Sadler, D. G. | Search bioRxiv

bioRxiv2022

Replicative ageing perturbs the metabolic signature of murine C2C12 skeletal myotubes

IntroductionChronological ageing is associated with mitochondrial dysfunction and increased reactive oxygen species (ROS) production in skeletal muscle. However, the effects of replicative ageing on skeletal muscle cellular metabolism are not well known. Using an established myoblast model of cellular (replicative) ageing, we investigated the impact of ageing on energy metabolism in murine C2C12 myotubes. MethodsControl (P7-11) and replicatively aged (P48-51) C2C12 myoblasts were differentiated over 72-120 h. Mitochondrial bioenergetics were investigated by respirometry and mitochondrial superoxide and cellular ROS were measured in the absence and presence of antimycin A (AA). Genes related to mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Intracellular metabolites were quantified using an untargeted 1H-NMR metabolomics pipeline. ResultsMitochondrial coupling efficiency (Control: 79.5 vs. Aged: 70.3%, P=0.006) and relative oxidative ATP synthesis (Control: 48.6 vs. Aged: 31.7%, P=0.022) were higher in control vs. aged myotubes, but rates of mitochondrial superoxide production were lower (Control: 2.4x10-5 {+/-} 0.4 x 10-5 vs. Aged: 9.7x10-5 {+/-} 1.6x10-5 RFU/sec/cell; P=0.035). Replicatively aged myotubes had greater mRNA expression of mfn2 and Tfam compared to control. Yet, Nrf2 and PGC-1 expression were 2.8-fold and 3.0-fold higher in control versus aged myotubes over 24 h and 48 h (P<0.05), respectively. Branched chain amino acids L-leucine, L-isoleucine and L-valine, and L-carnitine were less abundant in aged versus control myotubes. Conclusion(s)Replicative ageing is associated with bioenergetic uncoupling, increased ROS production and impaired amino acid metabolism. Our findings suggest that cellular mitochondrial dysfunction and altered energy metabolism may exacerbate the age-related decline in skeletal muscle mass and function.

cell biology↗

bioRxiv2021

(--)-Epicatechin alters reactive oxygen and nitrogen species production independent of mitochondrial respiration in human vascular endothelial cells

IntroductionVascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction and altered cell signalling. (-)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPIs effects remain unclear. Objective(s)Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function. MethodsHUVECs were treated with EPI (0-20 {micro}M) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting. ResultsMitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 {micro}M EPI treatment vs. CTRL (P<0.001). With AA, only 10 {micro}M EPI increased mitochondrial superoxide production vs. CTRL (25%, P<0.001). NO bioavailability was increased by 45% with 10 {micro}M EPI vs. CTRL (P=0.010). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 {micro}M EPI over 48 h vs. CTRL (P=0.015 and P=0.001, respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P=0.035 and P=0.011). Conclusion(s)EPI dose dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.

physiology↗