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Acute fluoride poisoning alters myocardial cytoskeletal and AMPK signaling proteins in rats
Acute fluoride poisoning alters myocardial cytoskeleton and AMPK signaling proteins in rats Lakshmikanthan Panneerselvam, Azhwar Raghunath, Ekambaram Perumal PII: DOI: Reference:
S0167-5273(16)32094-0 doi:10.1016/j.ijcard.2016.11.221 IJCA 24083
To appear in:
International Journal of Cardiology
Received date: Accepted date:
6 September 2016 7 November 2016
Please cite this article as: Panneerselvam Lakshmikanthan, Raghunath Azhwar, Perumal Ekambaram, Acute fluoride poisoning alters myocardial cytoskeleton and AMPK signaling proteins in rats, International Journal of Cardiology (2016), doi:10.1016/j.ijcard.2016.11.221
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ACCEPTED MANUSCRIPT Acute fluoride poisoning alters myocardial cytoskeleton and AMPK signaling proteins in rats.
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Lakshmikanthan Panneerselvam, Azhwar Raghunath, Ekambaram Perumal#
Molecular Toxicology Laboratory, Department of Biotechnology,
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Bharathiar University, Coimbatore, Tamil Nadu, India.
corresponding author:
Dr. Ekambaram Perumal
Molecular Toxicology Laboratory Department of Biotechnology Bharathiar University Coimbatore – 641 046 Email: [email protected] Telephone: 91-422-2428293 Fax: 91-422-2425706
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Abbreviations
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ACC- Acetyl coA carboxylase, AMPK-5'adenosine monophosphate-activated protein kinase,
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ATP-Adenosine Triphosphate, IF-Intermediate filaments, DAB -3,3’-Diaminobenzidine, TMB-
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3,3',5,5'-Tetramethylbenzidine.
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ACCEPTED MANUSCRIPT Abstract
Background: Our previous findings revealed that increased oxidative stress, apoptosis and
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necrosis were implicated in acute fluoride (F-) induced cardiac dysfunction apart from
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hypocalcemia and hyperkalemia. Cardiac intermediate filaments (desmin and vimentin) and
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cytoskeleton linker molecule vinculin plays an imperative role in maintaining the architecture of cardiac cytoskeleton. In addition, AMPK is a stress activated kinase regulates the energy
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homeostasis during stressed state. The present study was aimed to examine the role of
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cytoskeleton proteins and AMPK signaling molecules in acute F- induced cardiotoxicity in rats. Methods: In order to study this, male Wistar rats were treated with single oral doses of 45 and 90
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mg/kg F- for 24 h.
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Results: Acute F- intoxicated rats showed declined cytoskeletal protein expression of desmin,
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vimentin and vinculin in a dose dependent manner compared to control. A significant increase in phosphorylation of AMPKα (Thr172), AMPKß1 (Ser108) and Acetyl-coA carboxylase (ACC) (Ser79) in the myocardium and associated ATP deprivation were found in acute F- intoxicated rats. Further, ultra-structural studies confirmed myofibril lysis with interruption of Z lines, dilated sarcoplasmic reticulum and damaged mitochondrion were observed in the both the groups of F- intoxicated rats. Conclusion: Taken together, these findings reveal that acute F- exposure causes sudden heart failure by altering the expression of cytoskeletal proteins and AMPK signaling molecules.
Key words: Acute fluoride poisoning, AMPK, Cardiac dysfunction, Cytoskeletal disruption 3
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1. Introduction
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Fluorine (F) is a ubiquitous, highly reactive, electronegative element reacts with
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positively charged minerals and metal ions in the nature to form stable compounds such as sodium fluoride, calcium fluoride, aluminum, magnesium and hydrogen fluoride. Besides from
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its natural existence in the earth crust, the major anthropogenic source includes industrial wastes,
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electroplating, glass etching, rust removers, phosphate-fertilizers, aluminum plants [1] and domestic usage includes coal combustion, dental mouth washes and public water fluoridation
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system [2-3]. Accidental, occupational and suicidal (intentional) exposure of acute fluoride (F-) intoxication causes cardiac dysfunction which leads to sudden death in humans [4-6] and
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experimental animals [7]. It has been extensively studied that acute F- toxicity impairs cardiac function via decreased extracellular calcium (hypocalcemia), indirectly blocks the cardiac
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voltage gated sodium channels (hyponatremia) and increased intracellular K+ efflux (hyperkalemia) causes ventricular arrhythmias [4,8]. In our earlier study, we found oxidative stress, apoptosis and necrosis mediated cardiac dysfunction in acute F- intoxicated rats [9]. However, the underlying molecular mechanism in acute F- induced cardiac dysfunction needs to be studied.
Intermediate filaments (IFs) are the principal components in the myocardial cytoskeleton. Desmin and vimentin falls under the category of type III IFs predominantly present in the endothelial and fibroblast cells of the heart and muscles. Desmin and vimentin were the peculiar cardio-specific smooth muscle markers used for the prognosis in the development of cardiovascular disease and eventually heart failure. Desmin provides structural integrity to the
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ACCEPTED MANUSCRIPT cardiomyocytes by integrates each myofibrils one another at the Z disc of the sarcomere and anchored to the costamere of the sarcolemma [10]. Vimentin plays an important role in linking
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the cellular components with the nucleus and provides the stability to perform various cellular
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processes such as cell cycle, cell migration, cell spreading, and cell signaling [11]. In addition, the filamentous structure of vimentin form a network like apparatus helps in positioning of sub
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cellular organelles and prevents the slippage either from external or internal stress [12].
