Collagen architecture and biomechanics of gracilis and adductor longus muscles from children with cerebral palsy.

Cerebral palsy (CP) describes some upper motoneuron disorders due to non-progressive disturbances occurring in the developing brain that cause progressive changes to muscle. While longer sarcomeres increase muscle stiffness in patients with CP compared to typically developing (TD) patients, changes in extracellular matrix (ECM) architecture can increase stiffness. Our goal was to investigate how changes in muscle and ECM architecture impact muscle stiffness, gait and joint function in CP. Gracilis and adductor longus biopsies were collected from children with CP undergoing tendon lengthening surgery for hamstring and hip adduction contractures, respectively. Gracilis biopsies were collected from TD patients undergoing anterior cruciate ligament reconstruction surgery with hamstring autograft. Muscle mechanical testing, two-photon imaging and hydroxyproline assay were performed on biopsies. Corresponding data were compared to radiographic hip displacement in CP adductors (CPA), gait kinematics in CP hamstrings (CPH), and joint range of motion in CPA and CPH. We found at matched sarcomere lengths muscle stiffness and collagen architecture were similar between TD and CP hamstrings. However, CPH stiffness (R2 = 0.1973), collagen content (R2 = 0.5099) and cross-linking (R2 = 0.3233) were correlated to decreased knee range of motion. Additionally, we observed collagen fibres within the muscle ECM increase alignment during muscular stretching. These data demonstrate that while ECM architecture is similar between TD and CP hamstrings, collagen fibres biomechanics are sensitive to muscle strain and may be altered at longer in vivo sarcomere lengths in CP muscle. Future studies could evaluate the impact of ECM architecture on TD and CP muscle stiffness across in vivo operating ranges. KEY POINTS: At matched sarcomere lengths, gracilis muscle mechanics and collagen architecture are similar in TD patients and patients with CP. In both TD and CP muscles, collagen fibres dynamically increase their alignment during muscle stretching. Aspects of muscle mechanics and collagen architecture are predictive of in vivo knee joint motion and radiographic hip displacement in patients with CP. Longer sarcomere lengths in CP muscle in vivo may alter collagen architecture and biomechanics to drive deficits in joint mobility and gait function.

Alignment, cross linking, and beyond: a collagen architect's guide to the skeletal muscle extracellular matrix.

The muscle extracellular matrix (ECM) forms a complex network of collagens, proteoglycans, and other proteins that produce a favorable environment for muscle regeneration, protect the sarcolemma from contraction-induced damage, and provide a pathway for the lateral transmission of contractile force. In each of these functions, the structure and organization of the muscle ECM play an important role. Many aspects of collagen architecture, including collagen alignment, cross linking, and packing density affect the regenerative capacity, passive mechanical properties, and contractile force transmission pathways of skeletal muscle. The balance between fortifying the muscle ECM and maintaining ECM turnover and compliance is highly dependent on the integrated organization, or architecture, of the muscle matrix, especially related to collagen. While muscle ECM remodeling patterns in response to exercise and disease are similar, in that collagen synthesis can increase in both cases, one outcome leads to a stronger muscle and the other leads to fibrosis. In this review, we provide a comprehensive analysis of the architectural features of each layer of muscle ECM: epimysium, perimysium, and endomysium. Further, we detail the importance of muscle ECM architecture to biomechanical function in the context of exercise or fibrosis, including disease, injury, and aging. We describe how collagen architecture is linked to active and passive muscle biomechanics and which architectural features are acutely dynamic and adapt over time. Future studies should investigate the significance of collagen architecture in muscle stiffness, ECM turnover, and lateral force transmission in the context of health and fibrosis.

A mutation in desmin makes skeletal muscle less vulnerable to acute muscle damage after eccentric loading in rats.

