Scientific Programme
Physiology & Nutrition
IS-PN01 - Human skeletal muscle plasticity studied with single-nuclei RNA sequencing and novel spatial proteomic approaches
Date: 01.07.2025, Time: 13:30 - 14:45, Session Room: Castello 2
Description
Skeletal muscle adapts quickly to physiological stressors including aging, disuse, and exercise. Aging decreases muscle mass and strength, increasing falls, fractures, and critical illness risks. These conditions often lead to muscle disuse (bed rest or casting), exacerbating muscle atrophy and weakness and reducing quality of life. Exercise can counteract muscle deterioration. This symposium explores modern multi-omics techniques (single nuclei RNA sequencing (snRNA-seq), spatial proteomics, and mass-spectrometry based proteomics) to unravel molecular alterations in human aging, disuse and exercise. The goal is to understand the mechanisms underlying muscle deterioration in aging and disuse, paving the way for effective interventions improving quality of life. Prof Bottinelli will discuss human neuromuscular system degenerative changes in response to disuse, highlighting snRNA-seq potential for generating novel insights. Dr. Monti will explore the use of this technology to assess aging and sarcopenia-related alterations, coupled with spatial proteomics to examine muscle tissue reorganization at the protein level. Prof. Murgia will conclude by overviewing mass-spectrometry based proteomics use in understanding protein content changes in skeletal muscle disuse and exercise with unprecedented resolution. This session will interest researchers in human physiology, biology, neuroscience, sports medicine doctors, bioinformaticians, and health practitioners.
Chair(s)
Elena Monti
Stanford University, Microbiology and Immunology
United States
Speaker A
Roberto Bottinelli
University of Pavia, Department of Experimental Medicine
Italy
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Single nuclei transcriptomics to unravel the mechanisms of skeletal muscle homeostasis in disuse
Inactivity can be considered a pandemic, causing 5 million deaths per year, decreasing exercise tolerance and dramatically increasing disease risks. The major phenomena occur at the level of muscle (loss of mass and strength), nervous system (impairment in central and peripheral motor control) and metabolism (impaired oxidative metabolism and insulin resistance). So far, most studies aiming to clarify the mechanisms underlying muscle deterioration following disuse focused on structure and function of muscle fibers and on intracellular pathways controlling muscle mass, metabolism, and redox balance. However, a clear and comprehensive explanation of why loss of muscle mass and force occurs is still lacking, especially in humans. Global transcriptomics recently provided important clues on the mechanisms underlying muscle adaptations. However, such approach has some important limitations. It has been recently understood that up to 40% of all skeletal muscle nuclei are not from myofibers and that although satellite cells are the stem cells regenerating skeletal muscle fibers, other resident mononuclear cell populations (including Fibro-Adipogenic Progenitors (FAPs), endothelial cells and immune cells) play important roles not only in regeneration, but also in normal muscle homeostasis in conditions such as disuse and ageing. Global transcriptomic analysis fails to assess individual cells gene expression. Hence, the complex interactions among muscle fibers and mononuclear cells in muscle homeostasis are missed. One of the ways to address most of the above limitations is to extract all nuclei from muscle samples and perform single nuclei RNA-Seq (snRNA-Seq). The key advantages of such approach are: (i) define clusters of nuclei representing different mononuclear resident cell types and nuclei of muscle fibers (NMJ; myotendinous junction (MTJ); nuclei along the length of muscle fibers or myonuclei) by means of specific marker genes (ii) study gene expression of each cluster of nuclei, addressing the heterogeneity of gene expression among different cells and of different nuclei of muscle fibers. This approach should enable to address the interplay among the heterogeneous cell populations orchestrating muscle homeostasis. Prof Bottinelli will guide the audience through the journey to study human skeletal muscle deterioration following disuse with the above-mentioned approaches, the findings and limitations that each of them entail together with preliminary findings obtained applying snRNA-Seq on human muscle samples from young subjects before bed rest, after 10 and 21 days of BR an after 3 weeks recovery.
