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

Abstract

SummarySkeletal 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.

Poor old pores—The challenge of making and maintaining nuclear pore complexes in aging

“…C). For the specific proteome studies of aging yeast, mouse and rats that we compare in Fig. 3, we estimate, that the viability of the yeast replicative aging population is about 55% at the last time point measured (72 h, ~ 24 divisions) , the viability of the yeast chronologically aged population is ~ 60% (after 21 days) , and the mice and rat should have a survival of ~ 50–70% at the latest age time points (24 months) .…”

“…In general, there is a large degree of conservation of age‐associated changes across different eukaryotic species . To investigate whether NPCs change in similar ways in aging cells and tissues from different species, we extracted data on the abundance of Nups from six published proteome datasets, derived from aging budding yeast , rat , and mouse . The studies using baker's yeast addressed proteome changes that occur during aging in nondividing cells, chronological aging (Fig.…”

Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue

“…However, myonuclear proteins involved in these processes are difficult to study because of four technical limitations. First, skeletal muscle is dense, tightly packed with contractile proteins that make up more than 60% of proteins in the tissue (Deshmukh et al ., 2015; Cutler et al ., 2017). These high abundance contractile proteins eclipse the far less abundant nuclear proteins.…”

“…The isolated nuclei are intact, biochemically depleted of proteins from non-nuclear organelles, and 85% of nuclei are myonuclei. To isolate myonuclei more quickly we also optimized affinity-based purification using a myonuclear-specific nuclear envelope protein, Transmembrane Protein 38A (TMEM38A) (Bleunven et al ., 2008; Cutler et al ., 2017), and magnetic beads. This approach is more rapid and yields nuclei with comparable population purity to nuclear isolation by ultracentrifugation but with lower biochemical purity.…”

“…We used these purified nuclei for downstream applications including flow cytometry and mass spectrometry. We used this method to compare the myonuclear proteome of young and old mouse hindlimb muscles (Cutler et al ., 2017). This protocol may be applied to isolating myonuclei for a variety of downstream analyses such as flow cytometry, microscopy, Western blot, and proteomics.…”