Limitations in metabolic plasticity after traumatic injury are only moderately exacerbated by physical activity restriction

Physical inactivity (e.g., bedrest) after traumatic musculoskeletal injury impairs skeletal muscle function and metabolic flexibility. Volumetric muscle loss (VML) causes permanent loss of muscle tissue and is associated with chronic whole-body and local metabolic dysfunction, including reduced metabolic rate and altered mitochondrial function, even without changes in spontaneous ambulation. While restricted physical activity after VML has been shown to increase RER and lower lipid oxidation, its specific impact on the remaining uninjured muscle’s plasticity and metabolome relative to healthy controls is unclear. This study aimed to define how VML and reduced physical activity interact to influence whole-body metabolism, skeletal muscle function, and the metabolomic environment of the remaining muscle. The authors hypothesized that restricted physical activity combined with VML would worsen function of the muscle remaining and negatively impact whole-body metabolism.

Prior work demonstrates that VML induces chronic metabolic dysfunction at both whole-body and muscle levels: decreased metabolic rate, diurnal metabolic inflexibility, and mitochondrial impairments, which can be exacerbated by a Western diet. Activity restriction after VML increases RER and lowers lipid oxidation, mimicking clinical bedrest, but does not further impair muscle function beyond the injury. Physical activity/inactivity modulates the skeletal muscle metabolome; reduced activity can lead to accumulation of long-chain fatty acids and acylcarnitines, while activity acutely elevates β-oxidation-related metabolites (e.g., carnitine/acylcarnitines). Prolonged inactivity after traumatic injury is associated with chronic lipid oxidation dysfunction and polyamine alterations, which may worsen in VML. Dysregulation in carnitine shuttling and β-oxidation is linked to mitochondrial myopathy, and imbalances between fatty acid uptake and oxidation can drive ectopic lipid accumulation. These observations motivated assessing how physical activity restriction alters the metabolome and metabolic signaling in the muscle remaining after VML compared with uninjured controls.

Design: Adult male C57BL/6J mice (n=40; 8–12/group) were randomized at 13 weeks to unilateral posterior hindlimb VML or naïve controls, and further to standard cages (28×18×12.5 cm) or restricted cages (12.5×8.5×6.3 cm) for 8 weeks. Restricted cages reduce activity by ~40–50% and alter whole-body metabolism within a week. Assessments: At 6 weeks, 24-h physical activity and whole-body metabolism were measured using CLAMS (ambulation; metabolic rate; RER; carbohydrate and lipid oxidation) across 24-h, and 12-h active/inactive phases, including RER AUC and ΔRER (active–inactive). At 7 weeks, glucose tolerance tests (GTT) were conducted after a 6-h fast (2 mg/g glucose i.p.; 0–120 min). Muscle function: At 8 weeks, in vivo posterior compartment function was assessed under isoflurane anesthesia. The common peroneal nerve was severed to isolate posterior muscles, and the sciatic nerve was stimulated via percutaneous electrodes to obtain twitch and maximal isometric torque frequency curves (5–200 Hz). Contractile properties included time to peak, half-relaxation time, and ±dP/dt. Tissue collection: Gastrocnemius muscles were weighed, sectioned (proximal/mid/distal), snap-frozen at −80°C; a subset mid-belly including defect was embedded in OCT for histology. Liver and serum were collected. Histology: Ten-µm gastrocnemius cross-sections were stained with H&E for fiber number and morphology. NADH-tetrazolium reductase (NADH-TR) staining quantified oxidative fiber proportion in three standardized 1000×1000 µm ROIs: defect, border, and remaining muscle (and corresponding ROIs in naïve). Counting/classification was performed blinded using Fiji. Biochemistry/Signaling: Western blots on muscle and liver homogenates (4–15% TGX Stain-Free gels, PVDF) probed ACC, phospho-ACC (pACC), fatty acid synthase (FAS; liver), FABP4, and GLUT4, with stain-free total protein normalization. Muscle adiponectin (multiplex) and serum leptin (ELISA) were quantified. Mitochondrial content was assessed via citrate synthase (CS) activity; β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity was measured and normalized to CS. Metabolomics: Untargeted targeted-panel metabolomics of distal gastrocnemius (~50 mg) using Biocrates MxP Quant 500. Samples were isopropanol-extracted and analyzed on Sciex QTRAP 5500 or Agilent 6495C platforms (LC-MS/MS for small molecules; FIA-MS/MS for lipids/hexoses). Data processed with WebIDQ; tissue correction applied; between-plate normalization to QC. Statistics: Two-way ANOVA (injury × activity) with Tukey HSD for most endpoints; one-way ANOVA for RER AUC; three-way ANOVA (injury × activity × time) for GTT. Significance p<0.05. Metabolomics: excluded metabolites with >20% missing; KNN (k=10) imputation; log-transformation; GLM controlling for batch; Benjamini–Hochberg adjustment. Volcano thresholds: |log2FC|≥1 and −log10 p≥1.3 (p≤0.05).

