The impact of biochemical methods for single muscle fibre analysis

Biochemical methods for single muscle fibre analysis provide sensitive meas ures for elucidating muscle fibre heterogeneity. The understanding of the c omplexity of skeletal muscle fibres, initially based on qualitative histoch emistry and immunohistochemistry, has been greatly expanded by quantitative micromethods, such as microphotometry and microbiochemical assays. Assessm ent of metabolic enzyme activity levels has revealed pronounced scattering within and between different fibre types and has highlighted the use of spe cific enzyme activity ratios as discriminative measures. With the exception of type I fibres, metabolic properties are loosely coupled with molecular properties of the myofibrillar apparatus. As such, myosin heavy chain (MHC) isoforms appear to be the best choice for fibre type delineation. Among th e two available methods for MHC-based fibre type distinction, single fibre electrophoresis appears to be superior to immunohistochemistry. The electro phoretic separation of MHC isoforms in single fibres is quantitative and, a s opposed to immunohistochemistry, yields important information on MHC isof orm proportions in hybrid fibres. Histochemical staining for myofibrillar A TPase activity can, thus, be correlated in most cases with specific MHC iso form profiles. Single fibre studies have demonstrated a relationship betwee n ATP phosphorylation potential and MHC isoform complement. This relationsh ip corresponds to different tension costs and provides an additional ration ale for the MHC-based fibre type diversity and transitions. The combination of reverse transcriptase (RT) with polymerase chain reaction (PCR) has pro ved to be a highly sensitive tool and has extended single fibre analysis to the level of MHC mRNA isoforms. Application of RT-PCR techniques to single fibre fragments identified by their MHC protein isoform profile, provides insights at two levels of expression and, thus, has extended our knowledge on the plasticity of muscle and the dynamical state of muscle fibres.