QUANTITATIVE-ANALYSES OF MYOSIN HEAVY-CHAIN MESSENGER-RNA AND PROTEINISOFORMS IN SINGLE FIBERS REVEAL A PRONOUNCED FIBER HETEROGENEITY IN NORMAL RABBIT MUSCLES

A highly sensitive method of reverse-transcriptase polymerase chain re action (RT-PCR) was established to study myosin heavy-chain (MHC) mRNA isoform expression in single fibers of rabbit limb muscles. In combin ation with myofibrillar adenosine triphosphatase histochemistry and el ectrophoretic separation of MHC protein isoforms in fragments of the s ame fibers, the direct RT-PCR method identified the pMHC20-40 and pMHC 24-79 cDNA sequences as being specific to MHCIIb and MHCIId/x isoforms , respectively In addition, a direct RT-PCR was established for determ ining relative amounts of MHC mRNA isoforms by using a sequence specif ic to alpha-skeletal actin as an endogenous reference. Analyses of lar ge amounts of single fibers revealed an unexpected heterogeneity of th e fast fiber population with regard to numerous fibers coexpressing MH CIIb and MHCIId/x. Based on quantitative RT-PCR, the percentages of MH CIIb/MHCIId hybrid fibers amounted to approximately 55% in the deep po rtion of gastrocnemius, to 43% in the adductor magnus, and to 12% in p soas muscle. Moreover, the two MHC mRNA isoforms were nonuniformly dis tributed along the fiber length. Qualitative RT-PCR detected even high er amounts of hybrid fibers in the three muscles. The percentages of h ybrid fibers identified at the protein level were smaller in adductor magnus muscle (25%) and psoas muscle (5%), but equaled that of the mRN A analysis in gastrocnemius muscle (61%). The detection of high amount s of IIBD and IIDB fibers suggested that hybrid fibers represent funct ional elements within the fiber spectrum of normal muscles. Our observ ations on hybrid fibers reveal a heterogeneity within the fiber popula tion of normal muscles that has not been realized to date.