The central nervous system (CNS)-independent anti-bone-resorptive activity of muscle contraction and the underlying molecular and cellular signatures

Abstract

Background: Mechanisms by which muscle regulates bone are poorly understood.

Results: Electrically stimulated muscle contraction reversed elevations in bone resorption and increased Wnt signaling in bone-derived cells after spinal cord transection.

Conclusion: Muscle contraction reduced resorption of unloaded bone independently of the CNS, through mechanical effects and, potentially, nonmechanical signals (e.g. myokines).

Significance: The study provides new insights regarding muscle-bone interactions. Muscle and bone work as a functional unit. Cellular and molecular mechanisms underlying effects of muscle activity on bone mass are largely unknown. Spinal cord injury (SCI) causes muscle paralysis and extensive sublesional bone loss and disrupts neural connections between the central nervous system (CNS) and bone. Muscle contraction elicited by electrical stimulation (ES) of nerves partially protects against SCI-related bone loss. Thus, application of ES after SCI provides an opportunity to study the effects of muscle activity on bone and roles of the CNS in this interaction, as well as the underlying mechanisms. Using a rat model of SCI, the effects on bone of ES-induced muscle contraction were characterized. The SCI-mediated increase in serum C-terminal telopeptide of type I collagen (CTX) was completely reversed by ES. In ex vivo bone marrow cell cultures, SCI increased the number of osteoclasts and their expression of mRNA for several osteoclast differentiation markers, whereas ES significantly reduced these changes; SCI decreased osteoblast numbers, but increased expression in these cells of receptor activator of NF-κB ligand (RANKL) mRNA, whereas ES increased expression of osteoprotegerin (OPG) and the OPG/RANKL ratio. A microarray analysis revealed that ES partially reversed SCI-induced alterations in expression of genes involved in signaling through Wnt, FSH, parathyroid hormone (PTH), oxytocin, and calcineurin/nuclear factor of activated T-cells (NFAT) pathways. ES mitigated SCI-mediated increases in mRNA levels for the Wnt inhibitors DKK1, sFRP2, and sclerostin in ex vivo cultured osteoblasts. Our results demonstrate an anti-bone-resorptive activity of muscle contraction by ES that develops rapidly and is independent of the CNS. The pathways involved, particularly Wnt signaling, suggest future strategies to minimize bone loss after immobilization.

Keywords: Bone; CNS; Gene Expression; Microarray; Muscle and Bone Interaction; Osteoblasts; Osteoclast; Osteoporosis; Spinal Cord Injury; Wnt Pathway.

FIGURE 1.

Effects of 7 days of ES on muscle and bone. A, scheme of the experimental design. B, plantaris muscle weights. C, trabecular architecture assessed by micro-computed tomography. D, areal BMD (aBMD). Data are expressed as mean ± S.E. n = 5–12 animals per group for A, B, and D and n = 3–4 animals per group for C. **, p < 0.01 and ***, p < 0.001 versus the indicated group. BV/TV, bone volume over total volume; Tb.N, trabecular number; Tb.Th., trabecular thickness; Tb.Sp, trabecular spacing

FIGURE 2.

Muscle contraction by ES protected against SCI-induced resorption and reduced osteoclastogenesis. A, the effects of 7 days of ES begun at 16 weeks after SCI are shown for serum CTX. B, representative images of cultured osteoclasts (OC). C, numbers of multinucleated TRAP⁺ cells in ex vivo cultures. Data are expressed as mean ± S.E. n = 5–12 animals per group for A and n = 3–4 animals per group for B. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 versus the indicated group.

FIGURE 3.

SCI reduced serum osteocalcin and osteoblast differentiation ex vivo, but ES had no effect on these changes. A, serum osteocalcin levels. B and C, representative images of alkaline phosphatase-stained cells for CFU-f and von Kossa-stained cultures for CFU-ob, respectively. D, quantitation of stained cells shown in B and C. n = 5–12 per group for A and n = 3–4 per group for B and C. *, p < 0.05

FIGURE 4.

The changes of gene expression and signaling pathway in ex vivo cultured osteoblasts. A, mRNA levels were determined by real time PCR. n = 3–4 per group. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 versus indicated group. B, a pathways analysis of differentially expressed genes identified by Illumina microarray analysis was performed. Selected gene expression changes were also highlighted.

FIGURE 5.

The changes of gene expression and signaling pathway in ex vivo cultured osteoclasts. A, mRNA levels were determined by real time PCR. n = 3–4 per group. *, p < 0.05, **, p < 0.01, and ***, p < 0.001 versus indicated group. B, a pathways analysis of differentially expressed genes identified by Illumina microarray analysis was performed. Selected gene expression changes were also highlighted.