4243 The genes of four ‘adult’ skeletal muscle myosin heavy chain (MHC) isoforms have been found by genomic analysis in all mammalian species considered until now: one slow (or type 1) isoform and three fast isoforms called 2A, 2X and 2B. In small rodents such as mice and rats, in rabbits and in marsupials (Schiaffino and Reggiani, 1996; Zhong et al., 2001), all four MHC isoforms are expressed, giving origin to four distinct fibre types. By contrast, in humans, the MHC-2B gene identified in the genome is not expressed, and only 1, 2A and 2X MHC isoforms are expressed in skeletal muscles. The fibre type originally classified as 2B (therefore often called ‘conventional 2B’) contains MHC-2X (Smerdu et al., 1994). In some specialised human muscles, such as masseter, the expression of the 2B gene has been demonstrated by in situ hybridisation, but the corresponding protein has not been found (Horton et al., 2001). The MHC-2B RNA is abundant in masseter fibres, histochemically and immunohistochemically identified as type 2A fibres, but is rare in limb muscle and present in few fibres of the abdominal external oblique muscle (Horton et al., 2001). The inter-species diversity of the expression of the MHC- 2B isoforms between humans and laboratory animals has prompted further studies to assess the expression in various species of veterinarian or experimental interest, from monkey to dog, sheep and llama (for a review, see Reggiani and Mascarello, 2004). In all these species, only three fibre types and three MHC isoforms have been identified in skeletal muscles, two of them (types 1 and 2A) being very similar to the corresponding ones in rat and mouse and the third one being similar to 2X. Thus, the 2B fibre type seems to be specific only for small mammals and marsupials. A recent study suggests that in the genome of some species, such as the horse, only a pseudogene with a sequence similar to that of the MHC-2B gene is present (Chikuni et al., 2004a). There are, however, exceptions: in pigs, the 2B isoform is expressed at the protein level in several skeletal muscles, generally in hybrid 2X/B fibres (Da Costa et al., 2002; Toniolo et al., 2004), and in cattle we have recently shown the expression of MHC- 2B at the mRNA level in extraocular muscles (Maccatrozzo et al., 2004). The Journal of Experimental Biology 208, 4243-4253 Published by The Company of Biologists 2005 doi:10.1242/jeb.01904 This study aimed to analyse the expression of myosin heavy chain (MHC) isoforms in bovine muscles, with particular attention to the MHC-2B gene. Diaphragm, longissimus dorsi, masseter, several laryngeal muscles and two extraocular muscles (rectus lateralis and retractor bulbi) were sampled in adult male Bos taurus (age 18–24·months, mass 400–500·kg) and analysed by RT- PCR, gel electrophoresis and immunohistochemistry. Transcripts and proteins corresponding to eight MHC isoforms were identified: MHC- and MHC-/slow (or MHC-1), two developmental isoforms (MHC-embryonic and MHC-neonatal), three adult fast isoforms (MHC-2A, MHC-2X and MHC-2B) and the extraocular isoform MHC-Eo. All eight MHC isoforms were found to be co- expressed in extrinsic eye muscles, retractor bulbi and rectus lateralis, four (/slow, 2A, 2X, neonatal) in laryngeal muscles, three (/slow, 2A and 2X) in trunk and limb muscles and two (/slow and ) in masseter. The expression of MHC-2B and MHC-Eo was restricted to extraocular muscles. Developmental MHC isoforms (neonatal and embryonic) were only found in specialized muscles in the larynx and in the eye. MHC- was only found in extraocular and masseter muscle. Single fibres dissected from masseter, diaphragm and longissimus were classified into five groups (expressing, respectively, /slow, , slow and 2A, 2A and 2X) on the basis of MHC isoform electrophoretical separation, and their contractile properties [maximum shortening velocity (v 0 ) and isometric tension (P 0 )] were determined. v 0 increased progressively from slow to fast 2A and fast 2X, whereas hybrid 1–2A fibres and fibres containing MHC- were intermediate between slow and fast 2A. Key words: myosin, MHC isoforms, cattle, skeletal muscles, extraocular muscles, laryngeal muscles, RT-PCR, electrophoresis, immunohistochemistry. Summary Introduction Expression of eight distinct MHC isoforms in bovine striated muscles: evidence for MHC-2B presence only in extraocular muscles L. Toniolo 1 , L. Maccatrozzo 2 , M. Patruno 2 , F. Caliaro 2 , F. Mascarello 2, * and C. Reggiani 1 1 Dipartimento di Anatomia e Fisiologia Umana, Università di Padova, Italy and 2 Dipartimento di Scienze Sperimentali Veterinarie, Università di Padova, Italy *Author for correspondence (e-mail: masca@unipd.it) Accepted 28 September 2005 THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY