In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle.
It is well established that mutations in specific structural elements of the motor protein myosin are directly linked to debilitating diseases involving malfunctioning striated muscle cells. A potential way to study the relationship between myosin structure and function is to express exogenous myosin in vivo and determine contractile properties of the transgenic muscle cells. However, in vivo expression of functional levels of contractile proteins using transient transgenesis in skeletal muscle has not been demonstrated. Presently, we used in vivo gene transfer to express high levels of full-length myosin light chain (MLC) in skeletal muscle fibers of Rana pipiens. Anterior tibialis (AT) muscles were injected with cardiotoxin to cause degeneration and then injected at various stages of regeneration with plasmid expression vectors encoding full-length MLC1(f). In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1(f) expression averaged 22-43% of the endogenous MLC1(f). Trans-MLC1(f) expression was the same whether a small epitope tag was placed on the C- or N-terminus and was highly variable along individual fibers. Confocal microscopy of skinned fibers showed correct sarcomeric incorporation of trans-MLC1(f). The expression profile of myosin heavy chain isoforms 21 days after transfection was similar to normal AT muscle. These data demonstrate the feasibility of using in vivo gene transfer to probe the structural basis of contractile protein function in skeletal muscle. Based on these promising results, we discuss how further improvements in the level and consistency of myosin transgene expression may be achieved in future studies, and the therapeutic potential of plasmid gene transfer in regenerating muscle.
Robinson
D A
DA
University of California San Diego and Veterans Affairs Medical Center, San Diego, CA, USA.
Bremner
S N
SN
Sethi
K
K
Shah
S B
SB
Sirsi
S R
SR
Lutz
G J
GJ
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D018613:Microscopy, Confocal; D018482:Muscle, Skeletal / Q000378:metabolism*; D009136:Muscular Dystrophies / Q000628:therapy*; D018994:Myosin Light Chains / Q000235:genetics*; D010957:Plasmids; D011894:Rana pipiens; D012038:Regeneration; D019076:Transgenes
D018994:Myosin Light Chains