Esketamine induces embryonic and cardiac malformation through regulating the nkx2.5 and gata4 in zebrafish

doi:10.21203/rs.3.rs-4971063/v1

Download PDF

Article

Esketamine induces embryonic and cardiac malformation through regulating the nkx2.5 and gata4 in zebrafish

https://doi.org/10.21203/rs.3.rs-4971063/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Esketamine (EK) has been widely used in the treatment of depression, but the effects of EK prenatal treatment on embryonic heart development have been rarely reported. This study assesses the effects of varying concentrations of EK on embryonic development and cardiogenesis to determine the teratogenic concentration in the zebrafish model, centering on the interaction with the genes nkx2.5 and gata4 to elucidate the mechanism cardiac morphogenesis. Zebrafish embryos were classified into six distinct groups and exposed to either a vehicle or EK to ascertain the half lethal concentration (LC50) at 48 hours post-fertilization (hpf) and 72hpf through enumerating statistics on mortality rates. Embryonic and heart morphologies were assessed utilizing live embryo imaging techniques and stereo microscopy. Nkx2.5 and gata4 were identified via whole-mount in situ hybridization. Exposure to EK results in concentration- and time- dependent significant teratogenic effects on zebrafish embryos. The 48h- and 72h-LC50 of EK for zebrafish embryos were 0.31 (95% CI, 0.22, 0.38) mg·mL-1 and 0.17 (95% CI，0.11, 0.24) mg·mL-1, respectively. A significant reduction in heart rates and body length were observed and the distance between the sinus venosus and bulbar artery (SV-BA) was also found expanded, the pericardial edema area showed significant swelling, and the body axis curvature was more pronounced in the EK exposure groups. WISH analysis showed nkx2.5 staining intensity significantly decreased, while gata4 expression notably increased in direct proportion to EK concentration increase. Our findings suggest that exposure of zebrafish embryos to EK leads to embryonic and cardiac malformations, primarily due to the down-regulation of nkx2.5 and the over-expression of gata4. The insights advocate to maintain equilibrium and a compensatory mechanism in the spatiotemporal regulation of gene expression is of paramount importance.

Biological sciences/Developmental biology

Health sciences/Biomarkers

Health sciences/Cardiology

Health sciences/Medical research

Health sciences/Diseases/Reproductive disorders

Health sciences/Diseases/Cardiovascular diseases

Health sciences/Diseases/Cardiovascular diseases/Congenital heart defects

Health sciences/Diseases

Health sciences/Diseases/Psychiatric disorders

Health sciences/Diseases/Psychiatric disorders/Depression

Biological sciences/Drug discovery

Biological sciences/Drug discovery/Drug safety

Perinatal depression (PND) is a major depressive episode and public health problem during pregnancy¹, which has received much attention in recent years due to its severe social dysfunction. Generally, antidepressant drugs are considered as a common treat for PND in clinical practice. However, many of the antidepressants may cause fetus developmental malformation through the placental barrier or lactate so that a significant proportion of PND women have terminated the antidepressive therapy with the consideration of fetal dysplasia², congenital heart disease (CHD), which is one of serious adverse consequences.

Being the S-enantiomer of ketamine, EK is an emerging antidepressant drug (Fig .1A), which may rapidly relieve the symptoms of major depressive disorder (MDD) through inhibiting the NMDA receptor. In recent years, EK has received much attention in clinical anesthesia due to its twice as potent as the racemic ketamine, less reported adverse effects, and rapid acting antidepressant (RAAD) effect ³^,⁴. The safety of EK used in clinical antidepressant treatment for PND has not been evaluated and explored thoroughly. Recently, Wang reported on BMJ that a single low dose of EK after delivery reduced major depressive episodes by about three-quarters at 42 days postpartum⁵. However, whether the feasibility of EK exposure to PND women before delivery is still mysterious.

