Effect of zinc supplements in the attenuated

Effect of zinc supplements in the attenuated cardioprotective effect of ischemic preconditioning in hyperlipidemic rat heart Sunil Kumar Kansal, Uma Jyoti, Samridhi Sharma, Arun Kaura, Rahul Deshmukh & Sandeep Goyal Naunyn-Schmiedeberg's Archives of Pharmacology ISSN 0028-1298 Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-015-1105-6

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Author's personal copy Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-015-1105-6

ORIGINAL ARTICLE

Effect of zinc supplements in the attenuated cardioprotective effect of ischemic preconditioning in hyperlipidemic rat heart Sunil Kumar Kansal & Uma Jyoti & Samridhi Sharma & Arun Kaura & Rahul Deshmukh & Sandeep Goyal

Received: 30 November 2014 / Accepted: 9 February 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Hyperlipidemia is regarded as independent risk factor in the development of ischemic heart disease, and it can increase the myocardial susceptibility to ischemia-/reperfusion (I/R)-induced injury. Hyperlipidemia attenuates the cardioprotective response of ischemic preconditioning (IPC). The present study investigated the effect of zinc supplements in the attenuated cardioprotective effect of ischemic preconditioning in hyperlipidemic rat hearts. Hyperlipidemia was induced in rat by feeding high-fat diet (HFD) for 6 weeks then the serum lipid profile was observed. In experiment, the isolated Langendorff rat heart preparation was subjected to 4 cycles of ischemic preconditioning (IPC), then 30 min of ischemia followed by 120 min of reperfusion. Myocardial infarct size was elaborated morphologically by triphenyltetrazolium chloride (TTC) staining and biochemically by lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) release from coronary effluent and left ventricular collagen content. However, the effect of zinc supplement, i.e., zinc pyrithione (10 μM) perfused during reperfusion for 120 min, significantly abrogated the attenuated cardioprotective effect of ischemic preconditioning in hyperlipidemic rat heart whereas administration of chelator of this zinc ionophore, i.e., N,N,N′,N′tetrakis(2-pyridylmethyl)ethylene diamine (TPEN; 10 μM), perfused during reperfusion 2 min before the perfusion of zinc pyrithione abrogated the cardioprotective effect of zinc supplement during experiment in hyperlipidemic rat heart. Thus, the administration of zinc supplements limits the infarct size, LDH, and CK-MB and enhanced the collagen level which S. K. Kansal : U. Jyoti : S. Sharma : A. Kaura : S. Goyal (*) University Institute of Pharmaceutical Sciences & Research, Baba Farid University of Health Sciences, Faridkot, Punjab 151203, India e-mail: [email protected] R. Deshmukh Cardiovascular Division, ISF College of Pharmacy, Ferozpur Road, Ghal Kalan, Moga, Punjab 142001, India

suggests that the attenuated cardioprotective effect of IPC in hyperlipidemic rat is due to zinc loss during reperfusion caused by ischemia/reperfusion. Keywords Hyperlipidemia . Ischemic preconditioning . Zinc ionophore . Zinc chelator . Collagen

Introduction Hyperlipidemia is a state associated with elevation in the profile of lipid and cholesterol in plasma which results in accumulation of cholesterol in sarcolemmal and mitochondrial membranes and leads to development of various disorders including coronary artery disease and myocardial infarction (Onody et al. 2003; Puskas et al. 2004). Hyperlipidemia also increases the susceptibility of myocardium in ischemia- and reperfusion-induced injury (Hoshida et al. 1996; Jung et al. 2000) due to mast cell degranulation, increased postcapillary permeability (Kurose et al. 1997), and enhanced superoxide production (Ohara et al. 1995). During myocardium ischemia, the abrogation of oxygen supply and nutrient occurs, which leads to deplete the ATP, decrease the pH, lactate accumulation, and abrogation in Na+K+ATPase channel; enhanced calcium load leads to cell swelling (Collard and Gelman 2001; Buja 2005), but on prolonged ischemia, the cells turned at the end into necrosis. On the other hand, during reperfusion, the restoration of above conditions occurs except irreversible injury. However, during preconditioning, the short bouts of ischemia followed by reperfusion show myocardium endogenous adaptation phenomenon through enhanced résistance against longer bouts of ischemia by limiting the infarct size, neutrophils accumulation (Nakamura et al. 2000), and oxidative stress (Hausenloy et al. 2003) and inhibiting the apoptotic

