Lucas R. Smith

Collagen content does not alter the passive mechanical properties of fibrotic skeletal muscle in mdx mice

Many skeletal muscle diseases are associated with progressive fibrosis leading to impaired muscle function. Collagen within the extracellular matrix is the primary structural protein providing a mechanical scaffold for cells within tissues. During fibrosis collagen not only increases in amount but also undergoes posttranslational changes that alter its organization that is thought to contribute to tissue stiffness. Little, however, is known about collagen organization in fibrotic muscle and its consequences for function. To investigate the relationship between collagen content and organization with muscle mechanical properties, we studied mdx mice, a model for Duchenne muscular dystrophy (DMD) that undergoes skeletal muscle fibrosis, and age-matched control mice. We determined collagen content both histologically, with picosirius red staining, and biochemically, with hydroxyproline quantification. Collagen content increased in the mdx soleus and diaphragm muscles, which was exacerbated by age in the diaphragm. Collagen packing density, a parameter of collagen organization, was determined using circularly polarized light microscopy of picosirius red-stained sections. Extensor digitorum longus (EDL) and soleus muscle had proportionally less dense collagen in mdx muscle, while the diaphragm did not change packing density. The mdx muscles had compromised strength as expected, yet only the EDL had a significantly increased elastic stiffness. The EDL and diaphragm had increased dynamic stiffness and a change in relative viscosity. Unexpectedly, passive stiffness did not correlate with collagen content and only weakly correlated with collagen organization. We conclude that muscle fibrosis does not lead to increased passive stiffness and that collagen content is not predictive of muscle stiffness.

SMASH – semi-automatic muscle analysis using segmentation of histology: a MATLAB application

BackgroundHistological assessment of skeletal muscle tissue is commonly applied to many areas of skeletal muscle physiological research. Histological parameters including fiber distribution, fiber type, centrally nucleated fibers, and capillary density are all frequently quantified measures of skeletal muscle. These parameters reflect functional properties of muscle and undergo adaptation in many muscle diseases and injuries. While standard operating procedures have been developed to guide analysis of many of these parameters, the software to freely, efficiently, and consistently analyze them is not readily available. In order to provide this service to the muscle research community we developed an open source MATLAB script to analyze immunofluorescent muscle sections incorporating user controls for muscle histological analysis.ResultsThe software consists of multiple functions designed to provide tools for the analysis selected. Initial segmentation and fiber filter functions segment the image and remove non-fiber elements based on user-defined parameters to create a fiber mask. Establishing parameters set by the user, the software outputs data on fiber size and type, centrally nucleated fibers, and other structures. These functions were evaluated on stained soleus muscle sections from 1-year-old wild-type and mdx mice, a model of Duchenne muscular dystrophy. In accordance with previously published data, fiber size was not different between groups, but mdx muscles had much higher fiber size variability. The mdx muscle had a significantly greater proportion of type I fibers, but type I fibers did not change in size relative to type II fibers. Centrally nucleated fibers were highly prevalent in mdx muscle and were significantly larger than peripherally nucleated fibers.ConclusionsThe MATLAB code described and provided along with this manuscript is designed for image processing of skeletal muscle immunofluorescent histological sections. The program allows for semi-automated fiber detection along with user correction. The output of the code provides data in accordance with established standards of practice. The results of the program have been validated using a small set of wild-type and mdx muscle sections. This program is the first freely available and open source image processing program designed to automate analysis of skeletal muscle histological sections.

Targeting latent TGFβ release in muscular dystrophy

Blocking cleavage of latent TGFβ binding protein 4, a modifier of muscular dystrophy in humans and mice, inhibits release of latent TGFβ and decreases pathology in muscular dystrophy. Unhinging Muscle Injury Disruption of the muscle plasma membrane underlies muscle injury and muscular dystrophy. Latent TGFβ binding protein 4 (LTBP4) is a membrane-associated protein that sequesters latent TGFβ. Upon injury, the hinge region of LTBP4 undergoes proteolytic cleavage to release latent TGFβ. Excessive TGFβ release and activity accelerates muscular dystrophy and muscle injury. This hinge domain of LTBP4, and consequently TGFβ release, can be inhibited using antibodies that block proteolysis of LTBP4’s hinge domain. LTBP4 blocking antibodies can be applied as a biological approach to reduce muscle injury and the adverse consequences of muscular dystrophy. Latent transforming growth factor–β (TGFβ) binding proteins (LTBPs) bind to inactive TGFβ in the extracellular matrix. In mice, muscular dystrophy symptoms are intensified by a genetic polymorphism that changes the hinge region of LTBP, leading to increased proteolytic susceptibility and TGFβ release. We have found that the hinge region of human LTBP4 was also readily proteolysed and that proteolysis could be blocked by an antibody to the hinge region. Transgenic mice were generated to carry a bacterial artificial chromosome encoding the human LTBP4 gene. These transgenic mice displayed larger myofibers, increased damage after muscle injury, and enhanced TGFβ signaling. In the mdx mouse model of Duchenne muscular dystrophy, the human LTBP4 transgene exacerbated muscular dystrophy symptoms and resulted in weaker muscles with an increased inflammatory infiltrate and greater LTBP4 cleavage in vivo. Blocking LTBP4 cleavage may be a therapeutic strategy to reduce TGFβ release and activity and decrease inflammation and muscle damage in muscular dystrophy.

