Chemical structures of cocaine and MDMA, as well as the synthetic cathinones MDPV, methylone, and mephedrone.

Chamber-specific beat rate of zebrafish embryos exposed to the five illicit drugs after 2 h. All values represent the mean normalized beat rate versus control ± standard error of the mean (SEM) (n = 10–12 embryos per clutch, at least three clutches were analyzed; * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control values, # p < 0.05, ### p < 0.001 compared between heart chambers (atrium and ventricle)). Two-way ANOVA statistical results are displayed in the graphs.

Graphical representation of the percentage of embryos exposed to each drug concentration presenting AV block (ventricle beating half as often as the atrium), ventricular fibrillation (less than 2.5 times the atrial rate), or none of these effects (regular beating heart 1:1). Fisher’s Exact test * p < 0.05, *** p < 0.001.

Arrhythmicity index (AI) of zebrafish embryos treated with the illicit drugs for 2 h. AI was calculated as the heart period standard deviation normalized to the median heart period. Data points represent the average AI ± standard error of the mean (SEM) (n = 10–12 embryos per clutch, at least three clutches were analyzed; ** p < 0.01, *** p < 0.001 compared to control AI values, # p < 0.05 compared between heart chambers (atrium and ventricle)). Two-way ANOVA statistical results are displayed in the graphs. Data from embryos exposed to 500 µM of cocaine had no ventricle heartbeat; therefore, no AI could be calculated. For that reason, two-way ANOVA was performed considering up to 250 µM of cocaine. For drugs with no statistically significant interaction, significant comparisons between control AI values and treated are displayed, +++ p < 0.001.

Ventricular beat rate of zebrafish embryos exposed to the Cm of the five illicit drugs after 2 h. All values represent the mean normalized beat rate versus control ± standard error of the mean (SEM) (n = 10–12 embryos per clutch, at least three clutches were analyzed; * p < 0.05, ** p < 0.01, *** p < 0.001). t-test statistical results are displayed in the graphs.

Schematic overview of the cardiotoxic effects evaluated in the zebrafish embryo model for MDPV, cocaine, MDMA, mephedrone and methylone. The following values obtained from the zebrafish experiments have been represented for each endpoint: ventricle tachycardia (LOAEC), ventricle bradychardia (EC₅₀), atrium bradychardia (EC₅₀), AV block 2:1 (LOAEC), ventricular fibrillation (LOAEC), arrythmicity index (LOAEC). Grey areas represent in vivo relevant concentrations in humans in blood/plasma after recreative doses described in the literature [47,48], while blue areas represent in vivo relevant concentrations in humans in blood/plasma after lethal doses described in the literature [48,49,50,51].

Schematic representation of the approach used to calculate the interbeat intervals (heart period) from video recordings of zebrafish embryos. Color dots in the bottom right image indicate identified peaks (local maxima, red and minima, green) in data. Methodology adapted from [61].