• Users Online: 104
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
Year : 2016  |  Volume : 2  |  Issue : 3  |  Page : 123-130

Primary Screening for Proteins Differentially Expressed in the Myocardium of a Rat Model of Acute Methamphetamine Intoxication

1 Hubei Chongxin Forensic Identification Centre, Wuhan, Hubei 430000, China
2 Department of Forensic Medicine, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430030, China
3 Key Laboratory of Evidence Science, China University of Political Science and Law, Ministry of Education; Collaborative Innovation Center of Judicial Civilization, Beijing, Haidian 100192, China

Date of Web Publication30-Sep-2016

Correspondence Address:
Haidong Zhang
Key Laboratory of Evidence Science (China University of Political Science and Law), Ministry of Education, Beijing, Haidian 100192, China; Collaborative Innovation Center of Judicial Civilization, Beijing, Haidian 100192
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2349-5014.191460

Rights and Permissions

The mechanism of myocardial injury induced by the cardiovascular toxicity of methamphetamine (MA) has been shown to depend on alterations in myocardial proteins caused by MA. Primary screening of the expression of myocardial proteins in a rat model of MA intoxication was achieved by combining two-dimensional electrophoresis and mass spectrometry analyses, which revealed a total of 100 differentially expressed proteins. Of these, 13 displayed significantly altered expression. Moreover, Western blotting and real-time reverse transcription quantitative polymerase chain reaction analyses of several relative proteins demonstrated that acute MA intoxication lowers protein expression and mRNA transcription of aldehyde dehydrogenase-2 and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 10. In contrast, MA intoxication elevated the protein expression and mRNA transcription of heat shock protein family B (small) member 1. By combining behavioral assessments of experimental rat models with the histological and pathological changes evident in cardiomyocytes, a mechanism accounting for MA myocardial toxicity was suggested. MA alters the regulation of gene transcription and the subsequent expression of certain proteins that participate in myocardial respiration and in responding to oxidative stress, resulting in myocardial dysfunction and structural changes that affect the functioning of the cardiovascular system.

Keywords: Cardiotoxicity, methamphetamine, proteomics, toxicology

How to cite this article:
Qu G, Huang S, Liu L, Yu T, Bai R, Liu H, Song F, Wen Y, Zhang H. Primary Screening for Proteins Differentially Expressed in the Myocardium of a Rat Model of Acute Methamphetamine Intoxication. J Forensic Sci Med 2016;2:123-30

How to cite this URL:
Qu G, Huang S, Liu L, Yu T, Bai R, Liu H, Song F, Wen Y, Zhang H. Primary Screening for Proteins Differentially Expressed in the Myocardium of a Rat Model of Acute Methamphetamine Intoxication. J Forensic Sci Med [serial online] 2016 [cited 2022 Oct 2];2:123-30. Available from: https://www.jfsmonline.com/text.asp?2016/2/3/123/191460

  Introduction Top

Methamphetamine (N-methyl-1-phenylpropan-2-amine, MA) is a semi-synthetic cathinone derivative, possessing a similar chemical structure to amphetamine; it is highly addictive and produces a similar physiological effect. [1],[2] Sudden death caused by MA intoxication is common among MA addicts. [3] In addition to the neurotoxicity arising from sympathetic nerve excitability, [4] research has confirmed that MA produces pulmonary toxicity, hepatotoxicity, and cardiovascular toxicity. [5] MA abuse can not only lead to severe dysfunctions of the cardiovascular system, including arrhythmias, hypertension, and myocardial infarction, [6] but also cause structural changes within the myocardium, such as myocardial hypertrophy, fibrosis, and interstitial inflammatory infiltration. [7] Recent research on the mechanism of myocardial toxicity arising from acute MA intoxication has mainly focused on histological and pathological changes, and on the relative alterations in protein expression within the myocardium. [8],[9] Several of these proteins have been reported to be implicated in myocardial functioning - they include aldehyde dehydrogenase-2 (ALDH2), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 10 (NDUAA), heat shock protein 90-beta (HS90B), and HSP family B (small) member 1 (Hspb1). [10],[11],[12] Furthermore, MA has been found to promote reactions causing intracellular oxidative stress, [13],[14] and to upregulate inflammatory cytokines, including interleukin-2 (IL-2) and IL-6. [6],[15]

In this study, a combination of two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) analyses were applied to a primary screen on rat models to identify proteins with differential myocardial expression following acute MA intoxication. Moreover, the relative expression levels of certain proteins, and their encoding mRNAs, were determined by Western blotting and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses. Through these investigations, we aimed to explore the mechanism underlying MA-induced injury to the myocardium, and to provide directions for further investigations of cardiovascular toxicity.