Vinculin is a membrane associated adaptor molecule which links the actin to other focal
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adhesion molecules. It also acts as mechno-coupler and mainly used as an exclusive marker for both cell–cell and cell–extracellular matrix adhesion [13]. Previous studies demonstrates that F-,
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a potent activator of small G protein Rho/RhoK signaling molecules and phosphorylates myosin light chain proteins which disturbs the F-actin cytoskeleton in murine ameloblasts [14] and cell
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barrier dysfunction in endothelial cells [15]. In addition, F- induces cell proliferation, cell migration and matrix protein production in normal epithelial cells [16] and GH4C1 cells [17]. However, the involvement of cytoskeletal protein vinculin and IFs desmin and vimentin in acute F- induced cardiac dysfunction remains unexplored.
AMP-activated protein kinase (AMPK) is the key regulator of energy metabolism and important stress signaling molecules in the heart. AMPK activation is the key indicator of ATP depletion and increased AMP:ATP ratio in the heart. Moreover, AMPK is also activated by various pathophysiological conditions such as oxidative stress, hypoxia, hypoglycemia, starvation, heat shock and ischemia [18]. Moreover, the activation of AMPK results in stimulation of glucose uptake, glycolysis and fatty acid oxidation [19]. Interestingly, F- is well
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ACCEPTED MANUSCRIPT known to inhibit cellular respiration enzymes of glycolytic, Krebs cycle pathway leads to the depletion of ATP and causes disturbances in the cell membrane potential and membrane–
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cytoskeleton interactions [20-22]. However, the molecular events leading to activation of AMPK signaling proteins in the myocardium during acute F- exposure has not been elucidated yet. Thus,
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we hypothesize that F- causes changes in the expression level of cytoskeletal and AMPK
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molecules may plays a crucial role in the progression of cardiac dysfunction.
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2. Materials and methods
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2.1. Chemicals
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Sodium fluoride (Loba Chemie, Mumbai, India) and antibodies of AMPKα, Phospho-
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AMPKα (Thr172), AMPKβ1/2, Phospho-AMPKß1 (Ser108), Acetyl-CoA Carboxylase, Phospho-Acetyl-CoA Carboxylase (Ser79), GAPDH (Cell Signaling Technology, Inc.,USA) and Secondary antibodies conjugated with horseradish peroxidase (Merck, Darmstadt, Germany) were used in this study. Polyvinylidene fluoride (PVDF) membrane (Millipore Immobilon, Bedford, USA), PierceTM DAB Substrate kit, PierceTM TMB Substrate kit, and ATP determination kit (Thermo Fisher Scientific, Texas, USA) were also used in this study. All the other chemicals used in the experiments were of molecular grade unless mentioned otherwise.
2.2. Animals
Adult (3 months and 2 weeks old) male Wistar rats, weighing (140-150 g) were chosen for the experiment. The animals were purchased from Karpagam University, Coimbatore, India. 6
ACCEPTED MANUSCRIPT The animals were housed in vivarium at standard temperature (22 ± 3 º C) and humidity (55 ± 5%) in a normal 12 h light/dark cycle and supplemented with standard commercially available
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pelleted rat chow (Krish Scientific, Pvt. Ltd, Bangalore, India), and the tap water was supplied
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ad libitum. All the animals experiments were performed in this study were consonance with the ethical guidelines certified by committee for the purpose of control and supervision of
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experiments on animals (CPCSEA) and institutional animal ethical committee (Approval No:
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722/02/CPCSEA).
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2.3. Experimental design
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Group 1: Normal control rats were administrated orally equivalent volume of tap
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Water (0.2 ppm of F-).
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Group 2: Rats administrated with F- (45 mg/kg body weight (b.w), orally in a volume of 0.1 mL/100 g b.w. for 24 h.
Group 3: Rats administrated with F- (90 mg/kg b.w, orally in a volume of 0.1 mL/100 g b.w. for 24 h.
The LD50 value of acute F- toxicity was reported as 126.3 mg F-/kg in male rats [23]. In our previous study we reported that single oral doses of 45 and 90 mg/kg F- for 24 h induces myocardial oxidative stress, apoptosis and necrosis in male rats [9]. Based on the previous reports we selected both dose of 45 and 90 mg F-/kg to assess the changes in the myocardial cytoskeleton and AMPK signaling molecules in rat heart. After, 24 h the rats were sacrificed by
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ACCEPTED MANUSCRIPT cervical decapitation and the heart tissues were dissected out, rinsed in physiological saline and
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stored in -80 º C for further experiments.
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2.4. ATP determination
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Myocardial tissue was homogenized with ice cold lysis buffer (25 mM Tris, pH 7.5, 4
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mM EGTA, 1% Triton X-100, 10% glycerol, 2 mM dithiothreitol). Briefly, the homogenate was centrifuged at 12,000 g for 5 min and the supernatant was used to measure myocardial ATP by
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firefly luciferase-based ATP detection kit as per manufacturer’s protocol (Thermo Fisher Scientific, Texas, USA). The reaction was initiated by adding 10 µL of diluted supernatant with
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Multi-Mode Reader, USA).
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100 µL of ATP reaction buffer in a luminescence based multimode reader (Synergy H1, BioTek
2.5. Western blot analysis
Western blot analysis were performed in the myocardial tissue of control and Fintoxicated rats. The myocardial tissues were homogenized with ice cold RIPA lysis buffer and the total protein was measured using Bradford reagent with BSA as standard. 40 µg of protein sample was equally loaded in each lane separated by 10% w/v, SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked using 5% fat free BSA and incubated overnight with primary antibodies against rabbit anti-desmin, anti-vimentin, antivinculin, anti-AMPK, anti-phospho AMPKα (Thr172), anti-AMPKβ1/2, anti-phospho AMPKβ (Ser108) anti-ACC and anti-phospho ACC (Ser79). The proteins were detected using horseradish
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ACCEPTED MANUSCRIPT peroxidase-conjugated anti-rabbit secondary antibodies and visualized by TMB staining kit. GAPDH was used as the internal control to ensure equal sample loading. Densitometry analyses
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of immunoblots were quantified using ImageJ analysis software (NIH).
