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. 2015 Aug 6;148(1):183–191. doi: 10.1093/toxsci/kfv170

Ecstasy (MDMA) Alters Cardiac Gene Expression and DNA Methylation: Implications for Circadian Rhythm Dysfunction in the Heart

Christopher A Koczor 1,1, Ivan Ludlow 1, Robert S Hight II 1, Zhe Jiao 1, Earl Fields 1, Tomika Ludaway 1, Rodney Russ 1, Rebecca A Torres 1, William Lewis 1
PMCID: PMC4731408  PMID: 26251327

Abstract

MDMA (ecstasy) is an illicit drug that stimulates monoamine neurotransmitter release and inhibits reuptake. MDMA’s acute cardiotoxicity includes tachycardia and arrhythmia which are associated with cardiomyopathy. MDMA acute cardiotoxicity has been explored, but neither long-term MDMA cardiac pathological changes nor epigenetic changes have been evaluated. Microarray analyses were employed to identify cardiac gene expression changes and epigenetic DNA methylation changes. To identify permanent MDMA-induced pathogenetic changes, mice received daily 10- or 35-day MDMA, or daily 10-day MDMA followed by 25-day saline washout (10 + 25 days). MDMA treatment caused differential gene expression (p < .05, fold change >1.5) in 752 genes following 10 days, 558 genes following 35 days, and 113 genes following 10-day MDMA + 25-day saline washout. Changes in MAPK and circadian rhythm gene expression were identified as early as 10 days. After 35 days, circadian rhythm genes (Per3, CLOCK, ARNTL, and NPAS2) persisted to be differentially expressed. MDMA caused DNA hypermethylation and hypomethylation that was independent of gene expression; hypermethylation of genes was found to be 71% at 10 days, 68% at 35 days, and 91% at 10 + 25 days washout. Differential gene expression paralleled DNA methylation in 22% of genes at 10-day treatment, 17% at 35 days, and 48% at 10 + 25 days washout. We show here that MDMA induced cardiac epigenetic changes in DNA methylation where hypermethylation predominated. Moreover, MDMA induced gene expression of key elements of circadian rhythm regulatory genes. This suggests a fundamental organism-level event to explain some of the etiologies of MDMA dysfunction in the heart.

Keywords: MDMA, ecstasy, DNA methylation, microarray, circadian rhythm heart


MDMA (3,4-methylenedioxymethamphetamine, ecstasy) is an illicit drug that stimulates monoamine neurotransmitter release and inhibits neurotransmitter reuptake (Kalant, 2001). Ecstasy is used at clubs and ‘raves’ with the purpose of enhancing sensory stimulation. Its abuse is a new epidemic (Substance Abuse and Mental Health Services Administration, 2010; Lenton et al., 1997; Peroutka et al., 1988). MDMA exhibits acute cardiotoxicity including tachycardia and ventricular arrhythmia which are associated pathogenetically with cardiomyopathy (CM) (Battaglia et al., 1988; Bexis and Docherty, 2006; de la Torre et al., 2000; Freese et al., 2002; Green et al., 1995; Greene et al., 2003; Gudelsky and Nash, 1996; Mizia-Stec et al., 2008; Rattray, 1991; Rudnick and Wall, 1992; Shenouda et al., 2008, 2009). Conceptually, CM is likened to an energy-starved engine, where left ventricle hypertrophy is part of a continuum to CM (Ahmad et al., 2005; Neubauer, 2007).

Acute MDMA cardiotoxicity has been explored, but persistent genetic or cardiac pathological changes resulting from either chronic MDMA or after its use and subsequent abstinence have not been evaluated systematically. Clinically, MDMA users may be young adults (age 18–25) who are less likely to continue MDMA usage as they become older (Johnston et al., 2013). In studies of MDMA effects on the brain, relatively brief MDMA administration yielded persistent deleterious effects after its discontinuation. On that basis, it is reasonable to suggest that MDMA’s acute cardiac effects may be similarly durable (Finnegan et al., 1988; McCann et al., 2005). We posited that MDMA induces cardiotoxic events that relate to alterations in gene expression and epigenetics through DNA methylation, one of the most common epigenetic mechanisms for gene expression control. Previous studies in human CM identified pathogenetic changes in gene expression mediated by epigenetic DNA methylation modulation (Koczor et al., 2013).

