Fig. 1. Calcium imaging in zebrafish embryos. The developing trunk muscle (DTM) of 48 h post-fertilization (hpf) zebrafish embryos were used to test the rapid effects of cortisol on Ca²⁺ dynamics using the FURA-2AM method. The treatments included cortisol antibody to sequester cortisol, EGTA to chelate extracellular Ca²⁺ (eCa²⁺), BAPTA-AM (BAPTA) to chelate intracellular Ca²⁺ (iCa²⁺), and RU486 as a GR antagonist. Also, Ca²⁺ channel blockers, including nifedipine (Nife) to block L-type channel, verapamil (VM) to block T-type channel, cadmium as a non-specific blocker of plasma membrane Ca²⁺ channels, and CpD5J-4 to block the CRAC channels were utilized. See materials and methods for details.

Fig. 2. Cortisol rapidly stimulates [(Ca²⁺)i] wave in zebrafish muscle. (a) Representative images of the developing trunk muscle (DTM) loaded with the ratiometric dye FURA-2AM either with (100 ng/ml) or without the addition of cortisol. (b) A representative line graph of the temporal calcium dynamics in response to cortisol (CORT) addition over a 5 min period. FURA-2AM intensity was recorded at 5 s intervals. (c) Graph showing the ratiometric FURA-2AM intensity with different concentrations of cortisol (10, 50 and 100 ng/ml) at 30 s post-cortisol addition. All bars represent mean ± SEM (n = 4–5 independent embryos); bars with different letters are significantly different; one-way ANOVA; Holm Sidak post hoc test; p < 0.05.

Fig. 3. The cortisol-mediated [(Ca²⁺)i] increase is independent of glucocorticoid receptor. (a) Representative images of the developing trunk muscle (DTM) loaded with the ratiometric dye FURA-2AM and treated with either vehicle (CONTROL), cortisol (CORT), RU486 or a combination of cortisol and RU486 (RU486+CORT), as well as cortisol treatment of the DTM from GRKO zebrafish (GRKO + CORT). (b) Graph showing the ratiometric FURA-2AM intensity in the DTM treated with either cortisol, RU486 or a combination of cortisol and RU486 at 30 s post-cortisol exposure. (c) Graph showing the ratiometric FURA-2AM intensity in wildtype or GR knockout (GRKO) DTM at 30 s post-cortisol addition. All bars represent mean ± SEM (n = 4–5 independent embryos); bars with different letters are significantly different; one-way ANOVA; Holm Sidak post hoc test; p < 0.05.

Fig. 4. Cortisol-mediated [(Ca²⁺)i] rise involves both intracellular and extracellular Ca²⁺. (a) Representative images of the developing trunk muscle (DTM) loaded with the ratiometric dye FURA-2AM and treated with either vehicle (CONTROL), cortisol (CORT), EGTA, BAPTA-AM or a combination of cortisol with EGTA (EGTA + CORT) or BAPTA-AM (BAPTA-AM + CORT). (b) A bar graph showing the ratiometric intensity at 30 s with extracellular calcium chelator EGTA either with or without cortisol. (c) A bar graph showing the ratiometric intensity at 30 s with an intracellular chelator BAPTA-AM either with or without cortisol. All bars represent mean ± SEM (n = 4–5 independent embryos); bars with different letters are significantly different; one way ANOVA; Holm Sidak post hoc test; p < 0.05. Abbreviations: Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′- tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxy methyl ester (BAPTA-AM).

Fig. 5. Cortisol-mediated [(Ca²⁺)i] rise involves plasma membrane Ca²⁺ channels. (a) Representative images of the developing trunk muscle (DTM) loaded with the ratiometric dye FURA-2AM and treated with either vehicle (CONTROL), cortisol (CORT) or the Ca²⁺ channel blockers nifedipine (NIFE), verapamil (VM) or cadmium (CAD) either without or with cortisol (NIFE + CORT; VM + CORT; CAD + CORT). (b) A bar graph showing the ratiometric intensity at 30 s with the L-type calcium channel inhibitor, nifedipine either with or without cortisol. (c) A bar graph showing the ratiometric intensity at 30 s with the T-type calcium inhibitor verapamil either with or without cortisol. (d) A bar graph showing the ratiometric intensity at 30 s with non-specific calcium channel inhibitor cadmium either with or without cortisol. All bars represent mean ± SEM (n = 4–5 independent embryos); Bars with different letters are significantly different; one way ANOVA; Holm Sidak post hoc test; p < 0.05.

Fig. 6. Cortisol-mediated [(Ca²⁺)i] rise involves calcium release-activated calcium channel (CRACC) activation. (a) Representative images of the developing trunk muscle (DTM) loaded with the ratiometric dye FURA-2AM and treated with either vehicle (CONTROL), cortisol (CORT) without or with antibody for quenching (CORT + Ab), and CRACC inhibitor (CpD5J-4) without or with cortisol (CpD5J-4+CORT). (b) A bar graph showing the ratiometric intensity at 30 s with the CRACC inhibitor CpD5J-4 either with or without cortisol. (c) A bar graph showing the ratiometric intensity at 30 s with CORT either without or with the antibody (CORT + Ab). All bars represent mean ± SEM (n = 4–5 independent embryos); bars with different letters are significantly different; one way ANOVA; Holm Sidak post hoc test; p < 0.05.

Fig. 7. Cortisol rapidly affects Orai1 expression in the developing trunk muscle. (a) Spatial changes in Orai1 (ORAI-1), the pore forming subunit of CRACC, expression in zebrafish embryo. ORAI-1 (green), Caveolin-1(red) and DAPI (blue). ORAI-1 expression in the developing trunk muscle (DTM) is enhanced in the cortisol exposed muscles, and the colocalization between ORAI-1 and caveolin-1 is shown as an inset (yellow), and the white arrows indicate colocalization.

Fig. 8. Proposed model for the rapid cortisol action on [(Ca²⁺)i] in the developing trunk muscle (DTM) of zebrafish. We propose that cortisol-mediated rapid rise in [(Ca²⁺)i] occur via two pathways: i) A direct effect of cortisol by binding to the CRAC channel and modulating the pore (Orai-1) opening to allow extracellular Ca²⁺ (eCa²⁺) entry causing the rapid intracellular Ca²⁺ (iCa²⁺) rise, and ii) an indirect effect of cortisol that may include the activation of a yet unknown membrane GPCR resulting in either the gating of VGCC and/or the activation of PKA/PKC signaling leading to the release of Ca²⁺ from the sarcoplasmic reticulum (SR). The drop in SR Ca²⁺ stores activates the Ca²⁺ sensor STIM, which interacts with the CRAC channel to cause eCa²⁺ entry leading to a rise in [(Ca²⁺)i] in the DTM. Abbreviations: [(Ca²⁺)i] – cytosolic free calcium levels, CRAC- calcium release-activated Ca²⁺ channel, VGCC - voltage-gated Ca²⁺ channels, STIM-stromal interaction molecule, PKA-protein kinase A, PKC – protein kinase C, GPCR-G protein-coupled receptor.