Abstract Body: An estimated 24 million people in the US live with cardiac conduction deficits that impair normal electrical signal propagation, yet effective strategies to regenerate conduction pathways remain elusive in both clinical and preclinical settings. To address this, we developed “bioelectric threads,” thin 3D engineered tissues composed of 95% human induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-CMs) and 5% human cardiac fibroblasts (hCFs) extruded in a gelatin-fibrinogen composite hydrogel, to build new conduction paths across poorly conducting regions. Previous work showed our bioelectric threads successfully couple two engineered cardiac tissues in vitro, but conduction velocities (CVs) ~20-fold lower than that of native human myocardium (0.5 m/s) limit their efficacy at physiological heart rates. In this study we use combined uniaxial mechanical conditioning and optimized cell composition to improve tissue alignment and enhance cell-cell junctions. Immunostaining of α-actinin shows that static stretch to 40% strain significantly increases mean sarcomere length from 1.6 to 2.1 μm with enhanced structural alignment along the axis of stretch (SotaTool analysis). Optical mapping with a voltage-sensitive dye increased mean CV by 61% to 43.0 ± 7.5 mm/s and by 81% to 53.8 ± 12.0 mm/s at 1 and 2 weeks, respectively, when compared to unstretched controls (n=5). Based on the promise of static stretch, we have designed, built, and validated a custom, modular bioreactor for conditioning threads using Arduino-driven stepper motors to apply controlled 1 Hz cyclic stretch up to 40% with simultaneous 1 Hz electrical stimulation. To further enhance cell-cell junctions, ongoing work optimizes cell density through comparison of increased total cell input (10, 30, and 50 million cells/mL) versus hCF fraction (5, 10, and 15%) in driving tissue compaction. Improved cell density is expected to both increase gap junction formation and improve mechanotransduction for greater alignment in response to biophysical conditioning. Together, this multi-faceted approach with cell content optimization and biophysical conditioning aims to improve bioelectric thread properties toward clinical translation for conduction regeneration.