Background:

Performing unaccustomed high-intensity resistance exercise (REx) causes acute skeletal muscle (SkM) damage, which must be repaired to enable further training and muscle adaptation. Autophagy, a vital cellular quality-control process, facilitates rodent SkM repair by recycling damaged proteins and organelles via autophagolysosomal vesicles (Call and Nichenko, 2020). Whether this occurs in human SkM remains unclear as previous studies rely on static markers of autophagosomes (i.e. LC3-II/I ratio), which do not reflect autophagic flux (Acheson et al., 2025). Intriguingly, some nutraceutical compounds observed to accelerate human SkM recovery (Tanabe et al., 2015) also exert pro-autophagic effects in vitro (Zhang et al., 2016); however, it is unknown if this is the underpinning mechanism in vivo.

Aims:

We utilised a novel ex vivo autophagic flux assay to investigate whether damaging REx alters human SkM autophagic flux and if nutraceutical supplementation augments autophagy to aid recovery.

Methods:

Twenty young healthy untrained adults were randomised to consume a daily nutraceutical supplement containing curcumin, quercetin, rosehip, piperine, and spermidine (n=10), or a placebo (n=10) for 3-weeks before and 1-week after performing 5 sets of leg extensions and leg presses to failure (80%1RM). Markers of SkM damage, including lower body strength and creatine kinase (CK) in venous blood samples, were measured before, immediately (0h), 24h, 72h, and 1-week post-REx. Vastus Lateralis biopsies were donated before, 24h, and 72h. Tissue was immediately snap frozen or incubated in control or lysosomal inhibition (NH4Cl, leupeptin) medium for 1h to assess autophagic flux ex vivo. Autophagy-related proteins and myosin-heavy chain (MyHC) fragments were assessed in sarcoplasmic and myofibrillar SkM fractions via western blots, respectively. LC3-II autophagic flux was calculated as inhibited minus control conditions. Serum CK activity was analysed using an N-acetylcysteine-activated photometric assay. Data were analysed using a mixed-design ANOVA with Holm-Bonferroni corrected post-hoc tests if main or interaction effects were observed (p<0.05).

Results:

At 0h, 24h, and 72h post-REx, quadriceps pain increased whilst leg extensor peak isometric torque and jump height were attenuated (all p<0.05). Serum CK was elevated at all post-REx timepoints (all p<0.01). LC3-II flux was elevated at 72h (p=0.018) whilst traditional ‘static’ markers of autophagic flux (LC3-II/I ratio) remained unchanged. Cytosolic p62 content was increased at 24h and 72h (both p<0.01), suggesting increased ubiquitinated autophagic cargo alongside increased autophagic flux. Furthermore, peak MyHC fragmentation, a novel marker of contractile protein damage, was elevated 76% from baseline (p=0.009). Autophagy signalling proteins, including phosphorylated/total ULK1, Beclin-1, and ATG4 b, remained unchanged throughout, whereas the autophagy repressor BCL-2 was reduced at 72h vs 24h (p=0.012). Intriguingly, lysosomal markers LAMP-2 and v-ATPase B1/2 were reduced at 72h vs 24h (p<0.05), indicating that lysosome content was attenuated during elevated flux. Nutraceutical supplementation did not affect recovery or autophagy outcomes.

Conclusion:

Here, we show that unaccustomed REx increases human SkM autophagic flux, an observation not reflected in commonly utilised ‘static’ measures of autophagy. Furthermore, we posit that lysosome abundance could form an autophagy ‘bottleneck’ previously observed in mice (Call and Nichenko, 2020). Future studies should utilise ex vivo flux assays to understand how exercise, health, and disease modulate human SkM autophagy.