Desminopathy is the most common intermediate filament disease in humans. The most frequent mutation causing desminopathy in patients is a R350P DES missense mutation. We have developed a rat model with an analogous mutation in R349P Des. To investigate the role of R349P Des in mechanical loading, we stimulated the sciatic nerve of wild-type littermates (WT) (n = 6) and animals carrying the mutation (MUT) (n = 6) causing a lengthening contraction of the dorsi flexor muscles. MUT animals showed signs of ongoing regeneration at baseline as indicated by a higher number of central nuclei (genotype: P < .0001). While stimulation did not impact central nuclei, we found an increased number of IgG positive fibers (membrane damage indicator) after eccentric contractions with both genotypes (stimulation: P < .01). Interestingly, WT animals displayed a more pronounced increase in IgG positive fibers with stimulation compared to MUT (interaction: P < .05). In addition to altered histology, molecular signaling on the protein level differed between WT and MUT. The membrane repair protein dysferlin decreased with eccentric loading in WT but increased in MUT (interaction: P < .05). The autophagic substrate p62 was increased in both genotypes with loading (stimulation: P < .05) but tended to be more elevated in WT (interaction: P = .05). Caspase 3 levels, a central regulator of apoptotic cell death, was increased with stimulation in both genotypes (stimulation: P < .01) but more so in WT animals (interaction: P < .0001). Overall, our data indicate that R349P Des rats have a lower susceptibility to structural muscle damage of the cytoskeleton and sarcolemma with acute eccentric loading.

Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking.

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, cross linking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline cross links, was increased compared with the cross-linked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, whereas MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying extracellular matrix (ECM) structures and enhancing differentiation on substrates with less collagen cross linking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting cross linking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Passive stiffness of fibrotic skeletal muscle in mdx mice relates to collagen architecture.

KEY POINTS: The amount of fibrotic material in dystrophic mouse muscles relates to contractile function, but not passive function. Collagen fibres in skeletal muscle are associated with increased passive muscle stiffness in fibrotic muscles. The alignment of collagen is independently associated with passive stiffness in dystrophic skeletal muscles. These outcomes demonstrate that collagen architecture rather than collagen content should be a target of anti-fibrotic therapies to treat muscle stiffness. ABSTRACT: Fibrosis is prominent in many skeletal muscle pathologies including dystrophies, neurological disorders, cachexia, chronic kidney disease, sarcopenia and metabolic disorders. Fibrosis in muscle is associated with decreased contractile forces and increased passive stiffness that limits joint mobility leading to contractures. However, the assumption that more fibrotic material is directly related to decreased function has not held true. Here we utilize novel measurement of extracellular matrix (ECM) and collagen architecture to relate ECM form to muscle function. We used mdx mice, a model for Duchenne muscular dystrophy that becomes fibrotic, and wildtype mice. In this model, extensor digitorum longus (EDL) muscle was significantly stiffer, but with similar total collagen, while the soleus muscle did not change stiffness, but increased collagen. The stiffness of the EDL was associated with increased collagen crosslinking as determined by collagen solubility. Measurement of ECM alignment using polarized light microscopy showed a robust relationship between stiffness and alignment for wildtype muscle that broke down in mdx muscles. Direct visualization of large collagen fibres with second harmonic generation imaging revealed their relative abundance in stiff muscles. Collagen fibre alignment was linked to stiffness across all muscles investigated and the most significant factor in a multiple linear regression-based model of muscle stiffness from ECM parameters. This work establishes novel characteristics of skeletal muscle ECM architecture and provides evidence for a mechanical function of collagen fibres in muscle. This finding suggests that anti-fibrotic strategies to enhance muscle function and excessive stiffness should target large collagen fibres and their alignment rather than total collagen.

Strain-dependent dynamic re-alignment of collagen fibers in skeletal muscle extracellular matrix.

Collagen fiber architecture within the skeletal muscle extracellular matrix (ECM) is significant to passive muscle mechanics. While it is thought that collagen fibers re-orient themselves in response to changes in muscle length, this has not been dynamically visualized and quantified within a muscle. The goal of this study was to measure changes in collagen alignment across a range of muscle lengths and compare the corresponding alignment to muscle mechanics. We hypothesized that collagen fibers dynamically increase alignment in response to muscle stretching, and this change in alignment is related to passive muscle stiffness. Further, we hypothesized that digesting collagen fibers with collagenase would reduce the re-alignment response to muscle stretching. Using DBA/2J and D2.mdx mice, we isolated extensor digitorum longus (EDL), soleus, and diaphragm muscles for collagenase or sham treatment and decellularization to isolate intact or collagenase-digested decellularized muscles (DCMs). These DCMs were mechanically tested and imaged using second harmonic generation microscopy to measure collagen alignment across a range of strains. We found that collagen alignment increased in a strain-dependent fashion, but collagenase did not significantly affect the strain-dependent change in alignment. We also saw that the collagen fibers in the diaphragm epimysium (surface ECM) and perimysium (deep ECM) started at different angles, but still re-oriented in the same direction in response to stretching. These robust changes in collagen alignment were weakly related to passive DCM stiffness. Overall, we demonstrated that the architecture of muscle ECM is dynamic in response to strain and is related to passive muscle mechanics. STATEMENT OF SIGNIFICANCE: Our study presents a unique visualization and quantification of strain-induced changes in muscle collagen fiber alignment as they relate to passive mechanics. Using dynamic imaging of collagen in skeletal muscle we demonstrate that as skeletal muscle is stretched, collagen fibers re-orient themselves along the axis of stretch and increase their alignment. The degree of alignment and the increase in alignment are each weakly related to passive muscle stiffness. Collagenase treatments further demonstrate that the basis for muscle Extracellular matrix stiffness is dependent on factors beyond collagen crosslinking and alignment. Together the study contributes to the knowledge of the structure-function relationships of muscle extracellular matrix to tissue stiffness relevant to conditions of fibrosis and aberrant stiffness.

