Screening for chemicals with thyroid hormone-disrupting effects using zebrafish embryo.

Thyroid disruption is an increasingly recognized issue in the use and development of chemicals and new drugs, especially to help toxicologist to complement the reproductive and developmental toxicology information of chemicals. Still, adequate assessment methods are scarce and often suffer a trade-off between physiological relevance and labor- and cost-intensive assays. Here, we present a tiered approach for a medium-throughput screening of chemicals to identify their thyroid disrupting potential in zebrafish embryos as a New Approach Methodology (NAM). After identifying the maximum tolerated concentrations, we exposed zebrafish larvae to sub-adverse effect levels of the reference compounds benzophenone-2, bisphenol A, phenylthiourea, potassium perchlorate, propylthiouracil, and phloroglucinol to exclude any systemic toxicity. Applying the transgenic zebrafish line that carries a gene for the red fluorescence protein (Tg(tg:mCherry)) under the thyroglobulin promoter, we could identify the thyroid disrupting effects of the chemicals by a time and cost-effective image analysis measuring the fluorescence levels in the thyroid glands. Our observations could be confirmed by altered expression patterns of genes involved in the hypothalamus-pituitary-thyroid (HPT) axis. Finally, to anchor the observed thyroid disruption, we determined some changes in the Thyroid hormone levels of triiodothyronine (T3) and Thyroxine (T4) using a newly developed liquid chromatography mass spectrometric (LCMS) method. The presented approach carries the potential to extend the toolbox for legislative authorities and chemical producers for the assessment of thyroid-specific endocrine disruption and to overcome current challenges in the evaluation of endocrine disruptors.

Active GSK3ß and an intact ß-catenin TCF complex are essential for the differentiation of human myogenic progenitor cells.

Wnt-ß-catenin signalling is essential for skeletal muscle myogenesis during development, but its role in adult human skeletal muscle remains unknown. Here we have used human primary CD56Pos satellite cell-derived myogenic progenitors obtained from healthy individuals to study the role of Wnt-ß-catenin signalling in myogenic differentiation. We show that dephosphorylated ß-catenin (active-ß-catenin), the central effector of the canonical Wnt cascade, is strongly upregulated at the onset of differentiation and undergoes nuclear translocation as differentiation progresses. To establish the role of Wnt signalling in regulating the differentiation process we manipulated key nodes of this pathway through a series of ß-catenin gain-of-function (GSK3 inhibition and ß-catenin overexpression) or loss-of-function experiments (dominant negative TCF4). Our data showed that manipulation of these critical pathway components led to varying degrees of disruption to the normal differentiation phenotype indicating the importance of Wnt signalling in regulating this process. We reveal an independent necessity for active-ß-catenin in the fusion and differentiation of human myogenic progenitors and that dominant negative inhibition of TCF4 prevents differentiation completely. Together these data add new mechanistic insights into both Wnt signalling and adult human myogenic progenitor differentiation.

Impaired calcium calmodulin kinase signaling and muscle adaptation response in the absence of calpain 3.

Mutations in the non-lysosomal, cysteine protease calpain 3 (CAPN3) result in the disease limb girdle muscular dystrophy type 2A (LGMD2A). CAPN3 is localized to several subcellular compartments, including triads, where it plays a structural, rather than a proteolytic, role. In the absence of CAPN3, several triad components are reduced, including the major Ca(2+) release channel, ryanodine receptor (RyR). Furthermore, Ca(2+) release upon excitation is impaired in the absence of CAPN3. In the present study, we show that Ca-calmodulin protein kinase II (CaMKII) signaling is compromised in CAPN3 knockout (C3KO) mice. The CaMK pathway has been previously implicated in promoting the slow skeletal muscle phenotype. As expected, the decrease in CaMKII signaling that was observed in the absence of CAPN3 is associated with a reduction in the slow versus fast muscle fiber phenotype. We show that muscles of WT mice subjected to exercise training activate the CaMKII signaling pathway and increase expression of the slow form of myosin; however, muscles of C3KO mice do not exhibit these adaptive changes to exercise. These data strongly suggest that skeletal muscle's adaptive response to functional demand is compromised in the absence of CAPN3. In agreement with our mouse studies, RyR levels were also decreased in biopsies from LGMD2A patients. Moreover, we observed a preferential pathological involvement of slow fibers in LGMD2A biopsies. Thus, impaired CaMKII signaling and, as a result, a weakened muscle adaptation response identify a novel mechanism that may underlie LGMD2A and suggest a pharmacological target that should be explored for therapy.