title: A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo.
abstract: Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.
authors.affiliation: Departments of Medicine, University of Virginia, Charlottesville, Virginia 22908; Center for Skeletal Muscle Research at the Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Rhianna C
authors.initials: RC
authors.lastname: Laker
authors.affiliation: Departments of Medicine, University of Virginia, Charlottesville, Virginia 22908; Center for Skeletal Muscle Research at the Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Peng
authors.initials: P
authors.lastname: Xu
authors.affiliation: Departments of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Ryall
authors.affiliation: Department of Geriatric Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Alyson
authors.initials: A
authors.lastname: Sujkowski
authors.affiliation: Departments of Pharmacology, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Brandon M
authors.initials: BM
authors.lastname: Kenwood
authors.affiliation: Center for Skeletal Muscle Research at the Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908; Departments of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Kristopher H
authors.initials: KH
authors.lastname: Chain
authors.affiliation: Departments of Medicine, University of Virginia, Charlottesville, Virginia 22908; Center for Skeletal Muscle Research at the Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Royal
authors.affiliation: Departments of Pharmacology, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Kyle L
authors.initials: KL
authors.lastname: Hoehn
authors.affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854.
authors.forename: Monica
authors.initials: M
authors.lastname: Driscoll
authors.affiliation: Departments of Biology, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Paul N
authors.initials: PN
authors.lastname: Adler
authors.affiliation: Department of Geriatric Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wessells
authors.affiliation: Departments of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Saucerman
authors.affiliation: Departments of Medicine, University of Virginia, Charlottesville, Virginia 22908; Center for Skeletal Muscle Research at the Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908; Departments of Pharmacology, University of Virginia, Charlottesville, Virginia 22908; Departments of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908. Electronic address: zhen.yan@virginia.edu.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yan
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D002460:Cell Line; D004331:Drosophila melanogaster / Q000235:genetics; D017930:Genes, Reporter; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology*; D018384:Oxidative Stress; D010805:Physical Conditioning, Animal; D013050:Spectrometry, Fluorescence
keywords: Confocal Microscopy; Exercise; High-fat Diet; Mitochondria; Oxidative Stress; Reporter Gene; Skeletal Muscle