Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis

Skeletal muscle is a heterogeneous tissue comprised of fibers with different morphological, functional, and metabolic properties. Different muscles contain varying proportions of fiber types; therefore, accurate identification is important. A number of histochemical methods are used to determine muscle fiber type; however, these techniques have several disadvantages. Immunofluorescence analysis is a sensitive method that allows for simultaneous evaluation of multiple MHC isoforms on a large number of fibers on a single cross-section, and offers a more precise means of identifying fiber types. In this investigation we characterized pure and hybrid fiber type distribution in 10 rat and 10 mouse skeletal muscles, as well as human vastus lateralis (VL) using multicolor immunofluorescence analysis. In addition, we determined fiber type-specific cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, and α-glycerophosphate dehydrogenase (GPD) activity. Using this procedure we were able to easily identify pure and hybrid fiber populations in rat, mouse, and human muscle. Hybrid fibers were identified in all species and made up a significant portion of the total population in some rat and mouse muscles. For example, rat mixed gastrocnemius (MG) contained 12.2% hybrid fibers whereas mouse white tibialis anterior (WTA) contained 12.1% hybrid fibers. Collectively, we outline a simple and time-efficient method for determining MHC expression in skeletal muscle of multiple species. In addition, we provide a useful resource of the pure and hybrid fiber type distribution, fiber CSA, and relative fiber type-specific SDH and GPD activity in a number of rat and mouse muscles

Use of imaging biomarkers to assess perfusion and glucose metabolism in the skeletal muscle of dystrophic mice

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease that affects 1 in 3500 boys. The disease is characterized by progressive muscle degeneration that results from mutations in or loss of the cytoskeletal protein, dystrophin, from the glycoprotein membrane complex, thus increasing the susceptibility of contractile muscle to injury. To date, disease progression is typically assessed using invasive techniques such as muscle biopsies, and while there are recent reports of the use of magnetic resonance, ultrasound and optical imaging technologies to address the issue of disease progression and monitoring therapeutic intervention in dystrophic mice, our study aims to validate the use of imaging biomarkers (muscle perfusion and metabolism) in a longitudinal assessment of skeletal muscle degeneration/regeneration in two murine models of muscular dystrophy.</p> <p>Methods</p> <p>Wild-type (w.t.) and dystrophic mice (weakly-affected mdx mice that are characterized by a point mutation in dystrophin; severely-affected mdx:utrn-/- (udx) mice that lack functional dystrophin and are null for utrophin) were exercised three times a week for 30 minutes. To follow the progression of DMD, accumulation of ¹⁸F-FDG, a measure of glucose metabolism, in both wild-type and affected mice was measured with a small animal PET scanner (GE eXplore Vista). To assess changes in blood flow and blood volume in the hind limb skeletal muscle, mice were injected intravenously with a CT contrast agent, and imaged with a small animal CT scanner (GE eXplore Ultra).</p> <p>Results</p> <p>In hind limb skeletal muscle of both weakly-affected mdx mice and in severely-affected udx mice, we demonstrate an early, transient increase in both ¹⁸F-FDG uptake, and in blood flow and blood volume. Histological analysis of H&E-stained tissue collected from parallel littermates demonstrates the presence of both inflammatory infiltrate and centrally-located nuclei, a classic hallmark of myofibrillar regeneration. In both groups of affected mice, the early transient response was succeeded by a progressive decline in muscle perfusion and metabolism; this was also evidenced histologically.</p> <p>Conclusions</p> <p>The present study demonstrates the utility of non-invasive imaging biomarkers in characterizing muscle degeneration/regeneration in murine models of DMD. These techniques may now provide a promising alternative for assessing both disease progression and the efficacy of new therapeutic treatments in patients.</p