Intermittent Supplementation With Fisetin Improves Physical Function and Decreases Cellular Senescence in Skeletal Muscle With Aging: A Comparison to Genetic Clearance of Senescent Cells and Synthetic Senolytic Approaches

Abstract

Excess cellular senescence contributes to age-related increases in frailty and reductions in skeletal muscle strength. In the present study, we determined the efficacy of oral intermittent treatment (1 week on-2 weeks off-1 week on) with the natural flavonoid senolytic fisetin to improve frailty and grip strength in old mice. Further, the effects of fisetin on physical function were evaluated in young mice. We performed bulk RNA sequencing of quadricep skeletal muscle to determine the cell senescence-related signaling pathways modulated by fisetin. We also assessed the relative effects of fisetin on frailty and grip strength with aging in comparison with two other well-established approaches for the removal of senescent cells: (1) genetic-based clearance of excess senescent cells in old p16-3MR mice, a model that allows for clearance of p16-positive (p16+) senescent cells, and (2) oral intermittent treatment with the synthetic pharmacological senolytic ABT-263 in old mice. We found that fisetin mitigated the adverse changes in frailty and grip strength with aging. Fisetin had no effects in young mice. The improvements in frailty and grip strength in old mice were accompanied by favorable modulation of the skeletal muscle transcriptome, including lower abundance of cellular senescence-related genes (e.g., Cdkn1a and Ddit4). Improvements in frailty and grip strength with fisetin were comparable to those observed with genetic-based clearance of excess p16+ senescent cells and treatment with ABT-263. Taken together, our findings provide proof-of-concept support for fisetin as a senolytic strategy to improve physical function with aging.

Keywords: flavonoid; motor function; natural senolytic; senescence associated secretory phenotype; skeletal muscle senescence; transcriptome.

FIGURE 1

Oral treatment with fisetin attenuates differences in frailty index score and grip strength with aging in old mice. Experimental design (A). Frailty index score (B) and normalized grip strength (C). Squares = male mice, inverted triangles = female mice. Data presented as mean ± SEM. One‐way ANOVA corrected with Tukey's multiple comparisons test. p < 0.05: *both young groups; ^‡old vehicle. Images made with Biorender.com.

FIGURE 2

Global quadriceps skeletal muscle gene expression is favorably altered with fisetin treatment in old mice. Principal component analysis of skeletal muscle mRNA abundance with aging (A, B). Genes were determined differentially expressed (DEGs) using the parameters of Log2FoldChange > ± 0.5 and p < 0.05 (C, Old Vehicle vs. Young Vehicle; D, Old Fisetin vs. Young Vehicle). This was visualized via volcano plot. DEGs were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) to determine enriched molecular and physiological processes. Only significant KEGG pathways are shown (false discovery rate [FDR] < 0.2), * significant pathway in Young Vehicle vs. Old Vehicle comparison (E, Young Vehicle vs. Old Vehicle; F, Young Vehicle vs. Old Fisetin).

FIGURE 3

Fisetin treatment in old mice attenuates excess cellular senescent‐related gene expression in aged skeletal muscle. Individual values of differentially expressed genes (DEGs) relative to Young Vehicle (A, Old Vehicle vs. Young Vehicle; B, Old Fisetin vs. Young Vehicle); YV = Young Vehicle; OV = Old Vehicle; OF = Old Fisetin. Black boxes indicate the removal of an outlier (Grubbs test, α < 0.05) (A & B). Violin plot of transcript abundance of Cdkn1a (i.e., p21) in skeletal muscle analyzed by an ordinary one‐way ANOVA with Tukey's multiple comparisons test; ***p < 0.001; shown as Log₂ fold change; squares = male mice, inverted triangles = female mice (C). Differences in the relative abundance of genes associated with cellular senescence and related pathways in old mice with and without fisetin treatment compared with Young Vehicle, e.g., gene “X” = [{(Old Fisetin/Young Vehicle)—(Old Vehicle/Young Vehicle)}*100] (D). Individual values of DEGs in old mice treated with fisetin (OF) relative to OV. Black boxes indicate the removal of an outlier (Grubbs test, α < 0.05) (E). Violin plot of transcript abundance of Ddit4 (i.e., DNA damage inducible transcript 4 or Redd1); **p < 0.01; squares = male mice, inverted triangles = female mice (F).

FIGURE 4

Genetic‐based clearance of excess p16+ senescent cells mitigates age‐related changes in frailty index score and grip strength in old mice. Experimental design (A). Frailty index score (B) and normalized grip strength (C). Squares = male mice, inverted triangles = female mice. Data presented as mean ± SEM. One‐way ANOVA corrected with Tukey's multiple comparisons test. p < 0.05: *both young groups; ^‡old vehicle. Images made with Biorender.com.

FIGURE 5

Oral senolytic therapy with the synthetic pharmacological agent ABT‐263 lessens the age‐related differences in frailty index score and grip strength in young and old mice. Experimental design (A). Frailty index score (B) and normalized grip strength (C). Squares = male mice, inverted triangles = female mice. Data presented as mean ± SEM. One‐way ANOVA corrected with Tukey's multiple comparisons test. p < 0.05: *both young groups; ^‡old vehicle. Images made with Biorender.com.

FIGURE 6

Comparison of efficacy for improvements in frailty index score and grip strength. Frailty index score (A) and normalized grip strength (B). Squares = male mice, inverted triangles = female mice. Data are relative to old vehicle‐treated mice in each respective study. Data presented as mean ± SEM. One‐way ANOVA corrected with Tukey's multiple comparisons test.