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Endothelin receptor antagonist BQ-123 ameliorates myocardial ischemic-reperfusion injury in rats: A hemodynamic, biochemical, histopathological and electron microscopic evidence
Biomedicine & Pharmacotherapy 64 (2010) 639–646
Original Article
Endothelin receptor antagonist BQ-123 ameliorates myocardial ischemic-reperfusion injury in rats: A hemodynamic, biochemical, histopathological and electron microscopic evidence S.N. Goyal, S. Bharti, S. Arora, M. Golechha, D.S. Arya * Cardiovascular Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 January 2010 Accepted 16 June 2010 Available online 21 July 2010
We investigated the effect of BQ-123, a selective endothelin-A (ETA) receptor antagonist in ischemiareperfusion (IR) induced myocardial infarction (MI) with and without endothelin-1 (ET-1) challenge. MI was produced in rats by occlusion of left anterior descending coronary artery for 40 min and reperfusion for 120 min. ET-1 was administered immediately prior to coronary occlusion whereas vehicle or BQ-123 was administered 20 min after the occlusion. IR control group exhibited marked hemodynamic changes along with significant impairment of left ventricular functions. In addition, oxidative stress was increased, as evidenced by marked reduction in the activities of antioxidants and cardiac injury markers in myocardium. Furthermore, light microscopic and ultrastructural changes revealed myocardial necrosis, edema and inflammation. Prior administration of ET-1 acts synergistically with IR injury and further aggravates the impairment of ventricular functions, increased percent infarct area and decreased antioxidant levels. However, treatment with BQ-123 (1 mg/kg, IV) with or without ET-1 caused significant improvement in cardiac functions, percent infarct area, decreased malonaldehyde level, restored myocardial enzymes activities and maintained the redox status of the myocardium as compared to IR control group. Further, histopathological and ultrastructural studies reconfirmed the protective action of BQ-123. The results of present study suggest that ET-1 acting via ETA receptor may exaggerate myocardial damage produced by IR injury and selective blockade of ETA receptor by BQ-123 might offer potential cardioprotective action. ß 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Endothelin BQ-123 Oxidative stress Myocardial infarction Ischemia-reperfusion injury
1. Introduction Ischemia-reperfusion (IR) injury, as a result of coronary occlusion, initiates a sequel of cellular events which leads to impaired cardiac performance and complications resulting in irreversible cellular injury and death [1,2]. Many mediators, either of exogenous or endogenous origin, contribute to the pathogenesis of myocardial ischemic insult [3]. Endothelin (ET), which acts as one of the mediator, has been found to be elevated in patients of acute myocardial infarction (MI) [4]. ETs are the most potent vasoconstrictors and powerful mitogens, belong to family of three structurally related and distinctive isoforms ET-1, ET-2 and ET-3. ET-1, which is most abundant isoform in cardiovascular system, is released from cardiomyocytes and vascular cells on account of stimulus such as hypoxia or shear stress. Ischemic insult has been found to be one of the contributing factors for 2-6-fold increase in ET-1 release [5]. This increased biosynthesis of ET during ischemia
* Corresponding author. Tel.:+91 11 26594266; Fax: +91 11 26584121. E-mail address: [email protected] (D.S. Arya). 0753-3322/$ – see front matter ß 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2010.06.001
is considered as a major candidate for the low coronary re-flow phenomenon [6]. Various preclinical and clinical studies have shown significantly elevated levels of ET-1 during various other cardiovascular complications such as unstable angina, atherosclerosis and congestive heart failure [7,8]. The vasoconstrictor effect of ET-1 is mainly mediated through ETA and partly by ETB receptors [9]. Pro-inflammatory actions such as production of neutrophils, cytokines release from macrophages or monocytes leading to microcirculatory and tissue damage are also mediated through ETA receptors [10]. So as to reverse the pro inflammatory and other deteriorating effects of ET on myocardium, there have been growing evidences of ET to be used as target for therapeutic intervention. This approach resulted in development of specific ETA receptor antagonist or mixed ETA/ETB receptor antagonists. ETA receptor antagonist such as LU135252 and mixed ETA/ETB receptor antagonist bosentan has been reported to reduce infarct size following ischemia and reperfusion [11,12]. Moreover, ET receptor antagonists have also been shown to decrease mortality, improves myocardial performance and restoration of hemodynamics functions in experimental models of heart failure [5]. Conversely, there are also some experimental studies where an
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ETA receptor antagonist and mixed ETA/ETB receptor antagonists does not show any cardio protection [13,14]. BQ-123 a cyclic pentapeptide is a selective antagonist of ETA receptor and is used frequently in preclinical research and clinical trials of ET [15]. Furthermore, BQ-123 has also reduced infarct size in canine model of MI [16]. Therefore, keeping in view the aforementioned factors, the goal of present study was to evaluate the effect of BQ-123, a selective ETA receptor antagonist, in the absence and presence of ET-1 on hemodynamic, biochemical, infarct size, histopathological, and ultrastructural changes in IR model of MI and to understand the molecular mechanisms of its potential cardioprotective effects. In addition, we also investigated whether exogenous administration of ET-1 aggravates myocardial injury or not. 2. Materials and methods 2.1. Animals Male Wistar rats, weighing 150–200 g, were used in the study. Animals were obtained from the Central Animal House Facility of All India Institute of Medical Sciences, New Delhi, India. They were kept under standard laboratory conditions at 25 2 8C, relative humidity 60 15% and natural light-dark photoperiod. Commercial pellet diet (Ashirwad Industries Ltd., Chandigarh, India) and tap water were provided ad libitum. Animals were maintained in polypropylene cages, each containing a maximum of four animals. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee and conformed to the Indian National Science Academy (INSA) guidelines for the use and care of experimental animals. 2.2. Chemicals BQ-123 was purchased from Calbiochem, USA. Creatine kinase (CK-MB) isoenzyme detection kit was purchased from Logotech India Pvt. Ltd (Delhi, India). All other chemicals used in this study were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO, USA).
