ABSTRACT Background Previous research has shown that the conditional knockout of Slo1 in muscle leads to a reduction in muscle strength and inhibits myogenic differentiation. Exosomes are emerging as necessary mediators of the crosstalk between muscle and bones. The present study focused on the communication between muscle and bone to investigate the effects and mechanisms of skeletal muscle‐specific knockout of Slo1 on bone mass and bone metabolism. Methods Myf5‐Cre mice were mated with Slo1 flox/flox mice to construct skeletal muscle‐specific Slo1 knockout mice (CKO mice). The legs of the Slo1 CKO mice were isolated, and a series of examinations were conducted to monitor bone mineral density (BMD) and bone microstructure. Exosomes were extracted from C2C12‐shNC and C2C12‐shSlo1 cells and subjected to transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting (WB). The osteogenic effect of EXO‐shSlo1 was examined in vitro and in vivo by Alkaline phosphatase (ALP) staining, q‐PCR, WB and microCT. RNA sequencing (RNA‐seq) analysis of EXO‐shNC and EXO‐shSlo1 was used to identify differentially expressed microRNAs. Mimics and inhibitors of miR‐222‐3p were transfected into MC3T3‐E1 cells to induce differentiation. The predicted targets of miR‐222‐3p were examined with Luciferase, qPCR, and WB. Results In Slo1 CKO mice, the bone mass decreased, and the microstructure was disrupted. TEM, NTA and WB assays showed that Slo1‐silenced C2C12 cells secreted exosomes and localized to MC3T3‐E1 cells and bone tissue through the circulation. EXO‐shSlo1 inhibited osteogenesis both in vivo and in vitro, as demonstrated by the decreased ALP activity (~40%, p < 0.05), osteogenic marker expression (~30%, p < 0.05), mineral deposition in osteoblasts and BMD. qPCR was performed to confirm the exosome RNA‐seq results, which indicated that miR‐222‐3p was increased by three times in the EXO‐shSlo1 group compared with the EXO‐shNC group. Transfection of mimics or inhibitors of miR‐222‐3p in MC3T3‐E1 cells inhibited or improved osteogenic differentiation. Luciferase reporter assays revealed that miR‐222‐3p targets STAT3. The mRNA and protein level of STAT3 was affected by miR‐222‐3p. Through inhibiting miR‐222‐3p or upregulating STAT3, the EXO‐shSlo1‐mediated osteogenic inhibition of MC3T3‐E1 cells was ameliorated, as indicated by increased ALP activity and osteogenic marker expression. Conclusion Slo1 deficiency in muscle results in decreased bone mass mediated by exosomal miR‐222‐3p. MiR‐222‐3p binds and inhibits the transcription of STAT3 to suppress the differentiation of osteoblasts. Slo1‐silenced myoblasts‐derived exosomal miR‐222‐3p inhibit osteogenesis and cause bone loss through the miR‐222‐3p/STAT3 pathway.
Wang et al. (Mon,) studied this question.