  Materials and Methods Top

Establishment of a rat model and preparation of the myocardium

MA was provided by the Ministry of Public Security of the People's Republic of China. The rat model used in this study, for acute MA-induced myocardial injury, was identical to that established during previous research. [16]

A total of twenty healthy adult Sprague-Dawley male rats (obtained from the Experimental Animal Center of the Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei, China), weighing between 190 g and 210 g, were randomly divided into an MA treated group (Group MA) and a control group (n = 10, each group). All the rats were maintained at room temperature (27°C) with commercial rat chow and tap water provided ad libitum. The rats in Group MA were treated with MA every 2 h (a single dose, 10 mg/kg, i.p.), and then separated and placed in cage individually. MA was administered a total of 4 times. The rats in the control group were treated with saline (10 mg/kg, i.p.) instead. All of the rats were euthanized 24 h following the final administration.

The heart was immediately dissected and the aortic root excised, washed in phosphate buffer solution (PBS), and weighed after drying. A sample, ~0.2 cm thick, was excised from the apical portion of the heart, and immediately fixed in 4% paraformaldehyde. The left ventricle was rapidly cooled in liquid nitrogen and then stored at −80°C.

Histological analysis of the myocardium

Samples of myocardium were fixed for 24 h in paraformaldehyde, followed by dehydration, paraffinization, wax removal, embedding, slicing, and hematoxylin and eosin staining. Samples were then inspected by optical microscopy to analyze histological changes.

The ultrastructural changes in cardiomyocytes were visualized by transmission electron microscopy (TEM). Samples were fixed in 2.5% glutaraldehyde, followed by washing with PBS, and then samples were postfixed in 1% aqueous osmium tetroxide. Samples were then washed with deionized distilled water, dehydrated, infiltrated, embedded, and incubated for complete polymerization. Ultrathin sections were prepared using an ultramicrotome equipped with a glass knife (Leica Ultracut UCT-GA-D/E-1/100). Sections were mounted on copper grids, stained with saturated aqueous uranyl acetate, and then counterstained with Reynolds' lead citrate before observation by TEM (Hitachi, H-7500, Japan).

Extraction of cardiomyocyte proteins and analysis by two-dimensional electrophoresis and mass spectrometry

The protein content of the myocardium was extracted by cell lysis, then purified by acetone precipitation method and preserved at −80°C. A Bradford assay kit (Sangon Biotech, Shanghai, China) was used to measure the protein concentration of the samples.

Samples of myocardium were ground under liquid nitrogen and a portion (~100 mg) was homogenized on ice in 10 volumes of 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris pH 7.4, in the presence of cocktails containing phosphatase inhibitors and protease inhibitors ("Complete" tablets; Roche, Indianapolis, USA). Following centrifugation at 12,000 ×g, at 4°C for 45 min, the supernatant was decanted and the protein concentration of a 5 μL aliquot measured using a modified microtiter plate version of the Bradford assay (Sigma, Poole, Dorset, UK).