In order to address this issue, the present study systematically examines the toxicity of EK exposure on the development of embryos and cardiac function, utilizing zebrafish embryos as a model organism. The zebrafish is a convenient vertebrate model organism with a high degree of homology with human genes and organs⁶. Compared with common model mammals, like rats and mice, zebrafish benefits from the low cost, small size and external development. Therefore, zebrafish embryo has become an excellent model for development biology, basic medicine, neurological and toxicology research during the last decades⁷^,⁸. The heart, which has a simplified structure consisting of only one atrium and one ventricle⁹, is the first organ to form and function during zebrafish embryogenesis. The zebrafish heart, despite its anatomical divergence from the human heart, possesses analogous characteristics that encompass atria, ventricles, heart valves, and a conduction system¹⁰^,¹¹. The pivotal phase of cardiac development in human embryos spans from the second to the seventh week of gestation, aligning with the timeframe of approximately 24 hours to 2 days for zebrafish heart development, as documented in Lohr’ study¹². Therefore, we have conducted a methodical examination of the toxicity of EK exposure on both embryonic and cardiac development, utilizing the zebrafish model as a means to broaden the scope of EK prescription.

Embryonic development represents a highly orchestrated and finely regulated spatiotemporal dynamic process. In the course of development, embryos are required to sustain this dynamic process while simultaneously safeguarding it from disruption by external, non-endogenous factors. Exploring and avoiding the toxic effects of EK exposure so that provide scientific theoretical support for PND women is meaningful treatment protocols, which also be great significance to improve the health of the population.

In consideration of the aforementioned points, the objective of the current study is to clearly delineate the influences of EK on the zebrafish embryonic morphology as well as the functionality of the heart and preliminary explore the underlying mechanism by observing the expression patterns of nkx2.5 and gata4, which are cardiac development-sensitive genes, during the process of cardiac dysplasia.

Ethical statement

All experimental procedures were approved by the Institutional Animal Care and Use Committee of the South China University of Technology (Guangzhou, Guangdong, P.R China, approval number SCUT-2022-096) and were carried out in accordance with relevant guidelines and regulations. All the methods are performed in accordance with ARRIVE guidelines.

Zebrafish husbandry and embryo collection

The used zebrafish strains in this study are all from the Chinese Academy of Sciences institute of aquatic organisms. Four months old, transgenic and wild type zebrafish embryos were chosen. Healthy adult zebrafish were regularly fed brine shrimp twice a day. Water temperature maintained at 28.5±1℃. The salinity of the water was regulated by NaCl and maintained between 450 and 500μs/cm. A 14-h light:10-h dark cycle was maintained to mimic natural day-night patterns. The day before the experiment, adult male and female zebrafish were placed in the breeding box at a 1:1 ratio and kept overnight under dark conditions. The next morning at 6:30 am, male and female zebrafish were stimulated to mate and lay eggs. Zebrafish eggs were collected for subsequent experiments.

Esketamine exposure and experimental design

In order to study the EK on zebrafish embryonic development and the influence of cardiac toxicity, the collected fertilized embryos at 24h post-fertilization (hpf) were incubated in 6-well plate 24 hours with 50 embryos each well, six groups randomly incubated in a range of EK concentrations (0, 0.05, 0.1, 0.2, 0.4 and 0.8 mg·mL^-1) of EK (diluted with Hof water) through 48 hours, exposure solution was changed every 24 hours, each group was set at least three repeated experiments. Using the stereo microscope (Nikon, SMZ18, Japan) record once every 24 hours hatching rate and mortality. Esketamine and Hof water were used to dissolve the chemical and make stock solutions for this study. On the first day of the experiment, the egg shells were removed with a demembrane enzyme (1:10 configuration) and embryos that developed normally during the same period were selected. Holt buffer solution was used as negative control group. Zebrafish embryos were treated with the above protocol to determine its toxic effects. Through counting the mortality, heart rate, body length and hatchability in designed timepoints until 72hpf, the LC₅₀values were determined by Prism 8.0 software. Zebrafish embryos were placed in an incubator at 29℃ and cultured until the target time.

Determination of the optimal concentration exposure

Based on the mortality of zebrafish embryos that resulted from a series of different concentrations of EK exposure, we have calculated the half lethal concentration (LC₅₀) of EK in zebrafish embryo model at 48hpf and 72hpf. 0.05, 0.1 and 0.2 mg·mL^-1 were selected as the low, median and maximum (optimal) concentrations to treat zebrafish larvae for further revealing the teratogenic effects on cardiogenesis and embryonic development. Solution in each group were changed every 24 hours. The larvae were collected in the -80℃ condition for subsequent experiments. Combined with the mortality, hatching, and malformation phenotypes under a range of EK concentrations, we selected some optimal concentrations to further prove the mechanism of EK developmental cardiotoxicity through WISH tests.

