International Journal of Cardiovascular ResearchISSN: 2324-8602

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Research Article, Int J Cardiovas Res Vol: 6 Issue: 1

The Role of Estrogen and Nitric Oxide in the Prevention of Cardiac Arrhythmias in the Embryonic Zebrafish (Danio Rerio)

Jonathan Winalski* and James Turner
Department of Biology, Virginia Military Institute, Lexington, Virginia, USA
Corresponding Author : Jonathan Winalski
Department of Biology, Virginia Military Institute, Lexington, Virginia, USA
E-mail: [email protected]
Received: November 02, 2016 Accepted: November 29, 2016 Published: January 04, 2017
Citation: Winalski J, Turner J (2017) The Role of Estrogen and Nitric Oxide in the Prevention of Cardiac Arrhythmias in the Embryonic Zebrafish (Danio Rerio). Int J Cardiovasc Res 6:1. doi: 10.4172/2324-8602.1000295

Abstract

Nitric oxide (NO) is a gaseous messenger molecule found to be critical in the regulation of cardiomyocyte contraction and blood vessel vasodilation in the cardiovascular system (CVS) of a diverse variety of organisms. NO is released by catalyzing the enzymatic transformation of L-arginine to L-citrulline by NO synthase (NOS), which is found in four distinct isoforms in the human body. The most prominent isoform in the sarcoplasmic reticulum (SR) of the CVS; however, is neuronal NO (nNOS or NOS1). Research shows that NOS1 is critical to the contraction and relaxation of cardiomyocytes. More specifically, NOS1 is believed to protect the regulation of cardiomyocyte calcium (Ca2+) release. This phenomenon occurs through specific ryanodine influenced Ca2+ channels and receptors, which allows for excitation coupling to occur. It has been hypothesized by prior research that diastolic Ca2+ leaks due to abnormally open SR ryanodine channels increases the presence of ventricular arrhythmias. The main hypothesis offered up by this research focuses on the mechanism of action in which deprivation of nNOS and its upstream regulation by estrogen leads to ventricular arrhythmias in the embryonic zebrafish via a dysregulated S-nitrosylation pathway. It was also determined that this S-nitrosylation pathway is independent of soluble guanylyl cyclase (sGC)-GMP mediated pathway, which is normally responsible for exerting NO effects throughout the body. Additionally, the goal of this research was to identify a highly successful rescue treatment that would restore normal CVS activity to the embryos. The most successful treatment option was Dantrolene, a hydantoin derivative whose mechanism of action revolves around closing ryanodine channels. When populations of nNOS deprived fish became 100% arrhythmic, Dantrolene was entirely successful as a treatment paradigm. This data allows for a better understanding of not only the entire CVS as a whole, but potentially elucidates a better understanding of ventricular arrhythmias.

Keywords: Cardiac Arrhythmias; Embryonic Zebrafish; Estrogen; Nitric oxide

Keywords

Cardiac Arrhythmias; Embryonic Zebrafish; Estrogen; Nitric oxide

Introduction

Nitric oxide (NO) is an important gaseous messenger molecule found to be critical in the regulation of heart cardiomyocyte contraction and blood vessel vasodilation in the cardiovascular system (CVS) of a diverse variety of organisms [1]. NO is released by catalyzing the enzymatic transformation of L-arginine to L-citrulline by NO synthase (NOS), which is found in four distinct isoforms in the human body. The most prominent isoform in the sarcoplasmic reticulum (SR) of the CVS is neuronal NO (nNOS or NOS1) which is a dimer structurally composed of a calmodulin linked oxygenase reductase domain. NO was originally thought to only act at the cellular level through a soluble guanylyl cyclase (sGC) receptor pathway, which results in increasing the production of cyclic guanosine monophosphate (cGMP) which in turn activates the cGMP dependent protein kinases (PKG) [2]. However, a second NOmediated pathway has recently emerged. The S-nitrosylation pathway, a cGMP independent pathway which involves the incorporation of cysteine thiolnitrates into specific proteins, thus altering the peptides physiological conformation in the heart and other tissues [3].
Research shows that NOS1 is critical to the contraction and relaxation of cardiomyocytes [1,2]. More specifically, NOS1 is believed to protect the regulation of cardiomyocyte calcium (Ca2+) release which is mediated through the S-nitrosylation pathway. This phenomenon occurs through specific ryanodine influenced Ca2+ channels and receptors, which allows for excitation coupling to occur, the anatomical event responsible for completing cardiomyocyte contractions [2,4]. It has been hypothesized by prior research that diastolic Ca2+ leaks due to genetic mutations or dysregulated S-nitrosylation in the SR ryanodine channels increases the phenotypic presence of ventricular arrhythmias. Ventricular arrhythmias are serious indicators of sudden cardiac failure and accounts for over half of all deaths due to cardiovascular disease (CVD) worldwide [5].
A link between these specific ryanodine channel leaks and NO was noticed as researchers discovered S-nitrosoglutathione reductase, (GSNOR) an enzyme which removes NO groups as a part of regulated S-nitrosylation of Cysteine thiols [6]. Researchers noted that when NOS is inhibited GSNOR and subsequently S-Nitrosylation are also inhibited. Consequently, due to GSNOR enzymes being responsible for the regulation of a vascular tone and cardiac contractility, impaired enzymes, as well as, defective NOS, may simultaneously aid in the dysregulation of S-nitrosylation resulting in phenotypical response of a decreased binding affinity of the major FK binding protein 12.6. This decreased binding affinity of the specific FK protein, whose major role is the closing of the ryanodine channels results in Ca2+ leaks which have been correlated to cardiac arrhythmias [4,6,7].
Estrogen (E2) and its corresponding hormonal response system has been demonstrated to be expressed in the cardiovascular system of adult males and females [8]. E2 is a noted upstream regulator of NOS and has been suggested to provide pre-menopausal women a decreased risk of sudden cardiac failure [8]. However, the American Heart Association (AHA) noted that in 2013, that as the age of woman increases, so does her risk of cardiovascular disease [9]. The AHA proposed that due to E2’s ability to provide flexibility to the human blood vessels, that a link between decreased levels of E2, after menopause may be responsible for this post-menopausal rise in risk of female CVD. Pre-menopausal women who experience low levels of E2 or E2 deprivation entirely, may have inhibited aromatase function or production, the enzyme responsible for E2 synthesis. Our past research has shown that when aromatase is inhibited via an aromatase inhibitor (AI, 4-Androstene-3, 17-Dione) Sigma A-9630 MW 286.4) zebrafish demonstrate phenotypical responses and symptoms similar to that of congestive heart failure [8]. Specifically, in the same study we also noted that when E2 deprived zebrafish were exposed to E2 hormonal therapy phenotypical responses included: reduced blood vessel deterioration, protected heart function, and reduction of cardiac abnormalities, demonstrating a rescue from the congestive heart failure like symptoms [8]. However, when the same zebrafish were placed in co-treatment solution of E2 with either AI or nNOSI, all beneficial phenotypic responses of E2 hormonal therapy were nonexistent. This co-treatment study suggested E2 as an upstream regulator of the NOS pathway related to heart function.
