4.1.1. Cell Viability Assay

Cells were seeded in 96-well plates at 2 × <sup>10</sup><sup>4</sup> cells/well until adherent. Cells were treated with SpD for 24 h and then 10 μL of Cell Counting Kit-8 reagent (CCK-8, Dojindo Molecular Technologies, Kyushu, Japan) was added into each well for 20–30 min at 37 ◦C. The absorbance was read using a SpectraMax microplate reader (Molecular Devices, San Jose, CA, USA) at 450 nm.

## 4.1.2. Intracellular ATP Measurement

The ATP levels that are produced by cardiomyocytes were measured by the Luciferin-Luciferase reaction, according to the manufacturer's instructions (Cayman Chemicals, Ann Arbor, MI, USA). Cells were plated at 2 × 104 cells/well in 96-well plates and incubated with test compounds at 37 ◦<sup>C</sup> and 5% CO2 for 24 h. Cells were lysed using 50 μL of lysis buffer (Triton X100; 0.1%), mixed with 50 μL of ATP measurement solution containing Luciferin-Luciferase, and then incubated at room temperature for 10–15 min. The luminescence was read using a luminometer (SpectraMax M2e; Molecular Devices, Sunnyvale, CA, USA) and expressed as relative light units. The ATP levels of the control and drug-treated samples were compared to ensure that the reading was exclusive to the ATP produced by drug-treated cardiomyocytes.