Live embryos imaging and morphological analyses

Selective concentrations of EK exposure was conducted until 72 hpf and solutions were refreshed per 24h. After dehydration, the embryonic development and morphological changes of zebrafish were recorded under stereomicroscope. All survival zebrafish larvae were anesthetized with 0.01% tricaine and mounted in 1% low-melting agarose with 0.01% tricaine, and their phenotypes were observed and measured by stereomicroscope equipped with a digital camera (Olympus ZX) at 72hpf. Using ImageJ software to measure the body length (μm) , SV-BA distance (µm), and pericardial area (μm²) of zebrafish in each group. The larvae heart rate at stage 48hpf and 72hpf were counted for 1min after anesthesia. The assessment of cardiac morphology and function of zebrafish embryonic heart according to the treatment protocol published by Hoage and Glickman¹³^,¹⁴. 20 fish were taken from each group, and each experiment was repeated at least three times.

Whole-mount in situ hybridization analysis of CHD susceptive genes

Congenital heart disease susceptive genes nkx2.5 and gata4 were selected as the indexes for the cardiotoxicity of esketamine¹⁵. Whole-mount in situ hybridization (WISH) using digoxigenin-labeled antisense RNA probes for nkx2.5, gata4 were performed based on previous protocols¹⁶. Combined with the above mortality, hatching, and malformation phenotypes, the optimum concentrations were chosen to further prove the mechanism of EK developmental cardiotoxicity through WISH tests.

Statsitical Analyses

All experiments were conducted in triplicate to ensure the reliability and validity of the experimental results. Statistically differences were analyzed by one way ANOVA and the Dunnett’s multiple comparison (mean ± SEM). All statistical analysis using GraphPad Prism software (version 8.0) and SPSS software (version 25.0). P < 0.05 was considered statistically significant.

Determination of the optimal concentration

Esketamine exposure was started at 24hpf and ended at 72hpf, we observed the mortality at multiple designed timepoints to determine its toxicity effect on larvaes. Consistent with control group, zebrafish larvae survival rate was 100% at the concentration of both 0.05 and 0.1mg·mL^-1 following a long duration from 24 to 72 hpf to EK exposure. In 0.2,0.4 and 0.8mg·mL^-1 groups, all the survival rate decreased slowly followed the EK exposure duration expanded including 3, 6, 12, 24 and 48 hpe (hours-post-exposure). However, in 0.2 and 0.4 mg·mL^-1 groups, when EK exposure time reached to 48h, the survival rate decreased to 37% and 0%, respectively. In 0.8mg·mL^-1 group, all the zebrafish embryos died at the beginning of the second phase of the study (24hpe). Therefore, 0.4 and 0.8mg·mL^-1 are not suitable for further studying about the toxicity effect on zebrafish embryonic development due to the high mortality. The 24hpe-LC₅₀ value of EK was 0.31 mg·mL^-1, with a 95% confidence interval between 0.22 and 0.38 mg·mL^-1. The 48hpe-LC₅₀ value of EK was 0.17mg·mL^-1, with a 95% confidence interval between 0.11 and 0.24 mg·mL^-1.

The number of dead zebrafish embryos were calculated at the designed timepoints in each group. Based on the toxicity grading standards, EK is considered the median toxic to zebrafish embryos, and 0.2mg·mL^-1 concentration was considered as a low mortality and high teratogenic concentration for zebrafish embryos (optimum concentration), which is highly consistent with the appropriate concentration of ketamine in zebrafish toxicity test¹⁷. A range of three concentrations: 0.05, 0.1 and 0.2 mg·mL^-1 were chosen to determine the teratogenicity of EK on zebrafish embryos and the cardiac development defects. Zebrafish embryos were exposed to EK 24 h post-fertilization, the mortality, hatching rate and malformation rate showed concentration- and time-effect dependent manner. The above results are based on the pharmacodynamic ratio of EK and ketamine. Each treatment was repeated three times for analysis.

