01 | Investigating skeletal muscle-endothelial cell crosstalk in plasticity and disease

José Fernández-Martínez^1|2, A. Gensel^1|2, E. Germinario², R.E. Oberkersch³, M.M. Santoro³, B. Blaauw^1|2 | ¹Veneto Institute of Molecular Medicine, Padua, Italy; ²Department of Biomedical Sciences, University of Padua, Italy; ³Department of Biology, University of Padua, Italy. | José Fernández-Martínez and A. Gensel contributed equally to this work.

The maintenance of skeletal muscle mass and function is a major clinical challenge in aging, cancer, muscular dystrophies, and related conditions. While much attention over the past two decades has focused on intracellular pathways like Akt-mTORC1, the roles of muscle perfusion and paracrine signaling between muscle fibers and endothelial cells (ECs) remain poorly understood. Emerging evidence suggests a bidirectional crosstalk between these cell types. However, in vivo tools to study this interaction with cell-type specificity and temporal control are still limited. To address this, we developed an EC-specific MetRS mouse model expressing a mutant methionyl-tRNA synthetase (MetRS^L274G) under the tamoxifen-inducible Cdh5-CreERT2 promoter. This allows temporally controlled, EC-specific incorporation of the methionine analog azidonorleucine (ANL) into nascent proteins in vivo. Administered via drinking water, ANL selectively labels EC proteins within defined windows. Labeled proteins can be visualized and quantified using click chemistry–based FUNCAT or enriched by immunoprecipitation for downstream mass spectrometry–based proteomic analysis. Combined with tissue clearing and light sheet microscopy, this enables 3D mapping of endothelial protein synthesis across skeletal muscle. To validate the model, we induced angiogenesis via unilateral AAV-VEGF overexpression (2×10¹⁰ U/muscle) in the gastrocnemius muscle, using the contralateral limb as control. Our preliminary results demonstrate successful and specific endothelial protein labeling with clear spatial distribution across muscle tissue. Interestingly, VEGF-induced angiogenesis correlates with increased muscle mass, fiber size, and protein synthesis. We are now using the model to explore how controlled muscle growth or atrophy influences ECs, and how primary EC changes affect muscle physiology. This work aims to provide new insights into muscle–vascular communication relevant to muscle health and disease.