Dantrolene, a derivative of hydantoin, possesses a mechanism of action that relies on the biding of the amino-terminal sequence of dantrolene to ryanodine channels. Currently marketed and used as a drug to prevent malignant hyperthermia, a condition ensued by the onset of surgical anesthesia, dantrolene has been shown to block abnormally open ryanodine channels [10]. Various recent studies have shown that when Ca2+ leaks are present in mammals as a result of open ryanodine channels, leading to the onset of cardiac arrhythmias, dantrolene is a viable treatment option, restoring rhythmic heart function and improving Ca2+ influxes. However, only recently have studies been able to confirm these past corrective results of dantrolene on human in vitro cardiomyocytes [10].
Recent controversial research has called into question the role that L-Type Calcium Channels (LTCC) play in embryonic zebrafish cardiomyocyte contractility. A study done in 2014 produced results arguing that 80% of Ca2+ action potentials in cardiac excitation contraction coupling (ECC) in zebrafish can be attributed to LTCCs [11]. Logically speaking, this would mean that only the remaining 20% of action potentials in ECC can stem from SR ryanodine channels. Such results would be indicative of an LTCC dominant role dysregulation of s-nitrosylation, with minimal impact from any ryanodine channels [11]. Despite these claims, research done in 2015 sought to reaffirm the dominance of ryanodine channel’s impact in zebrafish ECC. Researchers showed that when verapamil, an
LTCC blocker was administered to zebrafish, a negative forcefrequency relationship in relation to isometric cardiac contractions could be observed; resulting in a decreased force contraction of 22 ± 7%. In the same study, suppression of SR Ca2+ via ryanodine channels resulted in a decreased force development of 54 ± 3% in ventricular myocardium, and 52 ± 8% in atrial myocardium. These results clearly indicate the significance of SR ryanodine Ca+2 release in embryonic zebrafish ECC [12].
The current research revolves around strengthening the already proposed link between NO and E2. It is a well-known fact that E2 production drops 90% after menopause occurs in female organisms. By association this would correlate with decreased levels of NO, hypothetically rendering the CV system vulnerable to sudden heart failure via cardiac arrhythmias. As stated in the aforementioned section, source attribution to the mechanism that most negatively impacts ECC would theoretically simultaneously exert profound effects on S-nitrosylation. To identify the mechanism of action being attributed to sudden cardiac arrhythmias would hopefully lead to a greater understanding of sudden heart failure, improving future treatment options, specifically those incorporating verapamil and dantrolene.
Recent results from our laboratory have been obtained using an evolving in vivo embryonic zebrafish model. The zebrafish is an ideal model organism for this study due to the fact that the animal’s entire genome has been mapped; simultaneously over 804 of the genes have a human orthologues, with 80% of 523 specific genes having a synteny relationship with the human genome [13]. In addition to a closed cardiovascular system, embryonic zebrafish are transparent and can demonstrative regenerative properties. Simultaneously, embryonic zebrafish can be treated through diluted working concentrations of various reagents added directly into their aqueous environment; these reagents can then readily dissolve into the zebrafish’s bodily tissue.
Our lab has already displayed a high level of correlation revolving around a link between NO and E2, and their effects on motility and the listless condition. The initial experiments conducted served to reinforce and solidify the proposed downstream production association of the two molecules. The main hypothesis being offered up by current research focuses on the mechanism of action in which deprivation of nNOSI and E2 leads to arrhythmias of embryological CV system of the embryonic zebrafish model organism, while simultaneously attempting to identify a treatment paradigm for said arrhythmias. Therefore it is my hypothesis that ventricular arrhythmias are mainly affected by the abnormal function of ryanodine channels stemming from dysregulated S-nitrosylation caused by NO deprivation. While LTCC may play a role in normal ECC homeostasis and CV function, such involvement is proposed to be statistically insignificant. As a result the most successful corrective treatment paradigm would be a low dosage of dantrolene.

