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Subacute ghrelin administration inhibits apoptosis and improves ultrastructural abnormalities in remote myocardium post-myocardial infarction
Biomedicine & Pharmacotherapy 101 (2018) 920–928
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Subacute ghrelin administration inhibits apoptosis and improves ultrastructural abnormalities in remote myocardium post-myocardial infarction
T
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Refaat A. Eida, , Mohamed Samir Ahmed Zakib, Mubarak Al-Shraima, Samy M. Eleawac, Attalla Farag El-kottd,h, Fahaid H Al-Hasheme, Muhammad Alaa Eldeenf, Hoja Ibrahimg, Hussain Alderag, Mahmoud A. Alkhateebg a
Department of Pathology, College of Medicine, King Khalid University, Abha, Saudi Arabia Department of Anatomy, College of Medicine, King Khalid University, P.O. 641, Abha, 61421, Saudi Arabia Department of Applied Medical Sciences, College of Health Sciences, PAAET, Kuwait d Department of Biology, College of Science, King Khalid University, P.O. 641, Abha, 61421, Saudi Arabia e Department of Physiology, College of Medicine, King Khalid University, P.O. 641, Abha, 61421, Saudi Arabia f Biology Department, Physiology Section, Faculty of Science, Zagazig University, Egypt g Department of Basic Medical Sciences, College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Saudi Arabia h Department of Zoology, College of Science, Damanhour University, Egypt b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Ghrelin Remote myocardium Remodeling MI Electron microscope
This study investigated the effect of ghrelin on cardiomyocytes function, apoptosis and ultra-structural alterations of remote myocardium of the left ventricle (LV) of rats, 21 days post myocardial infarction (MI). Rats were divided into 4 groups as a control, a sham-operated rats, a sham-operated+ghrelin, an MI + vehicle and an MI + ghrelin-treated rats. MI was induced by LAD ligation and then rats were recievd a concomitant doe of either normal saline as a vehicle or treated with ghrelin (100 μg/kg S.C., 2x/day) for 21 consecutive days. Ghrelin enhanced myocardial contractility in control rats and reversed the decreases in myocardial contractility and the increases in the serum levels of CK-MB and LDH in MI-induced rats. Additionally, it inhibited the increases in levels of Bax and cleaved caspase 3 and increased those for Bcl-2 in the remote myocardium of rat's LV, post-MI. At ultra-structural level, while ghrelin has no adverse effects on LV myocardium obtained from control or sham-treated rats, ghrelin post-administration to MI-induced rats reduced vascular formation, restored normal microfilaments appearance and organization, preserved mitochondria structure, and prevented mitochondrial swelling, collagen deposition and number of ghost bodies in the remote areas of their LV. Concomitantly, in remote myocardium of MI-induced rats, ghrelin enhanced endoplasmic reticulum intracellular organelles count, decreased number of atrophied nuclei and phagocytes, diminished the irregularity in the nuclear membranes and inhibited chromatin condensation. In conclusion, in addition to the physiological, biochemical and molecular evidence provided, this is the first study that confirms the anti-apoptotic effect of ghrelin in the remote myocardium of the LV during late MI at the level of ultra-structural changes.
1. Introduction
activate remodeling in remote surviving myocardium [3], all of which lead to chamber dilation, LV dysfunction, and eventually heart failure (HF) [2,4–6]. Indeed, after an acute MI, apoptosis is apparently silent while its progression leads to subtle progressive cell loss in hibernating myocardium leading to HF [7,8]. Even with the extensive use of the light microscopy and its related biochemical techniques, e.g. TUNEL labeling [9,10], several authors have shown that such techniques are still nonspecific to detect cardiomyocytes apoptosis during LV remodeling [7].
Post-myocardial infarction (MI) remodeling occurs in both ischemic and non-ischemic areas of the left ventricle (LV) [1,2]. In this regard, the initial acute and rapid cardiomyocytes loss occurring in the ischemic area is associated with provoking inflammation, fibrosis, and hypertrophy in remote myocardium [1,3]. These events, in addition to the presence of regional wall strain, but independently of any further ischemia, can initiate and propagate cardiomyocytes apoptosis and ⁎
Corresponding author. E-mail address: [email protected] (R.A. Eid).
https://doi.org/10.1016/j.biopha.2018.03.010 Received 19 December 2017; Received in revised form 1 March 2018; Accepted 5 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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2. Materials and methods
Indeed, TUNEL labeling may provide variable data as it could detect DNA fragmentation of any origin including oncotic and apoptotic myocytes as well as the DNA repair mechanisms and cardiac hypertrophy [11–15]. This may reflect an overestimation of apoptosis, resulting in misleading conclusions [15]. Similarly, further flawed results could be obtained in techniques that depend on DNA or protein extraction, e.g, PCR and western blotting as these samples may contain extracts of both myocyte and non-myocyte cardiac cells, including intravascular circulating cells, vascular and interstitial cells [15,16]. On the other hand, several authors and the Nomenclature Committee on Cell Death (NCCD) have recently revised the mechanisms of cell death in tissues and recommended that classification of cell death modalities should rely purely by providing solid molecular evidence and on the presence of certain ultra-structural morphological criteria, as detected by electron microscopy (EM) [7,17–19]. Although the ultra-structural examinations by EM are considered the gold standard for the diagnosis of cardiomyocytes apoptosis, only few but recent EM studies have investigated the ultra-structural apoptotic changes that occur during cardiac remodeling post MI or during HF [15]. In this regard, the majority of these studies, which were carried only on the ischemic area after acute MI, had consensus and have confirmed active features apoptosis including aberrant nuclei, condensation of chromatin, myofibrillar disarray or lysis, disorganization of sarcomeres, peri-nuclear cytoplasmatic vacuolation, intranuclear clumping and mitochondria clustering as well as disturbances with disorganized cristae [15,20–23]. However, the ultra-structural changes in remote areas of the LV during late cardiac remodeling post-MI remain largely unknown and need further investigations. Ghrelin, a gut-derived 28-amino acid peptide hormone, is an endogenous ligand for the growth hormone secretagogue receptor (GHSR) and is a potent stimulator of GH [24]. In addition to the stomach and the gut, smaller amounts of ghrelin is also produced by peripheral organs including the heart, lung, kidney, pancreas, placenta, hypothalamus, gonads, thyroid, adrenal and pituitary glands indicating the wide physiological roles within the body [25–27]. Recently, accumulative evidence has shown important roles of ghrelin in regulating cardiac function and protection against various forms of cardiac disorders. Interestingly, human endothelial cells can synthesize ghrelin, and ghrelin receptors have been found in human endothelial cells, vascular smooth muscle cells, and LV [28]. In both clinical and experimental trials in patients with HF who survived after MI and in animal models of MI and ischemia/reperfusion (I/R) induced cardiac injury, exogenous ghrelin administration reduced infarct size and LV enlargements, enhanced LV contractility, improved cardiac output and index, reduced apoptosis and attenuated LV remodeling [29–31]. In other cardiotoxicity induced models of HF in rats (e.g. doxorubicin and Ang. II), ghrelin increased the size of cardiomyocytes, prolonged their survival, reduced fibrosis and inhibited apoptosis [32,33]. In spite of these findings, the ultra-structural evidence to support an anti-apoptotic effect of ghrelin post-MI in both infarcted and non infarcted areas post-MI is completely lacking. Very recently, we have shown that ghrelin administration (100 μg/ kg S.C., 2x/day) for 21 days, starting one day post MI, preserved LV function and reduced apoptosis in remote myocardial tissue of LV of rats in a mechanism that involves inhibition of SOCS3 and activation of JAK2/STAT3 signaling, an effect that is mediated at least by inhibition of cardiac levels of IL-6 [34]. To further support the role of the cardioprotective effect of ghrelin on cardiac apoptosis during remodeling, this study was designed to investigate the ultra-structural changes that occur in surviving remote myocardium during late MI with or without ghrelin administration in parallel with alterations in cardiac function and molecular evidence of apoptosis.
