Early ketamine exposure results in cardiac enlargement and heart dysfunction in Xenopus embryos
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Ketamine is a commonly used clinical anesthetic and a popular recreational drug. However, with the exception of studies about the nervous system, studies about the effect of early ketamine exposure on embryos are rare. Xenopus laevis is a commonly used vertebrate model for assessing teratogenicity. Therefore, we treated Xenopus embryos with ketamine to evaluate its teratogenicity on embryos.
Xenopus embryos were treated with ketamine from stages 8 to 21. Embryonic and cardiac morphology were analyzed using living embryo imaging and whole-mount RNA in situ hybridization (WMISH). Heart function was measured by heart rate and ventricular shortening fraction (VSF). The mRNA expression levels of several heart development-related genes were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The protein expression levels of XMLC2, phospho-histone H3 (pH3) and histone H3 were determined by western blot.
Ketamine caused concentration-dependent increases in mortality and shortening of body length. At a dose of 0.5 mg/ml, ketamine exposure resulted in cardiac enlargement as the primary manifestation of several malformations: gut defects, a curved axis and shortened body length. Cardiac cells underwent increased proliferation. Moreover, the heart rate and ventricular shortening fraction were decreased, findings indicative of heart dysfunction. XMLC2 expression levels were down-regulated at stages 28, 32/33, 35/36 and 46.
Ketamine exposure during early development has teratogenic effects on Xenopus embryos. The heart enlargement and decreased VSF may result from the down-regulation of XMLC2 mRNA and protein levels. These findings provide new insight into the potential fetal defects induced by ketamine exposure during early pregnancy.
KeywordsKetamine Drug exposure XMLC2 Xenopus laevis Heart development
Modified Barth’s Saline
reverse transcription quantitative polymerase chain reaction
ventricular shortening fraction
whole-mount RNA in situ hybridization
Ketamine, a non-competitive inhibitor of N-methyl-D-aspartate (NMDA) receptors, is widely used for sedation, analgesia, and the induction and maintenance of anesthesia in humans, particularly in pediatric patients [1, 2]. Moreover, as a hallucinogenic agent, ketamine is increasingly used for non-medical purposes and is one of the most popular “club drugs” . Epidemiological data indicate that 30 % of ketamine abusers are female  and are often less than 27 years of age , which is childbearing age. Therefore, it is of great significance to study the effect of ketamine exposure during pregnancy on embryonic development. However, clinical data regarding its effect on fetuses are rare. Previous studies using animal models to investigate the effects of ketamine on embryogenesis have focused primarily on neurological issues during either the late embryonic period or the neonatal period [6, 7, 8, 9]. Herein, we examined the impact of ketamine exposure during early embryonic development.
Xenopus is a commonly used vertebrate model for studying embryonic development mechanisms and drug teratogenicity . It produces massive eggs, and the transparency of tadpoles facilitates the detection of internal organization defects. More importantly, Xenopus is similar to humans in terms of organ development, physiology and gene expression . Therefore, we used Xenopus to investigate the teratogenic effects of early ketamine exposure on embryos in this study.
Studies with Xenopus laevis were approved by the local ethics committee (Chongqing Medical University, Chongqing, China). X. laevis were maintained in a recirculating system at 21 ± 1 °C with a 12-h light–dark cycle. Fertilization was stimulated by the administration of human chorionic gonadotropin (HCG).
Fertilized oocytes were washed in 2 % L-Cysteine (pH 8.0) to remove the jelly coat. Xenopus laevis embryos were collected and staged according to Nieuwkoop and Faber .
Experiments were conducted in 0.3× Modified Barth’s Saline (MBS, 88 mM NaCl, 1 mM KCl, 0.7 mM CaCl2, 1 mM MgSO4, 2.5 mM NaHCO3, and 5 mM HEPES, pH 7.8). Working solutions of ketamine (Sigma, St Louis, MO, USA; cat.no.K2753) were dissolved in 0.3 × MBS, and the treatment was administered at various concentrations from stage 8 (before the gastrula stage) to stage 21 (complete neural tube closure). After treatment, the embryos were transferred to 0.3× MBS and cultured in six-well plates at 21 °C; each well contained 10 embryos in 5 ml of 0.3× MBS. The solution and drug were changed every 24 h. Each treatment group was tested 3 separate times.
