Restricted Physical Activity After Volumetric Muscle Loss Alters Whole-Body and Local Muscle Metabolism

Abstract

Volumetric muscle loss (VML) is the traumatic loss of skeletal muscle, resulting in chronic functional deficits and pathologic comorbidities, including altered whole-body metabolic rate and respiratory exchange ratio (RER), despite no change in physical activity in animal models. In other injury models, treatment with β₂ receptor agonists (e.g., formoterol) improves metabolic and skeletal muscle function. We first aimed to examine if restricting physical activity following injury affects metabolic and skeletal muscle function. Second, to enhance the metabolic and contractile function of the muscle remaining following VML injury through treatment with formoterol. Adult male C57Bl/6J mice (n=32) underwent VML injury to the posterior hindlimb compartment and were randomly assigned to unrestricted or restricted activity and formoterol treatment or no treatment; age-matched injury naïve mice (n=4) were controls for biochemical analyses. Longitudinal 24-hr physical activity and whole-body metabolism evaluations were conducted post-VML. In vivo muscle function was assessed terminally, and muscles were biochemically evaluated for protein expression, mitochondrial enzyme activity, and untargeted metabolomics. Restricting activity chronically post-VML had the greatest effect on physical activity and RER, reflected in reduced lipid oxidation, although changes were attenuated by formoterol treatment. Formoterol enhanced injured muscle mass, while mitigating functional deficits. These novel findings indicate physical activity restriction may recapitulate post-VML clinically, and adjunctive oxidative treatment may create a metabolically beneficial intramuscular environment while enhancing the injured muscle’s mass and force producing capacity. Further investigation is needed to evaluate adjunctive oxidative treatment with rehabilitation, which may augment the muscle’s regenerative and functional capacity following VML.

Graphical Abstract

This study evaluated the effects of physical activity restriction and treatment with a β₂ adrenergic receptor agonist, formoterol, on whole-body and local muscle metabolism function 8 weeks following volumetric muscle loss (VML) injury. Physical activity restriction decreased ambulation and metabolic rate while increasing the respiratory exchange ratio (RER), an indication of the diurnal use of substrates used as fuel, due to decreased lipid oxidation. In contrast, formoterol treatment increased injured muscle mass and function while improving glucose uptake and metabolic flexibility, the ability to transition efficiently between fuels over the course of the day. Neither treatment or activity restriction affected markers related to atrophy (MuRF1, Atrogin-1), hypertrophy (Akt), or regulators of mitochondrial biogenesis (PGC-1α).

Introduction

The metabolic flexibility of skeletal muscle is reflected in its ability to adapt to changes in metabolic demand and to efficiently transition between fuel sources depending on energetic need (Goodpaster & Sparks, 2017). The respiratory exchange ratio (RER), ranging from 0.7 to 1.0, is used to determine the proportion of substrate used as fuel, with higher values indicating greater carbohydrate oxidation and lower values greater lipid oxidation (Livesey & Elia, 1988). Metabolic inflexibility refers to the inability to transition efficiently between fuel sources and can result from various pathologies or injuries, indicated by a decreased range in diurnal RER fluctuations (Kelley et al., 1999; Muoio, 2014; Goodpaster & Sparks, 2017), and is associated with mitochondrial impairment (Muoio, 2014). Although skeletal muscle demonstrates remarkable plasticity following injury, such as ischemia-reperfusion- (Criswell et al., 2012) and crush- (Stratos et al., 2010) induced injuries, this ability is compromised in chronic or large-scale injuries (Greising et al., 2019). As properly functioning mitochondria are essential for maintaining and restoring skeletal muscle health and regulating energy metabolism, therapeutic interventions are needed to mitigate mitochondrial and metabolic impairments resulting from traumatic injury.

Mitochondria and skeletal muscle demonstrate a robust response to physical activity, as muscle contractions stimulate intracellular mitochondrial and protein synthesis signaling pathways (Gan et al., 2018). Beta-2 adrenergic receptor agonists mimic physical activity by activating these signaling pathways and stimulating expression of related factors, including PGC-1α and protein kinase B (Akt), while inhibiting proteolytic pathways. Independent of insulin signaling, β₂ adrenergic receptor agonists also regulate glucose homeostasis and uptake, primarily through mTOR activation and GLUT4 receptor translocation (Sato et al., 2014). The FDA-approved β₂ adrenergic receptor agonist, formoterol, improves mitochondrial function through activation of PGC-1α (Harcourt et al., 2007), the major regulator of mitochondrial biogenesis. Formoterol also enhances skeletal muscle hypertrophy in healthy controls (Ryall et al., 2006), and in models of spinal cord (Scholpa et al., 2019; Scholpa et al., 2021) and myotoxic (Ryall et al., 2008) injuries by eliciting an anabolic response through the PI3K/Akt/mTOR pathway. Concurrently, formoterol reduces gene and protein expression of signaling factors contributing to skeletal muscle atrophy and autophagy (Scholpa et al., 2019). In non-recoverable skeletal muscle injuries such as spinal cord and traumatic musculoskeletal injuries, formoterol treatment has proven beneficial for stimulating mitochondrial biogenesis and protein synthesis signaling and improving recovery of skeletal muscle mass and function (Scholpa et al., 2019; Scholpa et al., 2021; McFaline-Figueroa et al., 2022).

Traumatic skeletal muscle injury or surgical removal of skeletal muscle, termed volumetric muscle loss (VML) (Grogan & Hsu, 2011), overcomes the endogenous capacity for regeneration of lost tissue and recovery of muscle function (Corona et al., 2015; Corona et al., 2016). Acute and chronic structural and functional impairments of the remaining muscle are likely due to a combination of factors, including loss of contractile tissue, fibrosis (Greising et al., 2017; Hoffman et al., 2021), denervation (Corona et al., 2018; Sorensen et al., 2021), and altered metabolic function (Aurora et al., 2014; Greising et al., 2018; Southern et al., 2019; Dalske et al., 2021), among others (see for review: (Corona & Greising, 2016; Corona et al., 2016; Greising et al., 2016; Willett et al., 2016; Greising et al., 2020). The muscle remaining post-VML injury has reduced mitochondrial respiration (Greising et al., 2018; McFaline-Figueroa et al., 2022), decreased enzymatic activity within the mitochondrial electron transport chain (Aurora et al., 2014), and blunted PGC-1α gene and protein expression (Aurora et al., 2014; Southern et al., 2019), indicating alterations in mitochondrial signaling and oxidative function. Following VML, metabolic rate and RER are decreased chronically, despite no change in physical activity (Dalske et al., 2021). The inability of the remaining muscle to fully recover function following VML may be partially explained mechanistically by dysregulated mitochondrial function impacting the metabolic capacity of the remaining muscle. Restoring mitochondrial function by therapeutically targeting mitochondrial signaling may decrease the severity of whole-body and muscle-specific metabolic dysfunction.

