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. 2016 Oct 26;6(4):51.
doi: 10.3390/brainsci6040051.

The Relationship Between Estrogen and Nitric Oxide in the Prevention of Cardiac and Vascular Anomalies in the Developing Zebrafish (Danio Rerio)

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Free PMC article

The Relationship Between Estrogen and Nitric Oxide in the Prevention of Cardiac and Vascular Anomalies in the Developing Zebrafish (Danio Rerio)

Benjamin G Sykes et al. Brain Sci. .
Free PMC article

Abstract

It has been known that both estrogen (E2) and nitric oxide (NO) are critical for proper cardiovascular system (CVS) function. It has also been demonstrated that E2 acts as an upstream effector in the nitric oxide (NO) pathway. Results from this study indicate that the use of a nitric oxide synthase (NOS) inhibitor (NOSI) which targets specifically neuronal NOS (nNOS or NOS1), proadifen hydrochloride, caused a significant depression of fish heart rates (HR) accompanied by increased arrhythmic behavior. However, none of these phenotypes were evident with either the inhibition of endothelial NOS (eNOS) or inducible NOS (iNOS) isoforms. These cardiac arrhythmias could also be mimicked by inhibition of E2 synthesis with the aromatase inhibitor (AI), 4-OH-A, in a manner similar to that of nNOSI. In both scenarios, by using an NO donor (DETA-NO) in either NO + nNOSI or E2 + AI co-treatments, fish could be significantly rescued from decreased HR and increased arrhythmias. However, the addition of an NOS inhibitor (L-NAME) to the E2 + AI co-treatment fish prevented the rescue of low heart rates and arrhythmias, which strongly implicates the NO pathway as a downstream E2 targeted molecule for the maintenance of healthy cardiomyocyte contractile conditions in the developing zebrafish. Cardiac arrhythmias could be mimicked by the S-nitrosylation pathway inhibitor DTT (1,4-dithiothreitol) but not by ODQ (1H-[1-3]oxadiazolo[4,3-a]quinoxalin-1-one), the inhibitor of the NO receptor molecule sGC in the cGMP-dependent pathway. In both the nNOSI and AI-induced arrhythmic conditions, 100% of the fish expressed the phenotype, but could be rapidly rescued with maximum survival by a washout with dantrolene, a ryanodine Ca2+ channel receptor blocker, compared to the time it took for rescue using a control salt solution. In addition, of the three NOS isoforms, eNOS was the one most implicated in the maintenance of an intact developing fish vascular system. In conclusion, results from this study have shown that nNOS is the prominent isoform that is responsible, in part, for maintaining normal heart rates and prevention of arrhythmias in the developing zebrafish heart failure model. These phenomena are related to the upstream stimulatory regulation by E2. On the other hand, eNOS has a minimal effect and iNOS has little to no influence on this phenomenon. Data also suggests that nNOS acts on the zebrafish cardiomyocytes through the S-nitrosylation pathway to influence the SR ryanidine Ca2+ channels in the excitation-coupling phenomena. In contrast, eNOS is the prominent isoform that influences blood vessel development in this model.