Materials and Methodology

Zebrafish model and husbandry
Several zebrafish lines were used during this study. The AB wild type fish and transgenic gCMP embryos were bred and obtained from the Roanoke College Biology Department laboratory of Dr. Christopher Lassiter. The second transgenic line used for our experimental model was the Casper fish obtained from Caroline Biological Laboratories. The Casper fish were specifically chosen as they lack all body pigment and remain translucent into adulthood. Past research from our lab established a model based on exposing the zebrafish aged two days post fertilization (dpf) to whatever specific experimental treatment was being observed for a four day time period [8]. However, this model often yielded a low but significant percentage of phenotypical responses. To maximize the phenotypic response, the current study focused on creating a model that would yield maximum phenotypic changes under a shorter duration. Our current zebrafish model focuses on treating fish four to six dpf as opposed to two dpf. The shift to a later developmental age may be based on the increased dependence of the zebrafish on CVS function, correlating with the development of the sensory motor and autonomic nervous system in the cardiovascular region. Past research on zebrafish shows a detectable response to adrenergic stimulation of the vagal nerve at four days post fertilization through the use of digital video microscopy. [7] The updated model utilizing older embryonic fish was successful in repeatedly yielding higher phenotypical responses in an accelerated time period.
The husbandry of the zebrafish embryos began immediately upon their arrival, all viable embryos were removed and placed in an autoclaved embryo rearing salt solution (ERS) composed of 0.04 grams of CaCl2, 0.163 grams of MgSO4, 1.0 grams of NaCL, and 0.03 grams of KCL, all in deionized water. A working diluted solution of a 0.05% methylene blue solution was then added to the ERS solution, which serves as an antimicrobial, antibacterial agent. All solutions were freshly changed every 24 hours. All embryos were consistently incubated at a constant 28° Celsius. All reagents were obtained from Sigma-Aldrich unless noted otherwise, and prepared daily before being used.
Arrhythmia and heart rate analysis
All heart rates (HR) were measured using a Nikon TE 300 eclipse microscope unless noted otherwise. To standardize all data, arrhythmic heart function was defined in accordance with the National Heart, Blood, and Lung Institute as any heartbeat that “can beat too fast, too slow, or with an irregular rhythm.” The baseline for normal, healthy embryonic zebrafish heart rates was established at roughly 120-140 beats per minute (bpm) [14]. Any HR under 112 bpm was deemed arrhythmic. HR analysis was performed by placing a single embryonic fish on its side in a separate petri dish void of all fluids. The fish’s heart rate was then counted in a 30 second interval and converted to bpm.
nNOSI dose response and washout recovery
The first experiment conducted was a dose response study to the various concentrations of the nNOSI inhibitor (Hydrochloride Millipore SKF-525A MW 390.0) with the purpose of establishing a timeline of physiological responses, as well as assuring the highest percentage of survival and the desired arrhythmic phenotype. The basis of the experiment stemmed from past research [8]. where the zebrafish were exposed to a 50 μM nNOSI solution for a 24 hour time period beginning at two days post fertilizations. Since the current study was using older zebrafish it became necessary to observe if lower doses of nNOSI shifted phenotypical responses. Accordingly, the embryonic zebrafish were separated and treated in four distinct wells: an ERS control, 10 μM, 30 μM, and 50 μM nNOSI respectively. Motility of the zebrafish was visually checked every 30 minutes, while HR was determined every two hours for eight hours total at which point the arrhythmic phenotype was present at or near one hundred percent of the population. From this point an ERS washout was then done in an effort to determine if recovery from the arrhythmic phenotype could occur. The washouts were completed by placing all treated fish in ERS solutions for 24 hours. The embryonic fish were then subsequently checked at various time intervals throughout the aforementioned 24 hour period. The data from this experiment, indicated that, as in the previous study that 50 μM was the best dosage for creating the arrhythmic phenotype.
E2 deprivation and subsequent co-treatments
Following completion of the first experiment it became necessary to determine how E2 deprivation impacted systemic heart function [8]. It was hypothesized that, due to the downstream link between E2 and NO, that the inhibition of E2 synthesis should exhibit the same phenotypical arrhythmic response as fish placed in 50 μM of nNOSI. Proceeding with this hypothesis fish approximately five dpf were placed in 50 μM of an AI solution, approximately five hours later, AI fish heart rates were recorded. Additionally, it was then decided that an attempt to isolate the protective effects of E2 through various cotreatments was necessary in order to again strengthen the E2/nNOSI regulation loop. A series of E2/AI and E2/nNOSI co-treatments were utilized, with fish following the current five dpf model being incorporated into the experiment. Results were then gathered twenty four hours later.