Physical inactivity is associated with metabolic inflexibility and increased risk of chronic diseases, such as type II diabetes and metabolic syndrome, as well as inflammation, frailty, and mortality (Rynders et al., 2018). Over time, physical inactivity leads to skeletal muscle atrophy, weakness, and impaired metabolism. Due to the traumatic and complex nature of VML injury, patients in the clinic can undergo periods of reduced physical activity, which may exacerbate these outcomes. In animal models of VML injury, however, physical inactivity is not apparent. Despite no change in physical activity preclinically, muscle function is significantly reduced and does not recover, indicating a discordant relationship between physical activity and muscle function. Whole-body metabolism is also significantly altered chronically following VML, in part driven by increased lipid oxidation (Dalske et al., 2021). It is unknown if restricting physical activity following VML injury, which may recapitulate conditions clinically, affects whole-body metabolism, muscle-specific mitochondrial biogenesis and protein synthesis signaling, and muscle function. It also remains unknown whether treatment with the β₂ adrenergic receptor agonist, formoterol, can improve these outcomes, with or without physical activity restriction.

The primary goal of this work was to evaluate the impact of restricted physical activity on whole-body metabolism after VML, with a secondary goal to therapeutically enhance metabolic function of the muscle remaining after injury. Studies were designed to test the hypothesis that formoterol treatment will attenuate alterations in whole-body metabolism, enhance mitochondrial and protein synthesis signaling, and improve muscle function. It was expected that a VML-related disruption in the underlying metabolic function of remaining muscle will contribute to decreased metabolic rate and RER, which will be most pronounced when physical activity is restricted.

Methods

Ethical approval

All protocols were approved by the Institutional Animal Care and Use Committee at the University of Minnesota (#1803-35671A), in compliance with the Animal Welfare Act, the Implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals.

Experimental design

Adult male C57Bl/6J (n=44) mice 11 weeks of age were purchased from Jackson Laboratories (Stock #000664; Bar Harbor, ME). Mice were allowed at least a one-week acclimation period prior to initiating any part of the study. Mice were housed on a 12-hr light-dark cycle (light phase begins at 06:00) with ad libitum access to chow and water.

In a sub-set of mice (n=8) a pilot cross-over study was designed to examine the effect of restrictive cages on physical activity, RER, and metabolic rate. Mice were assigned to unrestricted (28x18x12.5 cm) or restricted (12.5x8.5x6.3 cm) activity for 1 week in each condition. Paired t-tests were performed for physical activity and metabolic outcomes between unrestricted and restricted activity groups. Mice ambulated ~50% less when activity was restricted (p<0.0001), Confirming the restricted cages effectively restricted activity, the following primary study was designed.

At ~13 weeks of age, the second sub-set of mice (n=32) underwent a full thickness VML injury to the posterior compartment muscle group (Figure 1). Immediately following surgery mice were randomly assigned to standard or restricted cages and standard chow or experimental chow formulated with formoterol. Mice were weighed weekly for the duration of the study. Approximately 1 week prior to VML and 2- and 6- weeks following VML all mice were assessed for physical and metabolic activity using the Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments). At 7 weeks post-VML glucose uptake was evaluated. Terminally 8 weeks post-VML (~21 weeks of age) in vivo muscle function was assessed, and skeletal muscles were harvested, frozen, and stored at −80°C for later analyses. Mice were euthanized with pentobarbital (>100mg/kg; s.q.). A final sub-set of mice (n=4) was used as non-injured, age-matched (~21 weeks of age) controls for all biochemical analyses.

Figure 1.

Experimental design schematic. Physical activity and metabolic monitoring were conducted longitudinally beginning 1 week prior to VML injury and at 2- and 6- weeks following VML. Glucose tolerance testing was conducted 7 weeks post-VML, and one week prior to the terminal timepoint. Terminally, muscle function was evaluated, and gastrocnemius muscles were saved for biochemical analysis.

Surgical creation of volumetric muscle loss (VML) injury

As described previously (Greising et al., 2018; Southern et al., 2019), a full thickness VML injury was surgically created to the posterior compartment muscle group (gastrocnemius, soleus, plantaris muscles). Mice received buprenorphine SR (2.0 mg/kg; s.q.) approximately 2 hours prior to surgery for pain management. Mice were anesthetized by isoflurane inhalation (~2.0%) under aseptic surgical conditions. A posterior-lateral incision was made through the skin to reveal the gastrocnemius muscle. Blunt dissection was used to isolate the posterior muscle compartment, and a metal plate was inserted between the tibia and the deep aspect of the soleus. A 4 mm punch biopsy (18.2±4.1 mg, ~15% volume loss of muscle) was performed on the middle third of the muscle compartment. Any bleeding was stopped with light pressure. Skin incisions were closed with 6.0 silk sutures and animals were monitored through recovery.

Adjunctive metabolic treatment

Mice in the formoterol treated group were given ad libitum access to a diet enriched with formoterol (Sigma #1286107) at a dose of 0.3 mg/kg/day immediately following VML surgery. This diet was composed of the same macro- and micro- nutrient content as the standard chow (Mod LabDiet #5053; LabDiet, St. Louis, MO) provided to mice in untreated groups.

Physical activity and whole-body metabolic evaluations

Physical activity and metabolic assessments were conducted as previously described (Dalske et al., 2021) with a few modifications. Briefly, metabolic data were collected in 10-min increments and physical activity counts, subsequently calculated as ambulatory distance (m), were collected in 10-sec increments over 24 hours and processed by the CLAMS system (Columbus Instruments) and data examination tool (Clax, v2.2.15; Columbus Instruments, Columbus, OH, USA). MATLAB (version R2020a, MathWorks, Natick, MA, USA) was used to calculate RER and metabolic rate averages and moving averages over the 24-hr periods. Prior to each test the system was calibrated and an air flow rate of 0.8 liters per minute was set. The reference gas was measured after each set of cage measurements. Mice were allotted a 24-hr acclimation period prior to the data collection period. All data were exported and used for analyses.

Glucose tolerance test

One week prior to harvest (7 weeks post-VML) intraperitoneal glucose tolerance testing was performed. Mice were fasted beginning at 08:00, and baseline blood glucose and subsequent injection of D-glucose saline solution (Sigma G7021; 2 mg/g, i.p.) occurred 6 hours later. The lateral tail vein was nicked with a 20 G needle, and blood glucose was measured with a glucometer (Freestyle Lite, Abbott) at 15-, 30-, 45-, 60-, 90-, and 120-min post-injection. Mice were continuously monitored, and any additional bleeding was stopped with light pressure.

