Connexin43 deficiency reduces the sensitivity of cortical bone to the effects of muscle paralysis

Abstract

We have previously shown that the effect of mechanical loading on bone is partly dependent on connexin43 (Cx43). To determine whether Cx43 is also involved in the effect of mechanical unloading, we have used botulinum toxin A (BtxA) to induce reversible muscle paralysis in mice with a conditional deletion of the Cx43 gene in osteoblasts and osteocytes (cKO). BtxA injection in hind limb muscles of wild type (WT) mice resulted in significant muscle atrophy and rapid loss of trabecular bone. Bone loss reached a nadir of about 40% at 3 weeks post-injection, followed by a slow recovery. A similar degree of trabecular bone loss was observed in cKO mice. By contrast, BtxA injection in WT mice significantly increased marrow area and endocortical osteoclast number, and decreased cortical thickness and bone strength. These changes did not occur in cKO mice, whose marrow area is larger, osteoclast number higher, and cortical thickness and bone strength lower relative to WT mice in basal conditions. Changes in cortical structure occurring in WT mice had not recovered 19 weeks after BtxA injection, despite correction of the early osteoclast activation and a modest increase in periosteal bone formation. Thus, BtxA-induced muscle paralysis leads to rapid loss of trabecular bone and to changes in structural and biomechanical properties of cortical bone, neither of which are fully reversed after 19 weeks. Osteoblast/osteocyte Cx43 is involved in the adaptive responses to skeletal unloading selectively in the cortical bone, via modulation of osteoclastogenesis on the endocortical surface.

Notch Signaling in Osteocytes Differentially Regulates Cancellous and Cortical Bone Remodeling

“…However, activation of Notch in osteocytes resulted in a unique phenotype that is in contrast to the osteopenic phenotype observed following the activation of Notch in osteoblasts. Because the unloading of the skeleton is characterized by decreased bone formation and increased resorption, leading to bone loss, we postulated that the Notch activation in osteocytes would preserve the bone mass in a model of disuse osteoporosis (37,39,40). Microarchitectural and histomorphometric analyses revealed a decrease in trabecular bone volume and an increase in osteoclast surface in BtxAparalyzed limbs of control mice but not of Notch-expressing mice.…”

“…Mice were sacrificed 3 weeks later, the known nadir of the effect of BtxA-induced immobilization on bone mass (37). Muscle paralysis was confirmed by the digital abduction test, and saline-injected mice scored 0, whereas Dmp1-Cre ϩ/Ϫ ;Rosa Notch and Rosa Notch controls injected with BtxA scored (means Ϯ S.E.…”

“…The contralateral limb was used as an internal control. Experimental and control mice were sacrificed 3 weeks post-BtxA injection, when bone loss following muscle paralysis reaches its nadir (37). The degree of muscle paralysis was assessed 3 days postinjection using the digital abduction scoring test, where the degree of abduction of the digits in the paralyzed limb is assessed and scored using a scale of 0 -4, with 0 reflecting normal muscle function and 4 indicating total paralysis (41).…”

“…Expression of NICD from the targeted Rosa26 locus occurs following the excision of the STOP cassette by Cre recombination of loxP sequences (35,36 Muscle Immobilization Protocol-The effect of muscle immobilization on the skeleton was tested in 1-month-old Dmp1-Cre ϩ/Ϫ ;Rosa Notch and littermate control male mice. For this purpose, control and experimental mice were injected with botulinum toxin A (BtxA) (Allergan Inc., Irvine, CA) at a dose of 2 units/100 g of body weight administered in equal amounts in the quadriceps and the triceps surae muscle groups or injected with saline under anesthesia using a ketamine/xylazine mixture intraperitoneally (37)(38)(39)(40). The contralateral limb was used as an internal control.…”

N-cadherin in osteolineage cells is not required for maintenance of hematopoietic stem cells

“…The conditional Cdh2 allele contains loxP sites surrounding exon 1 of the gene, which includes not only the translational start site but also approximately 1 kb upstream of the exon. We thus would predict that conditional deletion of Cdh2 would remove both the translational start site and several transcriptional elements important for 21 in WT controls, N-cadherin is expressed at high levels early during culture, but its expression decreases as the cells mature and express osteoblastspecific genes, such as tissue nonspecific alkaline phosphatase. In contrast, N-cadherin-deleted mice show no expression of N-cadherin at baseline or on differentiation ( Figure 1B).…”