Connecting the Dots: Linking Osteocyte Activity and Therapeutic Modulation of Sclerostin by Extending a Multiscale Systems Model.

The goal of this work was to extend a mathematical, multiscale systems model of bone function, remodeling, and health in order to explore hypotheses related to therapeutic modulation of sclerostin and quantitatively describe purported osteocyte activity within bone remodeling events. A pharmacokinetic model with first-order absorption and dual elimination pathways was used to describe the kinetics of romosozumab, a monoclonal antibody (mAb) against sclerostin. To describe total circulating sclerostin, an extended indirect response model of inhibition of offset was developed. These models were subsequently linked to the systems model, with sclerostin signaling changes in resorption and formation through established osteocyte-mediated mechanisms. The model proposes relative contributions of the osteocyte to the RANKL pool, a major player in feedback signaling, and is used to explore hypotheses surrounding attenuation of anabolic activity after multiple doses of sclerostin mAbs, a phenomenon whose mechanism is poorly understood.

Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts.

Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization of Ca2+ stores with bradykinin (BK) increased the cytosolic Ca2+ concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2(+)-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+ concentration ([Ca2+]mt) of 2.0 +/- 0.2 microM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mt increase, although it did not affect the [Ca2+]c transient. Basal [Ca2+]c and [Ca2+]mt in control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 +/- 1% in normal cells to 19 +/- 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mt transients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.

CGP37157 modulates mitochondrial Ca2+ homeostasis in cultured rat dorsal root ganglion neurons.

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na+/Ca2+ exchange, on depolarization-induced intracellular free Ca2+ concentration ([Ca2+]i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca2+]i transient resulted from mitochondrion-mediated [Ca2+]i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca2+. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC50 = 4 +/- 1 microM). This decrease in [Ca2+]i was followed by an increase in [Ca2+]i upon removal of the drug, suggesting that Ca2+ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca2+ influx at the concentrations required to see effects on [Ca2+]i buffering. Thus, CGP37157 inhibits mitochondrial Na+/Ca2+ exchange and directly inhibits voltage-gated Ca2+ channels, suggesting caution in its use to study [Ca2+]i regulation in intact cells.