Do Stimulants Promote Arrhythmic Risk: Insights From a Human‐Induced Pluripotent Stem Cell‐Derived Cardiomyocyte Model

ABSTRACT

Background

Stimulant medications are widely prescribed for ADHD. Although generally considered safe, their use in patients with underlying cardiac conditions remains a concern due to potential arrhythmic risk.

Objective

To evaluate the effects of commonly prescribed stimulants on key electrophysiological parameters associated with arrhythmia risk in chamber‐specific human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs).

Methods

Atrial and ventricular hiPSC‐CMs were plated on a multielectrode array (MEA) platform and exposed to clinically relevant plasma concentrations of methylphenidate, dextroamphetamine, atomoxetine, caffeine, or the β‐agonist isoproterenol. Extracellular field potentials were recorded over 48 h to assess acute and prolonged effects on conduction velocity (CV), beat rate (BR), and corrected field potential duration (FPDc).

Results

CV was largely preserved across all stimulant conditions in both atrial and ventricular hiPSC‐CMs, while isoproterenol produced a modest early increase. All stimulants transiently elevated BR during the first hour of exposure, followed by a progressive decline over time. Isoproterenol and caffeine induced robust and sustained increases in BR, confirming model responsiveness. FPDc was prolonged by all stimulant compounds, most prominently by atomoxetine, consistent with its known I_Kr inhibitory properties. Isoproterenol produced a rate‐dependent FPDc shortening, particularly in hiPSC‐vCMs.

Conclusion

At clinically relevant plasma concentrations, stimulants modulated automaticity and repolarization but had minimal impact on conduction in hiPSC‐CMs. The preservation of CV suggests that these compounds are unlikely to alter myocardial conduction at therapeutic doses. The observed effects on BR and FPDc warrant further mechanistic studies to understand how these changes may contribute to arrhythmia risk in susceptible populations.

At clinically relevant plasma concentrations, stimulant medications produced minimal effects on conduction velocity but modulated automaticity and repolarization in a hiPSC‐derived cardiomyocyte (hiPSC‐CM) model. These findings provide a foundation for future investigations of stimulant‐associated cardiac safety and support the utility of hiPSC‐CM platforms for evaluating cardiac responses to pharmacological agents.

1. Introduction

Attention deficit hyperactivity disorder (ADHD) is a prevalent neurodevelopmental disorder with onset typically in childhood with most individuals experiencing symptoms throughout their lifetime [1]. First‐line therapy for ADHD consists of stimulant medication combined with psychotherapy and skill‐building interventions [2]. Stimulant medication therapy is highly effective and considered beneficial in up to 70% of ADHD cases by managing inattention, hyperactivity, and impulsivity [3, 4]. Prescription rates for ADHD medications have increased significantly across all age groups in recent years [5]. Methylphenidate (MPH) and amphetamine are commonly used stimulant medications, often alongside atomoxetine (ATX), an established selective norepinephrine reuptake inhibitor [3, 6]. All three drugs are sympathomimetic amines that increase norepinephrine and dopamine concentration in the prefrontal cortex, stimulating the central nervous system (CNS) [3].

In 2006, Health Canada issued a safety warning regarding rare heart‐related risks associated with ADHD medications after rare reports of sudden deaths in children and adults on stimulants. In 2011, an FDA safety review similarly advised against the use of stimulant medication and ATX in patients with heart disease or intolerance to heart rate and blood pressure changes. Although the American Heart Association issued a statement in 2008 recommending children undergo a thorough cardiac history, physical exam, and electrocardiogram (Class IIa recommendation) prior to starting stimulant therapy [7], subsequent clarification with the American Academy of Pediatrics limited electrocardiogram screening for cases with concerning medical family history or cardiac findings [8]. Similarly, the Canadian ADHD Resource Alliance (CADDRA) guidelines recommend monitoring blood pressure and heart rate in all patients while limiting electrocardiogram monitoring only in patients with known or high‐risk cardiac conditions [9]. In addition, ADHD itself is independently associated with an increased risk of cardiovascular disease, a risk that is significant for all patients regardless of age or medication use [10, 11]. Despite acknowledgement of statistically significant cardiovascular changes and events associated with stimulant medication use, the clinical relevance of stimulant medication‐related changes remains largely unknown.

