Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Washington University in St. Louis

Persistent muscle atrophy in the chronically torn rotator cuff is a significant obstacle for treatment and recovery. Large atrophic changes are predictive of poor surgical and nonsurgical outcomes and frequently fail to resolve even following functional restoration of loading and rehabilitation. New insights into the processes of muscle atrophy and recovery gained through studies in developmental biology combined with the novel tools and strategies emerging in regenerative medicine provide new avenues to combat the vexing problem of muscle atrophy in the rotator cuff. Moving these treatment strategies forward likely will involve the combination of surgery, biologic/cellular agents, and physical interventions, as increasing experimental evidence points to the beneficial interaction between biologic therapies and physiologic stresses. Thus, the physical therapy profession is poised to play a significant role in defining the success of these combinatorial therapies. This perspective article will provide an overview of the developmental biology and regenerative medicine strategies currently under investigation to combat muscle atrophy and how they may integrate into the current and future practice of physical therapy.

Developmental Biology and Regenerative Medicine: Addressing the Vexing Problem of Persistent Muscle Atrophy in the Chronically Torn Human Rotator Cuff.

Fatty infiltration of the skeletal muscle is a common but poorly understood feature of many myopathies. It is best described in human muscle, where non-invasive imaging techniques and representative histology have been optimized to view and quantify infiltrating fat. However, human studies are limited in their ability to identify cellular and molecular mechanisms regulating fatty infiltration, a likely prerequisite to developing targeted interventions. As mechanistic investigations move to small animals, studies may benefit from new or adapted imaging tools optimized for high resolution and whole muscle quantification.

A novel method for the quantification of fatty infiltration in skeletal muscle.

Efficient repositioning of centralized nuclei after injury has long been assumed, with centralized nuclei frequently cited as indicators of ongoing regeneration. However, reports of centralized nuclei that persist after full recovery of fiber area and muscle force production call into question the time course of nuclear repositioning.

Evidence of induced muscle regeneration persists for years in the mouse.

Posttraumatic elbow contracture is clinically challenging because injury often disrupts multiple periarticular soft tissues. Tissue specific contribution to contracture, particularly muscle, remains poorly understood.

Muscle does not drive persistent posttraumatic elbow contracture in a rat model.

Accumulation of adipose tissue around and within muscles is highly correlated with reduced strength, functional limitations, and poor rehabilitative outcomes. Given the intimate physical contact between these tissues, paracrine cross-talk is a likely mediator of this association. The recent discovery that muscle-associated adipose tissue exhibits features of beige fat has suggested that this cross-talk may be modifiable, as beige fat can be stimulated to assume features of brown fat. In this work, we describe a novel intermuscular fat transplant model in the mouse rotator cuff to investigate cross-talk between muscle and adipose tissue. Specifically, we examine the role of transplanted fat phenotype on muscle regeneration by transplanting pieces of classical brown (interscapular), beige (inguinal), or white (epididymal) adipose tissue in conjunction with cardiotoxin injection to the adjacent supraspinatus muscle. Transplantation of brown fat, but not beige or white, significantly increased muscle mass, fiber cross-sectional area and contractile force production compared with sham injury. This effect was not seen when cardiotoxin was delivered to a distant muscle, or when adjacent muscles were injected with saline indicating that the effect is localized and specifically targeting the regenerative process. Thus, we conclude that local signaling between fat and muscle varies by phenotype and that brown fat supports regeneration. Clinical significance: Our findings suggest that the phenotype of muscle-associated fat could be a novel therapeutic target to modulate fat-muscle signaling. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1817-1826, 2019.

Brown Fat Promotes Muscle Growth During Regeneration.

: Skeletal muscle tissue explants have been cultured and studied for nearly 100 years. These cultures, which retain complex tissue structure in an environment suited to precision manipulation and measurement, have led to seminal discoveries of the extrinsic and intrinsic mechanisms regulating contractility, metabolism and regeneration. This review discusses the two primary models of muscle explant: isolated myofiber and intact muscle.: Relevant literature was reviewed and synthesized with a focus on the unique challenges and capabilities of each explant model.: Impactful past, current and future novel applications are discussed.: Experiments using skeletal muscle explants have been integral to our understanding of the fundamentals of muscle physiology. As they are refined and adapted, they are poised to continue to inform the field for years to come.

Skeletal muscle explants: ex-vivo models to study cellular behavior in a complex tissue environment.

Reversible ε-amino acetylation of lysine residues regulates transcription as well as metabolic flux; however, roles for specific lysine acetyltransferases in skeletal muscle physiology and function are unknown. In this study, we investigated the role of the related acetyltransferases p300 and cAMP response element-binding protein-binding protein (CBP) in skeletal muscle transcriptional homeostasis and physiology in adult mice.

p300 and cAMP response element-binding protein-binding protein in skeletal muscle homeostasis, contractile function, and survival.

Decreased lean muscle mass in the lower extremity in diabetic peripheral neuropathy (DPN) is thought to contribute to altered joint loading, immobility, and disability. However, the mechanism behind this loss is unknown and could derive from a reduction in size of myofibers (atrophy), destruction of myofibers (degeneration), or both. Degenerative changes require participation of muscle stem (satellite) cells to regenerate lost myofibers and restore lean mass. Determining the degenerative state and residual regenerative capacity of DPN muscle will inform the utility of regeneration-targeted therapeutic strategies.

Skeletal Muscle Regeneration in Advanced Diabetic Peripheral Neuropathy.

Muscle infiltration with adipose tissue (IMAT) is common and associated with loss of skeletal muscle strength and physical function across a diverse set of pathologies. Whether the association between IMAT and muscle weakness is causative or simply correlative remains an open question that needs to be addressed to effectively guide muscle strengthening interventions in people with increased IMAT. In the present studies, we demonstrate that IMAT deposition causes decreased muscle strength using mouse models. These findings indicate IMAT is a novel therapeutic target for muscle dysfunction.

