Universität zu Köln

Schmidt, Luisa ORCID: 0000-0002-9805-4941 (2024). Advanced Proteomics of Skeletal Muscles: Spatial and Single Fiber Insights into Metabolic Diversity. PhD thesis, Universität zu Köln.

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Abstract

In recent years, the importance of muscle health for overall body function has gained increasing recognition. Although whole proteome methods with protein extraction and LC-MS analysis provide accurate insights into different cell types and protein composition, the localization of specific proteins, including the extracellular matrix (ECM) and intercellular junctions, is limited. Here, we present a novel approach that preserves tissue spatial organization. By employing thin cryosectioning followed by LC-MS/MS analysis, we spatially map proteins across the tissue. This method enabled us to characterize the myotendinous junction (MTJ) in the soleus and diaphragm muscles, revealing 32 new marker proteins and the significant role of the renin-angiotensin system (RAS) in the muscle-tendon transition zone. Our approach facilitated the spatial characterization of 33 known marker proteins of the neuromuscular junction (NMJ) along the diaphragm axis and identified approximately 140 potential NMJ proteins, which we deconvoluted to either neuronal or muscle origin. Utilizing the generated protein profiles for a distance-based network analysis, we identified spatial structural changes in aged mice, such as NMJ broadening and loss of connectivity. Applying this method to TDP43 dependent ALS mouse models uncovered NMJ structural changes that traditional methods would have missed. Using our new approach, we identified around 10,000 proteins in skeletal muscle, generating more than 3,500 protein profiles across the skeletal muscles which will be a comprehensive repository for researchers focusing on skeletal muscle biology and related diseases. Additionally, isolating single muscle cells from the mouse soleus muscle in response to a high-fat diet (HFD) administration revealed significant metabolic impacts on different fiber types. Fast fibers were particularly affected by nutrient overexposure, leading to a loss of peripheral mitochondria throughout the muscle, as demonstrated by immunohistochemistry (IHC). Single-cell analysis of muscle fibers from bears during hibernation and active periods demonstrated evolutionary adaptations to minimize energy expenditure. Hibernating bears showed reduced myosin ATPase activity, decreased mitochondrial protein levels, and altered myosin light chain kinase (MYLK2) activity, suggesting mechanisms to preserve muscle function and prevent wasting during inactivity.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Schmidt, Luisa luisa.schmidt@uni-koeln.de orcid.org/0000-0002-9805-4941 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-743645
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	CECAD - Cluster of Excellence Cellular Stress Responses in Aging-Associated Diseases
Subjects:	Natural sciences and mathematics Chemistry and allied sciences
Uncontrolled Keywords:	Keywords Language Proteomics UNSPECIFIED Skeletal Muscle UNSPECIFIED
Date of oral exam:	29 July 2024
Referee:	Name Academic Title Krüger, Marcus Professor Hammerschmidt, Matthias Professor Dengjel, Jörn Professor
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/74364