Regional differences in oxidative metabolism and mitochondrial activity among cortical bone osteocytes

Abstract

Metabolic oxidative stress has been implicated as a cause of osteocyte apoptosis, an essential step in triggering bone remodeling. However, little is known about the oxidative behavior of osteocytes in vivo. We assessed the redox status and distribution of total and active mitochondria in osteocytes of mouse metatarsal cortical bone in situ. Multiphoton microscopy (MPM) was used to measure fluorescence of reduced pyridine nucleotides (NADH) under normoxic conditions and acutely following extreme (postmortem) hypoxic stress. Under non-hypoxic conditions, osteocytes exhibited no detectable fluorescence, indicating rapid NADH re-oxidation. With hypoxia, NADH levels peaked and returned to near baseline levels over 3 hours. Cells near the periosteal surface reached maximum NADH levels twice as rapidly as osteocytes near the mid-cortex, due to the time required to initiate NADH accumulation; once started, NADH accumulation followed a similar exponential relationship at all sites. Osteocytes near periosteal and endosteal bone surfaces also had higher mitochondrial content than those in mid-cortex based on immunohistochemical staining for mitochondrial ATPase-5A (Complex V ATPase). The content of active mitochondria, assessed in situ using the potentiometric dye TMRM, was also high in osteocytes near periosteum, but low in osteocytes near endocortical surfaces, similar to levels in mid-cortex. These results demonstrate that cortical osteocytes maintain normal oxidative status utilizing mainly aerobic (mitochondrial) pathways but respond to hypoxic stress differently depending on their location in the cortex, a difference linked to mitochondrial content. An apparently high proportion of poorly functional mitochondria in osteocytes near endocortical surfaces, where increased apoptosis mainly occurs in response to bone remodeling stimuli, further suggest that regional differences in oxidative function may in part determine osteocyte susceptibility to undergo apoptosis in response to stimuli that trigger bone remodeling.

Osteocyte calcium signals encode strain magnitude and loading frequency in vivo

“…Our studies used the third metatarsal (MT3) of the mouse foot. Previous studies in our group demonstrated that this bone is well-suited for direct visualization of cortical bone osteocytes in living animals by using MPM (20). Its tubular geometry lends itself to bending without creat- ing off-axis stresses.…”

“…To address the importance of vascular pressure to maintaining the fluid pressure of the LCS, MT3s from an additional group of mice (n = 3) were cyclically loaded to a single test strain (2,000 µ , 1 Hz) for 60 s while animals were alive and then again at 15 min postmortem (no vascular pressure but cells viable) and 60 min postmortem (severe mitochondrial stress) (20). Amplitude of osteocyte Ca 2+ response was attenuated markedly at 15 min after death compared with in vivo loading (Fig.…”

β-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor

“…Osteocytes have been imaged previously in vivo for active mitochondria using Tetramethylrhodamine, ethyl ester, TMRE , a cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by active mitochondria (Frikha-Benayed et al, 2016). These investigators suggested that osteocytes with impaired mitochondrial function may be more susceptible to apoptosis.…”

Carbonic anhydrase III protects osteocytes from oxidative stress

“…Metabolic stress, impaired metabolite transport, and hypoxic conditions have been proposed as major triggers of osteocyte apoptosis and subsequent bone remodeling. By using in vivo multiphoton microscopy, Frikha-Benayed et al (37) demonstrated that osteocytes in the cortical bone exhibit a well-defined spatial heterogeneity, and they respond to hypoxic stress differently depending on their location. Osteocytes near the periosteum have higher mitochondrial content than do osteocytes that are deeper in the cortex, but most of these mitochondria are poorly functional (37).…”

“…By using in vivo multiphoton microscopy, Frikha-Benayed et al (37) demonstrated that osteocytes in the cortical bone exhibit a well-defined spatial heterogeneity, and they respond to hypoxic stress differently depending on their location. Osteocytes near the periosteum have higher mitochondrial content than do osteocytes that are deeper in the cortex, but most of these mitochondria are poorly functional (37). Here, we report that CAIII expression is elevated in high-Sost/sclerostin-expressing osteocytes and protects these cells from hypoxic stress.…”