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2.6. Immunohistochemical analysis
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Immunohistochemical analysis was performed in the paraffin embedded unstained section of myocardial tissues were deparffinized in the xylene and followed by subsequent
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rehydration with different grades of ethanol. The section were placed with laboratory stainless pressure cooker in order to unmask the antigen by Heat induced epitope retrieval method.
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Endogenous peroxidase activity was quenched by treating the slides with 1% H2O2 solution. The slides were blocked with 5 % BSA for 1 h at room temperature to avoid the non-specific
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reactivity. The sections were incubated with primary antibody of rabbit anti-desmin (1:100), antivimentin (1:250), anti-vinculin (1:500), anti-phospho AMPKα (Thr172) (1:100) and antiphospho ACC (Ser79) (1:100). The proteins were detected using horseradish peroxidaseconjugated anti-rabbit, or anti-goat secondary antibodies. Immunoreactions were labeled using a DAB staining kit. Sections were counterstained with hematoxylin, dehydrated with different grade of ethanol and mounted with distrene plasticizer xylene. Immunoreactivity was quantitated in a blinded fashion for depicting the total number of positively stained cells.
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ACCEPTED MANUSCRIPT 2.7. TEM analysis
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For TEM analysis, the myocardial tissues were fixed in 2.5%, v/v glutaraldehyde in 0.1
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M cacodylate buffer, pH 7.4, w/v at 4 ºC. Myocardial tissues were processed with 1%, w/v cacodylate-buffered osmium tetroxide, dehydrated with differently graded ethanol series and
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embedded in an epoxy resin. Myocardial sections were stained with uranyl acetate and lead
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acetate solutions and mounted in the copper grid. The ultrathin myocardial sections were
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examined using a transmission electron microscope
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3. Statistical analysis
All the values were expressed as mean ± SD of six rats in each group. The data were
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analyzed with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test using SPSS (20.0) software package. p < 0.05 was considered to indicate statistical significance.
4. Results
4.1. Mortality and clinical observation All the animals were monitored throughout the experimental period for any clinical signs of toxicity. There was no death in the control and F- intoxicated rats. But we observed clinical symptoms such as hyper salivation, diarrhea and increased heartbeat.
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ACCEPTED MANUSCRIPT 4.2. Myocardial ATP determination
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Fig. 1 shows the levels of ATP in the myocardial tissues of control and experimental groups. The ATP level in the F- treated cardiac tissues was found to be decreased when
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compared to control. There was no significance difference between 45 and 90 mg F- treated rats.
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4.3. Effect of acute F- intoxication on cardiac cytoskeletal protein
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Fig. 2 and 3 depict the protein expression of cardiac cytoskeletal (Desmin, vimentin and vinculin) in control and F- intoxicated rats. The relative quantitative levels of protein expression
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of cardiac cytoskeletal proteins such as desmin, vimentin and vinculin were found to be significantly decreased in both western blotting (Fig. 2) and immunohistochemical studies (Fig.
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3) of both 45 and 90 mg F- treated rats when compared to control.
4.4. Effect of acute F- intoxication on myocardial AMPK signaling molecules
To determine whether AMPK activation restores the energy homeostasis in acute Finduced cardiotoxicity, we tested the changes in the protein expression level of AMPK signaling molecules by western blotting (Fig. 4) and immunohistochemical studies (Fig. 5). A significant increase in the expression levels of phosphorylated AMPKα (Thr172), AMPKβ1 (Ser108) and ACC (Ser79) was observed in the myocardium of both 45 and 90 mg F- treated rats when compared to control.
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ACCEPTED MANUSCRIPT 4.5. TEM analysis
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TEM analysis was performed to examine the cardiac ultra-structural changes in the
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cytoskeleton of control and F- intoxicated rats (Fig. 6). Cytoskeletal fragmentation and lysis of myofibrils with interruption of Z lines, dilated sarcoplasmic reticulum and distorted
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mitochondrion were also observed in both groups of rats treated with different doses of F- when
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compared to control.
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5. Discussion
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Acute F- intoxication causes sudden cardiac failure with hypocalcemia, hyperkalemia mediated ventricular fibrillation, oxidative stress, mitochondrial dysfunction, myocardial
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apoptosis and necrosis were briefly discussed in our previous study [9]. However, the underlying molecular mechanism in acute F- induced cardiac dysfunction needs to be studied in detail. The present study was aimed to elucidate the changes in the expression of cardiac cytoskeleton proteins (desmin, vimentin and vinculin) and AMPK signaling molecules in acute F- induced cardiac dysfunction. Here, we report for the first time that acute F- intoxication alters the expression of myocardial cytoskeletal proteins and AMPK signaling molecules.
F- has been reported to alter the actin cytoskeletal networks involved in the epithelial cell proliferation [16] and cell migration and matrix protein production in pituitary tumor cells [17]. In the present study, we examined whether cytoskeletal proteins were implicated in acute Finduced cardiac dysfunction in rats. F- intoxicated rats showed significant decline in desmin,
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ACCEPTED MANUSCRIPT vimentin and vinculin expression in the myocardium when compared to control. It has been reported that decreased expression pattern of desmin along with focal loss of contractile
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apparatus and myocardial apoptosis were observed in doxorubicin treated rats [24]. In a recent
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study, arsenic exposure increases the risk of heart failure by decreased expression of vimentin in murine coronary progenitor cells [25]. In addition, decreased vinculin proteome was associated
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with altered structural integrity of left ventricular myocardium in bisphenol intoxicated rats [26].
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protein were consistent with our findings.
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In addition, several cardiotoxic studies [27-29] reveals that altered expression of cytoskeleton
In addition to the cytoskeletal protein expression studies, TEM analysis revealed the
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ultra-structural detrimental changes in the myocardium of acute F- intoxicated rats. In the present study we observed extensive lysis of myofibrils with disruption of Z lines, dilated fragmented
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sarcoplasmic reticulum and distorted mitochondrion in the both doses of F- treated rats when compared to control. The ultra-structural morphological deformities such as myofibril lysis and distorted mitochondrial matrix in the present study were in consistent with our previous findings [9]. Thus, F- induced cardiac impairment is not only mediated through oxidative stress, apoptosis, necrosis and also through decreased expression of cardiac cytoskeletal proteins in the rat myocardium.