Myocardium-derived homogenates were prepared for 2-DE, as described previously. [17] An aliquot of each supernatant was precipitated with acetone and resuspended in a solution containing 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 20 mM dithiothreitol, and 0.5% (v/v) ampholytes. Samples, containing 250 mg of protein, were loaded onto nonlinear IPG strips (13 cm, pH 3-11, GE Healthcare, Chalfont St. Giles, UK) and focused using an "active rehydration" and isoelectric focusing protocol comprising: 150 Vh at 30 V, 300 Vh at 60 V, 500 Vh at 500 V, 1000 Vh at 1000 V, and 48,000 Vh at 8000 V, performed at 20°C on an IPGPhor II instrument (GE Healthcare), with a maximum of 50 mA per strip. IPG strips were equilibrated in 50 mM Tris-HCl pH 8.8, containing 6 M urea, 30% (v/v) glycerol, 70 mM SDS, and a trace of bromophenol blue. DTT (65 mM) was present as a reducing agent in the first equilibration and iodoacetamide (135 mM) in the second. Proteins were electrophoresed at 20°C through 16 cm linear 12% polyacrylamide gels; at a constant current of 15 mA per gel for 30 min before adjusting to 30 mA per gel until the tracking dye reached the bottom edge of the gel. Gels were washed and stained with colloidal Coomassie blue (Bio-Safe; Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The 2-DE maps were analyzed using ImageMaster 2D Platinum 5.0 software (GE Healthcare, Connecticut, USA).

Gel spots containing proteins of interest were excised and processed using an Xcise robot (Proteome Systems, North Ryde, Australia), as directed by the gel analysis software. Gel plugs were destained in changes of 25 mM ammonium bicarbonate, and were then dehydrated in 50% acetonitrile, before being incubated with 35 μL of 1.25 mg/mL porcine trypsin (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate. Peptide solutions were desalted and concentrated (Zip-tips; Millipore, Billerica, MA, USA), before being mixed with matrix (5 mg/mL α-cyano-4-hydroxycinnamic acid, in 50:50 acetonitrile and 0.1% trifluoroacetic acid) and spotted onto 384-well stainless steel target plates. A calibration mix (LaserBio Labs, Sophia Antipolis, France) consisting of angiotensin II (m/z 1046.2), angiotensin I (m/z 1296.5), neurotensin (m/z 1672.9), ACTH fragment (1-17) (m/z 2093.5), and ACTH fragment (18-39) (m/z 2465.19) was mixed 1:1 with matrix solution and spotted (0.5 μL) between every four sample wells. Peptide mass spectra were recorded using a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS (Axima-TOF2; Shimadzu Biotech, Manchester, UK) in "positive reflectron" mode over a mass/charge (m/z) range of 900-3000. Data were smoothed (Gaussian, 2 channel peak width), baseline subtracted (100 channel peak width), and then an adaptive (8.0×) threshold was applied. Peptide mass lists (restricted to 20 peptides over 900--3000 m/z) were produced using the peak selection tool of the instrument's Launchpad software (Version 2.8.4, Kratos Analytical, Manchester, UK), and searched against the Swiss-Prot database, which was restricted to the genus Rattus using the online MASCOT server (www.matrixscience.com). The enzyme specificity was set as "trypsin," allowing one missed cleavage, carbamidomethyl modification of cysteine (fixed), oxidation of methionine (variable), and an m/z error of ± 0.3 Da. After filtering the mass peak using Flex-Analysis (Bruker Daltonics Inc., Pennsylvania, USA), the collected proteins of interest were identified via the NCBI database using Bio-Tools software (Bruker Daltonics Inc., Pennsylvania, USA).

Western blotting and quantitative polymerase chain reaction analyses

Western blotting was performed on proteins from myocardial tissue that had been stored at −80°C, and which had been homogenized and harvested in rehydration buffer stock containing Tris (Sigma, USA).

The protein concentrations were determined by Lowry method. Aliquots of the supernatants were diluted in an equal volume of 5× electrophoresis sample buffer and boiled for 5 min. Protein lysates (40 μg) were separated on a 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After being blocked with 5% nonfat dry milk in Tris-buffered saline-Tween#20 at room temperature for 2 h, the membranes were incubated with 1/400 diluted primary antibodies (NDUAA, Boster, BA3676; ALDH2, Boster, BA3673-2; Hspb1, Boster, BA0361; Beta-actin, Santa Cruz, Sc1616r), respectively, at 4°C overnight and horseradish peroxidase conjugated IgG at 1:8000 dilution at room temperature for 2 h. The blotting was visualized with Western blotting luminol reagent (sc-2048, Santa Cruz Biotechnology) by Electrophoresis Gel Imaging Analysis System (MF-ChemiBIS 3.2, DNR Bio-Imaging Systems, ISR). Subsequently, densitometric analyses of the bands were semiquantitatively conducted using Scion Image Software (Scion Corporation, MD, USA).

