Preliminary Studies Towards the Examination of Hypoxia‐related Transcriptional Regulation of Ryanodine Receptor Activity in Pulmonary Arteries of Fetal and Newborn Sheep

Abstract

Before birth the pulmonary vasculature does not receive much blood supply and is relatively constricted. However, with birth the vasculature dilates and blood flow and oxygenation increase. Evidence indicates that pulmonary arterial dilation during birth is related to an increase in the activation of large conductance potassium channels that are activated by Ca2+ spark events after activation of ryanodine receptors (RyR) on the sarcoplasmic reticulum. This coupled mechanism is largely driven through activation of L-type Ca2+ channels (CaL), which stimulate RyRs. Depolarizing cells with 30 mM potassium activates CaL and drives RyR mediated Ca2+ events. Previous evidence from our group illustrates that long term maternal hypoxia disrupts Ca2+ sparks and causes pulmonary hypertension in newborn lambs. We intend to modulate key transcriptional processes to better understand the molecular mechanisms that disrupt Ca2+ spark, vasoreactivity, and pulmonary pressure following long term hypoxia. The current studies were therefore performed as a foundation for our upcoming studies that will examine transcriptional control of RyRs and potassium channels. We tested the hypothesis that we could replicate earlier work that showed membrane depolarization with 30 mM K increases Ca2+ spark activity through activation of RyRs. To address this hypothesis, pulmonary arterial myocytes of fetal and newborn sheep were examined in the presence and absence of 30K and 10 mM ryanodine, which is a RyR antagonist. The intracellular Ca2+ was recorded in myocytes of isolated pulmonary arteries that were loaded with Fluo-4 using line-scan techniques on a confocal microscope. Spatial and temporal characteristics of the events were analyzed using customized software. As predicted, 30K increased Ca2+ spark activity while ryanodine treatment decreased activity. This confirmatory evidence in fetus and newborns provides a platform for upcoming studies to evaluate the role of transcriptional regulation to cell excitability in response to maternal hypoxia.