Respiration, coupling and ROS in senescent human fibroblasts





Eveline Huetter, Kathrin Renner, Erich Gnaiger, Gerald Pfister and Pidder Jansen Duerr

Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck/Austria



Human cells in primary culture have a finite lifespan, a phenomenon termed "replicative senescence". After about 50 population doublings, cells stop proliferation and arrest irreversibly in the G1 phase of the cell cycle. Cellular energy metabolism is an important aspect of aging, as shown by life span extension through caloric restriction. Analysis of the glycolytic pathway in young and old cells revealed age-associated changes in the activity of several enzymes. Staining cells with the oxidant-sensitive dye dihydrorhodamine 123 showed that senescent fibroblasts exhibit oxidative stress, a possible consequence of metabolic imbalance. Based on these results, we wanted to know whether mitochondrial function is impaired in senescent cells. Mitochondrial respiratory function was analyzed by high resolution respirometry with the OROBOROS Oxygraph. The experimental regime started with routine respiration, followed by inhibition of ATP synthase with oligomycin, and uncoupling by stepwise titration of FCCP. Finally, respiration was inhibited by sequential addition of rotenone and antimycin A [1]. Respiration per cell was highly increased in old fibroblasts, owing to increased mitochondrial content (citrate synthase activity) in line with an increase in cell size. The capacity of the respiratory chain, reflected by uncoupled respiration per citrate synthase, is unchanged in old and young fibroblasts. Oligomycin-inhibited respiration, however, was significantly increased in senescent cells. Further, senescent cells exhibit a slightly decreased uncoupling control ratio, and a decreased ratio between uncoupled respiration and oligomycin-inhibited respiration. This indicates a lower coupling state of mitochondria in senescent fibroblasts.

[1] Huetter E, Renner K, Jansen-Duerr P, Gnaiger E (2002) Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption. Molec. Biol. Rep. 29: 83-87.



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