Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.04.20.488970

Replicative ageing perturbs the metabolic signature of murine C2C12 skeletal myotubes

Sadler, D. G.·Phelan, M. M.·Barlow, J.·Draijer:, R.·Jones, H.·Thijssen, D. H. J.·Stewart, C. E.
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sadler, D. G., Phelan, M. M., Barlow, J., Draijer:, R., Jones, H., Thijssen, D. H. J., Stewart, C. E.. 2022-04-21. Replicative ageing perturbs the metabolic signature of murine C2C12 skeletal myotubes. https://doi.org/10.1101/2022.04.20.488970

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Contact-phagy remodels ER-mitochondria contact sites

Membrane contact sites between the endoplasmic reticulum (ER) and other organelles coordinate cellular organization, metabolism and stress responses, yet how these interfaces are selectively remodeled and removed remains poorly understood. Here we identify a selective autophagy pathway that targets defined ER-mitochondria contact-site (ERMCS) subdomains for turnover, a process we term Contact-phagy. The ER-resident chaperone SIGMAR1 forms a functional complex with the ER-phagy receptor CCPG1, and SIGMAR1-CCPG1 nanoclusters define ERMCS-associated sites of autophagy initiation. Contact-phagy is selectively engaged by metabolic and mitochondrial stress and promotes turnover of defined contact-site components without broadly affecting general ER-phagy, mitophagy or bulk autophagy. At these sites, SIGMAR1-CCPG1 function is coupled to mitochondrial fission: MFF promotes DRP1 recruitment to ERMCS, whereas loss of CCPG1 or SIGMAR1 causes abnormal persistence and clustering of DRP1 and MFF, accompanied by altered mitochondrial remodeling. Loss of CCPG1 further disrupts ER-mitochondria organization and mitochondrial function in pancreatic tissue, while pancreatic cancer cells depend on CCPG1 and SIGMAR1 for organelle integrity and bioenergetic capacity. Together, our findings establish Contact-phagy as a selective pathway for the turnover of defined organelle contact site subdomains and suggest a broader principle by which interorganelle interfaces are maintained.

cell biology↗
bioRxiv2026

HUH-tag conjugation enables accessible, site-specific functionalization of DNA origami with signaling-competent proteins

DNA origami enables precise nanometer scale control over the number, spacing, and geometry of biomolecules, making it a powerful tool for dissecting spatially organized biological processes such as receptor signaling. However, functionalizing origami with proteins remains a bottleneck restricting the technology to specialized laboratories. Here we show that viral-derived HUH endonuclease tags, small (~10 kDa) domains that form a covalent phosphotyrosine bond with short unmodified ssDNA, provide a simple and cost-effective route to decorating DNA origami with functional proteins. Using interleukin-1{beta} (IL-1{beta}) as a model ligand, we generated recombinant IL-1{beta}-HUH, achieved efficient site-specific conjugation to unmodified ssDNA within minutes, and coupled the conjugates to a DNA origami pegboard with tunable stoichiometry. IL-1{beta}-functionalized origami triggered ligand-dependent signalling in living cells and allowed visualization of the recruitment of downstream signaling molecules, confirming that HUH-tag conjugation preserves functionality. Because this strategy requires only standard molecular biology equipment and unmodified DNA, it lowers a technical and financial barrier to DNA origami-based experimentation, opening the approach to a broader range of biological questions and laboratories outside the DNA nanotechnology field.

cell biology↗
bioRxiv2026

Multivalent BBSome-IFT proximities support IFT-A-IFT-B coordination in vivo

Intraflagellar transport (IFT) depends on coordinated movement of IFT-A and IFT-B, yet how these assemblies remain coupled within dynamic ciliary trains is unclear. Here, we combine endogenous fluorescent knock-in tagging, quantitative in vivo FLIM-FRET, structural modeling, and targeted mutagenesis to define IFT organization in C. elegans and mammalian cells. FLIM-FRET mapping identifies proximity networks within IFT-B and places IFT144 near IFT172 and IFT88 in intact cilia. Loss of BBSome function is associated with weaker IFT-A-IFT-B proximity signals and divergent subcomplex transport dynamics, consistent with a contribution of the BBSome to IFT coordination in vivo. Split-GFP complementation places BBSome components near IFT-A and IFT-B, while structural modeling nominates complementary charged surfaces on the IFT46-IFT52 dimer and BBS7/BBS9. Charge-reversal mutations targeting these surfaces impair BBSome recruitment and functional rescue. Together, these findings support a model in which multivalent BBSome-IFT proximities contribute to IFT-A-IFT-B coordination during ciliary transport.

cell biology↗