Chronic Kidney Disease is Associated With Attenuated Plasma Metabolome Response to Oral Glucose Tolerance Testing.

OBJECTIVE: Chronic kidney disease (CKD) is associated with decreased anabolic response to insulin contributing to protein-energy wasting. Targeted metabolic profiling of oral glucose tolerance testing (OGTT) may help identify metabolic pathways contributing to disruptions to insulin response in CKD. METHODS: Using targeted metabolic profiling, we studied the plasma metabolome response in 41 moderate-to-severe nondiabetic CKD patients and 20 healthy controls at fasting and 2 hours after an oral glucose load. We used linear mixed modeling with random intercepts, adjusting for age, gender, race/ethnicity, body weight, and batch to assess heterogeneity in response to OGTT by CKD status. RESULTS: Mean estimated glomerular filtration rate among CKD participants was 38.9 ± 12.7 mL/min per 1.73 m2 compared to 87.2 ± 17.7 mL/min per 1.73 m2 among controls. Glucose ingestion induced an anabolic response resulting in increased glycolysis products and a reduction in a wide range of metabolites including amino acids, tricarboxylic acid cycle intermediates, and purine nucleotides compared to fasting. Participants with CKD demonstrated a blunted anabolic response to OGTT evidenced by significant changes in 13 metabolites compared to controls. The attenuated metabolome response predominant involved mitochondrial energy metabolism, vitamin B family, and purine nucleotides. Compared to controls, CKD participants had elevated lactate:pyruvate (L:P) ratio and decreased guanosine diphosphate:guanosine triphosphate ratio during OGTT. CONCLUSION: Metabolic profiling of OGTT response suggests a broad disruption of mitochondrial energy metabolism in CKD patients. These findings motivate further investigation into the impact of insulin sensitizers and mitochondrial targeted therapeutics on energy metabolism in patients with nondiabetic CKD.

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.

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.

Distinct effects of different matrix proteoglycans on collagen fibrillogenesis and cell-mediated collagen reorganization.

The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.

Matrix Metalloproteinase 13 from Satellite Cells is Required for Efficient Muscle Growth and Regeneration.

BACKGROUND/AIMS: Cell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion. Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration. A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s. The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown. The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase. METHODS: Mice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice. We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13. We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals. Outcome measures included muscle morphology and function. RESULTS: Under basal conditions, Mmp13-/- mice did not exhibit histological or functional deficits in muscle. However, following acute injury, regeneration was impaired at 11 and 14 days post injury. Muscle hypertrophy caused by increased IGF-1 was blunted with minimal satellite cell incorporation in the absence of MMP-13. Mmp13-/- primary myoblasts displayed reduced migratory capacity in 2D and 3D, while maintaining normal proliferation and differentiation. Satellite cell specific deletion of MMP-13 recapitulated the effects of global MMP-13 ablation on muscle regeneration, growth and myoblast movement. CONCLUSION: These results show that satellite cells provide an essential autocrine source of MMP-13, which not only regulates their migration, but also supports postnatal growth and resolution of acute damage.

Impaired skeletal muscle mitochondrial bioenergetics and physical performance in chronic kidney disease.

The maintenance of functional independence is the top priority of patients with chronic kidney disease (CKD). Defects in mitochondrial energetics may compromise physical performance and independence. We investigated associations of the presence and severity of kidney disease with in vivo muscle energetics and the association of muscle energetics with physical performance. We performed measures of in vivo leg and hand muscle mitochondrial capacity (ATPmax) and resting ATP turnover (ATPflux) using 31phosphorus magnetic resonance spectroscopy and oxygen uptake (O2 uptake) by optical spectroscopy in 77 people (53 participants with CKD and 24 controls). We measured physical performance using the 6-minute walk test. Participants with CKD had a median estimated glomerular filtration rate (eGFR) of 33 ml/min per 1.73 m2. Participants with CKD had a -0.19 mM/s lower leg ATPmax compared with controls but no difference in hand ATPmax. Resting O2 uptake was higher in CKD compared with controls, despite no difference in ATPflux. ATPmax correlated with eGFR and serum bicarbonate among participants with GFR <60. ATPmax of the hand and leg correlated with 6-minute walking distance. The presence and severity of CKD associate with muscle mitochondrial capacity. Dysfunction of muscle mitochondrial energetics may contribute to reduced physical performance in CKD.

