Molecular cloning and mRNA expression analysis of carp embryonic, slow and cardiac myosin heavy chain isoforms.

Three embryonic class II myosin heavy chains (MYHs) were cloned from the common carp (Cyprinus carpio L.), MYH e m b 1 , MYH e m b 2 and MYH e m b 3 . MYH DNA clones were also isolated from the slow muscle of adult carp acclimated to 10°C (MYH S 1 0 ) and 30°C (MYH S 3 0 ). Phylogenetic analysis demonstrated that MYH e m b 1 and MYH e m b 2 belonged to the fast skeletal muscle MYH clade. By contrast, the sequence of MYH e m b 3 was similar to the adult slow muscle isoforms, MYH S 1 0 and MYH S 3 0 . MYH e m b 1 and MYH e m b 2 transcripts were first detected by northern blot analysis in embryos 61 h post-fertilization (h.p.f.) at the heartbeat stage, with peak expression occurring in 1-month-old juveniles. MYH e m b 1 continued to be expressed at low levels in 7-month-old juveniles when MYH e m b 2 was not detectable. MYH e m b 3 transcripts appeared at almost the same stage as MYH e m b 1 transcripts did (61 h.p.f.), and these genes showed a similar pattern of expression. Whole mount in situ hybridization analysis revealed that the transcripts of MYH e m b 1 and MYH e m b 2 were expressed in the inner part of myotome, whereas MYH e m b 3 was expressed in the superficial compartment. MYH S 1 0 and MYH S 3 0 mRNAs were first detected at hatching. In adult stages, the expression of slow muscle MYH mRNAs was dependent on acclimation temperature. MYH S 1 0 mRNA was expressed at an acclimation temperature of 10 and 20°C, but not at 30°C. In contrast, MYH S 3 0 mRNA was strongly expressed at all acclimation temperatures. The predominant MYH transcripts found in adult slow muscle and in embryos at hatching were expressed in adult fast muscle at some acclimation temperatures but not others. A MYH DNA clone was isolated from the cardiac muscle of 10°C-acclimated adult fish (MYH c a r d ). MYH c a r d mRNA was first detected at 61 h.p.f., but strong signals were only observed in the adult myocardium. The present study has therefore revealed a complex pattern of expression of MYH genes in relation to developmental stage, muscle type and acclimation temperature. None of the skeletal muscle MYHs identified so far was strongly expressed during the late juvenile stage, indicating further developmentally regulated members of the MYH II gene family remain to be discovered.