Perturbation of cytosolic calcium by 2‐aminoethoxydiphenyl borate and caffeine affects zebrafish myofibril alignment

Abstract

The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.

Embryonic exposure to 4‐methylimidazole leads to zebrafish myofibril misalignment

“…Exposure of zebrafish embryos to 2‐aminothoxyldiphenyl borate (2‐APB), a chemical can reduce cytosolic Ca 2+ levels, led to split and short myofibril phenotypes. On the contrary, embryos treated with caffeine, a reagent can enhance cytosolic Ca 2+ level, displayed twisted and wavy myofibril morphology . Our data indicated that 4‐MI‐treated zebrafish embryos exhibited wavy and misaligned myofibrils which are highly similar to those of zebrafish embryos with calcium imbalance.…”

“…During zebrafish myogenesis, myofibril alignment is regulated by several factors, including calcium concentration, nerve activity and MRFs. Previous reports demonstrated that proper cytosolic calcium concentration can regulate myod expression and myofibrils alignment . Exposure of zebrafish embryos to 2‐aminothoxyldiphenyl borate (2‐APB), a chemical can reduce cytosolic Ca 2+ levels, led to split and short myofibril phenotypes.…”

“…In the present study, we explored the teratogenic effects of 4‐MI exposure during zebrafish embryogenesis, with a particular focus on the muscle development. In this animal model, subtle changes in muscle fiber alignment can be easily observed by staining with specific monoclonal antibody and fluorescent probe . Therefore, the combination of immunofluorescence and ultrastructural analysis was used to explore the defective phenotype of 4‐MI‐treated zebrafish embryos.…”

“…In this animal model, subtle changes in muscle fiber alignment can be easily observed by staining with specific monoclonal antibody and fluorescent probe. 16,17 Therefore, the combination of immunofluorescence and ultrastructural analysis was used to explore the defective phenotype of 4-MI-treated zebrafish embryos. To further reveal the molecular mechanism of 4-MI-induced phenotypes, we next investigated the effect of 4-MI on the expression of musclespecific genes using whole-mount in situ hybridization and quantitative reverse transcription-polymerase chain reaction (RT-qPCR).…”

Zebrafish Model for Safety and Toxicity Testing of Nutraceuticals

“…A green fluorescent vascular endothelium transgenic line was used to assess the induced toxicity of caffeine during embryonic vascular development, but no defects were found in vasculogenesis; however, real-time PCR showed that caffeine treatment can disturb the angiogenesis of zebrafish embryos (Yeh et al, 2012). Interestingly, Wu et al (2015) exposed zebrafish embryos to both 2-APB and caffeine and found no malformed phenotypes, which was totally different from the results found with the phenotype treated with 2-APB alone. Increasing exposure time to caffeine may have triggered apoptosis, but neither significant mortality nor morphological changes could be detected.…”