2.3.4. Group 4: IR-ET- treated group. The rats were subjected to a protocol of 40 min LAD coronary artery ligation followed by 120 min reperfusion. ET-1 (dissolved in phosphate buffered saline) was administered at (100 pmol/kg; intravenously), as slow bolus immediately before the left anterior descending coronary artery occlusion. The number of animals studied in this group was 25. 2.3.5. Group 5: IR+ET+BQ-123- treated group. The rats were subjected to a protocol of 40 min LAD coronary artery ligation followed by 120 min reperfusion. ET-1 was administered at (100 pmol/kg; IV), as slow bolus immediately prior to coronary artery occlusion whereas BQ-123 (1 mg/kg in 5% NaHCO3, intravenously), was injected at as slow bolus after 20 min of occlusion. The number of animals studied in this group was 23. The dose selected of BQ-123 was based on the previous studies [17]. The experimental animals were examined at regular intervals throughout the course of the study and any changes in body weight and/or food and water intake were noted. 2.4. Myocardial ischemia-reperfusion procedure The detailed surgical procedure for LAD coronary artery occlusion-reperfusion and recording of hemodynamic parameters has been described in our previous study [2]. Myocardial ischemia was induced by one stage occlusion of the LAD for 40 min and reperfused for a period of 120 min. At the end of reperfusion period, all animals were sacrificed, and their hearts were excised and processed for biochemical, histopathological, ultrastructural and for infarct size (triphenyl tetrazolium chloride (TTC) staining) determination. For biochemical analyses, hearts were removed and stored in liquid nitrogen, whereas they were fixed in 10% buffered formalin for light microscopy studies. Small pieces of myocardial tissue (approximately 1–2 mm thick) were also immediately fixed in ice-cold Karnovsky’s fixative for 10–12 h for ultrastructural studies using electron microscopy. 2.5. Biochemical estimation
2.3. Experimental Protocol The animals were randomly divided into five groups. 2.3.1. Group 1: Sham group. The animals were subjected to the entire surgical procedure and thread was passed beneath the coronary artery but the left anterior descending (LAD) coronary artery was not ligated. The number of animals studied in this group was 18. 2.3.2. Group 2: IR-control group. The rats were subjected to 40 min LAD coronary artery ligation followed by 120 min of reperfusion induced myocardial injury. Vehicle was administered intravenously, as slow bolus 20 min after the left anterior descending coronary artery occlusion. The number of animals studied in this group was 23. 2.3.3. Group 3: IR- BQ-123- treated group. The rats were subjected to a protocol of 40 min LAD coronary artery ligation followed by 120 min reperfusion. BQ-123 was administered at (1 mg/kg in 5% NaHCO3, intravenously), as slow bolus 20 min. after the left anterior descending coronary artery occlusion. The number of animals studied in this group was 23.
A 10% homogenate of myocardial tissue was prepared in icechilled phosphate buffer (50 mM, pH 7.4), and an aliquot was used to estimate malondialdehyde (MDA), according to the method described by Ohkawa et al. [18], and reduced glutathione (GSH) content was measured by the method published by Moron et al. [19]. The homogenate was centrifuged at 5000 rpm for 20 min at 4 8C, and the supernatant was assayed for lactate dehydrogenase (LDH) [20], catalase [21], and superoxide dismutase (SOD) [22] activity, as well as protein content [23]. Creatine kinase-MB (CKMB) isoenzyme was estimated spectrophotometrically using a kit from Logotech, India. 2.6. Determination of infarct size The infarct size was determined as previously described by Singh et al. [24]. At the end of reperfusion period, monastral blue (0.5 ml/kg) was injected into the left atrium over 30 s to determine the in vivo area at risk. TTC staining was used to distinguish between viable and infarcted myocardium. At least six hearts from each group were examined by infarct size determination. 2.7. Light microscopy and ultrastructural studies Histology and ultrastructural studies were performed by methods described previously Loh et al. [25].
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2.8. Statistical analysis All numerical data in text, figures and tables are presented as mean S.D. Statistical analysis was performed by one-way analysis of variance (Anova) or repeated measures Anova when data were compared at different time points within a study group and for time courses between study groups, followed by the Bonferroni post-hoc test. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Mortality An overall mortality of 12% was observed during the study protocol. The loss of animals was due to ventricular arrhythmias during ischemia reperfusion period, due to excessive bleeding, or improper cannulation of blood vessels during surgery. However, no mortality was observed in sham group. 3.2. Hemodynamic parameters As depicted in Fig. 1, the IR-control group results in MAP of 113.4 10.42 mm Hg at 0 min which decreases significantly throughout 120 min of IR period and was finally 77.2 14.07 mm Hg (P < 0.001) as compared to sham group. In addition, administration of ET-1 to IR rat results in significant (P < 0.01) increase in MAP of 151 10.26 mm Hg as compared to IR control group. This increase in MAP elicited by ET-1 was significantly (P < 0.01) attenuated by BQ123 treatment. However, there was no significant difference in the pre occlusion baseline values of the sham (120.4 9.82 mm Hg) and alone BQ-123 (131.3 6.25 mm Hg) treated group. Whereas, IR injury caused significant decrease in HR and ET-1 with or without BQ123 had no significant changes on HR (data not shown). As compared with sham group, IR-control rats and ET-1 treated rats showed left ventricular dysfunction as indicated by a significant increase in LVEDP from 3.00 0.21 to 7 0.19 mm Hg and 3.1 0.25 to 8 0.14 mm Hg respectively. In addition, BQ-123 with or without ET-1, significantly decreased LVEDP (2.8 0.35 mm Hg and 3.4 0.29 mm Hg) (Fig. 2) when compared with their respective groups. Furthermore, there was significant decrease in IR control group in +LVdP/dtmax from 2550 80 to 1800 110 mm Hg/S and in LVdP/dtmax from 1650 82.59 to 1150 87.90 mm Hg/S as compared to sham group (Fig. 3). Similarly, in case of ET-1 group there was also significant decrease in +LVdP/dtmax from 2500 89.20
Fig. 2. Time-course of changes in left ventricular end-diastolic pressure (LVEDP) during ischemia (40 min) and reperfusion (120 min) period in the different experimental groups. Data are expressed as mean S.D. values (n = 18/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: *P < 0.001 versus sham group, #P < 0.01 versus IR control, y P < 0.05 versus IR control.
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Fig. 3. Time-course of changes in maximum a) positive rate of developed left ventricular pressure (+LVdP/dtmax) b) negative rate of developed left ventricular pressure (LVdP/dtmax) during ischemia (40 min) and reperfusion (120 min) period in the different experimental groups. Data are expressed as mean S.D. values (n = 18/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: *P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.01 versus IR control.
Fig. 1. Time-course of changes in mean arterial pressure (MAP) during ischemia (40 min) and reperfusion (120 min) period in the different experimental groups. Data are expressed as mean S.D. values (n = 18/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: * P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.01 versus IR control.
to 1700 74.75 and in LVdP/dtmax from 1600 98.25 to 1000 88.10 mm Hg/S as compared to sham group. Administration of BQ-123 (1 mg/kg) after 20 min of ischemia significantly (P < 0.01) increased LVdP/dtmax as compared to IR-control group. However,
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hemodynamic parameters showed more statistically significant results in BQ-123 group (P < 0.01) as compare to BQ-123 plus ET-1 group (P < 0.05).
