Roles Of3,4-methylenedioxymethamphetamine-induced Alteration Of Connexin43and Intracellular Ca²⁺ Homeostasis In Its Cardiotoxicity

BACKGROUND3,4-methylenedioxymethamphetamine （MDMA）, also known as ‘Ecstasy’, ispopular among the young adults around25years old in night clubs and rave parties.The research and clinical data have determined that MDMA can induce not onlyneurotoxicity but also hepatoxicity and cardiovascular toxicity. Acute MDMAadministration can cause markedly cardiovascular dysfunction, including heartbeatrate, blood pressure and myocardial oxygen consumption increase, which result inhypertension, cardiac arrhythmia, myocardial infarction and even heart failure or heartarrest. Previous studies show MDMA may induce cardiotoxicity through indirect ordirect ways. Alteration of monoaminergic systems via the neurotoxic actions ofMDMA has the potential to cause cardiovascular changes. The oxidative metabolismof MDMA can also cause the oxidative stress in myocardial cells. Although plenty ofevidences have demonstrated that MDMA induce cardiac function disorder, the directeffect on the cardiomyocytes is still unclear. Whether MDMA has any othercardiotoxicity mechanisms is still unknown.Myocardial gap junctions are widely known to play a significant role in thevelocity and the safety of impulse propagation in cardiac tissue, maintaining thenormal cardiac rhythm. Changes in gap junction proteins, including proteinexpression, distribution and phosphorylation status, may lead to fetal cardiacarrhythmia. However, there is no previous study focuses on the relationship betweenMDMA-induced arrhythmia and myocardial gap junctions alterations. Therefore,based on previous research findings, we firstly investigated the effect of MDMA onmyocardial gap junction proteins in this study. The results will further disclose thedirect mechanism of MDMA-induced cardiotoxicity. OBJECTIVE1. To observe the effect on rat’s electro-cardiac function after a single dose MDMAacute administration;2. To investigate the change of myocardial gap junction connexin43proteinexpressions induced by acute MDMA administration3. To investigate the alteration of total connexin43expression and Cx43mRNA levelin the primary cultured neonatal rats’ ventricular myocytes exposed to differentconcentration of MDMA;4. To observe the changes of the amount and the distribution of junctional connxin43in cultured ventricular myocytes treated with MDMA;5. To investigate the changes of phosphorylation status in connexin43Ser368ofcultured cells after exposure to different concentrations of MDMA.6. To test the changes of intracellular Ca²⁺oscillation patterns and calciumconcentration during MDMA exposure to the cultured ventricular myocytes.METHODS1. Ecstasy tablets were grinded into powder and then added pure water to solve itcompletely. Extraction of MDMA was performed by acid-base reactions. Thecomponents and purity of product was analyzed by GC-MS methods.2. In vivo study: a total of18male SD rats （SPF grade） were divided into two groupsrandomly. The rats in experimental group were administrated by20mg/kg MDMA i.p..Subsequently, ECG was detected and recorded by ECG detector. Rats treated withsaline were used as control group. All the rats were sacrificed24hours after druginjections. The heart tissues were taken and washed by saline, and then fixed in4%paraformaldehyde for48hours prior to paraffin embedded. Sections of heart tissueswere HE stained. Immunohistochemical staining was performed to investigate theexpression and distribution of connexin43proteins.3. in vitro study: newborn rats were sterilized and anesthetized. Cardiac ventricular was excised and shredded into tissue fragments. Step-wise digestion and90minutesdifferential adhesion were performed in order to enrich the culture with ventricularmyocytes. The cells were incubation in5%CO2at37. At the6thday, cultured cellswere added into10,100,1000μM MDMA with serum-free culture medium andincubated at37for1hour in order to establish MDMA intoxication models.Total membrane proteins of ventricular myocytes were extracted by using BioVisionprotein kit. Western blot was applied to test the change of total connexin43expressions. In addition, total RNA was extracted and the mRNA level of connexin43was quantitatively analyzed by application of Real time qPCR associated with2-（Δ（ΔCt））method.4. The junctional connexin43in cultured cells exposed to MDMA （10,100,1000μM）were investigated by immunofluorescent staining in order to see the change of Cx43level and distribution.5.1000μM MDMAwith serum-free culture medium was added onto the cultured cellsand incubated for1hour. Western blot was applied in order to test the alteration ofphosphorylation status in specific phosphorylated site Ser368of connexin43, as wellas the amount of PKCalpha. Besides, the intracellular calcium was label by Fluo-4and observed under confocal laser scanning microscope. The change of Ca²⁺oscillation patterns and the intracellular Ca²⁺concentration induced by MDMA weretest.STATISTICAL ANALYSISResults are shown as mean±SEM, multiple comparisons between groups wereanalyzed by the two-way ANOVA. For two group comparisons, unpaired Student’st-test was used. A value of p<0.05was considered significant. All the statisticalanalysis were performed by using Graph prism software.RESULTS1. MDMA which was extracted from Ecstasy tablets were determined that no other pharmacological active components existed. The purity of extraction was95%.2. Abnormal ECG findings were observed in experimental rats after a single doseMDMA acute administration, which was characterized by prolonged QRS duration,enhanced amplitudes associated with irregular changes. The heart rates of rats treatedwith MDMA showed significant changes with time. The average heart rate ofexperimental rats was significant decreased compared to control group.3. H&E staining showed unremarkable morphological findings. Atrophy, eosinophilicdegeneration, myocytolysis and vacuolization were seen in the rats after MDMAadministration.4. Two rats of experimental group died suddenly nearly2hours after MDMAinjection. IHC results showed the expression of connexin43was significantlydecreased in MDMA-treated rats compared to controls. In addition, it was observed indead rats that distributions of Cx43were strewn in longitudinally orientated arraysalong the lateral interfaces instead of confining to the sites of intercalated disks.5. In vitro study, significant decrease in total connexin43expression was found inMDMA-treated cultured cells associated with the obvious down-regulation of Cx43mRNA level. Immunofluorescent detection showed high concentration of MDMA ledto significant decrease of junctional connexin43and MDMA caused parallel reductionof N-cadherin level in cultured ventricular myocytes.6. high concentration of MDMA （1000μM） also increased the non-phosphorylatedconnexin43, and induced the increase of phosphorylated Cx43at site Ser368. Therewas no significant difference in PKCalpha between MDMA-treated and controlgroups. However, intracellular Ca²⁺oscillation patterns alterations and increase ofintracellular Ca²⁺concentrations were found induced by high concentration ofMDMA.CONCLUSIONS1. A single dose of MDMA acute administration can cause reduction of myocardialconnexin43and induce changes in heart rate and ECG patterns of rats. 2. MDMA directly affects the cardiomyocytes and results in decrease of totalconnexin43and abnormal connexin43distribution which may be related to thereduction of N-cadherin induced by MDMA.3. MDMA induces the changes in intracellular Ca²⁺oscillation patterns and increaseof Ca²⁺concentration, which may activate related protein kinases and up-regulate thephosphorylated Cx43Ser368.4. Our findings provide first evidence of MDMA-mediated changes in cardiac gapjunctions that may underlie MDMA-induced cardiac arrhythmia.