Abstract 134: Electrical Stimulation Improves Calcium Handling in Engineered Heart Tissue

Abstract

Purpose: Despite considerable advances in cardiac tissue engineering, morphology and functionality of engineered heart tissue (EHT) needs to be further improved, likely by making use of more “physiologic” engineering paradigms. We hypothesized that electrical stimulation may improve calcium handling in EHTs through enhanced maturation of the excitation coupling apparatus.

Methods: EHTs were prepared from neonatal rat ventricular heart cells and after one week, transferred to dynamic stretchers. EHTs were cultured with (2 or 4 Hz) or without (non-stimulated ‘NS’) electrical stimulation from day 7 to 13 (Ionoptix C-Pacer: 2 ms square biphasic pulse, 4 Volts). At day 13, functional (twitch tension at 2 Hz field stimulation) were performed, and EHTs were processed for Western Blot analysis. In addition, EHT-derived single cardiomyocytes loaded with fura-2 (1 µM) were analyzed using an Ionoptix setup to assess alterations in calcium handling.

Results: Culture under electrical enhanced twitch tension of EHTs (2 Hz vs. NS: 0.83 ± 0.05 vs. 0.62 ± 0.03, p<0.0001, n=8/group), lowered excitation thresholds, and increased upstroke velocity. Assessment of sarcoplasmic reticulum (SR) functionality by post-rest potentiation revealed improved calcium storage capacity in EHTs (NS vs. 2 Hz vs. 4 Hz: 10.2 ± 1.4 vs. 11.7 ± 1.4 vs. 17.8 ± 1.3 % at 30 s rest; p<0.05, n=7/7/5). Ryanodine (1 µM, to block SR-calcium release) reduced EHT twitch tension to 88.3 ± 2.1 % [NS] vs. 87.2 ± 2.4 % [2 Hz] vs. 75.1 ± 4.5 % [4 Hz] (n=8/group, p<0.05) baseline force, indicating greater reliance on the SR for force generation, particularly in the 4 Hz stimulated group. Ryanodine receptor and SERCA protein expression increased with 4 Hz stimulation. In isolated cardiomyocytes, 4 Hz stimulated cells displayed faster upstroke velocity and relaxation times, approaching that of cardiomyocytes isolated from day 13 postnatal rats.

Conclusions: Electrical stimulation of dynamically loaded EHTs contributes to increased functionality and appears to improve excitation-contraction coupling through enhanced Calcium storage and release capacity. This combined biomimetic stimulation method may overcome the sub-physiologic SR functionality of classical cell culture models.