Can We Prevent Atrophy After ACL Tears? A Novel Biological Approach Caroline N Wolfe, MD1, Jonathan P Gumucio, BS1,2, Roger K Khouri, BS1, Jeremy A Grekin, MS1, Asheesh Bedi, MD1, Christopher L Mendias, PhD, ATC1,2

Departments of 1Orthopaedic Surgery and 2Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor

Background

Methods

Results

Conclusions

•  With rates of up to 250,000 per year, anterior cruciate ligament (ACL) ruptures are one of the most common knee injuries to physically active individuals (1). Many patients who suffer ACL ruptures have persistent atrophy and weakness even after rehabilitation with deficits exceeding 30% at 6 months post reconstruction, when patients would typically be cleared to return to sport (2,3). Persistent weakness can lead to poor physical performance, increased risk for repeat injury as well as alter knee kinematics that my promote early onset osteoarthritis (4,5,6,7). While the majority of rehabilitation interventions targeted at preventing muscle atrophy after ACL-R are focused on neurogenic muscle weakness (7), almost no research have directly addressed the cellular and molecular mechanisms of atrophy and weakness in muscle fibers themselves.

•  Myostatin (GDF-8) is a member of the transforming growth factor β (TGF-β) superfamily of cytokines and functions to induce muscle fiber atrophy and weakness (8). Myostatin induces muscle atrophy by activating the ubiquitin-proteosome pathway and by blocking protein synthesis pathways activated by IGF-1 signaling (9,10). The therapeutic inhibition of myostatin has been shown to protect against atrophy, weakness and fibrosis in several different models of cancer cachexia and neuromuscular diseases (11,12). Previous studies using rodent models and patients have demonstrated upregulation of myostatin after ACL tear (3,13).

•  Our objective was to evaluate the ability of a bioneutralizing monoclonal antibody against myostatin (10B3, GlaxoSmithKline) to prevent muscle weakness after ACL tear. Using a preclinical rat model, we tested the hypothesis that blocking myostatin activity after an ACL tear will prevent atrophy of lower limb muscles and also protect against the loss in muscle maximum isometric force production.

Animals and Surgical Procedure. This study was approved by the University of Michigan IACUC, and followed PHS guidelines for the ethical treatment of animals. The left ACL was transected in 36 Male Fischer 344 rats (Charles River, Wilmington, MA) using techniques by Delfino and Colleagues (13). Four rats served as non-operative controls. At the time of surgery, rats received a single IP injection of a bioneutralizing anti-myostatin monoclonal IgG antibody (10B3, GlaxoSmithKline) at a dose of 30 mg/kg or a sham anti-cholera toxin monoclonal IgG antibody (E1-82.15, GlaxoSmithKline). Rats (N=8 per group) were sacrificed and tissue was harvested 7 days or 21 days after tear. Following removal of tissues, rats were euthanized by overdose of sodium pentobarbital followed by induction of bilateral pneumothorax. EDL Contractility. Contractile properties of extensor digitorum longus (EDL) muscles was performed as previously described (14). Briefly, EDL was removed from the rat and immediately placed in a bath that contained Krebs mammalian Ringer solution supplemented with 11 mM glucose and 0.3 mM tubocurarine chloride. The distal tendon of the EDL was tied to a dual-mode servomotor/force transducer (Aurora Scientific) and the proximal tendon tied to a fixed hook. Using square wave pulses delivered from platinum electrodes connected to a stimulator (Aurora Scientific), muscles were stimulated to contract Histology. EDL and vastus lateralis muscles were sectioned and stained with wheat germ agglutin (WGA) lectin conjugated to AlexaFluor 488 (WGA-AF488, Life Technologies) to identify extracellular matrix. ImageJ software (NIH, Bethesda, MD) was used to perform quantitative fiber cross-sectional area (CSA) measurements. Gene Expression. Total RNA was isolated from homogenized rectus femoris muscles using a miRNeasy kit (Qiagen) and reverse transcribed into cDNA (iScript, Bio-Rad). Quantitative PCR (qPCR) was conducted (iTaq SYBR green), and the 2-ΔΔCt technique was used to normalize the expression of RNA transcripts to the stable housekeeping gene β-actin, and each of the treatment groups was further normalized to control, non-operated muscles. Statistics. Data are presented as mean±SD. Differences between treatment groups were tested with a two-way ANOVA (α=0.05) followed by Fisher's LSD post-hoc sorting in GraphPad Prism 6.0 (La Jolla, CA).

Figure 1. Body mass. Changes in body mass between sham mAb and anti-myostatin mAb treated animals at (A) 7 or (B) 21 days after inducing ACL tear.