The RNA pellet was air-dried for 10 min and resuspended in 15 μl diethylpyrocarbonate-treated dH 2 O. Using 1 μl RNA samples, cDNA synthesis was performed in a 9 μl reaction mixture containing 2 μl MgCl2, 1 μl 10× RT buffer, 3.75 μl RNase-free dH 2 O, 1 μl dNTP mixture, 0.25 μl RNase inhibitor, 0.5 μl avian myeloblastosis virus reverse transcriptase (AMV-RT), and 0.5 μl Random 9 mers provided by TaKaRa RNA PCR Kit (AMV) Version 3.0 (RR019, Takara Biotechnology, Shiga, Japan). The reaction mixture was incubated at 30°C for 10 min twice, 42°C for 30 min, 99°C for 1 min, followed by 5 min at 5°C. The resulting cDNA was used for PCR with the sequence-specific primer pairs for NDUAA, ALDH2, Hspb1, and GAPDH [Table S1]. PCR amplification was performed in a 60 μl reaction mixture which contained 12.5 μl 5× PCR buffer, 36 μl sterile H 2 O, 0.5 μl TaKaRa Ex Taq, 0.5 μl forward primer, 0.5 μl reverse primer, and 10 μl cDNA. After initial denaturation at 94°C for 2 min, amplification consisting of denaturation at 94°C for 40 s, annealing at 60°C for 40 s, and extension at 72°C for 1 min was performed for 35 cycles for GAPDH and 36 cycles for CB2R. The amplified PCR products were identified using electrophoresis of 6 μl aliquots on a 2% agarose gel and were stained with Genefinder (204001, Bio-V, Xiamen, China). To exclude any potential genomic DNA contamination, each PCR was also performed without the RT step. No DNA amplification product was detected. All PCRs were repeated at least three times for each cDNA. For normalization of the amount of different cDNAs, GAPDH was used as an internal standard.


Data were expressed as means ± standard deviation and analyzed using SPSS for Windows 11.0 (IBM, New York, USA). The one-way ANOVA was used for data analysis between two groups. Difference associated with P < 0.05 was considered statistically significant.

  Results Top

Assessment of intoxication models

Symptoms such as hyperactivity, irritability, piloerection, retrograding, and head shaking became evident in the experimental rats within 10 min upon the first occasion of MA intoxication, and these symptoms reoccurred each time MA was administered during the investigation. Hyperactivity occurred most frequently with the second MA intoxication while with the third such event, irritability and retrograding occurred more frequently. In contrast, rats in the control group had none of these aberrant behaviors.

Pathological and ultrastructural changes in cardiomyocytes

When compared to the control group, pathological changes were evident in sections from Group MA cardiomyocytes. These changes included vacuolar degeneration, focal hypertrophy, and necrosis of the cardiomyocytes, along with some low-level interstitial inflammatory infiltration and proliferation of fibrous tissue.

Disordered arrangements of cardiomyocytes were also observed [Figure 1].
Figure 1: Pathological changes in cardiomyocytes. (a) Group methamphetamine. (b) The control group (H and E, ×200)

Click here to view

Relative to the control group, the mitochondria within rat cardiomyocytes from Group MA exhibit evident swelling, and their mitochondrial cristae have partially disappeared [Figure 2].
Figure 2: Ultrastructural changes in cardiomyocytes. (a and b) Control group, ×7800, 200 kV. (c and d) Group methamphetamine, ×5000, 200 kV

Click here to view

Differentially expressed cardiomyocyte proteins analyzed by two-dimensional electrophoresis and mass spectrometry

Analysis by 2-DE revealed that a maximum of 1685 protein spots could be identified in the control sample [Figure 3]a, of which 1252 protein spots were matched with a matching rate of 74.30% following repeated experiments on the sample. Among the 10 samples in the control group, 1081 spots were matched with a matching rate 64.15%. In Group MA, a sample gel map [Figure 3]b revealed as many as 1459 protein spots, of these 1259 spots (matching rate = 86.29%) could be matched upon repeated analysis. The matching rate was 68.20% (995 matched protein spots) over all ten samples from Group MA.
Figure 3: Gel maps obtained by two-dimensional electrophoresis on samples from the control group and Group methamphetamine (24 cm across; pH 3-10; silver staining; acidic end = left; basic end = right). (a) Gel map with 1685 protein spots from the control group. (b) Gel map with 1459 protein spots from Group methamphetamine