Effects of esketamine on embryonic and cardiac morphology in zebrafish

Assessment of heart phenotype via stereomicroscope (Fig .2). The pericardial area (μm²) under different concentrations EK exposure and control group. At 72hpf, mild pericardial edema was observed in the 0.05mg·mL^-1 group, and the degree of pericardial edema became more severe with the increase of concentration. The phenotype of pericardial edema was in a concentration-dependent manner. Different concentrations of EK exposure results in expanded SV-BA distance (μm), At 72hpf, mild lengthened SV-BA distance was observed in the 0.1mg·mL^-1 group, and the SV-BA distance became longer with the increase of concentration. The phenotype of SV-BA distance was also in concentration-dependent manner.

Phenotypes of zebrafish larvae exposed to different concentrations of EK

Exposure to esketamine induced dysplasia within the chevron-shaped somites, where the angle in the 0.2mg·mL^-1 group was significantly enlarged.The impact of varying concentrations of EK on the body axis length across different groups was examined, a gradual reduction in body axis length was observed, as the concentration increased. These morphogenesis presents a pronounced time and concentration-dependent manner.

The expression of nkx2.5 and gata4 by WISH analysis

As can be seen, combined with the above mortality, hatching, and malformation phenotypes, 0.05, 0.1, and 0.2 mg·mL^-1were selected for their low mortality and high malformation rates to further investigate the mechanism of EK’s developmental cardiotoxicity via WISH. Exposure to EK markedly decreased the expression of the CHD susceptible gene nkx2.5 (Fig .4A), gata4 otherwise was opposite (Fig .4B). As the concentration increases, zebrafish exhibit a median cardiac morphology and gradually develop cardiac looping defects, as demonstrated in the Zoom images (P ＜ 0.05). Figure 4A&B presents the outcomes derived from the application of WISH.

Previous studies have conclusively demonstrated the neurotoxic effects of EK exposure on pregnant rats and their offspring. Specifically, Zhang¹⁸has reported that EK exposure results in memory impairment in the offspring rats. However, it is noteworthy that only a limited number of studies have been conducted to specifically investigate the potentially fatal consequences and cardiac teratogenic effects resulting from exposure to EK on zebrafish embryos. In the current research endeavor, we have conducted a thorough investigation into the toxicological implications of EK exposure on zebrafish embryos, meticulously observing alterations in their developmental morphology and functionality. Our findings demonstrated the detrimental and cardiac teratogenic impacts of EK on zebrafish embryos and cardiogenesis (Fig .2&3), with these effects manifesting in a concentration-dependent and time-dependent fashion. The outcomes obtained serve as corroboration for the presupposition that both the morphology and functionality of the heart were subject to modulation by a specific range of EK concentrations. The concentrations we have identified as optimal, through the 48 and 72 hpf-LC₅₀ assessments, demonstrate high teratogenic potential and low mortality rates (Fig. 1B&D). These concentrations have notably altered the gross morphological development of the organisms, specifically impacting the pericardium (Fig. 2A&C), SV-BA distance (Fig. 2B&C), body length (Fig. 3A&B), and Chevron-shaped angles of zebrafish (Fig. 3C&D). The exposure to EK also led to a reduction in heart rates (Fig. 1C), and a positive correlation between these two factors was observed, which is in line with the research conducted by Yuan¹⁷.

As depicted in Fig .1B, the exposure to EK exhibits a concentration-dependent effect, wherein a discernible increase in concentration is observed when the level surpasses 0.1 mg·mL^-1. At the same time point, the statistical differences observed among the three groups were found to be significant. When the exposure time reached 24 hours, all embryos in the 0.8 mg·mL^-1 group succumbed to death, mirroring the fate of embryos in the 0.4 mg·mL^-1 group that perished after 48 hours of exposure. In order to proceed with our research, we have excluded the aforementioned two concentration groups, specifically 0.4 and 0.8mg·mL^-1, from further consideration.

In the control and 0.05 mg·mL^-1 groups, no instances of malformation were observed. At the identical timepoint, the incidence of deformity in the 0.2 mg·mL^-1group was notably elevated compared to the 0.1 mg·mL^-1 group, exhibiting a clear time-dependent trend. In this research endeavor, the teratogenic concentration of EK exposure that was selected aligns precisely with the ketamine concentrations reported by Guo, based on the similarity in their pharmacokinetic profiles¹⁹. The 72-hLC₅₀ of EK was approximately 0.17 mg·mL^-1, as determined through a rigorous analysis of survival and hatching rates (Fig .1B). Consequently, for the teratogenic analysis of zebrafish embryos, three specific concentrations of 0.05, 0.1, and 0.2 mg·mL^-1 were carefully selected.