In vivo muscle function

Muscle function of the posterior compartment was determined 8 weeks post-VML as previously described (Greising et al., 2018; Southern et al., 2019; Dalske et al., 2021). Briefly, mice were first anesthetized using inhaled isoflurane (1.5-2.0%) while body temperature was maintained at 37°C. The anesthetized mouse was positioned on its right side and the left foot was attached to the footplate of the dual-mode muscle lever system (300C-LR; Aurora Scientific, Aurora, Ontario, Canada) while the knee and hip were stabilized at 90°. Passive torque about the ankle was evaluated while the ankle was passively rotated 20° from neutral in both the plantar- and dorsi-flexion directions (total 40° of motion) under computer control of the servomotor. Passive torque was evaluated at 5° intervals. Active torque was evaluated after severing the common peroneal nerve to avoid recruitment of the anterior compartment. Stimulation of the sciatic nerve was conducted using Platinum-Iridium percutaneous needle electrodes. Optimal nerve stimulation was achieved and then a protocol to evaluate the torque frequency relationship was imitated (5, 10, 20, 40, 60, 80, 100, 150, and 200 Hz). Torque is expressed as mN·m per kg body weight.

Biochemical analyses

At the terminal time point, the gastrocnemius muscle of all animals was harvested and cut into thirds, encompassing the proximal, middle/defect, and distal regions of the muscle. The middle portion encompassing the VML defect region was weighed and homogenized in 10mM phosphate buffer (pH 7.4) with protease inhibitors at a ratio of 1:10 (mg/μl) and used for immunoblotting analyses. Total protein content was analyzed in triplicate and averaged using the Protein A280 setting on a NanoDrop One spectrophotometer (Thermo Scientific). The proximal portion was weighed and homogenized in 33mM phosphate buffer (pH 7.4) phosphate buffer at a ratio of 1:20 (mg/μl) and used for mitochondrial enzyme analyses. The distal portion was used for evaluation of metabolites.

Immunoblot analyses

Thirty or fifty μg of protein were separated by 4-15% or 4-20% SDS-PAGE, transferred onto a low fluorescence PVDF membrane, and immunoblotted. The following primary antibodies were used: MuRF1 (ECM Biosciences Cat# MP3401; Lot#5, RRID: AB_2208832, 1:1000), Atrogin-1 (ECM Biosciences Cat# AP2041; Lot#3, RRID:AB_2246979, 1:1000), Akt (Cell Signaling Technology Cat# 2920; Lot#8, RRID:AB_1147620, 1:2000), p-Akt (Cell Signaling Technology Cat# 9271; Lot#15, RRID:AB_329825, 1:1000), PGC-1α (Abcam Cat# ab54481; Lot#GR3393001, RRID:AB_881987, 1:1000), and Beta-2 adrenergic receptor (Abcam Cat# ab182136; Lot# GR3232710-13, RRID:AB_2747383, 1:10000). Primary antibodies were detected using a corresponding host- and isotype-specific fluorescence conjugated secondary antibody, DyLight 550 (Invitrogen, #SA5-10173) or DyLight 800 (Invitrogen, #SA5-10036). Immunoblots were blocked with 5% BSA, and primary and secondary antibodies were diluted in 5% BSA. Immunoblots were visualized with stain-free and fluorescence imaging using a ChemiDoc MP System (Bio-Rad Laboratories, Hercules, CA, USA) (Gürtler et al., 2013). Total protein was quantified in each lane using stain-free imaging, and the band of interest was identified at the molecular weight noted by the manufacturers’ technical information using the corresponding fluorescence image. The intensity of each band was normalized to total protein in each respective lane using Bio-Rad Laboratories Image Lab software (Hercules, CA). The gastrocnemius of age-matched injury naïve control mice was used for comparison and as a loading control on all immunoblots.

Mitochondrial enzyme analyses

Mitochondrial abundance was analyzed by citrate synthase activity from the reduction of 5,5’-dithio-bis (2-nitrobenzoic acid (DTNB, 0.773 mM) over time by measurement of absorbance at 412 nm as previously described (Southern et al., 2019; Dalske et al., 2021). β-nicotinamide adenine dinucleotide (NADH) oxidation was measured through the reduction of DCPIP at 600 nm. β-hydroxyacyl CoA dehydrogenase (β-HAD) activity was evaluated by incubating muscle homogenate in a buffer containing 100 mM triethanolamine, 0.451 mM NADH, and 5 mM EDTA, as previously described (McFaline-Figueroa et al., 2022). The reaction was started with acetoacetyl CoA (0.1 mM). β-HAD activity was monitored from the oxidation of NADH over time as determined by its absorbance at 340 nm (McFaline-Figueroa et al., 2022).

Untargeted metabolomic sample preparation and LC-MS/MS acquisition

Biocrates MxP^® Quant 500 (Biocrates Life Sciences, Innsbruck, Austria) commercially available kit enabled the quantification of up to 630 metabolites from 26 compound classes (e.g., amino acids, biogenic amines, bile acids, fatty acids, phosphatidylcholines, ceramides, di- and triglycerides). Lipids and hexoses were measured by flow injection analysis-tandem mass spectrometry (FIA-MS/MS) and small molecules were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The experimental metabolomics measurement technique is described in detail by patents EP1875401B1 (Ramsay et al., 2014a) and EP1897014B1 (Ramsay et al., 2014b). Briefly, a 56-well based sample preparation device was used to quantitatively analyze the metabolite profile in the distal portion of all experimental and injury naïve control gastrocnemius muscles. This device consists of inserts that have been impregnated with internal standards, along with a predefined sample amount. Subsequently, a phenyl isothiocyanate (PITC) solution was added to derivatize some of the analytes (e.g., amino acids). After derivatization completion, the target analytes were extracted with an organic solvent, followed by dilution. The obtained extracts were analyzed by FIA-MS/MS and LC-MS/MS methods using multiple reaction monitoring (MRM) for analyte detection.

Biocrates’ LC-MS/MS analysis was carried out using a Shimadzu LC-20AD XR (Shimadzu USA Manufacturing Inc Canby, Oregon) coupled to a Sciex QTRAP 5500 mass spectrometer (Sciex, Framingham, MA). Chromatographic separation was achieved using the Biocrates’ C18 separation column (4.5x 100mm, 1.7μM) maintained at 50°C. Mobile phases consisted of: A (0.2% formic acid in water) and B (0.2% formic acid in 95% acetonitrile). The flow rate of 0.8ml/min was maintained from 0-4.7 min, with a subsequent increase to 1.0 ml/min from 4.7-5.1 min, followed by a decrease to the initial flow rate (0.8ml/min) for the remainder of the negative ion run time, 5.1-5.8 min. The elution started with isocratic 100% A, 0-0.50 min; 0.5-2.0 min linear gradient 75-50% A; 2.0-3.0 min linear gradient 50-25% A; 3.0-3.5 min linear gradient 25-0%; 3.5-5.1 min isocratic hold 0% A; 5.1-5.8 min isocratic hold 100% A. For the FIA, flow rates were set at 0.03 ml/min, with an injection volume of 5 μl. The flow rate of 0.03 ml/min was maintained from 0-2.4 min, with a subsequent increase in flow rate to 0.20 ml/min for 2.4-3.0, and returned to the initial flow rate of 0.03 ml/min at 3.0 min. The elution maintained an isocratic hold of 100% B for the entirety of the FIA run time, 0-3.0 min.