AMPK is the master regulator of energy homeostasis in higher eukaryotes. Numerous studies have revealed that activation of AMPK signaling molecules, mainly occurs during increased energy deprivation (ATP level), intracellular rise of AMP, ADP ratio levels in the cardiac pathology of humans and experimental models [30-32]. Our previous study showed that
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ACCEPTED MANUSCRIPT F- causes cardiac dysfunction via induction of ROS, oxidative stress, apoptosis and necrosis in rat [9]. Induction of ROS production also serves as an upstream signal for the activation of
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AMPK signaling molecules in various cardiotoxicity studies [33-34]. It is well acknowledged that the F- is the potent inhibitor of glycolytic and Krebs cycle enzymes, which causes energy
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depletion [20]. Recent studies showed that F- impairs mitochondrial function results in decreased
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the intracellular ATP level in both in vivo [22] and in vitro model [35]. However, the activation
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of AMPK signaling molecules in F- induced cardiotoxicity is not known.
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In the present study, we found a significant increased phosphorylation of AMPKα (Thr 172), AMPKß1 (Ser108) and ACC (Ser79) in the myocardium of both 45 and 90 mg F - treated
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rats when compared to control. Earlier studies revealed that phosphorylation of AMPK and its downstream target protein ACC (ser79) enhances the oxidation of fatty acids in the
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mitochondria, which rescue the cardiomyocytes from the energy deprived state [19,36]. In addition, a significant decrease in myocardial ATP level were observed in F - intoxicated rats confirms the energy depletion. However, in our earlier study we observed oxidative stress, apoptosis and necrosis were might be due to pro-apoptotic action of AMPK dependent or independent mechanism in the myocardium of F- intoxicated rats. Further, in-depth molecular studies are needed to confirm the co-existences of both pro-survival and pro-apoptotic mechanism in the activation of AMPK signaling pathways against acute F- induced cardiac dysfunction. The limitation of present study speculates the absence of multiple time point observation of acute F- exposure and its pathophysiological changes in rat heart. Further, studies are needed
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ACCEPTED MANUSCRIPT to confirm with AMPK activators, inhibitors and their differential protein expression in acute Finduced cardiac dysfunction.
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6. Conclusion
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Acute F- poisoning alters the expression of cardiac cytoskeletal proteins and AMPK signaling pathway in cardiac physiology and to understand the molecular mechanism involved in
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pathology of cardiac dysfunction.
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Conflict of interest
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The authors declare that they have no conflict of interest
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Funding information
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This work was supported by the UGC-SAP DRS II:F-3-30/2013 and DST-FIST:SR/FST/LSI618/2014, New Delhi, India for the partial financial assistance. The author LP also extends his thanks to the Indian Council of Medical Research, New Delhi, for financial assistance in the form of a senior research fellowship (No. 45/25/2013/BMS/TRM). Acknowledgement
The authors would like to thank Dr. Prakash Dharmalingam, Houston Methodist Research Institute, Texas, USA for his valuable suggestion and comments throughout this study. The authors are also grateful to research fellows Mr. G. Vimal, K. Vishnu Vignesh, and Ms. S. Kirthika Bharathiar University, Coimbatore for their timely assistances throughout the work. The authors sincerely acknowledge Mrs. B. Rita, for her technical assistance in transmission electron
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ACCEPTED MANUSCRIPT microscopic studies in Wellcome Trust Research Laboratory, Department of Gastrointestinal
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Sciences, Christian Medical College and Hospital, Vellore, India.
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ACCEPTED MANUSCRIPT Figure legends Fig. 1 ATP level in the heart tissue of control and F- treated rats. Values are mean±SD of six rats
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in each group. *p<0.05 significantly different from control, +p<0.05, significantly different from
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45 mg F- treated rats (One way ANOVA followed by Tukey’s multiple comparison test).
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Fig. 2 Western blotting and its densitometric analysis of cardiac cytoskeletal proteins (desmin,
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vimentin and vinculin) in control and F- treated rats. GAPDH was used as loading control. Values represent mean±SD (n=3). *p<0.05 significantly different from control, +p<0.05,
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significantly different from 45 mg F- treated rats (One way ANOVA followed by Tukey’s
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multiple comparison test).
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Fig. 3 Representative micrographs of immunostained sections of myocardium in control and Ftreated rats. Myocardial sections were stained with antibodies specific for desmin, vimentin and
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vinculin and the number of immunopostively stained cells in ten fields/section (200x) was quantified by three independent observers in blinded fashion. The bar diagram shows the quantification of positively stained cells of control and F- treated rats. The values represent mean ± SD (n=3). *p < 0.05 significantly different from control, +p < 0.05, significantly different from 45 mg F- treated rats (One way ANOVA followed by Tukey's multiple comparison test). Fig. 4 Western blotting and its densitometric analysis of both total and phosphorylated myocardial AMPK signaling molecules in control and F- treated rats. GAPDH was used as loading control. Values represent mean±SD (n=3). *p<0.05 significantly different from control, +p<0.05, significantly different from 45 mg F- treated rats (One way ANOVA followed by Tukey’s multiple comparison test).
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ACCEPTED MANUSCRIPT Fig. 5 Representative micrographs of immunostained sections of myocardium in control and Ftreated rats. Myocardial sections were stained with antibodies specific for p-AMPKα (Th172), p-
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ACC (Ser79) and the number of immunopostively stained cells in ten fields/section (200x) was quantified by three independent observers in blinded fashion. Representative bar diagram shows
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quantification of positively stained cells of control and F- treated rats. Values represent mean±SD
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(n=3). *p<0.05 significantly different from control, +p<0.05, significantly different from 45 mg
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F- treated rats (One way ANOVA followed by Tukey’s multiple comparison test).
