Swimming induces bone loss via regulating mechanical sensing pathways in bone marrow

Bone is an organ capable of perceiving external mechanical stress in real time and respond dynamically via mechanosensing proteins such as Piezo1 and YAP/TAZ. Upon sensing the mechano-signals, cells within the bone matrix collaborate to coordinate bone formation and resorption, while bone marrow cells are also stimulated and mobilized. High-load exercise stimulates osteoblast differentiation and bone formation. However, the mechanism through which the low-load exercises on bone homeostasis is still unclear. In this work, we established a long-term swimming training model to unload the mechanical stress in mice. Throughout the training model, we observed a significant loss in trabecular bone mass, as evidenced by microCT scanning and histological staining. Single-cell sequencing of the tibial bone marrow tissue revealed a significant increase in the percentage of bone marrow neutrophils, along with alterations in integrins and the ERK1/2 signaling pathway. Notably, the changes in both Integrins and the ERK1/2 signaling pathway in macrophages were more pronounced than in other cell types, which not only suggests a mechanical adaptive response in these cells. Moreover, the involvement of integrins is also critical for the crosstalk between monocyte precusors and macrophages during swimming. Together, this study provide a resource of the alterations of bone marrow cell gene expression profile during swimming and highlights the importance of Integrins and the ERK1/2 signaling pathway in the bone marrow microenvironment after swimming. Overall design: Twelve 2-month-old male C57bl/6j mice, sourced from Shanghai Ninth People's Hospital, were housed in SPF conditions with free access to food and water, under a 12-hour light/dark cycle (07:00-19:00), at 22°C and 30-70% humidity. They were divided into swimming (n=6) and non-swimming (n=6) groups. The swimming group mice starting with 10 minutes of swimming on the first day and gradually increasing to 60 minutes per day within a week. Afterward, they swam for 60 minutes every day, five times a week, for a total of 12 weeks. All swimming training sessions were conducted in the afternoon. The non-swimming group mice did not undergo swimming training. After the previous training, three mice from the swimming group and three mice from the non-swimming group were euthanized. Bone marrow was flushed out from their tibiae using PBS, respectively. After dispersing the bone marrow tissue, dead cells were removed using a Dead Cell Removal Kit (130-090-101; Miltenyi Biotec) according to the manufacturer's instructions. Single-cell RNA sequencing (scRNA-seq) analysis of the tibial bone marrow cells was then performed using the 10x Genomics Chromium platform. We designed this experiment to explore the changes in bone marrow environment induced by mechanical stress unloading during swimming, and to provide an explanation for bone loss caused by swimming.