Statistical analysis

Analysis was performed with JMP Pro statistical software (version 16.0.0 SAS Institute, Cary, NC, USA) and GraphPad Prism (version 9.2.0, GraphPad Software, San Diego, CA, USA). Body mass and gastrocnemius mass were evaluated across all VML-injured groups by two-way ANOVA. Physical activity and whole-body metabolic data were analyzed over 24 hours for each group at 1 week pre- and at 2- and 6- weeks post-VML. Three-way ANOVA was used to examine the effects of activity and treatment on metabolic rate, RER, and lipid and carbohydrate oxidation across time. Percent changes were calculated for physical activity and metabolic outcomes for each animal from pre- to 2- and 6- weeks post-VML and analyzed by three-way ANOVA. The difference in RER (delta) between the active and inactive phases was calculated and analyzed by two-way ANOVA. Area under the curve (AUC) was calculated for 24-hr metabolic rate and RER and analyzed by one-way ANOVA. Three-way ANOVA was used to evaluate blood glucose across timed intervals during the glucose tolerance test. Two-way ANOVA compared blood glucose AUC and outcomes of muscle function across activity and treatment groups. One-way ANOVA evaluated terminal outcomes of protein expression and mitochondrial enzyme kinetics. When appropriate Tukey’s Honest Significant Difference post-hoc analyses were used to determine specific group significance.

Metabolite concentrations were calculated using appropriate mass spectrometry software, and data was imported into Biocrates MetIDQ^™ software (Biocrates Life Sciences AG, Innsbruck, Austria). Metabolite sums and ratios were calculated in the Biocrates MetaboINDICATOR^™ module with the purpose of associating metabolites with their related biological functions. Metabolites missing greater than 20% of measurements were excluded from the statistical analysis. Hypothesis testing was performed using independent t-tests to compare individual groups. The obtained p-values were used for statistical significance and raw values were used in transformations necessary for data visualizations.

Data are presented as mean±SD, with individual data points displayed per mouse. Statistical significance level was set at p≤0.05. During all evaluations the research team was blinded to the experimental groups.

Results

Animals

Mice underwent and recovered from the VML surgery. Immediately following VML, mice were randomized into one of four groups that varied by activity (unrestricted, restricted) and treatment (untreated, formoterol treated). No difference in body mass was observed across experimental groups up to 8 weeks following VML (Table 1). Body mass of injury naïve mice (30.2 ± 1.0 g) was not different than any experimental groups (p=0.884). Despite no body mass differences the mass of the gastrocnemius muscle was impacted by formoterol treatment (Table 1; main effect of treatment p=0.014). Untreated mice had the smallest normalized injured (left) gastrocnemius mass, which was not impacted by restricting activity (Table 1; p=0.020). When comparing normalized injured (left) gastrocnemius mass with those of the injury naïve controls (5.41 ± 0.18 mg/g), only mice that received formoterol treatment and with unrestricted activity had a similar mass while all others had a significantly lower normalized left gastrocnemius mass (p<0.0001). Similarly, evaluations of the right (uninjured) gastrocnemius mass revealed formoterol treatment increased mass compared to no treatment (Table 1). When comparing normalized uninjured (right) gastrocnemius mass with injury naïve controls (5.44 ± 0.05 mg/g), all groups had a similar mass to controls, but untreated mice with unrestricted activity had a lower normalized mass than formoterol treated mice with restricted activity (p=0.008). Overall, indicating a protective effect of formoterol treatment on muscle mass following VML injury.

Table 1.

Animal characteristics and weights

	Unrestricted		Restricted		Two-way ANOVA p-value
	VML	VML Formoterol	VML	VML Formoterol	Main Effect of Activity	Main Effect of Treatment	Interaction
	(n=8)	(n=8)	(n=8)	(n=8)
Pre-VML Body Mass (g)	27.2 ± 1.7	27.1 ± 2.4	26.6 ± 1.7	27.1 ± 1.7	0.631	0.711	0.657
2-week Body Mass (g)	26.7 ± 1.2	28.0 ± 1.5	26.7 ± 2.4	27.4 ± 1.6	0.638	0.119	0.638
6-week Body Mass (g)	30.0 ± 2.4	30.5 ± 2.6	28.4 ± 4.2	29.8 ± 2.3	0.282	0.349	0.665
Terminal Body Mass (g)	30.9 ± 2.2	31.6 ± 2.6	30.4 ± 3.2	30.7 ± 2.5	0.476	0.641	0.839
Right (uninjured) Gastrocnemius Mass (g)	159.4 ± 12.0‡§	176.6 ± 23.1	162.5 ± 7.6‡§	180.0 ± 11.3	0.537	0.002	0.981
Right (uninjured) Gastrocnemius Mass/Body Mass (mg/g)	5.16 ± 0.34‡§	5.59 ± 0.41	5.38 ± 0.46‡§	5.88 ± 0.33	0.072	0.002	0.776
Left (injured) Gastrocnemius Mass (mg)	130.4 ± 16.2‡§	158.5 ± 24.1	139.4 ± 14.5‡§	146.0 ± 19.0	0.794	0.014	0.117
Left (injured) Gastrocnemius Mass/Body Mass (mg/g)	4.21 ± 0.35‡§	5.01 ± 0.45	4.58 ± 0.23	4.75 ± 0.38	--	--	0.020

Data presented as mean±SD. Significantly different than ‡VML Unrestricted Formoterol

^§VML Restricted Formoterol

Physical and whole-body metabolic activity

One-week prior to VML surgery and randomization into experimental groups, mice ambulated 1.2±0.4 km daily. Supporting the pilot cross-over experiments (see Methods), paired analyses revealed restricting activity decreased ambulation ~41% by 6 weeks post-VML compared to pre-VML and ~28% compared to unrestricted activity, independent of treatment (Figure 2A; main effect of activity p<0.0001).

Figure 2. Physical activity and metabolic rate up to 6 weeks post-VML.