To test our hypothesis, we exposed inbred mice to MDMA parenterally and examined epigenetic, gross pathologic and pathophysiological events in the murine heart. Administered parenterally once daily, in ways that resembled protocols of others’ experiments, MDMA induced cardiac gene expression and DNA methylation changes (Colado et al., 2001; Johnson et al., 2000; O’Shea et al., 2001). Three MDMA administration protocols helped us identify the durability of MDMA-associated epigenetic and gene expression changes: daily (10 days) MDMA to mimic relatively short-term abuse, MDMA daily for 35 days to mimic long-term abuse, and 10-day MDMA+25 days washout with saline vehicle to mimic abuse followed by abstinence. Functional and structural parameters included echocardiographic measurements and cardiac pathological changes. Microarray data analysis of cardiac gene expression and epigenetic DNA methylation changes were analyzed followed by qPCR-validation of microarray data to confirm findings. Among the differentially expressed cardiac DNA methylation and gene expression changes identified following MDMA, the most prominent were those involving genes of the circadian rhythm pathways. This previously undiscovered finding in the MDMA-treated heart suggests a previously unexplored relationship between the psychological stimulatory effect of MDMA thought to be key to its addiction and the life-threatening cardiovascular effects seen in tragic cases of overdose.

MATERIALS AND METHODS

Reagents

All reagents were analytical grade (Sigma Aldrich, St. Louis, Missouri) unless otherwise indicated.

Mouse protocols

Wild-type C57Bl/6 mice were from Jackson Labs (Bar Harbor, Maine) and were bred at the Emory University Vivarium (AAALAC certified vivarium) where they were housed and treated in accordance with IACUC protocols and NIH guidelines. Mice were 8–12 weeks old, and both genders were used. MDMA (NIDA Drug Repository, RTP, North Carolina) was obtained under a Schedule 1 license to one of the investigators. MDMA was administered intraperitoneally in saline (40 mg/kg MDMA) or saline vehicle control daily for 10 or 35 days, respectively. This MDMA dose was selected because it is consistent with doses of MDMA that other investigators used experimentally in rodents (Colado et al., 2001; Johnson et al., 2000; O’Shea et al., 2001). For the 10+25 washout group, mice received 10 days of 40 mg/kg/day MDMA followed by 25 days of saline vehicle only. All terminal physiological, histopathological, and molecular changes were determined 24 h after final injection and before (echocardiography) or at the time of euthanasia.

Echocardiography

At the termination of each experimental protocol, mice were anesthetized with Avertin (0.25 mg/g of body weight) and weighed. Echocardiography was performed using VisualSonics Vevo 770 (VisualSonics, Toronto, Ontario, Canada). Results from 2-dimensional M-mode analysis along the short axis (at the level of the largest LV diameter) were used to determine LV end diastolic dimension (LVEDD), and ejection fraction. Wet heart weight was obtained at postmortem dissection and normalized to mouse body weight.

Histology

Following treatment, mice were terminated by cervical dislocation under avertin anesthesia, and hearts were removed, sectioned rapidly with a razor blade (2 mm sections), fixed in 10% neutral buffered formalin and embedded in paraffin. Six micrometer histological sections were processed with hematoxylin and eosin to highlight cytological changes, and images were collected at 40× magnification using a Nikon Eclipse E800M microscope (Nikon, Melville, New York).

