The influence of age, sex, and exercise on autophagy, mitophagy, and lysosome biogenesis in skeletal muscle

Physical characteristics of young and aged male, and female mice

To determine whether aging differentially impacted muscle mass in male and female mice, we first measured body mass (g), muscle mass (mg), and muscle mass corrected for body mass (mg/g) in the predominantly fast tibialis anterior (TA) and predominantly slow-twitch soleus (Sol) muscles (Table 1). Overall, body mass was 1.4-fold greater in aged mice versus young counterparts (p < 0.05). Main effects of age and sex effects were found. Post hoc tests revealed that both young and aged male mice were significantly larger than age-matched female counterparts (p < 0.05). Furthermore, aged male mice were 32% heavier than young males, and aged female mice were 28% heavier than young females (p < 0.05). Raw TA mass (mg) was lower in female mice (sex effect, p < 0.05) and TA mass was significantly less in young females versus young males (t-test, p < 0.05). When corrected for body mass, TA mass was 24% smaller in aged mice (p < 0.05), and a main effect of sex was observed in our separated analysis (p < 0.05). On average, TA mass/body mass (mg/g) was 28% lower in female mice (post hoc, p < 0.05) and 18% less in male mice (t-test, p < 0.05). Sol mass (mg) was not different between any groups. When corrected for body mass (mg/g), a significant 30% decrease in Sol mass with age was measured in sex-pooled data (p < 0.05). In a sex-separated analysis, a main effect of age and sex was observed (p < 0.05). Young females had a 1.4-fold larger Sol mass/body mass than young males (post hoc, p < 0.05). With age, male mice had a 24% decline in Sol mass/body mass (t-test, p < 0.05), whereas females displayed a 36% decline (post hoc, p < 0.05).

Table 1 Animal body weight and muscle characteristicsExercise capacity in young and aged, male and female mice

To determine if age and biological sex impact acute exercise capacity, we exposed a cohort of mice to an exhaustive bout of incremental exercise. In our sex-pooled comparison, aged mice ran for an average of 25 min less (t-test, p < 0.05, Fig. 1A) accounting for 645 m of less distance covered (t-test, p < 0.05, Fig. 1B). In sex-separated comparisons, a main effect of age and an interaction of age and sex were found in run time (p < 0.05, Fig. 1A). Further analysis revealed 34% and 43% declines in aged male and female mice versus their young counterparts, respectively (post hoc, p < 0.05, Fig. 1A). Effects of age, sex, and an interaction of the two variables were measured in distance to fatigue (p < 0.05, Fig. 1B). Run distance was reduced with age in both male and female mice (post hoc, p < 0.05, Fig. 1B). On average, young female mice ran 263 m more than young males (post hoc, p < 0.05, Fig. 1B), whereas aged females ran slightly less (28m) than aged males (post hoc, p < 0.05, Fig. 1B). Blood lactate was similarly increased with exercise in all groups (t-test, p < 0.05, Fig. 1C).

Fig. 1figure 1

Exercise capacity in young and aged, male and female mice. A Time to fatigue in minutes. B Distance to fatigue in meters. C Blood lactate (mM). Values are means ± SEM. The main effects are represented on a graph at p < 0.05. *p < 0.05, t-test between indicated groups. δ p < 0.05, post hoc significance. N = 5/male group, N = 4/female group

Mitochondrial parameters in young and aged, male and female mice

To understand the divergent endurance capacity with age and sex, we assessed mitochondrial parameters as these organelles are correlated with muscle fatigability. We examined respiration and H2O2 emission in permeabilized TA muscle fibers from all groups (Fig. 2A, B). We observed an overall effect of age, whereby aged muscle had lower respiratory capacity (3-way ANOVA, p < 0.05, Fig. 2A). Independent analyses were performed for each subsequent titration, and we measured a main effect of age for all respiratory measurements (2-way ANOVA, p < 0.05 Fig. 2A), apart from the Complex I-Basal condition. An interaction between age and sex was found in Complex II-Basal respiration (2-way ANOVA, p < 0.05 Fig. 2A); however, no post hoc significance was observed. Overall, no changes were measured in H2O2 emission in permeabilized fibers (Fig. 2B), but a trending effect of sex was measured in Complex II-active (2-way ANOVA, p = 0.09, Fig. 2B), with lower values in female samples.