Increased collagen cross-linking is a signature of dystrophin-deficient muscle

Collagen cross‐linking is a key parameter in extracellular matrix (ECM) maturation, turnover, and stiffness. We examined aspects of collagen cross‐linking in dystrophin‐deficient murine, canine, and human skeletal muscle.

Stem Cell Differentiation is Regulated by Extracellular Matrix Mechanics

Stem cells mechanosense the stiffness of their microenvironment, which impacts differentiation. Although tissue hydration anti-correlates with stiffness, extracellular matrix (ECM) stiffness is clearly transduced into gene expression via adhesion and cytoskeleton proteins that tune fates. Cytoskeletal reorganization of ECM can create heterogeneity and influence fates, with fibrosis being one extreme.

Masticatory muscles of mouse do not undergo atrophy in space

Muscle loading is important for maintaining muscle mass; when load is removed, atrophy is inevitable. However, in clinical situations such as critical care myopathy, masticatory muscles do not lose mass. Thus, their properties may be harnessed to preserve mass. We compared masticatory and appendicular muscles responses to microgravity, using mice aboard the space shuttle Space Transportation System‐135. Age‐ and sex‐matched controls remained on the ground. After 13 days of space flight, 1 masseter (MA) and tibialis anterior (TA) were frozen rapidly for biochemical and functional measurements, and the contralateral MA was processed for morphologic measurements. Flight TA muscles exhibited 20 ± 3% decreased muscle mass, 2‐fold decreased phosphorylated (P)‐Akt, and 4‐ to 12‐fold increased atrogene expression. In contrast, MAs had no significant change in mass but a 3‐fold increase in P‐focal adhesion kinase, 1.5‐fold increase in P‐Akt, and 50‐90% lower atrogene expression compared with limb muscles, which were unaltered in microgravity. Myofibril force measurements revealed that microgravity caused a 3‐fold decrease in specific force and maximal shortening velocity in TA muscles. It is surprising that myofibril‐specific force from both control and flight MAs were similar to flight TA muscles, yet power was compromised by 40% following flight. Continued loading in microgravity prevents atrophy, but masticatory muscles have a different set point that mimics disuse atrophy in the appendicular muscle.— Philippou, A., Minozzo, F. C., Spinazzola, J. M., Smith, L. R., Lei, H., Rassier, D. E., Barton, E. R. Masticatory muscles of mouse do not undergo atrophy in space. FASEB J. 29, 2769‐2779 (2015). www.fasebj.org

Regulation of fibrosis in muscular dystrophy

The production of force and power are inherent properties of skeletal muscle, and regulated by contractile proteins within muscle fibers. However, skeletal muscle integrity and function also require strong connections between muscle fibers and their extracellular matrix (ECM). A well-organized and pliant ECM is integral to muscle function and the ability for many different cell populations to efficiently migrate through ECM is critical during growth and regeneration. For many neuromuscular diseases, genetic mutations cause disruption of these cytoskeletal-ECM connections, resulting in muscle fragility and chronic injury. Ultimately, these changes shift the balance from myogenic pathways toward fibrogenic pathways, culminating in the loss of muscle fibers and their replacement with fatty-fibrotic matrix. Hence a common pathological hallmark of muscular dystrophy is prominent fibrosis. This review will cover the salient features of muscular dystrophy pathogenesis, highlight the signals and cells that are important for myogenic and fibrogenic actions, and discuss how fibrosis alters the ECM of skeletal muscle, and the consequences of fibrosis in developing therapies.

Mechanisms of Plastic Deformation in Collagen Networks Induced by Cellular Forces

Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis.

Influencing the secretion of myogenic factors from mesenchymal stem cells

Mounting evidence indicates that the regenerative effect of mesenchymal stem cells in skeletal muscle is related to the secretion of factors that stimulate resident myogenic cells. However, the environmental cues that affect the secreted factors of mesenchymal stem cells are not well understood. A recent publication demonstrated that secretion of factors is dependent on cell substrate, with mesenchymal stem cells grown on laminin providing more pro-myogenic factors than those grown on collagen, and that cellular strain may also play a role. Conditioned media from mesenchymal stem cells grown on laminin and subjected to strain provided the quickest and largest stimulation to myogenic cell proliferation. The influence of cell substrate and mechanical perturbation on mesenchymal stem cells therefore appears key to secretion of factors that support myogenesis.

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix—all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker &ggr;H2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage.