Testing Adjunctive Oxidative‐Modulators to Improve Functional Capacity in a Murine Model of Volumetric Muscle Loss

Abstract

Volumetric muscle loss injuries (VML) entail a major loss of tissue beyond the muscle's normal repair and regeneration capabilities, leading to severe structural and functional deficits. The purpose of this study was to test the effect of different pharmacological adjuvants on the oxidative capacity of VML-injured muscle. These adjuvants were selected to stimulate a variety of pathways (AMPK, G-coupled receptors, PPARα, and cGMP) that increase expression of PGC1-α, a transcription factor that regulates mitochondrial biogenesis. We hypothesize that increasing mitochondrial biogenesis will improve overall muscle function and make tissue receptive to future physical rehabilitation. C57BL6 mice underwent unilateral surgery to create a VML defect in the lower posterior hindlimb under anesthesia, as approved by the University of Georgia's Institutional Animal Care and Use Committee. Animals were divided randomly into untreated or treatment groups and received 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, 250 mg/kg/day), formoterol (FOR, 0.3 mg/kg/day), pioglitazone (PIO, 20 mg/kg/day), or sildenafil (SIL, 1 mg/kg/day) to target AMPK, G-coupled receptors, PPARα, or cGMP, respectively. All adjunctive treatments were milled into their diet for two months. Injury naïve mice were used as a baseline measurement. Peak isometric torque of the ankle plantarflexors and oxygen consumption of permeabilized muscle fibers were used to assess muscle function. Mice receiving FOR and AICAR had significantly higher gastrocnemius muscle wet mass (mg g-1 body mass) compared to untreated controls (p<0.05). Peak isometric torque in formoterol-treated mice was significantly greater over non-treated controls (p<0.01). Mitochondrial content was similar between injury naïve and FOR-treated mice, while there was significantly more mitochondria in AICAR and non-treated controls, as estimated through citrate synthase activity. Mitochondrial respiration normalized to content was higher in FOR treated VML muscle compared to all other experimental groups and comparable to injury naïve mice (p<0.005). While many studies have attempted the regeneration of tissue within the defect, few evaluate remaining tissue viability to improve its strength and function. Here we have shown that the treatment of VML-injured muscles by stimulating the G-coupled protein receptors leads to greater muscle mass and peak isometric torque. Although FOR treatment did not influence mitochondrial content, the muscle's metabolic function was significantly enhanced, suggesting an improvement in mitochondrial efficiency. Future investigations will combine this adjunctive pharmaceutical therapy with physical rehabilitation to magnify its effect. Combined with physical rehabilitation, it may offer better outcomes over current strategies and improve functional outcomes such as mobility, muscle strength, and overall quality of life.