2.1. Animals Disease-free adult Sprague−Dawley male rats (9 weeks, 260-270 g) were included in this study. All rats were supplied by the animal facility at King Khalid University, Abha, Saudi Arabia and they were always housed (n = 4 rats/cage) in their controlled rooms at 23 ± 3 °C and 40−65% humidity, on a 12-h light/dark cycle. All animals had free access to drinking water and normal chow (AIN-93G diet). All experimental protocols of this study were approved by the College of Medicine Ethics Committee which are in accordance with guidelines published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). 2.2. Experimental approach Rats were divided into five groups (n = 6/group) and treated as follows for 21consecutive days: 1) A control group: received normal saline (0.5 ml S.C.) and were not exposed to any surgery. 2) A sham-operated group: control rats which received normal saline (0.5 ml S.C.) but underwent surgical procedure similar to that used MI model rats but without tying the anterior descending coronary artery (LAD). 3) A sham-operated + ghrelin-treated group: sham-operated rats as in group 2, but then received ghrelin (100 μg/kg S.C., twice/day). 4) An MI-induced group: underwent a surgical procedure to ligate LAD and received normal saline (0.5 ml S.C.). 5) MI + ghrelin-treated group: MI-induced rats but then received ghrelin (100 μg/kg S.C. twice/day). Rat’s synthetic ghrelin (C147H245N45O42) powder was purchased from Sigma (Cat. No. ab120231) and was freshly prepared in distilled water to a final concentration of (100 μ/kg). All treatments were started immediately after induction of MI and continued for a period of 21 days. The period and dose period of selected dose was based on our previous study where we showed that exogenous ghrelin administration of the same dose for 21 days post MI preserved cardiac hemodynamics and inhibited cardiomyoctes death in remote myocardium of LV [34]. Similarly, other authors have shown increased apoptosis and fibrosis in the remote areas of LV starting from 21 day and up to 6 months [29,31,34,35]. In addition, the selected dose of ghrelin was based on previous research that confirmed a cardioprotective effect of ghrelin in remote and ischemic myocardium for a similar period of treatment [29,31,34,]. 2.3. Surgical procedure and induction of MI Induction of MI was done as described before by us [34]. In brief, all rats were anesthetized with i.p. administration of 1% solution of sodium pentobarbital (50 mg/kg). After successful ventilation (Harvard rodent ventilator, model 863, Harvard Apparatus, Holliston) of each rats, the heart was exposed and LAD coronary artery was ligated, 2–3 mm from its origin, using a 6–0 prolene suture. Then the chest was sutured with 6–0 sutures and chest air was vacuumed using a 3 ml syringe. All procedures during the surgery and and recovery were done on a heated pad and a thermal probe was used to monitor the animal's temperature. In addition, an eye lubricant ointment was applied to both eyes in order to prevent dryness. Induction of MI in each rat was confirmed by the ST-segment elevation of the rat's electrocardiogram (ECG) recorded with the help of PowerLab system (AD instrument, Australia) using 3 limbs electrodes. All procedures were done by the same surgeon and usually lasted between 20–25 min. Post-operation, all rats received routine intramuscular injections of buprenorphine-HCl (0.2 mg/kg) for 921
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Fig. 1. Hemodynamic parameters (A–D) and serum enzyme levels of (CK-MB, E) and lactate dehydrogenase (LDH, F) in all group of rats. Values are expressed as Mean ± SD for 6 rats in each group. Values were considered significantly different at P < 0.05. a:vs. control group, b:Vs. sham group. c:Vs. sham + ghrelin group, d:Vs. MI group.
5000 rpm for 10 min to collect serum which was stored at −20 °C and used later to determine levels of CK-MB and lactate dehydrogenase using a biochemical analyzer. Then after, the rat was killed and heart was rapidly collected on ice. LV from each heart was collected and the non-infarcted area was identified and isolated. Parts of the non-infarcted area were directly frozen at −80 °C for western blotting study, other parts were directly collected in either 10% buffered formalin or 2.5% glutaraldehyde (in 0.1 M cacodylate buffer).
analgesia and penicillin (1000 U) to prevent infection. 2.4. LV function measurement and blood and tissue collection At the end of the experimental procedure, animals received intravenous (i.v.) administration of heparin (1000 unit) and then were anesthetized again with 1% solution of sodium pentobarbital (50 mg/ kg). Next, all animals were ventilated and their chest were opened again. In each rat, a pressure catheter (SPR-320, instrument) was directly inserted into the LV by stab method. With the help of power lab software and recording system (LabChart 8.3 ML780 and PowerLab/ 8channels, AD Instruments Ltd., Australia), LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), maximal rate of rise in LV pressure (LV dP/dtmax), and maximal rate of decline in LV pressure (LV dP/ dtmin) were recorded for next 30 min. All readings were analyzed and average values were presented. Then, 1ml blood samples was directly collected from the right carotid artery into a plain tube, centrifuged at
2.5. Western blotting Whole proteins were extracted from each frozen LV using a Millipore extraction kit (Cat. No. 2140, Merck Millipore, USA). During the extraction, protease inhibitor was added to each sample to prevent protein degradation (Cat. No. P8340, Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations in all samples were measured by a Bradford assay kit. Western blotting procedure was performed as 922
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Fig. 2. Protein levels of type activated caspase-3, BCL-2, and BAX in the left ventricles of all groups of rats as detected by western blotting.Values are expressed as Mean ± SD for 6 rats in each group. Values were considered significantly different at P < 0.05. a:vs. control group, b:Vs. sham group. c:Vs. sham + ghrelin group, d:Vs. MI group.
1 cubic millimeter sizes, fixed in gluteraldehyde solution in 0.1 M sodium cacodylate buffer, pH 7.2, and placed in a thermal box cooled to 4 °C for 2 h. They were post-fixed in 1% osmium tetraoxide in a sodium cacodylate buffer and then dehydrated in ascending series of ethyl alcohol and embedded in Spurr’s resin. Ultrathin sections stained with uranyl acetate and lead citrate were examined by TEM (JEM-1011, Jeol Co., Japan) operated at 80 KV in the Electron Microscopy Unit, Pathology Department, College of Medicine, King Khalid University [37].
described preciously by us [34]. Then, protein samples from all groups (60 μg protein/well) were separated on SDS-PAGE and transferred manually to nitrocellulose membranes and incubated with primary antibodies against Bax (Cat. No 2772, 20 kDa, cell signaling technology), Cleaved caspase-3 (Cat. No 9662, 17, 19 and 35 kDa, cell signaling technology) and BCL-2 (Cat. No, 2876, 28 kDa, cell signaling technology) and β-Actin (C4, sc-47778, 43 kDa, Santa Cruz Biotechnology). Then, membranes were washed and incubated with their corresponding HRP-conjugated secondary antibody. The protein bands were detected by chemiluminescence (Pierce ECL reagents, Thermofisher, USA, Piscataway, NJ) and were quantified using C-DiGit Blot Scanner (LI-COR, USA) with the supplied Image Studio DiGits software.