Table 1 Myocardial malondialdehyde (MDA), CK-MB isoenzyme and LDH levels in ischemia-reperfusion model of myocardial infarction in rats. Groups
MDA (nmol/g tissue)
CK-MB (IU/mg protein)
LDH (IU/mg protein)
Sham IR-control IR+ET-1 IR+BQ-123 IR+ET-1+BQ-123
68.2 3.8 76.2 5.3* 84.5 9.5y 74.2 11.7# 75.2 7.4@
164.2 24.3 92.5 24.8* 56.7 2.9y 143.4 8.8# 111.6 11.2@
75.23 14.19 52.40 14.30* 42.06 9.24y 67.07 6.52# 55.23 5.78@
3.3. Biochemical parameters Tables 1 and 2 display the activities of lipid peroxidation, cardiac injury marker enzymes and antioxidant levels in an IR model of MI. In IR control group, the activities of CK-MB and LDH decreased significantly (P < 0.001) from 164.2 24.3 to 92.5 24.8 IU/mg protein and from 75.23 14.19 to 52.40 14.30 IU/mg protein respectively as compared to sham group. Administration of ET-1 further aggravates the injury as compared to IR group. Parallely, MDA level also increases significantly (P < 0.001) in IR group (76.2 5.3 nmol/g tissue) as compared to sham group (68.2 3.8 nmol/g tissue). Treatment of BQ-123 to IR group with or without ET-1 causes statistically significantly (P < 0.01) decrease in lipid peroxidation and restored the activities of CK-MB isoenzyme and LDH in myocardium as compared to IR-control group. In addition, IR-control group showed a significant (P < 0.001) reduction in the activities of myocardial SOD (from 8.5 3.7 to 4.1 2.6 U/mg protein), catalase (from 20.9 4.3 to 13.9 3.6 U/mg protein) and GSH levels (from 2.42 0.71 to 0.73 0.13 mg/g tissue) as compared to sham group. The decreased in antioxidant content caused by myocardial IR injury was further aggravated by ET-1 treatment (P < 0.05). Rats treated with BQ-123 exhibited a significant increase in these antioxidants when compared with their respective control groups (P < 0.01). 3.4. Infarct size evaluation Table 3 summarizes the mean area at risk, which is a major predictor of infarct size in models of regional ischemia. In the ET-1 group, the percent infarct area (58.0 1.47) was significantly higher than IR group (51.2 1.22). In the BQ-123 treated group with or without exogenous ET-1 treatment, the percent infarct area was significantly lower at 45.4 2.6 (P < 0.05) and 41.0 1.82 (P < 0.01) respectively. The percentage of mean area at risk that actually proceeded to infarction was found to be significantly smaller in the rats that received BQ-123 than in those that were untreated (IRcontrol) and ET-1 applied alone groups. When ET-1 was injected in the presence of the ETA antagonist, the marked inhibitory effects on cardiovascular function were not observed when compared with ET-1 treatment alone. 3.5. Histopathological evaluation In Fig. 4, microscopic histology revealed that the non-infarcted myocardium in the sham group was characterized by an organized pattern and shows normal architecture (Fig. 4a). The IR control and ET-1 alone treated groups showed severe myocardial membrane damage, edema and infiltration of inflammatory cells as compared to sham control. Significant myonecrosis with fibroblastic proliferation and presence of inflammatory cells were observed in the both the groups as compared to sham (Fig. 4b and 4c). BQ-123 treatment decreased myonecrosis, inflammatory cells, vacuolar changes and edema (Fig. 4d). However, the degree of myocardial damage in BQ-123 plus ET-1-treated rats was severe than BQ-123 alone treated rats (Fig. 4e). 3.6. Ultrastructural evaluation Fig. 5 shows the TEM images of the myocardium of drug and vehicle treated groups. Normal cardiac architecture was seen in electron micrographs of rats in sham group (Fig. 5a). Whereas IRcontrol group shows extensive myonecrosis, loss of lipid droplets
MDA: malondialdehyde, CK-MB: creatine kinase-MB, LDH: lactate dehydrogenase. Data are expressed as mean SD values (n = 6/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: * P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.05 versus IR control, @ P < 0.01 versus ET + IR group.
Table 2 Myocardial antioxidant activities in ischemia-reperfusion model of myocardial infarction in rats. Groups
SOD (U/mg protein)
CAT (U/mg protein)
GSH (mg/g tissue)
Sham IR-control IR+ET-1 IR+BQ-123 IR+ET-1+BQ-123
8.5 3.7 4.1 2.6* 2.7 1.2y 6.8 2.7# 4.54 7.6@
20.9 4.3 13.9 3.6* 11.8 9.3y 17.9 3.9# 16.9 5.10@
2.42 0.71 0.73 0.13* 0.41 0.11y 1.92 1.37# 1.21 1.78@
SOD: superoxide dismutase; CAT: catalase; GSH: reduced glutathione. Data are expressed as mean S.D. values (n = 6/group). Significance was determined by oneway Anova or repeated measures Anova followed by Bonferroni Post-hoc test: * P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.05 versus IR control, @ P < 0.01 versus ET + IR group. Table 3 Mean area at risk and infarct area in rats of different experimental groups. Groups
Mean area at risk (%)
Infarct area (%)
IR-control IR+ET-1 IR+BQ-123 IR+ET-1+BQ-123
42.14 1.4 40.25 2.09 42.12 1.1 41.54 1.56
51.2 1.22 58.0 1.47 41.0 1.82* 45.4 2.6*,y
Data are expressed as mean S.D. values (n = 6/group). *P < 0.01 versus IR control, y P < 0.05 versus BQ-123 treated group.
along with significant disruption of myofilaments and Z band architecture (Fig. 5b). Similar changes were also observed in alone ET-1 group (Fig. 5c). However, BQ-123 treatment to IR rats shows normal ultrastructure with mild separation of cristae without swelling and vacuolation (Fig. 5d). Changes in the BQ-123 plus ET1-treated group were not as significant (Fig. 5e). 4. Discussion The present study demonstrated that, BQ-123, a selective ETA receptor antagonist exhibits a cardioprotective effect by improving cardiac functions, bolstering endogenous defense systems, reducing myocardial lipid peroxidation and preserving myofibril structure and morphology. In addition, BQ-123 significantly reduced infarct size and offers protection against IR and IR plus ET-1 induced myocardial injury. However, the co-treatment with ET-1 significantly attenuated BQ-123 induced cardioprotection. Therefore, we have provided direct evidence that the ETA receptor plays a significant role in regulating oxidative stress and subsequent tissue injury in IR-induced MI. To our knowledge, this is the first ever study conducted to report the cardioprotective effect of BQ-123 on the basis of hemodynamic, histopathological and ultrastructural changes in IR-induced MI. It has been widely documented that ET-1 has a detrimental effect on the myocardium following ischemic insult [26]. To annul
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Fig. 4. Light photomicrographs of representative rat heart sections (H&E, 200 from different experimental groups: a: sham group; b: IR control group; c: ET-1+ IR group; d: IR+ BQ-123 group; e: IR+ ET-1+ BQ-123 treated group.
