Antisense suppression of skeletal muscle myosin light chain-1 biosynthesis impairs myofibrillogenesis in cultured myotubes.
Although the alkali or essential light chains of skeletal muscle myosin are not required for actin-activated myosin ATPase activity, these myosin subunits are necessary for force transmission with in vitro actin motility assays and are believed to stabilize the alpha-helical neck region of myosin subfragment-1. To probe the functions of the essential light chains during myofibril assembly, we used recombinant DNA procedures to deplete this light chain in cultured muscle. Retroviral expression vectors were constructed which encoded the exon-1 sequence of the myosin light chain-1 gene in antisense orientation. These vectors were applied to myogenic cells from embryonic chick and quail pectoralis muscle. Colonies expressing antisense RNA were selected in growth medium containing the neomycin analogue G-418, plus 5-bromo-2'-deoxyuridine (BrdU) and triggered to differentiate by removal of the latter. Expression of antisense myosin light chain-1 mRNA impaired muscle development. In the antisense cultures there were more mononucleated cells, fewer and smaller myotubes which had poorly developed myofibrils and high levels of diffusely staining myosin heavy chain, not apparent in control myotubes. Protein synthesis in the myotube cultures was analyzed by 35S-methionine labelling and two-dimensional gel electrophoresis. Except for a suppression of approximately 80% of myosin light chain-1f synthesis, the overall pattern of protein synthesis was not altered significantly. These studies suggest that retardation of myosin light chain-1f accumulation inhibits or delays myofibrillogenesis.
Nawrotzki
R
R
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021, USA.
Fischman
D A
DA
Mikawa
T
T
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002478:Cells, Cultured; D002642:Chick Embryo; D003370:Coturnix; D004274:DNA, Recombinant / Q000235:genetics; D005091:Exons; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009210:Myofibrils / Q000378:metabolism* / Q000648:ultrastructure; D009218:Myosins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014176:Protein Biosynthesis / Q000187:drug effects; D016372:RNA, Antisense / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*
D004274:DNA, Recombinant; D016372:RNA, Antisense; D009218:Myosins