Click here to view

Gel maps from both the control group and Group MA were compared. In the control group, 57 protein spots with a protein expression ratio over 1.5 (relative to Group MA) were identified and annotated as A1-A57 [Figure 4]a. In Group MA, 43 protein spots with a protein expression ratio over 1.5 (relative to the control group) were identified and annotated as B1-B43 [Figure 4]b.
Figure 4: Differentially expressed protein spots marked on two-dimensional electrophoresis gel maps (24 cm across; pH 3-10; silver staining; acidic end = Left; basic end = Right). (a) Gel map of a control group sample containing 57 differentially expressed protein spots (A1-A57). (b) Gel map of a Group methamphetamine sample containing 43 differentially expressed protein spots (B1-B43)

Click here to view

Moreover, 56 differentially expressed protein spots with ratio >2.5 between the control group and Group MA were detected in the maps, from which a total of 13 highly expressed protein spots were selected for identification by MALDI-TOF/TOF [Figure S1, S2 and [Table 1].
Table 1: Identification of selected differentially expressed proteins by matrix - assisted laser desorption/ionization tandem time-of-flight

Click here to view

Verifying the results of Western blotting and reverse transcription quantitative polymerase chain reaction

In this study, among the aforementioned 13 differentially expressed proteins that were identified, specific proteins (ALDH2, NDUAA, and Hspb1) were selected to verify the results using Western blotting and RT-qPCR analyses. As shown in [Table 2], Western blotting analysis indicated that upon acute MA intoxication, the expression levels of ALDH2 and NDUAA became significantly decreased while the expression level of Hspb1 became elevated.

C(t) values for the mRNA transcripts encoding ALDH2, NDUAA, and Hspb1 were analyzed following detection by RT-qPCR [Table 3]. Values for the 2−ΔΔC(t) were also calculated [Figure 5]. These values indicated that ALDH2 and NDUAA mRNA transcription levels become significantly downregulated while Hspb1 mRNA transcription levels become highly elevated, following acute MA intoxication, which is consistent with tendency of Western blotting results to vary.
Figure 5: Levels of mRNA transcription were determined by reverse transcription quantitative polymerase chain reaction. n = 10, *P < 0.01 versus the control group. MA: Methamphetamine, ALDH2: Aldehyde dehydrogenase-2, NDUAA: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 10, Hspb1: Heat shock protein family B (small) member 1

Click here to view
Table 2: Protein strip optical densities of aldehyde dehydrogenase-2, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 10, and heat shock protein family B (small) member 1

Click here to view
Table 3: C(t) values for the mRNA transcripts encoding ALDH2, NDUAA, and Hspb1

Click here to view

  Discussion Top

Differential protein expression within cardiomyocytes following acute methamphetamine administration

Comparison of 2-DE gel maps between the control group and Group MA revealed a total of 100 proteins that become differentially expressed in rat cardiomyocytes - with either elevated or downregulated expression - following exposure to MA. Among these, 13 proteins were detected with large variations in their expression; these were identified by MALDI-TOF/TOF [Table 1] and found to include such proteins such as ALDH2 and NDUAA, which play an important roles in respiration within cardiomyocytes. [10],[11] Other proteins identified in that analysis include HS90B and Hspb1, which are proteins with an important function in myocytes, where they protect against intracellular oxidative stress. [12] The pattern of differential protein expression identified in this study suggests that the cardiac injury induced by acute MA intoxication may arise mainly from respiratory dysfunction. Under such circumstances, protective proteins would be expected to be expressed to a greater extent, to restore balance to the intracellular environment.