Patients with pre‐existing arrhythmia syndromes, such as long QT syndrome (LQTS) and Wolff–Parkinson–White (WPW), warrant special consideration when prescribing stimulant medications [12]. These conditions represent two distinct electrophysiological substrates that may be influenced by sympathomimetic medications: abnormal repolarization and abnormal impulse propagation, respectively. In LQTS, medications that prolong repolarization may further extend the QT interval, heightening the risk of torsades de pointes and sudden cardiac death [13, 14]. In contrast, stimulant use in patients with WPW carries a theoretical risk of increasing conduction velocity (CV) through accessory pathways, which could increase susceptibility to tachyarrhythmias [15, 16, 17]. Previous reports suggest that stimulant‐induced catecholamine release may facilitate rapid conduction of atrial fibrillation through an accessory pathway to the ventricles, potentially degenerating into ventricular fibrillation and sudden cardiac arrest [18, 19]. Although enhanced accessory pathway conduction has been proposed as a potential mechanism, the direct effects of stimulant medications on myocardial impulse propagation remain unclear [12]. While WPW and LQTS are frequently discussed in the context of stimulant‐associated cardiac safety, the potential cardiac effects of stimulant medications may have broader relevance beyond these conditions.

Human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) have emerged as a valuable in vitro platform for evaluating drug‐induced electrophysiological effects and cardiac safety beyond traditional preclinical models [20, 21]. Previous studies have demonstrated that hiPSC‐CMs exhibit functional adrenergic signaling, whereby sympathomimetic stimulation increases spontaneous beat rate (BR), alters intracellular calcium (Ca²⁺) handling, and modulates repolarization properties in a dose‐dependent manner. For example, β‐adrenergic agonists such as isoprenaline have been shown to enhance automaticity and Ca²⁺ transient amplitude, supporting their utility for studying pharmacological modulation of cardiac electrophysiology [22, 23, 24]. Such findings highlight the ability of hiPSC‐CM models to capture physiologically relevant responses to compounds that influence sympathetic signaling.

Despite the widespread clinical use of CNS stimulants such as MPH, ATX, and dextroamphetamine (DEX), there remains limited experimental data describing their direct effects on cardiomyocyte electrophysiology. To address this gap, we used atrial and ventricular hiPSC‐CM monolayers to characterize the time‐dependent effects of clinically relevant stimulant compounds on CV, BR, and repolarization. These parameters were selected because they reflect key electrophysiological parameters relevant to stimulant‐associated cardiac safety concerns. In particular, CV is relevant to disorders of abnormal impulse propagation such as WPW, while repolarization is relevant to conditions characterized by delayed repolarization such as LQTS. By evaluating multiple electrophysiological endpoints using a hiPSC‐CM model, this study sought to provide insights into cardiac electrophysiology responses that may be relevant to individuals with underlying arrhythmia susceptibility.

2. Methods

A detailed description of the methods is provided in the Supporting Information.

2.1. Cell Culture

A commercial hiPSC line (WiCell, Madison, WI) was differentiated into atrial and ventricular CMs (Figure 1A). Ventricular differentiation was performed using a well‐established Wnt/β‐catenin modulation protocol involving temporal activation and inhibition of Wnt signaling with CHIR99021 and IWP‐4, respectively [25]. Atrial differentiation was induced by supplementation with retinoic acid on Days 4–6 of differentiation [25, 26]. These protocols utilizing temporal Wnt modulation reliably generate hiPSC‐CM monolayers with greater than 85% cardiac troponin T (TNNT2) expression [25, 26, 27]. Following differentiation, hiPSC‐CMs were metabolically enriched by culturing cells in glucose‐depleted medium supplemented with sodium l‐lactate from Day 12 to 15, which allows for selective purification of CM population from non‐CM cells. hiPSC‐CMs subsequently underwent metabolic maturation for 5 weeks using a fatty acid‐based maturation medium, previously described by Feyen et al. [28]. This maturation approach was designed to promote a metabolic shift from glycolysis to oxidative phosphorylation, which has shown to enhance mitochondrial oxidative capacity, Ca²⁺ handling, ion channel expression profile, and structural organization, improving functional maturity and reducing variability in baseline electrophysiological parameters [28]. Cultures were maintained at 37°C with 5% CO₂ throughout the duration of experiments.