Fig. 2figure 2

Mitochondrial respiration and reactive oxygen species in young and age, male and female mice. A Oxygen consumption rates, B H2O2 emission, and C oxygen consumption rates normalized to mitochondrial protein content in the indicated respiratory states. All values are reported as means ± SEM. For A and B, the main effects of a 3-way ANOVA are represented on the graph at p < 0.05. For AC, the main effects of 2-way ANOVA are represented on the graph at p < 0.05. δ p < 0.05, post hoc significance. N = 10/male group, N = 8/female group

To determine the effects of age and sex on mitochondrial protein content, we quantified levels of proteins derived from each complex of the electron transport chain (ETC) (Fig. 3). In the sex-grouped data, we found no significant differences in any ETC proteins, and a trending increase in both Complex-V (t-test, p = 0.058, Fig. 3B) and Complex-II protein (t-test, p = 0.087, Fig. 3B, E). A main effect of age was observed in both Complex-V (Fig. 3B) and Complex-II (Fig. 3E). Each independent complex (Fig. 3B–F) and total OXPHOS protein (Fig. 3G) exhibited a main effect of sex (p < 0.05), such that females had more mitochondrial protein. Furthermore, an interaction between age and sex was found in Complex-V (p < 0.05, Fig. 3B), Complex-II (p < 0.05, Fig. 3E), Complex-I (p < 0.05, Fig. 3F), and total OXHOS (p < 0.05, Fig. 3G) protein, whereby female muscle did not display decrements in mitochondrial protein content with age.

Fig. 3figure 3

Mitochondrial protein content in young and age, male and female mice. A Representative western blot from male (top panel) and female (bottom panel) mice for OXPHOS protein. BF Quantification of each independent mitochondrial protein. B Complex-V protein (ATP5A) protein, C Complex-III (UQCRC2) protein, D Complex-IV (MTCO1) protein, E Complex-II (SDH8), and F Complex I (NDUFB8) protein. G Quantification of total OXPHOS. All values were corrected to Ponceau stain (P.S), and values are reported as means ± SEM, in A.U. The main effects of 2-way ANOVA are represented on the graph at p < 0.05. δ p < 0.05, post hoc significance. *p < 0.05, t-test between indicated groups. N = 10/male group, 8/female group

We assessed independent differences between young males and females and measured 35%, 61%, 38%, and 28% more Complex-V (t-test, p < 0.05, Fig. 3B), Complex-III (post hoc, p < 0.05, Fig. 3C), Complex-II (t-test, p < 0.05, Fig. 3E), and total OXPHOS (t-test, p = 0.08, Fig 3G) protein in young females versus young males, respectively. The same comparison in aged male and female mice showed that each complex had between 1.8- and 2.1-fold more mitochondrial protein (p < 0.05, Fig. 3B–F) and 2.1-fold more total OXPHOS in females than in males (post hoc, p < 0.05, Fig. 3G).

We then explored independent differences between young and aged muscle from same-sex mice. In male mice, we observed no change in Complex-V (Fig. 3B) or Complex-II (Fig. 3E) but measured 17 to 39% decreases in all other mitochondrial protein content with age (Fig. 3C, D, F, G). In females, we measured no change in Complex-III (Fig. 3C), Complex-IV (Fig. 3D), or Complex-I (Fig. 3F) protein, but 33 to 47% increases were evident in the remaining mitochondrial proteins (Fig. 3B, E, G) with age in female mice.

To confirm whether respiratory function differed on a per/mitochondria basis, active respiration data from Fig. 2A was normalized to associated protein levels in Fig. 3 (Fig. 2C). We found that complex-specific respiratory function was lower in females in comparison to aged matched males (2-way ANOVA, p < 0.05). Additionally, capacity was reduced as a product of age in all respiratory states measured (2-way ANOVA, p < 0.05). Further analysis revealed that this was largely driven by the greater extent of loss in mitochondrial function with age in the female cohort (post hoc, p < 0.05). Specifically, normalized Complex-I Active respiration was unchanged in males but was reduced by 49% in females with age. Complex-II Active respiration was non-significantly reduced by 23% but was significantly lower in aged females versus young counterparts by 66%. Finally, Complex-I and II Active respiration was 20% lower in aged males versus young males (not significant) but was 48% lower in aged females versus young females.