Effects of acute methamphetamine intoxication on proteins associated with myocardial injury

Methamphetamine intoxication lowers the expression of aldehyde dehydrogenase-2

ALDH, which belongs to the ALDH family, catalyzes the oxidation of acetaldehyde to acetic acid. [18] ALDH2 is an enzyme specific to mitochondria, where it plays an important role in the metabolism of acetaldehyde, minimizing damage to cardiomyocytes by preventing oxidation by acetaldehyde on the cell membrane. [19] Moreover, ALDH2 is involved in cellular respiration where it ensures sufficient provision of the cofactor NADH. The expression of ALDH2 becomes significant elevated in rat cardiomyocytes during ischemic conditions; under these conditions, ALDH2 can help provide energy as well as protection. [20]

Research has found that acute MA poisoning can cause sympathetic excitation, leading to vasoconstriction and elevations in blood pressure, heart rate, and myocardial oxygen consumption. [21] These responses can trigger myocardial protection by upregulating the expression of protective proteins, including HSP and ALDH2, to resist myocardial injury caused by ischemia. [10]

In this experiment, following exposure to MA, the levels of both mRNA transcription and protein expression of ALDH2 became downregulated, which was unexpected since these are typically elevated myocardial ischemia. This finding suggests that the downregulation of ALDH2 expression might be directly induced by MA, contributing to mitochondrial damage. It is anticipated that further research will elucidate the precise mechanism.

Methamphetamine intoxication lowers the expression of NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 10

NDUAA is a subtype of NADH dehydrogenase, capable of transforming NADH into the cofactor NAD + , which is involved in the regulation of intracellular and extracellular signaling, the mobilization of calcium ions, and the activation of cellular apoptosis. [11] Within cardiomyocytes, NADH and NAD + are receptors/donors for hydride ions that play key roles in cellular energy metabolism and respiration. [22]

This study discovered that the level of both mRNA transcription and protein expression of NDUAA are lowered in cardiomyocytes following MA intoxication. This alteration in NDUAA expression may dysregulate the intracellular NAD + equilibrium, resulted in dysfunctional respiration and disordered metabolism, causing subsequent myocardial injury.

Methamphetamine intoxication stimulates expression of heat shock protein family B (small) member 1 expression

HSPs are intracellular proteins that are highly protective in stressed environments. HSPs include HSP70, HSP90, HSP100, and "small molecular HSPs," which are so named on account of their smaller molecular weights and structures. [23],[24] Hspb1, also called HSP27, is an important member of the small molecular HSPs. When myocytes suffer damage from exogenous free radicals, thermal stimulation, ischemia, or toxic substances, Hspb1 expression is stimulated to protect cardiomyocytes. [25]

Research has found that when the myocardium is under conditions of ischemia and stress, insufficient myocardial blood supply may lead to the accumulation of oxygen free radicals, which can cause myocardial injury. In this situation, Hspb1 expression becomes significantly increased, suppressing the occurrence of cellular apoptosis by scavenging oxygen free radicals and by maintaining the membrane potential of mitochondria. [26]

The findings of this current study agree with those of former studies: acute MA intoxication may trigger an imbalance in the internal cardiomyocyte environment, promoting a corresponding elevation in the levels of transcription and protein expression of Hspb1, which then plays its role in protecting the myocardium during the oxidative stress response under circumstances of respiratory dysfunction.

The mechanism underlying myocardial injury caused by acute methamphetamine intoxication

Physiological functioning of the myocardium is accompanied by an increase in the activity of the various enzymes involved in mitochondrial respiration, which enhance respiratory functioning and ensure a sufficient supply of energy. Consequently, mitochondria play an indispensable role in myocardial respiration and in the maintenance of cellular functions. [27] Changes to the intracellular environment caused by MA may activate corresponding oxidative stress reactions, leading to increased intracellular levels of oxygen free radicals, which can damage the mitochondrial membrane and cause changes to the structure and functioning of mitochondria. It has been reported in the scientific literature that acute MA poisoning can lead to significant increases in myocardial IL-6 and other inflammatory factors, [28] which then triggers the accumulation of inflammatory cells within the myocardium.

Results of the 2-DE and MS analyses in this study demonstrated that acute MA intoxication produced different effects on the regulation of numerous proteins, among which were many proteins associated with mitochondria and myocardial respiration. As previously mentioned, MA lowered the expression of ALDH2 and NDUAA, which could then lead to respiration dysfunction and mitochondria damage. Consistent with this interpretation, Hspb1 expression became correspondingly elevated.

In this investigation, histopathological and ultrastructural observations revealed that the mitochondria of rat cardiomyocytes appeared conspicuously swollen and that their cristae had partially disappeared. Such distinct pathological changes as vacuolar degeneration, focal hypertrophy, and necrosis were also observed in cardiomyocytes, together with low levels of interstitial inflammatory infiltration.

During acute administrations of MA in this experiment, behavioral observations confirmed that symptoms of hyperactivity, irritability, piloerection, retrograding, and head shaking were elicited in the experimental rats. These symptoms may be related to the excessive nervous system excitability caused MA, which increases the consumption of oxygen and ATP while causing a deficit in energy supplies.

By combining the findings of this study with those of related reports in the scientific literature, it is suggested that acute MA intoxication might alter the relative expression levels of proteins, including IL-6, thereby causing mitochondrial damage and pathological changes to the structure of the myocardium by inducing respiratory dysfunction accompanied by oxidative stress. The proposed scheme may inform a mechanistic description of the myocardial injury induced by MA.

Financial support and sponsorship

Supported by Beijing Natural Science Foundation (7132116).

Conflicts of interest

There are no conflicts of interest.

  References Top

Brecht ML, von Mayrhauser C, Anglin MD. Predictors of relapse after treatment for methamphetamine use. J Psychoactive Drugs 2000;32:211-20.  Back to cited text no. 1
Wang L, Qu G, Dong X, Huang K, Kumar M, Ji L, et al. Long-term effects of methamphetamine exposure in adolescent mice on the future ovarian reserve in adulthood. Toxicol Lett 2016;242:1-8.  Back to cited text no. 2
Inoue H, Ikeda N, Kudo K, Ishida T, Terada M, Matoba R. Methamphetamine-related sudden death with a concentration which was of a 'toxic level'. Leg Med (Tokyo) 2006;8:150-5.  Back to cited text no. 3
Koriem KM, Abdelhamid AZ, Younes HF. Caffeic acid protects tissue antioxidants and DNA content in methamphetamine induced tissue toxicity in Sprague Dawley rats. Toxicol Mech Methods 2013;23:134-43.  Back to cited text no. 4
Stumm G, Schlegel J, Schäfer T, Würz C, Mennel HD, Krieg JC, et al. Amphetamines induce apoptosis and regulation of bcl-x splice variants in neocortical neurons. FASEB J 1999;13:1065-72.  Back to cited text no. 5
Yu Q, Montes S, Larson DF, Watson RR. Effects of chronic methamphetamine exposure on heart function in uninfected and retrovirus-infected mice. Life Sci 2002;71:953-65.  Back to cited text no. 6
Giordano S, Darley-Usmar V, Zhang J. Autophagy as an essential cellular antioxidant pathway in neurodegenerative disease. Redox Biol 2013;2:82-90.  Back to cited text no. 7
Furst SR, Fallon SP, Reznik GN, Shah PK. Myocardial infarction after inhalation of methamphetamine. N Engl J Med 1990;323:1147-8.  Back to cited text no. 8
Shinone K, Tomita M, Inoue H, Nakagawa Y, Ikemura M, Nata M. Molecular-biological analysis of the effect of methamphetamine on the heart in restrained mice. Leg Med (Tokyo) 2010;12:79-83.  Back to cited text no. 9
Li X, Wang H, Qiu P, Luo H. Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain. Neurochem Int 2008;52:256-64.  Back to cited text no. 10
Garcia MM, Guéant-Rodriguez RM, Pooya S, Brachet P, Alberto JM, Jeannesson E, et al. Methyl donor deficiency induces cardiomyopathy through altered methylation/acetylation of PGC-1a by PRMT1 and SIRT1. J Pathol 2011;225:324-35.  Back to cited text no. 11
Cuerrier CM, Chen YX, Tremblay D, Rayner K, McNulty M, Zhao X, et al. Chronic over-expression of heat shock protein 27 attenuates atherogenesis and enhances plaque remodeling: A combined histological and mechanical assessment of aortic lesions. PLoS One 2013;8:e55867.  Back to cited text no. 12