To meticulously investigate the developmental toxicity of drugs on zebrafish embryos, we have selected the hatching rate, heart rate, pericardial area, body length, and SV-BA distance as the key characterized parameters for analysis. Our research concentrated on the initial stages of cardiac development in zebrafish, which serves as a model for the early fetal development process in humans. Notably, the aforementioned parameters were most optimally observed during this particular phase. Except for the two elevated concentrations of EK, specifically 0.4 mg·mL^-1 and 0.8 mg·mL^-1, which significantly induced pericardial edema and mortality at extremely high levels, we also discerned a consistent trend with Luís M Félix's reports²⁰, observing a marked slowing of heart rate as the concentration of EK increased within a particular range. Zebrafish embryos exposed to that range of EK concentrations at the early stages of cardiac development showed enlarged pericardial area and longer SV-BA distance, all of these results were consistent with previous studies²¹^{, 2}². All of these occurrences are attributable to the harmful consequences of exposure to EK. Based on the outcomes obtained, concentrations less than 0.2 mg·mL^-1 were deemed as the optimal selection for the investigation and analysis of teratological effects, specifically focusing on the examination of two pivotal genes related to CHD formation utilizing the WISH technique (Fig. 4A&B).

Given the prevalence of cardiac phenotype in morphant embryos, we employed nkx2.5 and gata4 markers to ascertain the presence of cardiac development disturbances, which have been validated to play a crucial role in the development of congenital heart defects (CHD) during fetal development. Numerous studies²³^,²⁴ have conclusively shown that nkx2.5 is localized within heart precursors, serving a crucial regulatory role in cardiac morphogenesis. This process is intimately tied to the migration of cardiac precursors and the subsequent formation of the heart tube, as well as being a contributing factor in the development of CHD. The mutation or abnormal expression of nkx2.5 leads to impairments in cardiac development, encompassing morphological abnormalities in both the ventricle and atrium. These abnormalities manifest as cardiac looping disorders, which are characterized by median cardiac morphology, and can ultimately culminate in CHD. The insights derived from our extensive evaluation of cardiac dysplasia are markedly apparent, likely attributable to the diminished expression of nkx2.5 subsequent to EK exposure. Martin’s research further reveals that during embryonic development, the exposure to external stimuli, notably drugs, has been observed to elicit a response within 24 hours, specifically by affecting or diminishing the expression of nkx2.5²⁵. In our research, the influence of EK exposure on nkx2.5 was found to be in alignment with this conclusion. Harrington²⁶ has discovered that the nkx2.5 loss-of-function model demonstrates an elevated heart rate, exhibiting a contrasting trend in heart rate performance in comparison to our findings. This disparity may stem from the EK cardiotoxicity, which holds a pivotal position in explaining our contradictory results.

Gata4 plays a pivotal role in heart development, and the occurrence of mutations within the gata4 gene can potentially result in abnormal cardiac development, leading to CHD²⁷. The expression of gata4 is gradually increased throughout the whole heart development process under normal developmental conditions²⁸. Vikas Gupta and his colleages²⁹ have observed that the zebrafish heart's responses to various stressors are interconnected through a shared mechanism, which involves the activation of gata4. The gata4-mediated stress response pathway is consistently employed during the growth process of zebrafish in order to confront external stimuli. This study conclusively demonstrated that as the concentration of EK increased, there was a corresponding augmentation in the expression of gata4. Furthermore, the regulation of gata4 holds a pivotal significance in the process of ventricle formation during both the juvenile stage of zebrafish morphogenesis and the heart regeneration in adult zebrafish³⁰. An additional study³¹ has likewise demonstrated a correlation between ASD and gata4 mutation. In this comprehensive investigation, we observed a marked augmentation in gata4 expression as the cardiac malformation in zebrafish became increasingly pronounced. This finding underscores a potentially significant correlation between the two phenomena.

