Cell tak reagent

The Seahorse Bioanalyzer (Agilent, Cheadle, UK) was used to measure bioenergetic flux in control and treated samples, to establish whether chemicals influenced this endpoint. Seahorse microplates (Agilent) were coated using CellTak reagent (Corning, UK). Cells pre-treated with the appropriate chemical for 4 or 23 h were transferred to coated microplates (400,000 cells/well) 1 h prior to assay commencement, with gentle centrifugation at 20×g to aid adhesion. Unbuffered Seahorse medium adjusted to pH 7.5 (Agilent) was used. The plate was then transferred to a non-CO₂ incubator for 25 min prior to addition of 425 µl medium and then incubated for a further 35 min to promote equilibration. Following routine calibration of the machine, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured simultaneously using the XF^e24 Seahorse Bioanalyzer to assess basal versus drug-induced perturbations.

Hearts were washed by retrograde perfusion through the aorta using cannulas hooked to 1.0 mL syringes. Individual hearts were washed with 5.0 mL HBSS solution then fixed with 3.0 mL of 4% PFA in PBS during 5 min. The hearts were washed again with 3.0 mL of HBSS and finally rinsed with 1.0 mL of enzyme solution (Collagenase type 2, Worthington; 4 mg/mL and Protease IV, Roche; 1 mg/mL in HBSS) and kept on ice until all hearts have been harvested. The cannulated hearts were then digested 3 times at 37 °C, 15 min each, with a change of digestion solution at each digestion cycle. The digested hearts were cleared of atria and blood vessels, and the ventricles were teased into small pieces with forceps and triturated with a wide-bore pipette. Undigested fragments were left to sediment and the supernatants containing single-cell suspensions were centrifuged at 18× g for 3 min to pellet CMs. For BrdU immunostaining, CM preparations were fixed for 10 min at room temperature in 4% PFA, washed and resuspended in PBS. Aliquots containing approximately 5 × 10⁴ isolated CMs were immobilized on microscope slides using Cell Tak reagent (Corning). After drying, the attached cells were permeabilized with 0.25% Triton X-100 and treated with 2.5 Units of DNase I per slide (New England Biolabs, Ipswich, MA, USA), and stained for BrdU and α-actinin as described for tissue sections.