• Activity restriction reduced ambulation by ~40% (p<0.0001) and lowered 24-h metabolic rate in VML-restricted mice (interaction p=0.032), driven by lower active-period metabolic rate (activity p=0.029).
• RER increased with activity restriction across 24 h (activity p=0.001), during both inactive (activity p=0.001) and active periods (interaction p=0.047). RER AUC was higher with restriction (activity p<0.0001) and with VML (injury p<0.0001). VML-restricted showed the greatest 24-h RER AUC (~31% average increase; p≤0.0001).
• ΔRER (active–inactive) was greater after VML (injury p=0.030), but restriction blunted the shift, indicating metabolic inflexibility.
• Lipid oxidation: largely unaltered over 24 h (injury p=0.068), but lower during the active period in injured mice (injury p=0.020). Carbohydrate oxidation increased overall with restriction and VML (24-h interaction p=0.001; active-period interaction p<0.0001). In the abstract: naïve restriction raised RER by ~5%; with VML, carbohydrate oxidation was ~23% greater than VML alone, lipid oxidation largely unchanged.
• Glucose tolerance: VML lowered blood glucose during GTT (injury p<0.0001) and reduced AUC (injury p=0.011), suggesting improved glucose tolerance, independent of activity.
• Muscle function: VML caused significant impairments in twitch and maximal isometric torque normalized to body mass (injury p<0.0001). Restriction modestly increased twitch torque (activity p=0.034) without affecting maximal isometric torque (activity p=0.518). Contractile properties differed due to injury (p≤0.023), not activity.
• Body/muscle mass: VML reduced gastrocnemius mass and mass normalized to body mass (injury p<0.0001). VML mice had a greater total body mass change over 8 weeks (~11 g; injury p<0.0001). Restricted mice had the lowest terminal body mass (activity p=0.026).
• Histology: Total fiber number was ~16% lower after VML (injury p=0.008). NADH-positive fibers were reduced overall with VML (~49% vs ~58% naïve; injury p=0.005), notably in defect and border regions (injury p≤0.043), with no differences in the remaining muscle region.
• Signaling: Total ACC and pACC levels were not different across groups (p≥0.216), but the pACC:ACC ratio was higher with VML-restriction (injury p=0.031; interaction p=0.046), consistent with ACC inactivation and potential reduction in fatty acid synthesis. Liver FAS, muscle GLUT4, and FABP4 were unchanged (p≥0.117, p=0.564, p=0.668, respectively).
• Metabolomics: 242 metabolic signatures detected. Activity restriction alone (naïve-restricted vs naïve) did not significantly alter the metabolome. VML vs naïve: 45 upregulated and 9 downregulated signatures; 93% of upregulated were triglycerides containing long-chain fatty acids. GABA increased ~1.5-fold after VML. VML-restricted vs naïve: 54 signatures altered; 91% overlap with VML alone. Downregulated ratios indicated β-oxidation dysregulation (e.g., hydroxylated acylcarnitines/acylcarnitines, acetylcarnitine/carnitine, carnitine esterification). Sum of triglycerides was >20-fold upregulated in both VML groups; di-/tri-glyceride ratio decreased.
• Enzymes/hormones: Muscle adiponectin increased ~57% with injury (p<0.0001). Serum leptin unchanged (p≥0.178). CS activity unchanged (p≥0.241). β-HAD activity normalized to CS decreased with injury (p=0.011), indicating reduced fatty acid oxidation capacity.

The study shows that although physical activity restriction reduces ambulation and increases reliance on carbohydrates systemically (higher RER), the dominant driver of muscle metabolic alterations is the VML injury itself. VML led to significant deficits in muscle quantity and quality, reduced oxidative capacity (fewer NADH-positive fibers in defect/border), and impaired fatty acid oxidation capacity (lower β-HAD/CS), with accompanying accumulation of triglyceride metabolites. ACC phosphorylation (higher pACC:ACC) with VML plus restriction suggests decreased fatty acid synthesis and potentially increased mitochondrial fatty acid transport; however, impaired β-oxidation likely prevents complete oxidation, favoring triglyceride accumulation. Whole-body lipid oxidation was reduced during active periods after VML, while carbohydrate oxidation and glucose tolerance improved, implying a systemic substrate preference shift possibly driven by local muscle fatty acid oxidation deficits. Metabolomics revealed that restriction alone did not alter the remaining muscle metabolome, and >90% of metabolite changes in VML-restricted were shared with VML alone, emphasizing that VML, rather than reduced activity, governs the local metabolic signature. Together, these findings address the hypothesis by showing only moderate exacerbation from activity restriction, with VML injury principally constraining metabolic plasticity via fatty acid oxidation impairment and triglyceride accumulation.

VML injury primarily dictates alterations in whole-body metabolism and the muscle metabolome, whereas chronic physical activity restriction only moderately exacerbates certain metabolic features. VML reduces oxidative capacity and β-oxidation (lower β-HAD/CS), increases systemic carbohydrate utilization (higher RER), and drives a marked accumulation of triglyceride metabolites in the remaining muscle. Physical activity restriction further elevates RER and increases the pACC:ACC ratio but does not substantially change the local muscle metabolome beyond VML’s effects. These results add to the VML injury sequelae by indicating an exhausted capacity of the remaining muscle to oxidize fatty acids, potentially leading to triglyceride accumulation. Future work should delineate mechanisms of impaired carnitine shuttling/CPT-1 regulation, resolve region-specific mitochondrial dysfunction near the defect, determine time courses of fat accumulation and oxidation recovery, and evaluate therapeutic strategies (e.g., metabolic/rehabilitative interventions) to restore fatty acid oxidation and improve long-term muscle function and metabolic health.

• The reduced physical activity model (smaller cage size) may not fully recapitulate clinical sedentarism; preservation of muscle function in naïve-restricted mice suggests modeling limitations.
• Many biochemical and respiration assessments rely on tissue from the remaining muscle, which may under-represent deficits at the border/defect regions where oxidative capacity was more impaired.
• Only adult male C57BL/6J mice were studied, limiting generalizability across sexes, ages, and strains.
• Metabolomics was performed on gastrocnemius muscle only and may not capture systemic or other muscle-group metabolic changes.
• The study duration (8 weeks) may not reveal longer-term adaptations to restriction or recovery post-VML.