Upon observation of the aforementioned phenomenon, it is intriguing to note that as the concentration of EK rises, the expression of nkx2.5 undergoes downregulation, whereas the gata4 gene displays a contrasting tendency towards upregulation. Gata4 has the ability to interact with the C-terminal auto-inhibitory region, thereby releasing the active domain of nkx2.5. This process could potentially serve as a compensatory mechanism in response to the downregulation of nkx2.5 expression induced by EK exposure³². Tong’s research³³ demonstrated that nkx2.5 engages in an interaction with gata4, and any imbalance in this interaction can lead to the development of severe cardiac abnormalities. Nkx2.5 and gata4 possess the ability to enable and interact with each other within cardiomyocytes. It is noteworthy that mutations in gata4 may give rise to defective interactions with nkx2.5, ultimately resulting in the development of CHD³⁴. Another investigation has revealed that the nkx2.5 mutation could potentially be linked to Tetralogy of Fallot³⁰, a highly prevalent congenital heart defect, which may also be influenced by gata4 mutations³⁵. Gata4 and nkx2.5 are key synergistic regulators of atrial natriuretic factor and B-type natriuretic peptide gene, which play critical role in repairing and remodelling cardiogenesis³⁶^,³⁷.

Carl O. Brown has disclosed that the incorporation of gata transcription factors can potentially trigger the early expression of nkx2.5, thereby playing a crucial role in the development of cardiac progenitors³⁰. Daniel Durocher and his colleagues had reported that the nkx2.5 and gata4 provide cooperative crosstalk, which may present a paradigm for transcription factor interaction during cardiogenesis³⁸. Nkx2.5 serves as a pivotal gata4 cofactor, both of which are indispensable for the development of the heart, and their mutations are intricately linked to congenital heart disease (CHD). Wang and colleages³⁹speculated the nkx2.5 lies in the upstream of gata4, and also prior expression to gata4 in the cardiogenic area at embryonic period. The functional cooperation of gata4 and nkx2.5 plays crucial role in the cardiogenetic process⁴⁰. Nkx2.5, as a cofactor of gata4, has been shown to be able to recruit gata4 to cardiac gene promoters⁴¹, but our findings are the gata4 overexpression is unidentical to that of nkx2.5. Therefore, to ascertain the detailed functional interaction of nkx2.5 and gata4 still need further exploration. Based on these discoveries, we speculate that the over-expression of gata4 might be a result of the nkx2.5 down-regulation, there may be a compensatory mechanism between nkx2.5 and gata4 to correct pathological conditions during cardiac development.

We furthermore exhibited that as the concentration augmented, zebrafish embryos gradually manifested a median cardiac morphology phenotype. The initiation of cardiac looping and the process of tube formation may potentially be impeded by exposure to EK, particularly in instances where an imbalance arises between the expression levels of nkx2.5 and gata4 (Fig. 4 Zoom pictures).

In Conclusion, our research confirms that 48h and 72h-LC₅₀ of EK and its teratogenic effects on embryos and cardiac development in time- and concentration-dependent manner. The selective concentration of EK exposure results in compromised cardiac morphology and function in zebrafish embryos, potentially due to an imbalance in the expression of cardiogenesis-susceptive genes, such as gata4 and nkx2.5, along with disruptions in the network. Our findings highlight the risks of trans-placental and latex exposure to EK for fetuses and pregnant women, which indicates that prudence should be exercised in employing EK to treat antepartum depression.

Acknowledgments We extend our sincere gratitude to the esteemed members of the Qiang Wang laboratory, including Yiming Hou and Yuting Zhu, South China University of Technology, Guangzhou, Guangdong, P.R. China, 510006.

Funding The funding for this work has been provided by the Guangzhou Municipal Science and Technology Project of China, specifically allocated to J.T.&S.L. under the grant number 2024A03J0195&2024A04J5069. Additionally, support has also been received from the Guangzhou Medical University Students Innovation Ability Improvement Plan Project, which has granted to J.T. with the project code 02-408-2304-02122XM. University scientific research project of Guangzhou Education Bureau, which has granted to J.T. with the project code 2024312101.

Conflicts of interest The authors have declared that no competing interests exist.

