Molecular mechanisms underlying adverse effects of dexamethasone and betamethasone in the developing cardiovascular system

Abstract

Antenatal glucocorticoids accelerate fetal lung maturation and reduce mortality in preterm babies but can trigger adverse effects on the cardiovascular system. The mechanisms underlying off-target effects of the synthetic glucocorticoids mostly used, Dexamethasone (Dex) and Betamethasone (Beta), are unknown. We investigated effects of Dex and Beta on cardiovascular structure and function, and underlying molecular mechanism using the chicken embryo, an established model system to isolate effects of therapy on the developing heart and vasculature, independent of effects on the mother or placenta. Fertilized eggs were treated with Dex (0.1 mg kg^-1 ), Beta (0.1 mg kg^-1 ), or water vehicle (Control) on embryonic day 14 (E14, term = 21 days). At E19, biometry, cardiovascular function, stereological, and molecular analyses were determined. Both glucocorticoids promoted growth restriction, with Beta being more severe. Beta compared with Dex induced greater cardiac diastolic dysfunction and also impaired systolic function. While Dex triggered cardiomyocyte hypertrophy, Beta promoted a decrease in cardiomyocyte number. Molecular changes of Dex on the developing heart included oxidative stress, activation of p38, and cleaved caspase 3. In contrast, impaired GR downregulation, activation of p53, p16, and MKK3 coupled with CDK2 transcriptional repression linked the effects of Beta on cardiomyocyte senescence. Beta but not Dex impaired NO-dependent relaxation of peripheral resistance arteries. Beta diminished contractile responses to potassium and phenylephrine, but Dex enhanced peripheral constrictor reactivity to endothelin-1. We conclude that Dex and Beta have direct differential detrimental effects on the developing cardiovascular system.

FIGURE 1

Effect of Dex and Beta on biometry in the chicken embryo. Data are mean ± SEM for chicken embryos at day 19 of incubation following vehicle (Control: n = 10), dexamethasone (Dex: n = 10), or betamethasone (Beta: n = 15) treatment at day 14 of incubation. (A) Absolute embryo weight in grams; (B) Embryo weight relative to initial egg weight; (C) Absolute heart weight in grams; (D) Heart weight relative to body weight. Significant differences are (p < .05): *versus Control; †Beta versus Dex. One‐way ANOVA with Tukey post hoc test.

FIGURE 2

Effect of Dex and Beta on cardiac function in the chicken embryo. Data are mean ± SEM for chicken embryos at day 19 of incubation following vehicle (Control: n = 10), dexamethasone (Dex: n = 10), or betamethasone (Beta: n = 9) treatment at day 14 of incubation. (A) Heart rate; (B) Cardiac cycle duration (overall histogram describes overall cycle duration, black histogram is the diastolic component, and white histogram is the systolic component); (C) Left ventricular developed pressure (LVDP); (D) Contractility index; (E) Left ventricular end diastolic pressure (LVEDP); (F) Left ventricular relaxation time constant (Tau); (G,H) Left ventricular inotropic responses to isoprenaline and carbachol (Control: open symbols; Dex: blue; and Beta: red). Significant differences are (p < .05): *versus Control; ^†Beta versus Dex. One‐way ANOVA with Tukey post hoc test. One‐way ANOVA with Tukey post hoc test for Panels A–F; Two‐way RM ANOVA with Tukey post hoc test for Panels G and H.

FIGURE 3

Effect of Dex and Beta on cardiovascular morphology in the chicken embryo. Data are mean ± SEM for chicken embryos at day 19 of incubation following vehicle (Control: n = 7–8), dexamethasone (Dex: n = 6–8), or betamethasone (Beta: n = 5–10) treatment at day 14 of incubation. (A) Ratio of the lumen to wall volume in the left ventricle; (B) Ratio of the lumen to wall volume in the right ventricle; (C) Aortic wall thickness in micrometers; (D) Aortic lumen diameter in micrometers; (E) Width of cardiomyocytes in micrometers; (F) Cardiomyocyte volume in cubic micrometers; (G) Percentage of mononucleated cardiomyocytes; (H) Cardiomyocyte nuclei density per cubic millimeter. Significant differences are (p < .05): *versus Control; ^†Beta versus Dex. One‐way ANOVA with Tukey post hoc test.

