Excess TGF-β mediates muscle weakness associated with bone metastases in mice

Authors

David L Waning, Khalid S Mohammad, Steven Reiken, Wenjun Xie, Daniel C Andersson, Sutha John, Antonella Chiechi, Laura E Wright, Alisa Umanskaya, Maria Niewolna, Trupti Trivedi, Sahba Charkhzarrin, Pooja Khatiwada, Anetta Wronska, Ashley Haynes, Maria Serena Benassi, Frank A Witzmann, Gehua Zhen, Xiao Wang, Xu Cao, G David Roodman, Andrew R Marks & Theresa A Guise

Abstract

Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases—representing breast, lung and prostate cancers, as well as multiple myeloma—exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-β, released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-β signaling, TGF-β release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer–associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-β activity. Thus, pathological TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production.

Link to Article

http://dx.doi.org/10.1038/nm.3961

The Role of Muscle Loading on Bone (Re)modeling at the Developing Enthesis

Authors

Alexander M. Tatara, Justin H. Lipner, Rosalina Das, H. Mike Kim, Nikunj Patel, Eleni Ntouvali, Matthew J. Silva, Stavros Thomopoulos

Abstract

Muscle forces are necessary for the development and maintenance of a mineralized skeleton. Removal of loads leads to malformed bones and impaired musculoskeletal function due to changes in bone (re)modeling. In the current study, the development of a mineralized junction at the interface between muscle and bone was examined under normal and impaired loading conditions. Unilateral mouse rotator cuff muscles were paralyzed using botulinum toxin A at birth. Control groups consisted of contralateral shoulders injected with saline and a separate group of normal mice. It was hypothesized that muscle unloading would suppress bone formation and enhance bone resorption at the enthesis, and that the unloading-induced bony defects could be rescued by suppressing osteoclast activity. In order to modulate osteoclast activity, mice were injected with the bisphosphonate alendronate. Bone formation was measured at the tendon enthesis using alizarin and calcein fluorescent labeling of bone surfaces followed by quantitative histomorphometry of histologic sections. Bone volume and architecture was measured using micro computed tomography. Osteoclast surface was determined via quantitative histomorphometry of tartrate resistant acid phosphatase stained histologic sections. Muscle unloading resulted in delayed initiation of endochondral ossification at the enthesis, but did not impair bone formation rate. Unloading led to severe defects in bone volume and trabecular bone architecture. These defects were partially rescued by suppression of osteoclast activity through alendronate treatment, and the effect of alendronate was dose dependent. Similarly, bone formation rate was increased with increasing alendronate dose across loading groups. The bony defects caused by unloading were therefore likely due to maintained high osteoclast activity, which normally decreases from neonatal through mature timepoints. These results have important implications for the treatment of muscle unloading conditions such as neonatal brachial plexus palsy, which results in shoulder paralysis at birth and subsequent defects in the rotator cuff enthesis and humeral head.

Link To Article

http://dx.doi.org/10.1371/journal.pone.0097375

The interaction of force and repetition on musculoskeletal and neural tissue responses and sensorimotor behavior in a rat model of work-related musculoskeletal disorders

Authors

Mary F Barbe, Sean Gallagher, Vicky S Massicotte, Michael Tytell, Steven N Popoff, and Ann E Barr-Gillespie

Abstract

Background

We examined the relationship of musculoskeletal risk factors underlying force and repetition on tissue responses in an operant rat model of repetitive reaching and pulling, and if force x repetition interactions were present, indicative of a fatigue failure process. We examined exposure-dependent changes in biochemical, morphological and sensorimotor responses occurring with repeated performance of a handle-pulling task for 12 weeks at one of four repetition and force levels: 1) low repetition with low force, 2) high repetition with low force, 3) low repetition with high force, and 4) high repetition with high force (HRHF).

Methods

Rats underwent initial training for 4–6 weeks, and then performed one of the tasks for 12 weeks, 2 hours/day, 3 days/week. Reflexive grip strength and sensitivity to touch were assayed as functional outcomes. Flexor digitorum muscles and tendons, forelimb bones, and serum were assayed using ELISA for indicators of inflammation, tissue stress and repair, and bone turnover. Histomorphometry was used to assay macrophage infiltration of tissues, spinal cord substance P changes, and tissue adaptative or degradative changes. MicroCT was used to assay bones for changes in bone quality.

Results

Several force x repetition interactions were observed for: muscle IL-1alpha and bone IL-1beta; serum TNFalpha, IL-1alpha, and IL-1beta; muscle HSP72, a tissue stress and repair protein; histomorphological evidence of tendon and cartilage degradation; serum biomarkers of bone degradation (CTXI) and bone formation (osteocalcin); and morphological evidence of bone adaptation versus resorption. In most cases, performance of the HRHF task induced the greatest tissue degenerative changes, while performance of moderate level tasks induced bone adaptation and a suggestion of muscle adaptation. Both high force tasks induced median nerve macrophage infiltration, spinal cord sensitization (increased substance P), grip strength declines and forepaw mechanical allodynia by task week 12.