this effect, we demonstrated that, a specific antagonist of the ETA receptor i.e. BQ-123 ameliorates the myocardial injury in rat model of MI. In corroboration with our findings, Ozdemir et al. [5] also reported that selective ETA receptor blockade reduces the infarct size and oxidant injury. Since blocking of ETB receptor does not preclude myocardial injury, this led to ratiocination that ETA receptor mediates the action of ET that lead to stimulation of inflammatory and thrombotic cascades leading to myocardial injury. Therefore we used specific receptor antagonist against the major form ET receptors present in human cardiomyocytes rather than using mixed ETA/ETB antagonists [27]. As no single hemodynamic variable can reliably anticipate the outcome of myocardial ischemia and the effectiveness of a therapeutic intervention, so cluster of hemodynamic indices were used to explain the cardiac functions. The present study demonstrated that ET-1 treatment increased arterial and ventricular dysfunction. Whereas, BQ-123 treatment prevented IR and ET1 plus IR-induced cardiac dysfunction as evidenced by restoration of mean arterial pressures. It also improved left ventricular
function by increasing inotropic (+LVdP/dtmax, marker of myocardial contraction) and lusitropic (LVdP/dtmax, marker of myocardial relaxation) states of the heart. In addition, it ameliorated IR and ET-1-induced increased in LVEDP, a marker of pre-load, which again reflects an improvement of left ventricular function. This finding is consistent with that of an earlier study by Cernacek et al. [28]. Moreover, bosentan, a mixed ETA and ETB receptor antagonist, significantly improved the left ventricular developed pressure, dP/ dtmax, end-diastolic pressure and coronary flow during reperfusion in isolated rat hearts subjected to global ischemia [6]. One possibility as postulated by Wang et al. [29] that these antagonists do possess vasodilation property and hence prevents the development of no-reflow phenomenon, which resulted in restitution of hemodynamic parameters. Furthermore, heart rate is not changed significantly in BQ-123 treated groups when compare with IR control group. This may also contribute to the beneficial effect of BQ-123 as a cardioprotective agent, as a small decrease in HR has been said to ameliorate the left ventricular filling. Parallel to our findings, Ozdemir et al. [5] has also reported
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Fig. 5. Electronmicrograph of representative rat myocardium sections (magnification 3500 ) from different experimental groups: a: sham group; b: IR control group; c: ET1+ IR group; d: IR+ BQ-123 group; e: IR+ ET-1+ BQ-123 treated group.
similar finding with BQ-123 treatment. Hence, our results suggest protective action of BQ-123 on the myocardium by improving cardiac functionality in spite of ET-1 administration in rats. From these findings it was also revealed that both endogenous and exogenous ET-1 significantly increases the infarct area. Moreover the activation of cardiac endothelial system due to ET-1 also results in expansion of myocardial infarct size ratio, which was evident from the present investigation also [30]. It is well established that post-acute MI, ET-1 levels are several times higher in the scar area in myocardium [31]. Therefore, administration of BQ-123 to IR and IR plus ET-1 groups significantly reduces the infarct area. It is interesting to recall that BQ-123 reduced the infarct size in canine model of coronary occlusion and reperfusion [16]. In contrast to above-mentioned studies there are also some reports where ET antagonists did not reduce infarct size in IR induced myocardial injury [32]. Apart from hemodynamic and infarct parameters, myocardium specific CK-MB isoenzyme and LDH activity has been employed as estimate of myocardial injury after coronary artery occlusion and
reperfusion [2]. In consonance with previously reported studies, we also observed a decrease in activities of these enzymes following IR. Besides, administration of ET-1 further declines the levels of these enzymes in ischemic and reperfusion injury. However, administration of BQ-123 significantly restored the activities of CK-MB isoenzyme and LDH in the myocardium following ET-1 challenge. Earlier, Singh et al. [24] has reported that, bosentan significantly restored CK-MB isoenzyme activity in IRinduced MI. Therefore, cardioprotection exerted by BQ-123 might be due to preservation of stability, cellular integrity and constraining the leakage of CK-MB isoenzyme and LDH through membranes. Lipid peroxidation is an important pathogenic event in myocardial injury and accumulation of lipid hydroperoxides reflects damage of the cardiac constituents. Production of reactive oxygen species (ROS) during ischemia and reperfusion leads to enhanced lipid peroxidation in the myocardium [33]. In present study, exogenously delivered ET-1 acts synergistically with IR induced oxidant injury and worsened the cardiac dysfunction
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observed after reperfusion, thereby suggesting that post-ischemic dysfunction results, at least in part, from ET-1 induced excessive MDA production in myocardium. Findings of the present study show that, administration of BQ-123 after 20 min of ligation significantly decreased the MDA content as compare to IR and IR plus ET-1 groups. Similarly, in accordance with our results, several other studies have also reported that an ETA antagonist improved the heart function, by decreased MDA content in the ischemic myocardium [5,34]. It is well known that IR produces free radical moieties, which initiate a cascade of events leading to the formation of superoxide anions that’s lead to endothelial dysfunction. Free radical scavenging antioxidant such as SOD, catalase and GSH are the first line of cellular defense systems in improving the myocardial oxidative imbalance [35]. The decrease activities of these antioxidants in the heart following ET-1 administration further elucidates excessive generation of superoxide and hydrogen peroxides, resulting in myocardial injury and also leads to ingestion of these endogenous antioxidants [5]. Similarly, many studies have demonstrated that in the event of IR, the tissue levels of SOD and/or catalase increases so as to protect cells from the detrimental effects of ROS [35]. In present study, we observed that decreased activities of SOD, catalase and GSH level in ET-1 challenged rats were significantly ameliorated by BQ-123 treatment. These findings are in accordance with Hong et al. [34], which also showed selective ETA receptor antagonist improves the imbalance in the anti-oxidant status in rat model of IR injury. Furthermore, coronary endothelial dysfunction has been prevented by SOD [36]. Therefore one of the possibilities of BQ-123 as a cardioprotective agent might be due to its antioxidant activity, which could exert a beneficial effect against pathological alterations caused by free radical in MI. To further elucidate the effect of BQ-123 on IR induced myocardial injury light microscopic study was performed. Histological study shows the myocardial injury in IR group and ET-1 treatment further aggravated the myocardial injury. Treatment with BQ-123 showed a well preserved normal morphology of cardiac muscle with no evidence of cellular influx, edema and were more effective in preserving tissue integrity as compared to IR and IR plus ET-1 groups which reconfirms the cardioprotective action of BQ-123. Singh et al. [24] reported similar type of protection with bosentan, a mixed ETA and ETB in IR induced MI. Likewise, TEM studies also demonstrated that BQ-123 decreases myonecrosis and showed normal ultrastructure of myocardium. These data further confirmed the cardioprotective role of BQ-123 in IR injury. In the light of these findings, finally it can be concluded that ET-1 acting via ETA receptor not only aggravates hemodynamic parameters but also accounts for the imbalance in antioxidant status, lipid peroxidation and increased infarct size along with the myocardial disorganization. Following administration of ETA receptor antagonist BQ-123 results in the improvement of the above said parameters and can also be used as an important pharmacological tool for salvaging myocardium following IR injury. Conflict of interest statement All authors have no conflict of interest. References [1] Jennings RB, Reimer KA. The cell biology of acute myocardial ischemia. Annu Rev Med 1991;42:225–46. [2] Mohanty IR, Arya DS, Gupta SK. Withania somnifera provides cardioprotection and attenuates ischemia-reperfusion induced apoptosis. Clin Nutr 2008;27:635–42. [3] Grover GJ, Sleph PG, Fox M, et al. Role of endothelin-1 and big endothelin-1 in modulating coronary vascular tone, contractile function and severity of ischemia in rat hearts. J Pharmacol Exp Ther 1992;263:1074–82.