Rescue of DNA damage after constricted migration reveals a mechano-regulated threshold for cell cycle.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.

Constricted migration modulates stem cell differentiation.

Tissue regeneration at an injured site depends on proliferation, migration, and differentiation of resident stem or progenitor cells, but solid tissues are often sufficiently dense and constricting that nuclei are highly stressed by migration. In this study, constricted migration of myoblastic cell types and mesenchymal stem cells (MSCs) increases nuclear rupture, increases DNA damage, and modulates differentiation. Fewer myoblasts fuse into regenerating muscle in vivo after constricted migration in vitro, and myodifferentiation in vitro is likewise suppressed. Myosin II inhibition rescues rupture and DNA damage, implicating nuclear forces, while mitosis and the cell cycle are suppressed by constricted migration, consistent with a checkpoint. Although perturbed proliferation fails to explain defective differentiation, nuclear rupture mislocalizes differentiation-relevant MyoD and KU80 (a DNA repair factor), with nuclear entry of the DNA-binding factor cGAS. Human MSCs exhibit similar damage, but osteogenesis increases-which is relevant to bone and to calcified fibrotic tissues, including diseased muscle. Tissue repair can thus be modulated up or down by the curvature of pores through which stem cells squeeze.

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.

Matrix metalloproteinase 13 is a new contributor to skeletal muscle regeneration and critical for myoblast migration.

Efficient skeletal muscle repair and regeneration require coordinated remodeling of the extracellular matrix (ECM). Previous reports have indicated that matrix metalloproteinases (MMPs) play the pivotal role in ECM remodeling during muscle regeneration. The goal of the current study was to determine if the interstitial collagenase MMP-13 was involved in the muscle repair process. Using intramuscular cardiotoxin injections to induce acute muscle injury, we found that MMP-13 expression and activity transiently increased during the regeneration process. In addition, in muscles from mdx mice, which exhibit chronic injury, MMP-13 expression and protein levels were elevated. In differentiating C2C12 cells, a murine myoblast cell line, Mmp13 expression was most pronounced after myoblast fusion and during myotube formation. Using pharmacological inhibition of MMP-13 to test whether MMP-13 activity is necessary for the proliferation, differentiation, migration, and fusion of C2C12 cells, we found a dramatic blockade of myoblast migration, as well as a delay in differentiation. In contrast, C2C12 cells with stable overexpression of MMP-13 showed enhanced migration, without affecting myoblast maturation. Taken together, these results support a primary role for MMP-13 in myoblast migration that leads to secondary effects on differentiation.

Reduced satellite cell population may lead to contractures in children with cerebral palsy.

AIM: Satellite cells are the stem cells residing in muscle responsible for skeletal muscle growth and repair. Skeletal muscle in cerebral palsy (CP) has impaired longitudinal growth that results in muscle contractures. We hypothesized that the satellite cell population would be reduced in contractured muscle. METHOD: We compared the satellite cell populations in hamstring muscles from participants with CP contracture (n=8; six males, two females; age range 6-15y; Gross Motor Function Classification System [GMFCS] levels II-V; 4 with hemiplegia, 4 with diplegia) and from typically developing participants (n=8; six males, two females, age range 15-18y). Muscle biopsies were extracted from the gracilis and semitendinosus muscles and mononuclear cells were isolated. Cell surface markers were stained with fluorescently conjugated antibodies to label satellite cells (neural cell adhesion molecule) and inflammatory and endothelial cells (CD34 and CD4 respectively). Cells were analyzed using flow cytometry to determine cell populations. RESULTS: After gating for intact cells a mean of 12.8% (SD 2.8%) were determined to be satellite cells in typically developing children, but only 5.3% (SD 2.3%; p<0.05) in children with CP. Hematopoietic and endothelial cell types were equivalent in typically developing children and children with CP (p>0.05) suggesting the isolation procedure was valid. INTERPRETATION: A reduced satellite cell population may account for the decreased longitudinal growth of muscles in CP that develop into fixed contractures or the decreased ability to strengthen muscle in CP. This suggests a unique musculoskeletal disease mechanism and provides a potential therapeutic target for debilitating muscle contractures.

