Author(s): TAYLOR, D., LUDLOW, A., REISMAN, E., GARNHAM, A., HOFFMAN, N., HAWLEY, J., CARUANA, N., BISHOP, D., Institution: VICTORIA UNIVERSITY, Country: AUSTRALIA, Abstract-ID: 805

INTRODUCTION:
While exercise promotes a wide range of health benefits that contribute to the prevention and management of disease, the underlying molecular mechanisms remain incompletely understood. This is a critical knowledge gap, as understanding how exercise drives health-promoting adaptations is essential for optimising exercise prescriptions and developing novel therapeutic strategies. One underexplored area of investigation is the effect of exercise on alternative gene splicing. Although many studies have quantified exercise-induced changes in total gene expression, most overlook that exercise may also alter mRNA splicing and the relative abundance of individual isoforms, with potential consequences for downstream protein translation. To address this gap, we investigated alternative splicing by quantifying changes in exon expression, splicing events, and isoform usage, and by comparing these with gene-level differential expression across the most comprehensive post-exercise biopsy time course in humans to date.
METHODS:
RNA sequencing (~24 M 100 bp paired-end reads) was performed on muscle biopsies collected before, during, and 0, 3, 6, 9, 12, 24, and 48 h after a single session of high-intensity interval exercise in 20 men (27.3 ± 6.3 y; 179.6 ± 10.2 cm; 81.5 ± 12.5 kg) and 20 women (27.6 ± 5.6 y; 166.5 ± 7.8 cm; 68.0 ± 9.9 kg) who were healthy, untrained, and matched for cardiorespiratory fitness. Isoform-specific changes in genes that were alternatively spliced but not differentially expressed at the gene level were validated by quantitative PCR.
RESULTS:
Using our novel three-tiered analysis approach, we show that exercise induces extensive alternative splicing in a time-dependent manner. Alternative splicing increased steadily up to 6 h post-exercise, as reflected by differential exon usage (94,214 exons across 4,349 genes), differential splicing events (12,249 events across 3,536 genes), and isoform switching (4,765 isoforms across 651 genes), followed by a decline at 9, 12, and 24 h and a subsequent rise at 48 h. This pattern suggests that alternative splicing is a key mechanism underlying both the early and later adaptive responses to exercise. Genes with differential exon usage and splicing events were enriched in pathways related to autophagy, translation, oxidative phosphorylation, cytoskeletal organisation, and even RNA splicing itself. Notably, several genes underwent consequential isoform switches predicted to alter protein function or translation within each pathway, with the expression of specific isoforms changing even when total gene expression remained unchanged at specific time points.
CONCLUSION:
Collectively, these data reveal additional complexity and new layers of molecular regulation in the adaptive response to exercise, highlighting that reliance on total gene expression alone may obscure changes in isoform-specific expression, downstream protein translation, and, consequently, skeletal muscle adaptation.