FIGURE 1.

Schematic overview of the experimental workflow. (A) Cardiac differentiation protocol based on temporal modulation of the canonical Wnt/β‐catenin signaling pathway. (B) Multielectrode array (MEA) workflow for electrophysiological assessment.

2.2. Compound Preparation

Stimulant medications used in this study, including MPH, ATX, and DEX, were provided by the hospital's clinical pharmacy, while caffeine (CAFF) and isoproterenol (ISO, positive control) were obtained commercially (Sigma‐Aldrich, Saint Louis, MO). MPH, ATX, and DEX are expected to be stable under physiological conditions [29, 30, 31]. Stimulants were selected as commonly prescribed ADHD medications, and experimental concentrations were chosen to reflect therapeutic blood plasma levels [5]. Table 1 contains further information on each compound, reported C _max values, and experimental concentrations used in this study.

TABLE 1.

Pharmacological characteristics of the selected stimulants.

Compound	Mechanism of action	Reported physiological C max range	Experimental concentration
Methylphenidate hydrochloride [32]	CNS stimulant; sympathomimetic effect	Therapeutic effects at plasma levels of 8–40 ng/mL, with a maximal efficacy around 10 ng/mL [33] After 0.30 mg/kg (at ~2 h): 10.8 ng/mL (children), 7.8 ng/mL (adults) [32]	0.04 μM [~10 ng/mL]
Atomoxetine hydrochloride [34]	Non‐stimulant; sympathomimetic effect	Pediatric single dose of 10 mg: 80–212 ng/mL [35] 20–45 mg twice daily regimen: 174–1221 ng/mL [35]	3 μM [~875 ng/mL]
Dextroamphetamine sulfate [36]	CNS stimulant; sympathomimetic effect	Single 5 mg dose: 11.5 ng/mL [37] Triple 5 mg dose: 36.6 ng/mL at ~3 h [36]	0.10 μM [~36.6 ng/mL]
Caffeine	Positive chronotropic and inotropic effect	Up to 12 μg/mL, at half‐life of 2–8 h [38] Ingestion of 500 mg: ~20 μg/mL [38, 39]	103 μM [~20 μg/mL]
Isoprenaline hydrochloride	Positive chronotropic and inotropic effect	Therapeutic C max level has not been reported in the literature Common in vitro study doses ranges from 100 nM to 1 μM [40, 41]	1 μM

2.3. Multielectrode Array (MEA) Assay

Electrophysiological recordings were performed using a Maestro Pro MEA system (Axion Biosystems, Atlanta, GA) (Figure 1B). hiPSC‐CMs were plated on 48‐well Cytoview MEA plates (M768‐tMEA‐48 W, Axion Biosystems, Atlanta, GA) and allowed up to 7 days to recover and reform a synchronized syncytium. A complete medium change was performed 3 h before baseline recordings. Field potentials (FPs) were recorded from spontaneously beating monolayers in a time‐dependent manner including physiological baseline prior to addition of stimulant compounds, and at 0.5, 1, 3, 6, 9, 12, 18, 24, and 48 h after compound addition to assess both acute and prolonged effects of MPH, ATX, DEX, CAFF, and ISO. Data acquisition was performed with AxIS Navigator software (Axion Biosystems, Atlanta, GA). FPs were recorded for 1.5 min at 12.5 kHz sampling frequency. Electrophysiological parameters analyzed include BR, field potential duration (FPD), and CV. To account for rate‐dependent variability in repolarization, FPD was normalized to a standard cycle length using Fridericia's correction (field potential duration corrected [FPDc]) [20, 42].