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Fig. 6 Representative micrographs shows TEM images of myocardial section. Normal architecture of cytoskeletal myofibrils with Z lines, sarcoplasmic reticulum and mitochondrion in
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the control rat heart. Fragmented cytoskeleton shows myofibril lysis with interruption of Z lines,
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dilated sarcoplasmic reticulum and distorted mitochondrion in both 45 mg F- and 90 mg F-
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treated rat heart (Scale bar: 200 nm, 26500x and 16500x).
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S0167-5273(16)32094-0 doi:10.1016/j.ijcard.2016.11.221 IJCA 24083
To appear in:
International Journal of Cardiology
Received date: Accepted date:
6 September 2016 7 November 2016
Please cite this article as: Panneerselvam Lakshmikanthan, Raghunath Azhwar, Perumal Ekambaram, Acute fluoride poisoning alters myocardial cytoskeleton and AMPK signaling proteins in rats, International Journal of Cardiology (2016), doi:10.1016/j.ijcard.2016.11.221
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Acute fluoride poisoning alters myocardial cytoskeleton and AMPK signaling proteins in rats.
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Lakshmikanthan Panneerselvam, Azhwar Raghunath, Ekambaram Perumal#
Molecular Toxicology Laboratory, Department of Biotechnology,
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Bharathiar University, Coimbatore, Tamil Nadu, India.
corresponding author:
Dr. Ekambaram Perumal
Molecular Toxicology Laboratory Department of Biotechnology Bharathiar University Coimbatore – 641 046 Email: [email protected] Telephone: 91-422-2428293 Fax: 91-422-2425706
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Abbreviations
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ACC- Acetyl coA carboxylase, AMPK-5'adenosine monophosphate-activated protein kinase,
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ATP-Adenosine Triphosphate, IF-Intermediate filaments, DAB -3,3’-Diaminobenzidine, TMB-
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3,3',5,5'-Tetramethylbenzidine.
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ACCEPTED MANUSCRIPT Abstract
Background: Our previous findings revealed that increased oxidative stress, apoptosis and
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necrosis were implicated in acute fluoride (F-) induced cardiac dysfunction apart from
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hypocalcemia and hyperkalemia. Cardiac intermediate filaments (desmin and vimentin) and
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cytoskeleton linker molecule vinculin plays an imperative role in maintaining the architecture of cardiac cytoskeleton. In addition, AMPK is a stress activated kinase regulates the energy
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homeostasis during stressed state. The present study was aimed to examine the role of
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cytoskeleton proteins and AMPK signaling molecules in acute F- induced cardiotoxicity in rats. Methods: In order to study this, male Wistar rats were treated with single oral doses of 45 and 90
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mg/kg F- for 24 h.
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Results: Acute F- intoxicated rats showed declined cytoskeletal protein expression of desmin,
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vimentin and vinculin in a dose dependent manner compared to control. A significant increase in phosphorylation of AMPKα (Thr172), AMPKß1 (Ser108) and Acetyl-coA carboxylase (ACC) (Ser79) in the myocardium and associated ATP deprivation were found in acute F- intoxicated rats. Further, ultra-structural studies confirmed myofibril lysis with interruption of Z lines, dilated sarcoplasmic reticulum and damaged mitochondrion were observed in the both the groups of F- intoxicated rats. Conclusion: Taken together, these findings reveal that acute F- exposure causes sudden heart failure by altering the expression of cytoskeletal proteins and AMPK signaling molecules.
Key words: Acute fluoride poisoning, AMPK, Cardiac dysfunction, Cytoskeletal disruption 3
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1. Introduction
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Fluorine (F) is a ubiquitous, highly reactive, electronegative element reacts with
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positively charged minerals and metal ions in the nature to form stable compounds such as sodium fluoride, calcium fluoride, aluminum, magnesium and hydrogen fluoride. Besides from
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its natural existence in the earth crust, the major anthropogenic source includes industrial wastes,
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electroplating, glass etching, rust removers, phosphate-fertilizers, aluminum plants [1] and domestic usage includes coal combustion, dental mouth washes and public water fluoridation
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system [2-3]. Accidental, occupational and suicidal (intentional) exposure of acute fluoride (F-) intoxication causes cardiac dysfunction which leads to sudden death in humans [4-6] and
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experimental animals [7]. It has been extensively studied that acute F- toxicity impairs cardiac function via decreased extracellular calcium (hypocalcemia), indirectly blocks the cardiac
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voltage gated sodium channels (hyponatremia) and increased intracellular K+ efflux (hyperkalemia) causes ventricular arrhythmias [4,8]. In our earlier study, we found oxidative stress, apoptosis and necrosis mediated cardiac dysfunction in acute F- intoxicated rats [9]. However, the underlying molecular mechanism in acute F- induced cardiac dysfunction needs to be studied.
Intermediate filaments (IFs) are the principal components in the myocardial cytoskeleton. Desmin and vimentin falls under the category of type III IFs predominantly present in the endothelial and fibroblast cells of the heart and muscles. Desmin and vimentin were the peculiar cardio-specific smooth muscle markers used for the prognosis in the development of cardiovascular disease and eventually heart failure. Desmin provides structural integrity to the
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ACCEPTED MANUSCRIPT cardiomyocytes by integrates each myofibrils one another at the Z disc of the sarcomere and anchored to the costamere of the sarcolemma [10]. Vimentin plays an important role in linking
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the cellular components with the nucleus and provides the stability to perform various cellular
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processes such as cell cycle, cell migration, cell spreading, and cell signaling [11]. In addition, the filamentous structure of vimentin form a network like apparatus helps in positioning of sub
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cellular organelles and prevents the slippage either from external or internal stress [12].