Gene expression analysis

Gene expression analysis was performed as previously detailed by our group in previous articles (Koczor et al., 2013). Total RNA was extracted from at least 3 mouse hearts from each 2 × 2 groups using the Fibrous Tissue RNeasy kit (Qiagen, Germantown, Maryland). Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies Corp, Grand Island, New York), labeled with Cy3, and hybridized overnight to a 12 × 135 kb human expression array (Roche Nimblegen, Indianapolis, Indiana). Arrays were washed, and scanned using Roche Nimblegen MS200 scanner. Images were analyzed using Nimblescan software as directed by the manufacturer, including RNA normalization and generation of expression data. Expression results were analyzed by t test using Bioconductor for R, with each set compared with saline controls. Differentially expressed genes were defined operationally with a p < .05 and 1.5-fold change in gene expression compared with saline controls. Heatmaps were generated using R. Gene ontology was performed using DAVID bioinformatics database (Huang da et al., 2009). Microarray array data (raw and processed) were deposited into NIH/NCBI Gene Expression Omnibus (GEO) and can be accessed under GEO series GSE68216.

Quantitative RT-PCR

RNA was isolated as described earlier, and single-stranded cDNA was synthesized using SuperScript III (Life Technologies). qPCR was performed using SYBR green dye and a LightCycler 480 system (Roche) using manufacturer protocols. Primers were obtained from Integrated DNA Technologies (Coralville, Iowa) and are shown in Supplementary Table S1. Individual sample results were normalized to controls ACTB and RN18S (Qiagen). Results are expressed as fold change of saline controls.

DNA methylation analysis

DNA methylation analysis was performed as described by us previously (Koczor et al., 2013). Briefly, total cellular DNA was extracted and sonicated to an average fragment size of 200–500 bp. A sample of DNA was set aside for later normalization (denoted ‘Input’), and then a portion of the sonicated DNA was enriched using the MethylCollector Ultra kit (Active Motif, Carlsbad, California) following the manufacturer’s directions. Both the methylated and input DNA were amplified using whole genome amplification (Sigma-Aldrich). Samples of the methylated and input DNA were validated for enrichment. For DNA methylation analysis, Nimblegen 2.1M Deluxe Promoter Arrays were utilized (Roche). DNA was labeled with Cy5 and Cy3 dyes to distinguish methylated and input DNA, and DNA hybridized to arrays overnight. Arrays were scanned using a MS200 scanner and were analyzed using proprietary Nimblescan software. Final analysis included a p-score of the detected methylated DNA peak and annotation to the probe location. DNA methylation results were analyzed using Bioconductor for R. Differentially methylated gene promoter regions were identified as those with a p < .05 by t test (compared with appropriate saline controls) and peak score change >1. Gene ontology was performed using DAVID bioinformatics database. Microarray array data (raw and processed) were deposited into NIH/NCBI GEO and can be accessed under GEO series GSE68216.

Statistical analysis

Statistical analyses (other than those used for microarray analyses described earlier) were performed using GraphPad Prism 5.0 (Graphpad, La Jolla, California). Each experiment was analyzed using a one-way ANOVA or t-test where appropriate; p < 0.05 was deemed significant. Survival data was analyzed using a Log-rank test. The data are presented as mean ± SEM; all experiments were performed at least in triplicate.

RESULTS

Physiology

Mice were treated with MDMA under the protocols described graphically in Figure 1. Analyses of 10-day MDMA compared that treated group to its vehicle treated control. However, in the longer duration experiments (10 + 25- and 35-day MDMA groups, respectively), each of those treatment groups were compared with 35 days vehicle controls. Results showed a 14.3% increase in normalized heart weight in mice treated with MDMA for 10 days compared with those measurements from vehicle controls (Fig. 2A). In the longer term experiments, mice receiving MDMA daily for 35 days also exhibited a 13.3% increase in normalized heart weight compared with respective controls. However, for the cohort of mice that received 10-day MDMA followed by 25-day saline vehicle ‘washout’, heart weights were similar to those of controls, suggesting that removal of MDMA treatment could revert the heart to its weight in the unexposed state. Functionally, echocardiographic measurement found no change in LVEDD or ejection fraction resulted from any of the MDMA administration paradigms (Fig. 2B and C, respectively).

FIG. 1.

FIG. 1.

Experimental timelines. Experiments were designed to identify the effects of MDMA following short- and long-term use. MDMA was administered once daily for 10 days in the short-term group and once-daily for 35 days in the long-term group. Saline controls were used for both. To identify permanent cardiac changes following short-term MDMA, another 35-day group was used where mice received 10 days of MDMA followed by 25 days saline (termed 10 + 25-day group).