3. Results 3.1. Cardiac function and serum enzymes levels
2.6. Light microscopy The hemodynamic parameters presented in Fig. 1 describe the average mean changes as analyzed after continuous recording for 30 min on the last day of the experimental procedure, in each rat. In sham-operated rats, ghrelin administration only and significantly increased LVSP (122.8 ± 12.54 vs. 151.4 ± 12.14) (Fig. 1A). Twentyone day’s post-MI and as compared to control rats, MI-induced rats had significant lower values of LVSP (122.8 ± 12.54 vs.48.8 ± 15.69, Fig. 1A), dp/dtmax (5232 ± 992.5 vs. 2220 ± 380.9, Fig. 1C) and dp/ dtmin (3042 ± 842.2 vs 1428 ± 347.1, Fig. 1D) and higher levels of LVEDP (7.46 ± 1.92 vs. 23.48 ± 3.26, Fig. 1B). However, ghrelin administration for 21 days to MI-induced rats significantly decreased values of LVEDP (12.12 ± 1.89, Fig. 1B) and significantly increased those for of LVSP (79.0 ± 9.05, Fig. 1A), dp/dtmax (4083 ± 405.1,
For light microscopy, collected ventricular specimens were kept in 10% neutral buffered formalin for 24–48 h, dehydrated in graded alcohol series, cleared in xylene, and embedded in paraffin wax. Paraffinized specimens were sectioned at 4 μm and then stained with hematoxylin and eosin (H&E), examined under light microscope (Olympus CX31, Japan) and photographed using digital camera (Olympus, Camedia-5060, Japan) [36]. 2.7. Transmission electron microscopy (TEM) For TEM, heart specimens from both control and treated rats were immediately preserved in 2.5% gluteraldehyde, trimmed and diced into 923
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associated with a decrease in density of the entire muscle cell accentuated the darker intercalary disc. Intracellular spaces appeared between myofilaments in the perinuclear region and around the mitochondria (Fig. 5A–D). Later, these spaces were increased in size and number. Also, the bundles of myofilaments showed longitudinal cracking and separation. The separation of myofilaments was random since the size of the resulting bundles was variable. Moreover, a revealed markedly change is mitochondria swelling and disappearance of their cristae or with loss of cristae spatial organization (Fig. 5A–D). In some instances, the limiting membrane was altered, and the overall appearance was similar to that of ruptured mitochondria. Again, due of the separation of myofibrils and surrounding loss of density in the later preparations, the system was more clearly seen with swelling and enlargement of the vesicles and tubules. Nuclear alterations were detectable. The nucleoplasm showed condensation of heterochromatin with irregularity in the nuclear membrane (Fig. 5D). Moreover, transmission electron micrographs of the remote myocardium of LV in MI-induced rats showed necrotic myocardial cells with phagocytic vacuoles (Fig. 6A and B), irregular heterochromatic nuclei and plenty of ghost bodies, shrinkage blood vessel and atrophied nuclei were squeezed in the interstitium between myocardial cells (Fig. 6D). Degenerated cytoplasm with damaged myofibrils, fragmentation of muscle bands and swollen plus damaged mitochondria were noticed (Fig. 6A–D). Distinct collagen bundles were clearly demonstrated inside myocardial cells and in the interstitium (Fig. 6B and C). However, transmission electron micrographs of the remote myocardium of LV of MI + ghrelin-treated rats showed improvement of myocardial structure with dense cytoplasm which were filled with healthy myofibrils, intact muscle bands and mitochondria with moderate changes in its cytoplasm (Fig. 7A and B).
Fig. 1C) and dp/dtmin (2453 ± 400.9, Fig. 1D) as compared to MI-induced rats. On the other hand, normal sera levels of LDH and CK-BM were seen between control, sham-operated and sham-operated +ghrelin treated groups. However and as compared to control or shamoperated rats, MI-induced rats had significantly higher sera levels of both CK-MB (628.8 ± 93.54 vs. 181.0 ± 36.3, Fig. 1E) and LDH (2031 ± 220.2 vs.732.8 ± 103.8, Fig. 1F). On the other hand, ghrelin administration to MI-induced rats for 21 days significantly decreased levels of both CK-MB (281.1 ± 36.74) and LDH (1028.0 ± 153.5) in the sera of these treated rats (Fig. 1E and F). 3.2. Western blotting Detected bands of cleaved caspase-3, Bax and Bcl-2 as well as the reference protein, β-actin were within expected sizes (Fig. 2). Protein levels were presented as a relative expression of the loading control, βactin, which remained constant in the cardiomyocytes of the LV of all groups of rats. Interestingly, levels of Bcl-2 were significantly increased while levels of Bax and cleaved casopase-3 were significantly decreased in the LV of sham-operated rats treated with ghrelin. However, significant elevations in the protein levels of both Bax and cleaved caspase-3 with parallel significant decreases in the levels of Bcl-2 were detected in the remote myocardial tissue of LV of MI-induced rats after 21 days, as compared to control or sham-operated rats. However, as compared to MI-induced rats, MI + ghrelin-treated rats showed significantly inhibited levels of Bax and cleaved caspase-3 and significantly increased levels of Bcl-2 in the remote areas of their LV, 21 days post-MI. 3.3. Light microscopy
4. Discussion
Photomicrographs obtained from control, sham-operated and shamoperated+ghrelin rats showed normal branched cardiac muscle cells which were seen to contain nuclei (Fig. 3A–C). Photomicrographs obtained from MI-induced rats showed signs of both necrosis and apoptosis of the myocardium as evident by the presence of pyknotic nuclei and fragmentation of myocardial tissue (Fig. 3D and E). However, Photomicrographs obtained from MI + ghrelin treated group showed little changes of the architecture in the form of normal myofibrils and other fibrils appeared disrupted (Fig. 3F).
The exclusive findings of this study describe the ultra-structural changes that occur in the remote areas of LV of rats during cardiac remodeling, post experimentally induced MI in rats with or without ghrelin treatments. Generally, cell death or necrosis of cardiac myocytes may occur by the process of apoptosis, oncosis, or autophagy [15]. Our findings clearly demonstrate that modalities of cell death in these remote areas of LV of rats, 21 days post MI are due to activation of apoptosis and necrosis. While necrosis was evident by elevations of cardiac function enzymes (CK-MB and LDH) in the sera and histological alterations in remote myocardial tissues of the LV of MI-induced rats, apoptosis has been also confirmed in the same tested area by means of western blotting that showed enhanced expression of Bax and cleaved caspase-3 and was supported further by means of electron microscopy. Even parts of our curent data support our and other previously reported findings on the cardioprotective and anti-apoptotic roles of ghrelin during cardiac remodeling post MI [29–31,34], these data are the first that confirm these findings at the level of ultra-structure. In the current study, supporting to the our previous findings [34], hemodynamic parameters measured on day 21 days post-MI showed impaired LV contractility and lusitropy function as evident by the significant increases in LVEDP, significant decreases in LVdP/dtmax and LVSP, markers of myocardial contraction and by the significant decreases in LVdP/dtmin, a marker of myocardial relaxation and lusitropy [29–31,34]. The first observations of the cardioprotective effect of ghrelin were the observation that ghrelin administration to sham-operated rats increased LV inotropy without affecting LVEDP and significantly restored contractility and cardiac function parameters in MI rats. These findings are not surprising with the long history of the cardioprotective effect of ghrelin in various pathological conditions including I/R injury, MI, HF and drug-induced toxicities [29–33]. Such effect has been demonstrated previously in our labs in MI-induced-rats using the same rat's species, ghrelin dose, and treatment period [34]. In this regards, it has been suggested that ghrelin can exert its
3.4. Electron microscopy Ultrastructural findings of control, sham-operated and sham + ghrelin treated groups were depicted in Fig. 6. No significant alterations were detected between these three groups and their LV showed normal structures. The bundles of myofilaments are occasionally branched and contraction bands were generally in the region of the “Z” band (Fig. 4A–C). These were the result of placing pieces of muscle directly in fixative with no attempt to stretch or maintain fiber length. In most longitudinal sections, a dense “H” zone appeared halfway between two “Z” bands. Myofilaments coursed through a given cell and terminated at intercalary discs. The myofibrils were frequently surrounded by mitochondria (Fig. 4A–C). Usually, a mitochondrion was found in a branching manner. Mitochondria were found throughout the heart muscle fiber. They were located about the nucleus at either pole. They were also found in long chains between bundles of myofilaments (Fig. 4C). The nucleus was bounded by a double membrane (Fig. 4A–C). Also, nucleoplasm was evenly distributed, without dense aggregates. The nuclear material itself was comprised of a random array of filamentous structures and denser granules. Nucleolar substance frequently appeared as an aggregate of closely packed granules (Fig. 4A–C). A definite accentuation of the intercalary discs was evident in the LV specimens obtained from MI-induced rats (Fig. 5A–D). Adjacent to the disc, an intracellular pale zone appeared which contained a few randomly placed fine filamentous structures. The vacuole formation 924
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Fig. 3. Histopathological evaluation of the LVs obtained from all group of rats. (400X). Micrographs A, B & C are taken from the control, sham-operated and sham-operated+ghrelin treated rats, respectively showing normal cardiac muscle cells which are seen to contain nuclei (N) and branched fibers (arrows). Micrographs D and E are taken from MI-induced group showing necrosis and apoptosis of the myocardium that are evident by the presence of pyknotic nuclei (N) and fragmentation of myocardial tissue (arrows). Micrograph F is taken from rats MI + ghrelin treated group showing normal nuclei (N) with little changes of the architecture in the form of normal myofibrils and other fibrils appeared disrupted (arrow).