Conclusions

Although not consistent in all tissues, we found several significant interactions between the critical musculoskeletal risk factors of force and repetition, consistent with a fatigue failure process in musculoskeletal tissues. Prolonged performance of HRHF tasks exhibited significantly increased risk for musculoskeletal disorders, while performance of moderate level tasks exhibited adaptation to task demands.

Link to Article

http://dx.doi.org/10.1186/1471-2474-14-303

NF1 is a critical regulator of muscle development and metabolism

Authors

Kate Sullivan, Jad El-Hoss, Kate G. R. Quinlan, Nikita Deo, Fleur Garton, Jane T. C. Seto, Marie Gdalevitch, Nigel Turner, Gregory J. Cooney, Mateusz Kolanczyk, Kathryn N. North, David G. Little and Aaron Schindeler

Abstract

There is emerging evidence for reduced muscle function in children with Neurofibromatosis type 1 (NF1). We have examined three murine models featuring NF1 deficiency in muscle to study the effect on muscle function as well as any underlying pathophysiology. The Nf1+/- mouse exhibited no differences in overall weight, lean tissue mass, fiber size, muscle weakness as measured by grip strength, or muscle atrophy-recovery with limb disuse, although this model lacks many other characteristic features of the human disease. Next, muscle-specific knockout mice (Nf1muscle−/-) were generated and they exhibited a failure to thrive leading to neonatal lethality. Intramyocellular lipid accumulations were observed by electron microscopy (EM) and Oil Red O staining. More mature muscle specimens lacking Nf1 expression taken from the limb-specific Nf1Prx1−/- conditional knockout line showed a 10-fold increase in muscle triglyceride content. Enzyme assays revealed a significant increase in the activities of oxidative metabolism enzymes in the Nf1Prx1−/- mice. Western analyses showed increases in the expression of Fatty Acid Synthase (FAS) and the hormone Leptin, as well as decreased expression of a number of fatty acid transporters in this mouse line. These data support the hypothesis that NF1 is essential for normal muscle function and survival and are the first to suggest a direct link between NF1 and mitochondrial fatty acid metabolism.

Link to Article

http://dx.doi.org/10.1093/hmg/ddt515

Increased Serum and Musculotendinous Fibrogenic Proteins following Persistent Low-Grade Inflammation in a Rat Model of Long-Term Upper Extremity Overuse

Authors

Helen G. L. Gao, Paul W. Fisher, Alex G. Lambi, Christine K. Wade, Ann E. Barr-Gillespie, Steven N. Popoff, Mary F. Barbe

Abstract

We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.

Link to Article

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0071875

Performance of Repetitive Tasks Induces Decreased Grip Strength and Increased Fibrogenic Proteins in Skeletal Muscle: Role of Force and Inflammation

Authors

Samir M. Abdelmagid, Ann E. Barr, Mario Rico, Mamta Amin, Judith Litvin, Steven N. Popoff, Fayez F. Safadi, Mary F. Barbe

Abstract

This study elucidates exposure-response relationships between performance of repetitive tasks, grip strength declines, and fibrogenic-related protein changes in muscles, and their link to inflammation. Specifically, we examined forearm flexor digitorum muscles for changes in connective tissue growth factor (CTGF; a matrix protein associated with fibrosis), collagen type I (Col1; a matrix component), and transforming growth factor beta 1 (TGFB1; an upstream modulator of CTGF and collagen), in rats performing one of two repetitive tasks, with or without anti-inflammatory drugs. To examine the roles of force versus repetition, rats performed either a high repetition negligible force food retrieval task (HRNF), or a high repetition high force handle-pulling task (HRHF), for up to 9 weeks, with results compared to trained only (TR-NF or TR-HF) and normal control rats. Grip strength declined with both tasks, with the greatest declines in 9-week HRHF rats. Quantitative PCR (qPCR) analyses of HRNF muscles showed increased expression of Col1 in weeks 3–9, and CTGF in weeks 6 and 9. Immunohistochemistry confirmed PCR results, and also showed greater increases of CTGF and collagen matrix in 9-week HRHF rats than 9-week HRNF rats. ELISA, and immunohistochemistry revealed greater increases of TGFB1 in TR-HF and 6-week HRHF, compared to 6-week HRNF rats. To examine the role of inflammation, results from 6-week HRHF rats were compared to rats receiving ibuprofen or anti-TNF-α treatment in HRHF weeks 4–6. Both treatments attenuated HRHF-induced increases in CTGF and fibrosis by 6 weeks of task performance. Ibuprofen attenuated TGFB1 increases and grip strength declines, matching our prior results with anti-TNFα. Performance of highly repetitive tasks was associated with force-dependent declines in grip strength and increased fibrogenic-related proteins in flexor digitorum muscles. These changes were attenuated, at least short-term, by anti-inflammatory treatments.

Link to Article

http://dx.doi.org/10.1371/journal.pone.0038359