Abstract 20622: Combined Treatment of Microgroove- Induced Alignment, Triiodothyronine and Electrical Conditioning Promotes Functional Maturation of Human Pluripotent Stem Cell- Derived Cardiomyocytes in Engineered Cardiac Monolayers

Human pluripotent stem cell (hPSC) derived cardiomyocytes (CMs) and their engineered tissues display low anisotropic ratio (AR) and conduction velocity (CV), high spontaneity and poor calcium handling. Non cell autonomous factors such as hormonal and microenvironmental cues have been shown to promote their maturation. However, one cue is often insufficient to promote multiple changes. We hypothesized that combined treatment of microgroove (M), triiodothyronine (T3) and electrical conditioning (EC) exerts synergistic effects to promote functional maturation in engineered human cardiac tissues. hPSC-CMs were seeded onto M or Flat substrates to form human ventricular cardiac monolayers (hvCMLs). T3 was treated continuously and/or EC was introduced 5 days before measurement. hvCMLs w/o T3 and EC were defined as untreated(UN).AR of M-hvCMLs with 8, 10 and 15 μm lines (8M,10M and 15M) were increased (1.21 ± 0.03, n=8; 1.40 ± 0.04, n=7; 1.64 ± 0.06, n=7) when compared to Flat UN (1.02 ± 0.02, n=9), and it further improved in 10M T3-EC group (1.63 ± 0.03, n=6). This change in AR was due to the increase in longitudinal CVs (Flat UN: 4.34 ± 0.19 cm/s, n=9; 10M UN: 5.36 ± 0.38 cm/s, n=7; 10M T3-EC: 8.06 ± 0.45 cm/s, n=6). M-T3-EC treatment raised I Na current density as shown by patch clamping (At -30mV: Flat UN: -9.05 ± 2.55 ρA/ ρF, n=9; 10M T3-EC: -24.09 ± 5.24 ρA/ ρF, n=11). After M-T3-EC treatment, spontaneous AP firing rate of the dissociated cells reduced from 1.33 ± 0.08 Hz (Flat UN, n=22) to 0.78 ± 0.06 Hz (n=24) in whole cell patch clamping. They also showed a reduced I f current density (At -110mV: Flat UN: -4.91 ± 0.34 ρA/ ρF, n= 16; 10M T3-EC: -3.956 ± 0.23 ρA/ ρF, n=21). This was corroborated by a 70% decrease in spontaneous contraction rate in T3-EC hvCMLs with T3 and EC showing an intermediate effect, suggesting synergism between T3 and EC. M-T3-EC treatment reduced the time to peak of calcium transient and time of 50% decay, indicating improvement in calcium handling. qPCR data showed that the functional changes in M-T3-EC treated hvCMLs were associated with the downregulation of HCN4 and upregulation of SCN1B, PLN and ATPA2A. To conclude, combined treatment of M-T3-EC not only induces quiescence, but also promotes a more mature action potential and calcium handling profile in hvCMLs.