Co-expression of Multiple Myosin Heavy In Addition to a Tissue-Specific One, in Extraocular Musculature Chain Genes,

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using Sl-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated 2,10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac ~-MHC), cardiac (eMHC), and EOM (X 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors. Myosin heavy chain (MHC), j the main component of the thick filament of the sarcomere, is encoded by a highly conserved multigene family of 7-10 members (47) of which several appear to be clustered onto a single chromosome in mouse (16, 35) and human (35). Each different sarcomeric MHC gene displays a pattern of expression that is both tissue specific and developmentally regulated (39, 43, 46, 50). Protein and mRNA studies have demonstrated that myosin isozymes follow an embryonic ~ neonatal ~ adult transition pattern during mammalian (50, 73) and avian (2, 23, 37, 66) skeletal muscle development. In the cardiac muscle of small mammals, myosin isozyme switches occur in an embryonic Abbreviations used in this paper: EOM, extraocular musculature; MHC, myosin heavy chain; nt, nucleotide(s). adult pattern (24, 36, 59). The mechanisms regulating these myosin isozyme transitions and their relevance to muscle physiology are not fully understood (see reference 69 for discussion). Cellular and hormonal environments of muscle are known to significantly affect the phenotypic expression and transition of MHC isoforms. Both the cardiac and skeletal muscle isomyosin switches are subject to control by thyroid hormone. Hypothyroidism of adult mammals leads to an increased transcription of the cardiac ~-MHC isoform in ventricular cardiac tissue (14, 19, 20, 36, 60); slow muscle-type MHC isoforms are quantitatively increased in adult rat skeletal muscle after thyroidectomy (12, 22, 31, 67). Developmental isoform transitions are also inhibited by hypothyroidism and accelerated by hyperthyroidism (12, 14, 22, 74). 618 THE JOURNAL OF CELL BIOLOGY VOLUME 101 August 1985 618 629 © The Rockefeller University Press • 0021-9525/85]08/0618/12 $1.00 on Jne 5, 2017 D ow nladed fom Published August 1, 1985