At the time of tissue construct formation, spontaneous and electrically paced twitch forces (10 V, 0.1 s) were measured within a thermostated water bath (37°C) using a high-sensitivity isometric force transducer (MLT0202, ADInstruments, Colorado Springs, CO, USA) connected to a quad bridge amplifier (FE224, ADInstruments). ECG signals were recorded using a Bio Amp (FE136, ADInstruments) as previously described.39 (link) Data were acquired through a 16-channel PowerLab system (PL3516/P, ADInstruments). The ECG of the tissue construct was detected by inserting a needle cathode (MLA1213, ADInstruments) into the center of the construct and a needle anode in one of the four construct corners. The media immersing the tissue was used as a ground. The contractile twitch force was measured by attaching the force transducer arm to one free-corner of the square construct, while the other three ends of the square construct were held fixed by pins. Pre-tension was adjusted using a micro-manipulator (Radnoti LLC, Monrovia, CA, USA), set at 1000–2000 μN, force during spontaneous contraction was recorded. LabChart (ADInstruments) was used for data analysis. Electrical pacing was performed by attaching stimulating electrodes (MLA0320, ADInstruments) to the edge of the tissue and pacing at frequencies of 0.25 and 1–5 Hz (in 1-unit increments).