and DAP, respectively) and subsequent increase in mean arterial pressure (MAP), a marker of after load. Serum cardiac troponins and cytosolic enzyme activities such as CK-MB, LDH and AST are very sensitive and specific diagnostic markers of acute myocardial infarction [19, 20]. Our results confirmed previous studies reporting that isoproterenol induced significant increases in serum troponin-T level and CK-MB, LDH and AST activities which reflected the severe damage in myocardium tissues [20, 21]. HT pre- and co-treatment reduced the activity of the marker enzymes in serum of isoproterenol-induced rats. It confirmed that HT could maintain membrane integrity, so restricting the leakage of these enzymes. Previous study clearly evidenced that administration of HT to normal rats showed no toxicity effect in tolerant dose, and there was no significant difference between
Cardiovasc Toxicol Table 4 Effect of hydroxytyrosol (HT) on serum lipids profile
Groups
Control
Isop
Isop ? HT1
T-Ch
1.81 ± 0.12
2.35 ± 0.10*
2.04 ± 0.16*,#
1.86 ± 0.05#,@
1.20 ± 0.10*
,#
0.77 ± 0.11#,@
,#
0.92 ± 0.03*,#,@
,#
0.79 ± 0.02#,@
TG
0.72 ± 0.05
LDL-c
0.84 ± 0.06
HDL-c
0.82 ± 0.05
1.50 ± 0.14* 0.61 ± 0.05*
Isop ? HT2
0.92 ± 0.11* 1.13 ± 0.18* 0.72 ± 0.04*
Total cholesterol (T-ch), triglycerides (TG), low density lipoprotein-cholesterol (LDL-c) and high density lipoprotein-cholesterol (HDL-c) in serum. Values are given as mean ± SD for group of eight rats Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. # P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
ACE activity in serum (UI/L)
90 *
80 70
*#
60
#@
50 40 30 20 10 0
Control
Isop
Isop+HT1
Isop+HT2
Groups
Fig. 5 ACE activity in serum of normal and experimental rats. Values are given as mean ± SD for group of eight rats. Statistically, values are represented as follows: *P \ 0.05 significant differences compared to controls. #P \ 0.05 significant differences compared to isoproterenol group. @P \ 0.05 significant differences compared to isoproterenol-treated group with 2 mg/kg of HT
normal rats treated with HT and untreated normal group in all tested parameters such as heart weight, ratio, cardiac enzymes markers, oxidative stress statute and myocardium histology [18]. Our histopathological findings of the isoproterenol-induced myocardial infarction showed large infarcted zone with edema, necrosis and obvious separation of cardiac myofibrillar and inflammatory cells which explained the ST-segment elevation observed in the ECG findings. The encroachment of the injured myocardium by neutrophils during ischemia could be considered as the major source of free radicals [22]. However, HT pre- and co-treatment (2 and 5 mg/kg, respectively) showed clear protective effects in isoproterenol-treated groups. Furthermore, lipids have a major role in cardiovascular diseases through the development of hyperlipidemia, hence increasing atherosclerosis risks. Else, it could lead to modify the composition, structure and stability of the cellular membranes. In the present study, a significant increase in serum pancreatic lipase activity was observed which led to
considerable rise in serum T-Ch, TG and LDL-c and remarkable decreased in HDL-c in isoproterenol-induced myocardial infarcted rats. These changes in lipids profile could be attributed to cardiac cyclic adenosine monophosphate that enhanced lipid biosynthesis during the hypoxic condition in isoproterenol-induced myocardial infarcted rats [23]. In fact, lipase is secreted from the pancreas, transported to small intestine and hydrolyzes triglycerides nonabsorbable into absorbable monoglycerides and free fatty acids absorbable by small intestine. Hence, inhibition of lipase activity by HT explains the significant reduced uptake of triglycerides from the circulation of infarcted rats which is confirmed in our previous study [24]. Queenthy and John [7] revealed the positive correlation between the risk of developing ischemic heart disease and serum LDL-c level. In this regard, previous investigations have demonstrated that HT can correct dyslipidemia, improve triglyceride metabolism, enhance endothelial function, increase antioxidant potential and decrease low density lipoprotein-cholesterol (LDL-c), which is an important cardiovascular diseases risk factor due to its role in atherosclerosis [25, 26]. Accumulating evidence suggests that the cardiac rennin–angiotensin system (RAS) is activated during the LV remodeling process after acute myocardial infarction [27, 28]. Our data evidenced that serum ACE activity of isoproterenol-induced rats underwent a significant increase accompanied with elevated heart weight ratio (hypertrophy) indicative of ventricular remodeling process. It includes infarct expansion as well as compensatory reactive hypertrophy and dilation of the noninfarcted LV [28]. Preand co-treatment of isoproterenol-induced myocardial infarction in rat at moderate dose of HT caused a significant decrease of serum ACE activity with marked reduce in heart weight ratio indicative of cardioprotective effect preventing the increased risk of infarct expansion and LV remodeling that follows myocardial infarction. Actually, there is evidence from several experimental studies that the cardiac tissue-type rennin–angiotensin system is activated after myocardial infarction and failure [28, 29]. However, ACE inhibition has been revealed to prevent cardiac
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remodeling and to prolong survival in experimental myocardial infarction. This process has been related to the reduced levels of circulating and tissue angiotensin II which is involved in the development of vascular and ventricular remodeling. Hence, ACE inhibitor increases tissue bradykinin accumulation, and the bradykinin has antigrowth effects and reduces vasomotor [30]. In conclusion, the present study provides experimental evidence that oral pre- and co-treatment with HT (2 and 5 mg/kg) evidence cardioprotective effect in isoproterenolinduced myocardial infarction in rats by improving heart weight, lipids profile and cardiac dysfunctional markers as well as demonstrating the beneficial role of HT as ACE inhibitor to prevent cardiac remodeling and failure. Acknowledgments This research was supported by the Tunisian Ministry of Higher Education and Scientific Research and the Tunisian Ministry of Public Health. Authors wish to thank Mrs. Sadok Slema, Mbarek Nasri and Chedly Tmar for their assistance and cooperation. Conflict of interests
The authors declare no conflict of interest.
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