Kyle S. Martin

Sphingosine 1-phosphate receptor 3 regulates recruitment of anti-inflammatory monocytes to microvessels during implant arteriogenesis

Endothelial cells play significant roles in conditioning tissues after injury by the production and secretion of angiocrine factors. At least two distinct subsets of monocytes, CD45+CD11b+Gr1+Ly6C+ inflammatory and CD45+CD11b+Gr1−Ly6C− anti-inflammatory monocytes, respond differentially to these angiocrine factors and promote pathogen/debris clearance and arteriogenesis/tissue regeneration, respectively. We demonstrate here that local sphingosine 1-phosphate receptor 3 (S1P3) agonism recruits anti-inflammatory monocytes to remodeling vessels. Poly(lactic-co-glycolic acid) thin films were used to deliver FTY720, an S1P1/3 agonist, to inflamed and ischemic tissues, which resulted in a reduction in proinflammatory cytokine secretion and an increase in regenerative cytokine secretion. The altered balance of cytokine secretion results in preferential recruitment of anti-inflammatory monocytes from circulation. The chemotaxis of these cells, which express more S1P3 than inflammatory monocytes, toward SDF-1α was also enhanced with FTY720 treatment, but not in S1P3 knockout cells. FTY720 delivery enhanced arteriolar diameter expansion and increased length density of the local vasculature. This work establishes a role for S1P receptor signaling in the local conditioning of tissues by angiocrine factors that preferentially recruit regenerative monocytes that can enhance healing outcomes, tissue regeneration, and biomaterial implant functionality.

Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle Abnormalities, Cachexia, and Exercise Intolerance in Mice with Congestive Heart Failure

Background—Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide–dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of nitric oxide–inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. Methods and Results—We demonstrated that systemic administration of endogenous nitric oxide donor S-nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, as well as the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis (muscle creatine kinase [MCK]-EcSOD) in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF (&agr;-myosin heavy chain–calsequestrin), MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced HF. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria, and vascular rarefaction in skeletal muscle. Conclusions—EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF.

Multiscale models of skeletal muscle reveal the complex effects of muscular dystrophy on tissue mechanics and damage susceptibility

Computational models have been increasingly used to study the tissue-level constitutive properties of muscle microstructure; however, these models were not created to study or incorporate the influence of disease-associated modifications in muscle. The purpose of this paper was to develop a novel multiscale muscle modelling framework to elucidate the relationship between microstructural disease adaptations and modifications in both mechanical properties of muscle and strain in the cell membrane. We used an agent-based model to randomly generate new muscle fibre geometries and mapped them into a finite-element model representing a cross section of a muscle fascicle. The framework enabled us to explore variability in the shape and arrangement of fibres, as well as to incorporate disease-related changes. We applied this method to reveal the trade-offs between mechanical properties and damage susceptibility in Duchenne muscular dystrophy (DMD). DMD is a fatal genetic disease caused by a lack of the transmembrane protein dystrophin, leading to muscle wasting and death due to cardiac or pulmonary complications. The most prevalent microstructural variations in DMD include: lack of transmembrane proteins, fibrosis, fatty infiltration and variation in fibre cross-sectional area. A parameter analysis of these variations and case study of DMD revealed that the nature of fibrosis and density of transmembrane proteins strongly affected the stiffness of the muscle and susceptibility to membrane damage.

Agent-based computational model investigates muscle-specific responses to disuse-induced atrophy

Skeletal muscle is highly responsive to use. In particular, muscle atrophy attributable to decreased activity is a common problem among the elderly and injured/immobile. However, each muscle does not respond the same way. We developed an agent-based model that generates a tissue-level skeletal muscle response to disuse/immobilization. The model incorporates tissue-specific muscle fiber architecture parameters and simulates changes in muscle fiber size as a result of disuse-induced atrophy that are consistent with published experiments. We created simulations of 49 forelimb and hindlimb muscles of the rat by incorporating eight fiber-type and size parameters to explore how these parameters, which vary widely across muscles, influence sensitivity to disuse-induced atrophy. Of the 49 muscles modeled, the soleus exhibited the greatest atrophy after 14 days of simulated immobilization (51% decrease in fiber size), whereas the extensor digitorum communis atrophied the least (32%). Analysis of these simulations revealed that both fiber-type distribution and fiber-size distribution influence the sensitivity to disuse atrophy even though no single tissue architecture parameter correlated with atrophy rate. Additionally, software agents representing fibroblasts were incorporated into the model to investigate cellular interactions during atrophy. Sensitivity analyses revealed that fibroblast agents have the potential to affect disuse-induced atrophy, albeit with a lesser effect than fiber type and size. In particular, muscle atrophy elevated slightly with increased initial fibroblast population and increased production of TNF-α. Overall, the agent-based model provides a novel framework for investigating both tissue adaptations and cellular interactions in skeletal muscle during atrophy.

