Developmental patterns of expression and coexpression of myosin heavy chains in atria and ventricles of the avian heart

Monoclonal antibodies (mAbs), electrophoresis, immunoblotting, and immunohistochemistry were used to determine the molecular properties of cardiac myosin heavy chain (MHC) isoforms and the regions of the developing chicken heart in which they were expressed. Adult atria expressed three electrophoretically distinct MHCs that reacted specifically with mAbs F18, F59, or S58. During embryonic Days 2-4, when the atrial and ventricular chambers are forming, MHCs that reacted with mAbs F18, F59, or S58 were expressed in both the atria and ventricles. The atria continued to express MHCs that reacted with mAbs F18, F59, or S58 at all stages of development and in the adult. In the ventricles, expression of the MHCs reacting with these mAbs was found to be developmentally regulated. By embryonic Day 16, MHC(s) reacting with mAb F18 had disappeared from the developing ventricles, whereas MHCs reacting with S58 and F59 continued to be expressed throughout the ventricles. As development continued, MHC(s) reacting with S58 in the ventricle became restricted to expression in only the ventricular conducting system. MHC(s) reacting with F59 were expressed in both the ventricular myocytes and the ventricular conducting system throughout development and in the adult. Thus, in contrast to the embryonic chicken heart where at least three MHC isoforms were expressed in both the atria and ventricles, we found in the adult chicken heart that-at a minimum-three MHC isoforms were expressed in the atria, two MHC isoforms were expressed in the ventricular conducting system, and one MHC isoform in the ventricular myocardium. MHC isoform expression in the developing avian heart appears to be more complex than previously recognized.     

Smooth muscle myosin is composed of homodimeric heavy chains.

Vertebrate smooth muscle myosin heavy chains (MHCs) exist as two isoforms with molecular masses of 204 and 200 kDa (MHC204 and MHC200) that are generated from a single gene by alternative splicing of mRNA (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). A dimer of two MHCs associated with two pairs of myosin light chains forms a functional myosin molecule. To investigate the isoform composition of the MHCs in native myosin, antibodies specific for MHC204 were generated and used to immunoprecipitate purified bovine aortic smooth muscle myosin from a solution containing equal amounts of each isoform. MHC204 quantitatively removed from this mixture was completely free of MHC200. Immunoprecipitation of the supernatant with an antiserum that recognizes both isoforms equally well revealed that only MHC200 remained. We conclude that only homodimers of MHC204 and MHC200 exist under these conditions. A method is described for the purification of enzymatically active MHC204 and myosin on a protein G-agarose high performance liquid chromatography column containing immobilized MHC204 antibodies. We show, using an in vitro motility assay, that the movement of actin filaments by myosin containing 204-kDa heavy chains (0.435 +/- 0.115 microns/s) was not significantly different from that of myosin containing 200-kDa heavy chains (0.361 +/- 0.078 microns/s) or from myosin containing equal amounts of each heavy chain isoform (0.347 +/- 0.082 microns/s).     

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.     

Myosin heavy chain composition of single fibres from normal human muscle.

Electrophoretic analysis in the presence of 33% glycerol of purified myosin from normal human muscle shows three distinct protein bands which are identified as type 1, 2B, and 2A myosin heavy chain (MHC) isoforms by affinity-purified polyclonal antibodies. Analysis of MHC of single human muscle fibres shows that human muscles contain a large population of fibres showing the coexistence of type 2A and 2B MHC.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1-day-posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross-reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross-reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin. Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to from the contractile ring for cytokinesis.