Volumetric muscle loss (VML) injuries result in a non-recoverable loss of muscle tissue and function due to trauma or surgery. Reductions in physical activity increase the risk of metabolic comorbidities over time, and it is likely that VML may reduce whole-body activity. However, these aspects remain uncharacterized following injury. Our goal was to characterize the impact of VML on whole-body physical activity and metabolism, and to further investigate possible muscle-specific metabolic changes. Adult male C57Bl/6J (n = 28) mice underwent a standardized VML injury to the posterior compartment of the hind limb, or served as injury naïve controls. Mice underwent longitudinal evaluation of whole-body physical activity and metabolism in specialized cages up to three times over the course of 8 weeks. At terminal time points of 4- and 8-weeks post-VML in vivo muscle function of the posterior compartment was evaluated. Additionally, the gastrocnemius muscle was collected to understand histological and biochemical changes in the muscle remaining after VML. The VML injury did not alter the physical activity of mice. However, there was a noted reduction in whole-body metabolism and diurnal fluctuations between lipid and carbohydrate oxidation were also reduced, largely driven by lower carbohydrate utilization during active hours. Following VML, muscle-specific changes indicate a decreased proportion of fast (i.e., type IIb and IIx) and a greater proportion of slow (i.e., type I and IIa) fibers. However, there were minimal changes in the capillarity and metabolic biochemical activity properties of the gastrocnemius muscle, suggesting a miss-match in capacity to support the physiologic needs of the fibers. These novel findings indicate that following VML, independent of changes in physical activity, there is whole-body diurnal metabolic inflexibility. Supporting future investigations into the chronic and overlooked co-morbidities of VML injury.
Volumetric muscle loss (VML) is the traumatic loss of muscle, resulting in long‐term functional deficits and various pathologic comorbidities. The metabolic consequences, whole‐body and muscle‐specific, of VML are unclear. We hypothesized that VML would decrease physical activity, respiratory exchange ratio (RER), and metabolic rate. A subset of mice underwent VML injury to the plantarflexors, 24‐hr whole‐body physical and metabolic activity was measured longitudinally. Prior to VML, 24‐hr ambulation was 1.3km/day, RER was 0.91, and metabolic rate was 19kcal/kg/hr; by 6 weeks post‐VML, daily ambulation did not change but 24‐hr RER and metabolic rate decreased (0.88 and 17kcal/kg/hr p≤0.036). Intriguingly, active to inactive‐phase RER, a marker of metabolic flexibility (fat vs. carbohydrate oxidation), was less post‐ compared to pre‐VML injury suggesting whole‐body metabolic inflexibility post‐VML. Additionally, in a cross‐over design, a subset of mice were assigned to restricted (12.5x8.5x6.3cm) or standard cages for 1‐wk, to model clinical conditions. When activity was restricted ambulation decreased ~50% and 24‐hr metabolic rate decreased ~23% (p≤0.002). In contrast, 24‐hr RER increased ~4% (p<0.001), suggesting greater carbohydrate utilization and supporting future use of this restrictive model in combination with VML. We next hypothesized a VML‐related disruption in the underlying oxidative physiology of the muscle remaining after VML contributed to whole‐body metabolic inflexibility. Additional mice were evaluated at 1‐, 4‐ and 8‐weeks following VML or sham procedure. Various oxidative, contractile, and physiology evaluations of the gastrocnemius muscle were completed. 1‐week post‐VML, permeabilized myofibers from injured limbs had a decreased conductance of electrons through the electron transport chain with fats as a fuel, but not carbohydrates; providing a potential molecular mechanism to whole‐body metabolic inflexibility. Interestingly, contractile properties support a slower myofiber phenotype that is typically more ready to use fat substrates for fuel. There was a VML‐induced slowing of torque production and elevated twitch:tetani ratio. Maximum torque generated in VML injured muscles was significantly reduced compared to uninjured (334±28 vs. 557±66mN·m). Although there was no impact of VML on mitochondria content following injury there was a flattening of the capillary distribution per fiber with an impact at both 4‐ and 8‐weeks post‐VML. The average cross‐sectional area of myofibers shifted right, with an increasing proportion of MyHCslow expressing fibers, again supporting slowing of the muscle following VML. The most salient findings herein suggest metabolic activity and flexibility are negatively impacted following VML injury despite a shift toward a slower phenotype. Metabolic inflexibility in humans is known to be associated with greater risk of comorbidities and cardiovascular disease, findings herein support a potential increased risk for VML‐afflicted patients to develop various chronic dis...
