Spatially distinct ECM-producing fibroblasts and myonuclei orchestrate early adaptation to mechanical loading in the human muscle-tendon unit

Mechanical loading drives structural and functional improvements in muscle and tendon, protecting against injury at their interface, at the myotendinous junction (MTJ), and within the tendon matrix. However, the early cellular and molecular events that initiate these adaptations in humans remain poorly understood. To investigate this, we applied single-nucleus RNA sequencing and in situ hybridization to map the acute transcriptional response of the human muscle-tendon unit to a single bout of eccentric resistance exercise, with a focus on extracellular matrix (ECM) regulation. We identified four transcriptionally distinct fibroblast subtypes expressing key ECM components, including <i>COL1A1</i> and <i>DCN</i>. Three of these subtypes were localized to the tendon and responded to exercise: two were spatially restricted to the collagen fascicles or the MTJ, while the third, enriched in the interfascicular matrix (IFM), exhibited the strongest response. This IFM population, marked by <i>PDGFRA</i>, upregulated <i>PRG4</i> and <i>VCAN</i>, ECM genes linked to tissue lubrication and resilience. In parallel, exercise induced dynamic ECM regulation in myonuclei, particularly in a distinct subset of type II myonuclei at the MTJ, that expanded in number and robustly upregulated <i>COL22A1</i>, a collagen essential for MTJ integrity. Together, these findings uncover a spatially organized, cell type-specific program of ECM remodeling in response to mechanical load, offering new insight into the early molecular events of human muscle-tendon adaptation.<b>NEW & NOTEWORTHY</b> We provide the first high-resolution, in vivo single-nucleus map of the acute human muscle-tendon response to mechanical loading. Within 4 hours of resistance exercise, spatially distinct tendon fibroblasts and myonuclei activate coordinated extracellular matrix programs. Interfascicular fibroblasts upregulate <i>PRG4</i> and <i>VCAN</i> in the tendon, while myonuclei at the myotendinous junction induce <i>COL22A1</i> and <i>LAMA2</i>. These findings reveal a new principle of human mechanobiology, where exercise rapidly engages niche-specific programs to initiate extracellular matrix adaptation.