Autophagy ablation in skeletal muscles worsens sepsis-induced muscle wasting, impairs whole-body metabolism, and decreases survival

Abstract

Septic patients frequently develop skeletal muscle wasting and weakness, resulting in severe clinical consequences and adverse outcomes. Sepsis triggers sustained induction of autophagy, a key cellular degradative pathway, in skeletal muscles. However, the impact of enhanced autophagy on sepsis-induced muscle dysfunction remains unclear. Using an inducible and muscle-specific Atg7 knockout mouse model (Atg7^iSkM-KO), we investigated the functional importance of skeletal muscle autophagy in sepsis using the cecal ligation and puncture model. Atg7^iSkM-KO mice exhibited a more severe phenotype in response to sepsis, marked by severe muscle wasting, hypoglycemia, higher ketone levels, and a decreased in survival as compared to mice with intact Atg7. Sepsis and Atg7 deletion resulted in the accumulation of mitochondrial dysfunction, although sepsis did not further worsen mitochondrial dysfunction in Atg7^iSkM-KO mice. Overall, our study demonstrates that autophagy inactivation in skeletal muscles triggers significant worsening of sepsis-induced muscle and metabolic dysfunctions and negatively impacts survival.

Keywords: Genetics; Human metabolism; Musculoskeletal medicine.

Graphical abstract

Figure 1

Muscle-specific deletion of Atg7 inhibits basal autophagy in skeletal muscles (A) mRNA expressions of Atg7 in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery. Data presented as fold change relative to sham Atg7^f/f mice. (B) Representative immunoblots of Atg7, p62/SQSTM1, and LC3B proteins. GAS = gastrocnemius, PL = plantaris, SOL = soleus, TA = tibialis anterior. (C and D) Immunostaining and quantification of the proportion of p62/SQSTM1 positive fibers in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery. Scale bars = 20μm. (E) mRNA expressions of Atg7 in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after CLP. 18S levels used as a control. (F) Representative immunoblots of ATG7, p62/SQSTM1, and LC3B proteins in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. (G–I) Densitometric analyses of ATG7, p62/SQSTM1, and LC3B protein levels in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. (J and K) Immunostaining for p62/SQSTM1 in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after CLP surgery. Scale bars = 20 μm, inset scale bar = 10 μm. (L and M) mRNA expressions of various autophagy-related genes (M) and lysosomal cathepsin in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. Gaba refers to Gabarapl1 and Becn1 refers to Beclin1. N = 6–8 group. Data in panels A, D, E, G, H, I, K, L, and M are presented as mean ± SEM. Number of animals indicated within bars, where applicable. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).

Figure 2

Autophagy inactivation exacerbates disease severity (A) Percent change in body mass of female (left panel) and male (right panel) Atg7^f/f and Atg7^iSkM−KO mice in response to acute sepsis. Body mass measured prior to sham surgery or CLP then 48 h later. (B) Percent change in body mass of female Atg7^f/f and Atg7^iSkM−KO mice in response to prolonged sepsis. Body mass measured prior to sham surgery or CLP then daily over a 6-day period. (C and D) Percent change in lean (C) and fat (D) muscle mass of female Atg7^f/f and Atg7^iSkM−KO mice in response to prolonged sepsis. Mass measured prior to sham surgery or CLP then daily over a 4-day period. (E) Clinical severity score (CSS) for female Atg7^f/f and Atg7^iSkM−KO mice in response to prolonged sepsis. CSS measured prior to sham surgery or CLP then daily over a 6-day period. (F) Percent survival of female Atg7^f/f and Atg7^iSkM−KO mice in response to prolonged sepsis. Survival was monitored over a 6-day period following sham surgery or CLP. No mortality of sham Atg7^iSkM−KO mice was observed. Comparisons of curves indicated by ∗∗p < 0.001. Data in panels A–E are presented as mean ± SEM. Number of animals indicated in bars, where applicable. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).

Figure 3

Impact of 48 h of sepsis on skeletal muscle mass, myofiber fiber size, and contractility (A and B) Muscle mass loss in TA and GAS muscles of female (A) and male (B) Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. Data are presented as percent change relative to sham Atg7^f/f mice. (C–E) Myofiber size of TA muscles of female (C and E) and male (D) Atg7^f/f and Atg7^iSkM−KO mice 48 h (C and D) or 6 days (E) after sham surgery or CLP. (F) Isometric tension of TA muscles of female and male Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. N = 20–23 per group). (G) Fatigue in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. Fatigue curves presented as percent change from tetanus 1 (initial force) to tetanus 60, n = 17 of anima, sham Atg7^f/f; n = 14, sham Atg7^iSkM−KO; n = 11, CLP Atg7^f/f; n = 12, CLP Atg7^iSkM−KO. Data in panels A–G are presented as mean ± SEM. Number ls indicated in bars, where applicable. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).