Keywords: blood vessel development; embryonic zebrafish; estrogen; heart arrhythmias; nitric oxide.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effects of various treatments on fish heart rates (HR) in beats/minute (bpm) after 4 days of treatment beginning at 48 h post fertilization (hpf). (A) This figure indicates that both AI (50 µM) and general (g) NOSI (15 mM) significantly reduces (p < 0.001) HR; (B) These data indicate that gNOSI + NO (50 µM) co-treatment significantly rescues (** p < 0.05) HR when compared to that of gNOSI; (C) On the other hand, gNOSI can prevent the rescue of HR caused by E2 (10 nM) replacement therapy. Specifically, if gNOSI is added to the AI + E2 treatment paradigm, the rescue of HR was significantly eliminated (p < 0.001). All bars = ± SD. Asterisks indicate significant differences between treatment groups and controls.
Figure 2
Figure 2
The effects of various treatments on fish heart rates (HR) in beats/minute (bpm) after 4 days of treatment beginning at 48 h post fertilization (hpf). (A) The only significant decrease in HR among the three NOS isoform inhibitors was induced by the nNOSI treatment (p < 0.001). The nNOSI (50 µM) treatment is shown to closely mimic the significant decrease in HR initiated by the gNOSI (15 mM) treatment; (B) This figure shows a dose–response analysis for varying concentrations of nNOSI on fish HRs. ERS (control embryo rearing solution) was used as a control to measure the effectiveness of the different nNOSI concentrations. Specifically, data shows that nNOSI at 30 and 50 μM lowered HR in a dose dependent manner when compared to the 10 µM concentration (p < 0.05 for both). Note that gNOSI also significantly reduced HR in a manner similar to that of nNOSI; (C) This figure shows the HR rescue effect initiated by combining both nNOSI or AI (50 µM) with DETA-NO (50 µM) as an NO donor (p < 0.002 for nNOSI + DETA-NO vs. nNOSI and p < 0.001 for AI (50 µM) + DETA vs. AI). Double labeled asterisk group indicate a significant difference from their single asterisk counterparts. All bars = ± SD.
Figure 3
Figure 3
The dose–response effects of nNOSI on zebrafish treated at 2 and 6 dpf and analyzed for heart rate and arrhythmia phenotypes at 4 dpf and 8 h post treatment respectively. (A) A depiction of the actual temporal events leading to the significant decrease (* p < 0.01) in heart rates (HR) elicited by both 30 and 50 µM nNOSI over the 8 h treatment period in 6 dpf fish. Most significantly, note that 100% of the treated fish populations recover from both the arrhythmic and HR phenotypes when nNOSI is washed out at 8 h of treatment with the ERS control solution (asterisk indicates the time of washout); (B) The arrhythmic phenotype in 6 dpf fish can be induced in 100% of the fish population treated with 50 µM nNOSI compared to only 50% affected with a 30 µM concentration both of which are significantly different from each other and that of the ERS controls (* p < 0.001). However, no phenotypic expression is induced with a 10 µM treatment which is not significantly different from that of the ERS control fish (p < 0.001); (C) The effects of different nNOSI concentrations on arrhythmic behavior in fish treated at 2 dpf and analyzed at 4 dpf. Note that the lower concentrations produce the most consistent arrhythmic behavior (p < 0.002 for 10 μM and p < 0.02 for 30 μM). Also, note that the % arrhythmias in this younger embryonic population (10%–15%) are much less than that seen in the 6 dpf treated fish (compare with Figure 3B above) but are significantly greater (p < 0.001) than that of the ERS controls. Bars represent ± SD. Single asterisks represent significant differences between treated groups and controls. Double asterisks indicate a significant difference between dose treatments.
Figure 4
Figure 4
An analysis of arrhythmic behavior under various treatment conditions in the embryonic zebrafish. (A) Both AI (50 µM) and nNOSI (50 µM) fish treated at 6 dpf and analyzed 8 h later caused 100% of the population to exhibit the arrhythmic phenotype; (B) The demonstration that the arrhythmic phenotype can be attributed to the NO/sGC/cGMP-independent pathway inhibition in fish treated at 6 dpf and analyzed 8 h later. Specifically, fish treated with 100 µM DTT, which reverses S-nitrosylation protein modification, elicits the arrhythmic phenotype in 100% of the fish population. In contrast, no arrhythmic phenotypic expression is evident with a 50 µM ODQ (1H-[1–3]oxadiazolo[4,3-a]quinoxalin-1-one) treatment, which blocks the activity of sGC, and is no different than that of the ERS controls (p > 0.05).
Figure 5
Figure 5
The effects of various treatments on selected representative fish hearts as measured by Ca2+ flux patterns in the Tg(cmlc2:gCaMP) line of transgenic fish after 4 days of treatment beginning at 48 h post fertilization (hpf). (A) This figure shows the Ca2+ flux that takes place in a control zebrafish heart treated in ERS solution. Note the regularity of the CA2+ flux spike patterns represented by the peaks and valleys. For example, this regular spike interval occurs every 0.4 s (see comparisons in Figure 3D. below); (B) This figure shows the calcium flux in a zebrafish heart treated with the aromatase inhibitor (AI) 4-androstene-3,17-dione. Note that peaks are even farther apart than that of nNOSI and ERS controls. The time difference between peaks ranges between 1.4–1.8 s, which are more than four times longer than ERS controls; however, the spike patterns still remain more regular than their nNOSI treated counterparts (see comparisons in Figure 3C,D below); (C) This figure shows a more pronounced arrhythmia behavior exhibited by irregular calcium flux patterns in a zebrafish heart treated with 50 μM nNOSI. When compared to ERS controls, the interval differences between peaks were observed to occur over a more extensive and irregular range of 0.6–1 s; (D) This table quantifies calcium flux values for the three fish seen in Figure 5A–C above. Specifically, the Ca2+ peak values/min for all three fish matches very closely the actual measured heart rates (beats/minute) for comparable treatment groups throughout the study with the AI and nNOSI HRs in the arrhythmic range (<120 bpm, 45 ± 8 and 75 ± 10 bpm respectively, compared to 150 ± 10 for controls) and were further confirmed by their increased and more irregular Ca2+ spike intervals.
Figure 6
Figure 6
The effects of ERS and dantrolene (10 µM) washout on the recovery of arrhythmic fish previously treated with nNOSI. Note that by 24 min after washout approximately 75% of dantrolene treated fish had recovered from the arrhythmic phenotype while in ERS treated fish recovery was insignificant (p < 0.001). By 30 min after ERS washout, although approximately 60% of the fish had recovered from the arrhythmic phenotype, they were still significantly behind the 90% recovery for the dantrolene treated fish (p < 0.05). However, by 60 min after washout, both treated fish group HRs had returned to normal heart rate levels. Bars = ± SD. Asterisk indicates significant differences between both 24 and 30 min measures.
Figure 7
Figure 7
The effects of various treatments in TG(fli1:EGFP) y1/+y1 (AB) transgenic fish on average tail caudal artery (CA) diameter as measured from confocal z-stack photomicrographs after 4 days of treatment beginning at 48 h post fertilization (hpf). (A) CA diameter is significantly decreased in gNOSI treated fish when compared to that of controls (* p < 0.001). In addition, all three NOSI isoforms also cause a significant decrease in CA diameter (p < 0.05; Bar = ± SD); (B) In order to ensure that measurements of all vessels including the CA were collected in the same location in every confocal- imaged photograph, a standard reference point (arrow) of a distance (875 µm) from the tail region was used. Bar equals 100 µm.
Figure 8
Figure 8
The effects of various treatments in TG(fli1:EGFP) y1/+y1 (AB) transgenic fish on average tail blood vessel measurements calculated from confocal z-stack photomicrographs after 4 days of treatment beginning at 48 h post fertilization (hpf). (A) This figure indicates that the number of intersegmental vessel (SE) abnormal bifurcations increases significantly when treated with gNOSI (15 mM) or eNOSI (5 µM). Both treatments were significantly more effective than either nNOSI (50 µM) or iNOSI (10 µM) in causing this anomaly (* p < 0.001); (B) The number of SE misconnections increases significantly when treated with gNOSI compared to the control values (* p < 0.001). Also, gNOSI was significantly more effective than eNOSI, nNOSI, and iNOSI in causing this vessel anomaly (* p < 0.001); (C,D) Confocal z-stack imagery of the vasculature shows either retarded growth or deterioration, particularly in the dorsal longitudinal anastomotic vessel (DLAV) and vertebral artery (VA) vessels (arrows) in the various treated groups mentioned in A–B above which appear patchy or absent (arrows) when compared to control fish as seen in D; (E) Treatment effects on vertebral artery (VA) development demonstrates significant numbers of misconnections when treated with gNOSI and eNOSI compared to the control values (* p < 0.001). Also, note that both eNOSI and gNOSI were significantly more effective than nNOSI and iNOSI in perpetuating this vessel anomaly (* p < 0.001). Bars = ± SD. The data indicated in A, B, and D above was collected from the last 875 µM of the caudal area.

