Inhibition of CERS1 in skeletal muscle exacerbates age-related muscle dysfunction

Reviewer #1 (Public Review):

Summary: The authors identified that genetically and pharmacological inhibition of CERS1, an enzyme implicated in ceramides biosynthesis worsen muscle fibrosis and inflammation during aging.

Strengths: the study points out an interesting issue on excluding CERS1 inhibition as a therapeutic strategy for sarcopenia. Overall, the article it's well written and clear.

Weaknesses: Many of the experiments confirmed previous published data, which also show a decline of CERS1 in ageing and the generation and characterization of a muscle specific knockout mouse line. The mechanistic insights of how the increased amount of long ceramides (cer c24) and the decreased of shorter ones (cer c18) might influence muscle mass, force production, fibrosis and inflammation in aged mice have not been addressed.

Reviewer #2 (Public Review):

Summary:

The manuscript by Wohlwend et al. investigates the implications of inhibiting ceramide synthase Cers1 on skeletal muscle function during aging. The authors propose a role for Cers1 in muscle myogenesis and aging sarcopenia. Both pharmacological and AAV-driven genetic inhibition of Cers1 in 18-month-old mice lead to reduced C18 ceramides in skeletal muscle, exacerbating age-dependent features such as muscle atrophy, fibrosis, and center-nucleated fibers. Similarly, inhibition of the Cers1 orthologue in C. elegans reduces motility and causes alterations in muscle morphology.

Strengths:

The study is well-designed, carefully executed, and provides highly informative and novel findings that are relevant to the field.

Weaknesses:

The following points should be addressed to support the conclusions of the manuscript.

1. It would be essential to investigate whether P053 treatment of young mice induces age-dependent features besides muscle loss, such as muscle fibrosis or regeneration. This would help determine whether the exacerbation of age-dependent features solely depends on Cers1 inhibition or is associated with other factors related to age-dependent decline in cell function. Additionally, considering the reported role of Cers1 in whole-body adiposity, it is necessary to present data on mice body weight and fat mass in P053-treated aged-mice.

2. As grip and exercise performance tests evaluate muscle function across several muscles, it is not evident how intramuscular AAV-mediated Cers1 inhibition solely in the gastrocnemius muscle can have a systemic effect or impact different muscles. This point requires clarification.

3. To further substantiate the role of Cers1 in myogenesis, it would be crucial to investigate the consequences of Cers1 inhibition under conditions of muscle damage, such as cardiotoxin treatment or eccentric exercise.

4. It would be informative to determine whether the muscle defects are primarily dependent on the reduction of C18-ceramides or the compensatory increase of C24-ceramides or C24-dihydroceramides.

5. Previous studies from the research group (PMID 37118545) have shown that inhibiting the de novo sphingolipid pathway by blocking SPLC1-3 with myriocin counteracts muscle loss and that C18-ceramides increase during aging. In light of the current findings, certain issues need clarification and discussion. For instance, how would myriocin treatment, which reduces Cers1 activity because of the upstream inhibition of the pathway, have a positive effect on muscle? Additionally, it is essential to explain the association between the reduction of Cers1 gene expression with aging (Fig. 1B) and the age-dependent increase in C18-ceramides (PMID 37118545).

Addressing these points will strengthen the manuscript's conclusions and provide a more comprehensive understanding of the role of Cers1 in skeletal muscle function during aging.