Speaker B
Elena Monti
Stanford University, Microbiology and Immunology
United States
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A transcriptomic and spatial proteomic atlas of human aging and sarcopenia
Aging is associated with a progressive decrease in muscle mass and strength. Sarcopenia is the clinical manifestation of the extreme age-related muscle atrophy and weakness, and affects approximately 50 million people worldwide. Severe consequences of sarcopenia include increased frailty and reduction in ability to perform basic life functions, leading to higher risk of falls, increased healthcare costs, institutionalization, and mortality risk. Diagnosing and treating sarcopenia is challenging because of the complex pathophysiology of sarcopenia, which is a multifactorial syndrome. Current research lacks comprehensive studies investigating the mechanisms involved in sarcopenia development and progression in humans, which hinder the ability to identify biomarkers for its diagnosis and effective drugs to slow down its progression. Dr. Monti will present data showing transcriptomic (snRNA-seq, single cell RNA-seq, ATAC-seq) and proteomic changes observed in aging and late aging as well as data personally generated from 67 muscle biopsies from 15 young (18-35 years old) and 52 aged (>70 years old) stratified for sarcopenia according to the most updated guidelines. These samples were processed using snRNA-seq and, for the first time, CODEX spatial proteomics technologies, with the aim to generate the first matched transcriptomic and spatial proteomic atlas of human aging and sarcopenia, encompassing changes in muscle cell type composition and spatial interaction and changes in gene and protein expression. Evidence that will be presented show increased muscle denervation and transcriptomic changes in the neuromuscular junction nuclei, altered mitochondria biogenesis, complexes and metabolism confirmed by western blot analyses are aging signature exacerbated by sarcopenia. Further, sarcopenia leads to increase in immune and pro-fibrotic cells (fibro-adipogenic progenitors, FAPs), alterations in their transcriptome and cell-cell spatial interaction. The lab Dr. Monti currently works in has demonstrated with prostaglandin E2 (PGE2) to be a beneficial molecule for muscle regeneration. Decrease in PGE2 and increase in its degrading enzyme 15-PGDH were shown in old mice, and 15-PGDH inhibition rejuvenated muscle strength, force and mitochondria in old mice. Dr. Monti will show evidence for 15-PGDH to be upregulated specifically in sarcopenic patients muscles, and novel insights derived from snRNA-seq and CODEX that show how the increase in this enzyme may be implicated in sarcopenia development.
Speaker C
Marta Murgia
Universita' degli studi di Padova, Dipartimento di Scienze Biomediche
Italy
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Resistance training and muscle disuse. Enhancing Muscle Plasticity Analysis with Deep Visual Proteomics
Skeletal muscle plasticity is a highly complex phenomenon, as this tissue is composed of multinucleated cellular units, slow and fast muscle fibers, with different molecular and physiological properties. In her talk, prof. Murgia will describe single fiber proteomic workflows and their limitations. She will present a novel mass spectrometry-based proteomic approach, Deep Visual Proteomics (DVP), allowing to tackle the analysis of single human muscle fibers from a spatial angle. DVP allows to analyze subcellular compartments of muscle fibers in a fiber type-resolved manner. DVP uses high-resolution imaging to guide automated laser-capture microdissection (LMD) of tissue sections, which is then followed by MS-based proteomic profiling. At the core of the workflow is artificial intelligence (AI)-guided image analysis, that allows single-cell/single shape classification and subsequent LMD-based isolation. We applied DVP to frozen sections of human skeletal muscle, with various fiber type-resolving staining as well as fluorescent lectins to mark fiber borders and guide AI-based segmentation. Compared to previous single fiber proteomic workflows, based on fibers freshly dissected under a stereomicroscope, there are at least three major practical advantages of the new DVP-based workflow on frozen tissue: i) minimal handling time from biopsy collection to freezing, making it potentially compatible with the analysis of highly reversible post-translational modifications; ii) no loss of highly soluble cytosolic proteins in the isolation solution and iii) easier multicentric sample collection due to the compatibility of the workflow with frozen tissue. The added conceptual value of DVP is that it allows to carry out proteomics of muscle subcellular compartments in a fiber type-resolved manner. Prof. Murgia will provide some data concerning the application of this workflow to study skeletal muscle plasticity in response to different activity conditions and loading states.