Gene therapy-based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle has been reported to generate potent short-term pacemaker function in various animal models. Here, we investigated the impact of adeno-associated virus (AAV)-mediated long-term expression of TBX18, and compared the outcomes to those of the pacemaker ion channel Hcn2. Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, non-fibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete AV-block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activity nor augmented Hcn2-mediated pacing. In conclusion, at functionally non-fibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development.

Although flavonoids from Scutellaria baicalensis are well known for their antioxidant properties, the active constituents and biological effects of its congeneric species, Scutellaria indica L. (S. indica), remain unclear. In this study, we investigated the antioxidant functions and cytoprotective effects of 5,2′,5′-trihydroxy-7,8-dimethoxyflavanone (TDF), a flavanone constituent of S. indica, against oxidative stress. Among six Scutellaria-derived compounds screened, TDF produced the highest antioxidant response element (ARE)-reporter activity in HepG2 cells, and it upregulated the expression of Nrf2 target genes in HaCaT cells. In an H₂O₂-induced oxidative stress model, TDF suppressed ROS and reversed the H₂O₂-induced reduction in total Nrf2 protein. Additionally, TDF showed SOD-like activity and modest elastase-inhibitory activity in cell-free assays and was classified as a non-irritant in a 3D-reconstructed human epidermis model. Overall, these results indicate that TDF is a bioactive constituent of S. indica that protects keratinocytes against oxidative stress, and that this protective effect is associated with the regulation of the Nrf2/ARE pathway.