Volumetric muscle loss (VML) injury occurs when a substantial volume of skeletal muscle is abruptly removed and results in significant long‐term functional limitations. Following VML injury, patients likely undergo periods of low or no mobility and are expected to have reduced physical activity. Immobility and lack of physical activity leads to impaired muscle function, and increased all‐cause mortality and risk for chronic disease. It is plausible that inactivity and immobility negatively affect outcomes following VML injury, exacerbating functional impairments. Our previous work supports an ~25% lower oxygen consumption rate in permeabilized muscle fibers of the muscle remaining after VML up to 4 months post‐injury, but how this may impact whole body metabolism is unclear. Therefore, the purpose of this study was to characterize whole body metabolic and physical activity following VML injury. We hypothesized that metabolism and physical activity would be impaired following VML. To elucidate the interplay between metabolism, activity, and muscle function following VML injury, adult male C57BL/6 mice (n=7) underwent a full‐thickness multi‐muscle VML injury (removal of ~22mg) to the gastrocnemius, soleus, and plantaris, or sham surgery (n=7). Twenty‐four hour whole body metabolic and physical activity was assessed pre‐, and 2 and 6 weeks post‐VML (or sham) injury. Contractile activity of the ankle plantarflexors was assessed terminally at 8 weeks post‐VML. Prior to randomization into experimental groups metabolic rate was ~18.5kcal/kg/hr and there was no difference in any metabolic or activity measure (p≥0.21), and both groups gained ~3g over the 8 week period. Whole body metabolism was evaluated for the 24‐hour period and isolated by the 12 hours of active and inactive time; respiratory exchange ratio (RER), an indirect measure of muscle oxidative capacity, was not different across experimental groups (p≥0.76) but was significantly decreased with time (main effect p≤0.01). Two weeks post‐VML mice tended to become hypermetabolic with a metabolic rate ~14% greater than sham. On average, mice ambulated ~1.2 km per 24‐hour period and contrary to the hypothesis, there was no difference in activity levels (p≥0.23) between sham and VML injured mice. Terminally the plantarflexor muscle group of the VML‐injured mice had ~1.4 fold greater maximal passive torque (indicator of muscle stiffness) and ~40% less maximal active isometric torque. These findings indicate limited changes whole body metabolic and physical activity levels in a model of VML injury, contrary to our hypothesis. Future studies will limit physical activity levels in this model to better recapitulate the low or no mobility expected following VML injury clinically. Support or Funding Information Supported by W81XWH‐18‐1‐0710 & T32AR050938
Volumetric muscle loss (VML) injury, due to abrupt and significant loss of tissue, exceeds the endogenous regenerative ability of the skeletal muscle, leading to long‐term functional impairment and disability. There is a well‐established physiologic relationship between skeletal muscle mass and metabolism, but the implications of VML injury on this relationship are widely unexplored. Recent investigations have identified oxidative and mitochondrial dysfunction associated with VML injury; however, the local metabolic myofiber response is not completely understood. We hypothesized that significant metabolic and contractile limitations would be observed in the remaining myofibers following VML injury, suggestive of motor unit reorganization. To evaluate the metabolic properties following VML, adult C57BL/6 male mice (n=16) underwent a sham procedure or multi‐muscle full thickness VML injury to the gastrocnemius, soleus, and plantaris muscles (21.1±0.7mg). Two months post‐VML, terminal histologic evaluation of capillarity, mitochondrial content, fibrotic deposition, and myosin heavy chain distribution was completed. Additionally, maximal contractile function and force‐frequency response of the posterior muscle compartment were evaluated. Maximum passive torque at 20 degrees dorsiflexion was significantly greater in the VML group (5.5±0.22 mN·m) compared to sham (2.3±0.13 mN·m). Maximum isometric torque was significantly less in VML (334±28 mN·m) compared to sham (557±66 mN·m). VML injury resulted in slower torque production and elevated twitch:tetani ratio, perhaps indicating a slower muscle fiber phenotype in the tissue remaining. Collectively, this work suggests that motor unit reorganization may alter muscle fiber phenotype following VML injury, likely limiting long‐term muscle functionality. Support or Funding Information Supported by W81XWH‐18‐1‐0710 & T32AR050938.
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