Control of the heart rate of rat embryos during the organogenic period

Abstract

The aim of this study was to gain insight into whether the first trimester embryo could control its own heart rate (HR) in response to hypoxia. The gestational day 13 rat embryo is a good model for the human embryo at 5-6 weeks gestation, as the heart is comparable in development and, like the human embryo, has no functional autonomic nerve supply at this stage. Utilizing a whole-embryo culture technique, we examined the effects of different pharmacological agents on HR under normoxic (95% oxygen) and hypoxic (20% oxygen) conditions. Oxygen concentrations ≤60% caused a concentration-dependent decrease in HR from normal levels of ~210 bpm. An adenosine agonist, AMP-activated protein kinase (AMPK) activator and K_ATP channel opener all caused bradycardia in normoxic conditions; however, putative antagonists for these systems failed to prevent or ameliorate hypoxia-induced bradycardia. This suggests that the activation of one or more of these systems is not the primary cause of the observed hypoxia-induced bradycardia. Inhibition of oxidative phosphorylation also decreased HR in normoxic conditions, highlighting the importance of ATP levels. The β-blocker metoprolol caused a concentration-dependent reduction in HR supporting reports that β₁-adrenergic receptors are present in the early rat embryonic heart. The cAMP inducer colforsin induced a positive chronotropic effect in both normoxic and hypoxic conditions. Overall, the embryonic HR at this stage of development is responsive to the level of oxygenation, probably as a consequence of its influence on ATP production.

Keywords: ATP; bradycardia; embryo; embryonic heart rate; hypoxia; in vitro.

Figure 1

Effects of differing oxygen levels on HR of GD13 rat embryo. Notes: (A) HR (bpm) had significantly decreased compared to starting HR from 30 to 120 minutes when oxygen levels were <80%. (B) HR (bpm) recovered to control levels following return to 95% oxygen. Data are presented as bpm ± SEM. Abbreviation: SEM, standard error of the mean; bpm, beats per minute; HR, heart rate; GD, gestational day.

Figure 2

Effects on HR of GD 13 rat embryo in 95% oxygen after 1 hour. Notes: (A) 2,4-Dinitrophenol induced a concentration-dependent decrease in embryonic HR. (B) N6-cyclopentyladenosine (CPA) induced a concentration-dependent decrease in embryonic HR. (C) Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) induced a decrease in embryonic HR. (D) Pinacidil induced a concentration-dependent decrease in embryonic HR. Changes in HR (expressed as percent change from starting HR). Data are presented as mean ± SEM. *P<0.05 when compared to control. Abbreviations: HR, heart rate; SEM, standard error of the mean; GD, gestational day.

Figure 3

Effects on HR of GD 13 rat embryo in 20% oxygen after 1 hour. Notes: (A) Dorsomorphin did not prevent the drop in HR induced by 20% oxygen. (B) Glibenclamide did not prevent the drop in HR induced by 20% oxygen. Changes in HR (expressed as percent change from starting HR). Data are presented as mean ± SEM. *P<0.05 when compared to control. Abbreviations: HR, heart rate; SEM, standard error of the mean; GD, gestational day.

Figure 4

Effects on HR of GD 13 rat embryo after 1 hour. Notes: (A) DMOG and cobalt chloride induced minimal changes in HR in 95% oxygen. (B) IOX2 or BAY 87-2243 did not prevent drop in heart rate in 20% oxygen. Change in HR (expressed as percent change from starting HR). Data are presented as mean ± SEM. *P<0.05 when compared to control. Abbreviations: HR, heart rate; SEM, standard error of the mean; GD, gestational day; DMOG, dimethyloxalylglycine; IOX2, N-[[1,2-dihydro-4-hydroxy-2-oxo-1-(phenylmethyl)-3-quinolinyl]carbonyl]glycine; BAY 87-2243, 1-cyclopropyl-4-[4-[(5-methyl-3-{3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl}-1H-pyrazol-1-yl)methyl]pyridin-2-yl]piperazine.

Figure 5

Effects on HR of GD 13 rat embryo Notes: (A) Metoprolol induced a concentration-dependent decrease in embryonic HR in 95% oxygen after 1 hour. (B) Colforsin induced an increase in embryonic HR in 95% oxygen compared to control after 10 minutes but not 60 minutes. (C) Colforsin (3.6 and 7.2 mM) reduced the drop in embryonic HR in 20% oxygen compared to control after 10 and 60 minutes. (D) Heart rate decreased significantly within the first 15 minutes in 20% oxygen and 12 mM CPA compared to 95% oxygen. Change in HR (expressed as percent change from starting HR). Data are presented as mean ± SEM. *P<0.05 when compared to control. Abbreviations: HR, heart rate; SEM, standard error of the mean; AICAR, aminoimidazole-4-carboxyamide ribonucleoside; CPA, N6-cyclopentyladenosine; PIN, pinacidil.