Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis

Abstract

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.

Figure 1. Representative images of rat red tibialis anterior (RTA) muscle showing MHC expression as well as SDH and GPD activity staining.

Panel A, rat muscle serial cross-section incubated with a primary antibody cocktail against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. Shown are type I (blue), type IIA (green), type IIB (red), type IIX (unstained), and type IIAX (intermediate green) fibers. Panel B, rat muscle serial cross-section incubated with a primary antibody cocktail against MHCIIa (SC-71) and MHCIIx (6H1), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. This confirms the presence of type IIA (green) fibers, as well as confirms that the unstained fibers and intermediate green stained fibers in Panel A are type IIX (purple) and type IIAX fibers (green and purple), respectively. Panel C, rat muscle serial cross-section showing SDH activity staining. Panel D, rat muscle serial cross-section showing GPD activity staining. Bar represents 50 µm.

Figure 2. Representative images of mouse red gastrocnemius (RG) muscle showing MHC expression as well as SDH and GPD activity staining.

Panel A, mouse muscle serial cross-section incubated with a primary antibody cocktail against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. Shown are type I (blue), type IIA (green), type IIB (red), type IIX (unstained), and type IIXB (intermediate red) fibers. Panel B, mouse muscle serial cross-section incubated with a primary antibody cocktail against MHCIIa (SC-71) and MHCIIx (6H1), followed by incubation with appropriate fluorescent-conjugated secondary antibodies. This confirms the presence of type IIA (green) fibers, as well as confirms that the unstained fibers and intermediate red stained fibers in Panel A are type IIX (purple) and type IIXB (purple and red) fibers, respectively. Panel C, mouse muscle serial cross-section showing SDH activity staining. Panel D, mouse muscle serial cross-section showing GPD activity staining. Bar represents 50 µm.

Figure 3. Representative images of rat, mouse, and human muscle showing type I/IIA hybrid fibers.

Panels A–C (rat RG), D–F (mouse soleus), and G–I (human VL) are images of muscle cross-sections incubated concurrently with primary antibodies against MHCI (BA-F8) and MHCIIa (SC-71) showing type I (blue), type IIA (green), and type I/IIA (blue and green) fibers. Bars represent 50 µm.

Figure 4. Bubble plot showing SDH activity, GPD activity, and CSA for each fiber type in mouse, rat, and human skeletal muscles.

Panel A, relative fiber type-specific SDH activity, GPD activity, and CSA for ten mouse skeletal muscles. Panel B, relative fiber type-specific SDH activity, GPD activity, and CSA for ten rat skeletal muscles. Panel C, relative fiber type-specific SDH activity, GPD activity, and CSA for human VL muscle. Data presented in this figure are from Tables 3, 4, and 5. Bubble size represents the relative CSA within a species. SDH and GPD activity are expressed relative to the values obtained in type I fibers (soleus for mouse and rat, VL for human) and assigned a reference value of 1.0.

Figure 5. Representative images of human muscle showing MHC expression as well as SDH and GPD activity staining.

Panels A–D, merged and single channel images of a human vastus lateralis (VL) muscle serial cross-section incubated with an antibody cocktail (BA-F8, SC-71, and 6H1). Shown are type I (blue), type IIA (strong green), type IIX (strong red and intermediate green), and type IIAX (intermediate/strong green and intermediate red) fibers. *Note that the lower intensity staining for SC-71 in type IIX fibers is indicative of non-specific cross-reactivity (see results and discussion for further details). Panels E–H, merged and single channel images of a human VL muscle serial cross-section incubated with an antibody cocktail (BA-F8, BF-35, and 6H1). Shown are type I (blue), type I and type IIA (intermediate green and strong green, respectively), type IIX (strong red), and type IIAX (intermediate/strong green and intermediate red) fibers. Panel I, human VL muscle serial cross-section showing SDH activity staining. Panel J, human VL muscle serial cross-section showing GPD activity staining. Bar represents 50 µm.