Exploring skeletal muscle tolerance and whole-body metabolic effects of FDA-approved drugs in a volumetric muscle loss model

Abstract

Volumetric muscle loss (VML) is associated with persistent functional impairment due to a lack of de novo muscle regeneration. As mechanisms driving the lack of regeneration continue to be established, adjunctive pharmaceuticals to address the pathophysiology of the remaining muscle may offer partial remediation. Studies were designed to evaluate the tolerance and efficacy of two FDA-approved pharmaceutical modalities to address the pathophysiology of the remaining muscle tissue after VML injury: (1) nintedanib (an anti-fibrotic) and (2) combined formoterol and leucine (myogenic promoters). Tolerance was first established by testing low- and high-dosage effects on uninjured skeletal muscle mass and myofiber cross-sectional area in adult male C57BL/6J mice. Next, tolerated doses of the two pharmaceutical modalities were tested in VML-injured adult male C57BL/6J mice after an 8-week treatment period for their ability to modulate muscle strength and whole-body metabolism. The most salient findings indicate that formoterol plus leucine mitigated the loss in muscle mass, myofiber number, whole-body lipid oxidation, and muscle strength, and resulted in a higher whole-body metabolic rate (p ≤ 0.016); nintedanib did not exacerbate or correct aspects of the muscle pathophysiology after VML. This supports ongoing optimization efforts, including scale-up evaluations of formoterol treatment in large animal models of VML.

Keywords: formoterol; muscle function; neuromusculoskeletal injury; nintedanib; skeletal muscle injury.

FIGURE 1

Treatment effects on body mass, muscle mass, and myofiber size were evaluated in the injury naïve cohort. (a) Over 4 weeks, body mass was significantly impaired with nintedanib, which was more pronounced with the high dose (main effect of group p < 0.001; main effect of time p = 0.545). (b) Gastrocnemius muscle mass (p = 0.002) and (c) tibialis anterior (TA) muscle mass (p = 0.003) normalized to body mass were less with high‐dose nintedanib. (d) Extensor digitorum longus (EDL) myofiber cross‐sectional (CSA) was not different between groups (p = 0.500), while (e) the high‐dose nintedanib impacted soleus muscle CSA (p = 0.018). (f) Representative EDL muscle sections, stained with Masson's Trichrome, across treatment groups and control. Scale bar is 100 μm. Data are mean ± SD; each data point represents an individual mouse. Significantly different from *control; ^†high‐dose nintedanib.

FIGURE 2

Physical activity and whole‐body metabolism were evaluated 6 weeks after VML. (a) Total ambulatory distance over 24 h was similar across groups (p = 0.070). (b) Formoterol plus leucine resulted in a higher 24‐h metabolic rate (p < 0.001). (c) Although 24‐h RER was similar across groups (p = 0.172), RER was elevated during the 12‐h active (p = 0.024) and inactive (p = 0.043) periods for nintedanib and formoterol plus leucine‐treated mice, respectively. (d) Carbohydrate oxidation was similar across groups over 24 h (p = 0.181) and during the 12‐h active period (p = 0.512) but was higher during the 12‐h inactive period following formoterol plus leucine treatment (p = 0.006). (e) VML‐untreated and nintedanib‐treated mice had a lower 24‐h lipid oxidation, while formoterol plus leucine treatment mitigated this decline (p = 0.005). Data are mean ± SD; each data point represents an individual mouse. Significantly different from *Naïve; ^§VML untreated; ^‡VML + nintedanib; ^†VML + formoterol + leucine.

FIGURE 3

Treatment effects on body mass, muscle mass, and myofiber number and size were evaluated in the VML cohort. (a) Body mass increased over 8 weeks for all experimental groups but was smaller in both treatment groups (main effect of time p < 0.001; main effect of treatment p < 0.001). (b) Formoterol plus leucine‐treated mice had a higher gastrocnemius muscle mass normalized to body mass than VML‐untreated mice (p = 0.004). (c) Representative trichrome‐stained mid‐belly gastrocnemius muscle sections across groups; scale bar is 500 μm. (d) Mid‐belly gastrocnemius myofiber number following VML (p = 0.009). (e, f) Gastrocnemius myofiber average cross‐sectional area (CSA) distribution was different in all experimental groups compared to injury naïve, and in nintedanib‐treated versus formoterol (p < 0.001), but mean CSA was similar across groups (p = 0.534). Data are mean ± SD; each data point represents an individual mouse. Significantly different than ^§ week 1; ^‡week 2; *naïve; ^†VML + formoterol + leucine.

FIGURE 4

Evaluation of markers related to myogenesis and atrophy were measured 8 weeks following VML injury. (a) Representative stain‐free blot image displaying total lane protein with molecular weights of protein markers noted. (b) Corresponding fluorescent bands for each group and molecular weight for each marker. Bands were normalized to total protein in each respective lane to quantify relative protein expression compared to naïve. There were no differences in gastrocnemius muscle protein content of (c) Akt (p = 0.768), (d) myostatin (p = 0.723), (e) MyoD1 (p = 0.493), or (f) Myf5 (p = 0.383) across groups. Data are mean ± SD; each data point represents an individual mouse.