Postnatal myosin heavy chain isoform expression in normal mice and mice null for IIb or IId myosin heavy chains

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that alpha MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.     

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.     

Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development

Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or intrinsic "clock." Taken together, these results suggest that, unlike embryonic and neonatal MyHCs, the expression of slow MyHC in vivo at different developmental stages during gestation is not the result of commitment to a distinct myoblast lineage, but is largely determined by the environment.     

Spatial and temporal changes in myosin heavy chain gene expression in skeletal muscle development

Seven myosin heavy chains (MyHC) are expressed in mammalian skeletal muscle in spatially and temporally regulated patterns. The timing, distribution, and quantitation of MyHC expression during development and early postnatal life of the mouse are reported here. The three adult fast MyHC RNAs (IIa, IIb, and IId/x) are expressed in the mouse embryo and each mRNA has a distinct temporal and spatial distribution. In situ hybridization analysis demonstrates expression of IIb mRNA by 14.5 dpc, which proceeds developmentally in a rostral to caudal pattern. IId/x and IIa mRNAs are detectable 2 days later. Ribonuclease protection assays demonstrate that the three adult fast genes are expressed at approximately equal levels relative to each other in the embryo but at quite low levels relative to the two developmental isoforms, embryonic and perinatal. Just after birth major changes in the relative proportions of different MyHC RNAs and protein occur. In all cases, RNA expression and protein expression appear coincident. The changes in MyHC RNA and protein expression are distinct in different muscles and are restricted in some cases to particular regions of the muscle and do not always reflect their distribution in the adult.     

Comparative sequence analysis of the complete human sarcomeric myosin heavy chain family: implications for functional diversity.

The conventional myosin motor proteins that drive mammalian skeletal and cardiac muscle contraction include eight sarcomeric myosin heavy chain (MyHC) isoforms. Six skeletal MyHCs are encoded by genes found in tightly linked clusters on human and mouse chromosomes 17 and 11, respectively. The full coding regions of only two out of six mammalian skeletal MyHCs had been sequenced prior to this work. In an effort to assess the extent of sequence diversity within the human MyHC family we present new full-length coding sequences corresponding to four additional human genes: MyHC-IIb, MyHC-extraocular, MyHC-IIa and MyHC-IIx/d. This represents the first opportunity to compare the full coding sequences of all eight sarcomeric MyHC isoforms within a vertebrate organism. Sequence variability has been analyzed in the context of available structure/function data with an emphasis on potential functional diversity within the family. Results indicate that functional diversity among MyHCs is likely to be accomplished by having small pockets of sequence diversity in an otherwise highly conserved molecule.     

A distinct subclass of mammalian striated myosins: structure and molecular evolution of "superfast" or masticatory myosin heavy chain

"Superfast" or masticatory myosin is the molecular motor in the powerful and specialized jaw-closing muscles of carnivores, folivores, and frugivores. This myosin presumably underpins the unusual high force and moderate shortening velocity of muscle fibers expressing it. Here, we report the cloning and sequencing of the cDNA encoding the full-length masticatory myosin heavy chain (MyHC) from cat temporalis muscle. This was obtained by immunoscreening a cDNA expression library and RACE-PCR (rapid amplification of cDNA ends–PCR). Sequence comparisons at the DNA and amino acid levels show that masticatory MyHC has less than 70% homology to known striated MyHCs, compared with 87–96% between other mammalian fast isoforms themselves. Nucleotide substitution rates at the nonsynonymous sites between masticatory MyHC and other mammalian striated MyHCs are considerably higher than between these striated MyHCs themselves. Phylogenetic analysis revealed that masticatory MyHC diverged from invertebrate MyHC before the avian cardiac MyHC subclass and the mammalian fast/developmental and slow/cardiac MyHC subclasses. Masticatory MyHC is thus a distinct new subclass of vertebrate striated myosins. The early divergence from invertebrate MyHC, combined with immunochemical evidence of its expression in reptilian and shark jaw-closing muscles, suggests that masticatory MyHC evolved in early gnathostomes, driven by benefits derived from powerful jaw closure. During the mammalian radiation, some taxa continued to express it, while others adapted to new types of food and eating habits by replacing masticatory MyHC with more appropriate isoforms normally found in limb and cardiac muscles.