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Original Article
Endothelin receptor antagonist BQ-123 ameliorates myocardial ischemic-reperfusion injury in rats: A hemodynamic, biochemical, histopathological and electron microscopic evidence S.N. Goyal, S. Bharti, S. Arora, M. Golechha, D.S. Arya * Cardiovascular Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110029, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 January 2010 Accepted 16 June 2010 Available online 21 July 2010
We investigated the effect of BQ-123, a selective endothelin-A (ETA) receptor antagonist in ischemiareperfusion (IR) induced myocardial infarction (MI) with and without endothelin-1 (ET-1) challenge. MI was produced in rats by occlusion of left anterior descending coronary artery for 40 min and reperfusion for 120 min. ET-1 was administered immediately prior to coronary occlusion whereas vehicle or BQ-123 was administered 20 min after the occlusion. IR control group exhibited marked hemodynamic changes along with significant impairment of left ventricular functions. In addition, oxidative stress was increased, as evidenced by marked reduction in the activities of antioxidants and cardiac injury markers in myocardium. Furthermore, light microscopic and ultrastructural changes revealed myocardial necrosis, edema and inflammation. Prior administration of ET-1 acts synergistically with IR injury and further aggravates the impairment of ventricular functions, increased percent infarct area and decreased antioxidant levels. However, treatment with BQ-123 (1 mg/kg, IV) with or without ET-1 caused significant improvement in cardiac functions, percent infarct area, decreased malonaldehyde level, restored myocardial enzymes activities and maintained the redox status of the myocardium as compared to IR control group. Further, histopathological and ultrastructural studies reconfirmed the protective action of BQ-123. The results of present study suggest that ET-1 acting via ETA receptor may exaggerate myocardial damage produced by IR injury and selective blockade of ETA receptor by BQ-123 might offer potential cardioprotective action. ß 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Endothelin BQ-123 Oxidative stress Myocardial infarction Ischemia-reperfusion injury
1. Introduction Ischemia-reperfusion (IR) injury, as a result of coronary occlusion, initiates a sequel of cellular events which leads to impaired cardiac performance and complications resulting in irreversible cellular injury and death [1,2]. Many mediators, either of exogenous or endogenous origin, contribute to the pathogenesis of myocardial ischemic insult [3]. Endothelin (ET), which acts as one of the mediator, has been found to be elevated in patients of acute myocardial infarction (MI) [4]. ETs are the most potent vasoconstrictors and powerful mitogens, belong to family of three structurally related and distinctive isoforms ET-1, ET-2 and ET-3. ET-1, which is most abundant isoform in cardiovascular system, is released from cardiomyocytes and vascular cells on account of stimulus such as hypoxia or shear stress. Ischemic insult has been found to be one of the contributing factors for 2-6-fold increase in ET-1 release [5]. This increased biosynthesis of ET during ischemia
* Corresponding author. Tel.:+91 11 26594266; Fax: +91 11 26584121. E-mail address: [email protected] (D.S. Arya). 0753-3322/$ – see front matter ß 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2010.06.001
is considered as a major candidate for the low coronary re-flow phenomenon [6]. Various preclinical and clinical studies have shown significantly elevated levels of ET-1 during various other cardiovascular complications such as unstable angina, atherosclerosis and congestive heart failure [7,8]. The vasoconstrictor effect of ET-1 is mainly mediated through ETA and partly by ETB receptors [9]. Pro-inflammatory actions such as production of neutrophils, cytokines release from macrophages or monocytes leading to microcirculatory and tissue damage are also mediated through ETA receptors [10]. So as to reverse the pro inflammatory and other deteriorating effects of ET on myocardium, there have been growing evidences of ET to be used as target for therapeutic intervention. This approach resulted in development of specific ETA receptor antagonist or mixed ETA/ETB receptor antagonists. ETA receptor antagonist such as LU135252 and mixed ETA/ETB receptor antagonist bosentan has been reported to reduce infarct size following ischemia and reperfusion [11,12]. Moreover, ET receptor antagonists have also been shown to decrease mortality, improves myocardial performance and restoration of hemodynamics functions in experimental models of heart failure [5]. Conversely, there are also some experimental studies where an
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ETA receptor antagonist and mixed ETA/ETB receptor antagonists does not show any cardio protection [13,14]. BQ-123 a cyclic pentapeptide is a selective antagonist of ETA receptor and is used frequently in preclinical research and clinical trials of ET [15]. Furthermore, BQ-123 has also reduced infarct size in canine model of MI [16]. Therefore, keeping in view the aforementioned factors, the goal of present study was to evaluate the effect of BQ-123, a selective ETA receptor antagonist, in the absence and presence of ET-1 on hemodynamic, biochemical, infarct size, histopathological, and ultrastructural changes in IR model of MI and to understand the molecular mechanisms of its potential cardioprotective effects. In addition, we also investigated whether exogenous administration of ET-1 aggravates myocardial injury or not. 2. Materials and methods 2.1. Animals Male Wistar rats, weighing 150–200 g, were used in the study. Animals were obtained from the Central Animal House Facility of All India Institute of Medical Sciences, New Delhi, India. They were kept under standard laboratory conditions at 25 2 8C, relative humidity 60 15% and natural light-dark photoperiod. Commercial pellet diet (Ashirwad Industries Ltd., Chandigarh, India) and tap water were provided ad libitum. Animals were maintained in polypropylene cages, each containing a maximum of four animals. The study protocol was reviewed and approved by the Institutional Animal Ethics Committee and conformed to the Indian National Science Academy (INSA) guidelines for the use and care of experimental animals. 2.2. Chemicals BQ-123 was purchased from Calbiochem, USA. Creatine kinase (CK-MB) isoenzyme detection kit was purchased from Logotech India Pvt. Ltd (Delhi, India). All other chemicals used in this study were of analytical grade and purchased from Sigma Chemicals (St. Louis, MO, USA).