Differential encoding of mammalian proprioception by voltage-gated sodium channels.

Animals that require purposeful movement for survival are endowed with mechanosensory neurons called proprioceptors that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we have identified distinct and nonredundant roles for two voltage-gated sodium channels (NaVs), NaV1.1 and NaV1.6, in mammalian proprioception. Deletion of NaV1.6 in somatosensory neurons (NaV1.6cKO mice) causes severe motor deficits accompanied by complete loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target NaV1.1 (NaV1.1cKO). In NaV1.6cKO animals, loss of proprioceptive feedback caused non-cell-autonomous impairments in proprioceptor end-organs and skeletal muscle that were absent in NaV1.1cKO mice. We attribute the differential contribution of NaV1.1 and NaV1.6 in proprioceptor function to distinct cellular localization patterns. Collectively, these data provide the first evidence that NaV subtypes uniquely shape neurotransmission within a somatosensory modality.

The extracellular matrix of dystrophic mouse diaphragm accounts for the majority of its passive stiffness and is resistant to collagenase digestion.

The healthy skeletal muscle extracellular matrix (ECM) has several functions including providing structural integrity to myofibers, enabling lateral force transmission, and contributing to overall passive mechanical properties. In diseases such as Duchenne Muscular dystrophy, there is accumulation of ECM materials, primarily collagen, which results in fibrosis. Previous studies have shown that fibrotic muscle is often stiffer than healthy muscle, in part due to the increased number and altered architecture of collagen fibers within the ECM. This would imply that the fibrotic matrix is stiffer than the healthy matrix. However, while previous studies have attempted to quantify the extracellular contribution to passive stiffness in muscle, the outcomes are dependent on the type of method used. Thus, the goals of this study were to compare the stiffness of healthy and fibrotic muscle ECM and to demonstrate the efficacy of two methods for quantifying extracellular-based stiffness in muscle, namely decellularization and collagenase digestion. These methods have been demonstrated to remove the muscle fibers or ablate collagen fiber integrity, respectively, while maintaining the contents of the extracellular matrix. Using these methods in conjunction with mechanical testing on wildtype and D2.mdx mice, we found that a majority of passive stiffness in the diaphragm is dependent on the ECM, and the D2.mdx diaphragm ECM is resistant to digestion by bacterial collagenase. We propose that this resistance is due to the increased collagen cross-links and collagen packing density in the ECM of the D2.mdx diaphragm. Taken altogether, while we did not find increased stiffness of the fibrotic ECM, we did observe that the D2.mdx diaphragm conveyed resistance against collagenase digestion. These findings demonstrate how different methods for measuring ECM-based stiffness each have their own limitations and can produce different results.