FIG. 2.

FIG. 2.

Cardiac physiology, histology, and survival. Echocardiographic measurements and gross heart measurements were obtained at completion of the experiment. A, Wet heart weight showed a significant 14.3% increase following 10-day MDMA and 13.3% increase following 35-day MDMA. In the 10 + 25-day group, heart weights were normal compared with saline controls. *p < .05 by t test in the 10-day group and by 1-way ANOVA in 35-day group. B, No change in LVEDD was observed following any MDMA treatment paradigm. C, No change in ejection fraction was observed following any MDMA treatment paradigm. D, A 47% decrease in survival was observed in the 35-day MDMA mice compared with saline controls. No change in survival was observed in the 10- or 10 + 25-day MDMA groups. * p < .05 by Log-rank test. E–H, Photomicrographs of LV samples from C57B6 mice treated with MDMA. At sacrifice, a sample of heart was immersion fixed in neutral buffered formalin overnight at room temperature, processed using automated tissue processing, embedded, sectioned at 6 µm and stained with hematoxylin and eosin. Bar indicates 20 µm. E, LV cardiac myocytes from hearts of mice treated with saline for 10 days. no evidence of cytological change. F, LV cardiac myocytes from mice treated with MDMA for 10 days showing focal nuclear hypertrophy (black arrow). G, LV cardiac myocytes from hearts of mice treated with saline for 35 days. No evidence of cytological abnormality. H, LV cardiac myocytes from hearts of mice treated with MDMA for 35 days. Nuclear hypertrophy (black arrow) and multifocal contraction band changes (blue arrow). Full color version available online.

Survival data from this relatively short-term study revealed some interesting points. No change in survival was observed in murine cohorts treated with 10-day MDMA compared with that of saline controls (data not shown). However, a significant decrease in survival occurred precipitously in the long-term MDMA treatment group that received continuous 35-day MDMA treatment compared with saline vehicle controls (Fig. 2D). Of the 15 MDMA-treated mice (35 days), nearly half died, and almost all experienced spontaneous ‘sudden death’ within days of the termination of the experiments (35 days). One died on treatment day 29; 6 died on treatment day 33. These interesting data may suggest significant preservation of cardiac function with long-term MDMA even in face of increased heart weight along with a profound decrease (nearly 50%) of survival that appears to be related to sudden death.

Histological examination of cardiac changes verified hypertrophic changes following MDMA (Fig. 2E–H). Nuclear hypertrophy was increased in cardiomyocytes following 10-day MDMA (Fig. 2F) and 35-day MDMA (Fig. 2H). Contraction bands were observed following 35-day MDMA but were not present following 10-day MDMA, indicating progressive cardiac damage from MDMA. No nuclear hypertrophy or contraction bands were observed in 10 + 25-day experimental mice (data not shown), which again suggest attenuation of MDMA-induced cardiac hypertrophy following cessation.

Cardiac Gene Expression

Experiments were designed to identify groups of differentially regulated genes and enable cross evaluation between experimental sets. Using microarray technology, we analyzed gene expression profiles for changes resulting from MDMA use. Statistically significant changes in cardiac gene expression (p < .05, fold change > 1.5) in 10-, 35-day MDMA and 10 + 25 days experimental sets are provided in Supplementary Table S2. Following 10-day MDMA, 752 genes were differentially expressed, of which 77% were downregulated (Fig. 3A). For the 10 + 25 days ‘washout’ group, 113 differentially expressed genes were identified, with 12% downregulated (Fig. 3B). Analysis identified 558 differentially expressed genes in the 35-day MDMA group, with 76% downregulated (Fig. 3C). Pathway analysis identified enrichment of (Kyoto Encyclopedia of Genes and Genomes) KEGG-annotated pathways in experimental group, including MAPK pathways, Wnt signaling, and circadian rhythms (Table 1). Of note, the circadian rhythm pathway was enriched in both the 10- and 35-day MDMA groups but not in the 10 + 25-day group.

FIG. 3.