[14,45,46,]. However, apoptosis is an energy-dependent process of cell death where apoptotic myocytes are removed by macrophages through phagocytosis without triggering inflammation and peripheral circulatory levels of major markers of cell death such as troponin levels, CKMB and LDH are usually within the normal range [14,15,45,46]. Apoptosis in myocardium has been described as a major mechanism of the heart-induced dysfunction in various animal models including I/ R injury [47,48], post-MI LV remodeling [49] and heart failure [9,50]. Similarly, autophagic vacuoles are commonly seen in failing human hearts [44]. Furthermore, even not well characterized, the common form of oncotic cell death that has been described in acute MI is coagulative necrosis [39,51]. In the current study, light microscopy, biochemical and molecular findings are clear indications for the involvements of both necrosis and apoptosis in the cardiomyocyte death in remote non-infarcted areas of MI-induced rats during cardiac remodeling, 21 days post-MI. In this study, a significant elevation in the circulatory levels of LDH and CK-MB with histological alterations in the form of myofibril degenerations and fragmentation, pyknosis in most nuclei was seen in the remote areas of the LV, 21 days post-MI in MI-induced rats. In addition, the LV of MI-induced rats showed significant increases in protein levels of cleaved caspase 3 and Bax and significant decreases in levels of Bcl-2, major markers of cell apoptosis. Interestingly, ghrelin administration to
cardiovascular effect via its potent vasodilator effect (enhancement of NO production and decreasing peripheral vascular resistance) [38] as well as by its anti-apoptotic and anti-fibrotic effects [29–34]. In support to our current and previous findings [34], acute administration of ghrelin (100 μg/kg sc, twice daily) for 2 weeks post MI significantly reduced left ventricular end diastolic pressure and significantly reduced the enlargement of LV in rats [31]. However, associated with these changes it was of our interest to investigate the common modality of cell death in the remote areas of LVs post-MI in both MI-induced rats received the vehicle or treated with ghrelin. Hence, histological, molecular and ultrastructural approaches were considered as recommended by Nomenclature Committee on Cell Death (NCCD) as well as other authors [7,17–19]. In general, cardiomyocytes necrosis is well characterized by cellular swelling, coagulation of the sarcoplasmic proteins and nuclear loss and is usually associated with the release of intracellular contents such as troponin, CK-MB and LDH and triggered by an inflammatory response and fibrosis [15,39,40]. On the other hand, autophagy is a cellular degradation process to remove damaged organelles and cytoplasmic proteins [41] and occurs when damaged mitochondria and cytoplasmic proteins become included within the lysosomes leading to the generation of autophagocytic bodies [42–44]. Removal of autophagocytic necrotic cells occurs through phagocytosis with no inflammation 925
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Fig. 4. Transmission electron micrographs of the control (A and B), sham-operated (C) and sham + ghrelin treated rats (D). These micrographs showing normal myocyte structure with dense cytoplasm filled with healthy myofibrils and its striated pattern with clear bands (Z and H) and mitochondria (m). A clear nucleus (N) with its nucleolus (nu) and blood vessels (BV) with its endothelium (En) are also seen.
the vehicle or ghrelin by means of electron microscopy. According to several authors, to detect myocardial apoptosis by electron microscopy, the following criteria should be reported including cell shrinkage, apoptotic body formation, chromatin condensation, vacuoles formation, mitochondriosis, myofibrillar disarray or lysis and phagocytosis [22,52]. On the other hand, ultra-structural characteristics that describe necrosis include cell swelling, sarcomeric proteins coagulation, karyolysis, pyknosis and karyorrhexis as well as granulocytes and macrophage infiltration [15]. In control or sham-operated rats, proteinaceous-like materials were
sham-operated rats significantly enhanced protein levels of Bcl-2 and lowered those related to Bax and cleaved caspase-3, indicating it is the ability to inhibit apoptosis. Indeed, in MI-treated rats, ghrelin significantly restored normal histological architectures and concomitantly increased protein levels of Bcl-2 and decreased Bax and cleaved caspase-3 protein levels. However, since light microscopy and even analysis of protein levels (in whole tissue homogenates) are not enough to determine cell death modality in cell or tissues, our main target in this study is to characterize cell death modality in remote myocardium of MI rats received
Fig. 5. Transmission electron micrographs of the remote myocardium of Left ventricles in MI-induced rats. These micrographs showing degenerated cytoplasm with discontinuation and lysis of some myofibrils (stars), blebs (arrows) on the periphery cell membranes, myelin figures (MF), lipofuscin particles (L) and pyknotic nuclei (N) with condensation of its heterochromatin (Chr) with irregularity in the nuclear membranes. Fragmentation of muscle bands (Z and H) and mitochondria (m) are markedly abnormal in shape, with abnormal cristae, areas of loss of matrix, and localized high densities. Proteinaceous-like materials (white asterisks) in the interstitium, vacuoles (V), Blood vessels (BV) and endothelium (En) are clearly seen.
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Fig. 6. Transmission electron micrographs of the remote myocardium of Left ventricles in MI-induced rats. These micrographs showing necrotic myocardial cells with phagocytic vacuoles (V), irregular heterochromatic nuclei (N) and plenty of ghost bodies (Gb), shrinkage blood vessel (BV) and atrophied nucleus were squeezed in the interstitium between myocardial cells. Degenerated cytoplasm with damaged myofibrils (stars), fragmentation of muscle bands (Z and H bands) and swollen or damaged mitochondria (m). Distinct collagen bundles (F) are clearly demonstrated inside myocardial cells (Fig B) and in the interstitium (Fig C).
like particles, which may result in apoptosis because of proteasomal inhibition [54]. Interestingly, all these ultra-structural alterations of both necrosis and apoptosis were diminished in the remote areas of LV of MI-induced rats treated with ghrelin. Hence and in conclusion, even it wasn’t of our interest to investigate the molecular mechanism by which ghrelin prevents necrosis and apoptosis post MI in survival myocardium during cardiac remodeling in late MI, these findings are the first one to describe the modality of cell death that occurs in the remote myocardium during cardiac remodeling and describes the ultra-structural improvements in these areas after ghrelin administration.
clearly seen in the interstitium with distinct collagen bundles. The electron microscopy from the control or sham-operated rat's hearts revealed a typical interphase area between the intact myocardium, with collagen deposition dispersed among them. The interphase zone demonstrated cells with different intracellular structures. In the majority of the cells, clusters of myofibrils anchored to well-developed Z-lines and structures resembling the morphological characteristics of mature intact cardiomyocytes were evident [53]. Supporting to biochemical, histological and molecular evidences, ultra-structural changes of the current study of both necrosis and apoptosis were also evident in the remote myocardium of the LV of MI –induced rats. There was clear disorganization of the myocardium with degenerated and discontinuation of cytoplasm, lysis of some myofibrils, fragmentation of muscle bands, loss of cell membranes in some myocytes with swollen and abnormal mitochondria and cristae. There were also abundant pyknotic nuclei and bundles of collagen fibers. In addition, blebs were seen at the periphery cell membranes. Irregularity in the nuclear membranes with chromatin condensation, atrophied nuclei, vacuolated mitochondria, limited endoplasmic reticulum, lesser intracellular organelles, and the presence of phagocytes were also seen. Additionally, we have found an enhanced accumulation of lipofuscin-
Conflict of interest The authors declare no conflict of interest. Acknowledgements The authors extend their appreciation to the deanship of Scientific Research at King Khalid Unviersity, Abha, KSA for funding this work through the research groups program under grant number (R.G.P.1/1/
Fig. 7. Transmission electron micrographs of the remote myocardium of Left ventricles in MI + ghrelin-treated rats. These micrographs showing marked improvement of normal myocardial structure with dense cytoplasm which filled with healthy myofibrils, intact muscle bands (Z and H) and mitochondria with moderate changes (arrows) in the cytoplasm.