Dantrolene and verapamil’s effects on both AI and nNOSI treated fish
The first of several dantrolene experiments were simply designed based on determining a working concentration that was safe and viable for zebrafish, but also to simply observe if there was any potential for a successful rescue to occur through its use. Dantrolene currently functions as a viable treatment option, both orally and intravenously, for the symptoms of malignant hypothermia, one of which is irregular heartbeat [10,15]). After eight hours of exposure to 50 μM of nNOSI, in which cardiac arrhythmias were deemed present in one hundred percent of the population, a stock solution of dantrolene was prepared by placing 0.003 grams of dantrolene powder in 1 mL of DMSO. A 10 μM working concentration of dantrolene was then prepared by adding 5 μL of the stock solution to 40 mLs of ERS. Next, the arrhythmic population of zebrafish was divided into two rescue categories: an ERS washout, and a dantrolene washout. All fish were then placed in their prospective solutions, approximately 13 fish in the ERS washout and 20 fish in the Dantrolene rescue. Forty minutes later, the fish were analyzed for recovery from their listless/ arrhythmic condition. Approximately 30 hours later, heart rate, presence of arrhythmias, and survival were checked for both treatment rescue categories. The following procedure was repeated exactly as stated when comparing AI fish to ERS fish. Recent repetitions had determined that the estimated time for the rescuing techniques to be at or near one hundred percent complete at approximately 30 minutes, thus the analysis was adjusted within this time period.
In order to be able to successfully identify whether similar doses of dantrolene or verapamil would be the more successful option in treating the ventricular arrhythmia phenotype, verapamil was placed under the same initial testing conditions experience by dantrolene. Verapamil is a drug comparable to dantrolene which is taken both oral and intravenously, functioning to slow the release of calcium into cardiomyocytes and subsequent blood vessels. The body responds to this inhibition by decreased levels of vasoconstriction and a decreased heartbeat, which allows for increased oxygen absorbance. We first needed to determine if verapamil could even be used as a treatment option under our current embryonic zebrafish model. We placed a healthy zebrafish in line with the lab’s current model in a 10 μM dosage of verapamil for 24 hours in order to observe the potential impact of verapamil on the healthy CV system. Once we were confident in the minimal impacts of verapamil on a normal function CV system, verapamil was used in place if dantrolene in an attempt to rescue arrhythmic zebrafish.
In the final co-treatment experiment of E2/AI and E2/nNOSI both treatment paradigms were incorporated into the experiment, meaning that four total populations of fish were observed. When arrhythmias were induced into the subsequent populations, one population received a 10 μM treatment of dantrolene, the other population receiving verapamil.
Calcium probe
In order to determine if it was indeed the dantrolene treatment responsible for the prompt recovery from the arrhythmic phenotype, as opposed to the ERS dilution factor, a calcium probe was initiated. Specifically, fish were treated overnight, for approximately 17 hours with the 50 μM AI solution to illicit the maximum arrhythmic phenotype. Following this the AI fish were deemed arrhythmic and listless, were placed in 5 mL of a Ca+2 Orange probe for two hours in a dark room. Simultaneously, groups of ERS fish were also placed in the Ca+2 Orange probe to serve as controls. After completion of the two hour incubation, a fish was briefly rinsed in fresh ERS and was analyzed for levels of probe fluorescence using a Nikon Ti Eclipse microscope. A fluorescent AI treated fish was selected and again rinsed in fresh ERS to prevent background fluorescence. Upon observing the high levels of cellular Ca+2, the same fish began a washout in a 10 μM solution of dantrolene. The fish was then rinsed in fresh ERS and observed approximately 30 minutes later for levels of orange probe fluorescence.
ODQ and DTT inhibitors of the NO pathways
The goal of this experiment was to determine which NO pathway was involved with protection and regulation of the zebrafish from the arrhythmic phenotype. The S-nitrosylation pathway was inhibited by 100 μM of DTT (Dithiothreitol, Promege), an oxidizing reagent responsible for reversing the thiol incorporation events of nitrosylation. In turn ODQ, an inhibitor of the major NO receptor sGC in the NO/cCMP/PKG dependent pathway, was used at a concentration of 50 μM. Several key phenotypical responses were recorded including: onset of arrhythmias, decrease in motility, and loss of circulation. This procedure was repeated on 8 individual fish until data analysis was completed.
Data analysis was done via Microsoft Excel, with the standard one way analysis of variance (ANOVA) test being used in order to determine statistical significance. P values were recorded and compared to the null hypothesis when needed. Results determined to be statistically significant have been denoted with an asterisk.

