In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle

D. A. Robinson, S. N. Bremner, K. Sethi, S. R. Sirsi, G. J. Lutz

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 MLC1f. In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1f expression averaged 22-43% of the endogenous MLC1f. Trans-MLC1f 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-MLC1f. 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.

Original languageEnglish (US)
Pages (from-to)347-357
Number of pages11
JournalGene Therapy
Volume12
Issue number4
DOIs
StatePublished - Feb 1 2005

Fingerprint

Myosin Light Chains
Myosins
Anura
Skeletal Muscle
Plasmids
Contractile Proteins
Muscles
Muscle Cells
Cardiotoxins
Genes
Rana pipiens
Gene Transfer Techniques
Striated Muscle
Myosin Heavy Chains
Skeletal Muscle Fibers
Transgenes
Confocal Microscopy
Transfection
Epitopes
Regeneration

Keywords

  • Cardiotoxin
  • Motor protein
  • Regeneration
  • Transient transgenesis

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle. / Robinson, D. A.; Bremner, S. N.; Sethi, K.; Sirsi, S. R.; Lutz, G. J.

In: Gene Therapy, Vol. 12, No. 4, 01.02.2005, p. 347-357.

Research output: Contribution to journalArticle

Robinson, D. A. ; Bremner, S. N. ; Sethi, K. ; Sirsi, S. R. ; Lutz, G. J. / In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle. In: Gene Therapy. 2005 ; Vol. 12, No. 4. pp. 347-357.
@article{68d730d557b84ee1b2f189d20312bbd1,
title = "In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle",
abstract = "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 MLC1f. In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1f expression averaged 22-43{\%} of the endogenous MLC1f. Trans-MLC1f 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-MLC1f. 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.",
keywords = "Cardiotoxin, Motor protein, Regeneration, Transient transgenesis",
author = "Robinson, {D. A.} and Bremner, {S. N.} and K. Sethi and Sirsi, {S. R.} and Lutz, {G. J.}",
year = "2005",
month = "2",
day = "1",
doi = "10.1038/sj.gt.3302411",
language = "English (US)",
volume = "12",
pages = "347--357",
journal = "Gene Therapy",
issn = "0969-7128",
publisher = "Nature Publishing Group",
number = "4",

}

TY - JOUR

T1 - In vivo expression of myosin essential light chain using plasmid expression vectors in regenerating frog skeletal muscle

AU - Robinson, D. A.

AU - Bremner, S. N.

AU - Sethi, K.

AU - Sirsi, S. R.

AU - Lutz, G. J.

PY - 2005/2/1

Y1 - 2005/2/1

N2 - 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 MLC1f. In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1f expression averaged 22-43% of the endogenous MLC1f. Trans-MLC1f 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-MLC1f. 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.

AB - 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 MLC1f. In fibers from the most robustly transfected muscles 3 weeks after plasmid injections, trans-MLC1f expression averaged 22-43% of the endogenous MLC1f. Trans-MLC1f 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-MLC1f. 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.

KW - Cardiotoxin

KW - Motor protein

KW - Regeneration

KW - Transient transgenesis

UR - http://www.scopus.com/inward/record.url?scp=14644444161&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=14644444161&partnerID=8YFLogxK

U2 - 10.1038/sj.gt.3302411

DO - 10.1038/sj.gt.3302411

M3 - Article

C2 - 15538392

AN - SCOPUS:14644444161

VL - 12

SP - 347

EP - 357

JO - Gene Therapy

JF - Gene Therapy

SN - 0969-7128

IS - 4

ER -