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38). [28]
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Subacute ghrelin administration inhibits apoptosis and improves ultrastructural abnormalities in remote myocardium post-myocardial infarction
T
⁎
Refaat A. Eida, , Mohamed Samir Ahmed Zakib, Mubarak Al-Shraima, Samy M. Eleawac, Attalla Farag El-kottd,h, Fahaid H Al-Hasheme, Muhammad Alaa Eldeenf, Hoja Ibrahimg, Hussain Alderag, Mahmoud A. Alkhateebg a
Department of Pathology, College of Medicine, King Khalid University, Abha, Saudi Arabia Department of Anatomy, College of Medicine, King Khalid University, P.O. 641, Abha, 61421, Saudi Arabia Department of Applied Medical Sciences, College of Health Sciences, PAAET, Kuwait d Department of Biology, College of Science, King Khalid University, P.O. 641, Abha, 61421, Saudi Arabia e Department of Physiology, College of Medicine, King Khalid University, P.O. 641, Abha, 61421, Saudi Arabia f Biology Department, Physiology Section, Faculty of Science, Zagazig University, Egypt g Department of Basic Medical Sciences, College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Saudi Arabia h Department of Zoology, College of Science, Damanhour University, Egypt b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Ghrelin Remote myocardium Remodeling MI Electron microscope
This study investigated the effect of ghrelin on cardiomyocytes function, apoptosis and ultra-structural alterations of remote myocardium of the left ventricle (LV) of rats, 21 days post myocardial infarction (MI). Rats were divided into 4 groups as a control, a sham-operated rats, a sham-operated+ghrelin, an MI + vehicle and an MI + ghrelin-treated rats. MI was induced by LAD ligation and then rats were recievd a concomitant doe of either normal saline as a vehicle or treated with ghrelin (100 μg/kg S.C., 2x/day) for 21 consecutive days. Ghrelin enhanced myocardial contractility in control rats and reversed the decreases in myocardial contractility and the increases in the serum levels of CK-MB and LDH in MI-induced rats. Additionally, it inhibited the increases in levels of Bax and cleaved caspase 3 and increased those for Bcl-2 in the remote myocardium of rat's LV, post-MI. At ultra-structural level, while ghrelin has no adverse effects on LV myocardium obtained from control or sham-treated rats, ghrelin post-administration to MI-induced rats reduced vascular formation, restored normal microfilaments appearance and organization, preserved mitochondria structure, and prevented mitochondrial swelling, collagen deposition and number of ghost bodies in the remote areas of their LV. Concomitantly, in remote myocardium of MI-induced rats, ghrelin enhanced endoplasmic reticulum intracellular organelles count, decreased number of atrophied nuclei and phagocytes, diminished the irregularity in the nuclear membranes and inhibited chromatin condensation. In conclusion, in addition to the physiological, biochemical and molecular evidence provided, this is the first study that confirms the anti-apoptotic effect of ghrelin in the remote myocardium of the LV during late MI at the level of ultra-structural changes.
1. Introduction
activate remodeling in remote surviving myocardium [3], all of which lead to chamber dilation, LV dysfunction, and eventually heart failure (HF) [2,4–6]. Indeed, after an acute MI, apoptosis is apparently silent while its progression leads to subtle progressive cell loss in hibernating myocardium leading to HF [7,8]. Even with the extensive use of the light microscopy and its related biochemical techniques, e.g. TUNEL labeling [9,10], several authors have shown that such techniques are still nonspecific to detect cardiomyocytes apoptosis during LV remodeling [7].
Post-myocardial infarction (MI) remodeling occurs in both ischemic and non-ischemic areas of the left ventricle (LV) [1,2]. In this regard, the initial acute and rapid cardiomyocytes loss occurring in the ischemic area is associated with provoking inflammation, fibrosis, and hypertrophy in remote myocardium [1,3]. These events, in addition to the presence of regional wall strain, but independently of any further ischemia, can initiate and propagate cardiomyocytes apoptosis and ⁎
Corresponding author. E-mail address: [email protected] (R.A. Eid).
https://doi.org/10.1016/j.biopha.2018.03.010 Received 19 December 2017; Received in revised form 1 March 2018; Accepted 5 March 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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2. Materials and methods
Indeed, TUNEL labeling may provide variable data as it could detect DNA fragmentation of any origin including oncotic and apoptotic myocytes as well as the DNA repair mechanisms and cardiac hypertrophy [11–15]. This may reflect an overestimation of apoptosis, resulting in misleading conclusions [15]. Similarly, further flawed results could be obtained in techniques that depend on DNA or protein extraction, e.g, PCR and western blotting as these samples may contain extracts of both myocyte and non-myocyte cardiac cells, including intravascular circulating cells, vascular and interstitial cells [15,16]. On the other hand, several authors and the Nomenclature Committee on Cell Death (NCCD) have recently revised the mechanisms of cell death in tissues and recommended that classification of cell death modalities should rely purely by providing solid molecular evidence and on the presence of certain ultra-structural morphological criteria, as detected by electron microscopy (EM) [7,17–19]. Although the ultra-structural examinations by EM are considered the gold standard for the diagnosis of cardiomyocytes apoptosis, only few but recent EM studies have investigated the ultra-structural apoptotic changes that occur during cardiac remodeling post MI or during HF [15]. In this regard, the majority of these studies, which were carried only on the ischemic area after acute MI, had consensus and have confirmed active features apoptosis including aberrant nuclei, condensation of chromatin, myofibrillar disarray or lysis, disorganization of sarcomeres, peri-nuclear cytoplasmatic vacuolation, intranuclear clumping and mitochondria clustering as well as disturbances with disorganized cristae [15,20–23]. However, the ultra-structural changes in remote areas of the LV during late cardiac remodeling post-MI remain largely unknown and need further investigations. Ghrelin, a gut-derived 28-amino acid peptide hormone, is an endogenous ligand for the growth hormone secretagogue receptor (GHSR) and is a potent stimulator of GH [24]. In addition to the stomach and the gut, smaller amounts of ghrelin is also produced by peripheral organs including the heart, lung, kidney, pancreas, placenta, hypothalamus, gonads, thyroid, adrenal and pituitary glands indicating the wide physiological roles within the body [25–27]. Recently, accumulative evidence has shown important roles of ghrelin in regulating cardiac function and protection against various forms of cardiac disorders. Interestingly, human endothelial cells can synthesize ghrelin, and ghrelin receptors have been found in human endothelial cells, vascular smooth muscle cells, and LV [28]. In both clinical and experimental trials in patients with HF who survived after MI and in animal models of MI and ischemia/reperfusion (I/R) induced cardiac injury, exogenous ghrelin administration reduced infarct size and LV enlargements, enhanced LV contractility, improved cardiac output and index, reduced apoptosis and attenuated LV remodeling [29–31]. In other cardiotoxicity induced models of HF in rats (e.g. doxorubicin and Ang. II), ghrelin increased the size of cardiomyocytes, prolonged their survival, reduced fibrosis and inhibited apoptosis [32,33]. In spite of these findings, the ultra-structural evidence to support an anti-apoptotic effect of ghrelin post-MI in both infarcted and non infarcted areas post-MI is completely lacking. Very recently, we have shown that ghrelin administration (100 μg/ kg S.C., 2x/day) for 21 days, starting one day post MI, preserved LV function and reduced apoptosis in remote myocardial tissue of LV of rats in a mechanism that involves inhibition of SOCS3 and activation of JAK2/STAT3 signaling, an effect that is mediated at least by inhibition of cardiac levels of IL-6 [34]. To further support the role of the cardioprotective effect of ghrelin on cardiac apoptosis during remodeling, this study was designed to investigate the ultra-structural changes that occur in surviving remote myocardium during late MI with or without ghrelin administration in parallel with alterations in cardiac function and molecular evidence of apoptosis.