A) Restricted activity decreased physical activity as a percentage of baseline ambulation at 2- and 6- weeks post-VML (main effect of activity p<0.0001; treatment p=0.446; time p=0.085; interactions p≥0.116). B) Metabolic rate was impacted by activity and time (interaction p=0.040; main effect of activity p=0.0004; time p=0.002; treatment p=0.575) specifically at 2 weeks post-VML. C) The metabolic rate percentage change from pre-VML was greater in mice with unrestricted activity vs. restricted at 2- and 6- weeks (interaction activity x time p=0.036; main effect of activity p<0.0001; time p=0.001; treatment p=0.147). D) Over 24 hrs., metabolic rate fluctuations at 6 weeks were evaluated. E) Quantified by area under the curve (AUC), mice treated with formoterol with restricted activity had the lowest metabolic rate (p=0.001). Significantly different than § VML Restricted Formoterol; * restricted activity at 2 weeks; ∫∫ unrestricted activity at 6 weeks; ∯ restricted activity at 6 weeks. Individual data points represent a single mouse. Graphs displaying only mean±SD include n=8 per group.

At baseline, 24-hr metabolic rate was 18.3±1.2 kcal/hr. At 2 weeks post-VML, mice with unrestricted activity demonstrated the highest metabolic rate, which was reduced by 6 weeks post-VML (Figure 2B; main effect of time p=0.002, main effect of activity p=0.0004). Restricting activity decreased metabolic rate ~10% by 6 weeks post-VML, independent of treatment (Figure 2C; interaction activity x time p=0.036). Formoterol treatment combined with restricting activity had the greatest impact in lowering metabolic rate over 24-hr (Figure 2D-E; p<0.001), corresponding to reduced ambulation.

Prior to VML, 24-hr RER was 0.88±0.02, and lipid and carbohydrate oxidation rates were 1.12±0.26 and 5.12±0.90 g/min, respectively. By 6 weeks post-VML, RER was lowest for mice with unrestricted activity and highest for untreated mice with restricted activity, evaluated as an average (Figure 3A; interaction activity x time p=0.006; activity x treatment p=0.034) and a percentage of pre-VML (Figure 3B; interaction activity x time p=0.016). Suggesting a hypermetabolic state when activity is limited following injury. Similarly, restricting activity without treatment resulted in the highest area under the curve for 24-hr RER, while unrestricted activity without treatment resulted in the lowest (Figure 3C-D; p<0.0001). Formoterol treatment increased 24-hr RER compared to no treatment with unrestricted activity, but decreased RER compared to no treatment with restricted activity (Figure 3C-D). Formoterol also increased the change in RER between the active and inactive phases of the light cycle (Figure 3E; main effect of treatment p=0.012). Suggesting formoterol attenuates large changes in RER resulting from either VML injury or restricted activity and improves metabolic flexibility.

Figure 3. Respiratory exchange ratio (RER) and carbohydrate and lipid oxidation up to 6 weeks post-VML.

A) Mice with restricted activity had the highest RER at 6 weeks post-VML (interaction activity x time p=0.006; main effect of activity p<0.0001; time p=0.156; treatment p=0.691). Considering both activity and treatment, untreated mice with restricted activity had the highest RER, while untreated mice with unrestricted activity had the lowest. Formoterol treated mice had similar RER independent of activity (interaction activity x treatment p=0.034). B) The RER percentage change from pre-VML was greatest for mice with restricted activity at 2- and 6- weeks and was reduced by 6 weeks for unrestricted mice (interaction activity x time p=0.016). C) Over 24 hrs., RER fluctuations at 6 weeks were evaluated. D) Quantified by the AUC, mice with restricted activity had the highest RER, while untreated mice with unrestricted activity had the lowest (p<0.0001). E) The delta RER between active and inactive periods was greater with formoterol treatment than untreated, indicating enhanced metabolic flexibility (main effect of treatment p=0.012). F) By 6 weeks, mice with restricted activity had the lowest lipid oxidation (interaction activity x time p=0.002; activity x treatment p=0.023; main effect of activity p<0.0001; time p=0.082; treatment p=0.802) and G) a reduced lipid oxidation percentage change (interaction activity x time p=0.023; main effect of activity p<0.0001; time p=0.027; treatment p=0.856). H) Independent of activity and treatment, there were similar carbohydrate oxidation rates across time points (interaction activity x time p=0.007; main effect of activity p=0.496; time p=0.934; treatment p=0.309) and I) as a percentage of pre-VML (interaction activity x time p=0.022; main effect activity p=0.205; time p=0.297; treatment p=0.053). Significantly different than † VML; ‡ VML Formoterol; § VML Restricted Formoterol; ∫ unrestricted activity at 2 weeks; * restricted activity at 2 weeks; ∯ restricted activity at 6 weeks. Individual data points represent a single mouse. Graphs displaying only mean±SD include n=8 per group.

Increased RER at 6 weeks post-VML is largely due to reduced lipid oxidation when activity is restricted compared to unrestricted (Figure 3F). With unrestricted activity, RER was reduced primarily due to a ~24% increase in lipid oxidation (Figure 3F; interaction activity x time p=0.002). Evaluated as a percentage of pre-VML, restricting activity, regardless of treatment, increased RER 2.3% due to a ~21% reduced lipid oxidation (Figure 3G; interaction activity x time p=0.023). No differences in carbohydrate oxidation across groups were observed up to 6 weeks post-VML despite an interaction between activity and time (Figure 3H-I; p≤0.022).

Glucose tolerance testing

Glucose uptake is impaired in conditions of chronic disease and sedentarism (Rynders et al., 2018) but can be improved with β₂ adrenergic receptor treatment such as formoterol (Sato et al., 2014). Across groups, blood glucose was highest at 15- and 30-min post-injection (Figure 4A; main effect of time p<0.0001). Up to 60-min following injection, blood glucose plateaued for the untreated VML group while it declined with formoterol treatment. These observations were reflected in a lower blood glucose for formoterol treatment, independent of activity, indicating greater clearance of glucose from the blood than no treatment (Figure 4B; p=0.001). Suggesting formoterol enhances glucose uptake potentially through upregulation of related receptors or improved insulin regulation.

Figure 4. Glucose tolerance testing 7 weeks following injury.

A) Blood glucose was impacted by activity and treatment (interaction p=0.045) and by time (main effect p<0.0001). B) Over the course of two hours following injection of D-glucose solution, total blood glucose AUC was lower with formoterol (main effect of treatment p=0.001). n=8 per group.

In vivo muscle function

Formoterol-treated mice demonstrated greater maximal isometric torque normalized to body mass than untreated mice, regardless of activity (Figure 5; main effect of treatment p=0.049). This data corresponds with the significantly greater gastrocnemius masses in formoterol-treated mice, independent of activity (Table 1). Although passive torque about the ankle joint was not impacted (Table 2). Maximal twitch and torque tracings were evaluated for contractile properties. There were no differences across treatment or activity groups for contractile properties, including time-to-peak twitch, half-relaxation time, and rates of contraction and relaxation (Table 2). Additionally, submaximal relative to maximal torques were not different across groups (Table 2). These data suggest torque gains following formoterol treatment may be due to recovery of gastrocnemius muscle mass.