Figure 2. 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 3. 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 4. (A) Atrophy and hypertrophy- and (B) ECM-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.

A

B

•  Persistent muscle atrophy and weakness limit the full functional recovery of patients following ACL tear. •  The results of this study indicate that myostatin inhibition may be a promising therapeutic strategy to prevent the loss in muscle size and strength

after ACL tear. •  While the mechanism of action is not entirely clear, based on the observed changes in expression of atrogin-1, MuRF-1, and MUSA-1 (which are

ubiquitin ligases that are rate limiting steps in protein breakdown), and IGF-Ea, IGF-Eb and 18S rRNA (which are genes that promote muscle protein synthesis), it is possible that the targeted inhibition of myostatin preserves force production by limiting the expression of proteolytic enzymes and inducing hypertrophy-related genes in the post-acute atrophy phase.

•  Although further studies are needed, the results from this preclinical model of ACL tears suggest that therapeutic inhibition of myostatin may help prevent muscle atrophy in patients who suffer joint injuries.

Supported by NIH grants R01-AR063649 and F31-AR065931, and an Orthopaedic Research and Education Foundation Resident Research Grant

References 1.  Griffin LY, Albohm MJ, Arendt EA, et al. (2006). Understanding and preventing noncontact anterior cruciate ligament injuries: a review of the Hunt Valley II meeting. Am J Sports Med 34(9):1512-1532. 2.  Ingersoll CD, Grindstaff TL, Pietrosimone BG, Hart JM. (2008). Neuromuscular consequences of anterior cruciate ligament injury. Clin Sports Med 27(3):383-404. 3.  Mendias CL, Lynch EB, Davis ME, Sibilsky Enselman ER, Harning JA, Dewolf PD, Makki TA, Bedi A. (2013). Changes in circulating biomarkers of muscle atrophy, inflammation, and cartilage turnover in patients undergoing anterior cruciate ligament reconstruction

and rehabilitation. Am J Sports Med 41(8):1819-26. 4.  Andriacchi TP, Mündermann A, et al. (2004). A framework for the in vivo pathomechanics of osteoarthritis at the knee. Ann Biomed Eng. 32(3):447-57. 5.  Lohmander LS, Ostenberg A, Englund M, Roos H. (2004). High prevalence of knee osteoarthritis, pain, and functional limitations in female soccer players twelve years after anterior cruciate ligament injury. Arthritis Rheum 50(10):3145-52. 6.  Palmieri-Smith RM, Thomas AC, Wojtys EM. (2008). Maximizing quadriceps strength after ACL reconstruction. Clin Sports Med 27(3):405-24. 7.  Palmieri-Smith RM, Thomas AC. A Neuromuscular Mechanism of Posttraumatic Osteoarthritis Associated with ACL Injury (2009) Exerc Sport Sci Rev July;37(3):147-53 8.  Gumucio JP, Mendias CL. (2013). Atrogin-1, MuRF-1, and sarcopenia. Endocrine 43(1):12-21. 9.  Yang W, Zhang Y, Li Y,Wu Z & Zhu D (2007). Myostatin induces cyclin D1 degradation to cause cell cycle arrest through a phosphatidylinositol 3-kinase/AKT/GSK-3β pathway and is antagonized by insulin-like growth factor 1. J Biol Chem 282, 3799–3808. 10.  Mendias CL, Kayupov E, Bradley JR, Brooks SV, Claflin DR. (2011). Decreased specific force and power production of muscle fibers from myostatin-deficient mice are associated with a suppression of protein degradation. J Appl Physiol 111(1):185-91. 11.  Bogdanovich S, Perkins KJ, Krag TO, Whittemore LA, Khurana TS. (2005)nMyostatin propeptide-mediated amelioration of dystrophic pathophysiology. FASEB J Apr;19(6):543-9. 12.  Murphy KT, Chee A, Gleeson BG, Naim T, Swiderski K, Koopman R & Lynch GS (2011). Antibody-directed myostatin inhibition enhances muscle mass and function in tumor-bearing mice. Am J Physiol Regul Integr Comp Physiol 301, R716–R726. 13.  Delfino GB, Peviani SM, Durigan JLQ, et al. (2013). Quadriceps muscle atrophy after anterior cruciate ligament transection involves increased mRNA levels of atrogin-1, muscle ring finger 1, and myostatin. Am J Phys Med Rehabil. 92(5):411-419. 14.  Mendias CL, Lynch EB, Gumucio JP, Flood MD, Rittman DS, Van Pelt DW, Roche SM, Davis CS. (2015) Changes in skeletal muscle and tendon structure and function following genetic inactivation of myostatin in rats. J Physiol April 15;593(8):2037-52.