Autophagy-related protein expression in aged muscle

To evaluate how aging and biological sex affect the autophagy-lysosome system, we measured upstream autophagy proteins in whole muscle quadriceps samples (Fig. 4A–C). In combined-sex groups, aging led to a significant 44% increase in Beclin1 protein (t-test, p < 0.05, Fig. 4B) and a trending 47% increase in ATG-7 protein (t-test, p = 0.09, Fig. 4C). When the sexes were analyzed separately, no main or interaction effects were measured in Beclin1 protein (Fig. 4B), but a main effect of both age and sex was found in ATG-7 protein (2-way ANOVA, p < 0.05, Fig. 4C), whereby aging and female muscle displayed increased protein expression.

Fig. 4figure 4

Upstream autophagic proteins in young and age, male and female mice. A. Representative western blots for Beclin1 and ATG7. B Quantification of Beclin1 protein in combined and sex-separated groups. C Quantification of ATG7 protein in combined and sex-separated groups. All values were corrected to GAPDH and are reported as means ± SEM, in A.U. The main effects of 2-way ANOVA are represented on a graph at p < 0.05. δ p < 0.05, post hoc significance. *p < 0.05, t-test between indicated groups. N = 10/male group, 8/female group

Independent differences between the groups were then examined for these autophagy proteins. Beclin1 protein was significantly increased by 36% in aged males versus young counterparts (t-test, p < 0.05, Fig. 4B), whereas female mice displayed no age effect (Fig. 4B). ATG-7 protein was unchanged in both sexes independently; however, both young (t-test, p < 0.05, Fig. 4C) and aged female mice (post hoc, p < 0.05, Fig. 4C) contained ~2-fold more ATG-7 protein in comparison to age-matched male mice.

Autophagosomal-associated protein content in male and female mice with age

We next wanted to explore how markers of mature autophagosome content were changed in whole muscle samples with age and biological sex in skeletal muscle. We also assessed the impact of exercise in these murine groups. We first measured LC3-II/I as markers of the ratio of mature to immature autophagosomes, respectively. Only minor changes were observed in our combined-group analysis (2-way ANOVA, p = 0.09, Fig. 5B), with no main effects or post hoc significance in our sex-separated groups. We observed an overall effect of age on p62 levels in our combined group (2-way ANOVA, p < 0.05, Fig. 5C) and a trending 37% increase in p62 protein in our young versus aged sedentary animals (t-test, p = 0.085, Fig. 5C). In the sex-separated data, a significant main effect of age was observed, along with an interaction between age and acute exercise (3-way ANOVA, p < 0.05, Fig. 5C). When we assessed the influence of age and exercise in independent sexes, a main effect of age was evident in both males and females (2-way ANOVA, p < 0.05, Fig. 5C). Independent analyses revealed a significant 33% decrease in p62 protein with exercise in young males (t-test, p < 0.05, Fig. 5C) and a 25% increase with exercise in young females (t-test, p = 0.05, Fig. 5C).

Fig. 5figure 5

Autophagosomal proteins in sedentary and acute-exercised young and age, male and female mice. A Representative western blots for p62, LC3-I, and LC3-II. B Quantification of LC3-II/I protein in combined and sex-separated groups. C Quantification of p62 protein in combined and sex-separated groups. All values were corrected to GAPDH and are reported as means ± SEM, in A.U. The main effects of a 3-way ANOVA are represented on the graph at p < 0.05. The main effects of 2-way ANOVA are represented on a graph at p < 0.05. #p < 0.05, main effect age. δ p < 0.05, post hoc significance. *p < 0.05, t-test between indicated groups. N = 5/male group, 4/female group

Mitophagic protein content in whole muscle of young and aged male and female mice

To determine if age and sex impact mitophagy in skeletal muscle, we first probed for the mitophagy markers BNIP3 and Parkin in whole muscle samples (Fig. 6A–C). In the sex-combined group, there were 4.8-fold and 3.6-fold increases in aged muscle BNIP3 and Parkin protein, respectively (t-test, p < 0.05, Fig. 6B, C). In sex-separated comparisons, a main effect of age was observed in BNIP3 protein (2-way ANOVA, p < 0.05, Fig. 6B), and post hoc comparisons revealed similar, significant increases in aged muscle BNIP3 protein vs sex-matched young counterparts (post hoc, p < 0.05, Fig. 6B). A main effect of both age and sex was found in Parkin protein, whereby females, both young and old, had more Parkin than their sex-matched, young, counterparts (2-way ANOVA, p < 0.05, Fig. 6C). Aging in both sexes led to large increases in Parkin protein (male: t-test, p < 0.05; female: post hoc, p < 0.05; Fig. 6C), suggesting a high capacity for the triggering of mitophagy in aging muscle.

