Investigations of an inducible intact dystrophin gene excision system in cardiac and skeletal muscle in vivo

Addeli Bez Batti  Angulski; John Bauer; Houda Cohen; Kazuhiro Kobuke; Kevin P Campbell; Joseph M Metzger

doi:10.1038/s41598-020-67372-0

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Investigations of an inducible intact dystrophin gene excision system in cardiac and skeletal muscle in vivo

Journal article

Open access

Peer reviewed

Investigations of an inducible intact dystrophin gene excision system in cardiac and skeletal muscle in vivo

Addeli Bez Batti Angulski, John Bauer, Houda Cohen, Kazuhiro Kobuke, Kevin P Campbell and Joseph M Metzger

Scientific reports, Vol.10(1), pp.10967-10967

07/03/2020

DOI: 10.1038/s41598-020-67372-0

PMCID: PMC7335168

PMID: 32620803

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Abstract

We sought here to induce the excision of a large intragenic segment within the intact dystrophin gene locus, with the ultimate goal to elucidate dystrophin protein function and stability in striated muscles in vivo. To this end, we implemented an inducible-gene excision methodology using a floxed allele approach, demarcated by dystrophin exons 2-79, in complementation with a cardiac and skeletal muscle directed gene deletion system for spatial-temporal control of dystrophin gene excision in vivo. Main findings of this study include evidence of significant intact dystrophin gene excision, ranging from ~ 25% in heart muscle to ~ 30-35% in skeletal muscles in vivo. Results show that despite evidence of significant dystrophin gene excision, no significant decrease in dystrophin protein content was evident by Western blot analysis, at three months post excision in skeletal muscles or by 6 months post gene excision in heart muscle. Challenges of in vivo dystrophin gene excision revealed acute deleterious effects of tamoxifen on striated muscles, including a transient down regulation in dystrophin gene transcription in the absence of dystrophin gene excision. In addition, technical limitations of incomplete dystrophin gene excision became apparent that, in turn, tempered interpretation. Collectively, these findings are in keeping with earlier studies suggesting the dystrophin protein to be long-lived in striated muscles in vivo; however, more rigorous quantitative analysis of dystrophin stability in vivo will require future works in which more complete gene excision can be demonstrated, and without significant off-target effects of the gene deletion experimental platform per se.

Gene Targeting - methods

Muscular Dystrophy, Animal - genetics

Gene Expression - drug effects

Male

Muscle, Skeletal - metabolism

Gene Knockdown Techniques - methods

Dystrophin - deficiency

Gene Deletion

Muscle, Skeletal - drug effects

Myocardium - metabolism

Mice, Inbred mdx

Muscular Dystrophy, Animal - metabolism

Female

Protein Stability

Mice, Inbred C57BL

Tamoxifen - toxicity

Mice, Knockout

Animals

Dystrophin - genetics

Tamoxifen - pharmacology

Heart - drug effects

Mice

Muscular Dystrophy, Duchenne - metabolism

Muscular Dystrophy, Duchenne - genetics

Cardiomyopathies - chemically induced

Details

Title: Subtitle: Investigations of an inducible intact dystrophin gene excision system in cardiac and skeletal muscle in vivo
Creators: Addeli Bez Batti Angulski - Department of Integrative Biology and Physiology, University of Minnesota Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN, 55455, USA
John Bauer - Department of Integrative Biology and Physiology, University of Minnesota Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN, 55455, USA
Houda Cohen - Department of Integrative Biology and Physiology, University of Minnesota Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN, 55455, USA
Kazuhiro Kobuke - Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA, 52242, USA
Kevin P Campbell - Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA, 52242, USA
Joseph M Metzger - Department of Integrative Biology and Physiology, University of Minnesota Medical School, 6-125 Jackson Hall, 321 Church Street SE, Minneapolis, MN, 55455, USA. metzgerj@umn.edu
Resource Type: Journal article
Publication Details: Scientific reports, Vol.10(1), pp.10967-10967
DOI: 10.1038/s41598-020-67372-0
PMID: 32620803
PMCID: PMC7335168
NLM abbreviation: Sci Rep
ISSN: 2045-2322
eISSN: 2045-2322
Publisher: England
Grant note: U54 NS053672 / NINDS NIH HHS T32 AG029796 / NIA NIH HHS R01 HL138490 / NHLBI NIH HHS
Language: English
Date published: 07/03/2020
Academic Unit: Neurology; Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984068385302771

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