Infiltration of intramuscular adipose tissue impairs skeletal muscle contraction.

Since the first isolation of mesenchymal stem cells from lipoaspirate in the early 2000s, adipose tissue has been a darling of regenerative medicine. It is abundant, easy to access, and contains high concentrations of stem cells (ADSCs) exhibiting multipotency, proregenerative paracrine signaling, and immunomodulation-a winning combination for stem cell-based therapeutics. While basic science, preclinical and clinical findings back up the translational potential of ADSCs, the vast majority of these used cells from a single location-subcutaneous abdominal fat. New data highlight incredible diversity in the adipose morphology and function in different anatomical locations or depots. Even in isolation, ADSCs retain a memory of this diversity, suggesting that the optimal adipose source material for ADSC isolation may be application specific. This review discusses our current understanding of the heterogeneity in the adipose organ, how that heterogeneity translates into depot-specific ADSC characteristics, and how atypical ADSC populations might be harnessed for regenerative medicine applications. While our understanding of the breadth of ADSC heterogeneity is still in its infancy, clear trends are emerging for application-specific sourcing to improve regenerative outcomes.

Harnessing adipose stem cell diversity in regenerative medicine.

In response to chronic injury, the muscles of the rotator cuff (RC) experience a unique degeneration characterized by extensive fatty infiltration and loss of contractile function. Human studies suggest this degeneration is also a feature of RC sarcopenia and may precede RC injury. In this study, we investigated whether RC muscles exhibit a similar unique sarcopenia in the mouse.

A unique sarcopenic progression in the mouse rotator cuff.

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

p300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes.

Tenotomy, or the severing of then tendinous connection between muscle and bone, is an experimental model frequently used to assess muscular changes in response to unloading with retraction. It is most translationally relevant to rotator cuff (RC) tendon tears as these frequently progress to chronic muscle retraction and thus most recent tenotomy animal models have used RC muscles. Tenotomy induces chronic changes to muscle architecture including reduced muscle mass, muscle length, fiber length and pennation angle. However, most RC studies evaluating the physiological consequences of tenotomy do not account for changes in architecture, in part because these are difficult to measure in RC muscles. This is a critical omission as architectural changes can dramatically impact muscle force generating capacity. In this work, we develop a geometric model to predict changes in fiber length and pennation angle of the mouse supraspinatus and infraspinatus muscles given a measured muscle length. We then validate this model with detailed architectural measurements in tenotomized muscles and show a close match between predicted and experimental values. Finally, using this model, we find that predicted changes in architecture cannot explain the force deficit in tenotomized muscle. The contributions of this work are 1) a simple geometric model that predicts changes in architecture with retraction, validated in the mouse RC but with potential application across muscles and species and 2) data indicating that architectural adaptation is not solely responsible for the force deficit with tenotomy which suggests future research should focus on intrinsic changes to the myofiber.

Geometric modeling predicts architectural adaptations are not responsible for the force deficit following tenotomy in the rotator cuff.

Adipose tissue secretes numerous cytokines (termed 'adipokines') that have known or hypothesized actions on skeletal muscle. The majority of adipokines have been implicated in the pathological link between excess adipose and muscle insulin resistance, but approximately half also have documented in vitro effects on myogenesis and/or hypertrophy. This complexity suggests a potential dual role for adipokines in the regulation of muscle mass in homeostasis and the development of pathology. In this study, we used lipodystrophic 'fat-free' mice to demonstrate that adipose tissue is indeed necessary for the development of normal muscle mass and strength. Fat-free mice had significantly reduced mass (∼15%) and peak contractile tension (∼20%) of fast-twitch muscles, a slowing of contractile dynamics and decreased cross-sectional area of fast twitch fibres compared to wild-type littermates. These deficits in mass and contractile tension were fully rescued by reconstitution of ∼10% of normal adipose mass, indicating that this phenotype is the direct consequence of absent adipose. We then showed that the rescue is solely mediated by the adipokine leptin, as similar reconstitution of adipose from leptin-knockout mice fails to rescue mass or strength. Together, these data indicate that the development of muscle mass and strength in wild-type mice is dependent on adipose-secreted leptin. This finding extends our current understanding of the multiple roles of adipokines in physiology as well as disease pathophysiology to include a critical role for the adipokine leptin in muscle homeostasis. KEY POINTS: Adipose-derived cytokines (adipokines) have long been implicated in the pathogenesis of insulin resistance in obesity but likely have other under-appreciated roles in muscle physiology. Here we use a fat-free mouse to show that adipose tissue is necessary for the normal development of muscle mass and strength. Through add-back of genetically modified adipose tissue we show that leptin is the key adipokine mediating this regulation. This expands our understanding of leptin's role in adipose-muscle signalling to include development and homeostasis and adds the surprising finding that leptin is the sole mediator of the maintenance of muscle mass and strength by adipose tissue.

Gretchen A. Meyer

Program in Physical Therapy,
Departments of Neurology,
Orthopaedic Surgery and Biomedical Engineering,
Departments of Neurology, Orthopaedic Surgery and Biomedical Engineering

Washington University in St. Louis

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Gretchen A. Meyer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Program in Physical Therapy,Departments of Neurology,Orthopaedic Surgery and Biomedical Engineering,Departments of Neurology, Orthopaedic Surgery and Biomedical Engineering

Washington University in St. Louis

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Program in Physical Therapy,
Departments of Neurology,
Orthopaedic Surgery and Biomedical Engineering,
Departments of Neurology, Orthopaedic Surgery and Biomedical Engineering