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| Status |
Public on Jun 05, 2022 |
| Title |
Aging exacerbates neuromuscular junction disruption after injury that stimulates pro-inflammatory and diminishes pro-regenerative factor expression in macrophages and fibroadipogenic progenitors. |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Aging is associated with delayed skeletal muscle repair and regeneration. Loss of innervation occurs after degenerative muscle injury, however, the extent of denervation, whether the kinetics of reinnervation changes with aging, and the cellular consequences of neuromuscular junction (NMJ) disruption in adult and aged muscle have not been explored. Here we show after degenerative muscle injury progressive denervation and delayed reinnervation in aged compared to adult muscle. Using confocal microscopy and 3-D image rendering we found a relationship between innervation and myofiber size in aged regenerating muscle. Although timely inhibition of pro-inflammatory Ccr2 activity after degenerative muscle injury improved aged regenerated myofiber size, this was not associated with an increase in reinnervation. In contrast, single cell RNA sequencing (scRNASeq) analysis revealed denervation triggers an inflammatory response in target muscles regardless of age; however, pro-inflammatory signaling predominated in aged macrophages and fibroadipogenic progenitors (FAPs) compared to a pro-regenerative response in adult cells. Thus, denervation and delayed reinnervation of NMJs after injury is a feature of persistent pro-inflammatory signals associated with delayed repair and regeneration of aged muscle.
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| Overall design |
Young (3 months) and aged (25 months) C57BL/6J mice were administered nerve crush injury. 5 days post injury, the TA muscles were isolated using surgical scissors, minced and digested with Collagenase type II (0.2%) and Dispase II (2.5U/mL) in 10mL of DMEM per muscle. Samples were placed on rocker in a 37˚C incubator for 1 hour and mixed by pipette every 30 minutes. The enzymes within the slurry were then inactivated by addition of 20% heat-inactivated fetal bovine serum (HI-FBS) in Ham’s F10 media. The solution was passed through a 70um cell strainers, centrifuged, washed, and counted. The isolated cells were then stained with Propidium Iodide (PI) and viable cells isolated using fluorescent-activated cell sorting (FACS). Cells from each mouse were then labeled with cellular hash tags using CellPlex reagents (10x Genomics), pooled from two mice according to age (young and aged) into each lane and 10,000-16,000 cells were loaded into the 10x Genomics chromium single cell controller lane. Single cells were then captured in nanoliter-scale gel bead-in-emulsions (GEMs), lysed, RNA captured, and cDNAs prepared using the single cell 3′ v3 Protocol as per manufacturer’s instructions. Each sample was sequenced on a NovaSeq 6000 (Illumina) with 26 bases for read1 and 98x8 bases for read2.
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| Contributor(s) |
Yang BA |
| Citation missing |
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| Submission date |
Jun 02, 2022 |
| Last update date |
Jun 07, 2022 |
| Contact name |
Carlos Andres Aguilar |
| E-mail(s) |
caguilar@umich.edu
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| Organization name |
University of Michigan, Ann Arbor
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| Department |
Biomedical Engineering
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| Lab |
Aguilar Lab
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| Street address |
2800 Plymouth Rd
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA844967 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE205395_RAW.tar |
114.7 Mb |
(http)(custom) |
TAR (of TAR) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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