(H-293) DMP1 Lineage Connexin43 Deficiency Attenuates Decreases in Skeletal Muscle Strength and Fracture Callus Mineralization During Hindlimb Unloading

Preclinical studies have shown that musculoskeletal unloading by hindlimb unloading (HLU) decreases hindlimb bone and skeletal muscle mass and alters fracture healing, with some similarities to aging ^1-4. Our lab has shown that deletion of connexin 43 (Cx43), the predominate gap junction protein in bone, in early osteoblasts/osteocytes (Osteocalcin-Cre) limits bone loss during hindlimb unloading but delays bone formation during normal ambulatory fracture healing.^{5, 6} Emerging evidence also suggests that bone cell derived Cx43 may play a key role in regulating bone-muscle crosstalk. For example, deletion of Cx43 in early and more mature osteoblasts and osteocytes (Col1a1 and Dmp1 lineage cells) reduces muscle mass and strength during natural aging^{7, 8} However, the role that mature osteoblast/osteocyte Cx43 deficiency plays in bone-muscle crosstalk and fracture healing during HLU remains unknown. We hypothesized that mature osteoblast and osteocyte Cx43 deficiency (Dmp1-Cre) impairs bone healing and hindlimb musculoskeletal tissue mass and strength during normal ambulatory conditions, but is protective during hindlimb unloading.

At DPF14, qPCR demonstrated significant, knockdown of Gja1 expression in uninjured cortical bone (-33%) but not in soleus or gastrocnemius (Fig 1), confirming bone-specific deletion. HLU mice demonstrated significantly reduced body mass compared to controls at DPF14 regardless of genotype (Fig 2; -8.0% vs. +0.4%). HLU and Cx43 deficiency synergistically decreased normalized gastrocnemius mass and soleus independent of Cx43 deficiency (Fig 2). Surprisingly, Cx43 deficiency increased in vivo tetanic torque overall and attenuated decreases seen with HLU compared to WT (Fig 2). With HLU, both genotypes showed significant diaphyseal and metaphyseal bone loss, with diaphyseal bone loss significantly increased due to Cx43 deficiency compared to WT (Fig 3A). Fractured WT femurs showed reduced callus mineralization with HLU and Cx43 deficiency, but HLU induced decreases in callus woven bone volume fraction were blunted in Cx43 cKO mice (Fig 3B). Our results suggest that loss of Cx43 in mature osteoblasts and osteocytes may protect against HLU induced decreases in skeletal muscle and fracture callus mineralization in vivo, confirming our hypothesis. However, contrary to our hypothesis, Dmp1-Cre Cx43 deficiency didn’t alleviate HLU induced decreases in uninjured bone microarchitecture. Our Cx43 cKO HLU-induced metaphyseal results are in contrast to our previous studies using Ocn-Cre⁵ and may arise due to differences in mouse sex utilized or inclusion of a fracture injury. Nonetheless, these differences in uninjured bone, suggest that Cx43 deficiency’s protective effects on bone during unloading by HLU may be due to deletion in earlier osteoblast lineage cells. Our results of attenuated loss of skeletal muscle torque and callus mineralization with Cx43 deficiency during HLU are novel and exciting. Previous results suggest that loss of Cx43 in early and more mature osteoblasts and osteocytes may delay fracture healing during ambulatory conditions by upregulating sclerostin expression¹². Furthermore, Cx43 deficiency has been shown to increase slow-twitch fibers in adjacent skeletal muscle.¹³ Since HLU has been shown to cause preferential fast-twitch fiber and sclerostin upregulation^{14, 15}, these mechanisms will be evaluated in future work in our model.

This work is supported by the Translational Research Institute through NASA Cooperative Agreement NNX16AO69A. Services in support of the research project were provided by the VCU Massey Cancer Center Tissue Data and Acquisition and Analysis Shared Resource and the Transgenic/Knockout Mouse Shared Resource, [Health Communication and Digital Innovation Shared Resource core], supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059.

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