Concurrent with the exponential surge in plastic production, plastic pollution has become increasingly pervasive(Lehner et al., 2019). Nanoplastics (NPs) (1nm to 1000nm) with colloidal properties, are an emerging pollutant of concern(Gigault et al., 2021). Recent reports confirm the presence of NPs in food chains and the environment, leading to human exposure through inhalation, ingestion, and dermal contact(Lehner et al., 2019). After exposure, NPs can accumulate in organs, causing a spectrum of toxic effects, including cytotoxicity and genotoxicity in animals and human cells(Meng et al., 2023). Previous studies have reported NP presence in animals led to impaired cardiac contractility, prompting our investigation into their impact on human cardiac health using an in-vitro disease model.
Cardiomyocytes were derived from human embryonic stem cells (hESC-CMs) following an established protocol(Lian et al., 2013). Metabolically restrictive media was used to achieve hESC-CMs maturation mimicking the adult human heart(Correia et al., 2017). Following the experimental timeline in Figure 1a using GFP-tagged NPs, we demonstrated via fluorescence imaging and flow cytometry analysis that 50nm NPs were readily taken up and evenly distributed within cells, while 500nm NPs formed large aggregates (Figure 1b & 1c). Consequently, 50nm NPs were used in subsequent experiments. Exposing CMs to different NP concentrations (10, 20 and 40 µg/ml) over 7 days (Figure 1d), we demonstrated that saturation uptake occurred by day 3 with 20 and 40 µg/ml NPs (Figure 1e). Flow cytometry results (Figure 1f) mirrored this trend, where 10 µg/ml showed a significant increase from 25.0 ± 3.29% to 41 ± 1.06%. In contrast, 20 and 40 µg/ml NPs only saw marginal increases, from 74 ± 2.13% to 81 ± 1.66% and 87.7 ± 0.46% to 92 ± 0.76%, respectively. Consistently, a similar trend was observed for mean fluorescence intensity (Figure 1f).
Next, we examined the specific effects of NPs on hESC-CMs. Quantitative polymerase chain reaction (qPCR) revealed a dose-dependent upregulation of endoplasmic reticulum (ER) stress starting from day 5 onwards (Figure 2a). Consistent with Feng et al., upregulation of catalase (CAT) was observed in cells treated with 20 and 40 µg/ml NPs, suggesting an increase in oxidative stress independent of the superoxide dismutase (SOD)pathway (Figure 2a)(Feng et al., 2022). Complementing the qPCR findings, MitoSOX assay revealed an increase in oxidative stress in a dose-dependent manner on day 5 and 7 (Figure 2b). Apoptosis, commonly observed alongside oxidative stress, examined through cleaved caspase-3 levels, was increased across all timepoints and became significant on day 7 (Figure 2c).
As the ion channels associated with CM contraction rhythms are disrupted by NPs, we explored the electrophysiological health of NP treated hESC-CMs(McCarthy et al., 2011). Using GCaMP6s calcium reporter hESC-CMs, we observed increased irregularities in contraction rhythm (Figure 2d)(Pang et al., 2022). By day 7 of NPs treatment, CMs were visually inconsistent in their contraction rhythm (Figure 2e). Quantification of the contraction rhythm showed significant increase in beat-to-beat and calcium transient duration variability on day 7, while variability in repolarisation was significantly increased by day 5 (Figure 2f). These findings were consistent with prior work on neonatal rat CMs treated with NPs, where Roshanzadeh et al. noted NPs adsorption to CM membranes interfered with Ca2+ transduction and reduced intracellular Ca2+(Roshanzadeh et al., 2021). Overall, the findings indicate that NP exposure predisposes to arrhythmia due to disrupted Ca2+ signalling, resulting in irregularities during CM repolarisation.
Conclusively, our data suggests that 50nm NPs infiltrate and accumulate within hESC-CMs, leading to cytotoxic phenotypes such as ER stress, oxidative stress, and reduced cell viability, contributing to arrhythmia. Given NPs' small size and hydrophobicity, they may impede body clearance mechanisms, resulting in biopersistence, raising concerns about long-term toxic effects(Forest & Pourchez, 2023). Future research can investigate the mechanistic basis to develop preventive measures against NPs' adverse effects on the heart and other organs.