INTRODUCTION
Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed therapy for depression.1 1–4 5 6 7–12
SSRIs inhibit the serotonin transporter, preventing reuptake of serotonin (5-HT).13 13 14 15,16 17,18 serotonin receptors important in ventricular development and cardiovascular regulation.19 2B receptor, which is expressed on the cell surface of cardiomyocytes, is also critical for normal cardiac development .20 2B receptor in mice leads to increased embryonic and neonatal death. In surviving 5-HT2B receptor null mice, cardiomyocyte proliferation is reduced and dilated cardiomyopathy develops in adulthood.20 2B receptor in mice leads to ventricular hypertrophy because of an increase in both cardiomyocyte number and size.21
We and others have demonstrated adverse cardiac effects in animal models with disrupted serotonin signaling, from either SSRI or serotonin-norepinephrine reuptake inhibitor exposure during development.22–25 22 cardiac development . Specifically, offspring exposed to sertraline in utero had reductions in neonatal cardiomyocyte cross-sectional area and proliferation, decreased expression of the 5-HT2B receptor, and reduced ventricular size in adulthood.23
Given the prevalence of SSRI use during pregnancy, it is important to better understand the effects of SSRIs on cardiac development . However, there are limitations to using rodent studies to determine whether the effects of SSRI exposure are direct or through a maternal or placental influence. Zebrafish provide an ideal model to address this issue because they develop externally. Zebrafish embryos develop in translucent eggs that can be easily visualized to monitor each stage of cardiac development after fertilization. Moreover, zebrafish embryos are easily amenable to pharmacologic administration and manipulation of gene expression.26
Here, we tested our hypothesis that sertraline exposure or 5-HT2B disruption in zebrafish embryos leads to reduced ventricular size in zebrafish . Using both the pharmacologic and genetic approaches, we were able to assess the role of serotonin signaling in heart development. Because the genetic networks that regulate vertebrate heart development are well conserved across species, our studies are expected to be highly relevant to human cardiac development .
METHODS
Zebrafish Strain, Maintenance, and Sertraline ExposureZebrafish were maintained as previously described.27 28 zebrafish lines were used: AB*/Tuebingen, Tg(cmlc2:GFP) ,29 Tg(nkx2.5:Zsyellow) ,30 and Tg(cmlc2:nls-EGFP) .31 Zebrafish embryos were exposed daily to either 1% dimethyl sulfoxide (DMSO) for vehicle control or the SSRI sertraline (5–20 µM in 1% DMSO) from 5.5 to 72 hpf or from 20 to 72 hpf.32,33 26
Mutant Lines
Zebrafish embryos carrying the mutant allele sa16649 of htr2b (htr2b sa16649 Zebrafish International Resource Center (ZIRC, Eugene, OR). This specific nonsense mutation incorporates a C-to-A nucleotide transversion in exon 3, introducing a premature stop which results in a loss of 211 amino acids. To genotype the sa16649 allele, DNA fragments were amplified using the following primer: forward, 5′-CCCTCGCAATCATGATGATCATTTACTTGC-3’; reverse, 5′-GATATGACCATTTTCTCTGGTGAAGCAAAAGC-3’. polymerase chain reaction (PCR) amplicons were digested with Hind-III restriction enzyme (New England BioLabs Inc, Ipswich, MA). Bands of 154 base pairs for wild type and 121 + 33 base pairs for homozygous mutants were detected using electrophoresis.
Live Images
To globally assess for toxicity, live embryos were mounted in 2.5% methylcellulose and bright-field images were taken on a Lecia M165FC stereomicroscope with a Leica DFC290 color digital camera at 32, 48, and 72 hpf during the exposure window.