Vinculin is a membrane associated adaptor molecule which links the actin to other focal
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adhesion molecules. It also acts as mechno-coupler and mainly used as an exclusive marker for both cell–cell and cell–extracellular matrix adhesion [13]. Previous studies demonstrates that F-,
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a potent activator of small G protein Rho/RhoK signaling molecules and phosphorylates myosin light chain proteins which disturbs the F-actin cytoskeleton in murine ameloblasts [14] and cell
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barrier dysfunction in endothelial cells [15]. In addition, F- induces cell proliferation, cell migration and matrix protein production in normal epithelial cells [16] and GH4C1 cells [17]. However, the involvement of cytoskeletal protein vinculin and IFs desmin and vimentin in acute F- induced cardiac dysfunction remains unexplored.
AMP-activated protein kinase (AMPK) is the key regulator of energy metabolism and important stress signaling molecules in the heart. AMPK activation is the key indicator of ATP depletion and increased AMP:ATP ratio in the heart. Moreover, AMPK is also activated by various pathophysiological conditions such as oxidative stress, hypoxia, hypoglycemia, starvation, heat shock and ischemia [18]. Moreover, the activation of AMPK results in stimulation of glucose uptake, glycolysis and fatty acid oxidation [19]. Interestingly, F- is well
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ACCEPTED MANUSCRIPT known to inhibit cellular respiration enzymes of glycolytic, Krebs cycle pathway leads to the depletion of ATP and causes disturbances in the cell membrane potential and membrane–
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cytoskeleton interactions [20-22]. However, the molecular events leading to activation of AMPK signaling proteins in the myocardium during acute F- exposure has not been elucidated yet. Thus,
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we hypothesize that F- causes changes in the expression level of cytoskeletal and AMPK
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molecules may plays a crucial role in the progression of cardiac dysfunction.
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2. Materials and methods
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2.1. Chemicals
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Sodium fluoride (Loba Chemie, Mumbai, India) and antibodies of AMPKα, Phospho-
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AMPKα (Thr172), AMPKβ1/2, Phospho-AMPKß1 (Ser108), Acetyl-CoA Carboxylase, Phospho-Acetyl-CoA Carboxylase (Ser79), GAPDH (Cell Signaling Technology, Inc.,USA) and Secondary antibodies conjugated with horseradish peroxidase (Merck, Darmstadt, Germany) were used in this study. Polyvinylidene fluoride (PVDF) membrane (Millipore Immobilon, Bedford, USA), PierceTM DAB Substrate kit, PierceTM TMB Substrate kit, and ATP determination kit (Thermo Fisher Scientific, Texas, USA) were also used in this study. All the other chemicals used in the experiments were of molecular grade unless mentioned otherwise.
2.2. Animals
Adult (3 months and 2 weeks old) male Wistar rats, weighing (140-150 g) were chosen for the experiment. The animals were purchased from Karpagam University, Coimbatore, India. 6
ACCEPTED MANUSCRIPT The animals were housed in vivarium at standard temperature (22 ± 3 º C) and humidity (55 ± 5%) in a normal 12 h light/dark cycle and supplemented with standard commercially available
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pelleted rat chow (Krish Scientific, Pvt. Ltd, Bangalore, India), and the tap water was supplied
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ad libitum. All the animals experiments were performed in this study were consonance with the ethical guidelines certified by committee for the purpose of control and supervision of
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experiments on animals (CPCSEA) and institutional animal ethical committee (Approval No:
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722/02/CPCSEA).
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2.3. Experimental design
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Group 1: Normal control rats were administrated orally equivalent volume of tap
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Water (0.2 ppm of F-).
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Group 2: Rats administrated with F- (45 mg/kg body weight (b.w), orally in a volume of 0.1 mL/100 g b.w. for 24 h.
Group 3: Rats administrated with F- (90 mg/kg b.w, orally in a volume of 0.1 mL/100 g b.w. for 24 h.
The LD50 value of acute F- toxicity was reported as 126.3 mg F-/kg in male rats [23]. In our previous study we reported that single oral doses of 45 and 90 mg/kg F- for 24 h induces myocardial oxidative stress, apoptosis and necrosis in male rats [9]. Based on the previous reports we selected both dose of 45 and 90 mg F-/kg to assess the changes in the myocardial cytoskeleton and AMPK signaling molecules in rat heart. After, 24 h the rats were sacrificed by
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ACCEPTED MANUSCRIPT cervical decapitation and the heart tissues were dissected out, rinsed in physiological saline and
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stored in -80 º C for further experiments.
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2.4. ATP determination
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Myocardial tissue was homogenized with ice cold lysis buffer (25 mM Tris, pH 7.5, 4
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mM EGTA, 1% Triton X-100, 10% glycerol, 2 mM dithiothreitol). Briefly, the homogenate was centrifuged at 12,000 g for 5 min and the supernatant was used to measure myocardial ATP by
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firefly luciferase-based ATP detection kit as per manufacturer’s protocol (Thermo Fisher Scientific, Texas, USA). The reaction was initiated by adding 10 µL of diluted supernatant with
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Multi-Mode Reader, USA).
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100 µL of ATP reaction buffer in a luminescence based multimode reader (Synergy H1, BioTek
2.5. Western blot analysis
Western blot analysis were performed in the myocardial tissue of control and Fintoxicated rats. The myocardial tissues were homogenized with ice cold RIPA lysis buffer and the total protein was measured using Bradford reagent with BSA as standard. 40 µg of protein sample was equally loaded in each lane separated by 10% w/v, SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked using 5% fat free BSA and incubated overnight with primary antibodies against rabbit anti-desmin, anti-vimentin, antivinculin, anti-AMPK, anti-phospho AMPKα (Thr172), anti-AMPKβ1/2, anti-phospho AMPKβ (Ser108) anti-ACC and anti-phospho ACC (Ser79). The proteins were detected using horseradish
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ACCEPTED MANUSCRIPT peroxidase-conjugated anti-rabbit secondary antibodies and visualized by TMB staining kit. GAPDH was used as the internal control to ensure equal sample loading. Densitometry analyses
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of immunoblots were quantified using ImageJ analysis software (NIH).
