Poster U1 - Electromechanical Evaluation of the Pulsatile Properties of Cardiac Spheroid under Contact Deformation

Introduction: Myocardial sheets and cardiac spheroids (CSs), cultured myocardial tissue, have been widely investigated for myocardial regeneration for severe heart failure. One of the problems that may arise during transplantation is the development of arrhythmias due to interactions between tissues in different beating states. Therefore, it is extremely important to understand the electrophysiological and mechanical beating properties of CSs in order to ensure the therapeutic efficacy of transplantation and to prevent arrhythmias. Recently, it has been reported that the beating energy and beating frequency of CSs can be altered by compressive deformation. It is thought that the changes in the "contact area" and the "internal stress and strain distribution" resulting from the three-dimensional structure affect the beating force and the beating cycle of CSs. It is inevitable that contact between CSs and the patient's heart, as well as resulting deformation, will occur during the implantation of CSs. The unpredictable changes in CS beating properties caused by such contact may be one of the factors that lead to the development of arrhythmogenesis. In this study, we analyzed the electromechanical window (EMW), the arrhythmia risk index, by means of mechanical and electrical multimodal measurement of the changes in beating characteristics caused by contact deformation of the CS, and evaluated the effects of contact deformation on the synchronous beating of the CS.

Materials and

Methods: 36,000 cells of iPS cell-derived cardiomyocytes and 12,000 cells of normal adult ventricular fibroblasts were co-cultured in U-bottom 96-well plates to produce CSs with a diameter of approximately 500 µm. The beating force and extracellular field potential of the CSs were simultaneously measured using a laboratory-made multimodal measurement system, as illustrated in Fig. 1. This system consists of two units: a unit for measuring the beating force and a unit for measuring the extracellular filed potential. The beating force measurement unit employs a leaf spring (spring constant: 6.78 N/m) and a displacement sensor, and an extracellular field potential measurement unit employing an electrode probe (Φ0.1, Pt) and a differential amplifier. In this study, extracellular field potential was defined as the potential difference between the culture medium and the CS surface. The CS contacted both the leaf spring and the electrode probe, and the contact state of the CS was controlled by moving the leaf spring in the y-axis direction, coupled to the beating force measurement module. With the above system, the CS was deformed stepwise at compression ratios of 10 and 30% with respect to the initial diameter, and the extracellular field potential and the beating force were simultaneously measured in each contact state.

Results, Conclusions, and Discussions: Results & Disucussions: Figure. 2 shows alternations in the extracellular field potential and the beating force in a CS acquired in the simultaneous series at each contact state. In both contact states, the upward peak of the extracellular field potential (shown in solid line) occurs approximately 0.05 seconds before that of the beating force (shown in broken line). This phenomenon is consistent with the depolarization phase, which is primarily attributed to the influx of Na+ ions. Subsequently, as the beating force increased, the extracellular field potential decreased and exhibited a downward peak, reflecting the influx of Ca2+ ions into the CS. This influx leads to calcium-induced calcium release, enabling actin-myosin cross-bridging. As the beating force began to decrease, the extracellular filed potential continued to decline, reflecting the ongoing repolarization process. The return of the beating force and the extracellular potential indicates the completion of the repolarization phase and the cessation of CS beating. A comparison of the amplitudes of the extracellular field potential and the beating force in the presence of 10% and 30% compression revealed a clear increase in both variables with compression. This suggests that the compression enhanced the influx of ions flowing into the tissue per unit time, which in turn increased the beating force. The electromechanical window (EMW) was evaluated by measuring the time difference between the end of variability in the extracellular potential and that in the beating force in Fig. 2 (Fig. 3). Previous studies have demonstrated that the EMW is shortened when arrhythmogenic agents such as E4031 are administered to cultured cardiomyocytes. The results of this experiment indicate that deformation can affect CS synchrony.

Conclusions: In this study, we developed a system that can simultaneously measure the beating force and extracellular field potential of a CS under contact deformation. The obtained waveforms demonstrate the feasibility of evaluating the effects of contact deformation on the electrical and mechanical pulsatile characteristics of the CS and the synchronization of the beating.

Acknowledgements (Optional): ・This work was supported by JSPS KAKENHI Grant Number 22K19914
・iPS cell line (253G1, HPS0002) was provided by Riken BRC through the National BioResource Project of the Ministry of Education, Culture, Sports,
Science and Technology.