FIG. 3.

Differential cardiac gene expression following MDMA. Differentially expressed cardiac genes were identified (p < .05 and fold change > 1.5) following (A) 10-day MDMA, (B) 10-day MDMA+ 25-day saline, or (C) 35-day MDMA. 752 genes were differentially expressed following 10-day MDMA, with 77% downregulated. 113 genes were differentially expressed in the 10 + 25-day group, with 12% downregulated. 558 genes were differentially expressed following 35-day MDMA, with 76% downregulated. Differentially expressed genes are identified in Supplementary Table S2.

TABLE 1.

KEGG Pathway Analysis

Term Count Genes p-Value Benjamini
10-day MDMA Pathway Enrichment
Circadian rhythm 5 NPAS2, CRY2, PER3, ARNTL, CLOCK 0.00083 0.11050
MAPK signaling pathway 20 IL1R1, TAOK3, ELK1, MAPK11, MAPK10, PPM1B, TAB2, PRKCB, ATF2, CDC25B, DUSP5, RPS6KA5, ACVR1B, CRKL, ARRB2, RASGRP2, PDGFRA, MAPK9, NFATC2, MAP2K7 0.00232 0.10337
Wnt signaling pathway 13 WNT5A, PPP2R1B, ROCK1, ROCK2, PPP2R5C, CREBBP, MAPK10, TCF7L2, PRKCB, LRP6, MAPK9, WIF1, NFATC2 0.00600 0.19118
35-day MDMA Pathway Enrichment
Circadian rhythm 5 NPAS2, PER1, PER3, ARNTL, CLOCK 0.00033 0.04368

Multi-set analysis identified genes associated with MDMA administration protocols. Multi-set analysis was used to identify early/late and temporary/durable expression changes related pathophysiologically to cardiac function (Fig. 4). Figure 4A highlights grouping of genes between experimental sets and their interpreted results. Genes present in each section of Figure 4B are tabulated in Supplementary Table S3. Of the 1314 genes that were differentially expressed in all groups, only 116 were shared between at least 2 groups (Sections D–G, Fig. 4B). This sharing was independent of fold change in gene expression. Only 1 gene (Ubxn10) was significantly altered between 10 and 10 + 25-day groups, suggesting that genes differentially expressed following 10-day MDMA revert back to normal following a 25 days saline washout period of abstinence (Section D). Analysis of 10 + 25- and 35-day MDMA differentially expressed genes identified 31 shared genes (Section E). Of these genes, all 31 showed parallel gene expression directionality (ie, each of the genes was either downregulated in both groups or upregulated in both groups). Three genes were downregulated and 28 upregulated compared with controls (Section E, Fig. 4B). Pathway analysis of the kind we employed was unable to identify significant interaction between these 31 cardiac genes. Comparing 10- and 35-day MDMA differentially expressed genes, 83 genes were shared between groups while 82 of those genes also showed parallel differential gene expression; 69 were downregulated, 13 were upregulated (Section F, Fig. 4B). Exploring these 82 genes further through pathway analysis, we identified significant enrichment in the circadian rhythm pathway, with 4 downregulated genes identified (ARNTL, CLOCK, NPAS2, and Per3) and no upregulated genes. Finally, Myh7 was differentially expressed in all 3 groups, but had variable directionality of expression change (Section G). Myh7 was upregulated in the 10- and 35-day MDMA groups, whereas it was downregulated in the 10 + 25-day group. The explanation for this observation requires further exploration.

FIG. 4.

FIG. 4.

Multiset analysis of MDMA-induced differential gene expression. Genes were identified by whether they appeared in individual groups (ie, 10-, 35-day MDMA, etc.) or were differentially expressed in multiple groups. A, Interpretation of gene grouping. Genes that appeared each of the sections were identified as being related to certain responses to MDMA. For example, if a gene appeared in Section D (the region shared between 10-day MDMA and the 10 + 25-day group), then that gene was present following short-term MDMA and was still differentially expressed following 25-day saline in the 10 + 25-day group. B, The number of differentially expressed genes that were present in each of the functional groups. Of the 1314 gene differentially expressed in all MDMA treatment groups, only 116 genes (9%) showed up in multiple groups (Sections D, E, F, and G). Lists of genes in each group are presented in Supplementary Table S3.