FIGURE 4

Effect of Dex and Beta on cardiac molecular pathways in the chicken embryo. Data are mean ± SEM for chicken embryos at day 19 of incubation following vehicle (Control: n = 6–11), dexamethasone (Dex: n = 7), or betamethasone (Beta: n = 7) treatment at day 14 of incubation. (A) Protein carbonylation; (B) Protein expression of HSP70; (C) Protein expression of HSP27; (D) Protein expression of the ratio of pERK to ERK, the ratio of pSAPK/JNK to SAPK/JNK, the protein expression of HSP60, and the protein expression of PDI; (E) mRNA expression of MKK3, p16, p38, and p53; (F) mRNA expression of CDK2; (G) Protein expression of cleaved Caspase‐3; (H) GR protein levels relative to tubulin. Significant differences are (p < .05): *versus Control; One‐way ANOVA with Tukey post hoc test.

FIGURE 5

Effect of Dex and Beta on peripheral vascular reactivity in the chicken embryo. Data are mean ± SEM for chicken embryos at day 19 of incubation following vehicle (Control: n = 5–10), dexamethasone (Dex: n = 6–10), or betamethasone (Beta: n = 6–10) treatment at day 14 of incubation. (A) Vasorelaxant response to SNP (Control: open symbols; Dex: blue; and Beta: red); (B) Area above the curve for vasodilatation in response to SNP; (C) Vasorelaxant response to ACh (Control: open symbols; Dex: blue; and Beta: red); (D) Maximal vasodilatation in response to ACh; (E) Area for the NO‐dependent relaxation in response to ACh following treatment of the vessels with the NOS inhibitor L‐NAME; (F) Area for the NO‐independent relaxation in response to ACh following treatment of the vessels with the NOS inhibitor L‐NAME; (G) Constrictor response curves in response to K⁺ (Control: open symbols; Dex: blue; and Beta: red); (H) Area under the curve for vasoconstriction in response to K⁺; (I) Constrictor response curves in response to PE (Control: open symbols; Dex: blue; and Beta: red); (J) Area under the curve for vasoconstriction in response to PE; (K) Constrictor response curves in response to ET‐1 (Control: open symbols; Dex: blue; and Beta: red); (L) Maximal vasoconstriction in response to ET‐1. Significant differences are (p < .05): *versus Control; ^†Beta versus Dex. One‐way ANOVA with Tukey post hoc test. One‐way ANOVA with Tukey post hoc test for Panels B, D, E, F, G, J, and L; Two‐way RM ANOVA with Tukey post hoc test for Panels A, C, G, I and K.

FIGURE 6

Effect of Dex and Beta on indices of lung maturation in the chicken embryo. Data are mean ± SEM for chicken embryos at day 19 of incubation following vehicle (Control: n = 5–8), dexamethasone (Dex: n = 6), or betamethasone (Beta: n = 6) treatment at day 14 of incubation. (A) Diffusion distance from air capillary lumen to nearest vessel lumen in micrometers; (B) Air capillary diameter in micrometers; (C) Protein expression of Caveolin‐1; (D) Protein expression of SP‐B. Significant differences are (p < .05): *versus Control; One‐way ANOVA with Tukey post hoc test.

FIGURE 7

Data suggest that in Dex‐treated chicken embryos, oxidative stress signaling through pERK and JNK pathways may lead to p38‐mediated activation of caspase‐3, promoting cardiomyocyte hypertrophy. Conversely, in Beta‐treated chicken embryos, sustained activation of GR signaling through pERK/JNK/MKK3 pathways may lead to p53‐mediated repression of CDK2, triggering cellular senescence. This may explain the markedly reduced cardiomyocyte density, the dilated cardiomyopathy phenotype, yielding weak hearts with significant evidence of systolic and diastolic dysfunction in Beta‐ compared to Dex‐treated embryos.