Pharmacological inhibition of myostatin protects against skeletal muscle atrophy and weakness after anterior cruciate ligament tear

Abstract

Anterior cruciate ligament (ACL) tears are among the most frequent knee injuries in sports medicine, with tear rates in the US up to 250,000 per year. Many patients who suffer from ACL tears have persistent atrophy and weakness even after considerable rehabilitation. Myostatin is a cytokine that directly induces muscle atrophy, and previous studies rodent models and patients have demonstrated an upregulation of myostatin after ACL tear. Using a preclinical rat model, our objective was to determine if the use of a bioneutralizing antibody against myostatin could prevent muscle atrophy and weakness after ACL tear. Rats underwent a surgically induced ACL tear and were treated with either a bioneutralizing antibody against myostatin (10B3, GlaxoSmithKline) or a sham antibody (E1-82.15, GlaxoSmithKline). Muscles were harvested at either 7 or 21 days after induction of a tear to measure changes in contractile function, fiber size, and genes involved in muscle atrophy and hypertrophy. These time points were selected to evaluate early and later changes in muscle structure and function. Compared to the sham antibody group, 7 days after ACL tear, myostatin inhibition reduced the expression of proteolytic genes and induced the expression of hypertrophy genes. These early changes in gene expression lead to a 22% increase in muscle fiber cross-sectional area and a 10% improvement in maximum isometric force production that were observed 21 days after ACL tear. Overall, myostatin inhibition lead to several favorable, although modest, changes in molecular biomarkers of muscle regeneration and reduced muscle atrophy and weakness following ACL tear. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2499-2505, 2017.

Keywords: myostatin; GDF-8; anterior cruciate ligament; atrophy; muscle contractility.

Figure 1. Histology

Muscle fiber cross-sectional areas of (A) Extensor digitorum longus and (B) Vastus lateralis muscles from sham mAb and anti-myostatin mAb treated animals 7 or 21 days after inducing ACL tear. Values are mean±SD, N≥7 for each group. Dashed line indicates mean values from control, uninjured animals. Differences tested with a two-way ANOVA (α=0.05) followed by Fisher’s LSD post-hoc sorting. Letters above bar graphs indicate post-hoc sorting differences.

Figure 2. Extensor digitorum longus (EDL) muscle mass and in vitro contractility measurements

(A) Muscle mass, (B) physiological cross-sectional area (PCSA), (C) Maximum isometric force, (D) Specific force (maximum isometric force normalized to PCSA) of EDL muscles from sham mAb and anti-myostatin mAb treated animals 7 or 21 days after inducing ACL tear. Values are mean±SD, N≥7 for each group. Dashed line indicates mean values from control, uninjured animals. Differences tested with a two-way ANOVA (α=0.05) followed by Fisher’s LSD post-hoc sorting. Letters above bar graphs indicate post-hoc sorting differences.

Figure 3. Atrophy and hypertrophy-related gene expression

Gene expression, measured by quantitative PCR, from rectus femoris muscles. Target genes were normalized to the expression of the stable housekeeping gene β-actin, and then further normalized to the relative expression of that gene in control muscle from an uninjured knee. Any expression value greater than 1 indicates an upregulation compared to control muscle, and values less than 1 indicates a downregulation compared to control muscle. Values are mean±SD, N≥7 for each group. Differences tested with a two-way ANOVA (α=0.05) followed by Fisher’s LSD post-hoc sorting. Letters above bar graphs indicate post-hoc sorting differences.

Figure 4. Extracellular matrix-related gene expression

Gene expression, measured by quantitative PCR, from rectus femoris muscles. Target genes were normalized to the expression of the stable housekeeping gene β-actin, and then further normalized to the relative expression of that gene in control muscle from an uninjured knee. Any expression value greater than 1 indicates an upregulation compared to control muscle, and values less than 1 indicates a downregulation compared to control muscle. Values are mean±SD, N≥7 for each group. Differences tested with a two-way ANOVA (α=0.05) followed by Fisher’s LSD post-hoc sorting. Letters above bar graphs indicate post-hoc sorting differences.