Figure 5. Maximal isometric torque 8 weeks following injury.

Mice with formoterol treatment demonstrated greater maximal isometric torque normalized to body mass, independent of activity (main effect of treatment, p=0.049). For comparison only, data from previously published (Dalske et al., 2021) age and sex matched control mice is ~670 mN•m/kg, highlighting the VML-induced impact on function. Individual data points represent a single mouse.

Table 2.

In vivo contractile parameters

	Unrestricted		Restricted		Two-way ANOVA p-value
	VML	VML Formoterol	VML	VML Formoterol	Main Effect of Activity	Main Effect of Treatment	Interaction
	(n=7)	(n=6)	(n=7)	(n=6)
Maximal Passive Torque at 20° Dorsiflexion (mN·m/kg)	193.8±46.4	194.6±61.3	163.6±51.0	169.0±46.2	0.180	0.879	0.910
40Hz/Peak (%)	37.4±9.6	43.2±21.9	41.8±6.2	31.0±8.0	0.441	0.623	0.108
60Hz/Peak (%)	63.0±12.5	69.2±15.4	69.2±13.2	64.7±13.2	0.881	0.874	0.325
Time to Peak Twitch (s)	0.021±0.001	0.022±0.004	0.020±0.002	0.020±0.001	0.201	0.739	0.864
½ Relaxation Time (s)	0.014±0.001	0.014±0.002	0.013±0.001	0.013±0.001	0.242	0.808	0.931
+dP/dt (mN·m/s)	195.9±85.6	196.7±65.9	207.3±57.3	312.7±43.1	0.589	0.891	0.914
−dP/dt (mN·m/s)	−201.5±91.3	−259.4±42.8	−238.7±85.9	−270.5±57.7	0.415	0.137	0.657

Data presented as mean±SD.

Protein expression

The effects of activity and treatment on the protein expression of atrophy-, protein synthesis-, and mitochondria-related signaling markers were evaluated in the remaining muscle at the defect site. There were minimal differences in protein expression across experimental groups for atrophy- (MuRF1, Atrogin-1) and protein synthesis- (Akt/pAkt) related markers and for the mitochondrial regulator PGC-1α (Figure 6A-F; p≥0.235). However, there was a significant reduction in the protein expression of β₂ adrenergic receptors for all VML mice with unrestricted activity compared to injury naïve controls (Figure 6G; p=0.015), although this effect was not different across VML-injured groups. Results suggest VML injury reduces β₂ adrenergic receptor protein expression at the injury site but does not chronically impact protein expression of markers related to atrophy and protein synthesis signaling.

Figure 6. Protein expression of atrophy, protein synthesis, and mitochondrial biogenesis markers at the VML defect site.

A) Representative stain-free blot displaying total protein, and corresponding fluorescent bands and molecular weight for each marker. Bands were normalized to total protein in each respective lane to quantify relative protein expression compared to injury naïve control. Relative protein expression of the atrophy markers B) MuRF1 and C) Atrogin-1 (p≥0.505). Relative expression of the D) total and E) phosphorylated protein synthesis marker Akt, and the corresponding ratio of F) phosphorylated to total Akt (p≥0.235). G) Relative protein expression of the mitochondrial regulator PGC-1α (p=0.591). H) Relative β₂ adrenergic receptor protein expression was lower in mice with unrestricted activity (p=0.015). Significantly different than *Injury Naïve Control. Abbreviations: C, injury naïve control; VML, volumetric muscle loss; VML Form, VML Formoterol. Individual data points represent a single mouse.

Mitochondrial enzymatic activity

Biochemical evaluation of the proximal gastrocnemius muscle determined the effects of activity and treatment on mitochondrial enzymatic activity of the muscle remaining following injury. Independent of activity, formoterol treatment decreased citrate synthase activity within the muscle, indicating a reduction in mitochondrial abundance (Table 3; p=0.019). Similarly, ß-HAD, the third enzyme of ß-oxidation, was reduced following formoterol treatment (p=0.011), but this difference was non-significant when normalized to citrate synthase activity (Table 3; p=0.124). Pyruvate dehydrogenase (PDH) activity, measured as joules of energy produced from NADH, was greatest with no treatment in conjunction with unrestricted activity and lowest when activity was restricted (Table 3; p=0.001), even when normalized to citrate synthase activity (Table 3; p=0.002). These results suggest a heightened oxidative state in the muscle remaining following VML with unrestricted activity and a greater glycolytic state or reliance on lipid oxidation when activity is limited. Pyruvate dehydrogenase activity was not impacted by formoterol treatment compared to injury naïve control muscles. However, restricting activity mitigated the heightened pyruvate dehydrogenase activity observed when physical activity was unrestricted. Similar to whole-body RER, results suggest treatment with formoterol attenuates large fluctuations in metabolic enzymatic activity resulting either from VML or activity restriction.

Table 3.

Metabolic enzymatic activity of the gastrocnemius muscle

		Unrestricted		Restricted
	Injury Naïve Control	VML	VML Formoterol	VML	VML Formoterol	One-Way ANOVA p-value
	(n=4)	(n=8)	(n=8)	(n=8)	(n=8)
Citrate Synthase (μmol/min/g)	675.9±110.0	546.0±66.9	534.7±76.3*	572.2±76.4	513.7±62.3*	0.019
ß-HAD (μmol/min/g)	19.4±2.8	15.9±2.0	14.8±2.5*	17.2±2.9	14.4±2.0*	0.011
ß-HAD/CS (μmol/min/g)	21.3±1.3	21.6±1.5	20.4±1.2	22.2±1.5	20.7±1.6	0.124
NADH (pmols)	53928±8015	53627±6246	48296±7930	53072±6984	53627±6246	0.150
JNADH (pmols/s•mg)	48.3±8.6	49.6±9.9	46.2±7.0	39.3±7.7†	37.2±9.5*†‡	0.001
JNADH/CS (pmols/s•mg)	54.8±16.2	68.9±18.4	64.8±11.3	51.2±9.8*	53.9±13.2†	0.002

ß-HAD, 3-hydroxyacyl-CoA dehydrogenase; CS, citrate synthase; NADH, nicotinamide adenine dinucleotide; JNADH, joules of energy produced from NADH. Data presented as mean±SD. Significantly different than *Injury Naïve Control

