Max E. Davis

Transforming growth factor-beta induces skeletal muscle atrophy and fibrosis through the induction of atrogin-1 and scleraxis.

Introduction: Transforming growth factor‐beta (TGF‐β) is a well‐known regulator of fibrosis and inflammation in many tissues. During embryonic development, TGF‐β signaling induces expression of the transcription factor scleraxis, which promotes fibroblast proliferation and collagen synthesis in tendons. In skeletal muscle, TGF‐β has been shown to induce atrophy and fibrosis, but the effect of TGF‐β on muscle contractility and the expression of scleraxis and atrogin‐1, an important regulator of muscle atrophy, were not known. Methods: We treated muscles from mice with TGF‐β and measured force production, scleraxis, procollagen Iα2, and atrogin‐1 protein levels. Results: TGF‐β decreased muscle fiber size and dramatically reduced maximum isometric force production. TGF‐β also induced scleraxis expression in muscle fibroblasts, and increased procollagen Iα2 and atrogin‐1 levels in muscles. Conclusion: These results provide new insight into the effect of TGF‐β on muscle contractility and the molecular mechanisms behind TGF‐β–mediated muscle atrophy and fibrosis. Muscle Nerve 45: 55–59, 2012

Rotator cuff tear reduces muscle fiber specific force production and induces macrophage accumulation and autophagy

Full‐thickness tears to the rotator cuff can cause severe pain and disability. Untreated tears progress in size and are associated with muscle atrophy and an infiltration of fat to the area, a condition known as “fatty degeneration.” To improve the treatment of rotator cuff tears, a greater understanding of the changes in the contractile properties of muscle fibers and the molecular regulation of fatty degeneration is essential. Using a rat model of rotator cuff injury, we measured the force generating capacity of individual muscle fibers and determined changes in muscle fiber type distribution that develop after a full thickness rotator cuff tear. We also measured the expression of mRNA and miRNA transcripts involved in muscle atrophy, lipid accumulation, and matrix synthesis. We hypothesized that a decrease in specific force of rotator cuff muscle fibers, an accumulation of type IIb fibers, and an upregulation in fibrogenic, adipogenic, and inflammatory gene expression occur in torn rotator cuff muscles. Thirty days following rotator cuff tear, we observed a reduction in muscle fiber force production, an induction of fibrogenic, adipogenic, and autophagocytic mRNA and miRNA molecules, and a dramatic accumulation of macrophages in areas of fat accumulation.

MMP inhibition as a potential method to augment the healing of skeletal muscle and tendon extracellular matrix

The extracellular matrix (ECM) of skeletal muscle and tendon is composed of different types of collagen molecules that play important roles in the transmission of forces throughout the body, and in the repair and regeneration of injured tissues. Fibroblasts are the primary cells in muscle and tendon that maintain, repair, and modify the ECM in response to mechanical loading, injury, and inactivity. Matrix metalloproteinases (MMPs) are enzymes that digest collagen and other structural molecules, which are synthesized and excreted by fibroblasts. MMPs are required for baseline ECM homeostasis, but disruption of MMP regulation due to injury or disease can alter the normal ECM architecture and prevent proper force transmission. Chronic injuries and diseases of muscles and tendons can be severely debilitating, and current therapeutic modalities to enhance healing are quite limited. This review will discuss the mechanobiology of MMPs, and the potential use of MMP inhibitors to improve the treatment of injured and diseased skeletal muscle and tendon tissue.

Inhibition of 5-LOX, COX-1, and COX-2 Increases Tendon Healing and Reduces Muscle Fibrosis and Lipid Accumulation After Rotator Cuff Repair

