Mitochondrial assay

Mitochondria isolation from rat hearts or cardiomyocytes was performed according to the methods as previously reported [16, 21]. In briefly, myocardial tissue (30 mg) from the apical of fresh harvested rat heart was minced and homogenized by a motor-driven homogenizer in the isolation buffer containing 250 mM sucrose, 0.1 mM KCL, 1 mM Na₂EDTA, and 0.1 mM Tris-HCl with pH 7.4. The homogenate was centrifuged at 800×g for 10 min under 4 °C, and the supernatant was centrifuged again at 10,000×g for 10 min under 4 °C. The pellet was washed with isolation buffer and gently resuspended in buffer (30 μl) containing 0.1 mM KCL and 0.1 mM Tris-HCl for mitochondrial assays. Cultured cardiomyocytes in each well of 6-well plates were respectively homogenized mechanically after digestion with trypsin and suspended in 0.5 ml ice-cold hypotonic buffer containing 10 mM HEPES, 10 mM MgCl₂ and 42 mM KCl. The supernatant was centrifuged at 800×g for 5 min under 4 °C, and the resultant supernatant was centrifuged again at 15,000×g for 15 min under 4 °C. The mitochondrial pellet was resuspended in buffer (30 μl) containing 0.25 mM sucrose and 10 mM Tris with pH 7.0 for mitochondrial assays.

Mitochondrial biogenesis was evaluated by mtDNA content, which was mirrored by the copy number ratio of mitochondrial genes like cytochrome C oxidase subunit I (COX I) to the nuclear genes such as β-actin indirectly [21]. Briefly, the total genomic DNA of heart tissue or cells was extracted with proteinase K digestion followed by phenol-chloroform extraction. PCR amplification was carried out using the extracted total genomic DNA as templates and specific primers of COX I and β-actin genes (Table 1) at the reaction conditions of PCR (Table 2).

PCR primer sequences

Sense: 5′-GCCTAGATGTAGACACCCGAGCC-3′

Anti-sense: 5′-CGACG AGGTATCCCTGCTAATCC-3’

Sense: 5’-TAAAGACCTCTATGCCAACACAGT-3′

Anti-sense: 5′-CACGATGGAGGGGCCGGACTCATC-3’

Sense: 5’-AAGGTCCCCAGGCAGTAGAT-3′

Anti-sense: 5′-GCGGTCTCTCAGTTCTGTCC-3’

Sense: 5’-GAGCACTGTCGAAGCCTACA-3′

Anti-sense: 5′-GAGGTCGTCTGTCATGAGGT-3’

Sense: 5’-CTACCCACGGCAAGTTCAAC-3′

Anti-sense: 5′-CCAGTAGACTCCACGACATAC-3’

PCR reaction conditions

The content of mtATP was measured to reflect mitochondrial function according to the manufacturer’s instructions of ATP-dependent bioluminescence assay kit. The mixture of 30 μl of mitochondrial extract solution and 300 μl of lysis buffer was centrifuged at 12000×g for 10 min under 4 °C.The resulting supernatant (100 μl) was used for the analysis of ATP content, which was normalized to the protein content of the mitochondrial extract solution [21].