Author contributions J.T., S.L and R.Z. collaboratively designed and executed the majority of the experiments, with invaluable assistance from Q.O. contributed to the execution of the zebrafish behavioral experiments. J.T. and S.L. were responsible for devising the experimental protocols, interpreting the data obtained, and drafting the manuscript. Additionally, R.Z. and T.J. dissected the relevant tissues and conducted the WISH procedure. Throughout the process, H.H. offered their insights and provided crucial guidance in discussing the results.

Data availability All of the data presented in this study could be obtained from corresponding author.

Dagher, R. K. et al. Perinatal Depression: Challenges and Opportunities. J. Womens Health (Larchmt). 30 (2), 154–159 (2021).
Allbaugh, L. J. et al. Development of a screening and recruitment registry to facilitate perinatal depression research in obstetrics settings in the USA. Int. J. Gynaecol. Obstet. 128 (3), 260–263 (2015).
Gao, S. et al. Process for (S)-Ketamine and (S)-Norketamine via Resolution Combined with Racemization. J. Org. Chem. 85 (13), 8656–8664 (2020).
Daly, E. J. et al. Efficacy of Esketamine Nasal Spray Plus Oral Antidepressant Treatment for Relapse Prevention in Patients With Treatment-Resistant Depression: A Randomized Clinical Trial. JAMA Psychiatry. 76 (9), 893–903 (2019).
Wang, S. et al. Efficacy of a single low dose of esketamine after childbirth for mothers with symptoms of prenatal depression: randomised clinical trial. Bmj. 385, e078218 (2024).
Howe, K. et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496 (7446), 498–503 (2013).
He, J. H. et al. Zebrafish models for assessing developmental and reproductive toxicity. Neurotoxicol Teratol. 42, 35–42 (2014).
Mi, P. et al. Melatonin protects embryonic development and maintains sleep/wake behaviors from the deleterious effects of fluorene-9-bisphenol in zebrafish (Danio rerio). J. Pineal Res. 66 (1), e12530 (2019).
Bakkers, J. Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovasc. Res. 91 (2), 279–288 (2011).
Stainier, D. Y., Lee, R. K. & Fishman, M. C. Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development. 119 (1), 31–40 (1993).
Beis, D. et al. Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development. Development. 132 (18), 4193–4204 (2005).
Lohr, J. L. & Yost, H. J. Vertebrate model systems in the study of early heart development: Xenopus and zebrafish. Am. J. Med. Genet. 97 (4), 248–257 (2000).
Glickman, N. S. & Yelon, D. Cardiac development in zebrafish: coordination of form and function. Semin Cell. Dev. Biol. 13 (6), 507–513 (2002).
Hoage, T., Ding, Y. & Xu, X. Quantifying cardiac functions in embryonic and adult zebrafish. Methods Mol. Biol. 843, 11–20 (2012).
Kinnunen, S. et al. Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5. PLoS One. 10 (12), e0144145 (2015).
Alexander, J., Stainier, D. Y. & Yelon, D. Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning. Dev. Genet. 22 (3), 288–299 (1998).
Yuan, W. et al. Apoptotic mechanism of development inhibition in zebrafish induced by esketamine. Toxicol. Appl. Pharmacol. 482, 116789 (2024).
Zhang, L. M. et al. S-ketamine administration in pregnant mice induces ADHD- and depression-like behaviors in offspring mice. Behav. Brain Res. 433, 113996 (2022).
Matłoka, M. et al. Esketamine inhaled as dry powder: Pharmacokinetic, pharmacodynamic and safety assessment in a preclinical study. Pulm Pharmacol. Ther., 73–74 : p. 102127. (2022).
Félix, L. M., Antunes, L. M. & Coimbra, A. M. Ketamine NMDA receptor-independent toxicity during zebrafish (Danio rerio) embryonic development. Neurotoxicol Teratol. 41, 27–34 (2014).
Liu, J. & Stainier, D. Y. Zebrafish in the study of early cardiac development. Circ. Res. 110 (6), 870–874 (2012).