^†VML Unrestricted

^‡VML Unrestricted Formoterol

Metabolite investigation

Metabolites were evaluated in an untargeted approach to establish the pathophysiologic effects in the muscle remaining after VML injury. Currently there is no understanding of the changing regulation of metabolites after injury. Thus, data presented are comparisons between the injury naïve control and VML injured, unrestricted untreated, groups. In total, five-hundred-and-four metabolites were evaluated for significant expression and thirty-five were significantly expressed in VML unrestricted untreated group compared to injury naïve control (Figure 7A). Metabolites related to polyamine regulation, specifically putrescine (p=0.001) and spermidine (p=0.0002), were significantly upregulated in the VML unrestricted untreated group when compared to injury naïve control (Figure 7B). Similarly, the ratio of polyamine synthesis (p=0.001) and total sum of polyamines (p=0.002) were upregulated 1.9- and 2.1-fold, respectively (Figure 7C). Sum of polyamines was calculated as the sum of putrescine, spermidine, and spermine concentrations while polyamine synthesis was calculated using the formula (putrescine + spermidine + spermine)/ornithine. Arginine (p=0.001) and global arginine bioavailability ratio (GABR; p<0.0001) were significantly upregulated in VML unrestricted untreated (Figure 7B-C). Unrestricted untreated VML injury displayed upregulation of gamma-aminobutyric acid (GABA; p=0.008) and GABA synthesis (p=0.013) when compared to injury naïve control (Figure 7B-C). Given arginine is a precursor for polyamines, which also play a role in GABA synthesis, suggesting not only a dysfunction in polyamine metabolism, but a possible interconnectivity between these metabolic pathways following VML injury. Downregulation of nitric oxide synthase (NOS) activity was observed in VML unrestricted untreated group compared to injury naïve control (Figure 7C; p<0.0001). NOS activity was calculated as the ratio of citrulline/arginine. This untargeted metabolomics analysis confirmed that VML injury alone impacts the metabolite environment of the skeletal muscle chronically.

Figure 7. Metabolite changes between control and VML injured muscle.

A) Volcano plot of significantly up- and down-regulated metabolites with a >2.0-fold change between VML unrestricted untreated and injury naïve control. The dotted horizontal and longitudinal axes indicated the lower thresholds for statistical (p<0.05) and biological significance (2-fold change) of expression. B) Higher concentrations of spermidine, putrescine, GABA, and arginine (p≤0.008) were found following VML but no difference in spermine concentration (p=0.255). C) Metabolite sums and ratios were increased for polyamine and GABA synthesis, NO synthase activity, and GABR following VML (p≤0.013). Significantly different than *Injury Naïve Control. Abbreviations: GABA, gamma-aminobutyric acid; NO, nitric oxide; GABR, global arginine bioavailability ratio. Individual data points represent a single mouse.

Discussion

Skeletal muscle is a highly metabolic tissue that demonstrates robust plasticity following acute injury; however, in cases of chronic or large-scale injury, such as VML, the ability of the muscle to regenerate and recover metabolic and contractile function is impaired. Due to the severity and complexity of VML, patients in the clinic can undergo periods of reduced physical activity, which may have further deleterious consequences on metabolic function of the muscle and the body as a whole. Modeling physical activity restriction following VML may provide greater insight into whole-body physiological consequences of injury (Basten et al., 2022), as well as a model to evaluate new interventions. Recovery of the muscle following injury may be improved by treatment with the highly selective β₂ adrenergic receptor agonist, formoterol. Herein, physical activity restriction altered whole-body metabolism as a result of reduced lipid oxidation, while preserving local metabolic activity. Treatment with formoterol attenuated large fluctuations in whole-body metabolism and enhanced glucose uptake, while mitigating functional deficits in the muscle resulting from either injury or reduced physical activity. Results corroborate formoterol’s influence in enhancing skeletal muscle mass and function, while impacting metabolic function, in injury models of spinal cord (Scholpa et al., 2021), myotoxic (Ryall et al., 2008), and more recently VML (McFaline-Figueroa et al., 2022).

Normally, chronic restriction of physical activity maladaptively increases whole-body RER by decreasing whole-body lipid oxidation and increasing carbohydrate oxidation independent of energy balance (Stein & Wade, 2005; Bergouignan et al., 2006; Bergouignan et al., 2009). In addition to altered whole-body RER, physical inactivity (e.g., bedrest) and sedentarism significantly impairs insulin sensitivity and is directly correlated with lower content and activity of key proteins involved in glucose transport (Bergouignan et al., 2011). Hyperinsulinemia ensues, contributing to insulin resistance. Bedrest also leads to the accumulation of lipotoxic intermediaries, including triglycerides, diacylglycerols, and ceramides, contributing to insulin resistance and the onset of type II diabetes and obesity. Among other contributing factors, the maladaptive changes to physical inactivity can ultimately lead to metabolic inflexibility and metabolic syndrome (Bergouignan et al., 2011). In line with a recent report (Dalske et al., 2021), whole-body metabolic rate and RER were blunted following VML injury. In contrast, when activity was restricted, whole-body RER increased due to decreased lipid oxidation and elevated carbohydrate oxidation, similar to bedrest clinically as well as obesity. The large fluctuations in RER, due to either VML or physical activity restriction, were attenuated by treatment with formoterol, and the delta RER between the active and inactive periods was increased, indicating enhanced whole-body metabolic flexibility. Observations suggest that modeling VML injury with restricted activity may recapitulate expected clinical conditions, providing greater physiological insight into metabolic and functional limitations following VML, and formoterol treatment may be beneficial for partially mitigating negative whole-body metabolic consequences of injury.

Despite increased whole-body RER and carbohydrate oxidation following VML injury combined with physical activity restriction, the activity of PDH, the gateway enzyme between glycolysis and oxidative phosphorylation, at the local muscle level was similar to injury naïve controls and lower than VML injury alone (i.e., without activity restriction). Located in the mitochondrial matrix, PDH is responsible for converting pyruvate to acetyl CoA, which then enters the citric acid cycle as part of aerobic metabolism. Activity of PDH is sensitive to oxidative stress and dependent on the availability of various co-enzymes, including nicotinamide dinucleotide (NAD+) and flavin adenine dinucleotide (FAD). PDH is also inhibited by the accumulation of products such as acetyl CoA and NADH. Normally when physical activity is chronically reduced, whole-body metabolism maladaptively relies more heavily on the oxidation of carbohydrates rather than lipids, partially resulting from accumulation of products at the site of the PDH complex. While skeletal muscle as a whole significantly contributes to daily metabolic fluctuations, the discordant findings between whole-body and local muscle metabolism following VML injury may be explained by the fact that a single muscle does not solely drive whole-body metabolism, and the whole-body may be more greatly impacted by physical activity restriction or other factors such as hormones (i.e., insulin) than the injured muscle itself.