Enhanced Skeletal Muscle Expression of Extracellular Superoxide Dismutase Mitigates Streptozotocin-Induced Diabetic Cardiomyopathy by Reducing Oxidative Stress and Aberrant Cell Signaling

Background—Exercise training enhances extracellular superoxide dismutase (EcSOD) expression in skeletal muscle and elicits positive health outcomes in individuals with diabetes mellitus. The goal of this study was to determine if enhanced skeletal muscle expression of EcSOD is sufficient to mitigate streptozotocin-induced diabetic cardiomyopathy. Methods and Results—Exercise training promotes EcSOD expression in skeletal muscle and provides protection against diabetic cardiomyopathy; however, it is not known if enhanced expression of EcSOD in skeletal muscle plays a functional role in this protection. Here, we show that skeletal muscle–specific EcSOD transgenic mice are protected from cardiac hypertrophy, fibrosis, and dysfunction under the condition of type 1 diabetes mellitus induced by streptozotocin injection. We also show that both exercise training and muscle-specific transgenic expression of EcSOD result in elevated EcSOD protein in the blood and heart without increased transcription in the heart, suggesting that enhanced expression of EcSOD from skeletal muscle redistributes to the heart. Importantly, cardiac tissue in transgenic mice displayed significantly reduced oxidative stress, aberrant cell signaling, and inflammatory cytokine expression compared with wild-type mice under the same diabetic condition. Conclusions—Enhanced expression of EcSOD in skeletal muscle is sufficient to mitigate streptozotocin-induced diabetic cardiomyopathy through attenuation of oxidative stress, aberrant cell signaling, and inflammation, suggesting a cross-organ mechanism by which exercise training improves cardiac function in diabetes mellitus.

Computational Modeling of Muscle Regeneration and Adaptation to Advance Muscle Tissue Regeneration Strategies.

Skeletal muscle has an exceptional ability to regenerate and adapt following injury. Tissue engineering approaches (e.g. cell therapy, scaffolds, and pharmaceutics) aimed at enhancing or promoting muscle regeneration from severe injuries are a promising and active field of research. Computational models are beginning to advance the field by providing insight into regeneration mechanisms and therapies. In this paper, we summarize the contributions computational models have made to understanding muscle remodeling and the functional implications thereof. Next, we describe a new agent-based computational model of skeletal muscle inflammation and regeneration following acute muscle injury. Our computational model simulates the recruitment and cellular behaviors of key inflammatory cells (e.g. neutrophils and M1 and M2 macrophages) and their interactions with native muscle cells (muscle fibers, satellite stem cells, and fibroblasts) that result in the clearance of necrotic tissue and muscle fiber regeneration. We demonstrate the ability of the model to track key regeneration metrics during both unencumbered regeneration and in the case of impaired macrophage function. We also use the model to simulate regeneration enhancement when muscle is primed with inflammatory cells prior to injury, which is a putative therapeutic intervention that has not yet been investigated experimentally. Computational modeling of muscle regeneration, pursued in combination with experimental analyses, provides a quantitative framework for evaluating and predicting muscle regeneration and enables the rational design of therapeutic strategies for muscle recovery.

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 μm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

Muscle-derived extracellular superoxide dismutase inhibits endothelial activation and protects against multiple organ dysfunction syndrome in mice

