Cardiac Calcium Dynamics and Contractility Assay

Sarcometic contractions and calcium transients were measured as described previously [41 (link)]. The cardiomyocytes from the left ventricle (n = 16–23 in each group) were incubated with 2-μmol/L-Fura-2-AM Tyrode solution for 20 min and then washed with Tyrode solution repeatedly. The sarcomeric contractions and calcium transients were recorded in the Tyrode solution (control), 0.1-μmol/L-NPW Tyrode solution, and 0.1-μmol/L-NPB solution at 37 °C using the Ionoptix HyperSwitch Myocyte Calcium and Contractility System (IonOptix LLC, Westwood, CA, USA). The composition of the Tyrode solution was as follows (in mmol/L): NaCl 137, KCl 4.5, MgCl₂ 1, CaCl₂ 2, glucose 10, HEPES 5, and pH adjusted to 7.4 with NaOH. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). During the measurement, the cells were stimulated with a field stimulator (MyoPacer Field Stimulator, IonOptix LLC, Westwood, CA, USA) at a frequency of 1 Hz. The offline analysis was performed with IonWizard 6.5 software (IonOptix LLC, Westwood, CA, USA).