Salvianolic acid A protects H9C2 cardiomyocytes against hypoxia/reoxygenation injuryby regulating VDAC1

OBJECTIVE To investigate the role of voltage-dependent anion channel 1(VDAC1) in the protective effects of Salvianolic acid A(SAA) on H9C2 cardiomyocytes subjected to hypoxia/reoxygenation(H/R) injury.METHODS H9C2 cardiomyocytes were subjected to 4 h of hypoxia(1% O_2, 94% N_2, 5% CO_2, 37℃) followed by 4 h of reoxygenation(95% air, 5% CO_2, 37℃). SAA(1 umol·L~(-1))was administrated at the same time of reoxygenation. To examine whether the expression of VDAC1 is regulated by SAA, H9C2 cardiomyocytes were respectively transfected with VDAC1 si RNA and VDAC1 over expression plasmid. The viability of H9C2 cardiomyocytes was detected by Cell Counting kit-8 assay and further confirmed by detecting the level of intracel ular lactate dehydrogenase.Apoptosis were evaluated using an Annexin Ⅴ-FITC/propidiumiodide staining kit detected by flow cytometry.The level of mitochondrial ROS was measured using the fluorescent probe Mito SOX. The mitochondrial Ca~(2+) signal was monitored with a FRET-based Ca~(2+) indicator. Western blotting and q RT-PCR analysis were used to observe the protein and m RNA levels of VDAC1, and we use luciferase reporter assay to further explore whether SAA has a potential binding sites on VDAC1 promoter to perform the direct regulation on VDAC1. The oligomeric state of VDAC1 was explored by BRET2 assay. RESULTS H/R injury significantly increased the expression of VDAC1, and induced VDAC1 oligomerization. VDAC1 silencing could increase the viability of H/R-treated cardiomyocytes, and attenuate apoptosis as well as the increase on the level of mitochondrial ROS and Ca~(2+) uptake against H/R injury,which performed the same protective effects with SAA.Meanwhile, VDAC1 overexpression could aggravate cell injury. Taken together, VDAC1 was involved in the mechanisms of H/R-induced cell injury. SAA could significantly inhibit the expression of VDAC1 compared with H/R group, we also found that SAA exhibited a direct regulative effect in 3′-UTR of VDAC1 gene. Moreover, SAA could inhibit the oligomerization of VDAC1. Al these protective effects of SAA were abolished in H9C2 cardiomyocytes transfected with VDAC1 overexpression plasmid.CONCLUSION Our findings indicate that SAA can inhibit apoptosis and improve mitochondrial function by regulating the expression and oligomerization of VDAC1, which revealed VDAC1 as a novel potential target and mechanism for the cardioprotective effects of SAA.