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A guide to examining intramuscular fat formation and its cellular origin in skeletal muscle

Connor D. Johnson, Lylybell Y. Zhou, View ORCID ProfileDaniel Kopinke
doi: https://doi.org/10.1101/2022.03.06.483182
Connor D. Johnson
1Department of Pharmacology and Therapeutics, University of Florida, Gainesville 32610, FL, USA
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Lylybell Y. Zhou
1Department of Pharmacology and Therapeutics, University of Florida, Gainesville 32610, FL, USA
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Daniel Kopinke
1Department of Pharmacology and Therapeutics, University of Florida, Gainesville 32610, FL, USA
2Myology Institute, University of Florida College of Medicine, Gainesville, FL, USA
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  • ORCID record for Daniel Kopinke
  • For correspondence: dkopinke@ufl.edu
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ABSTRACT

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. Here, we describe a protocol that provides robust preservation of adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. We also outline how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in expression of adipogenic genes. Additionally, we will describe how our protocol can be adopted to visualize adipocytes by whole mount immunofluorescence of muscle samples. Finally, we will outline how to combine this protocol with genetic lineage tracing of Pdgfrα-expressing FAPs to study the adipogenic conversion of FAPs. Our protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes that, along with confirmation by RT-qPCR, allows for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, our analysis pipeline is the first step to improve our understanding of how FAPs differentiate into intramuscular fat and provides a framework to validate novel interventions to prevent fat formation.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 07, 2022.
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A guide to examining intramuscular fat formation and its cellular origin in skeletal muscle
Connor D. Johnson, Lylybell Y. Zhou, Daniel Kopinke
bioRxiv 2022.03.06.483182; doi: https://doi.org/10.1101/2022.03.06.483182
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A guide to examining intramuscular fat formation and its cellular origin in skeletal muscle
Connor D. Johnson, Lylybell Y. Zhou, Daniel Kopinke
bioRxiv 2022.03.06.483182; doi: https://doi.org/10.1101/2022.03.06.483182

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