2.4. Statistical Analysis

MEA recordings were processed using the AxIS Cardiac Analysis Tool (Axion Biosystems, Atlanta, GA). Only the electrodes with stable FP signals were included in the analysis. Baseline and temporal measurements were obtained from the same well, and stimulant‐induced changes were expressed as % change from baseline. Baseline comparisons of atrial versus ventricular hiPSC‐CMs (BR, FPDc, CV) were performed using independent Student's t‐test. Temporal effects of each stimulant were assessed using a mixed‐effects model with Greenhouse–Geisser correction and Dunnett's post hoc test for comparison to baseline. Statistical analyses were conducted in GraphPad Prism v10.4.0 (GraphPad Software, San Diego, CA), with significance defined as p < 0.05.

3. Results

3.1. Baseline Electrophysiological Properties of hiPSC‐CMs

To compare the baseline electrophysiological properties of atrial (aCMs) and ventricular (vCMs) hiPSC‐CMs, we analyzed BR, FPDc, and CV under spontaneous beating conditions (Figure 2). Representative FP traces from aCMs and vCMs are shown in Figure 2A, illustrating distinct electrophysiological profiles between the two cell types. aCMs exhibited a significantly higher BR compared to vCMs (Figure 2B; 128.2 ± 16.9 vs. 56.62 ± 12.9 bpm; p < 0.0001). Similarly, CV was significantly faster in aCMs than in vCMs (Figure 2D; 39.12 ± 7.355 vs. 21.41 ± 5.929 cm/s; p < 0.0001). In contrast, FPDc was significantly shorter in aCMs compared to vCMs (Figure 2C; 164.3 ± 19.19 vs. 339.8 ± 20.29 ms; p < 0.0001).

FIGURE 2.

Baseline electrophysiological differences between atrial (aCM) and ventricular (vCM) hiPSC‐CMs. (A) Representative field potential (FP) traces. (B) Beat rate (N _atrial = 60, N _ventricular = 51). (C) Corrected field potential duration, FPDc (N _atrial = 59; N _ventricular = 49). (D) Conduction velocity (N _atrial = 56, N _ventricular = 48). Data are presented as mean ± SD. Statistical significance is indicated as ****p < 0.0001.

These findings are consistent with the expected electrophysiological distinctions between these two CM subtypes, with aCMs demonstrating a faster intrinsic rhythm, greater CV, and shorter repolarization time [43, 44]. This baseline characterization provides a foundation for evaluating stimulant‐induced effects on hiPSC‐CM electrophysiology.

3.2. Effect of Stimulants on Atrial and Ventricular hiPSC‐CMs

We evaluated the effects of stimulant compounds (MPH, DEX, and ATX) and positive controls (ISO and CAFF) on aCMs and vCMs over a 48‐h treatment period. Time‐dependent changes in CV, BR, and FPDc are presented as % change from baseline values (Figure 3). Complete raw electrophysiological data (mean ± SD) for all compounds and timepoints are provided in Table S1 (aCMs) and Table S2 (vCMs). In addition, to complement quantitative electrophysiological analyses, Figure S1 provides representative FP traces from aCMs and vCMs at baseline and during stimulant treatments. Overlaid recordings at 0.5, 6, and 24 h illustrate the temporal evolution of depolarization and repolarization features in response to each stimulant. Our vehicle control experiments showed no significant time‐dependent changes in FP morphology, CV, BR, or FPDc (Figure S2).

FIGURE 3.

Temporal effect of stimulant treatments on electrophysiological parameters in atrial (aCM) and ventricular (vCM) hiPSC‐CMs. (A and B) Conduction velocity. (C and D) Beat rate. (E and F) Corrected field potential duration, FPDc. Data are presented as mean percentage change from baseline, error bars represent SD (aCM: N _MPH = 12, N _DEX = 12, N _ATX = 12, N _CAFF = 12, N _ISO = 12; vCM: N _MPH = 12, N _DEX = 10, N _ATX = 10, N _CAFF = 11, N _ISO = 8). Statistical significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

In aCMs, stimulants exerted minimal effects on CV (Figure 3A), with values remaining close to baseline across all time points. MPH, DEX, and ATX did not induce sustained changes, although MPH caused a small but significant early reduction in CV at 0.5 h (−12.4%, p < 0.05). In contrast, the positive control ISO produced a pronounced, transient increase in CV (+26% at 0.5 h, p < 0.01), while CAFF elicited only minor and short‐lived effects. A similar pattern was observed in vCMs (Figure 3B), where CV remained largely unchanged following stimulant treatments. Neither MPH, DEX, nor ATX resulted in significant alterations in CV. As in aCMs, ISO induced an early increase in CV (+31% at 0.5 h, p < 0.01), whereas CAFF did not produce appreciable effects.