Results and Data

nNOSI timeline experiment resulting in arrhythmic phenotypes
Figure 1, below shows the various effects of the tested μM concentrations of nNOSI on the heart rates of the zebrafish. The experiment was conducted in accordance with the updated zebrafish model with treatments beginning at six dpf as opposed to four dpf, and lasting a minimal of eight hours. As the period of time increases the noted significant (p<.05) trend is the reduction in bpm, regardless of concentration. It is worth noting that the fish in the 50 μM concentration of the nNOSI inhibitor began experiencing cardiac arrhythmias at approximately 5 hours post treatment. The percentage of zebrafish displaying the arrhythmic condition continued to (p<.05) increase until approximately eight hours. At this time point, the zebrafish were incredibly susceptible to heart failure. As a result of this, a washout rescue with the standard ERS aqueous solution was initiated. As it was observed, once the fish were placed in the ERS solution a recovery to a normal, homeostatic bpm range was observed. Statistical analysis confirms that the data is statistically significant, (p<.05) and that each concentration yielded different results at 8 hours post fertilization, such is denoted by the asterisk. However, a concentration of 50 μM nNOSI treatment was responsible for inducing the arrhythmic response in the majority of the treatment populations by reducing the heart rate to approximately 60bpm. The significance from this data allowed for a better understanding of the treatment timeline, allowing for us to better understand at which hour the phenotypical response was maximized, while simultaneously minimizing unnecessary fish causalities.
Figure 1: Depicts average heart rate (bpm) of the zebrafish (Danio Rerio) in various concentrations of a nNOSI inhibitor 6 days post fertilizations. Note that there is a significant reduction in the bpm for fished exposed to the 50 μM inhibitor, this significances the onset of the arrhythmic state. Bars represent standard error.
nNOSI dose response experiment resulting in arrhythmic phenotypes
The data collected from the nNOSI dose response is represented below and shows the percentage of arrhythmias found in each variable experimental population. Figure 2 directly correlates with Figure 1, showing that at approximately eight hours after the experimental treatment began that 50 μM had maximized the arrhythmic phenotypical response. This response at 50 μM was significantly higher (p<.05) than the 30 μm and 10 μM concentrations. The significance extrapolated from this experiment allowed for the identification of the most effective dose for inducing cardiac arrhythmias.
Figure 2: Depicted is the percentage of zebrafish population that demonstrated the arrhythmic phenotype as a result of the population’s exposure to each specific nNOSI concentration. Note that 100% of the fish exposed to the 50 μM nNOSI solution were arrhythmic, while the percentages in the subsequent lower doses of 30 μM and 10 μM are greatly reduced.
Figure 3 demonstrates that the AI dosage of 50 μM mimics that of the nNOSI response of inducing 100% of arrhythmias in the experimental populations. This data positively reinforces past experiments in our lab which first proposed the E2, NO regulation link.
Figure 3: Depicted is the percentage of embryonic zebrafish that displayed the arrhythmic phenotype by treatment in either 50 μM of nNOSI or an AI treatment. Note that 50 μM of AI mimics the results of nNOS inhibition by inducing 100% arrhythmias in the treatment population eight hours post treatment time.
Visual comparison of dantrolene accelerating cardiac recovery from arrhythmias
The data shown by Figure 4 compares the visual results of the dantrolene vs ERS recovery washout. The data clearly indicates that arrhythmic zebrafish placed in dantrolene were able to recover at a faster rate when compared to arrhythmic zebrafish placed in the normal aqueous ERS washout environment. It can be observed that the largest increase in recovery periods for the dantrolene rescue group occurred approximately between eleven and twenty four minutes. Likewise, the comparable ERS recovery group saw the largest increase in recovery rate later, between roughly twenty four minutes and half an hour. These visual results suggested that dantrolene was representative of a treatment paradigm that not only works faster than a general washout, but also more effectively. Regardless of the treatment solution, all experimentally arrhythmic zebrafish made a full recovery to a homeostatic range within sixty minutes.
Figure 4: Depicted is the comparison between the ERS washout and dantrolene rescue over a period of sixty minutes. Note that dantrolene treatment accelerates recovery by roughly thirteen minute compared to the ERS washout. The error bars represent the standard error.
Dantrolene Treatment Rescues Arrhythmic Zebrafish by Preventing Ca+2 Leakage
Figures 5A, 5B, 5C are the images captured from the microscope in various stages of the calcium probe experiment. Figure 5A shows a control fish placed in the binding probe with minimal fluorescence which indicates an absence of overt calcium leaks. This image is representative of a normal embryonic zebrafish with a properly function CV system and subsequent circulation loops.
Figure 5A: Control Fish under Texas Red Filter, 5mL of Ca+2 probe, +40% brightness.
Figure 5B: AI treated Fish under Texas Red Filter, 5mL of Ca+2 probe, +40% brightness.
Figure 5C: Same AI treated Fish as in 5B, after undergoing the dantrolene treatment, analyzed under Texas Red Filter, 5mLof Ca+2 probe, +40% brightness.
Figure 5B, shows one experimental fish, which was placed in one of the aforementioned inhibitory solutions. As one can observe, there are high levels of fluorescence which represents abnormal levels of calcium in the CVS’s SR. This fish was arrhythmic, with interrupted systemic circulation at the distal and caudal end of its body.
Figure 5C is the same AI treated fish from Figure 5B, yet this fish was placed a dantrolene treatment for thirty minutes. The fluorescence has dissipated meaning that dantrolene was successful in closing the abnormally open ryanodine channels which allowed for the influx of calcium into the CVS’s SR. The dissipation of calcium resulted in the restoration of circulation and CVS function eliminating the accompanying arrhythmias.
The clinical significance of this experimental directly confirms that the mechanism of dantrolene allows it to function as a possible treatment paradigm for cardiac abnormalities resulting from abnormally open ryanodine channels.
ERS, ODQ, DTT treatments resulting in arrhythmic phenotype identifies the dysregulated NO pathway
The final data set is displayed in Figure 6 and directly displays the correlation between the inhibition of the S-nitrosylation pathway and presence of arrhythmias in the experimental treatment groups. As one can observe the reducing agent DTT was able to induce one hundred percent of the zebrafish in the treatment group with arrhythmias, while ODQ a sGC inhibitor resulted in a negligible difference. It is worth noting that DTT elicited its effects on the CVS of all zebrafish regardless of dosage. The results below suggest the S-nitrosylation pathway as the main affecter of cardiovascular function in terms of the E2 NO regulatory relationship. To disrupt this pathway by any means, will negatively impact the concentrations of E2 and NO, thus resulting in the observed and recorded cardiac anomalies.
Figure 6: Depicted is the percentage of the population of embryonic zebrafish in ODQ/DTT treatments compared to ERS controls. The DTT yielded the highest percentage of arrhythmias over the shortest period of time, while none were observed in the ODQ or control treatments, ultimately suggesting the impairment of the S-nitrosylation pathway. The error bars represent standard error.

