Cx43 Overexpression in Osteocytes Prevents Osteocyte Apoptosis and Preserves Cortical Bone Quality in Aging Mice

Abstract

Young, skeletally mature mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and decreased bone strength, resembling the phenotype of old mice. Further, the expression of Cx43 in bone decreases with age, suggesting a contribution of reduced Cx43 levels to the age-related changes in the skeleton. We report herein that Cx43 overexpression in osteocytes achieved by using the DMP1-8kb promoter (Cx43OT mice) attenuates the skeletal cortical, but not trabecular bone phenotype of aged, 14-month-old mice. The percentage of Cx43-expressing osteocytes was higher in Cx43OT mice, whereas the percentage of Cx43 positive osteoblasts remained similar to wild type (WT) littermate control mice. The percentage of apoptotic osteocytes and osteoblasts was increased in aged WT mice compared to skeletally mature, 6-month-old WT mice, and the percentage of apoptotic osteocytes, but not osteoblasts, was decreased in age-matched Cx43OT mice. Aged WT mice exhibited decreased bone formation and increased bone resorption as quantified by histomorphometric analysis and circulating markers, compared to skeletally mature mice. Further, aged WT mice exhibited the expected decrease in bone biomechanical structural and material properties compared to young mice. Cx43 overexpression prevented the increase in osteoclasts and decrease in bone formation on the endocortical surfaces, and the changes in circulating markers in the aged mice. Moreover, the ability of bone to resist damage was preserved in aged Cx43OT mice both at the structural and material level. All together, these findings suggest that increased Cx43 expression in osteocytes ameliorates age-induced cortical bone changes by preserving osteocyte viability and maintaining bone formation, leading to improved bone strength.

Senescent and apoptotic osteocytes and aging: Exercise to the rescue?

“…Aging and physical inactivity are each associated with a decrease in lacunae that contain osteocytes, loss of directional orientation of lacunae, and the number and length of dendrites that connect through canniculi; all of which would interfere with the detection of strain or fluid-flow shear stress [25][26][27][28]. Connexin43, a gap junction protein involved in mechanotransduction, protects from osteocyte apoptosis, but also decreases with aging [29][30][31][32]. Alternatively, unloading could induce senescence indirectly through the effect of inactivity on reductions in myokines, adverse changes in whole body nutrient trafficking, and the resulting lipid accumulation in both osteoblasts and osteocytes.…”

Short-term pharmacologic RAGE inhibition differentially affects bone and skeletal muscle in middle-aged mice

“…4-(young, n=10) and 15-month-old mice (middle-aged, n=10) C57BL/6 female mice were obtained from National Institute on Aging (NIA) and received daily intraperitoneal injection of veh (1.7% DMSO) or 100μg/day (young mice) and 110μg/day (middle-aged mice) of AZ (DC Chemicals, Shanghai, P.R., China, cat.# DC8338), to account for body weight differences, for 28 days. We chose these 2 ages to determine the effect of RAGE inhibition at the age in which the mice reach peak bone mass (4 months of age), and later in life (15months of age), in which we showed there is already a significant deterioration, without reaching very low levels of bone mass and strength that might be difficult to reverse (Davis et al, 2018). Mice were assigned an ID number and the age/treatment were recorded in a database.…”

“…S5), but no other signs of distress were recorded. Young mice received IP injections of calcein (30 mg/kg; Sigma-Aldrich, Saint Louis, MO, USA) and alizarin red (50 mg/kg; Sigma) 7 and 2 days before sacrifice, respectively and aged mice received IP injections of calcein (30 mg/kg; Sigma-Aldrich, Saint Louis, MO, USA) and alizarin red (50 mg/kg; Sigma) 10 and 3 days before sacrifice, respectively, to allow for dynamic histomorphometric measurements (Davis et al, 2018). The longer duration between labels in aged mice was done to help facilitate the differentiation of the two labels given the slower bone formation rate.…”

“…Femora and lumbar vertebrae (L1-L3) were dissected and fixed in 10% neutral buffered formalin as previously published (Davis et al, 2018). Static histomorphometric analysis was performed on the cancellous surface of sequential methyl methacrylate embedded lumbar vertebrae sections stained with von Kossa/McNeal [osteoblasts] and TRAPase/Toludine blue [osteoclasts].…”

“…Dynamic histomorphometric measurements were performed in unstained plastic embedded femoral mid-diaphysis and lumbar vertebrae bone sections (Pacheco-Costa et al, 2014). Histomorphometric analysis was performed using OsteoMeasure high-resolution digital video system (OsteoMetrics Inc., Decatur, GA, USA) (Davis et al, 2018). The terminology and units used are those recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (Dempster et al, 2013).…”