Abstract Multiple organ dysfunction syndrome (MODS) is a detrimental clinical complication in critically ill patients with high mortality. Emerging evidence suggests that oxidative stress and endothelial activation (induced expression of adhesion molecules) of vital organ vasculatures are key, early steps in the pathogenesis. We aimed to ascertain the role and mechanism(s) of enhanced extracellular superoxide dismutase (EcSOD) expression in skeletal muscle in protection against MODS induced by endotoxemia. We showed that EcSOD overexpressed in skeletal muscle‐specific transgenic mice (TG) redistributes to other peripheral organs through the circulation and enriches at the endothelium of the vasculatures. TG mice are resistant to endotoxemia (induced by lipopolysaccharide [LPS] injection) in developing MODS with significantly reduced mortality and organ damages compared with the wild type littermates (WT). Heterogenic parabiosis between TG and WT mice conferred a significant protection to WT mice, whereas mice with R213G knock‐in mutation, a human single nucleotide polymorphism leading to reduced binding EcSOD in peripheral organs, exacerbated the organ damages. Mechanistically, EcSOD inhibits vascular cell adhesion molecule 1 expression and inflammatory leukocyte adhesion to the vascular wall of vital organs, blocking an early step of the pathology in organ damage under endotoxemia. Therefore, enhanced expression of EcSOD in skeletal muscle profoundly protects against MODS by inhibiting endothelial activation and inflammatory cell adhesion, which could be a promising therapy for MODS. Graphical abstract No caption available. HighlightsIncreased EcSOD in vital organs from skeletal muscle protects against MODS.EcSOD mutation mimicking human R213G SNP exacerbates MODS.Parabiosis and serum transfusion confirm causation of EcSOD in protection.The mechanism is inhibition of endothelial activation in endotoxemia.

Agent-based model illustrates the role of the microenvironment in regeneration in healthy and mdx skeletal muscle

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease with no effective treatment. Multiple mechanisms are thought to contribute to muscle wasting, including increased susceptibility to contraction-induced damage, chronic inflammation, fibrosis, altered satellite stem cell (SSC) dynamics, and impaired regenerative capacity. The goals of this project were to 1) develop an agent-based model of skeletal muscle that predicts the dynamic regenerative response of muscle cells, fibroblasts, SSCs, and inflammatory cells as a result of contraction-induced injury, 2) calibrate and validate the model parameters based on comparisons with published experimental measurements, and 3) use the model to investigate how changing isolated and combined factors known to be associated with DMD (e.g., altered fibroblast or SSC behaviors) influence muscle regeneration. Our predictions revealed that the percent of injured muscle that recovered 28 days after injury was dependent on the peak SSC counts following injury. In simulations with near-full cross-sectional area recovery (healthy, 4-wk mdx, 3-mo mdx), the SSC counts correlated with the extent of initial injury; however, in simulations with impaired regeneration (9-mo mdx), the peak SSC counts were suppressed relative to initial injury. The differences in SSC counts between these groups were emergent predictions dependent on altered microenvironment factors known to be associated with DMD. Multiple cell types influenced the peak number of SSCs, but no individual parameter predicted the differences in SSC counts. This finding suggests that interventions to target the microenvironment rather than SSCs directly could be an effective method for improving regeneration in impaired muscle. NEW & NOTEWORTHY A computational model predicted that satellite stem cell (SSC) counts are correlated with muscle cross-sectional area (CSA) recovery following injury. In simulations with impaired CSA recovery, SSC counts are suppressed relative to healthy muscle. The suppressed SSC counts were an emergent model prediction, because all simulations had equal initial SSC counts. Fibroblast and anti-inflammatory macrophage counts influenced SSC counts, but no single factor was able to predict the pathological differences in SSC counts that lead to impaired regeneration.

Spatial and age-related changes in the microstructure of dystrophic and healthy diaphragms

Duchenne muscular dystrophy (DMD) is a progressive degenerative disease that results in fibrosis and atrophy of muscles. The main cause of death associated with DMD is failure of the diaphragm. The diaphragm is a dome-shaped muscle with a fiber microstructure that differs across regions of the muscle. However, no studies to our knowledge have examined spatial variations of muscle fibers in dystrophic diaphragm or how aging affects those variations in DMD. In this study, diaphragms were obtained from mdx and healthy mice at ages three, seven, and ten months in the dorsal, midcostal, and ventral regions. Through immunostaining and confocal imaging, we quantified sarcomere length, interstitial space between fibers, fiber branching, fiber cross sectional area (CSA), and fiber regeneration measured by centrally located nuclei. Because DMD is associated with chronic inflammation, we also investigated the number of macrophages in diaphragm muscle cross-sections. We saw regional differences in the number of regenerating fibers and macrophages during the progression of DMD in the mdx diaphragm. Additionally, the number of regenerating fibers increased with age, while CSA and the number of branching fibers decreased. Dystrophic diaphragms had shorter sarcomere lengths than age-matched controls. Our results suggest that the dystrophic diaphragm in the mdx mouse is structurally heterogeneous and remodels non-uniformly over time. Understanding regional changes in dystrophic diaphragms over time will facilitate the development of targeted therapies to prevent or minimize respiratory failure in DMD patients.