Mesenchymal stromal cell spheroids in sulfated alginate enhance muscle regeneration.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is critically linked to the potency of the complex mixture of growth factors, cytokines, exosomes, and other biological cues that they secrete. The duration of cell-based approaches is limited by rapid loss of cells upon implantation, motivating the need to prolong cell viability and extend the therapeutic influence of the secretome. We and others demonstrated that the secretome is upregulated when MSCs are formed into spheroids. Although the efficacy of the MSC secretome has been characterized in the literature, no studies have reported the therapeutic benefit of in situ sequestration of the secretome within a wound site using engineered biomaterials. We previously demonstrated the capacity of sulfated alginate hydrogels to sequester components of the MSC secretome for prolonged presentation in vitro, yet the efficacy of this platform has not been evaluated in vivo. In this study, we used sulfated alginate hydrogels loaded with MSC spheroids to aid in the regeneration of a rat muscle crush injury. We hypothesized that the use of sulfated alginate to bind therapeutically relevant growth factors from the MSC spheroid secretome would enhance muscle regeneration by recruiting host cells into the tissue site. The combination of sulfated alginate and MSC spheroids resulted in decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions 2 weeks after injury. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreased fibrosis and increased functional regeneration of muscle. STATEMENT OF SIGNIFICANCE: The therapeutic efficacy of mesenchymal stromal cells (MSCs) for tissue regeneration is attributed to the complex diversity of the secretome. Cell-based approaches are limited by rapid cell death, motivating the need to extend the availability of the secretome. We previously demonstrated that sulfated alginate hydrogels sequester components of the MSC secretome for prolonged presentation in vitro, yet no studies have reported the in situ sequestration of the secretome. Herein, we transplanted MSC spheroids in sulfated alginate hydrogels to promote muscle regeneration. MSC spheroids in sulfated alginate decreased collagen deposition, improved myogenic marker expression, and increased neuromuscular junctions. These data indicate that MSC spheroids delivered in sulfated alginate represent a promising approach for decreasing fibrosis and increasing functional muscle regeneration.

Matrix stiffness and architecture drive fibro-adipogenic progenitors' activation into myofibroblasts.

Fibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle, but in muscle pathologies FAPs the are main source of excess extracellular matrix (ECM) resulting in fibrosis. Fibrotic ECM has altered mechanical and architectural properties, but the feedback onto FAPs of stiffness or ECM properties is largely unknown. In this study, FAPs' sensitivity to their ECM substrate was assessed using collagen coated polyacrylamide to control substrate stiffness and collagen hydrogels to engineer concentration, crosslinking, fibril size, and alignment. FAPs on substrates of fibrotic stiffnesses had increased myofibroblast activation, depicted by αSMA expression, compared to substrates mimicking healthy muscle, which correlated strongly YAP nuclear localization. Surprisingly, fibrosis associated collagen crosslinking and larger fibril size inhibited myofibroblast activation, which was independent of YAP localization. Additionally, collagen crosslinking and larger fibril diameters were associated with decreased remodeling of the collagenous substrate as measured by second harmonic generation imaging. Inhibition of YAP activity through verteporfin reduced myofibroblast activation on stiff substrates but not substrates with altered architecture. This study is the first to demonstrate that fibrotic muscle stiffness can elicit FAP activation to myofibroblasts through YAP signaling. However, fibrotic collagen architecture actually inhibits myofibroblast activation through a YAP independent mechanism. These data expand knowledge of FAPs sensitivity to ECM and illuminate targets to block FAP's from driving progression of muscle fibrosis.

Collagen cross-links scale with passive stiffness in dystrophic mouse muscles, but are not altered with administration of a lysyl oxidase inhibitor.

In Duchenne muscular dystrophy (DMD), a lack of functional dystrophin leads to myofiber instability and progressive muscle damage that results in fibrosis. While fibrosis is primarily characterized by an accumulation of extracellular matrix (ECM) components, there are changes in ECM architecture during fibrosis that relate more closely to functional muscle stiffness. One of these architectural changes in dystrophic muscle is collagen cross-linking, which has been shown to increase the passive muscle stiffness in models of fibrosis including the mdx mouse, a model of DMD. We tested whether the intraperitoneal injections of beta-aminopropionitrile (BAPN), an inhibitor of the cross-linking enzyme lysyl oxidase, would reduce collagen cross-linking and passive stiffness in young and adult mdx mice compared to saline-injected controls. We found no significant differences between BAPN treated and saline treated mice in collagen cross-linking and stiffness parameters. However, we observed that while collagen cross-linking and passive stiffness scaled positively in dystrophic muscles, collagen fiber alignment scaled with passive stiffness distinctly between muscles. We also observed that the dystrophic diaphragm showed the most dramatic fibrosis in terms of collagen content, cross-linking, and stiffness. Overall, we show that while BAPN was not effective at reducing collagen cross-linking, the positive association between collagen cross-linking and stiffness in dystrophic muscles still show cross-linking as a viable target for reducing passive muscle stiffness in DMD or other fibrotic muscle conditions.