Transcriptional abnormalities of hamstring muscle contractures in children with cerebral palsy.

Cerebral palsy (CP) is an upper motor neuron disease that results in a spectrum of movement disorders. Secondary to the neurological lesion, muscles from patients with CP are often spastic and form debilitating contractures that limit range of motion and joint function. With no genetic component, the pathology of skeletal muscle in CP is a response to aberrant complex neurological input in ways that are not fully understood. This study was designed to gain further understanding of the skeletal muscle response in CP using transcriptional profiling correlated with functional measures to broadly investigate muscle adaptations leading to mechanical deficits.Biopsies were obtained from both the gracilis and semitendinosus muscles from a cohort of patients with CP (n = 10) and typically developing patients (n = 10) undergoing surgery. Biopsies were obtained to define the unique expression profile of the contractures and passive mechanical testing was conducted to determine stiffness values in previously published work. Affymetrix HG-U133A 2.0 chips (n = 40) generated expression data, which was validated for selected transcripts using quantitative real-time PCR. Chips were clustered based on their expression and those from patients with CP clustered separately. Significant genes were determined conservatively based on the overlap of three summarization algorithms (n = 1,398). Significantly altered genes were analyzed for over-representation among gene ontologies and muscle specific networks.The majority of altered transcripts were related to increased extracellular matrix expression in CP and a decrease in metabolism and ubiquitin ligase activity. The increase in extracellular matrix products was correlated with mechanical measures demonstrating the importance in disability. These data lay a framework for further studies and development of novel therapies.

Muscle extracellular matrix applies a transverse stress on fibers with axial strain.

It is widely assumed that skeletal muscle contraction is isovolumic. This assumption has been verified at the single fiber and at the myofibril level. Model development and mechanical analyses often exploit this assumption when investigating skeletal muscle and evaluating muscle mechanical properties. This communication describes a method whereby individual muscle fibers and bundles of fibers, which include their constituent extracellular matrix (ECM), were tested to define the change in volume with axial strain. The results demonstrate that fibers are isovolumic, but bundles decrease in volume with strain. The loss of volume implicates a transverse force being applied to the fibers by the ECM. The nature and importance of this transverse force warrant further investigation.

Hamstring contractures in children with spastic cerebral palsy result from a stiffer extracellular matrix and increased in vivo sarcomere length.

Cerebral palsy (CP) results from an upper motoneuron (UMN)lesion in the developing brain. Secondary to the UMNl esion,which causes spasticity, is a pathological response by muscle - namely, contracture. However, the elements within muscle that increase passive mechanical stiffness, and therefore result in contracture, are unknown. Using hamstring muscle biopsies from pediatric patients with CP (n =33) and control (n =19) patients we investigated passive mechanical properties at the protein, cellular, tissue and architectural levels to identify the elements responsible for contracture. Titin isoform, the major load-bearing protein within muscle cells, was unaltered in CP. Correspondingly, the passive mechanics of individual muscle fibres were not altered. However, CP muscle bundles, which include fibres in their constituent ECM, were stiffer than control bundles. This corresponded to an increase in collagen content of CP muscles measured by hydroxyproline assay and observed using immunohistochemistry. In vivo sarcomere length of CP muscle measured during surgery was significantly longer than that predicted for control muscle. The combination of increased tissue stiffness and increased sarcomere length interact to increase stiffness greatly of the contracture tissue in vivo. These findings provide evidence that contracture formation is not the result of stiffening at the cellular level, but stiffening of the ECM with increased collagen and an increase of in vivo sarcomere length leading to higher passive stresses.