Figure 4

Impact of autophagy inactivation and sepsis on mitochondrial content and function (A) Mitochondrial oxygen consumption in permeabilized myofibers from GAS muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. Data normalized per mg of muscle. (B) Acceptor control ratio (ACR) (index of mitochondrial coupling efficiency) in GAS muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. (C) Representative immunoblots of respiratory chain subunits (OXPHOS) in GAS muscles of female Atg7^f/f and Atg7^−/− mice 48 h after sham surgery or CLP (n = 8 for each sham group, n = 5 each CLP group). Stain-free technology was used to normalize subunits. Significant increases in sham and septic Atg7^iSkM−KO mice suggest accumulation of impaired mitochondria. (D) mRNA expression of PGC1α in TA muscles of male Atg7^f/fand Atg7^−/−mice 48 h after sham surgery or CLP (n = 6-8pergroup). (E) Time of opening of the mPTP in permeabilized myofibers from GAS muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. Number of animals indicated in bars, where applicable. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).

Figure 5

Impact of autophagy inactivation and sepsis on markers of the ubiquitin-proteasome pathway (A) mRNA expressions of E3 ubiquitin ligases in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. (B–E) mRNA expressions of proteasome subunits genes in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. 18S levels used as a control. As fold change relative to sham Atg7^f/f. (F–G) Representative immunoblot and quantification of ubiquitinated protein levels in TA muscle of female Atg7^f/f and Atg7^iSkM−KO mice 48 h after sham surgery or CLP. GAPDH serves as loading control. Data in panels A–E and G are presented as mean ± SEM. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).

Figure 6

Effects of prolonged sepsis and autophagy inactivation on skeletal muscle transcriptome (A) Heatmap showing skeletal muscle gene expression signatures in Atg7^f/f and Atg7^iSkM−KO mice in response to prolonged sepsis (6 days). Colors indicate relative expression levels; red indicates high expression and blue indicates low expression. (B) Top five upregulated and downregulated pathways as identified through biometric analyses. FDR = false discovery ratio. (C) Representative immunoblots of pFoxO1, FoxO1, pFoxO3, and FoxO3 protein levels in TA skeletal muscles of female Atg7^f/f and Atg7^iSkM−KO mice 6 days after CLP showing decreased phosphorylation of FoxO1 and FoxO3 resulting from Atg7 knockout and sepsis. (D and E) Densitometric analyses of pFoxO1 and pFoxO3 proteins normalized to GAPDH in Atg7^f/f and Atg7^iSkM−KO mice 6 days after CLP. GAPDH or Ponceau serves as loading control. Number of animals indicated within bars. Data are presented as mean ± SEM. Knockout effect indicated by ∗p < 0.05, CLP Atg7^f/f vs. CLP Atg7^iSkM−KO. (F) mRNA expressions of E3 ubiquitin ligases in TA muscles of female Atg7^f/f and Atg7^iSkM−KO mice 6 days after sham surgery or CLP. 18S levels used as a control. Data are presented as fold change relative to sham Atg7^f/f and as mean ± SEM, n = 5–8 per group. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).

Figure 7

Effects of autophagy inactivation and sepsis on food intake, energy expenditure and substrate utilization (A–C) Average kinetic data for whole-body oxygen consumption (VO₂) and RER (VCO₂/VO₂) during day and night periods over a 48-h period after sham surgery or CLP. D) Food intake (gram of consumed food per kg of body mass per day. (E) Total movement (ambulatory activity). (F) Heat (energy expenditure) relative to daily body mass. Metabolic parameters measured by indirect calorimetry for 2 days prior to surgery and 4 days after sham surgery or CLP. Food intake and metabolic parameters expressed relative to daily body mass, given differences in body composition over time. PhenoMaster metabolic cage data measured on a dark (6:00 p.m. to 6:00 a.m.)/light (6:00 a.m. to 6:00 p.m.) cycle. Data are presented as mean ± SEM. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).

Figure 8

Autophagy inactivation in skeletal muscles exacerbates sepsis-induced metabolic impairments (A–C) Kinetics of core body temperature B) Whole blood glucose and C) β-OHB levels measured in Atg7^f/f and Atg7^iSkM−KO mice over a 6-day period between 6 and 8 a.m. Data are presented as mean ± SEM. ∗p < 0.05 vs. sham Atg7^f/f; #p < 0.05 for sepsis effect (i.e., sham Atg7^f/f vs. CLP Atg7^f/f or sham Atg7^iSkM−KO vs. CLP Atg7^iSkM−KO); †p < 0.05 for sepsis plus knockout effect (i.e., CLP Atg7^f/f vs. CLP Atg7^iSkM−KO).