S1. Isolation of myocardial mitochondrial and cytosolic fractions
The mitochondrial and cytosolic fractions were prepared as previously described [12] with minor modification. Left ventricles were removed from the heart and washed in phosphate-buffered saline (PBS) solution, then again in sucrose buffer (in mmol/L: 1.0 EDTA, 10 Tris-HCl [pH 7.4], 250 sucrose) and resuspended in the same buffer. After 1 hr of incubation on ice, tissues were lysed by use of a Dounce homogenizer with 30 strokes. Homogenates were centrifuged at 800 g at 4 ℃ for 10 min and the supernatants were recentrifuged at 1200 g at 4 ℃ for 10 min. After decanting, supernatants were recentrifuged at 10000 g at 4 ℃ for 15 min, and pellets were used as the mitochondrial fraction. Supernatants were further centrifuged at 100000 g for 1 hr at 4 ℃ in an ultracentrifuge, and the final supernatant was used as the soluble cytosolic fraction. Both mitochondrial and cytosolic fractions were then processed for immunoblotting. The functional integrity of isolated mitochondria was identified by the determination of succinate dehydrogenase activity as in our previous study [13].
S2. Trypan blue exclusion test and lactate dehydrogenase (LDH) activity assay
Cellular trypan blue exclusion was performed as described previously with minor modification [14]. Briefly, cardiomyocytes were washed with PBS, trypsinized in 0.25 mg/mL trypsin for 2 min, and then neutralized with fetal calf serum. Cells were centrifuged (1000 rpm for 10 min), the supernatant was aspirated, and myocytes were resuspended in 0.1 mL PBS. An equal volume of 0.4% trypan blue in PBS was added, and the proportion of blue cells/total cells was counted by scoring not less than 250 cells, three times per well, by use of a hemocytometer.
As a reliable marker of myocyte injury, intracellular LDH release into the incubation medium was measured by the oxidation of lactate to pyruvate. Essentially, the reaction mixture contained 30 mL of sodium lactate buffer, 10 mL of 0.5% nicotinamide adenine dinucleotide (NAD), 50 mL of 1 mol/L 2, 4-dinitrophenyl-hydrazine (DNPH), 500 mL of 0.4 mol/L NaOH and 20 mL of supernatant from culture medium. The absorbance of solution at 440 nm wavelength is directly proportional to the LDH activity in the sample. One unit of enzyme activity represents 1 μmol of pyruvate derived from 100 mL of sample within 15 min at 37 ℃.