Stimulants induced a transient increase in BR in aCMs (Figure 3C) during the first hour, most notably with MPH and DEX (MPH: +21.6% at 0.5 h, p < 0.001; DEX: +26.4%, p < 0.001), followed by a return to baseline over time. While not significant, ATX also produced an early modest rise in BR with a significant decline evident from 6 h onward (−12% at 6 h, p < 0.05; −21.4% at 48 h, p < 0.0001). Positive controls produced robust and sustained BR increases across all time points, eliciting the strongest effect at early time points (ISO: +70%–90%, p < 0.001; CAFF: +20%–30%, p < 0.0001) and a gradual return to baseline at later time points. In vCMs (Figure 3D), stimulants produced a similar early transient BR increase followed by a progressive decline. An initial BR increase was observed with MPH and DEX (MPH: +18.4% at 0.5 h, p < 0.0001; DEX: +26.4% at 0.5 h, p < 0.01). MPH induced the most marked suppression at later times (−16% to −23% by 12–24 h, p < 0.01). ATX caused the strongest sustained BR reduction (−20% to −32% between 6 and 48 h, p < 0.001). CAFF elicited a large early BR increase (+56% at 0.5 h, p < 0.001) but this effect diminished by 48 h. ISO showed the most robust BR elevation (up to +164% at 0.5 h, p < 0.0001), gradually declining but remaining elevated at 48 h.

FPDc in aCMs (Figure 3E) increased significantly in response to all stimulants. Both MPH and DEX caused a modest but consistent prolongation over the first 12 h (MPH: +6.6% at 6 h, p < 0.05; DEX: +8.3% at 6 h, p < 0.0001), while ATX induced a markedly larger and sustained increase in FPDc (up to +24% by 6 h, p < 0.0001). CAFF also progressively prolonged FPDc (up to +18.5% by 24 h, p < 0.001), whereas ISO induced transient rate‐dependent FPDc shortening at 0.5 h, but marked FPDc increase from 6 h onward (up to +24.3% at 12 h, p < 0.0001). vCM FPDc responses (Figure 3F) mirrored aCMs, with stimulants inducing consistent prolongation. The most prominent FPDc prolongation was observed at 18 h with ATX (+39%, p < 0.0001), DEX (+19.1%, p < 0.001), and MPH (+18.8%, p < 0.0001). CAFF also caused progressive FPDc prolongation (+30.8% at 18 h, p < 0.0001), while ISO induced marked and sustained rate‐dependent shortening (−36% to −26% within the first 6 h, p < 0.0001), with partial recovery toward baseline at later time.

Overall, our data demonstrate consistency in stimulant treatment response effects between aCMs and vCMs. We observed a minimal impact on CV, indicating that these compounds do not substantially alter impulse propagation in our hiPSC‐CM monolayer model. Instead, stimulant effects appear more closely related to automaticity and repolarization changes.

4. Discussion

In this study, we investigated the effects of clinically relevant concentrations of stimulant medications, including MPH, DEX, and ATX on atrial and ventricular hiPSC‐CMs using a MEA platform. Given clinical concerns regarding stimulant use in individuals with underlying cardiac conditions such as WPW and LQTS [12], our primary aim was to characterize the effects of these compounds on key electrophysiological parameters relevant to arrhythmia susceptibility, including CV, automaticity, and repolarization.

Our findings demonstrate that hiPSC‐CMs preserve distinct chamber‐specific electrophysiological properties, with aCMs exhibiting higher intrinsic BR, faster CV, and shorter repolarization duration compared to vCMs (Figure 2). These chamber‐specific differences in CV may reflect variations in intrinsic cellular electrophysiology, intracellular coupling, and experimental configuration, as conduction properties across hiPSC‐CM models can be influenced by differences in tissue architecture, maturation state, and connexin expression [43, 44]. These observations are consistent with known electrophysiological differences between atrial and ventricular myocardium and support the utility of chamber‐specific hiPSC‐CMs as a platform for studying drug effects on human CMs. Importantly, accessory pathways in WPW consist of ordinary working myocardium or large fascicles of cardiac muscle that bridge atrium to ventricle in areas where the fibrous layer is fenestrated or incomplete, and in some cases, may take the form of atrio‐fascicular tracts [45, 46, 47]. Our approach enabled assessment of stimulant‐induced changes in CV across atrioventricular myocardial tissue relevant to pre‐excitation syndromes.

