Localization and Quantification of ATPase, Ca2+ Transporting, Plasma Membrane 2 (Atp2b2) in the Developing Euthyroid and Hypothyroid Cochlea

Thyroid hormone (TH) is essential for auditory system development. Proper auditory function requires remodeling and transformation of cochlear structures during a critical developmental period. In rodent models, low TH during this timeframe delays remodeling and impacts ion channel function required for hearing to occur. TH receptors are ligand-dependent transcription factors that regulate expression of genes. Considering this, auditory deficits observed in hypothyroidism are thought to be largely the result of altered expression of specific TH receptor target genes. TH is reported to impact calcium signaling in several tissues, but whether regulators of calcium signaling are altered following developmental hypothyroidism is largely unexplored. In this report, we investigated the spatial and temporal expression of the Atp2b2 gene in a mouse model of developmental hypothyroidism. Timed-pregnant dams were treated with thyroid gland inhibitors to induce developmental TH insufficiency from embryonic day 12.5 (E12.5) through post-natal day 15 (P15). Untreated timed-pregnant dams served as euthyroid control. Control and experimental cochleae were collected over a developmental period from embryonic day 18.5 (E18.5) through P15. In a parallel study, P2 pups were injected with either saline or T3 and sacrificed 8 hours post-injection to observe the effects of acute T3 action on Atp2b2 expression. Cochleae were then processed to detect Atp2b2 mRNA by in situ hybridization or quantitative real-time PCR. Select cochleae were also processed to detect Atp2b2 protein (PMCA2) through immunofluorescence. In situ hybridization localized Atp2b2 mRNA to inner hair cells, outer hair cells, and spiral ganglion neurons as expected. However, Atp2b2 was also observed at lower levels in the greater epithelial ridge. Quantification of Atp2b2 mRNA by qRT-PCR revealed an effect of post-natal age and treatment on expression. In euthyroid animals, Atp2b2 expression gradually increased until P10, then decreased by P15. In hypothyroid animals, Atp2b2 mRNA was significantly decreased from P5 through P10 compared to euthyroid control. Atp2b2 mRNA expression was significantly increased in T3-injected mice compared to saline-injected mice. Immunofluorescence showed the presence of PMCA2 in the stereocilia of the outer hair cells and greater epithelial ridge. The reduction in Atp2b2 mRNA expression in the hypothyroid mice suggests that Atp2b2 is developmentally regulated, in part, by thyroid hormone signaling. This study characterizes the relationship between TH and Atp2b2 expression during early cochlear development while providing insight into abnormal cochlear development due to hypothyroidism.