In situ Hybridization
To evaluate cardiac development at specific stages, in situ hybridization was performed as previously described.34 ventricular myosin heavy chain (vmhc; 32, 48 hpf),35 nkx 2.5 (32 hpf),35 cardiac myosin light chain 2 (cmlc2; 48 hpf).36
Heart Dissection, Confocal Imaging, and Cardiomyocyte Counts
Using the Tg(cmlc2:nls-EGFP) 31 zebrafish line, embryos were treated with either 1% DMSO or sertraline from 5.5 to 72 hpf. Hearts were dissected at 48 hpf according to the methods described by Lombardo et al 37 Z -stacks were acquired at 0.5 µM intervals using the following settings: 1024 × 1024 pixels, 8 speed, 4 averaging. To quantify the number of cardiomyocytes in each image, ImageJ software was used and GFP-labeled cardiomyocytes were counted by 3 blinded investigators.
Proliferation Studies
Using the Tg(cmlc2:nls-EGFP) 31 zebrafish line, embryos were treated with either 1% DMSO or sertraline from 5.5 to 72 hpf. Hearts were dissected at 72 hpf according to the methods described as above.37 31
RNA Isolation and Quantitative Real-Time PCR
To determine the cardiac expression of htr2b , heart was dissected as described above from sertraline and 1% DMSO-treated embryos at 48 hpf and transferred into Trizol (Life Technologies, Grand Island, NY). To obtain adequate cardiac mRNA, approximately 100 embryos were used per group as suggested by Lombardo et al.37 g for 15 minutes at 4°C and the aqueous phase transferred to a new tube. An equal volume of 100% EtOH was added to each sample's aqueous phase, and samples were transferred to the Zymo RNA Clean and Concentrator-25 purification kit (Zymo Research, Irvine, CA). The manufacturer's protocol was followed, with RNA eluted in RNAse-free water. RNA concentration and purity were assessed by absorbance measurement. RNA integrity was assessed by electrophoresis on a 1% agarose gel. cDNAs were then synthesized using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, Hercules, CA). Quantitative real-time PCR was performed with the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Primers (forward: 5′-TGCTGGTAGGACTGCTGGTG-3′, reverse: 5′-TGATGGACGCAGTGGAGAAC-3′) were used to detect htr2b , and primers (forward: 5′-GAGAAGTTCGAGAAGGAAGC-3′, reverse: 5′-CGTAGTATTTGCTGGTCTCG-3′) were used to detect the housekeeping gene eef1a1a as a reference.
Echocardiography To assess long-term cardiovascular effects from embryonic exposure, baseline echocardiograms were performed beginning at 4 months of age on zebrafish that received either 1% DMSO or sertraline as embryos. Zebrafish embryos that received 15 µM sertraline from 5.5 to 72 hpf did not survive beyond 1 week of life, so subsequent sertraline doses were decreased to 5, 7.5, and 10 µM. Echocardiograms were performed as described38 Zebrafish were anesthetized with tricaine. Zebrafish were placed abdominal side facing upwards on a small sponge. The sponge allowed fish to be in stable position while keeping them moist with the water system. The zebrafish body was covered with a thin plastic sheet to protect the gills from ultrasound gel. The probe was applied gently to the chest, and ventricular long-axis and short-axis images were acquired and stored off-line for analysis blinded to treatment condition. Spongiform architecture of zebrafish myocardium precludes ultrasonographic delineation of the blood-endocardium interface. Ventriculographic measurements were therefore based on visual identification of epicardial borders only. Thus, for example, stroke volume is reported as the change in total ventricular volume, including myocardium, from end-diastole to end-systole. Fractional area of change was determined using ventricular long-axis images. Fractional shortening was determined using ventricular short-axis images. End-diastolic volume, stroke volume, and ejection fraction were determined by the bi-plane area-length method, using companion software supplied by the vendor (VisualSonics). The heart rate was determined using pulse-wave Doppler interrogation of ventricular inflow.
Analysis
To assess heart development at the various stages, all in situ hybridization images of the same type were acquired using the same settings, processed using the MetaMorph software, and edited and compiled using Adobe Photoshop and Adobe Illustrator software (San Jose, CA). Data were compiled from each individual embryo. To objectively describe data, image quantification was performed using ImageJ software. All quantification analyses show individual measurements and mean ± standard error of the mean. Statistical analyses were performed using the Student's t test. P < 0.05 was considered significant.