Quantitative RT-PCR

Based on the microarray data earlier, we validated the microarray findings of the genes of interest by focusing on those members of circadian rhythm and MAPK signaling pathways (Table 1). NPAS2, PER3, CLOCK, and ARNTL all displayed decreased mRNA abundance following 10-day MDMA (Fig. 5A). With the exception of PER3, these results are concordant with microarray results. Additionally, only ARNTL exhibited decreased mRNA abundance 35 days following MDMA treatment, with NPAS2, PER3, and CLOCK expression returning to normal.

FIG. 5.

FIG. 5.

qRT-PCR. Validation of targets was performed of select genes identified by pathway analysis. A, Four members of the circadian rhythm pathway were differentially expressed following 10- or 35-day MDMA by microarray data. qRT-PCR validated the 10-day results but could not validate all 35-day results. This was attributed to lower sensitivity in the qRT-PCR assay. B, Three members of the MAPK pathway were selected for further analysis and to validate the microarray results. MAPK9, MAPK11, and NFATC2 were all downregulated following 10-day MDMA but returned to normal expression in the 35-day group, which is in agreement with the microarray results.

We also validated the expression of 2 prominent MAPKs and one related downstream transcription factor: MAPK9 (JNK), MAPK11 (p38MAPK), and NFATC2. MAPK9 expression was significantly decreased following 10-day MDMA (Fig. 5B). Similarly, a downstream target of MAPK9, NFATC2, was also downregulated following 10-day MDMA. MAPK11 also showed downregulation, validating microarray results. All 3 MAPK-related genes showed a return toward normal gene expression following 35-day MDMA, suggesting this is an early response to MDMA administration or the suppression of transcription of these genes becomes attenuated with increased MDMA exposure.

DNA Methylation

DNA microarrays were utilized to assess gene promoter DNA methylation changes. First, DNA methylation changes were determined independent of gene expression changes to identify global trends in DNA methylation patterns. Results identified regions approximately 50 bp long with DNA methylation changes (termed peaks). Analysis of 10-day MDMA identified 57 315 significant differentially methylated peaks across 10 243 genes (Fig. 6A). Of these differentially methylated peaks, 71% were hypermethylated and 29% hypomethylated (Fig. 6B). In the 10 + 25-day group, 136 604 significant differentially methylated peaks were identified in 13 919 genes, with 68% of peaks hypermethylated and 32% hypomethylated. For the 35-day MDMA group, 25 943 significant differentially methylated peaks were identified in 6466 genes, with 91% of differentially methylated peaks hypermethylated and 9% hypomethylated. Results show MDMA causes both hyper- and hypomethylation of cardiac DNA, though the data obtained indicate an overall trend of hypermethylation of cardiac DNA following MDMA (Fig. 6B).

FIG. 6.

FIG. 6.

Global DNA methylation analysis. DNA methylation arrays were compared pair-wise to appropriate saline controls to identify a global analysis of gene promoter DNA methylation in each group. A, The number of significant differentially methylated peaks in each MDMA experimental group. B, The percentage of peaks hypermethylated and hypomethylated in each group. There were over 57 000 differentially methylated peaks following 10-day MDMA, of which 71% were hypermethylated. There were over 136 000 peaks in the 10 + 25-day group, with 68% hypermethylated. There were over 25 000 differentially methylated peaks following 35-day MDMA, of which 91% were hypermethylated.

A second analysis was performed to identify DNA methylation changes that correspond to gene expression changes in the MDMA-treated heart (Fig. 7). Results are tabulated in Supplementary Table S4. Following 10-day MDMA, 170 genes (22% of significant differentially expressed genes) displayed 514 differentially methylated peaks, of which, 82% were hypermethylated and 18% were hypomethylated (Fig. 7B). In the 10 + 25-day group, 54 genes (48% of significant differentially expressed genes) displayed 358 differentially methylated peaks, of which 91% were hypermethylated and 9% were hypomethylated (Fig. 7C). In the 35-day MDMA group, 93 genes (17% of significant differentially expressed genes) displayed 371 differentially methylated peaks, of which, 100% were hypermethylated (Fig. 7D). In a comparison of the 31 genes differentially expressed in 10 + 25 and 35-day groups (Fig. 4, Section E), 4 of these genes had differential DNA methylation (10 peaks total), and all peaks were hypermethylated.