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necrosis cell death (Fryer et al. 2001; Iliodromitis et al. 2007). It is well documented that cardioprotective effect of ischemic preconditioning (IPC) is attenuated in experimental hyperlipidemic rat heart (Giricz et al. 2006; Ungi et al. 2005) due to decreases in myocardial NO concentration (Hoshida et al. 1996), enhanced reactive oxygen species (ROS) (Onody et al. 2003; Puskas et al. 2004; Csont et al. 2007), apoptotic caspase-3 activation (Wang et al. 2002), and impaired activation of mitoK+ATP channels (Katakam et al. 2007), but the exact explored mechanism of attenuation of cardioprotective effect of IPC in hyperlipidemia is not clearly understood. It has been reported that the level of zinc decreased during reperfusion by excessive zinc loss in ischemic/reperfusion injury (Chanoit et al. 2008; Xu et al. 2014). Besides this finding, the lipid activates the PKC which triggers the zinc release, so, we envisage the hyperlipidemia model for study. The imbalanced homeostasis of zinc in the cell leads to various cardiovascular disorders (Little et al. 2010). It has also documented that zinc reduces the oxidative stress by decreasing the ROS production (Bray and Bettger 1990). Thus, the present study was designed to investigate the effect of zinc supplement in attenuated cardioprotective effect of ischemic preconditioning in hyperlipidemic rats.

Materials and methods The experimental protocol used in the present study was approved by institutional animal ethical committee (IAEC) and committee for the purpose of control and supervision of experiments on animals (CPCSEA) (Reg no. 816/PO/a/04/ CPCSEA) in accordance with the national guidelines on the use and care of laboratory animals in scientific research. All the animals were placed in an animal house and were exposed to cycle of 12 h light and 12 h darkness.

contains 3.80 kcal/g. The establishment of hyperlipidemia was assessed by collecting the blood sample from the retroorbital plexus and then estimating the level of total cholesterol (TC) by cholesterol oxidase peroxidase (CHOD-POD) method (Allain et al. 1974), triglycerides (TG) by glycerophosphate oxidase peroxidase (GPO-PAP) method (Werner et al. 1981), and HDL by polyethylene glycol method (Allain et al. 1974) in serum using commercially available kits. Isolated rat heart preparation Rats were administered with heparin 500 U; i.p. after 20 min, the animal was sacrificed by cervical dislocation, and the heart was rapidly excised and immediately mounted on Langendorff’s apparatus. The heart was enclosed by a double-walled jacket, the temperature of which was maintained to 37.8 °C by circulating the heated water. The preparation was retrogradely perfused at constant pressure of 80 mmHg with Krebs-Henseleit buffer (NaCl 118 mM; KCl 4.7 mM; CaCl2 2.5 mM; MgSO4·7H2O 1.2 mM; NaHCO3 25 mM; KH2PO4 1.2 mM; C6H12O6 11 mM), pH 7.4, bubbled with 95 % O2 and 5 % CO2 through peristaltic pump. IPC was produced by closing the inflow of K-H solution for 5 min followed by 5 min of reperfusion. Four such bouts were employed followed by index ischemia that was produced for 30 min followed by reperfusion for 120 min. Coronary effluent was collected after stabilization, immediately, 5 and 30 min after reperfusion for estimation of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), and collagen assay was done by measuring the absorbance of supernatant of the homogenized tissue. Assessment of myocardial injury

Zinc pyrithione (HiMedia Laboratories (P) Ltd., Mumbai, India) and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN) (Sigma Chemicals, St. Louis, MO, USA) were added to Krebs-Henseleit (K-H) solution. All other reagents in the study were of analytical grade and prepared freshly.

To determine the extent of I/R-induced myocardial injury, the release of LDH and CK-MB in coronary effluents was measured. The release of LDH in the coronary effluent was noted after stabilization, immediately after reperfusion and 30 min after reperfusion, and CK-MB was noted after stabilization and 5 min after reperfusion (Parikh and Singh 1999) using commercially available kit (Vital Diagnostics (P) Ltd., Mumbai, India). Values were expressed in international units per liter (IU/l).

Induction of experimental hyperlipidemia

Assessment of left ventricle collagen content

Wistar rats of either sex (200–300 g) were employed in the present study. Experimental hyperlipidemia was produced by feeding high-fat diet each 1 kg consisting of normal chow— 365 g, cholesterol—10 g, casein—250 g, lard—310 g, dl methionine—3 g, vitamins and mineral mix—60 g, yeast powder—1 g, and sodium chloride—1 g for 6 weeks. The high-fat diet (HFD) contained 5.33 kcal/g while the normal chow

The left ventricular tissue was used for estimation of collagen contents in the heart by measuring hydroxyproline concentration (Jamall et al. 1981). The tissue was homogenized in 4 ml of 6N HCl and hydrolyzed at 110 °C for 16 h. Dried hydrolysate was dissolved in 1000 μl of 50 % isopropanol and then into 30 μl of aliquot; 1.2 ml of 50 % isopropanol was added and incubated with 0.2 ml of 0.84 % chloramines-T in citrate