RESULTS
Sertraline-Exposed Zebrafish Embryos Demonstrate Decreased Heart Size and Cardiomyocyte Number
To initially assess whether sertraline exposure influenced development in zebrafish embryos, either sertraline (5–20 µM in 1% DMSO) or 1% DMSO (vehicle control) was added to daily E3 medium from 5.5 to 72 hpf (early exposure) or from 20 to 72 hpf (late exposure). Live imaging was performed at 32, 48, and 72 hpf. Initial dose titration curves indicated that sertraline doses of 20 µM frequently resulted in death of the embryos by 72 hpf. In addition, no developmental effects were seen with 5 µM sertraline dosing at 32, 48, and 72 hpf (data not shown). Pericardial edema, a nonspecific indicator of disrupted development, was observed in bright-field images in sertraline-treated embryos at both the early and late exposure time points (Fig. 1A ). This pericardial edema was observed in a dose-dependent manner. However, the phenotype was most notable with early exposure; approximately 60% of early exposure embryos treated with 15 µM sertraline developing pericardial edema by 72 hpf (Fig. 1B ).
FIGURE 1.: Sertraline exposure leads to pericardial edema. A, Live images of zebrafish embryos at 48 hours post fertilization (hpf) treated with 1% dimethyl sulfoxide (DMSO) or sertraline. Time sertraline exposure began is indicated in top right corner. Pericardial edema, a marker of disrupted development, is shown by the red arrow. B, The percentage of embryos with pericardial edema at 72 hpf after being treated with 1% DMSO or sertraline from 5.5 to 72 hpf or 20 to 72 hpf. Data shown as mean ± SEM. **P < 0.01 compared to control.
To evaluate the impact of exposure window on cardiac development , we assessed vmhc expression at 32 and 48 hpf in embryos treated with 1% DMSO or 15 µM sertraline beginning at 5.5–72 hpf or 20–72 hpf. As compared with 1% DMSO, the cardiac expression pattern of vmhc was reduced in sertraline-exposed embryo at 32 and 48 hpf, particularly among the embryos that were exposed at 5.5 hpf (Fig. 2 ). Based on these representative images and the percentage of sertraline-treated embryos with pericardial edema (Fig. 1B ), further experiments only used the 5.5 to 72 hpf exposure window.
FIGURE 2.: Zebrafish embryos exposed to sertraline from 5.5 to 72 hpf or 20 to 72 hpf demonstrated. Smaller ventricular size at 32 and 48 hpf. In situ hybridization depicts diminished expression of vmhc in sertraline-treated embryos treated from 5.5 to 72 hpf or 20 to 72 hpf compared with controls. At 32 and 48 hpf indicating reduced or delayed formation of the ventricle with sertraline exposure.
To evaluate ventricular size and number of cardiomyocytes, hearts were extracted from Tg(cmlc2:nls-EGFP) 31 zebrafish embryos treated with either 1% DMSO or 15 µM sertraline at 48 hpf. Representative confocal images are shown in Figure 3 . Sertraline-exposed embryos had a decrease in heart size (Fig. 3B vs. Fig. 3A ) with a significant decrease in the total number of cardiomyocytes compared with controls (sertraline-exposed 114.8 ± 3.8, control 166.4 ± 6.2, P < 0.01, n = 7 embryos/group counted by 3 independent, blinded investigators, Fig. 3C ). These data indicate that exposure to the SSRI sertraline reduces the cardiomyocyte number of zebrafish embryos, consistent with our prior findings in mice23 15,16 zebrafish , the overall number of proliferating cardiomyocytes was low in zebrafish embryos treated with 1% DMSO or sertraline (see Figure 1 , Supplemental Digital Content 1 , https://links.lww.com/JCVP/A835
FIGURE 3.: Sertraline exposure leads to decreased total numbers of cardiomyocytes. Hearts were dissected from Tg(cmlc2:nls-EGFP) embryos treated with 1% DMSO or 15 µM sertraline beginning at 5.5 hpf. Confocal images at 48 hpf suggested smaller heart size in sertraline-treated embryos (Figs. 1B vs. 1A). GFP-labeled cardiomyocytes were significantly reduced in sertraline-treated embryos (Fig. 1C, n = 21 counts/group; 7 embryos per group counted by 3 blinded, independent reviewers). Expression of htr2b, the gene encoding the 5-HT2B receptor, quantified by qRT-PCR was decreased in sertraline-exposed embryos. Compared with controls, the housekeeping gene eef1a1a was unchanged (Fig. 1D). **P < 0.01 compared with control.