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2.6. Immunohistochemical analysis
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Immunohistochemical analysis was performed in the paraffin embedded unstained section of myocardial tissues were deparffinized in the xylene and followed by subsequent
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rehydration with different grades of ethanol. The section were placed with laboratory stainless pressure cooker in order to unmask the antigen by Heat induced epitope retrieval method.
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Endogenous peroxidase activity was quenched by treating the slides with 1% H2O2 solution. The slides were blocked with 5 % BSA for 1 h at room temperature to avoid the non-specific
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reactivity. The sections were incubated with primary antibody of rabbit anti-desmin (1:100), antivimentin (1:250), anti-vinculin (1:500), anti-phospho AMPKα (Thr172) (1:100) and antiphospho ACC (Ser79) (1:100). The proteins were detected using horseradish peroxidaseconjugated anti-rabbit, or anti-goat secondary antibodies. Immunoreactions were labeled using a DAB staining kit. Sections were counterstained with hematoxylin, dehydrated with different grade of ethanol and mounted with distrene plasticizer xylene. Immunoreactivity was quantitated in a blinded fashion for depicting the total number of positively stained cells.
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ACCEPTED MANUSCRIPT 2.7. TEM analysis
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For TEM analysis, the myocardial tissues were fixed in 2.5%, v/v glutaraldehyde in 0.1
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M cacodylate buffer, pH 7.4, w/v at 4 ºC. Myocardial tissues were processed with 1%, w/v cacodylate-buffered osmium tetroxide, dehydrated with differently graded ethanol series and
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embedded in an epoxy resin. Myocardial sections were stained with uranyl acetate and lead
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acetate solutions and mounted in the copper grid. The ultrathin myocardial sections were
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examined using a transmission electron microscope
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3. Statistical analysis
All the values were expressed as mean ± SD of six rats in each group. The data were
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analyzed with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test using SPSS (20.0) software package. p < 0.05 was considered to indicate statistical significance.
4. Results
4.1. Mortality and clinical observation All the animals were monitored throughout the experimental period for any clinical signs of toxicity. There was no death in the control and F- intoxicated rats. But we observed clinical symptoms such as hyper salivation, diarrhea and increased heartbeat.
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ACCEPTED MANUSCRIPT 4.2. Myocardial ATP determination
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Fig. 1 shows the levels of ATP in the myocardial tissues of control and experimental groups. The ATP level in the F- treated cardiac tissues was found to be decreased when
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compared to control. There was no significance difference between 45 and 90 mg F- treated rats.
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4.3. Effect of acute F- intoxication on cardiac cytoskeletal protein
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Fig. 2 and 3 depict the protein expression of cardiac cytoskeletal (Desmin, vimentin and vinculin) in control and F- intoxicated rats. The relative quantitative levels of protein expression
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of cardiac cytoskeletal proteins such as desmin, vimentin and vinculin were found to be significantly decreased in both western blotting (Fig. 2) and immunohistochemical studies (Fig.
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3) of both 45 and 90 mg F- treated rats when compared to control.
4.4. Effect of acute F- intoxication on myocardial AMPK signaling molecules
To determine whether AMPK activation restores the energy homeostasis in acute Finduced cardiotoxicity, we tested the changes in the protein expression level of AMPK signaling molecules by western blotting (Fig. 4) and immunohistochemical studies (Fig. 5). A significant increase in the expression levels of phosphorylated AMPKα (Thr172), AMPKβ1 (Ser108) and ACC (Ser79) was observed in the myocardium of both 45 and 90 mg F- treated rats when compared to control.
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ACCEPTED MANUSCRIPT 4.5. TEM analysis
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TEM analysis was performed to examine the cardiac ultra-structural changes in the
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cytoskeleton of control and F- intoxicated rats (Fig. 6). Cytoskeletal fragmentation and lysis of myofibrils with interruption of Z lines, dilated sarcoplasmic reticulum and distorted
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mitochondrion were also observed in both groups of rats treated with different doses of F- when
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compared to control.
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5. Discussion
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Acute F- intoxication causes sudden cardiac failure with hypocalcemia, hyperkalemia mediated ventricular fibrillation, oxidative stress, mitochondrial dysfunction, myocardial
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apoptosis and necrosis were briefly discussed in our previous study [9]. However, the underlying molecular mechanism in acute F- induced cardiac dysfunction needs to be studied in detail. The present study was aimed to elucidate the changes in the expression of cardiac cytoskeleton proteins (desmin, vimentin and vinculin) and AMPK signaling molecules in acute F- induced cardiac dysfunction. Here, we report for the first time that acute F- intoxication alters the expression of myocardial cytoskeletal proteins and AMPK signaling molecules.
F- has been reported to alter the actin cytoskeletal networks involved in the epithelial cell proliferation [16] and cell migration and matrix protein production in pituitary tumor cells [17]. In the present study, we examined whether cytoskeletal proteins were implicated in acute Finduced cardiac dysfunction in rats. F- intoxicated rats showed significant decline in desmin,
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ACCEPTED MANUSCRIPT vimentin and vinculin expression in the myocardium when compared to control. It has been reported that decreased expression pattern of desmin along with focal loss of contractile
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apparatus and myocardial apoptosis were observed in doxorubicin treated rats [24]. In a recent
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study, arsenic exposure increases the risk of heart failure by decreased expression of vimentin in murine coronary progenitor cells [25]. In addition, decreased vinculin proteome was associated
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with altered structural integrity of left ventricular myocardium in bisphenol intoxicated rats [26].
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protein were consistent with our findings.
