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This study examined the effects of IGF-1 and the role of miRNA in glucose-induced mitochondrial dysfunction, the release of cytochrome-c and apoptosis in rat cardiomyocyte cell line H9C2.
Materials and methods
Cellcultureandreagents. The rat cardiomyocyte cell line H9C2 was purchased from ATCC (Manassas, VA), and cultured in DMEM. The cultures were supplemental with 10% fetal bovine serum and 100 μg/ml penicillin/streptomycin. IGF-1 was obtained from GroPep (Australia), and Scrambled 10Panx was obtained from Sigma.
QuantificationoffragmentedDNAincelldeathbyELISA. DNA fragmentation in cell death was analyzed using a cell death detection ELISA kit (Roche) according to the manufacturer’s instructions. To determine the effect of glucose on apoptosis, cells were exposed to different concentrations of glucose (5, 10, and 25 mM) for different time periods (24, 48, 72 and 96 h). After glucose exposure, cells were lysed in 100 μl of lysis buffer and centrifuged for 10 min at 1500 rpm. Triplicate 20 μl samples of supernatant were placed into the streptavidin-coated microtest plates for analysis. DNA fragmentation was quantified by measuring absorbance at 405 nm with a reference wavelength at 492 nm.