2.3.4. Group 4: IR-ET- treated group. The rats were subjected to a protocol of 40 min LAD coronary artery ligation followed by 120 min reperfusion. ET-1 (dissolved in phosphate buffered saline) was administered at (100 pmol/kg; intravenously), as slow bolus immediately before the left anterior descending coronary artery occlusion. The number of animals studied in this group was 25. 2.3.5. Group 5: IR+ET+BQ-123- treated group. The rats were subjected to a protocol of 40 min LAD coronary artery ligation followed by 120 min reperfusion. ET-1 was administered at (100 pmol/kg; IV), as slow bolus immediately prior to coronary artery occlusion whereas BQ-123 (1 mg/kg in 5% NaHCO3, intravenously), was injected at as slow bolus after 20 min of occlusion. The number of animals studied in this group was 23. The dose selected of BQ-123 was based on the previous studies [17]. The experimental animals were examined at regular intervals throughout the course of the study and any changes in body weight and/or food and water intake were noted. 2.4. Myocardial ischemia-reperfusion procedure The detailed surgical procedure for LAD coronary artery occlusion-reperfusion and recording of hemodynamic parameters has been described in our previous study [2]. Myocardial ischemia was induced by one stage occlusion of the LAD for 40 min and reperfused for a period of 120 min. At the end of reperfusion period, all animals were sacrificed, and their hearts were excised and processed for biochemical, histopathological, ultrastructural and for infarct size (triphenyl tetrazolium chloride (TTC) staining) determination. For biochemical analyses, hearts were removed and stored in liquid nitrogen, whereas they were fixed in 10% buffered formalin for light microscopy studies. Small pieces of myocardial tissue (approximately 1–2 mm thick) were also immediately fixed in ice-cold Karnovsky’s fixative for 10–12 h for ultrastructural studies using electron microscopy. 2.5. Biochemical estimation
2.3. Experimental Protocol The animals were randomly divided into five groups. 2.3.1. Group 1: Sham group. The animals were subjected to the entire surgical procedure and thread was passed beneath the coronary artery but the left anterior descending (LAD) coronary artery was not ligated. The number of animals studied in this group was 18. 2.3.2. Group 2: IR-control group. The rats were subjected to 40 min LAD coronary artery ligation followed by 120 min of reperfusion induced myocardial injury. Vehicle was administered intravenously, as slow bolus 20 min after the left anterior descending coronary artery occlusion. The number of animals studied in this group was 23. 2.3.3. Group 3: IR- BQ-123- treated group. The rats were subjected to a protocol of 40 min LAD coronary artery ligation followed by 120 min reperfusion. BQ-123 was administered at (1 mg/kg in 5% NaHCO3, intravenously), as slow bolus 20 min. after the left anterior descending coronary artery occlusion. The number of animals studied in this group was 23.
A 10% homogenate of myocardial tissue was prepared in icechilled phosphate buffer (50 mM, pH 7.4), and an aliquot was used to estimate malondialdehyde (MDA), according to the method described by Ohkawa et al. [18], and reduced glutathione (GSH) content was measured by the method published by Moron et al. [19]. The homogenate was centrifuged at 5000 rpm for 20 min at 4 8C, and the supernatant was assayed for lactate dehydrogenase (LDH) [20], catalase [21], and superoxide dismutase (SOD) [22] activity, as well as protein content [23]. Creatine kinase-MB (CKMB) isoenzyme was estimated spectrophotometrically using a kit from Logotech, India. 2.6. Determination of infarct size The infarct size was determined as previously described by Singh et al. [24]. At the end of reperfusion period, monastral blue (0.5 ml/kg) was injected into the left atrium over 30 s to determine the in vivo area at risk. TTC staining was used to distinguish between viable and infarcted myocardium. At least six hearts from each group were examined by infarct size determination. 2.7. Light microscopy and ultrastructural studies Histology and ultrastructural studies were performed by methods described previously Loh et al. [25].
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2.8. Statistical analysis All numerical data in text, figures and tables are presented as mean S.D. Statistical analysis was performed by one-way analysis of variance (Anova) or repeated measures Anova when data were compared at different time points within a study group and for time courses between study groups, followed by the Bonferroni post-hoc test. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Mortality An overall mortality of 12% was observed during the study protocol. The loss of animals was due to ventricular arrhythmias during ischemia reperfusion period, due to excessive bleeding, or improper cannulation of blood vessels during surgery. However, no mortality was observed in sham group. 3.2. Hemodynamic parameters As depicted in Fig. 1, the IR-control group results in MAP of 113.4 10.42 mm Hg at 0 min which decreases significantly throughout 120 min of IR period and was finally 77.2 14.07 mm Hg (P < 0.001) as compared to sham group. In addition, administration of ET-1 to IR rat results in significant (P < 0.01) increase in MAP of 151 10.26 mm Hg as compared to IR control group. This increase in MAP elicited by ET-1 was significantly (P < 0.01) attenuated by BQ123 treatment. However, there was no significant difference in the pre occlusion baseline values of the sham (120.4 9.82 mm Hg) and alone BQ-123 (131.3 6.25 mm Hg) treated group. Whereas, IR injury caused significant decrease in HR and ET-1 with or without BQ123 had no significant changes on HR (data not shown). As compared with sham group, IR-control rats and ET-1 treated rats showed left ventricular dysfunction as indicated by a significant increase in LVEDP from 3.00 0.21 to 7 0.19 mm Hg and 3.1 0.25 to 8 0.14 mm Hg respectively. In addition, BQ-123 with or without ET-1, significantly decreased LVEDP (2.8 0.35 mm Hg and 3.4 0.29 mm Hg) (Fig. 2) when compared with their respective groups. Furthermore, there was significant decrease in IR control group in +LVdP/dtmax from 2550 80 to 1800 110 mm Hg/S and in LVdP/dtmax from 1650 82.59 to 1150 87.90 mm Hg/S as compared to sham group (Fig. 3). Similarly, in case of ET-1 group there was also significant decrease in +LVdP/dtmax from 2500 89.20
Fig. 2. Time-course of changes in left ventricular end-diastolic pressure (LVEDP) during ischemia (40 min) and reperfusion (120 min) period in the different experimental groups. Data are expressed as mean S.D. values (n = 18/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: *P < 0.001 versus sham group, #P < 0.01 versus IR control, y P < 0.05 versus IR control.
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Fig. 3. Time-course of changes in maximum a) positive rate of developed left ventricular pressure (+LVdP/dtmax) b) negative rate of developed left ventricular pressure (LVdP/dtmax) during ischemia (40 min) and reperfusion (120 min) period in the different experimental groups. Data are expressed as mean S.D. values (n = 18/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: *P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.01 versus IR control.
Fig. 1. Time-course of changes in mean arterial pressure (MAP) during ischemia (40 min) and reperfusion (120 min) period in the different experimental groups. Data are expressed as mean S.D. values (n = 18/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: * P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.01 versus IR control.
to 1700 74.75 and in LVdP/dtmax from 1600 98.25 to 1000 88.10 mm Hg/S as compared to sham group. Administration of BQ-123 (1 mg/kg) after 20 min of ischemia significantly (P < 0.01) increased LVdP/dtmax as compared to IR-control group. However,
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hemodynamic parameters showed more statistically significant results in BQ-123 group (P < 0.01) as compare to BQ-123 plus ET-1 group (P < 0.05).