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In addition, several cardiotoxic studies [27-29] reveals that altered expression of cytoskeleton
In addition to the cytoskeletal protein expression studies, TEM analysis revealed the
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ultra-structural detrimental changes in the myocardium of acute F- intoxicated rats. In the present study we observed extensive lysis of myofibrils with disruption of Z lines, dilated fragmented
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sarcoplasmic reticulum and distorted mitochondrion in the both doses of F- treated rats when compared to control. The ultra-structural morphological deformities such as myofibril lysis and distorted mitochondrial matrix in the present study were in consistent with our previous findings [9]. Thus, F- induced cardiac impairment is not only mediated through oxidative stress, apoptosis, necrosis and also through decreased expression of cardiac cytoskeletal proteins in the rat myocardium.
AMPK is the master regulator of energy homeostasis in higher eukaryotes. Numerous studies have revealed that activation of AMPK signaling molecules, mainly occurs during increased energy deprivation (ATP level), intracellular rise of AMP, ADP ratio levels in the cardiac pathology of humans and experimental models [30-32]. Our previous study showed that
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ACCEPTED MANUSCRIPT F- causes cardiac dysfunction via induction of ROS, oxidative stress, apoptosis and necrosis in rat [9]. Induction of ROS production also serves as an upstream signal for the activation of
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AMPK signaling molecules in various cardiotoxicity studies [33-34]. It is well acknowledged that the F- is the potent inhibitor of glycolytic and Krebs cycle enzymes, which causes energy
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depletion [20]. Recent studies showed that F- impairs mitochondrial function results in decreased
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the intracellular ATP level in both in vivo [22] and in vitro model [35]. However, the activation
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of AMPK signaling molecules in F- induced cardiotoxicity is not known.
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In the present study, we found a significant increased phosphorylation of AMPKα (Thr 172), AMPKß1 (Ser108) and ACC (Ser79) in the myocardium of both 45 and 90 mg F - treated
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rats when compared to control. Earlier studies revealed that phosphorylation of AMPK and its downstream target protein ACC (ser79) enhances the oxidation of fatty acids in the
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mitochondria, which rescue the cardiomyocytes from the energy deprived state [19,36]. In addition, a significant decrease in myocardial ATP level were observed in F - intoxicated rats confirms the energy depletion. However, in our earlier study we observed oxidative stress, apoptosis and necrosis were might be due to pro-apoptotic action of AMPK dependent or independent mechanism in the myocardium of F- intoxicated rats. Further, in-depth molecular studies are needed to confirm the co-existences of both pro-survival and pro-apoptotic mechanism in the activation of AMPK signaling pathways against acute F- induced cardiac dysfunction. The limitation of present study speculates the absence of multiple time point observation of acute F- exposure and its pathophysiological changes in rat heart. Further, studies are needed
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ACCEPTED MANUSCRIPT to confirm with AMPK activators, inhibitors and their differential protein expression in acute Finduced cardiac dysfunction.
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6. Conclusion
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Acute F- poisoning alters the expression of cardiac cytoskeletal proteins and AMPK signaling pathway in cardiac physiology and to understand the molecular mechanism involved in
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pathology of cardiac dysfunction.
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Conflict of interest
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The authors declare that they have no conflict of interest
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Funding information
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This work was supported by the UGC-SAP DRS II:F-3-30/2013 and DST-FIST:SR/FST/LSI618/2014, New Delhi, India for the partial financial assistance. The author LP also extends his thanks to the Indian Council of Medical Research, New Delhi, for financial assistance in the form of a senior research fellowship (No. 45/25/2013/BMS/TRM). Acknowledgement
The authors would like to thank Dr. Prakash Dharmalingam, Houston Methodist Research Institute, Texas, USA for his valuable suggestion and comments throughout this study. The authors are also grateful to research fellows Mr. G. Vimal, K. Vishnu Vignesh, and Ms. S. Kirthika Bharathiar University, Coimbatore for their timely assistances throughout the work. The authors sincerely acknowledge Mrs. B. Rita, for her technical assistance in transmission electron
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ACCEPTED MANUSCRIPT microscopic studies in Wellcome Trust Research Laboratory, Department of Gastrointestinal
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Sciences, Christian Medical College and Hospital, Vellore, India.
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ACCEPTED MANUSCRIPT Figure legends Fig. 1 ATP level in the heart tissue of control and F- treated rats. Values are mean±SD of six rats
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45 mg F- treated rats (One way ANOVA followed by Tukey’s multiple comparison test).
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Fig. 2 Western blotting and its densitometric analysis of cardiac cytoskeletal proteins (desmin,
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Fig. 3 Representative micrographs of immunostained sections of myocardium in control and Ftreated rats. Myocardial sections were stained with antibodies specific for desmin, vimentin and
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vinculin and the number of immunopostively stained cells in ten fields/section (200x) was quantified by three independent observers in blinded fashion. The bar diagram shows the quantification of positively stained cells of control and F- treated rats. The values represent mean ± SD (n=3). *p < 0.05 significantly different from control, +p < 0.05, significantly different from 45 mg F- treated rats (One way ANOVA followed by Tukey's multiple comparison test). Fig. 4 Western blotting and its densitometric analysis of both total and phosphorylated myocardial AMPK signaling molecules in control and F- treated rats. GAPDH was used as loading control. Values represent mean±SD (n=3). *p<0.05 significantly different from control, +p<0.05, significantly different from 45 mg F- treated rats (One way ANOVA followed by Tukey’s multiple comparison test).
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ACCEPTED MANUSCRIPT Fig. 5 Representative micrographs of immunostained sections of myocardium in control and Ftreated rats. Myocardial sections were stained with antibodies specific for p-AMPKα (Th172), p-
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Fig. 6 Representative micrographs shows TEM images of myocardial section. Normal architecture of cytoskeletal myofibrils with Z lines, sarcoplasmic reticulum and mitochondrion in
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the control rat heart. Fragmented cytoskeleton shows myofibril lysis with interruption of Z lines,
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dilated sarcoplasmic reticulum and distorted mitochondrion in both 45 mg F- and 90 mg F-
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treated rat heart (Scale bar: 200 nm, 26500x and 16500x).
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