Abstracts

Rationale: Fracture risk is a serious comorbidity in epilepsy and may relate to the use of anti-epileptic medications (AED). Many AEDs inhibit ion channel function and the expression of these channels in osteoblasts raises the idea that altered bone signaling increases bone fragility. We aimed to: (1) precisely define the subtypes of voltage-activated sodium channels (NaV) expressed in mouse osteoblast, and (2) investigate the action of carbamazepine (CBZ) and phenytoin (PHT) on NaV channels.Methods: Immunocytochemistry and RNASeq were performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice. An expression threshold of 0.3 fragments per kilobase of exon per million fragments mapped (FPKM) was utilized for RNASeq analysis results. In addition whole-cell patch-clamp recordings using Patchliner® (Nanion Technologies, Munich, Germany) were made to identify the native currents expressed and also assess the actions of CBZ (50 μM) or PHT (50 μM).Results: NaV expression was demonstrated with immunocytochemistry, RNASeq and functionally with demonstration of robust TTX sensitive and voltage-activated inward currents. RNASeq analysis showed Scn2α (NaV1.2), Scn3α (NaV1.3), Scn7α (NaVi2.1), and Scn1β and Scn3β were expressed with FPKM levels above the nominated threshold. Application of CBZ or PHT resulted in significant inhibition of current amplitude: for CBZ 31.6 ± 5.9 % (n = 9; p<0.001), and for PHT 35.5 ± 6.9 %, n = 7; p<0.001).Conclusions: Mouse osteoblasts express Scn2α, Scn3α, Scn7α and beta subunits Scn1β and Scn3β, genes which encode both the alpha pore forming and beta accessory subunits, indicating that functional NaVs are likely present in mouse osteoblasts. Native NaV currents are blocked by CBZ and PHT supporting our hypothesis that AEDs can directly influence osteoblast function and potentially impact upon signaling during modeling and remodeling, and therefore upon bone strength. Funding Sources: The Royal Melbourne Hospital Victor Hurley Grants-in-Aid (2013) and MBC Postdoctoral Medical Research Fellowship at The Melbourne Brain Centre RMH (June 2012-2014) (NHMRC Centre for Research Excellence Grant 1001216). Dr. Sandra Petty is the Thwaites Gutch Fellow at Ormond College Parkville VIC Australia.