Anti-senescence role of heterozygous fumarate hydratase gene knockout in rat lung fibroblasts in vitro

Abnormalities in tricarboxylic acid (TCA) cycle function were related to a variety of pathological processes. Fumarate hydratase (FH) is a required enzyme in the TCA cycle. To explore the general influence of FH knockout, we isolated FH +/- rat and normal rat lung fibroblasts and cultured these cells in vitro . The isolated fibroblasts with the current method were rather homogeneous and were confirmed spindle in morphology, positive for vimentin and negative for α-SMA (α-smooth muscle actin). Sequencing of the PCR (polymerase chain reaction) products flanking the FH gene mutation verified the FH +/- status, and the FH gene and protein expression were confirmed to be reduced in the FH +/- cells. No sign of ageing for the FH +/- cells after 61 passages was observed, but the controls died out at this stage. Flow cytometry revealed increased S-phase and decreased G1/G0 proportions with significantly less early apoptosis in FH +/- cells compared to that in control cells. At the same time, increased glucose consumption, intracellular fumarate production and extracellular lactate secretion were verified in the FH +/- cells. Correspondingly, FH +/- cells showed a lower basal oxygen consumption rate (OCR) but a higher level of reactive oxygen species (ROS) production. Single cell cloning and cell line establishment were successfully performed with the FH +/- cells at the 84 th passage. All the above results indicate an important role for FH +/- in the longevity or immortality of the FH +/- cells, in which increased p53 and TERT (telomerase reverse transcriptase) protein expression, decreased p21 and p16 protein expression and negative SA-β-Gal (senescence-associated beta-galactosidase) were verified along with metabolic reprogramming.