Liu, X. et al. Protective Effects of Spermidine and Melatonin on Deltamethrin-Induced Cardiotoxicity and Neurotoxicity in Zebrafish. Cardiovasc. Toxicol. 21 (1), 29–41 (2021).
Jay, P. Y. et al. Cardiac conduction and arrhythmia: insights from Nkx2.5 mutations in mouse and humans. Novartis Found. Symp. 250, 276–279 (2003). 227 – 38; discussion 238 – 41.
McElhinney, D. B. et al. NKX2.5 mutations in patients with congenital heart disease. J. Am. Coll. Cardiol. 42 (9), 1650–1655 (2003).
Martin, E. D. et al. Plakoglobin has both structural and signalling roles in zebrafish development. Dev. Biol. 327 (1), 83–96 (2009).
Harrington, J. K. et al. Nkx2.5 is essential to establish normal heart rate variability in the zebrafish embryo. Am. J. Physiol. Regul. Integr. Comp. Physiol. 313 (3), R265–r271 (2017).
Xiang, R. et al. A novel mutation of GATA4 (K319E) is responsible for familial atrial septal defect and pulmonary valve stenosis. Gene. 534 (2), 320–323 (2014).
Afouda, B. A. Towards Understanding the Gene-Specific Roles of GATA Factors in Heart Development: Does GATA4 Lead the Way? Int. J. Mol. Sci., 23(9). (2022).
Gupta, V. et al. An injury-responsive gata4 program shapes the zebrafish cardiac ventricle. Curr. Biol. 23 (13), 1221–1227 (2013).
Brown, C. O. 3 et al. The cardiac determination factor, Nkx2-5, is activated by mutual cofactors GATA-4 and Smad1/4 via a novel upstream enhancer. J. Biol. Chem. 279 (11), 10659–10669 (2004).
Yang, X. Y. et al. Correlation between GATA4 gene polymorphism and congenital heart disease. Int. J. Clin. Exp. Med. 8 (9), 16733–16736 (2015).
Ellesøe, S. G. et al. Familial Atrial Septal Defect and Sudden Cardiac Death: Identification of a Novel NKX2-5 Mutation and a Review of the Literature. Congenit Heart Dis. 11 (3), 283–290 (2016).
Tong, Y. F. Mutations of NKX2.5 and GATA4 genes in the development of congenital heart disease. Gene. 588 (1), 86–94 (2016).
Granados-Riveron, J. T. et al. Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations. Congenit Heart Dis. 7 (2), 151–159 (2012).
Abhinav, P. et al. Somatic GATA4 mutation contributes to tetralogy of Fallot. Exp. Ther. Med. 27 (2), 91 (2024).
Pikkarainen, S. et al. GATA-4 is a nuclear mediator of mechanical stretch-activated hypertrophic program. J. Biol. Chem. 278 (26), 23807–23816 (2003).
Kinnunen, S. M. et al. Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction. Sci. Rep. 8 (1), 4611 (2018).
Durocher, D. et al. The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. Embo j. 16 (18), 5687–5696 (1997).
Wang, Y. et al. Transcription factors Csx/Nkx2.5 and GATA4 distinctly regulate expression of Ca2 + channels in neonatal rat heart. J. Mol. Cell. Cardiol. 42 (6), 1045–1053 (2007).
Shiojima, I. et al. Context-dependent transcriptional cooperation mediated by cardiac transcription factors Csx/Nkx-2.5 and GATA-4. J Biol Chem, 274(12): pp. 8231-9. (1999).
Linhares, V. L. et al. Transcriptional regulation of the murine Connexin40 promoter by cardiac factors Nkx2-5, GATA4 and Tbx5. Cardiovasc Res, 64(3): pp. 402 – 11. (2004).

No competing interests reported.

Download PDF

Editorial decision: Revision requested
18 Sep, 2024
Reviews received at journal
15 Sep, 2024
Reviews received at journal
13 Sep, 2024
Reviews received at journal
13 Sep, 2024
Reviewers agreed at journal
05 Sep, 2024
Reviewers agreed at journal
05 Sep, 2024
Reviewers agreed at journal
05 Sep, 2024
Reviewers invited by journal
05 Sep, 2024
Editor assigned by journal
05 Sep, 2024
Editor invited by journal
05 Sep, 2024
Submission checks completed at journal
04 Sep, 2024
First submitted to journal
24 Aug, 2024

You are reading this latest preprint version

Esketamine induces embryonic and cardiac malformation through regulating the nkx2.5 and gata4 in zebrafish

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Version 1