FIG. 7.

FIG. 7.

Gene promoter DNA methylation of differentially expressed genes. The DNA methylation changes were identified for the 1314 differentially expressed genes identified in Supplementary Table S2. A, Key to graphs. Each graph shows the number of differentially expressed genes that had no change in DNA methylation (grey) and the number of genes with changes in DNA methylation (blue). Of the genes in blue, the percentage of genes hypermethylated (green) or hypomethylated (red) are shown in the neighboring graph. B, 170 differentially expressed genes following 10-day MDMA also had associated DNA methylation changes, of which 71% were hypermethylated. C, 53 differentially expressed genes in the 10 + 25-day group displayed differentially promoter methylation, of which 91% were hypermethylated. D, In the 35-day MDMA group, 93 differentially expressed genes displayed 371 differentially methylated DNA regions, with all regions hypermethylated. Full color version available online.

Focusing on the 4 differentially expressed circadian rhythm genes noted earlier (ARNTL, CLOCK, NPAS2, and Per3), only 1 differentially methylated peak was identified (in Per3 and hypermethylated). For Myh7, 2 differentially methylated peaks were identified at 10 days, 3 peaks were identified in the 10 + 25-day group, and 1 peak was identified at 35 days (all hypermethylated). None of the differentially methylated peaks observed in the Myh7 promoter from each of the treatment cohorts were identical. Lastly, only 2 cardiac genes displayed significant DNA methylation peaks in each of the 3 MDMA experimental groups. Fam46b (a pseudogene for COX20) displayed the same hypermethylated peak in the 10-, 10 + 25-, and 35-day groups, whereas SLC25A36 (a mitochondrial pyrimidine nucleotide carrier) demonstrated 3 reproducible hypermethylated peaks.

DISCUSSION

Two overarching and important findings resulted from these studies. Our studies address pathogenetic cardiac changes that persist after relatively long-term MDMA abuse and relatively short-term MDMA use followed by abstinence. This is an important modeling approach since most MDMA users are young adults (age 18–25) who are less likely to continue MDMA abuse as they get older (Johnston et al., 2013). Our results here now show that MDMA induces DNA methylation (predominantly hypermethylation) and gene expression changes in the heart. These changes are associated with an increase in cardiac heart weight (14.3% increase following 10-day MDMA and 13.3% increase following 35-day MDMA). Cardiac HW:BW ratios are a sensitive indicator of cardiac hypertrophy, and our results show that this hypertrophy is reversible following cessation of MDMA (Fig. 2). These results were confirmed by histological examination of the tissues (Fig. 2E–H). Although DNA methylation and gene expression changes are intriguing by themselves, it may further reflect on the psychosocial aspects of MDMA abuse in which the stimulation to stay active relates to alterations of circadian rhythms that promote increased daylight activity and mobility.

Previously we identified hypermethylation of cardiac genomic DNA in LV samples from human CM. Although more studies will help unravel the relationship further, a pathophysiological link between DNA methylation changes and CM was concluded (Koczor et al., 2013). Studying MDMA cardiac effects in humans is not possible; however, results from current murine studies in vivo indicate MDMA causes hypermethylation of cardiac DNA prior to any measurable cardiac functional decline. This point argues that differential DNA methylation may be a sufficient cause for CM or a biomarker for CM if an appropriate surrogate tissue is available. It is important to recall that increased mortality and sudden death from MDMA (≈50%) was observed before termination of the experiment (Fig. 2D). Those MDMA-treated mice appeared to die after significant MDMA exposure which appeared to correlate with acute cardiorespiratory (sudden) death.