Live embryo imaging
After inducing anesthesia with tricaine mesylate for 1 min, the embryos at stage 46 were immediately recorded using a stereo fluorescence microscope Stereo Lumar V12 (Zeiss, Germany) and photographed in diastole upon reaching the end diastolic volume using an SMZ 1500 (Nikon, Japan) at 10× magnification for the whole embryos and at 60× magnification for the heart region. Embryo body lengths were measured three times using Image J software (ver 1.47, Bethesda, MD), and the averages were analyzed.
Heart rate and ventricular contractility analysis
After inducing anesthesia for 1 min, the embryos at stage 46 were randomly selected for heart rate counts. Ventricular beats were counted for 1 min. At least three measurements were obtained, and the average was used for statistical analysis.
Cardiac contractions were recorded with Stereo Lumar V12. The lengths of ventricles in diastolic and systolic conditions were measured to calculate the ventricular shortening fraction (VSF) [13, 14]: VSF = (ventricular length at diastole–ventricular length at systole)/ventricular length at diastole.
Whole-mount RNA in situ hybridization (WMISH)
Digoxigenin (DIG)-labeled antisense RNA probes for XNkx2.5, XTnIc and XMLC2 were constructed based on previous research , and WMISH was performed as previously described . Embryos were imaged using a Nikon SMZ 1500 stereomicroscope at 60× magnification for the heart region.
RNA isolation and reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from ten whole embryos or hearts per experimental condition using TRIzol Reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. cDNA synthesis was conducted using a PrimeScript RT Reagent Kit (Takara, Otsu, Shiga, Japan). RT-qPCR was performed in triplicate using 10-fold diluted cDNA and a SYBR Green I kit (Tiangen, Beijing, China) on a CFX96 real-time PCR machine. The following primers were used for RT-qPCR:
XNkx2.5: forward 5′-GGC TAC CAC CTC CAA GAC G-3′, reverse 5′-GTT GGA GTG GGC AGG GTA AG-3′;
XTnIc: forward 5′-TGA ATC CAC TGG GGC TGT TG-3′, reverse 5′-AGA GTA ACG GCC TTC GAA CA-3′;
MHC-alpha: forward 5′-TCA AGG CTG GTT TGT TGG GT-3′, reverse 5′-AAC CAG AAG GGC ATC TCT GC-3′;
XMLC2: forward 5′-GCG CAA TGG TCT TGC TCT TC-3′, reverse 5′-GAG ATC GTG GAG GGC AAA GT-3′;
GAPDH: forward 5′-TAG TTG GCG TGA ACC ATG AG-3′, reverse 5′-GCC AAA GTT GTC GTT GAT GA-3′.
The primers for gata4 and gata6b were used as described .
The PCR cycling conditions were as follows: 1 cycle at 95 °C for 3 min, 39 cycles at 95 °C for 10 s and 60 °C for 30 s. GAPDH mRNA was used as an endogenous control. The relative quantification was calculated using the 2-△△ct method.
Western blot analysis
Protein was extracted from ten whole embryos or 200 embryonic hearts per experimental condition, and western blotting was performed using standard procedures. Monoclonal mouse anti-human MLC2 from Novus (Littleton, CO; cat. no. NBP1-30249), polyclonal rabbit anti-human histone H3 from proteintech (Chicago, IL; cat. no. 17168-1-AP) and polyclonal rabbit anti-rat GAPDH from EnoGene (Nanjing, Jiangsu, China; cat. no.E12-052) were diluted by 1:2000. Polyclonal rabbit anti-human pH3 from Santa Cruz Biotechnology (Dallas, Texas; cat. no.sc-8656-R) was diluted by 1:500. GAPDH was applied as an internal reference. Immunoreactive bands were quantified by densitometry using ImageJ.
SPSS Statistics version 17.0 (Chicago, IL, USA) was used for statistical analysis. Comparisons among different concentration groups were made by one-way ANOVA. Comparisons between two groups were conducted using either unpaired t-tests or chi-square tests for different data. P-values <0.05 were considered statistically significant.
All experiments were repeated at least 3 times.