Fig. 6figure 6

Mitophagy protein content in the muscle and mitochondria in young and aged, male and female mice. A Representative western blots for BNIP3 and Parkin in whole muscle samples. B Quantification of BNIP3 protein in combined and sex-separated groups. C Quantification of Parkin protein in combined and sex-separated groups. Whole muscle values were corrected to GAPDH. Values are reported as means ± SEM, in A.U. The main effects of 2-way ANOVA are represented on a graph at p < 0.05. δ p < 0.05, post hoc significance. *p < 0.05, t-test between indicated groups. N = 10/male group, 8/female group

Lysosomal protein content in young and aged male and female mice

To assess the end-stage of the autophagy pathway, we evaluated lysosomal protein content in our groups (Fig. 7A–E). Lysosome-associated membrane protein 1 (Lamp1) levels were unchanged with age in the sex-combined group. Alternatively, vesicular ATPase (V-ATPase), mature Cathepsin B, and mature Cathepsin D were all upregulated by 3.6-, 4.0-, and 5.5-fold with age, respectively (t-test, p < 0.05, Fig.7C, D, E). When sex was separated, all lysosomal proteins showed a significant main effect of age (2-way ANOVA, p < 0.05, Fig. 7B–E). A main effect of sex was found in Lamp1 (2-way ANOVA, p < 0.05, Fig. 7B), vATPase (2-way ANOVA, p < 0.05, Fig. 7C), and mature Cathepsin D (2-way ANOVA, p < 0.05, Fig. 7E), whereby these proteins were higher in the female mice. An interaction between age and sex was found for mature Cathepsin D protein (two-way ANOVA, p < 0.05, Fig. 7E). Independent analyses for each protein confirmed significant 1.8–3.9-fold increases in all measured lysosomal proteins with age in the male mice (p < 0.05, Fig. 7B–E). In female mice, significant 4.4–6.5-fold increases were found with age in each lysosome protein (post hoc; p < 0.05, Fig. 7C–E), except for Lamp1. We quantified higher Lamp1 (post hoc, p < 0.05, Fig. 7B) and mature Cathepsin D (t-test, p < 0.05, Fig. 7D, E) in young female mice versus young male mice and elevated mature Cathepsin D in aged females compared to aged males (post hoc, p < 0.05, Fig. 7E).

Fig. 7figure 7

Lysosome proteins in young and age, male and female mice. A Representative western blots for Lamp1, vATPase, mature Cathepsin B, and mature Cathepsin D. B Quantification of Lamp1 protein in combined and sex-separated groups. C Quantification of vATPase protein in combined and sex-separated groups. D Quantification of mature Cathepsin B protein in combined and sex-separated groups. E Quantification of mature Cathepsin D protein in combined and sex-separated groups. All values were corrected to GAPDH and are reported as means ± SEM, in A.U. The main effects of 2-way ANOVA are represented on a graph at p < 0.05. δ p < 0.05, post hoc significance. *p < 0.05, t-test between indicated groups. N = 10/male group, 8/female group

We also measured the protein levels of TFEB and TFE3, transcription factors that control the autophagy-lysosome pathway (Fig. 8A–C). TFEB was 3.8-fold greater with age in the sex-combined analysis (t-test, p < 0.05, Fig. 8B). In the sex-separated analyses, TFEB protein exhibited main effects of age and sex, and an interaction existed between these variables (2-way ANOVA, p < 0.05, Fig. 8B). This protein was greater in aged, compared to young muscle, and muscle from females exhibited higher TFEB levels than in male counterparts, and this was amplified further with age. Specifically, TFEB protein was 1.8-fold greater in young females (t-test, p < 0.05, Fig. 8B) and 2.5-fold greater in aged females (t-test, p < 0.05, Fig. 8B) when compared to age-matched male counterparts. Compared to young, sex-matched animals, TFEB protein was 3.3-fold greater in aged males (t-test, p < 0.05, Fig. 8B) and 4.2-fold higher in aged females (post hoc, p < 0.05, Fig. 8B). Conversely, TFE3 was 1.5-fold greater in our sex-combined analysis (t-test, p < 0.05, Fig. 8C). In the sex-separated analyses, TFE3 protein exhibited main effects of age and interaction between age and sex (2-way ANOVA, p < 0.05, Fig. 8C). As such, TFE3 protein was increased 3.3-fold with age in male mice (post hoc, p < 0.05, Fig. 8C), an effect not seen in females. A trending sex difference was also measured in TFE3 protein, whereby young females contained 66% more than young males (t-test, p = 0.057, Fig. 8C).

