2.10. ATP assessment

Intracellular ATP levels were measured from harvested LV heart tissue from WT and frataxin KO mice that had been comprehensively perfused with ice-cold PBS. The assessment of ATP levels was performed using a luciferin-luciferase assay system (P1041 and E1500, Promega), as previously described [39]. In brief, tissue was homogenized in 250 μL of extraction solution (10 mmol/L KH₂PO₄, 4 mmol/L MgSO₄, pH 7.4), heated for 98 °C/4 min, and placed on ice. Then, 50 μL of sample was added to a 100 μL reaction solution (50 mmol/L NaAsO₂, 20 mmol/L MgSO₄, pH 7.4, and 800 μg of luciferin/luciferase enzyme). Luminosity at 259 nm was measured with an extinction coefficient of 15,400 mol/L⁻¹.cm⁻¹ using a Turner Designs TD 20/20 luminometer (Stratec Biomedical Systems, Germany) [39]. ATP levels were normalized to protein concentration using the Bradford protein assay and reported as μg/mg protein. Analysis of ATP levels was conducted by an investigator who was blinded to mouse group identities.