Several reports of the muscle following VML injury indicate a possible survival bias of type I muscle fibers (or selective loss of type II fibers), partially explaining alterations in the functional and metabolic capacity of the muscle chronically following VML (Corona et al., 2018; Chao et al., 2019; Mintz et al., 2020; Dalske et al., 2021). It is possible that restricting physical activity further alters these muscle fibers, as is the case during chronic muscle disuse. Treatment with formoterol may attenuate local fiber changes as evidenced by increased force production (i.e., greater force production of the type II fibers), independent of physical activity. Further analyses of how formoterol may alter the muscle’s phenotype to be more glycolytic is needed. Notably, chronic administration of β₂ adrenergic receptor agonists can improve glycolytic capacity of fibers, while enhancing the force-producing capacity of muscle (Sato et al., 2011; Ohnuki et al., 2013). In support, formoterol treatment decreased local muscle ß-HAD and citrate synthase activity, indicating reduced muscle-specific lipid oxidation and mitochondrial content, respectively, while PDH activity was similar to injury naïve controls. Concurrently, compared to no treatment following VML, formoterol improved blood glucose clearance, independent of physical activity, suggesting improved insulin sensitivity and supporting enhanced metabolic flexibility with treatment. This may be due to stimulating the insulin/insulin like growth factor-1 signaling pathway, which further promotes protein synthesis and skeletal muscle hypertrophy (Gonçalves et al., 2019). Overall, findings suggest a greater glycolytic state of the muscle with formoterol treatment, which may be beneficially counteracting the slowing of the muscle induced by VML injury.

Polyamine dysregulation has been identified in various pathological conditions but has been specifically discussed for its role in adipogenesis and the development of metabolic syndrome. Endogenous spermidine and spermine aid in the differentiation of preadipocytes and are increased during adipogenesis (Brenner et al., 2015). Our observations of increased sum of polyamine metabolites following VML support enhanced adipogenic mechanisms and the development of myosteatosis in the affected muscle. Polyamines, specifically spermidine, have also been implicated in the activation of the 5’ adenosine monophosphate-activated protein kinase (AMPK) pathway (Ni & Liu, 2021). Given the role of AMPK as an activator of PDH, it is possible that the observed upregulation of polyamines in VML-injured muscle could have downstream effects on both glucose and fatty acid oxidation. Significant upregulation of arginine and GABR following VML suggest sufficient substrate availability for the nitric oxide (NO) signaling pathway, as NO is produced from arginine. However, downregulation of NO synthase indicates a disruption in NO production (De Palma et al., 2014). Defective NO signaling may be an additional contributor to the blunted myogenic response following VML injury. Enhanced NO signaling has been reported in similar skeletal muscle injury models (Filippin et al., 2011; Sakurai et al., 2013), justifying further examination of this mechanism in a VML-injured muscle model.

Protein expression of various atrophy-, hypertrophy-, and mitochondria-related markers were evaluated to investigate the mechanistic effects of physical activity level and formoterol treatment following VML. Formoterol is known to promote protein synthesis by activating cyclic AMP-PKA signaling and stimulating downstream activation of Akt and mTOR, the major regulator of skeletal muscle hypertrophy. Concurrently, activation of Akt inhibits protein breakdown by directly phosphorylating FoxO and suppressing the ubiquitin-proteosome and autophagy-lysosome pathways (Joassard et al., 2013). This anabolic response is greater in magnitude in predominantly fast muscles than in slow muscles (Ryall et al., 2006; Sato et al., 2010). In contrast, physical inactivity promotes net protein breakdown, resulting in muscle atrophy. Observations revealed no significant alterations in protein expression across hypertrophy- and atrophy-related markers, regardless of physical activity or treatment status following VML. In models of spinal cord injury, protein expression of hypertrophy markers (e.g., phosphorylated Akt) are increased and atrophy markers (e.g., MuRF1) are decreased compared to untreated, and similar to control levels, up to 21 days following injury (Scholpa et al., 2019). It is possible that acute administration of formoterol alters protein expression initially but chronically its effect is moderated, as formoterol treatment increased normalized gastrocnemius mass up to 8 weeks following VML, similar to injury naïve controls. Further, despite the impact of formoterol in improving mitochondrial biogenesis and function, PGC-1α protein expression was unaffected by treatment, supporting a blunted response to treatment in line with previous reports following VML injury (Aurora et al., 2014; Greising et al., 2018; Southern et al., 2019). It is noteworthy that β₂ adrenergic receptor protein expression was reduced following VML but increased to injury naïve levels with physical activity restriction, though treatment with formoterol did not appear to impact these changes.

This work provides novel insight into the impact of physical activity restriction and adjunctive oxidative treatment on whole-body and local muscle metabolism and function following VML injury. First, findings indicate physical activity restriction may recapitulate clinical conditions following VML, making this model physiologically relevant for evaluating the pathophysiology of VML and the efficacy of various treatment strategies. Second, data suggest adjunctive oxidative treatment, specifically formoterol, may create a metabolically beneficial intra-muscular environment while enhancing the injured muscle’s mass and force producing capacity. As formoterol is FDA-approved and readily available in the clinic, its use in treating VML injury is a viable option. Further investigation is warranted to evaluate a combined regenerative rehabilitative approach incorporating formoterol treatment with rehabilitation, which may augment the muscle’s regenerative and functional capacity while enhancing whole-body metabolism following VML injury.

Key points:

• The natural ability of skeletal muscle to regenerate and recover function is lost following complex traumatic musculoskeletal injury, such as volumetric muscle loss (VML), and physical inactivity following VML may incur additional deleterious consequences for muscle and metabolic health.

• Modeling VML injury-induced physical activity restriction altered whole-body metabolism, primarily by decreasing lipid oxidation, while preserving local skeletal muscle metabolic activity.

• The β₂ adrenergic receptor agonist formoterol has shown promise in other severe injury models to improve regeneration, recover function and to enhance metabolism. Treatment with formoterol enhanced mass of the injured muscle and whole-body metabolism while mitigating functional deficits resulting from injury.

• Understanding of chronic effects of the clinically available and FDA-approved pharmaceutical formoterol could be a translational option to support muscle function after VML injury.

Biography

Christiana Raymond-Pope obtained her PhD in Kinesiology, with an emphasis in Exercise Physiology, at the University of Minnesota. Her doctoral research evaluated body composition and muscle function in previously injured athletes clinically. To gain further mechanistic knowledge of post-injury skeletal muscle repair and regeneration, she completed a postdoctoral fellowship within the University of Minnesota’s School of Kinesiology supported by a NIH- T32 grant through the Department of Orthopedic Surgery. She is presently a Research Associate within Dr. Sarah Greising’s laboratory investigating novel regenerative and rehabilitative interventions to improve muscle regeneration and function following traumatic musculoskeletal injuries and in disease conditions.

Data availability statement

The datasets used and/or analyzed during the current study are primarily presented in the current manuscript and are available from the corresponding author on reasonable request.