Discussion

The data from the initial nNOSI dose response experiment is clearly indicative of the fact that 50μM is the most effective dosage for maximizing phenotypical response, while simultaneously minimizing unnecessary fish casualties. The major change thus comes in the form of shifting the treatment window from 2-4dpf to 5-6dpf. The logic behind this directly correlates with the development of the vagus cranial nerve. Functionally speaking the vagus nerve supplies nerve bundles directly to the heart and lungs, thus representing a more completely developed neuro-cardiac connection in the zebrafish. This more complete connection is representative of the fact that the zebrafish’s autonomic nervous system has begun development beginning at 4dpf [7].
The successful conduction of the nNOSI and AI experiments represent perhaps the most revealing pathophysiological pieces of evidence gathered throughout this entire experiment. The data from these experiments directly reinforce our labs previous understanding of the relationship between NO and E2. We had previously understood that E2 is synthesized upstream from NO and thus proposed the hypothesis that the inhibition of one molecule would allosterically impact the production of the other [8]. Thus, this hypothesis was proven to be correct through AI and nNOSI exhibiting the same disruptive arrhythmic response when fish were placed in 50 μM of each solution for a minimal of 8 hours. Tying this data into the AHA study of 2013, which proposed that premenopausal women are more resistant to heart anomalies due to their increased E2 production, one can see that this hypothesis is consistent with these findings [9]. As a result of our experimental results, both past and present, it is a logical assumption to propose that females who have undergone menopause, resulting in decreased E2 production, also suffer from a NO deficiency as well. The subsequent decrease in NO may result in the dysregulation of the S-nitrosylation pathway, which as previous literature has suggested is responsibility for decreasing the affinity of FK binding protein 12.6 to ryanodine channels [6,4,7]. All of this data supports and validates the fact that E2 and NO are critical in the regulation of a homeostatic cardiovascular system, and suggest that the AHA further studies into the NO-E2 relationship.
The portion of the hypothesis that selected the S-nitrosylation pathway as the main effector received support through the use of the DTT experiment. DTT was shown to terminate 100% of the experimental population regardless of dosage, 10 μM-100 μM dosages were tested. The variable from this experiment was the length of time before cardiac anomalies would set in, ultimately resulting in death. The data gathered from this experiment is suggestive at best due to
DTT’s broad effective range. DTT acts as a common reducing agent, breaking disulfide linked bonds ultimately resulting in the denaturing of proteins. We were unable to restrict to the broad spectrum of DTT’s effects exclusively down to the just the S-nitrosylation pathway, ultimately rendering this experiments results suggestive at best. Multiple attempts to find a molecule that inhibits or reverses S-nitrosylation exclusively proved to be unsuccessful. The molecule in question was thioredoxin reductase, which past literature had shown to be effective at reversing S-nitrosylation. Unfortunately, a zebrafish analog was unable to be located, requiring the use of Rabbit-Tr2 which produced minimal results at best. Due to time constrains it was best decided to abandon thioredoxin altogether.
The initial dantrolene results show that there are no deleterious effects on a healthy zebrafish’s CV system as a result of the aforementioned dantrolene treatments. Additionally, the dantrolene results also visually confirm that a rescue from the arrhythmic phenotype is possible through chemical means. Initially we were unable to directly observe if dantrolene’s mechanism of action, the closing ryanodine channels, was actually causing the abnormal SR calcium to dissipate, of if we were simply removing the specimen from the experimental conditions by placing it in dantrolene solution. Henceforth the fluorescent calcium tagging experiment became the next logical step. The results from this experiment are incredibly exciting as no prior literature had even suggested that dantrolene could be used to treat NO/E2 deprived zebrafish. The photographs taken from the microscope directly indicate that by placing an arrhythmic fish, which had experienced aromatase or nNO inhibition, in 10 μM of dantrolene for a baseline of 30 minutes, will allow for recovery to occur. However, these photographs simultaneously represent the fact that dantrolene will close the abnormally open ryanodine channels that have resulted from NO/E2 inhibition, and thus ultimately restore normal, homeostatic calcium influxes and effluxes into and out of the sarcoplasmic reticulum. This functional restoration of ryanodine channels counteracts the inhibition of NO/E2 and corrected the arrhythmic nature of the zebrafish’s cardiovascular region, while also restoring normal blood circulation through the caudal region. These results broaden our understanding of the E2/NO link and potentially represent a class of treatment options that are currently absent from medicine all together.