In our hiPSC‐CM model, stimulant exposure produced minimal effects on CV across both cell types (Figure 3A & 3B). In cardiac tissue, CV is primarily determined by the kinetics of Na_v1.5‐mediated inward sodium current (I _Na), which underlies the rapid upstroke (V _max) of the action potential depolarization, the passive electrical coupling between CMs via gap junctions, primarily Cx43 in ventricular and Cx40 in atrial tissue, and electrotonic properties of the cardiac tissue network [48, 49, 50, 51, 52]. Modulation of these properties can alter impulse propagations; however, none of the stimulant compounds tested in this study produced sustained changes in CV under the examined conditions. ISO modestly increased CV at early time points, which is consistent with the known effect of β‐adrenergic receptor (AR) stimulation on cardiac electrophysiology. Previous studies have demonstrated that activation of β‐ARs using isoprenaline increases I _Na availability and enhances gap junctional conductance through protein kinase A (PKA)‐dependent signaling pathways [53, 54]. In contrast, a transient early decrease in CV was observed with MPH, which could reflect indirect ionic effects as MPH has no established direct ion channel targets at therapeutic concentrations [55]. ATX, on the other hand, has been shown to block Na_v1.5 channels in a state‐ and use‐dependent manner at concentrations within its therapeutic plasma range; yet we observed no significant CV effects with ATX exposure under our experimental conditions [56]. MEA‐derived conduction measurements may be influenced by electrode geometry and lack the spatial resolution necessary to characterize conduction heterogeneity. While not employed in this study, optical mapping would provide substantially higher spatial and temporal resolution for assessment of activation patterns and monolayer‐level conduction dynamics. While MEA is well suited for comparative drug screening and longitudinal studies, complementary optical mapping may provide greater sensitivity for detecting subtle conduction effects. Overall, none of the stimulants tested (MPH, DEX, ATX, and CAFF) produced significant or sustained effects on CV in aCM and vCM monolayers. These findings are consistent with previous consensus statements indicating that stimulant use in WPW does not appear to increase arrhythmic risk through conduction‐dependent mechanisms [57], and reinforce the importance of pathway‐specific electrophysiologic properties, rather than stimulant exposure, in risk stratification and clinical decision‐making.

While CV was largely preserved, all stimulant compounds produced a rapid, transient increase in BR, most prominently within the first hour of exposure (Figure 3C,D). This response was consistent across both aCMs and vCMs. Following the initial chronotropic response, BR declined progressively over time, potentially reflecting a time‐dependent attenuation of the adrenergic signal. Positive controls, ISO and CAFF, induced the most robust and sustained increases in BR, confirming the chronotropic responsiveness of the hiPSC‐CM model [58, 59]. The positive chronotropic effects of catecholaminergic stimulation in hiPSC‐CMs are mediated through β‐AR activation, which increases intracellular cAMP via adenylyl cyclase and activates PKA. This signaling enhances HCN₄‐mediated pacemaker currents (I _f) and L‐type Ca²⁺ current (I _Ca,L), and accelerates sarcoplasmic reticulum Ca²⁺ cycling, resulting in increased automaticity and faster BR [60, 61]. In our model, the magnitude of the chronotropic response to stimulants was substantially smaller than that observed with ISO, a direct non‐selective β‐AR agonist. Stimulants like MPH, DEX, and ATX enhance noradrenergic signaling at sympathetic nerve terminals through distinct presynaptic mechanisms. MPH inhibits both the norepinephrine and dopamine transporters (NET and DAT, respectively), increasing synaptic catecholamine availability. ATX acts selectively at NET, elevating synaptic norepinephrine. DEX acts primarily as a transporter substrate rather than a reuptake inhibitor, which promotes presynaptic catecholamine release through vesicular displacement (VMAT2‐mediated) and reverse transport through NET. The net effect of all three compounds is an increase in extracellular synaptic norepinephrine availability, representing their principal cardiac effect. Dopamine elevation, while pharmacologically relevant in the CNS, contributes minimally to cardiac adrenergic signaling [3, 62, 63, 64, 65]. The resulting β‐AR stimulation in CMs mimics physiological sympathetic activation, which may underlie the increased incidence of palpitations reported by patients taking these medications [66].