Maragos WF, Jakel R, Chesnut D, Pocernich CB, Butterfield DA, St. Clair D, et al. Methamphetamine toxicity is attenuated in mice that overexpress human manganese superoxide dismutase. Brain Res 2000;878:218-22.  Back to cited text no. 13
Giovanni A, Liang LP, Hastings TG, Zigmond MJ. Estimating hydroxyl radical content in rat brain using systemic and intraventricular salicylate: Impact of methamphetamine. J Neurochem 1995;64:1819-25.  Back to cited text no. 14
Yu Q, Zhang D, Walston M, Zhang J, Liu Y, Watson RR. Chronic methamphetamine exposure alters immune function in normal and retrovirus-infected mice. Int Immunopharmacol 2002;2:951-62.  Back to cited text no. 15
Kita T, Wagner GC, Nakashima T. Current research on methamphetamine-induced neurotoxicity: Animal models of monoamine disruption. J Pharmacol Sci 2003;92:178-95.  Back to cited text no. 16
Seemampillai B, Germack R, Felkin LE, McCormack A, Rose ML. Heat shock protein-27 delays acute rejection after cardiac transplantation: An experimental model. Transplantation 2014;98:29-38.  Back to cited text no. 17
Armstrong CT, Anderson JL, Denton RM. Studies on the regulation of the human E1 subunit of the 2-oxoglutarate dehydrogenase complex, including the identification of a novel calcium-binding site. Biochem J 2014;459:369-81.  Back to cited text no. 18
Muthumani M, Prabu SM. Silibinin potentially attenuates arsenic-induced oxidative stress mediated cardiotoxicity and dyslipidemia in rats. Cardiovasc Toxicol 2014;14:83-97.  Back to cited text no. 19
Doser TA, Turdi S, Thomas DP, Epstein PN, Li SY, Ren J. Transgenic overexpression of aldehyde dehydrogenase-2 rescues chronic alcohol intake-induced myocardial hypertrophy and contractile dysfunction. Circulation 2009;119:1941-9.  Back to cited text no. 20
Islam MN, Jesmine K, Kong Sn Molh A, Hasnan J. Histopathological studies of cardiac lesions after long term administration of methamphetamine in high dosage - Part II. Leg Med (Tokyo) 2009;11 Suppl 1:S147-50.  Back to cited text no. 21
Xiao L, Pimentel DR, Wang J, Singh K, Colucci WS, Sawyer DB. Role of reactive oxygen species and NAD(P) H oxidase in alpha(1)-adrenoceptor signaling in adult rat cardiac myocytes. Am J Physiol Cell Physiol 2002;282:C926-34.  Back to cited text no. 22
Marunouchi T, Abe Y, Murata M, Inomata S, Sanbe A, Takagi N, et al. Changes in small heat shock proteins HSPB1, HSPB5 and HSPB8 in mitochondria of the failing heart following myocardial infarction in rats. Biol Pharm Bull 2013;36:529-39.  Back to cited text no. 23
Tang S, Buriro R, Liu Z, Zhang M, Ali I, Adam A, et al. Localization and expression of Hsp27 and aB-crystallin in rat primary myocardial cells during heat stress in vitro. PLoS One 2013;8:e69066.  Back to cited text no. 24
Reddy VS, Kumar CU, Raghu G, Reddy GB. Expression and induction of small heat shock proteins in rat heart under chronic hyperglycemic conditions. Arch Biochem Biophys 2014;558:1-9.  Back to cited text no. 25
Walker S, Danton M, Peng EW, Lyall F. Heat shock protein 27 is increased in cyanotic tetralogy of Fallot myocardium and is associated with improved cardiac output and contraction. Cell Stress Chaperones 2013;18:269-77.  Back to cited text no. 26
Schep LJ, Slaughter RJ, Beasley DM. The clinical toxicology of metamfetamine. Clin Toxicol (Phila) 2010;48:675-94.  Back to cited text no. 27
Jung F, Palmer LA, Zhou N, Johns RA. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res 2000;86:319-25.  Back to cited text no. 28


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2], [Table 3]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Materials and Me...
Article Figures
Article Tables

 Article Access Statistics
    PDF Downloaded313    
    Comments [Add]    

Recommend this journal