Ketamine induces embryonic malformations
The effect of ketamine at three concentrations on embryonic death and malformation
17 (9.44 %)
16 (8.89 %)
29b (16.11 %)
52a,c (28.89 %)
4 (2.45 %)
117a (71.34 %)
129a,b (85.43 %)
108a (84.38 %)
Ketamine predominantly causes cardiac enlargement
Characteristics of ketamine-treated embryos (0.5 mg/ml) at stage 46
Ketamine (0.5 mg/ml)
17 (9.44 %)
29 (16.11 %)
3 (1.84 %)
124* (82.12 %)
2 (1.23 %)
116* (76.82 %)
1 (0.61 %)
8* (5.30 %)
1 (0.61 %)
7* (4.64 %)
3 (1.84 %)
30* (19.87 %)
1 (0.66 %)
Body length (mm)
10.53 ± 0.20
8.86 ± 0.86*
Because of the remarkable heart defects, we subsequently investigated the specific effect of ketamine on heart during embryonic development.
Ketamine increases cardiac cell proliferation
Ketamine results in embryonic heart dysfunction
Ketamine down-regulates XMLC2 expression
We studied cardiac morphology, cardiac function and the expression of genes associated with heart development after ketamine exposure primarily at stage 46. Because at this stage, the Xenopus embryonic heart has matured . Furthermore, there are three key stages for heart development: stage 28, heart tube formation; stage 32/33, contractile myocardial tube formation and looping initiation; and stage 35/36, obvious heart looping [18, 19]. Therefore, we chose these three stages to investigate the effect of ketamine exposure on early embryonic cardiomyogenesis.
Thus far, reports on the effects of early embryonic exposure to ketamine on organs or systems other than the nervous system are rare. An in vivo research demonstrated that ketamine exposure during the early development of zebrafish embryos caused concentration-dependent increases in anomalies and mortality; the most prominent deformities were enlarged organs and tail/spine anomalies , which is consistent with the results of our study. Additionally, we found that the shortening of embryonic body length is also in concentration-dependent manners.
At a dose of 0.5 mg/ml, the ketamine-treated embryos exhibited several malformations. However, the most prominent phenotype was an enlarged heart. Moreover, the cardiac cells underwent significantly increased proliferation. Our results also showed that at each detected stage of heart development, from heart tube formation to heart maturity, XMLC2 mRNA and protein expression levels were significantly down-regulated after ketamine exposure. It has been reported that MLC2, myosin regulatory light chain 2, is essential for normal myocardium development . A defect of MLC2 gene results in myocardial malformations , and missense mutations of MLC2 in humans lead to hypertrophic cardiomyopathy [22, 23], resulting in heart enlargement and decreased cardiac contractility. In addition, deletion of MLC2 in mice results in cardiac enlargement . Therefore, the cardiac enlargement phenotype caused by ketamine may result from the down-regulation of XMLC2 mRNA and protein levels.
Furthermore, our results showed that VSF, a sensitive indicator of cardiac contractility, sharply decreased after ketamine treatment. It has been reported that MLC2 protein has a function in the maintenance of cardiac contractility. Deletion of MLC2 causes a reduced left ventricular ejection fraction and abnormalities in myofibril assembly in embryonic mice, and results in death due to cardiac dysfunction at E12.5 . Additionally, knockdown of MLC2 in chick embryos results in poorly developed sarcomeres . Because the sarcomere is the basic contractile unit, sarcomere structural abnormalities affect cardiac contractility. Therefore, the weakened cardiac contractility may be due to reduced expression levels of XMLC2 mRNA and protein.
Ketamine has been reported to reduce heart rates in pregnant and infant monkeys , as well as newborn human infants . During zebrafish embryogenesis, ketamine exposure at both 26 and 52 h post-fertilization results in a reduced heart rate , which is consistent with our results. Therefore, the results of heart rate and ventricular shortening fraction demonstrate that ketamine causes embryonic cardiac dysfunction.
Ketamine exposure from stage 8 to stage 21 results in concentration-dependent increases in anomalies and mortality. After 0.5 mg/ml ketamine exposure, the most prominent malformation is heart enlargement. Besides, the decrease of heart rate and VSF indicates heart dysfunction. The enlarged heart and decreased VSF may result from the down-regulation of XMLC2 mRNA and protein levels. These findings provide insight into the potential fetal defects and mechanism of ketamine exposure. However, additional studies are required to evaluate the potential risk during very early pregnancy in mammals.
This project was supported by the National Natural Science Foundation of China (NSFC) Grant 81200440. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Movie of control embryo. Control embryo at stage 46, showing heart with rhythmic and strong contractions. 60× magnification. (MOV 7950 kb)
Movie of ketamine-treated embryo. Note that the heart size is bigger and heart rate is slower than control embryo. The amplitude of heart beat is decreased. 60× magnification. (MOV 5408 kb)
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