In addition to their effects on conduction and automaticity, we evaluated the impact of stimulant compounds on repolarization, as reflected by changes in FPDc. FPDc measured on the MEA platform is considered as an in vitro surrogate for CM repolarization time, analogous to the rate‐corrected QT interval on the surface ECG, and forms the basis of several pro‐arrhythmia screening platforms [23]. Stimulant exposure produced a consistent directional effect on FPDc across all stimulants, with ATX eliciting the most pronounced FPDc prolongation in both aCMs and vCMs (Figure 3E,F). While direct ionic currents were not measured, previous in vitro studies have shown that ATX directly inhibits hERG‐mediated rapid delayed rectifier potassium current (I _Kr) in a concentration‐dependent manner [67]. Because I _Kr is the primary mediator of Phase 3 cardiac repolarization, its inhibition delays repolarization which prolongs action potential duration and FPD measured on the MEA platform. Inhibition of hERG channels is a well‐established mechanism underlying drug‐induced QT prolongation [68], and likely contributes to the measurable FPDc prolongation observed in our study. MPH and DEX are not reported to exert direct effects on hERG or other cardiac ion channels at therapeutic plasma concentrations. The modest FPDc prolongation observed with these stimulants may, therefore, reflect indirect effects of elevated catecholamines rather than direct ion channel blockade. Increased catecholamine levels activate β‐AR signaling pathways that enhance intracellular Ca²⁺ cycling and modify repolarizing K⁺ currents (I _K ). While acute β‐AR stimulation typically shortens repolarization via PKA‐mediated augmentations of I _Ks (encoded by KCNQ1/KCNE1) and I _Ca,L, sustained adrenergic drive has been shown to induce electrophysiological remodeling, including altered repolarizing I _K or I _Ca balance, which can prolong FPD [69, 70]. At the model level, hiPSC‐CMs have a relatively immature electrophysiological phenotype with attenuated I_K expression, which may limit repolarization response to adrenergic stimulation. This may also account for failure of indirect β‐AR stimulants (MPH, DEX, and CAFF) to accelerate FPDc as seen with ISO, which shortened FPDc in vCMs, consistent with rate‐dependent repolarization effects [71]. Smaller FPDc changes with MPH, DEX, and CAFF may reflect subtle repolarization changes that are often clinically silent but could become relevant in susceptible individuals. The divergence in FPDc responses between stimulants and ISO may reflect distinction between indirect catecholamine elevation and direct receptor agonism in modulation of cardiac repolarization through distinct pathways, warranting further mechanistic investigation.

Given ongoing concerns regarding the use of stimulant medications in children with underlying cardiac conditions such as LQTS and WPW [12], our findings provide insight into the electrophysiological effects of these medications in a human‐relevant cardiomyocyte model. Although the present study focused on conduction, automaticity, and repolarization, the observed responses may have a broader relevance to other inherited or acquired arrhythmogenic conditions in which sympathetic stimulation contributes to arrhythmia susceptibility. Together, these findings support the use of hiPSC‐CM platforms for evaluating cardiac responses to stimulant medications and other sympathomimetic agents.

4.1. Study Limitations

Several limitations should be considered when interpreting these findings. While hiPSC‐CMs provide a valuable platform for in vitro electrophysiological modeling, they do not fully recapitulate mature adult myocardium. In addition, hiPSC‐CM monolayers lack autonomic innervation which may attenuate the full physiological response to indirectly acting stimulants such as MPH, DEX, and ATX. Nevertheless, because the developmental phenotype of metabolically matured hiPSC‐CMs still remains closer to pediatric than adult cardiomyocytes, this platform may provide a clinically relevant model for investigating stimulant‐induced responses in the population that is most frequently exposed to these medications.