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References

    1. Turgeon J.L., Carr M.C., Maki P.M., Mendelsohn M.E., Wise P.M. Complex actions of sex steroids in adipose tissue, the cardiovascular system, and brain: Insights from basic science and clinical studies. Endocr. Rev. 2006;27:575–605. doi: 10.1210/er.2005-0020. - DOI - PubMed
    1. Lima B., Forrester M.T., Hess D.T., Stamler J.S. S-nitrosylation in cardiovascular signaling. J. Am. Heart Assoc. 2010;106:633–646. doi: 10.1161/CIRCRESAHA.109.207381. - DOI - PMC - PubMed
    1. Bradley S., Tossell K., Lockley R., McDearmid J.R. Nitric oxide synthase regulates morphogenesis of zebrafish spinal cord motor neurons. J. Neurosc. 2010;30:16818–16831. doi: 10.1523/JNEUROSCI.4456-10.2010. - DOI - PMC - PubMed
    1. Pelster B., Grillitsch S., Schwerte T. NO as a mediator during the early development of the cardiovascular system in the zebrafish. Com. Biochem. Physiol. Part A. 2005;142:215–220. doi: 10.1016/j.cbpb.2005.05.036. - DOI - PubMed
    1. Hammond J., Balligand J.L. Nitric Oxide synthase and cyclic GMP signaling in cardiac myocytes: From contractility to remodeling. J. Mol. Cell. Cardiol. 2011;52:330–340. doi: 10.1016/j.yjmcc.2011.07.029. - DOI - PubMed
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