Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Ranapipiens

Abstract

Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS‐PAGE and only one adult hindlimb MHC isoform has been cloned. In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Ranapipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS‐PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co‐migrated with type 1 MHC. The combined data from immunohistochemistry and SDS‐PAGE suggests that Rana fibre types are composed of four different MHCs. Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno‐identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types. Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level. These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system. Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation.