Muscle aging in humans and animals is uneven: men lose fast fibers earlier, women after menopause. The molecular causes of these differences remain poorly understood, partly because no short-lived vertebrate model has been available. The African killifish lives 4-6 months yet displays all the hallmarks of mammalian muscle aging: atrophy, loss of stem cells, denervation. Previously, dietary restriction extended lifespan only in males; no data existed for females. The authors filled this gap by developing an intermittent fasting protocol suitable for both sexes and comparing the muscle response at the level of cells, fibers and the transcriptome.
Fish of the wild GRZ line were kept at 27°C, males individually and females in groups of three. Starting at 30-40 days after hatching, some were switched to the IF1 regimen: two days of fasting, one day of feeding. Another regimen, IF2 (4 days of fasting, 3 days of feeding), proved unsuccessful and was abandoned. Swimming performance was measured in a hydrodynamic flume, with flow speed gradually increased by 2 cm/s every minute. Muscle volume was assessed by micro-CT, and fiber number and size from stained sections of the tail musculature. For molecular analysis, muscle tissue was collected and RNA sequencing was performed both on whole tissue (bulk) and on individual nuclei (snRNA-seq). Cell-cell interactions were reconstructed with the CellChat program, mapping the human signaling pathway database onto the killifish genome via orthologs.
The IF1 regimen extended median lifespan by 53% in males and 21% in females. Body length decreased by 25% in males and 29% in females, and mass by 57% and 62% respectively. Fertility fell in both sexes. Despite their smaller size, old fasting fish swam faster than young controls: the gain in speed was 61% in males and 41% in females. The skeletal muscle index (the ratio of muscle volume to body mass) did not explain the improvement: in females it even declined after fasting. Histology showed that in males the number of small fibers increases with age (hyperplasia), while in females the size of large fibers increases (hypertrophy); fasting partially reverses both processes. According to snRNA-seq, in females the proportion of fast glycolytic and oxidative fibers falls with age while the proportion of slow fibers rises; IF1 restores the youthful ratio. In males the fiber composition barely changes. Analysis of intercellular signaling revealed that aging weakens interactions in both sexes, but more strongly in females. Fasting reactivates axon guidance pathways in females (netrin, ephrin, semaphorin) and ANGPTL, linked to lipid and glucose metabolism, in males. Bulk RNA sequencing identified 1573 genes altered by fasting in males and 769 in females. Shared by both sexes were the genes smyd1a/b and mylpfb, important for myofibril organization and contraction, along with activation of ribosome biogenesis.
This work is the first to systematically compare how male and female muscles respond to dietary restriction in a vertebrate. It shows that improved function is not necessarily tied to preserved mass: in females fasting even reduced muscle volume, yet swimming improved. This aligns with clinical data showing that strength and endurance predict health better than muscle size. The sex differences identified in signaling pathways (neuronal in females, metabolic in males) explain why one-size-fits-all diets may work differently. The killifish model allows such hypotheses to be tested quickly thanks to its short life cycle.
The immediate next step is to determine which hormonal or genetic factors drive the sex specificity. The role of sex steroids can be tested by removing gonads or administering hormones. It is also important to understand how fasting affects neuromuscular synapses: the axon guidance pathway data in females hint at restored innervation. Further experiments with knockout of smyd1a/b or mylpfb will show whether they are sufficient to improve function. Studying how the IF1 regimen affects lipid and glucose metabolism in male muscles is a promising avenue.
The results matter for gerontology and sports medicine: they justify the need for sex-specific dietary recommendations against sarcopenia. For pharmacology, they open the possibility of seeking fasting mimetics that act on different pathways in men and women. For the fundamental biology of aging, the killifish is becoming a reference model of sexual dimorphism.
Test whether the effects of IF1 persist under milder regimens (for example, 1 day of fasting every 2). Determine the contribution of sex hormones through gonadectomy and replacement therapy. Investigate the synaptic structure of neuromuscular contacts in old fish after fasting.
In the biology of aging, it remains an open question why females of many species live longer than males yet their muscles degrade differently. Our work shows that the sex-specific trajectories of muscle aging diverge at the level of the transcriptome and intercellular signaling, not merely quantitatively. This links the problem of sexual dimorphism in lifespan to the mechanisms that maintain muscle homeostasis. It is also still a mystery how dietary restriction simultaneously activates anabolic (ribosome biogenesis) and catabolic (autophagy) processes; our data on IGF and ANGPTL offer new leads.
The killifish Nothobranchius furzeri lives in ephemeral pools in Mozambique and Zimbabwe that dry up within a few months. Its eggs can survive complete desiccation in diapause for a year or more. In the laboratory the fish live 4-6 months, making them the shortest-lived vertebrates bred in captivity.
Not published on arXiv. Here are its page, the PDF and the formalization: you can check it yourself, not only read our account.