In the present study, CV was assessed using the MEA system. While the platform provides a non‐invasive, high‐throughput approach for repeated longitudinal measurements, its spatial resolution is lower than that of optical mapping, limiting the characterization of conduction heterogeneity, conduction block, and re‐entrant activity. MEA recordings do not directly resolve ionic mechanisms underlying repolarization changes, such as alterations in I _Kr, I _Ks, I _Ca,L, or intracellular Ca²⁺ handling. FPDc was calculated using the Fridericia correction to account for differences in spontaneous BR. However, rate correction cannot completely eliminate the influence of cycle length. While fixed‐rate pacing could reduce this confounding effect, reliable entrainment could not be consistently maintained across wells and recording time points due to robust spontaneous activity and variable pacing thresholds of hiPSC‐CMs in our study. Therefore, spontaneous recordings provided the most reproducible approach for our longitudinal comparison across all treatment groups. Finally, the present study evaluated a single concentration of each stimulant selected to reflect clinically relevant therapeutic plasma concentration. While this approach provides translational relevance, it does not fully capture the dynamic pharmacokinetic profile of these stimulants in vivo or potential concentration‐dependent effects.

Future work incorporating dose–response analyses, complementary patch‐clamp and optical mapping electrophysiology, and more advanced cardiac models, including co‐cultures with hiPSC‐derived neurons and disease‐specific hiPSC‐CMs harboring pathogenic variants, may provide additional mechanistic insight into the cellular basis of the electrophysiological responses observed in the present study and determine whether these effects translate into clinically meaningful arrhythmic risk in susceptible populations. Despite these limitations, the use of both atrial and ventricular hiPSC‐CMs, prolonged 48‐h stimulant exposure, clinically relevant stimulant concentrations, and simultaneous assessment of conduction, automaticity, and repolarization represent important strengths of this study.

5. Conclusion

This study demonstrates the utility of chamber‐specific hiPSC‐CMs as a physiologically relevant platform for evaluating the electrophysiological effects of stimulant medications, particularly in the context of conditions associated with increased arrhythmia susceptibility, such as WPW and LQTS. At clinically relevant plasma concentrations, MPH, DEX, ATX, and CAFF did not produce significant or sustained changes in CV, suggesting that these stimulants are unlikely to substantially alter conduction properties at therapeutic doses. However, the observed effects on automaticity and repolarization indicate that the electrophysiological actions of stimulant medications may extend beyond conduction and highlight the importance of further mechanistic studies to identify the cellular and ionic mechanisms underlying these responses. While caution is warranted when extrapolating in vitro findings to clinical practice, particularly given the developmental phenotype of hiPSC‐CMs, this study provides a foundation for future investigations into stimulant‐associated cardiac safety.

Funding

This research was funded by the British Columbia Children's Hospital Foundation.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Representative spontaneous field potential traces from hiPSC‐CM monolayers during stimulant exposure. Representative FP recordings from (A) atrial and (B) ventricular hiPSC‐CMs at baseline and after 0.5, 6, and 24 h of stimulant exposure. Stimulant compounds included methylphenidate (0.04 μM), dextroamphetamine (0.10 μM), atomoxetine (3 μM), caffeine (103 μM), isoprenaline (1 μM). FP, field potential; BL, baseline.

Figure S2: Temporal stability of electrophysiological parameters under the vehicle‐control conditions. (A) Representative field potential traces from atrial and ventricular hiPSC‐CMs. (B) Quantification of the temporal effect of Milli‐Q water (vehicle) on atrial hiPSC‐CMs, showing (from left to right) beat rate, corrected field potential duration (FPDc), and conduction velocity. (C) Corresponding temporal measurements for ventricular hiPSC‐CMs. Data are presented as mean ± SD (N = 5). Statistical significance is indicated as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. BL, baseline.

Table S1: Time‐dependent effects of stimulant treatments on electrophysiological parameters in atrial hiPSC‐CMs (mean ± SD).

Table S2: Time‐dependent effects of stimulant treatments on electrophysiological parameters in ventricular hiPSC‐CMs (mean ± SD).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.