2.1. Animals Disease-free adult Sprague−Dawley male rats (9 weeks, 260-270 g) were included in this study. All rats were supplied by the animal facility at King Khalid University, Abha, Saudi Arabia and they were always housed (n = 4 rats/cage) in their controlled rooms at 23 ± 3 °C and 40−65% humidity, on a 12-h light/dark cycle. All animals had free access to drinking water and normal chow (AIN-93G diet). All experimental protocols of this study were approved by the College of Medicine Ethics Committee which are in accordance with guidelines published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). 2.2. Experimental approach Rats were divided into five groups (n = 6/group) and treated as follows for 21consecutive days: 1) A control group: received normal saline (0.5 ml S.C.) and were not exposed to any surgery. 2) A sham-operated group: control rats which received normal saline (0.5 ml S.C.) but underwent surgical procedure similar to that used MI model rats but without tying the anterior descending coronary artery (LAD). 3) A sham-operated + ghrelin-treated group: sham-operated rats as in group 2, but then received ghrelin (100 μg/kg S.C., twice/day). 4) An MI-induced group: underwent a surgical procedure to ligate LAD and received normal saline (0.5 ml S.C.). 5) MI + ghrelin-treated group: MI-induced rats but then received ghrelin (100 μg/kg S.C. twice/day). Rat’s synthetic ghrelin (C147H245N45O42) powder was purchased from Sigma (Cat. No. ab120231) and was freshly prepared in distilled water to a final concentration of (100 μ/kg). All treatments were started immediately after induction of MI and continued for a period of 21 days. The period and dose period of selected dose was based on our previous study where we showed that exogenous ghrelin administration of the same dose for 21 days post MI preserved cardiac hemodynamics and inhibited cardiomyoctes death in remote myocardium of LV [34]. Similarly, other authors have shown increased apoptosis and fibrosis in the remote areas of LV starting from 21 day and up to 6 months [29,31,34,35]. In addition, the selected dose of ghrelin was based on previous research that confirmed a cardioprotective effect of ghrelin in remote and ischemic myocardium for a similar period of treatment [29,31,34,]. 2.3. Surgical procedure and induction of MI Induction of MI was done as described before by us [34]. In brief, all rats were anesthetized with i.p. administration of 1% solution of sodium pentobarbital (50 mg/kg). After successful ventilation (Harvard rodent ventilator, model 863, Harvard Apparatus, Holliston) of each rats, the heart was exposed and LAD coronary artery was ligated, 2–3 mm from its origin, using a 6–0 prolene suture. Then the chest was sutured with 6–0 sutures and chest air was vacuumed using a 3 ml syringe. All procedures during the surgery and and recovery were done on a heated pad and a thermal probe was used to monitor the animal's temperature. In addition, an eye lubricant ointment was applied to both eyes in order to prevent dryness. Induction of MI in each rat was confirmed by the ST-segment elevation of the rat's electrocardiogram (ECG) recorded with the help of PowerLab system (AD instrument, Australia) using 3 limbs electrodes. All procedures were done by the same surgeon and usually lasted between 20–25 min. Post-operation, all rats received routine intramuscular injections of buprenorphine-HCl (0.2 mg/kg) for 921
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Fig. 1. Hemodynamic parameters (A–D) and serum enzyme levels of (CK-MB, E) and lactate dehydrogenase (LDH, F) in all group of rats. Values are expressed as Mean ± SD for 6 rats in each group. Values were considered significantly different at P < 0.05. a:vs. control group, b:Vs. sham group. c:Vs. sham + ghrelin group, d:Vs. MI group.
5000 rpm for 10 min to collect serum which was stored at −20 °C and used later to determine levels of CK-MB and lactate dehydrogenase using a biochemical analyzer. Then after, the rat was killed and heart was rapidly collected on ice. LV from each heart was collected and the non-infarcted area was identified and isolated. Parts of the non-infarcted area were directly frozen at −80 °C for western blotting study, other parts were directly collected in either 10% buffered formalin or 2.5% glutaraldehyde (in 0.1 M cacodylate buffer).
analgesia and penicillin (1000 U) to prevent infection. 2.4. LV function measurement and blood and tissue collection At the end of the experimental procedure, animals received intravenous (i.v.) administration of heparin (1000 unit) and then were anesthetized again with 1% solution of sodium pentobarbital (50 mg/ kg). Next, all animals were ventilated and their chest were opened again. In each rat, a pressure catheter (SPR-320, instrument) was directly inserted into the LV by stab method. With the help of power lab software and recording system (LabChart 8.3 ML780 and PowerLab/ 8channels, AD Instruments Ltd., Australia), LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), maximal rate of rise in LV pressure (LV dP/dtmax), and maximal rate of decline in LV pressure (LV dP/ dtmin) were recorded for next 30 min. All readings were analyzed and average values were presented. Then, 1ml blood samples was directly collected from the right carotid artery into a plain tube, centrifuged at
2.5. Western blotting Whole proteins were extracted from each frozen LV using a Millipore extraction kit (Cat. No. 2140, Merck Millipore, USA). During the extraction, protease inhibitor was added to each sample to prevent protein degradation (Cat. No. P8340, Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations in all samples were measured by a Bradford assay kit. Western blotting procedure was performed as 922
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Fig. 2. Protein levels of type activated caspase-3, BCL-2, and BAX in the left ventricles of all groups of rats as detected by western blotting.Values are expressed as Mean ± SD for 6 rats in each group. Values were considered significantly different at P < 0.05. a:vs. control group, b:Vs. sham group. c:Vs. sham + ghrelin group, d:Vs. MI group.
1 cubic millimeter sizes, fixed in gluteraldehyde solution in 0.1 M sodium cacodylate buffer, pH 7.2, and placed in a thermal box cooled to 4 °C for 2 h. They were post-fixed in 1% osmium tetraoxide in a sodium cacodylate buffer and then dehydrated in ascending series of ethyl alcohol and embedded in Spurr’s resin. Ultrathin sections stained with uranyl acetate and lead citrate were examined by TEM (JEM-1011, Jeol Co., Japan) operated at 80 KV in the Electron Microscopy Unit, Pathology Department, College of Medicine, King Khalid University [37].
described preciously by us [34]. Then, protein samples from all groups (60 μg protein/well) were separated on SDS-PAGE and transferred manually to nitrocellulose membranes and incubated with primary antibodies against Bax (Cat. No 2772, 20 kDa, cell signaling technology), Cleaved caspase-3 (Cat. No 9662, 17, 19 and 35 kDa, cell signaling technology) and BCL-2 (Cat. No, 2876, 28 kDa, cell signaling technology) and β-Actin (C4, sc-47778, 43 kDa, Santa Cruz Biotechnology). Then, membranes were washed and incubated with their corresponding HRP-conjugated secondary antibody. The protein bands were detected by chemiluminescence (Pierce ECL reagents, Thermofisher, USA, Piscataway, NJ) and were quantified using C-DiGit Blot Scanner (LI-COR, USA) with the supplied Image Studio DiGits software.
