Fadia Haddad

A rationale for SDS-PAGE of MHC isoforms as a gold standard for determining contractile phenotype.

TO THE EDITOR: We believe that a better “gold standard” with regard to assessing fiber type, specifically the contractile phenotype, is with SDS-PAGE separation of MHC isoforms performed, optimally, on single isolated muscle fibers. The metabolic and contractile phenotype must be assessed separately, and the electrophoretic approach offers distinct advantages to ATPase and immunohistochemistry (IHC). While the latter methods are probably the most commonly used, there are limitations to these approaches described by Booth et al. (1). Many muscle fibers express heterogeneity of MHC isoforms. ATPase and IHC provide information only on the presence of a given isoform, not its proportional contribution. These methods are therefore only informative from a qualitative perspective. Our suggested methodology is feasible for most labs to perform and, importantly, is quantitative. It allows for determination of precise proportions of all MHC isoforms in a given muscle fiber. Additionally, it provides a definitive means for identifying the IIx MHC isoform, something that is currently problematic with respect to ATPase and IHC techniques. Neonatal and embryonic MHCs can also be distinctly separated and quantified by electrophoresis, when performed under optimized conditions (6). Another specific advantage of this methodology is that it allows for the prediction of functional properties of the muscle, i.e., forcevelocity relationship (4, 5). SDS-PAGE has been definitively shown to be a method sensitive to subtle or marked differences in MHC isoform expression in response to altered loading and hormone states, during development, and between muscles of different fiber types (2– 6). To prove that interventions impact on contractile phenotype, MHC isoform composition should be determined by the electrophoretic approach.: We believe that a better “gold standard” withregard to assessing fiber type, specifically the contractile phe-notype, is with SDS-PAGE separation of MHC isoforms per-formed, optimally, on single isolated muscle fibers. The met-abolic and contractile phenotype must be assessed separately,and the electrophoretic approach offers distinct advantages toATPase and immunohistochemistry (IHC). While the lattermethods are probably the most commonly used, there arelimitations to these approaches described by Booth et al. (1).Many muscle fibers express heterogeneity of MHC isoforms.ATPase and IHC provide information only on the presence ofa given isoform, not its proportional contribution. These meth-ods are therefore only informative from a qualitative perspec-tive.Our suggested methodology is feasible for most labs toperform and, importantly, is quantitative. It allows fordetermination of precise proportions of all MHC isoforms ina given muscle fiber. Additionally, it provides a definitivemeans for identifying the IIx MHC isoform, something thatis currently problematic with respect to ATPase and IHCtechniques. Neonatal and embryonic MHCs can also bedistinctly separated and quantified by electrophoresis, whenperformed under optimized conditions (6). Another specificadvantage of this methodology is that it allows for theprediction of functional properties of the muscle, i.e., force-velocity relationship (4, 5). SDS-PAGE has been defini-tively shown to be a method sensitive to subtle or markeddifferences in MHC isoform expression in response toaltered loading and hormone states, during development,and between muscles of different fiber types (2–6). Toprove that interventions impact on contractile phenotype,MHC isoform composition should be determined by theelectrophoretic approach.