Kinetic microplate reader

Compounds 1–5 were assayed (at concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, and 40 μg/mL) against a human keratinocyte (HaCaT) cell line (Catalogue Number: T0020001, AddexBio, San Diego, CA, USA) to determine the potential toxic effect to mammalian skin cells in vitro as described before [18 (link)]. Briefly, cells were cultured in DMEM supplemented with 10% FBS at 37°C with CO₂ (5%). Control cells received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells were incubated with the compounds (in triplicate) in a 96-well plate at 37°C with CO₂ (5%) for two hours prior to addition of the assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA). Absorbance readings (at OD₄₉₀) were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The quantity of viable cells after treatment with each compound was expressed as a percentage of the viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity data was analyzed via a one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).

Myofibrillar ATPase activity was determined by measuring inorganic phosphate release^{40 (link)}. Myofibrillar proteins were isolated and purified, as previously reported^{41 (link),42 (link)}, and suspended in a F60 buffer with 500 mM NaCl. These proteins were extracted from week 1 post-MI and sham rat heart samples, as well as heart samples from cMyBP-C transgenic mice (NTG, AAA, and DDD). Protein was then determined by Coomassie blue staining (Bio-Rad Laboratories, Hercules, CA) of SDS-PAGE gels (Supplementary Fig. S10). The reaction mixture contained 0.25–0.5 mg/mL myofibrillar proteins, 15 mM Tris–HCl, 10 mM KCl, 2 mM MgCl₂, 0.5 mM EGTA, and 5 mM ATP (pH 7.0). Assays were performed in a 96-well microtiter plate at pCa²⁺ concentrations (pCa) from 8.0 to 2.0 at 37 °C. The 120-µL reaction mixture was incubated at 37 °C for 15 min and centrifuged at 1500 rpm for 3 min. Each 30 µL of supernatant was transferred into a new 96-well microtiter plate and mixed with 170 µL distilled water (dH₂O). A 30 µL detection reagent (Phosphate Assay Kit, Abcam) was added to create a reaction to develop color after which the production of inorganic phosphate was determined calorimetrically at 625 nm using a kinetic microplate reader (Molecular Devices). Calcium–stimulated ATPase activity was calculated by subtracting the activity at pCa 2.0 from the activity at pCa 8.0.