3. Results 3.1. Cardiac function and serum enzymes levels
2.6. Light microscopy The hemodynamic parameters presented in Fig. 1 describe the average mean changes as analyzed after continuous recording for 30 min on the last day of the experimental procedure, in each rat. In sham-operated rats, ghrelin administration only and significantly increased LVSP (122.8 ± 12.54 vs. 151.4 ± 12.14) (Fig. 1A). Twentyone day’s post-MI and as compared to control rats, MI-induced rats had significant lower values of LVSP (122.8 ± 12.54 vs.48.8 ± 15.69, Fig. 1A), dp/dtmax (5232 ± 992.5 vs. 2220 ± 380.9, Fig. 1C) and dp/ dtmin (3042 ± 842.2 vs 1428 ± 347.1, Fig. 1D) and higher levels of LVEDP (7.46 ± 1.92 vs. 23.48 ± 3.26, Fig. 1B). However, ghrelin administration for 21 days to MI-induced rats significantly decreased values of LVEDP (12.12 ± 1.89, Fig. 1B) and significantly increased those for of LVSP (79.0 ± 9.05, Fig. 1A), dp/dtmax (4083 ± 405.1,
For light microscopy, collected ventricular specimens were kept in 10% neutral buffered formalin for 24–48 h, dehydrated in graded alcohol series, cleared in xylene, and embedded in paraffin wax. Paraffinized specimens were sectioned at 4 μm and then stained with hematoxylin and eosin (H&E), examined under light microscope (Olympus CX31, Japan) and photographed using digital camera (Olympus, Camedia-5060, Japan) [36]. 2.7. Transmission electron microscopy (TEM) For TEM, heart specimens from both control and treated rats were immediately preserved in 2.5% gluteraldehyde, trimmed and diced into 923
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associated with a decrease in density of the entire muscle cell accentuated the darker intercalary disc. Intracellular spaces appeared between myofilaments in the perinuclear region and around the mitochondria (Fig. 5A–D). Later, these spaces were increased in size and number. Also, the bundles of myofilaments showed longitudinal cracking and separation. The separation of myofilaments was random since the size of the resulting bundles was variable. Moreover, a revealed markedly change is mitochondria swelling and disappearance of their cristae or with loss of cristae spatial organization (Fig. 5A–D). In some instances, the limiting membrane was altered, and the overall appearance was similar to that of ruptured mitochondria. Again, due of the separation of myofibrils and surrounding loss of density in the later preparations, the system was more clearly seen with swelling and enlargement of the vesicles and tubules. Nuclear alterations were detectable. The nucleoplasm showed condensation of heterochromatin with irregularity in the nuclear membrane (Fig. 5D). Moreover, transmission electron micrographs of the remote myocardium of LV in MI-induced rats showed necrotic myocardial cells with phagocytic vacuoles (Fig. 6A and B), irregular heterochromatic nuclei and plenty of ghost bodies, shrinkage blood vessel and atrophied nuclei were squeezed in the interstitium between myocardial cells (Fig. 6D). Degenerated cytoplasm with damaged myofibrils, fragmentation of muscle bands and swollen plus damaged mitochondria were noticed (Fig. 6A–D). Distinct collagen bundles were clearly demonstrated inside myocardial cells and in the interstitium (Fig. 6B and C). However, transmission electron micrographs of the remote myocardium of LV of MI + ghrelin-treated rats showed improvement of myocardial structure with dense cytoplasm which were filled with healthy myofibrils, intact muscle bands and mitochondria with moderate changes in its cytoplasm (Fig. 7A and B).
Fig. 1C) and dp/dtmin (2453 ± 400.9, Fig. 1D) as compared to MI-induced rats. On the other hand, normal sera levels of LDH and CK-BM were seen between control, sham-operated and sham-operated +ghrelin treated groups. However and as compared to control or shamoperated rats, MI-induced rats had significantly higher sera levels of both CK-MB (628.8 ± 93.54 vs. 181.0 ± 36.3, Fig. 1E) and LDH (2031 ± 220.2 vs.732.8 ± 103.8, Fig. 1F). On the other hand, ghrelin administration to MI-induced rats for 21 days significantly decreased levels of both CK-MB (281.1 ± 36.74) and LDH (1028.0 ± 153.5) in the sera of these treated rats (Fig. 1E and F). 3.2. Western blotting Detected bands of cleaved caspase-3, Bax and Bcl-2 as well as the reference protein, β-actin were within expected sizes (Fig. 2). Protein levels were presented as a relative expression of the loading control, βactin, which remained constant in the cardiomyocytes of the LV of all groups of rats. Interestingly, levels of Bcl-2 were significantly increased while levels of Bax and cleaved casopase-3 were significantly decreased in the LV of sham-operated rats treated with ghrelin. However, significant elevations in the protein levels of both Bax and cleaved caspase-3 with parallel significant decreases in the levels of Bcl-2 were detected in the remote myocardial tissue of LV of MI-induced rats after 21 days, as compared to control or sham-operated rats. However, as compared to MI-induced rats, MI + ghrelin-treated rats showed significantly inhibited levels of Bax and cleaved caspase-3 and significantly increased levels of Bcl-2 in the remote areas of their LV, 21 days post-MI. 3.3. Light microscopy
4. Discussion
Photomicrographs obtained from control, sham-operated and shamoperated+ghrelin rats showed normal branched cardiac muscle cells which were seen to contain nuclei (Fig. 3A–C). Photomicrographs obtained from MI-induced rats showed signs of both necrosis and apoptosis of the myocardium as evident by the presence of pyknotic nuclei and fragmentation of myocardial tissue (Fig. 3D and E). However, Photomicrographs obtained from MI + ghrelin treated group showed little changes of the architecture in the form of normal myofibrils and other fibrils appeared disrupted (Fig. 3F).
The exclusive findings of this study describe the ultra-structural changes that occur in the remote areas of LV of rats during cardiac remodeling, post experimentally induced MI in rats with or without ghrelin treatments. Generally, cell death or necrosis of cardiac myocytes may occur by the process of apoptosis, oncosis, or autophagy [15]. Our findings clearly demonstrate that modalities of cell death in these remote areas of LV of rats, 21 days post MI are due to activation of apoptosis and necrosis. While necrosis was evident by elevations of cardiac function enzymes (CK-MB and LDH) in the sera and histological alterations in remote myocardial tissues of the LV of MI-induced rats, apoptosis has been also confirmed in the same tested area by means of western blotting that showed enhanced expression of Bax and cleaved caspase-3 and was supported further by means of electron microscopy. Even parts of our curent data support our and other previously reported findings on the cardioprotective and anti-apoptotic roles of ghrelin during cardiac remodeling post MI [29–31,34], these data are the first that confirm these findings at the level of ultra-structure. In the current study, supporting to the our previous findings [34], hemodynamic parameters measured on day 21 days post-MI showed impaired LV contractility and lusitropy function as evident by the significant increases in LVEDP, significant decreases in LVdP/dtmax and LVSP, markers of myocardial contraction and by the significant decreases in LVdP/dtmin, a marker of myocardial relaxation and lusitropy [29–31,34]. The first observations of the cardioprotective effect of ghrelin were the observation that ghrelin administration to sham-operated rats increased LV inotropy without affecting LVEDP and significantly restored contractility and cardiac function parameters in MI rats. These findings are not surprising with the long history of the cardioprotective effect of ghrelin in various pathological conditions including I/R injury, MI, HF and drug-induced toxicities [29–33]. Such effect has been demonstrated previously in our labs in MI-induced-rats using the same rat's species, ghrelin dose, and treatment period [34]. In this regards, it has been suggested that ghrelin can exert its
3.4. Electron microscopy Ultrastructural findings of control, sham-operated and sham + ghrelin treated groups were depicted in Fig. 6. No significant alterations were detected between these three groups and their LV showed normal structures. The bundles of myofilaments are occasionally branched and contraction bands were generally in the region of the “Z” band (Fig. 4A–C). These were the result of placing pieces of muscle directly in fixative with no attempt to stretch or maintain fiber length. In most longitudinal sections, a dense “H” zone appeared halfway between two “Z” bands. Myofilaments coursed through a given cell and terminated at intercalary discs. The myofibrils were frequently surrounded by mitochondria (Fig. 4A–C). Usually, a mitochondrion was found in a branching manner. Mitochondria were found throughout the heart muscle fiber. They were located about the nucleus at either pole. They were also found in long chains between bundles of myofilaments (Fig. 4C). The nucleus was bounded by a double membrane (Fig. 4A–C). Also, nucleoplasm was evenly distributed, without dense aggregates. The nuclear material itself was comprised of a random array of filamentous structures and denser granules. Nucleolar substance frequently appeared as an aggregate of closely packed granules (Fig. 4A–C). A definite accentuation of the intercalary discs was evident in the LV specimens obtained from MI-induced rats (Fig. 5A–D). Adjacent to the disc, an intracellular pale zone appeared which contained a few randomly placed fine filamentous structures. The vacuole formation 924
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Fig. 3. Histopathological evaluation of the LVs obtained from all group of rats. (400X). Micrographs A, B & C are taken from the control, sham-operated and sham-operated+ghrelin treated rats, respectively showing normal cardiac muscle cells which are seen to contain nuclei (N) and branched fibers (arrows). Micrographs D and E are taken from MI-induced group showing necrosis and apoptosis of the myocardium that are evident by the presence of pyknotic nuclei (N) and fragmentation of myocardial tissue (arrows). Micrograph F is taken from rats MI + ghrelin treated group showing normal nuclei (N) with little changes of the architecture in the form of normal myofibrils and other fibrils appeared disrupted (arrow).