Expression of htr2b, the Gene Encoding the Serotonin Receptor Subunit 5-HT2B , Is Decreased in Sertraline-Exposed Zebrafish Embryos
We previously demonstrated that sertraline exposure in pregnant mice leads to decreased cardiac expression of the 5-HT2B receptor.23 2B receptor has been shown to be critical to normal cardiac development because inactivation and overexpression of the 5-HT2B receptor affects cardiomyocyte proliferation and cardiac development .20,21 htr2b expression was decreased in sertraline-treated zebrafish embryos, we isolated RNA from hearts of sertraline-exposed and control embryos, synthesized cDNA, and then quantified htr2b mRNA levels by real-time PCR. Sertraline-exposed zebrafish hearts had decreased cardiac expression of htr2b mRNA compared with control embryos (n = ∼100 embryos/group, Fig. 3D ). These results suggest that sertraline exposure decreases expression of the RNA encoding the 5-HT2B receptor in zebrafish embryos, paralleling our findings in neonatal mice.
Ventricle Size Is Reduced by the Deletion of htr2b and Treatment With Sertraline
To determine whether deletion of htr2b would result in a decreased ventricle size, we acquired from the ZIRC stock center a zebrafish strain with a nonsense mutation near the start of exon 3 of htr2b (htr2b sa16649 ) (Fig. 4A ). Homozygous mutants are viable as adults. To assess the consequence of this mutation to htr2b transcript levels, we crossed 2 mutants and assessed htr2b mRNA levels in mutant embryos and in wild-type (WT) embryos from a separate clutch at 48 hpf. htr2b mRNA levels were at least 8-fold lower in the mutants in comparison with in WT embryos (n= ∼30 embryos/group, Fig. 4B ). This was unexpected because mutations in terminal exons do not traditionally induce non–sense-mediated decay.39 htr2b sa16649 40 htr2b sa16649 cmlc2 expression at 48 hpf in WT embryos treated with 1% DMSO or 15 µM sertraline and htr2b mutants treated with 1% DMSO or 15 µM sertraline. As compared with WT, the cardiac expression pattern of cmlc2 is reduced in mutants (Fig. 4C ). The spectrum of phenotypes observed in each of the 4 groups is demonstrated more clearly in Supplemental Digital Content 2 , (see Figure 2 , https://links.lww.com/JCVP/A836 2 in htr2b mutants treated with sertraline [WT + 1% DMSO (n = 23): 48,311 ± 1406, WT + sertraline (n = 30): 44,879 ± 1061, htr2b + 1% DMSO (n = 31): 44,751 ± 1907, htr2b + sertraline (n = 27)**: 38,633 ± 2029; **P < 0.01 compared with WT + 1% DMSO, Fig. 4D ].
FIGURE 4.: A, Schematic of exon structure of ZIRC htr2b mutant. B, qRT-PCR in ZIRC mutants confirmed loss-of-function of the htr2b gene. C, In situ hybridization of WT and ZIRC mutants treated with 1% DMSO (vehicle control) or sertraline. At 48 hpf, cardiac expression of cmlc2 is reduced in htr2b mutants compared with WT with a more pronounced reduction in ventricular size in mutants exposed to sertraline. Figures demonstrating representative images and outline of ventricular size using ImageJ are shown. D, Results of total pixel2 as calculated by ImageJ in treated embryos (n = 23 WT + DMSO, n = 30 WT + sertraline, n = 31 mutants + DMSO, n = 27 mutants + sertraline). **P < 0.01 compared with control.
