SBiochemical isolation of myonuclei employed to define changes to the myonuclear proteome that occur with aging

• Type of Material: Article
• Authors: Alicia A. Cutler, Emory University; Eric Dammer, Emory University; Duc M. Doung, Emory University; Nicholas Seyfried, Emory University; Anita Corbett, Emory University; Grace Pavlath, Emory University
• Language: English
• Date: 2017-08
• Publisher: Wiley Open Access
• Publication Version: Final Published Version
• Copyright Statement: © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1111/acel.12604
• Title of Journal or Parent Work: Aging Cell
• ISSN: 1474-9718
• Volume: 16
• Issue: 4
• Start Page: 738
• End Page: 749
• Grant/Funding Information: This work was supported by grants to GKP (AR062483), to AAC (AR067645), and a training grant (T32GM008367) from the National Institutes of Health.
• Keywords: PEPTIDE IDENTIFICATION; CELLS; PURIFICATION; Science & Technology; Cell Biology; nuclear isolation; skeletal muscle; REVEALS; pro-teome; PHOSPHORYLATION; brain; NUCLEI; Geriatrics & Gerontology; aging; Life Sciences & Biomedicine; QUANTIFICATION; FRACTIONATION; myonuclei; AGED SKELETAL-MUSCLE; GENE-EXPRESSION
• Research Categories: Biology, Cell; Health Sciences, Pharmacology; Chemistry, Biochemistry

Abstract

Skeletal muscle aging is accompanied by loss of muscle mass and strength. Examining changes in myonuclear proteins with age would provide insight into molecular processes which regulate these profound changes in muscle physiology. However, muscle tissue is highly adapted for contraction and thus comprised largely of contractile proteins making the nuclear proteins difficult to identify from whole muscle samples. By developing a method to purify myonuclei from whole skeletal muscle, we were able to collect myonuclei for analysis by flow cytometry, biochemistry, and mass spectrometry. Nuclear purification dramatically increased the number and intensity of nuclear proteins detected by mass spectrometry compared to whole tissue. We exploited this increased proteomic depth to investigate age-related changes to the myonuclear proteome. Nuclear levels of 54 of 779 identified proteins (7%) changed significantly with age; these proteins were primarily involved in chromatin maintenance and RNA processing. To determine whether the changes we detected were specific to myonuclei or were common to nuclei of excitatory tissues, we compared aging in myonuclei to aging in brain nuclei. Although several of the same processes were affected by aging in both brain and muscle nuclei, the specific proteins involved in these alterations differed between the two tissues. Isolating myonuclei allowed a deeper view into the myonuclear proteome than previously possible facilitating identification of novel age-related changes in skeletal muscle. Our technique will enable future studies into a heretofore underrepresented compartment of skeletal muscle.