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

Darin Bloemberg,Joe Quadrilatero,Rebecca Berdeaux

doi:10.1371/journal.pone.0035273

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.

Highlights

Skeletal muscle is a heterogeneous tissue containing fibers with diverse morphological and functional characteristics [1,2]
The tibialis anterior was separated into red (RTA) and white (WTA) portions, the gastrocnemius was separated into red (RG), white (WG), and mixed (MG) portions, whereas the vastus intermedius (VI) and the white vastus lateralis (WVL) were isolated from the quadriceps
Rat and Mouse Muscle myosin heavy chain (MHC) Expression Incubation of rat and mouse muscle cross-sections with an antibody cocktail resulted in positive staining of the same fibers as serial cross-sections incubated with individual antibodies (Supplementary Figure S1)

Summary

Introduction

Skeletal muscle is a heterogeneous tissue containing fibers with diverse morphological and functional characteristics [1,2]. Adult mammalian muscle can contain 4 major MHC isoforms; one slow isoform (MHCI) and three fast isoforms (MHCIIa, MHCIIx, MHCIIb). Human skeletal muscle does not contain MHCIIb [2,4,5]. Fibers expressing MHCI are termed type I fibers, whereas fibers expressing MHCIIa, MHCIIx, and MHCIIb are termed type IIA, type IIX, and type IIB fibers, respectively. ‘‘hybrid’’ fibers containing two MHC isoforms (i.e., type I/IIA, IIAX, IIXB) can be present in muscle [4,6]. See review by Schiaffino [7] for a recent perspective regarding skeletal muscle fiber types

Methods

Results

Conclusion