Table 1 Myocardial malondialdehyde (MDA), CK-MB isoenzyme and LDH levels in ischemia-reperfusion model of myocardial infarction in rats. Groups
MDA (nmol/g tissue)
CK-MB (IU/mg protein)
LDH (IU/mg protein)
Sham IR-control IR+ET-1 IR+BQ-123 IR+ET-1+BQ-123
68.2 3.8 76.2 5.3* 84.5 9.5y 74.2 11.7# 75.2 7.4@
164.2 24.3 92.5 24.8* 56.7 2.9y 143.4 8.8# 111.6 11.2@
75.23 14.19 52.40 14.30* 42.06 9.24y 67.07 6.52# 55.23 5.78@
3.3. Biochemical parameters Tables 1 and 2 display the activities of lipid peroxidation, cardiac injury marker enzymes and antioxidant levels in an IR model of MI. In IR control group, the activities of CK-MB and LDH decreased significantly (P < 0.001) from 164.2 24.3 to 92.5 24.8 IU/mg protein and from 75.23 14.19 to 52.40 14.30 IU/mg protein respectively as compared to sham group. Administration of ET-1 further aggravates the injury as compared to IR group. Parallely, MDA level also increases significantly (P < 0.001) in IR group (76.2 5.3 nmol/g tissue) as compared to sham group (68.2 3.8 nmol/g tissue). Treatment of BQ-123 to IR group with or without ET-1 causes statistically significantly (P < 0.01) decrease in lipid peroxidation and restored the activities of CK-MB isoenzyme and LDH in myocardium as compared to IR-control group. In addition, IR-control group showed a significant (P < 0.001) reduction in the activities of myocardial SOD (from 8.5 3.7 to 4.1 2.6 U/mg protein), catalase (from 20.9 4.3 to 13.9 3.6 U/mg protein) and GSH levels (from 2.42 0.71 to 0.73 0.13 mg/g tissue) as compared to sham group. The decreased in antioxidant content caused by myocardial IR injury was further aggravated by ET-1 treatment (P < 0.05). Rats treated with BQ-123 exhibited a significant increase in these antioxidants when compared with their respective control groups (P < 0.01). 3.4. Infarct size evaluation Table 3 summarizes the mean area at risk, which is a major predictor of infarct size in models of regional ischemia. In the ET-1 group, the percent infarct area (58.0 1.47) was significantly higher than IR group (51.2 1.22). In the BQ-123 treated group with or without exogenous ET-1 treatment, the percent infarct area was significantly lower at 45.4 2.6 (P < 0.05) and 41.0 1.82 (P < 0.01) respectively. The percentage of mean area at risk that actually proceeded to infarction was found to be significantly smaller in the rats that received BQ-123 than in those that were untreated (IRcontrol) and ET-1 applied alone groups. When ET-1 was injected in the presence of the ETA antagonist, the marked inhibitory effects on cardiovascular function were not observed when compared with ET-1 treatment alone. 3.5. Histopathological evaluation In Fig. 4, microscopic histology revealed that the non-infarcted myocardium in the sham group was characterized by an organized pattern and shows normal architecture (Fig. 4a). The IR control and ET-1 alone treated groups showed severe myocardial membrane damage, edema and infiltration of inflammatory cells as compared to sham control. Significant myonecrosis with fibroblastic proliferation and presence of inflammatory cells were observed in the both the groups as compared to sham (Fig. 4b and 4c). BQ-123 treatment decreased myonecrosis, inflammatory cells, vacuolar changes and edema (Fig. 4d). However, the degree of myocardial damage in BQ-123 plus ET-1-treated rats was severe than BQ-123 alone treated rats (Fig. 4e). 3.6. Ultrastructural evaluation Fig. 5 shows the TEM images of the myocardium of drug and vehicle treated groups. Normal cardiac architecture was seen in electron micrographs of rats in sham group (Fig. 5a). Whereas IRcontrol group shows extensive myonecrosis, loss of lipid droplets
MDA: malondialdehyde, CK-MB: creatine kinase-MB, LDH: lactate dehydrogenase. Data are expressed as mean SD values (n = 6/group). Significance was determined by one-way Anova or repeated measures Anova followed by Bonferroni Post-hoc test: * P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.05 versus IR control, @ P < 0.01 versus ET + IR group.
Table 2 Myocardial antioxidant activities in ischemia-reperfusion model of myocardial infarction in rats. Groups
SOD (U/mg protein)
CAT (U/mg protein)
GSH (mg/g tissue)
Sham IR-control IR+ET-1 IR+BQ-123 IR+ET-1+BQ-123
8.5 3.7 4.1 2.6* 2.7 1.2y 6.8 2.7# 4.54 7.6@
20.9 4.3 13.9 3.6* 11.8 9.3y 17.9 3.9# 16.9 5.10@
2.42 0.71 0.73 0.13* 0.41 0.11y 1.92 1.37# 1.21 1.78@
SOD: superoxide dismutase; CAT: catalase; GSH: reduced glutathione. Data are expressed as mean S.D. values (n = 6/group). Significance was determined by oneway Anova or repeated measures Anova followed by Bonferroni Post-hoc test: * P < 0.001 versus sham group, #P < 0.01 versus IR control, yP < 0.05 versus IR control, @ P < 0.01 versus ET + IR group. Table 3 Mean area at risk and infarct area in rats of different experimental groups. Groups
Mean area at risk (%)
Infarct area (%)
IR-control IR+ET-1 IR+BQ-123 IR+ET-1+BQ-123
42.14 1.4 40.25 2.09 42.12 1.1 41.54 1.56
51.2 1.22 58.0 1.47 41.0 1.82* 45.4 2.6*,y
Data are expressed as mean S.D. values (n = 6/group). *P < 0.01 versus IR control, y P < 0.05 versus BQ-123 treated group.
along with significant disruption of myofilaments and Z band architecture (Fig. 5b). Similar changes were also observed in alone ET-1 group (Fig. 5c). However, BQ-123 treatment to IR rats shows normal ultrastructure with mild separation of cristae without swelling and vacuolation (Fig. 5d). Changes in the BQ-123 plus ET1-treated group were not as significant (Fig. 5e). 4. Discussion The present study demonstrated that, BQ-123, a selective ETA receptor antagonist exhibits a cardioprotective effect by improving cardiac functions, bolstering endogenous defense systems, reducing myocardial lipid peroxidation and preserving myofibril structure and morphology. In addition, BQ-123 significantly reduced infarct size and offers protection against IR and IR plus ET-1 induced myocardial injury. However, the co-treatment with ET-1 significantly attenuated BQ-123 induced cardioprotection. Therefore, we have provided direct evidence that the ETA receptor plays a significant role in regulating oxidative stress and subsequent tissue injury in IR-induced MI. To our knowledge, this is the first ever study conducted to report the cardioprotective effect of BQ-123 on the basis of hemodynamic, histopathological and ultrastructural changes in IR-induced MI. It has been widely documented that ET-1 has a detrimental effect on the myocardium following ischemic insult [26]. To annul
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Fig. 4. Light photomicrographs of representative rat heart sections (H&E, 200 from different experimental groups: a: sham group; b: IR control group; c: ET-1+ IR group; d: IR+ BQ-123 group; e: IR+ ET-1+ BQ-123 treated group.
