Sertraline-induced 5-HT dysregulation in mouse cardiomyocytes and the impact on calcium handling

Figures

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  Figure 1.. Sertraline exposure model. C57BL/6J mice at the age of 10-wk old were used. The female mice either received saline or sertraline (intraperitoneal) each day for the 5 days before mating with males, and daily throughout pregnancy to the time of delivery. Pups were weighed immediately prior to daily administration of saline or sertraline from postnatal (PN) age 0 to 14 days. Pups of PN day 3 were euthanized for cardiomyocyte (CM) dissociation and culture. Pups of PN day 14 were euthanized for tissue analysis. Pups raised to adults were subjected to echocardiography at 10 wk of age. H&E, hematoxyline-eosin; miRNA, microRNA analysis; WB, Western blot.

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  Figure 2.. Sertraline exposure reduced body weights. A: individual scatter plots of body weights from two randomly selected mice per litter (plotting mean with range) were graphed in relation to postnatal age (PN 0–14) with saline (black circles) vs. sertraline (red circles). Box-plot of body weights (displaying min to max with all points) on PN day 21 (B) and at 10 wk of age (C), presenting data of saline (Female: open circles, Male: open triangles) vs. sertraline (Female: filled circles, Male: filled triangles). The figures presented the number of pups in each group. The significance between the sertraline and saline groups was determined through two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 3.. The effects of sertraline exposure on mouse heart weights at PN14 and 10 wk. Box-whiskers plot of heart weight (A), and Box-whisker plot of heart/body weight ratios (B). Individual data were plotted with min to max and show all points for saline (○) vs. sertraline (●). The figures displayed the pup count for each group. Significance between the sertraline and saline groups was established using two-way ANOVA with Tukey’s multiple comparison test. *P < 0.05.

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  Figure 4.. Sertraline affected Ca2+ oscillations in neonatal mouse cardiomyocytes (CMs). A: Fluo-4 Ca2+ indicator loaded in CMs (three cells are marked). B: raw Ca2+ oscillation signal traces from three cardiomyocytes obtained in a single sampling. The colors assigned (red, blue, and pink) correspond to the calcium signal traces of cell-1, cell-2, and cell-3 in A, respectively. C: the raw signal of cell-1 (red) was normalized to maximum. D: After setting reference initial time, to 10% of the Ca2+ upstroke to peak, the parameters characterizing the calcium transient dynamics of Ca2+ oscillation were extracted from the Fluo-4 signal (calcium transient) traces. They include period (peak-to-peak time), representing the duration of one complete oscillation (ms) (E); time-to-peak (TTP), the time from to to the peak of the oscillation (ms) (F); CaD30 time (the duration time from t0 to 30% of Ca2+ extrusion), characterizing a specific phase of the oscillation (ms) (G); CaD80 time (duration time from t0 to 80% of Ca2+ extrusion), capturing another phase of the oscillation (ms) (H); and decay time (the time from peak to 90% of Ca2+ extrusion), indicating the duration of the declining phase of the…

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  Figure 5.. Effect of sertraline on cardiac expression of calcium signaling genes. Hearts were harvested on postnatal (PN) day 14 from mouse pups treated with either sertraline or saline (n = 8 each group). Relative mRNA levels of key players regulating cardiac muscle contraction and relaxation within the intricate cardiomyocyte system, as determined by qPCR: Cacna1c (A), Calm1 (B), Casq2 (C), Jnt (D), Ncx1 (E), Pln (F), Ryr2 (G), Serca2a (H), Tnnc1 (I), Tnni3 (J), Tnnt2 (K), Tpm1 (L), and Trdn (M). Data are graphed as box plots (displaying min to max with all points) with ○ for saline, and ● for sertraline, respectively. Significance testing between the sertraline and saline groups was conducted using an unpaired, two-tailed t test. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 6.. Sertraline exposure affected the cardiac extracellular matrix. Hearts were harvested on postnatal (PN) day 14 from mouse pups treated with either sertraline or saline. A: mouse heart photos were captured using an AmScope Stereo Microscope (SM-1TSZZ-144S-10M) and software. Scale bar = 1 mm. Hematoxylin and eosin (H&E) stained sections were examined under a light microscope (Olympus BX-61) equipped with a ×10 eyepiece lens, along with a high-power objective lens (×40), scale bar = 20 µm (B); and with an oil immersion objective lens (×100) (C), scale bar = 10 µm. Nucleus is blue and cytoplasm is red, and collagen fibers show a varying red color. D: quantitative comparison of extracellular matrix (ECM) area fractions (%) between the sertraline and saline groups (16 slices total, n = 8 for sertraline and n = 8 for saline groups). Values of ECM area fractions were in means ± SE (%) in the graph. Significance between the sertraline and saline groups was determined through an unpaired, two-tailed t test. *P < 0.05.

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  Figure 7.. Sertraline exposure altered microRNAs (miRNAs) that epigenetically regulate gene expression in hearts. Hearts were harvested on postnatal (PN) day 14 from mouse pups treated with either sertraline or saline. A: volcano plot of miRNAs using log-plotting of P values against fold change, to visualize the results of a differential gene expression analysis. B: the relative mRNA expression levels of Slc6a4, Htr2a, and Htr2b in mouse hearts with sertraline exposure were significantly decreased relative to the saline control group (n = 8/group, *P < 0.01). C: the corresponding protein expression changes in mouse hearts were consistent with the effects on mRNA expression (n = 4/group, *P < 0.05, **P < 0.01). B and C were graphed using box-plot (displaying min to max with all points) with ○ for saline, and ● for sertraline, respectively. Of note, Slc6a4 and HTR2B were obtained using the same membrane. Statistical significance between the sertraline and saline groups was determined through an unpaired, two-tailed t test. *P < 0.05, **P < 0.01.

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  Figure 8.. Cardiac function analysis using echocardiography for adult mice with perinatal plus neonatal sertraline exposure vs. saline. A: heart rate. B: ejection fraction. C: representative echocardiographic images. A and B were graphed using box-plot (displaying min to max with all points) with ○ for female saline and Δ for male saline, and ● for female sertraline and ▴ for male triangles, n = 34 for each group, respectively. Statistical significance between the sertraline and saline groups was determined through an unpaired, two-tailed t test. *P < 0.05.