Background: The repair and restoration of function after chronic rotator cuff tears are often complicated by muscle atrophy, fibrosis, and fatty degeneration of the diseased muscle. The inflammatory response has been implicated in the development of fatty degeneration after cuff injuries. Licofelone is a novel anti-inflammatory drug that inhibits 5-lipoxygenase (5-LOX), as well as cyclooxygenase (COX)–1 and COX-2 enzymes, which play important roles in inducing inflammation after injuries. While previous studies have demonstrated that nonsteroidal anti-inflammatory drugs and selective inhibitors of COX-2 (coxibs) may prevent the proper healing of muscles and tendons, studies about bone and cartilage have demonstrated that drugs that inhibit 5-LOX concurrently with COX-1 and COX-2 may enhance tissue regeneration. Hypothesis: After the repair of a chronic rotator cuff tear in rats, licofelone would increase the load to failure of repaired tendons and increase the force production of muscle fibers. Study Design: Controlled laboratory study. Methods: Rats underwent supraspinatus release followed by repair 28 days later. After repair, rats began a treatment regimen of either licofelone or a vehicle for 14 days, at which time animals were euthanized. Supraspinatus muscles and tendons were then subjected to contractile, mechanical, histological, and biochemical analyses. Results: Compared with controls, licofelone-treated rats had a grossly apparent decrease in inflammation and increased fibrocartilage formation at the enthesis, along with a 62% increase in the maximum load to failure and a 51% increase in peak stress to failure. Licofelone resulted in a marked reduction in fibrosis and lipid content in supraspinatus muscles as well as reduced expression of several genes involved in fatty infiltration. Despite the decline in fibrosis and fat accumulation, muscle fiber specific force production was reduced by 23%. Conclusion: The postoperative treatment of cuff repair with licofelone may reduce fatty degeneration and enhance the development of a stable bone-tendon interface, although decreases in muscle fiber specific force production were observed, and force production in fact declined. Clinical Relevance: This study demonstrates that the inhibition of 5-LOX, COX-1, and COX-2 modulates the healing process of repaired rotator cuff tendons. Although further studies are necessary, the treatment of patients with licofelone after cuff repair may improve the development of a stable enthesis and enhance postoperative outcomes.

Elevation in Circulating Biomarkers of Cartilage Damage and Inflammation in Athletes With Femoroacetabular Impingement

Background: Femoroacetabular impingement (FAI) is one of the most common causes of early cartilage and labral damage in the nondysplastic hip. Biomarkers of cartilage degradation and inflammation are associated with osteoarthritis. It was not known whether patients with FAI have elevated levels of biomarkers of cartilage degradation and inflammation. Hypothesis: Compared with athletes without FAI, athletes with FAI would have elevated levels of the inflammatory C-reactive protein (CRP) and cartilage oligomeric matrix protein (COMP), a cartilage degradation marker. Study Design: Controlled laboratory study. Methods: Male athletes with radiographically confirmed FAI (n = 10) were compared with male athletes with radiographically normal hips with no evidence of FAI or hip dysplasia (n = 19). Plasma levels of COMP and CRP were measured, and subjects also completed the Short Form–12 (SF-12) and Hip Disability and Osteoarthritis Outcome Score (HOOS) surveys. Results: Compared with controls, athletes with FAI had a 24% increase in COMP levels and a 276% increase in CRP levels as well as a 22% decrease in SF-12 physical component scores and decreases in all of the HOOS subscale scores. Conclusion: Athletes with FAI demonstrate early biochemical signs of increased cartilage turnover and systemic inflammation. Clinical Relevance: Chondral injury secondary to the repetitive microtrauma of FAI might be reliably detected with biomarkers. In the future, these biomarkers might be used as screening tools to identify at-risk patients and assess the efficacy of therapeutic interventions such as hip preservation surgery in altering the natural history and progression to osteoarthritis.

Changes in Circulating Biomarkers of Muscle Atrophy, Inflammation, and Cartilage Turnover in Patients Undergoing Anterior Cruciate Ligament Reconstruction and Rehabilitation

Background: After anterior cruciate ligament (ACL) reconstruction, there is significant atrophy of the quadriceps muscles that can limit full recovery and place athletes at risk for recurrent injuries with return to play. The cause of this muscle atrophy is not fully understood. Hypothesis: Circulating levels of proatrophy, proinflammatory, and cartilage turnover cytokines and biomarkers would increase after ACL reconstruction. Study Design: Descriptive laboratory study. Methods: Patients (N = 18; mean age, 28 ± 2.4 years) underwent surgical reconstruction of the ACL after a noncontact athletic injury. Circulating levels of biomarkers were measured along with Short Form−12, International Knee Documentation Committee, and objective knee strength measures preoperatively and at 6 postoperative visits. Differences were tested using repeated-measures 1-way analysis of variance. Results: Myostatin, TGF-β, and C-reactive protein levels were significantly increased in the early postoperative period and returned to baseline. Cartilage oligomeric matrix protein levels decreased immediately after surgery and then returned to baseline. CCL2, CCL3, CCL4, CCL5, EGF, FGF-2, IGF-1, IL-10, IL-1α, IL-1β, IL-1ra, IL-6, myoglobin, and TNF-α were not different over the course of the study. Conclusion: An increase in potent atrophy-inducing cytokines and corresponding changes in knee strength and functional scores were observed after ACL reconstruction. Clinical Relevance: Although further studies are necessary, the therapeutic inhibition of myostatin may help prevent the muscle atrophy that occurs after ACL reconstruction and provide an accelerated return of patients to sport.

