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 were 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 mass
|
Combined
|
Male
|
Female
|
Statistics
|
Young
|
Aged
|
Young
|
Aged
|
Young
|
Aged
|
Body Mass (mg)
|
|
30.73 ± 1.27
|
41.35 ± 1.91*
|
34.65 ± 1.01A
|
45.72 ± 2.45*,B
|
25.83 ± 0.976C
|
35.89 ± 1.62*,A
|
# †
|
Muscle Mass (mg)
|
TA
|
51.41 ± 1.41
|
53.49 ± 3.95
|
54.605 ± 1.34A
|
58.70 ± 3.95A
|
47.43 ± 1.97A,D
|
46.98 ± 5.58A
|
†
|
Sol
|
10.32 ± 0.49
|
9.60 ± 0.76
|
10.01 ± 0.42A
|
9.95 ± 0.916A
|
10.51 ± 1.00A
|
9.15 ± 1.32A
|
|
Muscle Mass (mg/g body weight)
|
TA
|
1.70 ± 0.06
|
1.31 ± 0.10*
|
1.586 ± 0.05A, B
|
1.295 ± 0.12*, A,B
|
1.85 ± 0.08A,C
|
1.34 ± 0.17*, A, B, D
|
#
|
Sol
|
0.34 ± 0.03
|
0.24 ± 0.02*
|
0.29 ± 0.02A
|
0.22 ± 0.02*, A,
|
0.409 ± 0.04B
|
0.26 ± 0.04*,A
|
# †
|
Body mass (g), muscle mass (mg) and muscle mass corrected for body weight (mg/g) in combined and sex-separated young and aged muscle. Values are means ± SEM. #, p<0.05 main effect of age; †, p<0.05 main effect of sex; ⁋, p<0.05 interaction of age and sex. Different letters represent post-hoc significance at p<0.05. *p<0.05, t-test between young vs old within the same sex; d p<0.05 t-test between male and females at the same age. N=10/male group, N=8/female group. |
Exercise 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 minute less (t-test, p<0.05, Fig.1A) accounting for 645 meters 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 was 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 meters 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 increase with exercise in all groups (t-test, p<0.05, Fig. 1C).
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.
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 -II protein (t-test, p=0.087, Fig. 3B, E). A main effect of age was observed in both Complex-V (Fig. 3B) and -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. Further, 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.
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. 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 the same-sex mice. In male mice, we observed no change in Complex-V (Fig. 3B) or -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), -IV (Fig. 3D) or -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.
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. 4 A-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.
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 protein content in male and female mice with age.
We next wanted to explore how markers of mature autophagosome content are changed in whole muscle samples with age and biological sex in skeletal muscle. Since, these proteins have been shown to change with exercise, we also assessed the impact of exercise in these murine groups. We first measured LC3-II/I as markers of the ratio of mature:immature autophagosomes, respectively. We found a tendency of exercise to reduce LC3-II/I in our combined-group analysis (2-way ANOVA, p=0.09, Fig 5.B), 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).
Mitophagic protein content in whole muscle and isolated mitochondria
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. 5 B, 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 were 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 increases in Parkin protein (Male: t-test, p<0.05; Female: post-hoc, p<0.05; Fig. 6C).
We also explored whether LC3-II protein, a marker of mature autophagosomes was different in isolated mitochondria from young and old male and female mice (Fig. 6D,E). We observed no effect of age in either sex. Since we have previously reported that acute exercise can stimulate mitophagic breakdown, we assessed whether our exercise stimulus altered mitochondrially-localized LC3-II. A main effect of exercise was observed, whereby LC3-II protein was decreased with exercise by an average of 20% overall (3-way ANOVA, p<0.05, Fig. 6E).
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.7 C, D, E). When sex was separated, all lysosomal proteins showed a significant main effect of age (2-way ANOVA, p<0.05, Fig. 7 B-E). A main effect of sex was found in Lamp1 (2-way ANOVA, p<0.05, Fig. 7C), vATPase (2-way ANOVA, p<0.05, Fig. 7D) 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. 7 B-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. 7 C-E), except for Lamp1. We quantified higher Lamp1 (post-hoc, p<0.05, Fig 7. B) and mature Cathepsin D (t-test, p<0.05, Fig 7. D) 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 7. E).
We 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), whereby these proteins were greater in aged, versus young muscle, female muscle versus male, and the increase with age was larger in the female cohort. 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. 8B, C). 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 increase was also measured in Tfe3 protein, whereby young females contained 66% more than young males (t-test, p=0.057, Fig. 8C).
Influence of exercise on lysosome biosynthetic pathway
We also wished to explore whether exercise can activate lysosome biosynthesis pathways in both young and aged, male and female mice (Fig 9. A-D). Thus, we measured levels of nuclear Tfeb protein in all groups. Sedentary aged, sex-combined muscle exhibited 18% more nuclear Tfeb (post-hoc, p<0.05, Fig. 9A). In this sex-combined analysis, there was an interaction between age and exercise (2-way ANOVA, p<0.05, Fig. 9B). Following cessation of exercise, nuclear Tfeb was increased by 30%, whereas this was not evident in aged male or female muscle (post-hoc, p<0.05, Fig 9B). However, aged male and female muscle appeared to possess approximately 20% higher basal pre-exercise levels of Tfeb in the nucleus, compared to young counterparts (t-test, p=0.075 and p=0.077 respectively, Fig. 9B). In response to exercise, young male mice enhanced nuclear Tfeb by 40% (post-hoc, p<0.05, Fig. 9B), whereas females only upregulated nuclear content by 16% (t-test, p=0.064, Fig. 9B). Thus, the fold-change in nuclear Tfeb with exercise was greater in males, compared to females (t-test, p<0.05, Fig 9C).
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 increase promoter activity with exercise (2-way ANOVA, p=0.09, Fig. 9D). There was also a main effect of age (2-way ANOVA, p<0.05, Fig. 9D), whereby Tfeb promoter activity was reduced with age. Exercise enhanced Tfeb promoter activity in both young (t-test, p=0.069, Fig. 9D) and aged (t-test, p<0.05, Fig. 9D) muscle by 1.9-and 2.5-fold, respectively.