Conclusions

In summation, the main conclusions to be taken away from this experiment all revolve around the NO-E2 synthesis relationship, one first suggested by our lab. The best dosage to inhibit nNOS and aromatase is 50 μM of the appropriate inhibitor. Maximum results will be obtained when treating 5-6 days post fertilization, for a minimal of at least 8 hours. Treating any earlier than the recommended treatment time will result in data that is inconsistent and unable to be reproduced. Subsequently, it has been suggested that inhibition of both E2/NO will dysregulate the post-transcriptional modulatory pathway of S-Nitrosylation ultimately opening abnormal levels of calcium ryanodine channels. The phenotypical outcome of these channels remaining open will be cardiac arrhythmias resulting from high concentrations of calcium flowing through the open ryanodine channels into the sarcoplasmic reticulum of cardiomyocytes. The most effective chemical treatment for this induced condition in embryonic zebrafish is exposure to dantrolene, a reagent shown to close ryanodine channels. Such an exposure will result in the elimination the extra calcium in the sarcoplasmic reticulum, ultimately resulting in the restoration of normal cardiac function to include correction of circulation anomalies.

Acknowledgements

I would like to thank first and foremost Col. James Turner ’65 for welcoming me into his lab as an inexperienced yet curious sophomore, he gave me the opportunity to develop myself as a scientist and learn more in three years and two summers than I ever have before. Col. Turner also graciously provided me with not only resources, but provided guidance throughout my thesis, without his assistance; I doubt much would have been accomplished during my cadetship. Always willing to provide gas money for trips to the Roanoke lab, or quick with an offer for dinner, Col. Turner’s kindness and generosity truly made Lexington and my time at
VMI during the summer and academic year feel like home. I would like to thank Ms. Julie Lozier for all her assistance in the lab. Julie was always ready to share her wealth of knowledge and experience and for that I am forever grateful, her guidance was instrumental to my success. I would like to thank Col. Wade Bell for all the knowledge he injected into lab discussions, as the source of the calcium probe experiments, his support was critical in the overall development of my thesis and hypothesis. Col. Bell’s timely assistance and execution in data collection allowed me to maximize the content of my thesis. Last but not least, I would like to thank Cadet
Vania Murcia ’17 for all of her assistance in zebrafish husbandry and experiments, she was a huge part of my success in undergraduate research, and always made my days in the lab pass a little bit faster.

Help Received Statement

Over the past three years of conducting research that led to the development of this thesis I received a large amount of aid and support from multiple individuals and sources:
Col. James Turner ’65 who aided in the forming of experiments, data collection, the proof reading of all drafts and presentations, all in the fulfillment of his role as my mentor. Additionally, Col. Turner’s suggestions and edits were at times directly inserted into this paper, verbatim.
Col. Wade Bel who aided in the forming and conduction of experiments
Julie Lozier for her aid in conducting experiments
My fellow VMI cadets, both past and present, for aiding in the conduction of experiments, providing me with formatting templates for presentations and submitted documents such as thesis updates.
All literature cited in my Bibliography, some citations were composed utilizing Bibme.org.

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