Reduced muscle fiber force production and disrupted myofibril architecture in patients with chronic rotator cuff tears

BACKGROUND A persistent atrophy of muscle fibers and an accumulation of fat, collectively referred to as fatty degeneration, commonly occur in patients with chronic rotator cuff tears. The etiology of fatty degeneration and function of the residual rotator cuff musculature have not been well characterized in humans. We hypothesized that muscles from patients with chronic rotator cuff tears have reduced muscle fiber force production, disordered myofibrils, and an accumulation of fat vacuoles. METHODS The contractility of muscle fibers from biopsy specimens of supraspinatus muscles of 13 patients with chronic full-thickness posterosuperior rotator cuff tears was measured and compared with data from healthy vastus lateralis muscle fibers. Correlations between muscle fiber contractility, American Shoulder and Elbow Surgeons (ASES) scores, and tear size were analyzed. Histology and electron microscopy were also performed. RESULTS Torn supraspinatus muscles had a 30% reduction in maximum isometric force production and a 29% reduction in normalized force compared with controls. Normalized supraspinatus fiber force positively correlated with ASES score and negatively correlated with tear size. Disordered sarcomeres were noted, along with an accumulation of lipid-laden macrophages in the extracellular matrix surrounding supraspinatus muscle fibers. CONCLUSIONS Patients with chronic supraspinatus tears have significant reductions in muscle fiber force production. Force production also correlates with ASES scores and tear size. The structural and functional muscle dysfunction of the residual muscle fibers is independent of the additional area taken up by fibrotic tissue. This work may help establish future therapies to restore muscle function after the repair of chronically torn rotator cuff muscles.

Simvastatin reduces fibrosis and protects against muscle weakness after massive rotator cuff tear.

BACKGROUND Chronic rotator cuff tears are a common source of shoulder pain and disability, and patients with chronic cuff tears often have substantial weakness, fibrosis, inflammation, and fat accumulation. Identifying therapies to prevent the development of these pathologic processes will likely have a positive impact on clinical outcomes. Simvastatin is a drug with demonstrated anti-inflammatory and antifibrotic effects in many tissues but had not previously been studied in the context of rotator cuff tears. We hypothesized that after the induction of a massive supraspinatus tear, simvastatin would protect muscles from a loss of force production and fibrosis. METHODS We measured changes in muscle fiber contractility, histology, and biochemical markers of fibrosis and fatty infiltration in rats that received a full-thickness supraspinatus tear and were treated with either carrier alone or simvastatin. RESULTS Compared with vehicle-treated controls, simvastatin did not have an appreciable effect on muscle fiber size, but treatment did increase muscle fiber specific force by 20%. Simvastatin also reduced collagen accumulation by 50% but did not affect triglyceride content of muscles. Several favorable changes in the expression of genes and other markers of inflammation, fibrosis, and regeneration were also observed. CONCLUSIONS Simvastatin partially protected muscles from the weakness that occurs as a result of chronic rotator cuff tear. Fibrosis was also markedly reduced in simvastatin-treated animals. Whereas further studies are necessary, statin medication could potentially help improve outcomes for patients with rotator cuff tears.