Sertraline Exposure Did Not Result in Long-Term Cardiovascular Changes in Volume Status or Function as Measured by Echocardiography
Whereas our previous work in mice showed that sertraline exposure in utero led to decreased shortening fraction and stroke volume in adult mice,23 zebrafish embryos did not affect adult cardiovascular status. At age 4 months, echocardiograms were performed in adult zebrafish with embryonic exposure to 1% DMSO or sertraline. A representative picture of echocardiography images in the long-axis and short-axis for systole and diastole is shown in Figure 5 . No differences were observed in any of the measured echocardiography parameters between zebrafish embryos treated with 1% DMSO or sertraline (n = 29 controls, 39 sertraline, Fig. 6 ). In addition, there was no difference in the overall heart rate (Fig. 6 ). It is important to note that because embryos exposed to sertraline doses of 15 µM did not survive after the exposure window ended (after 72 hpf), we modified our dosing regimen to 5–10 µM during the 5.5–72 hpf exposure window. For most experiments in this article, sertraline doses of 15 µM were used. After this modification, there were no differences in the number of zebrafish deaths between the sertraline and DMSO exposure groups from the end of the exposure window (72 hpf) to age 4 months (data not shown). All 3 sertraline dosing groups were combined into one group for the purpose of comparison because of the small number in each individual dosing group. However, when evaluated by individual concentrations, a dose-dependent trend toward decreased ejection fraction was observed with increased doses of sertraline [ejection fraction—5 µM sertraline (n = 9): 56.3 ± 5.1%, 7.5 µM sertraline (n = 13): 54.7 ± 4.2%, 10 µM sertraline (n = 17): 44.67 ± 3.7%].
FIGURE 5.: Echocardiography in a zebrafish treated with vehicle (1% DMSO). Diastolic (red) and systolic (green) epicardial silhouettes were identified visually, in long-axis and short-axis planes, assisted by video playback, which distinguished epicardium from pericardium (blue arrows). Spongiform architecture of ventricular myocardium prevents identification of fluid-endocardial interfaces. Pulse-wave Doppler interrogation of ventricular inflow (inset, diastole) and ventricular outflow (inset, systole) served to confirm the location of the atrioventricular plane and to calculate heart rate. Yellow scale bars = 200 µM.
FIGURE 6.: Echocardiograms were performed in sertraline and 1% DMSO-treated embryos at age 4 months (n = 29 control, n = 39 sertraline). No differences were observed in any of the echocardiography measurements between the 2 groups.
DISCUSSION
The use of SSRIs to treat depression during pregnancy has increased over the past decade, and SSRIs are now prescribed to 10% of pregnant women in the United States.1–4 7–12 cardiac development . To the best of our knowledge, this is one of the first studies investigating the cardiac effects of sertraline exposure in zebrafish embryos, a valuable model system for the study of heart development.
The major finding of this study is that sertraline-exposed zebrafish embryos have decreased total cardiomyocyte number and ventricular size. These data are consistent with our prior work demonstrating reductions in ventricular size in an animal model of in utero SSRI exposure and a small cohort of term infants exposed to SSRIs in utero.23,41 15,16,23
Another important finding in this study is the reduced expression of cardiac htr2b in sertraline-treated zebrafish embryos. The htr2b gene encodes the 5-HT2B receptor that is expressed on the cell surface of cardiomyocytes and is critical for normal cardiac development .20 2B receptor in mice leads to increased embryonic and neonatal death.20 2B receptors that survive embryogenesis have reduced cardiomyocyte proliferation and develop cardiomyopathy.20 2B receptor in offspring of pregnant mice treated with sertraline.23 zebrafish , we again demonstrated that htr2b levels were significantly decreased in hearts dissected from sertraline-exposed embryos compared with those dissected from controls. Because these embryos can survive even in the presence of severe cardiac defects, zebrafish serve as an important model system to better understand the impact of disrupted 5-HT signaling on cardiac development .
