M1254

The apex of the heart was homogenized using a glass dounce homogenizer at 4°C for 3 minutes in isolation buffer (200 mM Mannitol [M9647; Sigma], 60 mM sucrose [S7903; Sigma], 5 mM KH₂PO₄ [P5379; Sigma], 5 mM MOPS [M1254; Sigma], 1 mM EGTA [E4378; Sigma], and 3 mg of Nagarse protease [P8038; Sigma]). After 3 minutes, isolation buffer with 0.1% w/w BSA (A6003; Sigma) was added and the homogenate was centrifuged at 8000 × g for 10 min at 4°C. The pellet was resuspended in isolation buffer with BSA and centrifuged at 8000 × g for 10 min at 4°C. The resulting pellet was resuspended in isolation buffer with BSA and centrifuged at 700 × g for 10 min at 4°C. The supernatant was collected and centrifuged at 8000 × g for 10 min. The pellet was resuspended in isolation buffer with BSA and mitochondrial concentration was measured using a citrate synthase (CS) assay as previously published by Oroboros.³¹ Briefly, purified mitochondria were placed in a solution containing 100 mM Tris pH 8.1 (T1503, Sigma), 0.25% triton X-100 (T8532, Sigma), 0.31 mM acetyl CoA (A2181; Sigma), 0.1 mM DTNB (D218200, Sigma), and 0.5 mM oxaloacetate (04126, Sigma) at 30°C. Immediately, absorbance was measured at a wavelength of 412 nm.

Mitochondrial respiration was measured from purified mitochondria using a high resolution Oxygraph O2k (Oroboros Instruments GmbH; Innsbruck, Austria). Mitochondria were added to chambers containing a respiration buffer (90 mM KCl [P4504; Sigma], 5 mM K₂HPO₄ [P5504; Sigma], 50 mM MOPS [M1254; Sigma], 1 mM EGTA [E4378; Sigma], and 0.1% w/w BSA [A6003; Sigma], pH 7.2). The substrate of choice was added to the chambers (carbohydrate: 5 mM pyruvate [P8574; Sigma], 2 mM malate [M1000; Sigma], and 5 mM NaCl [S9888; Sigma]; fatty acid: 0.010 mM palmitoyl-L-carnitine [PLC; P1645; Sigma], 2 mM malate, and 10 mM NaCl). After mitochondria and substrate were added, 0.5 mM K-ADP (A5285; Sigma) was added to each chamber.

SC medium was prepared with deionized water, Yeast Nitrogen Base without Amino Acids (Sigma-Aldrich; Y0626), d-(+)-glucose (Sigma-Aldrich; G5767), Yeast Synthetic Dropout (−) Leu (Sigma-Aldrich; Y1376), and 2% l-leucine (Sigma-Aldrich; L8000). A 1 M 3-(N-Morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich; M1254) solution, adjusted to pH 7.00 with 10 M NaOH (Sigma-Aldrich; 72068), was added to the medium for a final concentration of 50 mM MOPS. The pH of the medium was adjusted to pH 7.00 with 10 M NaOH (Sigma-Aldrich; 72068). The SC was then filter sterilized with a 0.22 μm filtration system (Corning^®; 431096) and stored at 23°C. After 3 days, the medium was moved to 4°C overnight. The SC was then refiltered into sterile glass bottles (Pyrex) with a 0.22 μm filtration system (Corning; 431096) to remove precipitated crystals. Some bottles were frozen overnight at −20°C, thawed at room temperature, and filter sterilized again to remove further precipitation, and others were stored at 23°C as unfrozen controls.