Microarray technology was used to identify differentially expressed genes following MDMA. Experiments enabled us to classify these genes affected by continuous MDMA use (10 and 35 days continuous MDMA) or those that may be considered durable responders to MDMA administration and subsequent abstinence (10-day MDMA but not after 35-day MDMA). Interestingly, genes involved in essential biological rhythms, so called circadian rhythm genes, are more affected by continuous MDMA administration. In contrast, MAPK gene dysfunction did not persist after early MDMA administration followed by abstinence. The observed difference in gene expression profiles may suggest different mechanisms of cardiac dysfunction from MDMA depending on dose and duration, but additional studies are required to clarify this relationship.

Importantly, it is interesting to speculate a relationship between disturbance of circadian rhythms described as deleterious CNS effects following MDMA and the cardiovascular events related to MDMA here (Colbron et al., 2002; Dafters and Biello, 2003). Our findings here suggest that those CNS effects may be extended analogously to the cardiovascular system and possibly other susceptible organ systems. Microarray data indicated circadian rhythm gene expression was altered (ARNTL, CLOCK, NPAS2, and Per3) following 10 and 35 days continuous MDMA. In the washout experiments, removal of MDMA (as seen in the 10 + 25-day group) reverted expression profiles to control patterns. qRT-PCR only validated the 10-day MDMA effects on ARNTL, CLOCK, and NPAS2 gene expression. This apparent contradiction may be an effect of microarray overestimation of gene expression changes, lack of sensitivity in the qRT-PCR assay, or combinations of factors. Studies that quantitatively analyze protein abundance and function of these circadian rhythm gene products will help determine the molecular and functional changes elicited by MDMA administration.

Our results demonstrate reduced expression of ARNTL, CLOCK, and NPAS2 following MDMA administration. Based on previous studies, CLOCK and ARNTL form a heterodimer necessary for physiological circadian rhythms (Vitaterna et al., 1994). Experimentally generated homozygous mutant mice for CLOCK develop arrhythmias, a known feature of MDMA toxicity in humans (Henry et al., 1992). Although NPAS2 may serve as a CLOCK substitute, its decreased expression following MDMA may result in ARNTL without a biochemical partner and help promote arrhythmia on that basis (DeBruyne et al., 2007). Whether the changes in cardiac circadian gene expression is caused directly by interaction of MDMA with cardiac circadian control or is a secondary effect of CNS neurohumoral effects caused by MDMA neurotoxicity also requires experimental investigation, but is beyond the scope of this initial article.

Gene expression and DNA methylation microarrays identified persistent changes in cardiac genes following MDMA. By comparing those from 35-day MDMA to those of 10 + 25 days washout group, 31 genes were differentially expressed and showed similar expression directionality (Fig. 4, Section E). All of these genes were differentially expressed as a result of short-term (10 days) MDMA; their expression did not change whether MDMA was continued or replaced by saline injections. The interrelationship or molecular impact of these gene expression changes is unknown, but their continued presence following MDMA supports a hypothesis that MDMA induces durable changes in cardiac gene expression. DNA methylation changes were documented to occur in mouse cardiac DNA of both 10 + 25 and 35 days cohorts. Of the 31 genes, 4 displayed DNA methylation changes. A total of 10 peaks were identified in their respective gene promoters; all were hypermethylated and were present in both the 10 + 25 and 35 days cohorts.

In summary, MDMA administration to inbred mice increased heart cardiac mass by >10% following as little as 10 days or as much as 35-day MDMA. Mice exposed to 10 d MDMA followed by 25 days saline washout displayed normal heart size. No change in cardiac function was observed. MDMA reduced survival by almost 50% after 33 days daily treatment. MDMA reduces gene expression following its administration (either 10 or 35 days), and the circadian rhythm pathway appears significantly impacted in the heart by MDMA use. DNA methylation changes correlated with almost 1 in 5 differentially expressed genes, showing a trend toward hypermethylation in the gene promoters of differentially expressed genes and a global trend toward hypermethylation.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

This work was supported by the National Institute on Drug Abuse (DA030996 to W.L.).

Supplementary Material

Supplementary Data

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