Studying zebrafish embryos allows us to further evaluate the link between SSRIs and the 5-HT2B receptor. We found that both sertraline exposure and disruption of the 5-HT2B receptor affected ventricle development. Notably, this effect on ventricle development was most prominent when the htr2b mutant was also treated with sertraline. Because ventricle size was significantly reduced in embryos lacking htr2b treated with sertraline, this indicates there is a relationship between SSRIs and the 5-HT2B receptor. It has been proposed that the 5-HT2B receptor is critical to the action of SSRIs.42,43 42 2B receptors in rats causing them to postulate that the 5-HT2B receptor may positively modulate serotonergic activity required for the therapeutic actions of SSRIs. Our findings suggest that SSRIs do not act exclusively through the 5-HT2B receptor to affect heart development because htr2b mutants were affected by treatment with sertraline. The serotonergic system is complex with several serotonin receptors important in ventricular development and cardiovascular regulation, particularly 5-HT2A , 5-HT4 , and the serotonin transporter.19 2B receptor and pharmacologic inhibition of the serotonin transporter, but SSRIs could function through different receptors. There are 7 different 5-HT receptors and at least 15 subpopulations. Notably, the 5-HT2A receptor is expressed in the heart and mediates a hypertrophic response to 5-HT.44 4 receptor is expressed in fetal ventricles, and this receptor mediates the inotropic response to 5-HT late in development.45 46 2B receptor. These additional serotonin receptors and signaling pathways may be important to better understand the cardiac effects observed in models of SSRI exposure with disruptions to the 5-HT2B receptor.
An alternative hypothesis for our findings is that sertraline exposure delays development. Fraher et al32 23,47,48 49,50 51–57 zebrafish embryos were not observed in our bright-field images, we cannot exclude this as a possibility for our findings. The phenotype of reduced ventricular size in sertraline-exposed embryos was not corrected at 72 hpf, but we did not observe later time points. Although no differences were observed in volume status by echocardiography at age 4 months, our findings are confounded because we had to reduce our sertraline dosing regimen for embryos to survive long enough to obtain echocardiograms.
Our study has several important limitations. First, the dosing regimen for zebrafish embryos may exceed clinical dosing regimens.32 zebrafish embryos are not well established, and similar findings were observed in a preclinical mouse model with sertraline levels consistent with fetal exposure.23 htr2b translation may have important implications in cardiac development . It may be important to investigate a larger loss-of-function mutation in the htr2b gene.
CONCLUSIONS
We have demonstrated that sertraline exposure in zebrafish embryos leads to decreases in cardiomyocyte number, reduces the expression of cardiac htr2b mRNA levels, and reduces ventricular size. Importantly, in our previous study using mouse models, we found reduced cardiomyocyte number, cardiomyocyte proliferation, and stroke volumes after sertraline exposure during development. In addition, both models suggest an important role of the 5-HT2B receptor in SSRI-associated cardiac defects. However, this model indicates that the SSRI sertraline does not act exclusively through the 5-HT2B receptor. Zebrafish serve as an ideal model system to study cardiac development for multiple reasons including external development apart from mother, easy visualization, and easy pharmacologic and genetic manipulation. We can now focus on the mechanistic pathways for these associations and other serotonin receptor-mediated pathways. In pursuing this research, we furthermore hope to provide new insights into the impact of maternal depression treatment on offspring cardiac development .
ACKNOWLEDGMENTS
The authors acknowledge Anthony Zhang and Blake Bonde for their assistance with experiments in this project and Alyssa Bosko for design and technical performance of the echocardiographic studies. The authors acknowledge the Caroline Burns and C. Geoffrey Burns Laboratory at Massachusetts General Hospital for the Tg (cmlc2:nls-EGFP) zebrafish line.
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