this effect, we demonstrated that, a specific antagonist of the ETA receptor i.e. BQ-123 ameliorates the myocardial injury in rat model of MI. In corroboration with our findings, Ozdemir et al. [5] also reported that selective ETA receptor blockade reduces the infarct size and oxidant injury. Since blocking of ETB receptor does not preclude myocardial injury, this led to ratiocination that ETA receptor mediates the action of ET that lead to stimulation of inflammatory and thrombotic cascades leading to myocardial injury. Therefore we used specific receptor antagonist against the major form ET receptors present in human cardiomyocytes rather than using mixed ETA/ETB antagonists [27]. As no single hemodynamic variable can reliably anticipate the outcome of myocardial ischemia and the effectiveness of a therapeutic intervention, so cluster of hemodynamic indices were used to explain the cardiac functions. The present study demonstrated that ET-1 treatment increased arterial and ventricular dysfunction. Whereas, BQ-123 treatment prevented IR and ET1 plus IR-induced cardiac dysfunction as evidenced by restoration of mean arterial pressures. It also improved left ventricular
function by increasing inotropic (+LVdP/dtmax, marker of myocardial contraction) and lusitropic (LVdP/dtmax, marker of myocardial relaxation) states of the heart. In addition, it ameliorated IR and ET-1-induced increased in LVEDP, a marker of pre-load, which again reflects an improvement of left ventricular function. This finding is consistent with that of an earlier study by Cernacek et al. [28]. Moreover, bosentan, a mixed ETA and ETB receptor antagonist, significantly improved the left ventricular developed pressure, dP/ dtmax, end-diastolic pressure and coronary flow during reperfusion in isolated rat hearts subjected to global ischemia [6]. One possibility as postulated by Wang et al. [29] that these antagonists do possess vasodilation property and hence prevents the development of no-reflow phenomenon, which resulted in restitution of hemodynamic parameters. Furthermore, heart rate is not changed significantly in BQ-123 treated groups when compare with IR control group. This may also contribute to the beneficial effect of BQ-123 as a cardioprotective agent, as a small decrease in HR has been said to ameliorate the left ventricular filling. Parallel to our findings, Ozdemir et al. [5] has also reported
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Fig. 5. Electronmicrograph of representative rat myocardium sections (magnification 3500 ) from different experimental groups: a: sham group; b: IR control group; c: ET1+ IR group; d: IR+ BQ-123 group; e: IR+ ET-1+ BQ-123 treated group.
similar finding with BQ-123 treatment. Hence, our results suggest protective action of BQ-123 on the myocardium by improving cardiac functionality in spite of ET-1 administration in rats. From these findings it was also revealed that both endogenous and exogenous ET-1 significantly increases the infarct area. Moreover the activation of cardiac endothelial system due to ET-1 also results in expansion of myocardial infarct size ratio, which was evident from the present investigation also [30]. It is well established that post-acute MI, ET-1 levels are several times higher in the scar area in myocardium [31]. Therefore, administration of BQ-123 to IR and IR plus ET-1 groups significantly reduces the infarct area. It is interesting to recall that BQ-123 reduced the infarct size in canine model of coronary occlusion and reperfusion [16]. In contrast to above-mentioned studies there are also some reports where ET antagonists did not reduce infarct size in IR induced myocardial injury [32]. Apart from hemodynamic and infarct parameters, myocardium specific CK-MB isoenzyme and LDH activity has been employed as estimate of myocardial injury after coronary artery occlusion and
reperfusion [2]. In consonance with previously reported studies, we also observed a decrease in activities of these enzymes following IR. Besides, administration of ET-1 further declines the levels of these enzymes in ischemic and reperfusion injury. However, administration of BQ-123 significantly restored the activities of CK-MB isoenzyme and LDH in the myocardium following ET-1 challenge. Earlier, Singh et al. [24] has reported that, bosentan significantly restored CK-MB isoenzyme activity in IRinduced MI. Therefore, cardioprotection exerted by BQ-123 might be due to preservation of stability, cellular integrity and constraining the leakage of CK-MB isoenzyme and LDH through membranes. Lipid peroxidation is an important pathogenic event in myocardial injury and accumulation of lipid hydroperoxides reflects damage of the cardiac constituents. Production of reactive oxygen species (ROS) during ischemia and reperfusion leads to enhanced lipid peroxidation in the myocardium [33]. In present study, exogenously delivered ET-1 acts synergistically with IR induced oxidant injury and worsened the cardiac dysfunction
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observed after reperfusion, thereby suggesting that post-ischemic dysfunction results, at least in part, from ET-1 induced excessive MDA production in myocardium. Findings of the present study show that, administration of BQ-123 after 20 min of ligation significantly decreased the MDA content as compare to IR and IR plus ET-1 groups. Similarly, in accordance with our results, several other studies have also reported that an ETA antagonist improved the heart function, by decreased MDA content in the ischemic myocardium [5,34]. It is well known that IR produces free radical moieties, which initiate a cascade of events leading to the formation of superoxide anions that’s lead to endothelial dysfunction. Free radical scavenging antioxidant such as SOD, catalase and GSH are the first line of cellular defense systems in improving the myocardial oxidative imbalance [35]. The decrease activities of these antioxidants in the heart following ET-1 administration further elucidates excessive generation of superoxide and hydrogen peroxides, resulting in myocardial injury and also leads to ingestion of these endogenous antioxidants [5]. Similarly, many studies have demonstrated that in the event of IR, the tissue levels of SOD and/or catalase increases so as to protect cells from the detrimental effects of ROS [35]. In present study, we observed that decreased activities of SOD, catalase and GSH level in ET-1 challenged rats were significantly ameliorated by BQ-123 treatment. These findings are in accordance with Hong et al. [34], which also showed selective ETA receptor antagonist improves the imbalance in the anti-oxidant status in rat model of IR injury. Furthermore, coronary endothelial dysfunction has been prevented by SOD [36]. Therefore one of the possibilities of BQ-123 as a cardioprotective agent might be due to its antioxidant activity, which could exert a beneficial effect against pathological alterations caused by free radical in MI. To further elucidate the effect of BQ-123 on IR induced myocardial injury light microscopic study was performed. Histological study shows the myocardial injury in IR group and ET-1 treatment further aggravated the myocardial injury. Treatment with BQ-123 showed a well preserved normal morphology of cardiac muscle with no evidence of cellular influx, edema and were more effective in preserving tissue integrity as compared to IR and IR plus ET-1 groups which reconfirms the cardioprotective action of BQ-123. Singh et al. [24] reported similar type of protection with bosentan, a mixed ETA and ETB in IR induced MI. Likewise, TEM studies also demonstrated that BQ-123 decreases myonecrosis and showed normal ultrastructure of myocardium. These data further confirmed the cardioprotective role of BQ-123 in IR injury. In the light of these findings, finally it can be concluded that ET-1 acting via ETA receptor not only aggravates hemodynamic parameters but also accounts for the imbalance in antioxidant status, lipid peroxidation and increased infarct size along with the myocardial disorganization. Following administration of ETA receptor antagonist BQ-123 results in the improvement of the above said parameters and can also be used as an important pharmacological tool for salvaging myocardium following IR injury. Conflict of interest statement All authors have no conflict of interest. References [1] Jennings RB, Reimer KA. The cell biology of acute myocardial ischemia. Annu Rev Med 1991;42:225–46. [2] Mohanty IR, Arya DS, Gupta SK. Withania somnifera provides cardioprotection and attenuates ischemia-reperfusion induced apoptosis. Clin Nutr 2008;27:635–42. [3] Grover GJ, Sleph PG, Fox M, et al. Role of endothelin-1 and big endothelin-1 in modulating coronary vascular tone, contractile function and severity of ischemia in rat hearts. J Pharmacol Exp Ther 1992;263:1074–82.
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