Abstract
We have shown that the MAPK signaling protein ERK is phosphorylated in the ventricles of chronically hypertensive fetuses. We do not know if ERK activation is responsible for cardiomyocyte proliferation, enlargement or terminal differentiation occurring in these fetuses. As a preliminary step towards determining the role of ERK in fetal cardiac growth, we sought to determine if ERK phosphorylation could be inhibited acutely and chronically in the fetal heart using the MEK inhibitor U0126.
Methods: Near-term fetal sheep were surgically instrumented with vascular catheters and allowed to recover. U0126 was dissolved in DMSO at 1mg/ml for intravascular administration then diluted in Krebs-Henseleit buffer to 5μg/ml. To determine if U0126 administration blocked acute fetal cardiac ERK phosphorylation, 0.2mg/kg/hr of U0126 or vehicle was infused for 24 hours. Twenty minutes following injection of 6μg/kg angiotensin II (angII), fetal hearts were excised and expeditiously frozen in liquid nitrogen. To determine if U0126 administration blocked chronic fetal cardiac ERK phosphorylation, 0.2mg/kg/hr of U0126 or vehicle was infused for 4 days. These fetuses also received an infusion of plasma protein (∼13g/day), which we have shown to cause arterial and venous hypertension, cardiomyocyte growth, and cardiac ERK phosphorylation. At the conclusion of the experiment, a biopsy of the left ventricle (LV) was freshly prepared for protein analysis. Levels of protein expression and phosphorylation were measured by Western blot analysis. Data were normalized as phosphorylated-to-total protein.
Results: U0126 did not block the fetal arterial blood pressure or reflex heart rate responses to angII. U0126 blocked acute ERK phosphorylation following stimulation with angII. U0126 also blocked chronic ERK phosphorylation during arterial and venous hypertension induced by plasma infusion. Furthermore, isolated cardiomyocytes from the U0126-infused fetus did not display the same level of ERK phosphorylation as the vehicle-infused fetus after ex vivo stimulation with angII.
Discussion: U0126 does not alter fetal pressor response to angiotensin II or plasma infusion. Despite the poor solubility of U0126 in aqueous solutions, this MEK inhibitor can be successfully administered to the sheep fetus via intravascular catheters to block acute and chronic cardiac ERK phosphorylation.
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Jonker, S., Louey, S., Thornburg, K. et al. 21 In vivo Administration Of U0126 to the Sheep Fetus Blocks Acute and Chronic Cardiac ERK Phosphorylation.. Pediatr Res 60, 494 (2006). https://doi.org/10.1203/00006450-200610000-00043
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DOI: https://doi.org/10.1203/00006450-200610000-00043