Fig. 8figure 8

Regulation of lysosome biosynthetic pathways in young and aged, male and female mouse muscle with exercise. A Representative western blots for TFEB and TFE3 protein. B Quantification of TFEB protein in combined and sex-separated groups. C Quantification of TFE3 protein in combined and sex-separated groups. D Representative western blots for TFEB protein in nuclear and cytosolic fractions in sedentary and exercised, young and aged, male and female mice. E % nuclear TFEB protein in combined and sex-separated male and female mice. F Fold-change in nuclear TFEB protein in each group examined. G TFEB promoter activity (luciferase; RLU) in young and aged, sedentary, and exercised mice. Values in B and C were corrected to GAPDH and are reported as means ± SEM. N = 10/male group, 8/female group. Line break in representative blot is different sections from the same blot. In DF, cytosolic values were corrected to α-tubulin and nuclear values were corrected to H2B and reported as mean ± SEM, in A.U. N = 5/male group, 4/female group. In G, N = 6/group. The main effects of 2-way ANOVA are represented on graph at p < 0.05. δ p < 0.05, post hoc significance. *p < 0.05, t-test between indicated groups

Fig. 9figure 9

Summary of sex differences in the skeletal muscle of young and aged mice and the response to acute exhaustive exercise. Muscle from young males (top left panel) contains fewer mitochondria of higher functionality, less lysosomes, and their transcriptional regulator TFEB in comparison to young females (top right panel). With aging, there are decrements in mitochondrial content and function in males (bottom left). Alternatively, in females, mitochondrial content was relatively unchanged, although function was reduced. These changes were associated with a greater upregulation in the autophagy-lysosome system in females with age. With exercise, there was an induction of autophagy and nuclear TFEB in young males that was not present in young females or aged animals of either sex. Overall, exercise was capable of enhancing TFEB promoter activity. Font size represents the relative amount of TFEB; thickness of black arrows represents the relative contribution of the autophagy-lysosome system at baseline; red front dictates change with exercise (↑ represents increase, = represents no change). Orange mitochondria are healthy, and green are unhealthy

Influence of exercise on lysosome biosynthetic pathway

We wished to explore whether exercise could activate lysosome biosynthesis pathways in both young and aged, male and female muscle (Fig. 8D–G)Thus, we measured the percent of nuclear TFEB protein in all groups. Muscle from aged sedentary, sex-combined mice contained 18% more nuclear TFEB (t-test, p < 0.05, Fig. 8E). In this sex-combined analysis, there was an interaction between age and exercise (2-way ANOVA, p < 0.05, Fig. 8E). Specifically, following the cessation of exercise, nuclear TFEB was increased by 30% in the young sex-combined cohort (post hoc, p < 0.05, Fig. 8E), which was not evident in the muscle from aged animals. In the sex-separated analysis, aged male and female muscle appeared to possess approximately 20% higher basal levels of TFEB in the nucleus, compared to young counterparts (t-test, p = 0.075 and p = 0.077, male and female, respectively, Fig. 8E). In response to exercise, young male mice enhanced nuclear TFEB by 40% (post hoc, p < 0.05, Fig. 8E), which was modestly greater than the effect observed in females (t-test, p = 0.064, Fig. 8E; t-test, p < 0.05, Fig. 8F).

We also utilized a TFEB-luciferase promoter activity assay to determine whether exercise stimulates TFEB transcriptional activity. This analysis could only be completed in sex-combined groups. Overall, there was a trending main effect of increased promoter activity with exercise (2-way ANOVA, p = 0.09, Fig. 8G). There was also a main effect of age (2-way ANOVA, p < 0.05, Fig. 8G), whereby TFEB promoter activity was reduced with age. A trending 1.9-fold increase in TFEB promoter activity in young mice was observed (t-test, p = 0.069, Fig. 8G), whereas a significant 2.5-fold increase was measured in the aged cohort as a result of acute exercise (t-test, p < 0.05, Fig. 8G).

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