Drugs and chemicals


preparation was perfused with K-H solution and allowed for 10 min stabilization in all groups. Group 1 (sham control), the isolated Langendorff rat heart preparation was perfused with K-H buffer solution for 140 min without inducing sublethal ischemia and reperfusion. Group 2 (ischemia/reperfusion control), the isolated Langendorff rat heart preparation was perfused for 40 min with K-H buffer solution, then the isolated preparation was induced to 30 min index ischemia followed by 120 min of reperfusion. Group 3 (ischemic preconditioning control), the isolated Langendorff rat heart preparation was subjected to four bouts of 5 min ischemia followed by 5 min reperfusion with K-H buffer solution. Group 4 (zinc pyrithione (10 μM) treated ischemic preconditioning rat heart), the isolated Langendorff rat heart preparation was subjected to IPC as described in group 3, but zinc pyrithione (10 μM) was administrated through K-H buffer solution during 120 min reperfusion period. Group 5 (ischemic preconditioning in hyperlipidemic rat heart), the isolated Langendorff rat heart preparation was subjected to IPC as described in group 3. Group 6 (zinc pyrithione (10 μM) treated ischemic preconditioning in hyperlipidemic rat heart), the isolated Langendorff rat heart preparation was subjected to IPC as described in group 3, but zinc pyrithione (10 μM) was administrated through K-H buffer solution during 120 min reperfusion period. Group 7 (TPEN, Zn 2+ chelator, and zinc pyrithione administrated in IPC in hyperlipidemic rat heart), the isolated Langendorff rat heart preparation was subjected similarly as described in group 6, but the TPEN is

buffer at pH 6.0 for 10 min. At room temperature, then, 1 ml Ehrlich’s reagent was added and mixture was incubated for 25 min at 60 °C. The absorbance of sample solution was measured at 560 nm under UV spectrometer. Myocardial infarct size The heart was removed from the Langendorff apparatus. Both the atria and root of aorta were excised, and ventricles were kept overnight at −4 °C. Frozen ventricles were sliced into uniform sections of 2–3 mm thickness. The slices were incubated in 1 % 2,3,5-triphenyltetrazolium-chloride (TTC) for 30 min at 37 °C in 0.2 M Tris chloride buffer (prepared by dissolving 7.27 g of tris-(hydroxymethyl)-methylamine and 5.27 g of sodium chloride in water, adjusting pH up to 7.4 finally diluting up to 1000 ml with distilled water) (Fishbein et al. 1981). The TTC stain reacts with dehydrogenase in the presence of co-factor NADH, converts it into formazan pigment which stains the viable cell to brick red, and the infarcted cell lost its dehydrogenase enzyme, which remains unstained. The slices are then placed between the glass plates to measure the infarct size by the volume method (Chopra et al. 1992). Experimental protocol All the experimental groups in the study contain n=6 animals, and experimental design was represented diagrammatically in Fig. 1. During experiment, the isolated Langendorff rat heart Fig. 1 Diagrammatic representation of experimental protocol. S, I, R, IPC, HL, Zn, TPEN represents stabilization, ischemia, reperfusion, ischemic preconditioning, hyperlipidemia, zinc supplement, N,N,N′,N′tetrakis(2-pyridylmethyl)ethylene diamine, respectively

Experimental Protocol Group I: Sham Control

Group II: I/R Control

Group III: IPC Control

Group IV: Zn + IPC

Group V: IPC + HL

Group VI: Zn + IPC + HL

Group VII: TPEN + Zn + IPC + HL


administrated at the first 2 min of 120 min reperfusion period followed by zinc pyrithione for the remaining time. Statistical analysis Result values were expressed as mean±S.D. (n=6 animals/ groups). The data obtained from various groups were statistically analyzed using one-way ANOVA, followed by Tukey’s multiple comparison test. A p value of less than 0.05 was considered to be statistically significant.

Result Effect of hyperlipidemic diet on serum lipid profile After administration of HFD for 6 weeks, the serum total cholesterol of hyperlipidemic rats (252.17±14.72) as compared to normal rats (67.90±7.64), triglycerides of hyperlipidemic rats (241.51±12.24) as compared to normal rats (53.84 ± 5.01), low-density lipoprotein of hyperlipidemic rats (171.34±17.98) as compared to normal rats (32.11±3.97), and high-density lipoprotein of hyperlipidemic rats (26.86± 7.52) as compared to normal rats (53.31±5.38) was significantly increased in hyperlipidemic rats (Fig. 2). Effect of IPC on ischemia-/reperfusion-induced myocardial injury in normal and hyperlipidemic rat hearts There is a significant increase in the infarct size in normal rats when subjected to 30 min global ischemia and 120 min reperfusion as compared to normal rats with no I/R. The IPC significantly decreases the ischemia-/reperfusion-induced myocardial infarct size as compared to normal rats with I/R, and similarly, the zinc-treated IPC significantly decreases the ischemia-/reperfusion-induced myocardial infarct size as compared to normal rats with IPC. However, the IPC effect

Fig. 2 Effect of high-fat diet on serum level of total cholesterol, triglycerides, low-density lipoprotein, and high-density lipoprotein. The results are expressed as mean±S.D. *p