Inhibition of p38 mitogen-activated protein kinase signaling reduces fibrosis and lipid accumulation after rotator cuff repair

BACKGROUND The repair of rotator cuff tears is often complicated by fatty degeneration, which is the combination of lipid accumulation, fibrosis, inflammation, and muscle weakness. A signaling molecule that plays a central role in these processes is p38 mitogen-activated protein kinase (MAPK). The purpose of this study was to evaluate the ability of a small molecule inhibitor of p38 MAPK, SB203580, to reduce fatty degeneration in a preclinical model of rotator cuff injury and repair. MATERIALS AND METHODS Adult rats underwent a bilateral supraspinatus tenotomy that was repaired 30 days later. Rats were treated with SB203580 or vehicle every 2 days, with injections beginning 3 days before surgery and continuing until 7 days after surgery. Two weeks after surgical repair, muscles were analyzed using histology, lipid profiling, gene expression, and permeabilized muscle fiber contractility. RESULTS Inhibition of p38 MAPK resulted in a nearly 49% reduction in fat accumulation and a 29% reduction in collagen content, along with changes in corresponding genes regulating adipogenesis and matrix accumulation. There was also a marked 40% to 80% decrease in the expression of several proinflammatory genes, including IL1B, IL6, and COX2, and a 360% increase in the anti-inflammatory gene IL10. No differences were observed for muscle fiber force production. CONCLUSION Inhibition of p38 MAPK was found to result in a significant decrease in intramuscular lipid accumulation and fibrosis that is usually seen in the degenerative cascade of rotator cuff tears, without having negative effects on the contractile properties of the rotator cuff muscle tissue.

Muscle Injury Induced At the Time of Acute and Chronic Rotator Cuff Repair

Objectives: Rotator cuff tears are a common source of shoulder pain and disability. Even after surgical repair of these tears, many patients continue to have reduced shoulder function and a progression of intramuscular fatty degeneration. Previous work in a sheep model demonstrated that a slow, progressive lengthening of chronically torn cuff muscles followed by surgical repair of the tendon reversed fatty degeneration and improved muscle function. As patients with chronic cuff tears often experience a shortening of the muscle fibers, it is possible that repairing the torn tendon to its original anatomical footprint induces a massive stretch-induced muscle injury that could contribute to poor outcomes after repair. Evans Blue Dye (EBD) is a water soluble, membrane impermeable dye, that is used to identify muscle fibers that have suffered an injury to their plasma membrane. We hypothesized that, compared with acutely torn and repaired rotator cuff muscles, the substantial stretch that is required to repair a chronically torn and shortened rotator cuff would result in significant muscle fiber damage. Methods: This study was approved by our IACUC. Male Sprague-Dawley retired breeder rats (n=30) were placed into 5 groups: control, sham surgery, acute tear no repair, acute tear and repair, chronic tear no repair and chronic tear and repair, with each modality performed bilaterally (Fig 1A). An intraperitoneal injection of 1% EBD was administered 24 hours before the harvest surgery. At harvest, the supraspinatus muscles of each rat were removed and the length and mass of each muscle was measured. To determine EBD+ fibers the supraspinatus muscles were snap frozen in OCT and the muscles were cryosectioned and incubated with WGA-Lectin-AF488 to mark the extracellular matrix (ECM), DAPI to identify nuclei and EBD was used to identify the fibers with a damaged sarcolemma (Fig 1B-C). A one-way ANOVA (p<0.05) and Tukey’s post-hoc sorting was used to evaluate the differences between groups. Results: Compared with controls, rats that had a chronic supraspinatus tear but did not undergo repair had a 35% decrease in muscle mass compared to controls, while rats that had a chronic tear and also underwent repair had a 24% decrease in wet mass compared to controls (Fig 1D). Both groups of rats that underwent a chronic tear experienced an approximate 20% decrease in supraspinatus length when compared to the muscle length of other groups (Fig 1E). In the sham, acute and chronic tear and repair groups there were a greater number of EBD+ fibers in the mid-distal region of the muscle than in the mid-proximal region (Fig 1F). At the mid-distal end of the control and chronic tear no repair groups there were very few EBD+ fibers, in the sham 27% of the fibers were EBD+ and the remaining groups had almost 90% EBD+ fibers (Fig 1F). At the mid-proximal portion of the muscle the chronic tear and repair group had nearly 70% EBD+ fibers while none of the other groups showed a significant increase in EBD+ fibers over the control group (Fig 1G). Conclusion: A massive injury to muscle fibers is induced when the tendons of chronically torn rotator cuffs are repaired to their anatomical footprint. The repair of chronically torn cuff muscles results in extensive injury throughout the muscle that may contribute to poor long-term surgical outcomes even in the setting of a structurally intact repair. Therapeutic interventions that protect the muscle fiber plasma membrane may improve outcomes in patients with rotator cuff tears.