Importance of Spatial Arrangement of Cardiomyocyte Network for Precise and Stable On-Chip Predictive Cardiotoxicity Measurement.

One of the advantages of human stem cell-derived cell-based preclinical screening is the reduction of the false negative/positive misjudgment of lead compounds for predicting their effectiveness and risks during the early stage of development. However, as the community effect of cells was neglected in the conventional single cell-based in vitro screening, the potential difference in results caused by the cell number and their spatial arrangement differences has not yet been sufficiently evaluated. Here, we have investigated the effect of the community size and spatial arrangement difference for cardiomyocyte network response against the proarrhythmic compounds from the viewpoint of in vitro cardiotoxicity. Using three different typical types of cell networks of cardiomyocytes, small cluster, large square sheet, and large closed-loop sheet were formed in shaped agarose microchambers fabricated on a multielectrode array chip simultaneously, and their responses were compared against the proarrhythmic compound, E-4031. The interspike intervals (ISIs) in large square sheets and closed-loop sheets were durable and maintained stable against E-4031 even at a high dose of 100 nM. In contrast, those in the small cluster, which fluctuated even without E-4031, acquired stable beating reflecting the antiarrhythmic efficacy of E-4031 from a 10 nM medium dose administration. The repolarization index, field potential duration (FPD), was prolonged in closed-loop sheets with 10 nM E-4031, even though small clusters and large sheets remained normal at this concentration. Moreover, FPDs of large sheets were the most durable against E-4031 among the three geometries of cardiomyocyte networks. The results showed the apparent spatial arrangement dependence on the stability of their interspike intervals, and FPD prolongation, indicating the importance of the geometry control of cell networks for representing the appropriate response of cardiomyocytes against the adequate amount of compounds for in vitro ion channel measurement.

Tetrabromobisphenol A and hexabromocyclododecane, brominated flame retardants, trigger endoplasmic reticulum stress and activate necroptosis signaling in PC12 cells.

Tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCD) are brominated flame retardants commonly used in a variety of industrial and consumer products. In this study, we performed RNA sequencing analysis of PC12 cells to clarify the mechanisms by which TBBPA and HBCD induce neurotoxicity. Differential expression analysis demonstrated that 636 and 271 genes were differentially expressed after TBBPA and HBCD treatment, respectively. Gene Ontology (GO) enrichment analysis revealed that genes annotated with the GO term "endoplasmic reticulum unfolded protein response" were upregulated in both TBBPA- and HBCD-treated groups. Furthermore, protein expression of endoplasmic reticulum stress markers, such as HSPA5 and DDIT3, as well as cleaved caspase-3, an apoptosis marker, were induced by TBBPA and HBCD. We also found that the cytotoxicity induced by TBBPA and HBCD was blocked by necrostatin-1, a necroptosis inhibitor, indicating the contribution of necroptosis. Our findings provide new insight into the mechanisms of toxicity induced by these chemicals.