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 disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Infiltration of intramuscular adipose tissue impairs skeletal muscle contraction.

KEY POINTS: 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. ABSTRACT: Intramuscular adipose tissue (IMAT) is associated with deficits in strength and physical function across a wide array of conditions, from injury to ageing to metabolic disease. Due to the diverse aetiologies of the primary disorders involving IMAT and the strength of the associations, it has long been proposed that IMAT directly contributes to this muscle dysfunction. However, infiltration of IMAT and reduced strength could both be driven by muscle disuse, injury and systemic disease, making IMAT simply an 'innocent bystander.' Here, we utilize novel mouse models to evaluate the direct effect of IMAT on muscle contraction. First, we utilize intramuscular glycerol injection in wild-type mice to evaluate IMAT in the absence of systemic disease. In this model we find that, in isolation from the neuromuscular and circulatory systems, there remains a muscle-intrinsic association between increased IMAT volume and decreased contractile tension (r2  > 0.5, P < 0.01) that cannot be explained by reduction in contractile material. Second, we utilize a lipodystrophic mouse model which cannot generate adipocytes to 'rescue' the deficits. We demonstrate that without IMAT infiltration, glycerol treatment does not reduce contractile force (P > 0.8). Taken together, this indicates that IMAT is not an inert feature of muscle pathology but rather has a direct impact on muscle contraction. This finding suggests that novel strategies targeting IMAT may improve muscle strength and function in a number of populations.

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

BACKGROUND: 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. RESULTS: Here, we describe a novel method to evaluate fatty infiltration, developed for use with mouse muscle. In this methodology, muscle cellular membranes and proteins are removed via decellularization, but fatty infiltrate lipid is spared, trapped in its native distribution in a transparent extracellular matrix construct. This lipid can then be stained with visible or fluorescent dyes and imaged. We present three methods to stain and evaluate lipid in decellularized muscles which can be used individually or combined: (1) qualitative visualization of the amount and 3D spatial distribution of fatty infiltration using visible lipid soluble dye Oil Red O (ORO), (2) quantitative analysis of individual lipid droplet metrics (e.g., volume) via confocal imaging of fluorescent lipid soluble dye boron-dipyrromethene (BODIPY), and (3) quantitative analysis of total lipid content by optical density reading of extracted stained lipid. This methodology was validated by comparing glycerol-induced fatty infiltration between two commonly used mouse strains: 129S1/SvlmJ (129S1) and C57BL/6J (BL/6J). All three methods were able to detect a significant increase in fatty infiltrate volume in the 129S1 muscle compared with that in BL/6J, and methods 1 and 2 additionally described a difference in the distribution of fatty infiltrate, indicating susceptibility to glycerol-induced fatty infiltration is strain-specific. CONCLUSIONS: With more mechanistic studies of fatty infiltration moving to small animal models, having an alternative to expensive non-invasive imaging techniques and selective representative histology will be beneficial. In this work, we present a method that can quantify both individual adipocyte lipids and whole muscle total fatty infiltrate lipid.