Jones, S. W.; Hasoon, M.; Adair, K.; Shigdar, S.; Hemmings, K.; Henstock, J.; Brownridge, P.; McArdle, C.; Neri, G.; Blackler, W.; Olentsenko, G.; Jones, A. R.; Eyers, C.; Hoettges, K.; Jackson, M. J.; McArdle, A.

Age-related loss of skeletal muscle mass and function, or sarcopenia, presents a growing clinical challenge, mirroring the accelerated muscle atrophy seen in microgravity. This study, part of the UK Space Agency's MicroAge Mission, aimed to investigate microgravity-induced proteomic changes in 3D human skeletal muscle constructs and assess whether mitochondrial Heat Shock Protein 10 (HSP10) overexpression could modulate these responses. Constructs derived from control human AB1167 myoblasts and AB1167 myoblasts that were transduced to overexpress HSP10, were flown to the International Space Station (ISS), with a ground reference experiment (GRE) conducted post-flight. Proteomic analysis using mass spectrometry and bioinformatics revealed significant alterations in metabolic, structural, and mitochondrial protein profiles after microgravity exposure. Microgravity caused downregulation of key proteins involved in energy metabolism, stress responses and structural integrity, while upregulating catabolic and apoptotic enzymes. Many of these modifications parallel previously reported changes in protein composition of muscle with ageing on earth. Overexpression of HSP10 attenuated the effects of microgravity, with fewer proteins showing significant changes and reduced disruption to mitochondrial and cytoskeletal components. Pathway analysis indicated that HSP10 overexpression preserved mitochondrial protein expression, particularly in the matrix, and promoted mitochondrial gene expression and translation under microgravity conditions. Notably, 284 proteins altered by microgravity in unmodified muscle constructs remained stable in HSP10-overexpressing constructs, suggesting a protective effect. MitoCarta 3.0 analysis confirmed that HSP10 expression modulated protein responses at the mitochondrial level, mitigating declines in bioenergetic proteins that are typically associated with microgravity. Collectively, the findings demonstrate that microgravity induces extensive proteomic remodelling in human muscle, which is partially offset by HSP10 overexpression. These results offer insights into muscle atrophy in spaceflight and suggest that targeting mitochondrial stress pathways via chaperone modulation may be a viable strategy to combat sarcopenia and disuse-induced muscle loss on Earth and in space.