[14,45,46,]. However, apoptosis is an energy-dependent process of cell death where apoptotic myocytes are removed by macrophages through phagocytosis without triggering inflammation and peripheral circulatory levels of major markers of cell death such as troponin levels, CKMB and LDH are usually within the normal range [14,15,45,46]. Apoptosis in myocardium has been described as a major mechanism of the heart-induced dysfunction in various animal models including I/ R injury [47,48], post-MI LV remodeling [49] and heart failure [9,50]. Similarly, autophagic vacuoles are commonly seen in failing human hearts [44]. Furthermore, even not well characterized, the common form of oncotic cell death that has been described in acute MI is coagulative necrosis [39,51]. In the current study, light microscopy, biochemical and molecular findings are clear indications for the involvements of both necrosis and apoptosis in the cardiomyocyte death in remote non-infarcted areas of MI-induced rats during cardiac remodeling, 21 days post-MI. In this study, a significant elevation in the circulatory levels of LDH and CK-MB with histological alterations in the form of myofibril degenerations and fragmentation, pyknosis in most nuclei was seen in the remote areas of the LV, 21 days post-MI in MI-induced rats. In addition, the LV of MI-induced rats showed significant increases in protein levels of cleaved caspase 3 and Bax and significant decreases in levels of Bcl-2, major markers of cell apoptosis. Interestingly, ghrelin administration to
cardiovascular effect via its potent vasodilator effect (enhancement of NO production and decreasing peripheral vascular resistance) [38] as well as by its anti-apoptotic and anti-fibrotic effects [29–34]. In support to our current and previous findings [34], acute administration of ghrelin (100 μg/kg sc, twice daily) for 2 weeks post MI significantly reduced left ventricular end diastolic pressure and significantly reduced the enlargement of LV in rats [31]. However, associated with these changes it was of our interest to investigate the common modality of cell death in the remote areas of LVs post-MI in both MI-induced rats received the vehicle or treated with ghrelin. Hence, histological, molecular and ultrastructural approaches were considered as recommended by Nomenclature Committee on Cell Death (NCCD) as well as other authors [7,17–19]. In general, cardiomyocytes necrosis is well characterized by cellular swelling, coagulation of the sarcoplasmic proteins and nuclear loss and is usually associated with the release of intracellular contents such as troponin, CK-MB and LDH and triggered by an inflammatory response and fibrosis [15,39,40]. On the other hand, autophagy is a cellular degradation process to remove damaged organelles and cytoplasmic proteins [41] and occurs when damaged mitochondria and cytoplasmic proteins become included within the lysosomes leading to the generation of autophagocytic bodies [42–44]. Removal of autophagocytic necrotic cells occurs through phagocytosis with no inflammation 925
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Fig. 4. Transmission electron micrographs of the control (A and B), sham-operated (C) and sham + ghrelin treated rats (D). These micrographs showing normal myocyte structure with dense cytoplasm filled with healthy myofibrils and its striated pattern with clear bands (Z and H) and mitochondria (m). A clear nucleus (N) with its nucleolus (nu) and blood vessels (BV) with its endothelium (En) are also seen.
the vehicle or ghrelin by means of electron microscopy. According to several authors, to detect myocardial apoptosis by electron microscopy, the following criteria should be reported including cell shrinkage, apoptotic body formation, chromatin condensation, vacuoles formation, mitochondriosis, myofibrillar disarray or lysis and phagocytosis [22,52]. On the other hand, ultra-structural characteristics that describe necrosis include cell swelling, sarcomeric proteins coagulation, karyolysis, pyknosis and karyorrhexis as well as granulocytes and macrophage infiltration [15]. In control or sham-operated rats, proteinaceous-like materials were
sham-operated rats significantly enhanced protein levels of Bcl-2 and lowered those related to Bax and cleaved caspase-3, indicating it is the ability to inhibit apoptosis. Indeed, in MI-treated rats, ghrelin significantly restored normal histological architectures and concomitantly increased protein levels of Bcl-2 and decreased Bax and cleaved caspase-3 protein levels. However, since light microscopy and even analysis of protein levels (in whole tissue homogenates) are not enough to determine cell death modality in cell or tissues, our main target in this study is to characterize cell death modality in remote myocardium of MI rats received
Fig. 5. Transmission electron micrographs of the remote myocardium of Left ventricles in MI-induced rats. These micrographs showing degenerated cytoplasm with discontinuation and lysis of some myofibrils (stars), blebs (arrows) on the periphery cell membranes, myelin figures (MF), lipofuscin particles (L) and pyknotic nuclei (N) with condensation of its heterochromatin (Chr) with irregularity in the nuclear membranes. Fragmentation of muscle bands (Z and H) and mitochondria (m) are markedly abnormal in shape, with abnormal cristae, areas of loss of matrix, and localized high densities. Proteinaceous-like materials (white asterisks) in the interstitium, vacuoles (V), Blood vessels (BV) and endothelium (En) are clearly seen.
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Fig. 6. Transmission electron micrographs of the remote myocardium of Left ventricles in MI-induced rats. These micrographs showing necrotic myocardial cells with phagocytic vacuoles (V), irregular heterochromatic nuclei (N) and plenty of ghost bodies (Gb), shrinkage blood vessel (BV) and atrophied nucleus were squeezed in the interstitium between myocardial cells. Degenerated cytoplasm with damaged myofibrils (stars), fragmentation of muscle bands (Z and H bands) and swollen or damaged mitochondria (m). Distinct collagen bundles (F) are clearly demonstrated inside myocardial cells (Fig B) and in the interstitium (Fig C).
like particles, which may result in apoptosis because of proteasomal inhibition [54]. Interestingly, all these ultra-structural alterations of both necrosis and apoptosis were diminished in the remote areas of LV of MI-induced rats treated with ghrelin. Hence and in conclusion, even it wasn’t of our interest to investigate the molecular mechanism by which ghrelin prevents necrosis and apoptosis post MI in survival myocardium during cardiac remodeling in late MI, these findings are the first one to describe the modality of cell death that occurs in the remote myocardium during cardiac remodeling and describes the ultra-structural improvements in these areas after ghrelin administration.
clearly seen in the interstitium with distinct collagen bundles. The electron microscopy from the control or sham-operated rat's hearts revealed a typical interphase area between the intact myocardium, with collagen deposition dispersed among them. The interphase zone demonstrated cells with different intracellular structures. In the majority of the cells, clusters of myofibrils anchored to well-developed Z-lines and structures resembling the morphological characteristics of mature intact cardiomyocytes were evident [53]. Supporting to biochemical, histological and molecular evidences, ultra-structural changes of the current study of both necrosis and apoptosis were also evident in the remote myocardium of the LV of MI –induced rats. There was clear disorganization of the myocardium with degenerated and discontinuation of cytoplasm, lysis of some myofibrils, fragmentation of muscle bands, loss of cell membranes in some myocytes with swollen and abnormal mitochondria and cristae. There were also abundant pyknotic nuclei and bundles of collagen fibers. In addition, blebs were seen at the periphery cell membranes. Irregularity in the nuclear membranes with chromatin condensation, atrophied nuclei, vacuolated mitochondria, limited endoplasmic reticulum, lesser intracellular organelles, and the presence of phagocytes were also seen. Additionally, we have found an enhanced accumulation of lipofuscin-
Conflict of interest The authors declare no conflict of interest. Acknowledgements The authors extend their appreciation to the deanship of Scientific Research at King Khalid Unviersity, Abha, KSA for funding this work through the research groups program under grant number (R.G.P.1/1/
Fig. 7. Transmission electron micrographs of the remote myocardium of Left ventricles in MI + ghrelin-treated rats. These micrographs showing marked improvement of normal myocardial structure with dense cytoplasm which filled with healthy myofibrils, intact muscle bands (Z and H) and mitochondria with moderate changes (arrows) in the cytoplasm.
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