TP53INP2-dependent activation of muscle autophagy ameliorates sarcopenia and promotes healthy aging

Abstract

Sarcopenia is a major contributor to disability in older adults, and thus, it is key to elucidate the mechanisms underlying its development. Increasing evidence suggests that impaired macroautophagy/autophagy contributes to the development of sarcopenia. However, the mechanisms leading to reduced autophagy during aging remain largely unexplored, and whether autophagy activation protects from sarcopenia has not been fully addressed. Here we show that the autophagy regulator TP53INP2/TRP53INP2 is decreased during aging in mouse and human skeletal muscle. Importantly, chronic activation of autophagy by muscle-specific overexpression of TRP53INP2 prevents sarcopenia and the decline of muscle function in mice. Acute re-expression of TRP53INP2 in aged mice also improves muscle atrophy, enhances mitophagy, and reduces ROS production. In humans, high levels of TP53INP2 in muscle are associated with increased muscle strength and healthy aging. Our findings highlight the relevance of an active muscle autophagy in the maintenance of muscle mass and prevention of sarcopenia.Abbreviation: ATG7: autophagy related 7; BMI: body mass index; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ROS: reactive oxygen species; TP53INP2: tumor protein p53 inducible nuclear protein 2; WT: wild type.

Keywords: Sarcopenia; aging; autophagy; mitophagy; muscle atrophy.

Figure 1.

TP53INP2 protein expression decreases during aging and is associated with sarcopenia and unhealthy aging in humans. (a) quantification of TP53INP2 protein expression in muscle biopsies from young and aged human subjects. (b) correlation of muscle TP53INP2 protein expression with age in human subjects. (c) correlation of muscle TP53INP2 protein expression with hand grip strength in human subjects. (d) correlation of Charlson comorbidity index (CCI) values and hand grip strength in human subjects. (e) correlation of muscle TP53INP2 protein expression with CCI in human subject. (f) TP53INP2 protein expression in aged unhealthy and healthy subjects. Data are expressed as box and whiskers showing minimum to maximum data. Statistical analyses were performed by mann-whitney t-test (A), spearman correlation analysis (B, C, D and E) and ANOVA followed by Krustal-Wallis test (F).

Figure 2.

Overexpression of TP53INP2 in mouse muscle enhances autophagy and protects against sarcopenia and age-related metabolic disease. (a) Representative western blot (WB) and quantification of TP53INP2 protein expression in gastrocnemius muscle from young (4–6-months old) and old (22–24-months old) mice (n = 11). (b) Representative WB and quantification of the autophagy marker LC3-II in gastrocnemius muscle from old WT and TP53INP2 transgenic mice untreated or treated with colchicine (n = 5–6). (c) quantification of the percentage of age-induced reduction in the weight of the quadriceps, gastrocnemius and tibialis muscles in WT and TP53INP2 transgenic mice (n = 9–12). (d) quantification of the percentage of age-induced reduction in cross-sectional area (CSA) in gastrocnemius from WT and TP53INP2 transgenic mice (n = 8–10). (e) quantification of the percentage of age-induced loss of muscle performance in WT and TP53INP2 transgenic mice (n = 5–7). (f) glucose tolerance test (GTT) in old WT and TP53INP2 transgenic mice (n = 5–7). (g) Representative WB image and quantification of LC3-II protein levels in muscle mitochondria from old WT and TP53INP2 transgenic mice untreated or treated with colchicine (n = 4–6). Data are expressed as mean ± SE. *p < 0.05. Statistical analyses were performed by t-test (A, C, D and E), and two-way ANOVA (B, F and G).

Figure 3.

Acute overexpression of TP53INP2 in old mice improves muscle atrophy. (a) Representative WB and quantification of TP53INP2 protein expression in gastrocnemius muscle from old mice (24 months old) injected with control (AAV-null) or TP53INP2 overexpressing (AAV-TP53INP2) adeno-associated viruses (n = 5). (b) quantification of cross-sectional area (CSA) of gastrocnemius muscle from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). (c) fiber CSA distribution from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). (d) mRNA expression of atrophy-related genes in gastrocnemius from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). (e) mRNA expression of autophagy-related genes in gastrocnemius from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). (F) Representative WB and quantification of p-EIF4EBP1/EIF4EBP1 in gastrocnemius from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). (G) Representative WB and quantifiation of K48-ubiquitinated proteins in gastrocnemius from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5) (H) Representative WB and quantification of LC3-II in mitochondrial fractions from gastrocnemius from old mice transduced with AAV-null or AAV-TP53INP2 adeno-associated viruses and untreated or treated with chloroquine (n = 3–5). (I) Quantification of H₂O₂ levels in gastrocnemius muscle from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). (J) Representative WB and quantification of TOMM20 in gastrocnemius from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). (K) Quantification of mitochondrial DNA (mtDNA) levels in gastrocnemius from old mice injected with AAV-null or AAV-TP53INP2 adeno-associated viruses (n = 5). Data are expressed as mean ± SE. *p < 0.05. Statistical analyses were performed by t-test (A-G, I-K), and two-way ANOVA (H).