No abstract

Since its inception more than four decades ago, the Mitochondrial Free Radical Theory of Aging (MFRTA) has served as a touchstone for research into the biology of aging. The MFRTA suggests that oxidative damage to cellular macromolecules caused by reactive oxygen species (ROS) originating from mitochondria accumulates in cells over an animal’s lifespan and eventually leads to the dysfunction and failure that characterizes aging. A central prediction of the theory is that the ability to ameliorate or slow this process should be associated with a slowed rate of aging and thus increased lifespan. A vast pool of data bearing on this idea has now been published. ROS production, ROS neutralization and macromolecule repair have all been extensively studied in the context of longevity. We review experimental evidence from comparisons between naturally long- or short-lived animal species, from calorie restricted animals, and from genetically modified animals and weigh the strength of results supporting the MFRTA. Viewed as a whole, the data accumulated from these studies have too often failed to support the theory. Excellent, well controlled studies from the past decade in particular have isolated ROS as an experimental variable and have shown no relationship between its production or neutralization and aging or longevity. Instead, a role for mitochondrial ROS as intracellular messengers involved in the regulation of some basic cellular processes, such as proliferation, differentiation and death, has emerged. If mitochondrial ROS are involved in the aging process, it seems very likely it will be via highly specific and regulated cellular processes and not through indiscriminate oxidative damage to macromolecules.

Most mammalian tissue cells experience oxygen partial pressures in vivo equivalent to 1–6% O2 (i.e., physioxia). In standard cell culture, however, headspace O2 levels are usually not actively regulated and under these conditions are ~18%. This drives hyperoxia in cell culture media that can affect a wide variety of cellular activities and may compromise the ability of in vitro models to reproduce in vivo biology. Here, we review and discuss some specific O2-consuming organelles and enzymes, including mitochondria, NADPH oxidases, the transplasma membrane redox system, nitric oxide synthases, xanthine oxidase, and monoamine oxidase with respect to their sensitivities to O2 levels. Many of these produce reactive oxygen and/or nitrogen species (ROS/RNS) as either primary end products or byproducts and are acutely sensitive to O2 levels in the range from 1% to 18%. Interestingly, many of them are also transcriptional targets of hypoxia-inducible factors (HIFs) and chronic cell growth at physioxia versus 18% O2 may alter their expression. Aquaporins, which facilitate hydrogen peroxide diffusion into and out of cells, are also regulated by HIFs, indicating that O2 levels may affect intercellular communication via hydrogen peroxide. The O2 sensitivities of these important activities emphasize the importance of maintaining physioxia in culture.

Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.

No abstract

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

No abstract

Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/β-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3β) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5–1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3β (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2–2.5 fold, p < 0.05), a result associated with an increase in total β-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (−86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.